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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Colorectal cancer is the third most common cause of cancer-related deaths and the second most prevalent (after breast cancer) in the western world. High metastatic relapse rates and severe side effects associated with the adjuvant treatment have urged oncologists and clinicians to find a novel, less toxic therapeutic strategy. Considering the limited success of the past clinical trials involving peptide vaccine therapy to treat colorectal cancer, it is necessary to revise our knowledge of the immune system and its potential use in tackling cancer. This review presents the efforts of the scientific community in the development of peptide vaccine therapy for colorectal cancer. We review recent clinical trials and the strategies for immunologic monitoring of responses to peptide vaccine therapy. We also discuss the mechanisms underlying the therapy and potential molecular targets in colon cancer.Colorectal cancer (CRC) is the third most common cause of cancer-related deaths among men and women in the western world [1,2]. Europe has the highest incidence of CRC with an estimated 412,900 new cases in 2006 [1] closely followed by the USA with 145,290 colorectal cancers in 2008 [3]. With a prevalence of around 2.4 million cases, CRC ranks as the second most prevalent, after breast cancer. The discrepancy between incidence and prevalence reflects high rates of survival due to early detection and effective management [4]. Treatment with curative intent invariably involves surgical resection for stages I-III, followed by 5-fluorouracil-based adjuvant therapy for stages II–III. The latter is considered an essential part of the treatment due to the high metastatic relapse rates (75%) within 3 years after resection [5]. Although the adjuvant treatment substantially improves survival, it is associated with side effects that affect overall health and the quality of life [6]. Therefore it is necessary to develop a novel, less toxic adjuvant therapy to treat the disease, especially disseminated CRC.Since the discovery of tumour antigens in 1991, immunotherapy has become a potential alternative to conventional chemotherapy [7]. In 2010, immunotherapy in colon cancer started to become a reality with several clinical trials involving peptide vaccine therapy underway. Here we review recent advancements in the development of peptide vaccine therapy, including the clinical trials, and discuss the mechanisms underlying the therapy and potential molecular targets in colon cancer. The immunologic monitoring of cellular immune responses following peptide vaccine therapy is also discussed. Immunotherapy is based on the principle of aberrant expression of proteins by tumours, which either over-express ubiquitous proteins or express proteins that unusually occur in the original tissue of the cancer. Tumour cells that bear these antigens can be distinguished from the normal tissue by the immune system in the same way that bacteria or virus-infected cells are. Recognition of an unusual antigen starts an immune response aimed at elimination of the cell and generation of antigen-specific immune cells that provide long-lasting immunity. However, immune-mediated regression of cancer hardly ever takes place in real life due to the low effectiveness of wild-type antigens (generated by the tumour) in stimulating host immune responses. The aim of vaccine therapy is to provide a highly immunogenic antigen, such as peptides, which are capable of stimulating immune system to mount a cytotoxic attack against the tumour cells. The desired effect would be stabilization or even regression of the disease.Following an inoculation, the circulating peptides are degraded in the circulation and the antigenic products are endocytosed by antigen-presenting cells (APC), which migrate to lymph nodes where they present the antigen in a processed form to T cells. These are at the forefront of the battle against the tumour. There are two main types of T cells according to the surface markers and function. CD8+ T cells, also known as cytotoxic T lymphocytes (CTL), are responsible for direct killing of tumour cells. They induce apoptosis by release of granzymes and perforins or by presenting Fas ligand (FasL) to Fas death receptor on the target cells. Another consequence of granzyme release is target cell necrosis [8]. CD4+ T cells differentiate further upon activation. CD4+ T cells type 1 act as helper cells that secrete cytokines to recruit more CTLs, as well as other members of the innate immune system such as macrophages and natural killer cells. The interaction between individual cells is mediated by two major histocompatibility complex (MHC) proteins, MHC classes I and II (also known as HLA I and II), present on the surface of cells. The antigenic protein from the circulation is processed by cellular machinery into a several-amino acid-peptide that can enter the MHC molecule for presentation to T cell receptor (TCR). MHC class I is responsible for presentation of the vaccine-derived peptide between APC and naïve CTLs. Primed CTLs are able to recognize the genuine tumour antigen presented by the MHC I on the surface of the tumour. Thus activated CTL sends out a death signal to the tumour.MHC class II is responsible for “the talk” between APC and CD4+ T cells and subsequent generation of helper T cells. Cytokines secreted by helper T cells, such as IL-2, are essential for activation of strong cytotoxic pathways and potentiation of anti-tumour response [9]. All in all, the CTL encounter with the peptide is at the heart of peptide vaccine therapy. More details of interaction between individual cells can be found in Figure 1.The mechanism of anti-tumour effect of peptide vaccine therapy: introduction of vaccine to the bloodstream; processing and presentation of the peptide by the antigen-presenting cell (APC) in a lymph node resulting in activation of CD4+ helper T cells and CD8+ cytotoxic T cells; interaction between MHC I molecule on APC and T cell receptor (TCR) during antigen presentation facilitated by CD8 molecule; generation of tumour-specific CTLs capable of lysing tumour cells: degranulation of CTL following recognition of tumour antigen and Fas-mediated transduction of death signal to the tumour.Various immune cells are normally found infiltrating the mass of a tumour, some of which are specialized to recognize the tumour-associated antigens. Although there is evidence of a positive correlation between the number of these cells and good prognosis, their effect on the tumour is considered insignificant [10,11]. It has been proposed that tumours are capable of developing ways to evade immune system or down-modulate immune responses [12]. One such mechanism is expression of FasL, which is a ligand for Fas receptor present on CTL. Fas functions as a death receptor which, upon binding of its ligand expressed on the tumour surface, induces CTL to undergo apoptosis. Additionally, expression of FasL provides a degree of resistance to Fas-induced apoptosis in tumour cells themselves [13].Another mechanism involves expression of altered peptide ligands for T cell receptors (TCR) by the tumour. “Altered Peptide Ligand” (APL) are analogs of immunogenic peptides in which the TCR contact sites have been manipulated [14]. Recognition of a peptide ligand by CTL leads to lysis of the tumour cell. However, ligands that are slightly altered retain their ability to bind the TCR but the outcome of this interaction is different. The altered peptides may be related to the agonist ligand on the basis of their structural homology. Thus, partially activating APL is a subset of the antagonists and thereby modulates the activity of CTL [15].Oncogenic signalling pathways within tumour cells and immunologic checkpoints in the tumour microenvironment also play a crucial role in promoting immunologic tolerance. For example, tumour releases factors that induce inhibition of both innate and adaptive antitumor immunity. Stat3 activation in tumours, as well as Braf activation, can induce release of factors such as IL10 that induce Stat3 signalling in NK cells, granulocytes, inhibiting their tumouricidal activity. Stat3 is also activated within conventional dendritic cells (CDC) in the tumour, converting them to toleragenic DC, which can induce T cell anergy and possibly regulatory T cells (Treg). Plasmacytoid DC (PDC) or PDC-related cells in the tumour microenvironment upregulate indoleamine 2,3-dioxygenase (IDO), an enzyme that metabolizes tryptophan. T cells are very sensitive to tryptophan depletion. Tumours can express co-inhibitory B7 family members, such as B7-H1 and B7-H4, which downregulate T cell activation and/or cytolytic activity. They can also induce B7-H1 and B7-H4 expression on tumour associated macrophages (TAM). Related immature myeloid cells or myeloid suppressor cells can further inhibit antitumor T cells via production of NO by the enzyme arginase. [16,17].Regulatory T cells are an important inhibitor of antitumor immunity. T cell activation in the absence of appropriate co-stimulatory signals leads to T cell anergy and generation of induced regulatory T cells (Treg). Treg, characterized by the FoxP3 transcription factor, upregulate a number of cell membrane molecules, including Lag3, CTLA4, GITR, and neuropilin. Treg can inhibit effector T cell activation and function via T-T inhibition or inhibition of antigen presenting cells [17].Finally, cancers avoid recognition by the immune system by means of defective antigen presentation. This can be achieved by reduced expression of HLA type I (MHC I), which is a common event in colorectal cancer [18], as well as, reduced expression of antigen-processing machinery or tumour-associated antigen itself.Immune-competence of cancer patients who receive chemotherapy is already partially compromised. The design of peptide vaccines is aimed at overcoming the described problems by delivering highly immunogenic antigens combined with adjuvants that stimulate the immune system.Special design of the amino acid sequence of a peptide can enhance the interaction with the TCR. This usually involves creation of a peptide epitope with single amino acid substitutions, which exhibits improved binding to MHC or affinity for the TCR. Such manipulations are capable of potentiating secretion of interleukin 2 (IL-2, cytokine immune system signaling molecule), which is a potent immune-boosting cytokine. Other modifications, involving substitution at the peptide terminals, results in improved bio-stability and reduced degradation by seric proteases [19]. Effectiveness of the vaccine can also be improved by the peptide delivery system. The employment of vectors such as liposomes and dendritic cells with potent antigen-presenting properties has been exploited with success [20]. Use of adjuvants is another way to increase immunogenicity of a peptide. These adjuvants include IL-2 (immune system signaling molecule belonging to cytokine family) which activates CTL, GM-CSF which stimulates APC, as well as incomplete Freund’s adjuvant (IFA) and heat shock protein (hsp) 90 [9]. Novel solutions for adjuvant formulations come from the field of nanotechnology. Poly-γ-glutamic acid adjuvant nanoparticles used in colorectal cancer mouse model have been found to exert immune-boosting effect similar to that of complete Freund’s adjuvant which is considered the strongest adjuvant available. These nanoparticles have the advantage of causing little or no liver and kidney toxicity [21].The use of synthetic peptides is a fairly recent endeavour. In the early days of immune therapy tumour antigens were delivered to the patient in a form of tumour lysates. Nowadays tumour lysates are being pulsed into dendritic cells (DC) as a way of facilitating peptide presentation. Another recent development is the use of a retrovirus carrying the antigen-encoding gene, which inserts itself in the host genome leading to production of large quantities of the antigen [22]. Against this background, peptide therapy bears several advantages. Firstly it provides maximal essential component as opposed to tumour lysates where the antigen is diluted in a bulk of biological mass. Secondly, monitoring of immune response is easier with peptide-based therapy as it requires evaluation of only one cytotoxic T lymphocyte (CTL) type, which is specific for the peptide; while tumour lysates carry multiple antigens which make immune monitoring much more complicated. Furthermore, laboratory-based synthesis of peptides offers virtually unlimited possibilities for modifications and design enhancement. Last but not least, the process of generation of peptide vaccine and its use in the clinic is fairly cost-effective. The main drawback is the human leukocyte antigen (HLA)-type restricted nature of the therapy. HLA molecules are expressed in a multitude of super-types and individual antigens are only capable of interacting with a particular HLA-super type. In the clinical context this means that only patients carrying the required HLA allele will be able to respond to the therapy. This substantially limits the application of the therapy. Moreover, use of self-antigens carries a risk of inducing autoimmunity. This kind of therapy is also contraindicated in patients with existing autoimmune diseases, where exposure of the over-reactive immune system to a highly immunogenic antigen can have unpredictable consequences. An example of that comes from trials on immune therapy in Parkinson’s disease where the patients’ condition was reported to be aggravated following vaccination against a neuroprotein [22].Two aspects of response to peptide therapy are commonly evaluated in clinical setting: clinical outcome as the primary endpoint and stimulation of cell-mediated toxicity as a secondary endpoint. Several techniques have been used to measure the frequency and activity of antigen-specific CTLs, including ELISPOT assay, flow cytometry, RT-PCR, HLA/epitope tetramer assay. This is often combined with evaluation by means of serum cytokines and chromium (υ¹Cr) release assays, and proliferation studies [23]. The υ¹Cr release assays has a number of disadvantages, including low sensitivity, poor labelling and high spontaneous release of isotope from some tumour target cells. ELISPOT assays are used to assess secretion of proteins by CTLs which are indicative of an activated state, such as, INF-γ, granzyme B, IL-2 [24]. The assays are performed using peripheral blood mononuclear cells (PBMC) isolated from patient blood sample and the result is expressed as the number of reactive CTLs per 100,000 PBMC [23].Following T-cell receptor recognition of antigenic peptide–MHC class I complexes on the surface of target cells, CTLs induce target cell apoptosis through directed exocytosis of perforin and granzymes. The cytotoxic signalling leads to the activation of the caspase cascade that can be measured using flow cytometer. The assay involved labelling of P815, EL4 and T2 lymphoma cells with a cell tracker dye DDAO-SE and staining permeabilized cells with antibody against cleaved (activated) caspase-3. This assay proved to be robust and reliable in evaluating antigen-specific CTL [25].Another approach is to use flow cytometry for detection of apoptosis in CTL target cells (for example cytomegalovirus bearing appropriate HLA molecule). During apoptosis, phosphatidylserine (PS) is externalized on the surface of the cells and is available for binding of annexin V. Both granule marker and annexin V assays allow evaluation of the cytotoxic potential of tumour-specific CTLs [8].By contrast, HLA/epitope tetramer assay is used for mere enumeration of the antigen-specific CTLs. However, when combined with intracellular cytokine staining it gives a complete picture of quantity and function of the TCLs.Lastly, RT-PCR has been used to evaluate cytokine gene expression, including INF-gamma and granzyme B, by CTLs [25,26]. Cells are harvesting from patients who have been administered peptide vaccination followed by RNA extracted from it. cDNA is synthesised and RT-PCR analysis are performed using forward and reverse primers for IFNγ or CD8, The synthesised cDNA is further validated by the measurement of Gene expression using ABI Prism 7700 Sequence Detection System.Ideally the immune response should correlate with the primary endpoints, which reflect the disease progression. Sadly this has been exceptionally rare in the clinical trials so far. In addition to patient survival, the levels of tumour markers are measured using fluorescence-based immunoassays for monitoring of tumour growth and response to therapy [27]. Also, delayed-type hypersensitivity reactions are commonly performed and evaluated for macroscopic changes at the injection site and lymphocytic infiltration of the skin biopsy material [28]. Finally, CT scan has been employed for evaluation of liver metastases [29]. Tumour self-antigens are broadly categorized according to their specificity for the tumour. Differentiation tumour antigens present in both tumour and its original tissue, but are expressed at higher levels in the tumour. Tumour-specific antigens occur in tumours cells but are absent in normal adult tissues. Tumour/testis antigens are additionally expressed in the gonads. Unique tumour antigens constitute a separate group. These arise from nucleotide deletions or alternative splicing and characterize each single tumour. The choice of each one of these targets for vaccine therapy carries its specific risks as well as benefits. Targeting differentiation antigens, owing to their presence outside the tumour, can elicit a degree of adverse reaction in the healthy tissue. An example of that comes from clinical trials in melanoma where use of peptide vaccines directed against proteins involved in melanin biosynthesis leads to development of skin depigmentation (vitiligo). With regards to unique antigens, some evidence speaks in favour of their higher immunogenicity. The immune response against unique antigens has been found to persist for longer following tumour resection compared with shared tumour-specific antigens [30]. However, this notion remains controversial, as the data on unique antigens is scarce. On top of that, identification of unique antigens would require sequencing of the whole genome for every single tumour, which is not feasible in the present day. Nonetheless, a patient-tailored vaccine therapy is an appealing perspective. Tumour-specific antigens have been the most popular targets for vaccine therapy in general and in the context of colon cancer in particular. It must be noted that immunogenicity of colorectal cancers is considerably lower compared to the most immunogenic among cancers, i.e., melanoma. Nonetheless, the few antigen-based vaccines that have been developed show the ability to induce antigen-specific CTL and cause reduction in the tumour size in animal studies. Translation of these findings to a clinical setting was not completely successful. One particular pattern of response to peptide vaccine therapy prevails across all clinical studies. That is, strong induction of antigen-specific CTLs is not associated with improved clinical outcome. Frequently reported side effects of the therapy are: ulceration at the injection site, fever, fatigue, nausea, anorexia. However, serious adverse reactions are very rare. The protocols commonly require 3 or more injection courses at 2–3 weeks intervals and patients who show satisfactory response are offered continuation of the therapy. It has been argued that the lag between the first jab and the clinically relevant response is too long, especially in the case of advanced cancer patients who may not survive the necessary time. Below is a brief review of selected antigen targets in colon cancer including their biological role, results of in vivo studies and clinical trials. The remainder are summarized in the Table 1. Antigen targets in colon cancer, their biological role, results of in vivo studies and clinical trials for peptide vaccination.CEA-specific CTL responses were augmentedAntigen-specific proliferation of splenocytes and secretion of Th1 cytokines increasedSurvival rate increasedIn the therapeutic setting, tumour burden was significantly reducedIn the prophylactic setting, tumour was completely rejectedInhibition of tumour growth in a dose and route dependent mannerRepression of CRC lung metastasis in a dose dependent mannerInduction of colon carcinoma-specific CTLs in 52% patientsTwo-year overall survival and disease-free survival were significantly improvedIncreased cellular immune responses to the tumour and the vaccinated peptideDose-dependent responsesEphrin type-A receptor 2 (EphA2) is a member of a large tyrosine kinase receptor family. Eph receptors play an important role in oncogenesis [42] and tumour angiogenesis [43]. It has been found that EphA2 is over-expressed in colorectal carcinomas [44] and other various cancers [45]. The fact that highest level of EphA2 expression is observed in metastatic lesions makes EphA2 a high-priority target for immunotherapy [21]. Its utility as a tumour antigen has been evaluated in a liver metastasis mouse model transfected with EphaA2-positive colon cancer tumour. Immunization-induced antigen-specific CTL was associated with a degree of protection against the tumour expansion. A recent study using EphA2-derived peptide in combination with amphiphilic nanoparticles in a murine model demonstrated high a level of immunity against colorectal cancer liver metastases following immunization. Interestingly the study also found the novel nanoparticle-based adjuvant to be more beneficial in terms of effectiveness and toxicity [21]. Survivin is a protein which inhibits cancer cell apoptosis, is highly expressed during embryological development, and becomes undetectable in adult tissues [46]. A study of 171 cases of colorectal cancer found its abundant expression in more than 50% with no expression in adjacent normal tissue [47]. One of two variants of the protein, surviving-2B, was found to possess a peptide capable of binding HLA-A24. The peptide induced HLA-A24-restricted cytotoxic T cells, which subsequently exhibited high toxicity against HLA-A24-positive survivin-2B-positive cancer in vitro [48]. In 2003, the first clinical trial involving survivin-2B was conducted in advanced and recurrent colorectal cancer patients [24]. The therapy resulted in an increased proportion of peptide-specific CTL in the general population of circulating CTLs from 0.09% to 0.35%. However, this was not accompanied by significantly improved clinical outcome. One out of 17 patients showed minor reduction of tumour size and 6 patients had a reduced CEA marker confined to the duration of the therapy [29,35,48,49,50].SART3 is a tumour-rejection antigen which is expressed in more than 70% colorectal cancers but is not present in normal non-malignant tissues [51]. It was one of the first targets for vaccine therapy tested in a setting of a Phase I clinical trial. Myiagi et al. used a vaccine formulation combining two SART3 antigenic epitopes recognized by HLA-A24-restricted CTLs with IFA in colorectal cancer patients. Immunization resulted in significant induction of tumour-specific CTL in 7 of 11 patients. Two patients presented with induction of INF-γ following the sixth vaccination time. However, augmentation of cellular immunity was not associated with improved clinical outcome. Likewise, no induction of IgG or IgE specific for the peptides was observed. An adverse reaction at the injection site manifested as itching and redness occurred in 6 patients. The median number of vaccination times was 8 and the median observation period was 5 months. Despite the somewhat promising result no further trials involving SART antigen has been published since 2001 [52].Carcinoembryonic antigen (CEA) is a useful tumour marker which is correlated with tumour burden in the mouse metastasis model and has been used to monitor CRC metastasis development [53]. A mouse model for colorectal cancer was used to evaluate its suitability as a target for peptide vaccine therapy alone and in combination with an antibody that mimics a specific CEA epitope. Transgenic mice carrying tumours positive for CEA and HLA-A2 were vaccinated with dendritic cells pulsed with a CTL epitope of CEA. Complete tumour regression was observed in as many as 25% of mice. In the cured mice, no tumour recurrence was observed for 90 days. Use of the antibody leads to antigen presentation by both MHC I and II, whereby it stimulates CD4+ helper cells. The use of CEA-mimicking antibody was associated with production of INF-γ by CD4+, which is thought to improve the expression of MHC class I by the tumour. This approach resulted in increased proliferation and cytokine-production by tumour-specific CTLs and a complete sustained regression of the tumour in 67.5% mice. Overall, immunization with CEA resulted in enhanced immune response and survival. Further improvement in both endpoints was seen with the combination therapy. This constitutes an interesting case for employment of both antibody-mediated and cell-mediated immunity to achieve greater anti-tumour response. It also consolidates the importance of T helper cells in stimulation of cytotoxic killing of the tumour cells [54]. MUC-1 is an epithelial membrane antigen, which bound to the apical membrane of the secretory cells. It has been found that MUC-1 is expressed in more than 70% colorectal cancers and correlates with poor prognosis [55]. An early clinical trial involving mucin-like peptides was conducted in 1996 with unremarkable results and no further clinical investigations followed. However, recent animal experiments using MUC1 peptide-based vaccine therapy yielded promising results [36]. The researchers used a cocktail of strong adjuvants: MHC class II-restricted pan helper peptide, unmethylated CpG oligodeoxynucleotide, and GM-CSF. In addition to stimulating antigen-specific CTL, the vaccine elicited abundant secretion of INF-gamma by activated helper T cell type 1. This resulted in significant reduction of an established tumour. Interestingly, the vaccination was completely successful in preventing growth of a tumour transfected after immunization. This opens a window for the use of the therapy in the prophylaxis of colorectal cancer [28,36]. We have discussed some of the selected antigen targets in colon cancer including their biological role, results of in vivo studies and clinical trials. The remainder are summarized in the Table 1.Development of a novel, less toxic therapeutic strategy to treat advanced and disseminated colorectal cancer has been a goal for oncologists and clinicians for many years. The discovery of tumour antigens in 1991 has opened a window for the use of immune therapy in the treatment of cancer. In the last two decades, a great deal of effort has been made to identify the molecular targets for immune therapy in colorectal cancer; some of those targets proved effective in a pre-clinical setting. Moreover, the immune therapy has been shown to cause minimal side effects in a clinical setting, which makes it a desirable alternative to the current systemic treatments. Its ability to induce strong and specific anti-tumour immune responses in patients has been well documented using the current immunologic monitoring techniques. However, these findings did not correlate with improved clinical outcome. This calls for more accurate strategies for immune monitoring and could become the next direction for research.The author declares no conflict of interest.

Med-MDPI/vaccines/vaccines-01-01-00017.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Although mentioned in the UK pandemic plan, essential service providers were not among the priority groups. They may be important targets of future influenza pandemic vaccination campaigns. Therefore, we conducted a cross-sectional survey among 380 employees from West Midlands police headquarters and 15 operational command units in the West Midlands Area during December 2009–February 2010 to identify factors affecting intention to accept the pandemic influenza A (H1N1) vaccine. One hundred and ninety nine (52.4%) employees completed the questionnaire. 39.7% were willing to accept the vaccine. The most common reasons for intention to accept were worry about catching Swine Flu (n = 42, 53.2%) and about infecting others (n = 40, 50.6%). The most common reason for declination was worry about side effects (n = 45, 57.0%). The most important factor predicting vaccine uptake was previous receipt of seasonal vaccine (OR 7.9 (95% CI 3.4, 18.5)). Employees aged <40 years, males, current smokers, and those who perceived a greater threat and severity of swine flu were also more likely to agree to the vaccine. The findings of this study could be used to improve future pandemic immunization strategies. Targeted education programs should be used to address misconceptions; the single most important factor which might lead to a large improvement in uptake is to allay concern about side effects.In March 2009 the first cases of pandemic influenza A H1N1 virus were recorded [1]. The World Health Organization classified this outbreak, in June 2009, as phase 6 [2], indicating the start of a global pandemic. As a result of this, countries began implementing their pandemic plans [3]. On 21 October 2009 the UK began its national influenza pandemic vaccination program in preparation for the expected second wave of influenza infections [4].In comparison to seasonal influenza, the pandemic form was associated with higher hospitalization rates and mortality in younger adults under the age of 65 years, particularly those with underlying medical conditions [5]. It was also associated with more severe disease and increased complications in young children and pregnant women. This was the basis for the selection of target groups vaccinated in the pandemic H1N1 (2009) influenza vaccination program [4]. Although the UK Pandemic Plan stated that one of its goals was to “minimise disruption to health and other essential services” [3], essential service providers (such as the police and fire services) were not included in the priority vaccination groups and were not eventually offered vaccine during the H1N1 pandemic. However, it may be necessary to vaccinate them in future pandemics.Although uptake of both seasonal and pandemic influenza vaccine is known to be sub-optimal [5,6], the vast majority of studies exploring the determinants of influenza vaccine uptake are among healthcare workers. Doubts about efficacy, inadequate information, perception of not being at risk, vaccine safety and fear of side effects are the most prevalent reasons for vaccine declination [7,8,9,10,11,12].There is currently no literature addressing the question of vaccination acceptance among essential service providers. In the event of inclusion of this group in a future pandemic influenza vaccination program, it will be critical to understand how to maximize uptake.The aim of this study, therefore, was to identify the factors contributing to the likely acceptance or declination of pandemic influenza A (H1N1) vaccination among police workers in the West Midlands, UK.A questionnaire-based, cross sectional study among a population of West Midlands (WM) Police employees to identify factors affecting intention to receive pandemic influenza A (H1N1) 2009 vaccine, carried out during the winter pandemic of 2009-10.Following approval from the Occupational Health Department at the WM Police Headquarters, visits to all 21 Operational Command Units in the West Midlands were made from December 2009 to February 2010. Fifteen centers granted permission to distribute the questionnaires, which were left for one week at each station, before being collected.Twenty questionnaires were distributed to each participating Operational Command Unit, and 80 questionnaires were distributed in the Occupational Health Department at Police Headquarters to be completed only once by any of the staff employed by the WM Police Service. In total 380 questionnaires were distributed. All questionnaires were self administered and anonymous.The questions were selected after research of similar literature which provided direction towards potentially significant factors. Standard questions were used where available, these were based upon those used in available literature. A pilot study was carried out on 20 volunteers to improve comprehension.The primary outcome of the questionnaire was to determine the intention to have the pandemic influenza A (H1N1) 2009 vaccine. Participants who had already received the vaccine or those who would receive the vaccine if offered were classified as “intending to accept” the vaccine. In addition to this, the questionnaire also collected information on: sociodemographic factors, job title (later classed into office/non-office based jobs), number of dependents, personal or family illness, history of vaccination, history of pandemic influenza A (H1N1) infection, and general health beliefs, in particular those regarding the influenza A (H1N1) pandemic (See Appendix Figure A1).Data were analyzed using STATA version 11. Univariate associations were analyzed between intention to accept vaccine and important covariates. Factors found to be statistically significant (p < 0.005) or clinically important were entered into a multiple logistic regression model. Questions on health beliefs were measured on a 5-point likert scale but collapsed into 3 categories. Scores of 1/2 were classified as low, score 3 as medium, and scores 4/5 as high. Sensitivity analyses were undertaken to exclude those who had received the vaccine already.Between December 2009 and February 2010, 380 questionnaires were distributed and 206 completed (response rate 54.2%). Seven questionnaires were excluded as the respondents did not respond to the question about vaccine acceptance, leaving 199 for analysis (Figure 1).One hundred and five (52.8%) respondents were female (Table 1), their mean age was 38 (Range: 18–63) and 176 (88.4%) were of white ethnicity. The majority had a non-office based job (n = 149, 74.9%) and 57.1% education to at least A’level standard or equivalent. One hundred and twenty four (62.3%) had never smoked although 22 (11.0%) were current smokers. Thirty-two (16.1%) reported a long term illness and more than two-thirds exercised at least once per week. Of the respondents 86 (43.2%) reported seasonal influenza infection in the past, 90 (45.3%) had ever received a seasonal influenza vaccine (39 (43.3% of these reported side effects)). Twenty-eight (14.1%) reported having had “swine flu” during the 2009 pandemic.Flow chart illustrating response to questionnaire.* Numbers from: Sigurdsson J. and Mulchandani R. Police Service Strength. England and Wales, 30 September 2009. Home Office Statistical Bulletin [13].Characteristics of respondents.Characteristics of the sample were similar to WM police [13] in terms of the age and ethnicity distribution, although in our sample we had many more female respondents than in the police force as a whole. There were fewer smokers in our sample compared with the WM population [14] (data were not available on smoking in the West Midlands police) (Appendix Table A1).Eighty-one (40.7%) respondents felt there was a low threat of pandemic influenza A (H1N1) infection to the public, 90 (45.2%) felt there was a medium threat and only 28 (14.1%) felt there was a high threat to the public. Approximately half (n = 94, 49.8%) of respondents believed there was a low probability of catching pandemic influenza A (H1N1) virus and only 12% (n = 24) felt there was a high probability. Correspondingly, most (n = 93, 47.5%) felt the threat to their health was low, while 23.6% (n = 47) felt that the threat to their health was high. The majority of respondents (139, 69.8%) felt the media had overestimated the threat of the pandemic virus (Table 2).Respondents’ attitudes to Pandemic Influenza A (H1N1) 2009 virus.Of the 199 respondents, 14 (7.0%) had already received the vaccine, a further 65 (32.7%) said they would accept the vaccine if offered, and 80 (40.2%) said they would decline. 40 (20.1%) were still unsure if they would accept or not. Overall, therefore, intention to receive the vaccine was 79/199 (39.7%). The remaining analyses are based on the 159 respondents who either stated yes or no.The most common reasons for accepting the vaccine (Figure 2A) included worry about catching “Swine flu” (n = 42, 53.2%), infecting others (n = 40, 50.6%), and missing work (n = 20, 25.3%) (Figure 2A). Sixteen (20.3%) would accept because they would follow advice from employers/occupational health/Department of Health. The overwhelming reason for declination of the vaccine (Figure 2B) was worry regarding potential side effects (n = 45, 57.0%). However, 9 (11.4%) would/did decline because it was inconvenient, 9 (11.4%) were worried about the vaccine causing “Swine Flu”, 5 (6.3%) would decline because they had already had seasonal influenza vaccine, 7 (8.9%) had doubts about vaccine efficacy and 4 (5.1%) would decline because they had already had pandemic influenza infection that year. Other reasons stated by respondents for declination were: respondents not perceiving themselves to be at risk, simply not wanting to be vaccinated, fate, needle phobia, belief that “Swine flu” is only a threat to those in poor health, or contraindications to the vaccine itself.(A) Reasons reported by police workers for intention to accept the Pandemic (H1N1) 2009 vaccine; (B) Reasons reported by police workers for intention to decline the Pandemic (H1N1) 2009 vaccine.On univariate analysis (Table 3), three determinants related to either demographics or past medical history were significantly associated with the intention to accept the vaccine: non office-based employment (OR: 2.24, 95% CI: 1.06–4.72), having received a seasonal influenza vaccine in the past (OR: 4.08, 95% CI: 2.1–7.93), and history of pandemic influenza A (H1N1) infection (OR: 2.81, 95% CI: 1.03–7.68).Determinants associated with intention to accept the Pandemic Influenza A (H1N1) 2009 vaccine.* Includes 153 observations with complete data; Model 1 adjusted for age, sex, smoking status and prior receipt of influenza vaccine; Model 2 adjusted for age, sex, smoking status, prior receipt of influenza vaccine, ethnicity and type of job; Results in bold indicate p < 0.05.Three determinants related to health beliefs and perceptions were significantly associated with the intention to accept the vaccine (Table 4). The belief that “swine flu” posed a high threat to the public was significantly associated with acceptance (OR: 4.64 95% CI: 1.65–13.07). The belief that “swine flu” was a high risk to the respondents’ health was also significantly associated (OR: 3.01 95% CI: 1.36–6.68). In addition, if respondents felt there was a high likelihood of “swine flu” infection, this was significantly associated with intention to accept the vaccine (OR: 4.27 95% CI: 1.42–12.83).After adjustment (model 1) for age, sex and smoking status, participants who had ever received seasonal influenza vaccine remained significantly more likely to accept the pandemic influenza A (H1N1) vaccine (OR: 7.92, 95% CI 3.38–18.53), with employees over the age of 40 (OR: 0.36 (0.16–0.82), and females (OR: 0.47 (0.23–0.99)) significantly less likely to accept the vaccine, and current smokers more likely (OR: 4.89 (1.05, 22.72)). Additional inclusion of ethnicity and type of job (model 2) suggested a trend towards employees of non-white ethnicity and a job outside the office being more likely to accept the vaccine, although these results were not statistically significant. Excluding participants who had actually received the vaccine (leaving only those stating “intentions”) produced similar results.Adjustment by age, sex, smoking status and prior receipt of vaccine in a model including determinants related to health beliefs and perceptions highlighted the importance of belief that pandemic influenza A (H1N1) virus was a high threat to the public (OR: 4.44 (1.4, 14.7)), they had a high likelihood of catching the virus (OR: 5.07 (1.44–17.93)) and that “swine flu” was a serious problem to health (OR: 2.86 (1.14–7.15)) to acceptance of the pandemic vaccine (Table 4).Determinants related to health beliefs and perceptions associated with intention to accept the Pandemic Influenza A (H1N1) 2009 vaccine.* Includes 153 observations with complete data; Model adjusted for age, sex, smoking status and prior receipt of influenza vaccine; Results in bold indicate p < 0.05.This study showed that 39.7% of the police employees we sampled had already been vaccinated or would accept the pandemic Influenza A (H1N1) vaccine. Those who stated that they would decline the vaccine if offered numbered 40.2%, and the remainder were unsure.The willingness to receive pandemic Influenza A H1N1 vaccine in this study was found to be 39.7% which is near equivalent to the uptake rate seen in frontline healthcare workers in England of 40.3% in the period leading up to March 2010 [5]. These figures are notably different to those seen in the seasonal influenza vaccination program prior to the pandemic. In healthcare workers, the uptake rate of seasonal vaccine was only 16.5% during the 2008-9 season, compared with over 70% among over 65 year olds [15].Multivariate analysis identified history of seasonal influenza vaccination as the strongest determinant of positive intention to receive pandemic Influenza A H1N1 vaccine. This result concurs with a number of studies concerning Influenza H5N1 [10,16] and H1N1 [12,17], and also other studies of seasonal influenza uptake [8]. Research into pandemic influenza uptake amongst healthcare workers and general population groups reveals conflicting information about the effects of age on uptake, although generally older employees are more likely to receive vaccine [11,12]. In our study we found that younger employees were more likely to accept. Consistent with the weight of evidence [11], we also found that males were more likely to intend to receive the vaccine. It is possible also that non-white ethnicity (as found in other studies [11]), being a current smoker and working outside of an office environment were positive predictors of vaccine uptake, but small numbers may have limited statistical significance.Attitudes and perceptions of disease have been shown to determine one’s health protective behaviors, of which immunization is a key example. This is explained by the Health Belief Model [18] (HBM) which states that “health-related action depends on the simultaneous occurrence of three classes of factors” which include:The existence of sufficient health concern or motivation;Belief of susceptibility to a serious health problem or its complications;Belief that the benefit of an action outweighs its possible disadvantages.Our analysis showed that perceived high risk of infection and perceived severity of “Swine Flu” both to one’s health and the public were shown to be significant determinants of acceptance of pandemic Influenza A H1N1 vaccine. This is a prime example of the HBM in clinical practice and concurs with other literature concerning pandemic [11,16,19] and seasonal influenza vaccination [20]. The converse of this model is also true, and is demonstrated in our results and those of other authors. [8,10,11,21,22] We found that worry about side effects was four times as influential in determining declination of the vaccine as other factors including: inconvenience, and doubts about safety and efficacy. For these individuals the perceived disadvantages outweigh the benefits, and thus no health protective action, immunization, is taken.Consistent with most population groups, most respondents rated the pandemic as a medium or low threat, and this is likely to be a major reason for suboptimal vaccine uptake.To our knowledge, this is the only study addressing the issue of vaccine acceptance in essential service providers. The characteristics of our study sample were broadly comparable to the overall WM police [13] (or WM population) [14], although in our sample there were many more women. Since women were less likely to indicate acceptance of the vaccine, we may therefore have underestimated likely uptake rates. Social acceptability and interviewer bias were limited through the use of self administered, anonymous questionnaires.The small sample size and low response rate limited the statistical power of this study. 380 questionnaires were distributed with a response rate of 54.2% accounting for 2.0% of the sample population. Other than the characteristics stated above there was no way of determining the views of non-responders. Responder bias may have caused an overestimate of vaccine acceptance; those not interested in being vaccinated may have been less likely to participate in the study. This could have been further compounded by lack of promotion of questionnaires.Questionnaire distribution was targeted at Operational Command Units only; smaller stations were not included in this study. There could be differences between the employees of each. We attempted to counteract this by distributing questionnaires to the Occupational Health Department of WM police where all employees have access. Additionally, use of short questionnaires limited the breadth and depth of responses in certain areas of enquiry.Our study population was limited to the police force; therefore, our results may not be applicable to those in other essential services.The findings of this study can be used to improve immunization strategies if vaccination of essential service providers is implemented in future influenza outbreaks. It is clear that health perceptions and attitudes play a major role in influencing the decision to be vaccinated. Targeted education programs could be implemented to address the misconceptions held by many individuals about health issues and their management; this is particularly applicable in the case of immunization and for pandemic vaccine the key issue appears to be worry about side effects. Wider dissemination of studies depicting accurate portrayal of the side-effect risks and reassurance of the benefits of vaccination could substantially improve uptake among the 40% “decliners” and the 20% “unsures”. Other strategies that have been shown to increase uptake rates among healthcare workers which could be included include: use of mobile services to provide flexible delivery of vaccine [23], opt-out systems [24], and reminder schemes [25].We also advise that future research is carried out within both the police and fire service, using a larger sample size; this could be achieved through use of employee distribution lists. Active promotion or incentives could be used to increase response rate. Furthermore, it would be beneficial to obtain qualitative data through interviews of study subjects or focus groups.Despite some limitations in the power of the study, it is reassuring that acceptance rates of pandemic vaccine among the police force would be at least as good as healthcare workers and substantially better than that seen among healthcare workers in seasonal influenza years. Further exploration of why females and those of older ages (and possibly white ethnicity) are more reluctant to be vaccinated would be useful. In the meantime, it is important to make sure that the public and especially essential workers have confidence in the information about the severity of pandemics, the benefits and disadvantages of vaccination, and that media portrayal is accurate.Questionnaire.Comparison of Baseline Characteristics between study sample and total WM police [13].* Data taken from Office of National Statistics due to unavailability of data for West Midlands Police Service [14].The authors declare no conflict of interest.We are grateful to the West Midlands Police Service for providing access to their work force and John Woolley (Duty of Care Manager at WMP Occupational Health).

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+Present Address: Department of Pathology, University of Washington, Seattle, WA 98195, USAPresent Address: Department of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USAThis article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness. Tuberculosis (TB) remains an enormous global health problem despite the vaccination of more than 100 million newborns annually with Bacillus Calmette-Guérin (BCG), the current live-attenuated vaccine against TB [1,2]. The high prevalence of HIV infection in some countries combined with the rising incidence of infection caused by extensively drug-resistant strains of Mycobacterium tuberculosis threaten to make a dire global TB situation even worse.BCG has been used as a vaccine against TB for 9 decades. It was identified in 1921 as an attenuated mutant of M. bovis and was cultivated for decades in laboratories throughout the world. During this time it underwent divergent evolution and the currently-available BCG daughter strains (substrains) differ from each other and from the original BCG vaccine, which no longer exists [3,4]. BCG provides 80% protection against miliary and meningeal TB in childhood and the routine vaccination of newborns in much of the world is estimated to prevent about 40,000 cases annually [5,6]. In early studies, BCG was also highly efficacious against pulmonary TB with about 80% protection over the first 2 decades and up to 50% protection was still evident 6 decades after vaccination [7,8,9]. However, the effectiveness of the BCG daughter strains against pulmonary TB appears to have declined over time for reasons that continue to be debated [10,11]. Pulmonary TB is much more common than the disseminated forms of TB and accounts for the vast majority of the TB global burden. Pulmonary TB is also the contagious form of TB and thus an effective vaccine against pulmonary TB should reduce all forms of TB.In an effort to restore BCG’s ability to protect against pulmonary TB, we have enhanced BCG’s immunogenicity by reducing the activity and secretion of microbial antioxidants [12]. This approach differs markedly from the more common strategy of modifying BCG by increasing its production of prominent antigens and adding recombinant toxins or cytokines [13,14,15]. However our approach is well-founded in the context of reports that mycobacterial antioxidants suppress host immune responses and there is growing evidence that they also promote the pathogenesis of granulomatous inflammation [16,17,18,19,20,21,22]. Furthermore, it appears that as BCG was cultivated in vitro for decades it increased its production of antioxidants by a process involving duplication of regions of chromosomal DNA and other mutations [4], and this increase may partly explain the decline in its protective efficacy against pulmonary TB over time [11,12]. Thus, although we have not specifically undone the multiple mutations that arose in BCG during decades of in vitro cultivation, reducing the activity of microbial antioxidants makes our modified BCG more like the early BCG vaccine that was effective in preventing pulmonary TB.In the present investigation we make four modifications to BCG including reducing the activity of glutamine synthetase (GlnA1), a secreted mycobacterial enzyme implicated in immune evasion [23,24,25], thereby producing 4dBCG with diminished antioxidant and GlnA1 activity. We then compare 4dBCG to BCG in vivo including an assessment of the persistence of vaccine strains in the spleens of mice. We also use a vaccination-challenge model to assess protection against hematogenous dissemination and lung pathology after aerosol inoculation of vaccinated mice with M. tuberculosis. We find that although 4dBCG is cleared more rapidly than BCG from the spleens of mice, it provides greater protection against dissemination of an aerosolized challenge with M. tuberculosis and also alters lung histopathology. The clinical implications of these findings are discussed in the context of the difficulties in correlating results in mice with results in man.Details of the construction of 3dBCG, with three genetic modifications of the parent BCG Tice vaccine strain, have been previously reported [12]. Two of the modifications involved allelic inactivation of sigH, the oxidative stress sigma factor [20,26,27] and secA2, the secretion channel for iron co-factored superoxide dismutase (SodA) [17,18]. A third modification involved recombinant expression of a dominant-negative ∆H28∆H76 mutant of sodA that reduced SodA activity by more than 90% compared to the parent vaccine. GlnA1 is another microbial factor implicated in immune suppression that has been shown to confer resistance to killing of M. tuberculosis by human macrophages [24,25]. As GlnA1 is essential for the growth of M. tuberculosis [28] we partially lowered GlnA1 activity by using dominant-negative interference techniques while preserving potentially important epitopes for immune recognition [29]. First we deleted two amino acids that are important for enzymatic activity, an aspartic acid at position 50 and glutamic acid at position 327 (Figure 1A). Then we inserted the allele encoding the ∆D50∆E327 GlnA1 monomer on a plasmid vector into 3dBCG to yield 4dBCG. Immunoblotting demonstrated that enzyme activity was reduced 8-fold in 4dBCG, verifying a dominant-negative effect (Figure 1B,C).Construction of 4dBCG. (A) Hexameric ring representing half of the enzymatically-active dodecameric form of GlnA1 (left) and enlargement (right) of the area within the rectangle to show the deleted amino acids in relationship to the active site manganese atom, represented by the black dot, with amino acids numbered according to the glutamine synthetase of Salmonella [30]. As the active site comprises residues from adjacent monomers, insertion of one ∆D50∆E327 dnGlnA1 monomer into the ring is predicted to inactivate two active sites. (B) SDS-PAGE (upper) and immunoblot (lower) for GlnA1 of lysates of Bacillus Calmette-Guérin (BCG), 3dBCG and 4dBCG. Lanes are labeled on the figures as L (lysate) and AS (ammonium sulfate-treated lysate) for each strain, kDaM = Kilodalton markers. (C) Graph of a representative experiment comparing the glutamine synthetase activity of undiluted and diluted AS preparations from 3dBCG and 4dBCG as determined by monitoring A540 over time. BCG daughter strains that exhibit relative invasiveness and persistence in animal models such as BCG Pasteur 1173P2 and BCG Danish 1331 cause more adverse effects in man than less virulent BCG substrains such as BCG Tokyo 172 [1,31,32]. In the context of the recent withdrawal of the recombinant BCG vaccine candidate Aeras-422 from human clinical trials due to adverse effects [33,34], assessment of the virulence of the live vaccine strains has become an increasingly important part of the initial evaluation of a live vaccine candidate.To determine whether or not our modifications altered the in vivo persistence of vaccine bacilli, we administered 2 × 107 cfu of BCG or 4dBCG intravenously to C57Bl/6 mice. At day 60 post-inoculation the mean titer of BCG was lower, albeit not significantly, than the mean titer at day 2 post-inoculation p = 0.07, Figure 2). In contrast, the number of 4dBCG bacilli fell by more than 95% over the same period of time (p = 0.01) and was 20-fold less than the titer of BCG at day 60 post-inoculation (p = 0.002). In summary, 4dBCG did not persist as well as the parent BCG vaccine in the spleens of mice. The reduced persistence of 4dBCG may partly reflect a slower intrinsic growth rate combined with greater unmasking and activation of innate and adaptive host responses that mediate the clearance of live mycobacteria from host organs.Spleen titers of BCG and 4dBCG in C57Bl/6 mice. Mean ± SEM spleen titers on day 2, day 30, and day 60 after IV inoculation of mice with 2 × 107 cfu of BCG or 4dBCG. Each value represents four to six mice. The P value represents the comparison of the BCG and 4dBCG groups at 60 days. The day 0 value is estimated as 10% of the original IV inoculum.For nearly 6 decades it has been appreciated that BCG substrains differ in their ability to interfere with the hematogenous dissemination of a challenge dose of M. tuberculosis in animal models [35]. Furthermore, relatively virulent BCG substrains capable of persisting in relatively high titer in the organs of mice protect best in small animal models and thus have been considered by some authorities to have greater vaccine potency [35,36,37,38,39,40,41]. Accordingly, we wondered whether our modifications to BCG that reduced the in vivo persistence of 4dBCG would also reduce the ability of 4dBCG to protect against hematogenous dissemination.We compared mice vaccinated with BCG or 4dBCG to control mice that were vaccinated with phosphate-buffered saline (PBS), using 15 mice per vaccination arm. At 8 weeks after subcutaneous vaccination with 2 × 107 cfu of the vaccine strains, mice were challenged by aerosol with 100 cfu of M. tuberculosis strain Erdman S-1. To assess hematogenous dissemination, we determined spleen cfu counts at 8 weeks post-aerosol challenge. At this time, PBS-vaccinated mice harbored a median of 1.8 × 105 bacilli in the spleen. CFU titers were 5.8-fold lower in BCG-vaccinated mice (median, 3.1 × 104 cfu) and 18-fold lower in 4dBCG-vaccinated mice (median, 1.0 × 104 cfu, Figure 3).Lung cfu titers were also determined. PBS-vaccinated mice exhibited a median of 9.7 × 104 cfu at 8 weeks post-challenge and titers were 3.6-fold lower in BCG-vaccinated mice (median, 2.7 × 104 cfu) and 7.5-fold lower in 4dBCG-vaccinated mice (median, 1.3 × 104 cfu). These results paralleled the findings in the spleen and represent the growth over time of the original aerosolized inoculums of bacteria within the lung combined with bronchogenic and hematogenous dissemination to other parts of the lung. Results at 26 weeks are described below (Section 2.5)Titer of M. tuberculosis challenge bacilli in the spleens and left lungs of C57Bl/6 mice. Mice received aerosol challenge with 100 cfu of M. tuberculosis strain Erdman S-1. The whole spleens and left lungs were assessed for cfu titer while the right lungs were processed for histopathology as shown in Figure 4, Figure 5, Figure 6 and Figure 7. The individual data points (circles) and median (bar) for each vaccination arm are shown at 8 weeks (top, six mice per vaccination arm) and 26 weeks (bottom, eight or nine mice per vaccination arm) post-challenge. The dotted lines extend the bar representing the median value of the PBS-vaccinated and BCG-vaccinated groups and are marked P* and P**, respectively. Analysis of spleen and lung results at 8 weeks and lung results at 26 weeks by 1 way ANOVA demonstrated that the median values of the groups varied significantly by Kruskal-Wallis test. The groups were also compared using the Mann-Whitney test and values in the row labeled P* above the panel indicate the comparison of each vaccinated group against the phosphate-buffered saline (PBS) group. The values in the row labeled P** indicate the comparison of the 4dBCG and BCG groups.CFU counts are an incomplete measure of vaccine-induced protection against pulmonary TB, and thus we also evaluated the effect of vaccination upon lung pathology. To display these results objectively, we took low power photomicrographs (×2 magnification) of H&E-stained sections of lung tissue covering about 70% of the lung tissue on each slide for three mice per vaccination arm (Figure 4). We marked the parts of the low-power photomicrographs that were enlarged further to display representative histopathologic features. Thus the reader can visualize the representative features in the context of the whole lung cross-sections. As shown in Figure 4, regions of relatively unconsolidated lung parenchyma were observed in all three vaccination arms. Mice vaccinated with PBS exhibited relatively more inflammation and consolidation of lung tissue at 8 weeks post-aerosol challenge with M. tuberculosis compared to mice vaccinated with BCG and mice vaccinated with 4dBCG.Low-power photomicrographs of sections of lung tissue. (Upper left panel, labeled “Slides”) Photographs of the microscope slides containing H&E-stained lung sections are shown from right lungs of three mice from each of the PBS, BCG, and 4dBCG vaccination arms at 8 weeks post-aerosol challenge with 100 cfu of M. tuberculosis strain Erdman S-1. Three rectangles are labeled a, b, and c and cover approximately 70–80% of the lung tissue on each slide. (Panels labeled “PBS”, “BCG”, and “4dBCG”) Three low-power photomicrographs of the lung tissue were taken through the 2× microscope objective and correspond to the three rectangles in each lung labeled a, b, and c in the “slides” panel. Within these photomicrographs, the black rectangles highlight lymphohistiocytic nodules in relatively consolidated regions of lung tissue, which are enlarged further and displayed in Figure 5. The white rectangles in the 4dBCG group of mice outline lung tissue containing bronchus-associated lymphoid tissue (BALT) and are displayed in Figure 7.The most apparent difference in lung histopathology between vaccination arms involved the lymphohistiocytic nodules, which stain dark blue due to the density of nuclei and scant cytoplasm within lymphocytes (Figure 4 and Figure 5). They were most numerous and well-developed in the PBS vaccination arm, often exhibiting a circumferential or near-circumferential ring of lymphocytes around epithelioid cells that had replaced normal alveolar architecture (Figure 5 and Figure 6). Also staining dark blue was bronchus-associated lymphoid tissue (BALT). BALT typically can be distinguished from lymphohistiocytic nodules within the lung parenchyma by its association with bronchovascular structures (Figure 5 and Figure 7) and was observed in all 3 vaccination arms. Compared to BCG, the lungs of mice vaccinated with 4dBCG exhibited smaller collections of lymphocytes within the lung parenchyma with less disruption of alveolar architecture as demonstrated by better preservation of the alveolar walls and fewer epithelioid cells (Figure 5 and Figure 6). There was also more generalized eosinophilic staining of the lungs of mice vaccinated with 4dBCG (Figrue 4), due in part to extravasated RBCs and edema fluid.Due to weight loss in PBS-vaccinated mice the experiment was limited to 26 weeks post-challenge. At this time the mice vaccinated with BCG or 4dBCG exhibited spleen cfu titers that were not significantly lower than cfu titers in the PBS vaccination arm (Figure 3). Lung histopathology revealed extensive granulomatous inflammation in all 3 groups of mice (not shown). Lung cfu titers in the 4dBCG vaccination arm were significantly lower than titers in the PBS vaccination arm (median values of 5.0 × 104 cfu and 1.5 × 105 cfu, respectively, p = 0.004).Lymphohistiocytic nodules in the lung parenchyma. Mid-power (×10 microscope objective) photomicrographs of regions of lung consolidation within the tissue sections in Figure 4 are displayed at a greater magnification. Most of the areas that stain dark blue in the PBS and BCG groups represent dense collections of lymphocytes within the lung parenchyma that are accompanied by lymphohistiocytic infiltration and consolidation of the alveolar spaces. Examples of these regions are indicated with arrows (black arrowheads) and the white rectangles outline regions of lung tissue that are enlarged further in Figure 6. Lymphoid aggregates were also observed in the lung parenchyma in the 4dBCG group of mice however they were typically smaller with less dense alveolar infiltrate compared to the PBS and BCG groups. The arrows with white arrowheads indicate BALT, which is distinguished from lymphohistiocytic nodules in the lung parenchyma by their association with blood vessels and bronchi. High-power views of lymphohistiocytic nodules. Consolidated lung tissue with many epithelioid cells containing pale oval nuclei and eosinophilic cytoplasm are observed in the PBS-vaccinated mice and to a lesser degree in the BCG-vaccinated mice. The alveolar spaces adjacent to the smaller lymphoid nodules in the 4dBCG-vaccinated mice are edematous, contain relatively fewer epithelioid cells, and exhibit greater preservation of alveolar wall architecture than in the PBS- and BCG-vaccinated mice.Bronchus-associated lymphoid tissue (BALT). The panels display ×10 enlargements of the lung sections from mice vaccinated with 4dBCG as represented by the white rectangles in Figure 4. BALT was prominent along the central bronchovascular structures in all three groups of mice (see Figure 5) and its association with blood vessels and bronchi distinguishes BALT from collections of lymphocytes in the lung parenchyma.The results of these experiments support efforts to improve the safety and effectiveness of BCG by targeting microbial factors that suppress host immune responses. We previously have reported that 3dBCG, with 3 attenuating modifications, surpasses the parent BCG Tice vaccine in the induction of primary and secondary immune responses and in the clearance of challenge bacilli in a memory-immune model of subcutaneous vaccination followed by intravenous challenge [12]. Other groups of TB vaccine investigators have reported that the immunogenicity and protective efficacy of BCG can be improved by disrupting sapM and zmp1, microbial genes that encode, respectively, an acid phosphatase and zinc metalloprotease implicated in inhibition of phagosome maturation [42,43,44,45]. Collectively, there is now substantial evidence that the immunogenicity of BCG can be improved by targeting immune suppressive microbial factors.The present investigation further explores this strategy for improving BCG and demonstrates that despite diminished persistence in vivo 4dBCG surpasses the parent BCG Tice vaccine in protecting mice against the dissemination of an aerosolized challenge of M. tuberculosis from the lung to the spleen. Vaccination with 4dBCG also altered lung histopathology to result in fewer and less well-developed lymphohistiocytic nodules within the lung parenchyma at 8 weeks post-challenge. At 26 weeks post-challenge mice vaccinated with 4dBCG had significantly fewer bacilli in the lungs than control mice. The biological mechanisms that underlie the improved protection observed with 4dBCG have not been fully explored, however oxidants and glutamate are signaling molecules that activate antigen-presenting cells and T cells [46,47,48,49,50], promote apoptosis and apoptosis-associated cross presentation of bacterial antigens to induce CD8+ T cell responses [49,51,52], and influence the balance between Th1 and Th17 responses [53,54]. Thus our modifications to BCG may have enhanced protection by unmasking protective host responses that are suppressed by the parent BCG vaccine.There are several issues worthy of discussion regarding how these results fit into the TB vaccine literature and their implications for human vaccination. First, it is noteworthy that we used a relatively high dose of 4dBCG and BCG, 2 × 107 cfu, which is greater than the 3.7 × 104 cfu to 3 × 106 cfu typically used for human vaccination [1]. The ability of some BCG substrains to induce protection with doses as low as 101 cfu has been interpreted by some experts as indicative of vaccine potency [32,55,56,57,58] such that vaccines with this capability including Pasteur 1173P2 and Danish 1331 are called “strong” strains whereas those without this capability including Tokyo 172 are called “weak” [59,60]. The biological reason for this difference has traditionally been attributed to differences in the ability of the live vaccine to multiply within the host and the BCG vaccines exhibiting the smallest allergenic, i.e., DTH-inducing, doses also exhibit greater lethality in golden hamsters and cause more adverse effects in man [32,59,60,61,62]. As 4dBCG was less capable of persisting in the spleens of mice than the parent BCG Tice vaccine it behaves more like the “weak” BCG daughter strains and accordingly we used a relatively high dose for vaccination. It is possible that a smaller dose of 4dBCG would not confer as much protection. Despite this uncertainty, we have concerns about the ability of some BCG substrains to induce protection with a low dose; in the context of a modern understanding of host-pathogen interactions this capability indicates that the vaccine strain is able to suppress the host responses needed to eradicate live mycobacteria, which is not a good property for a vaccine.A second issue is that the protection induced by vaccination with 4dBCG waned over time. This was not unexpected as the waning of protection is characteristic of vaccination with BCG in small animal models [63]. Recent results involving C57Bl/6 mice and the same lot of the Erdman challenge strain used in our study found that vaccination with BCG Pasteur conferred protection at 4 and 10 weeks post-aerosol challenge, but not at 20 weeks [64]. Thus, although the magnitude of protection induced by BCG and 4dBCG in our study declined over time, these results are consistent with prior experience and our observation of continued, albeit reduced, protection at 26 weeks post-challenge in the 4dBCG vaccination arm exceeds the duration of protection usually observed in this model. Extrapolation from such results to expectations regarding vaccine effectiveness in man is difficult however it is important to remember that whereas C57Bl/6 mice invariably develop progressive infection that damages the lung [65] 90% of human hosts control aerogenic infection with M. tuberculosis for the duration of their lives. Accordingly, the real goal of vaccination against M. tuberculosis is to induce in the 10% of persons who are relatively susceptible to the development of active TB the type(s) of immune responses that protect 90% of us. As the immune system of man exhibits important differences from the immune system of mice [66] and is superior in restricting the growth of mycobacteria in vivo [21,67], it is possible that the modest enhancements in protection we observed in mice may correlate with enhanced protection against pulmonary TB in man.A third issue is that although vaccination altered lung histopathology and 4dBCG induced greater changes than BCG in comparison with control mice, the implications of these findings are unclear. In C57Bl/6 mice, the dense aggregates of lymphocytes in the lung parenchyma are comprised primary of B cells and surrounded by macrophages [68,69]. Defects in CXCL13 and IL-23 signaling have been associated with fewer and smaller B cell follicles in mice along with impaired containment of M. tuberculosis [70], suggesting that the follicles contribute to protection. Yet most human hosts are able to resolve the granulomatous lesions of primary infection with M. tuberculosis, which are often marked by foci of calcification within the lung parenchyma and hilar lymph nodes to form a Ghon complex [71]. Thus the presence of fewer and smaller lymphohistiocytic nodules in 4dBCG-vaccinated mice could represent a defect in the formation of these structures or instead reflect the unmasking of host responses involved in resolving granulomatous inflammation. There is growing appreciation in man that immune surveillance by CD8+ T cells helps to prevent latent TB infection from developing into active pulmonary TB [72,73,74]. Although such responses are not expressed well enough in mice to prevent eventual lung destruction, they may tip the balance towards the resolution of granulomatous inflammation and the containment of infection in human hosts who are intrinsically better than mice at controlling M. tuberculosis.The fourth issue is a question that is at the heart of any attempt to improve BCG. Is it better to administer a relatively virulent live vaccine that persists in vivo such that immune effector cells are already present when the host subsequently becomes infected with M. tuberculosis or instead is it better to use a less immune suppressive vaccine that induces host immune responses that not only eradicate the vaccine strain but also produce memory immunity that can be recalled years later when the host becomes infected? The current BCG daughter strains provide some insight into this important question. More than 40 years ago, two relatively “strong” BCG daughter strains, Pasteur 1173P2 and Danish 1331, were selected for evaluation in a large randomized prospective clinical trial of BCG vaccination in India on the basis of their relative ranking in animal models together with concern about the possible over-attenuation of BCG [32,58,60,61,75]. Yet neither vaccine was effective [76]. Although this was the only randomized trial to compare the protective efficacy of different BCG vaccines, it has subsequently been suggested that case-control studies in which a BCG substrain is replaced by another BCG substrain during the study interval can provide insight into their relative effectiveness. This type of analysis demonstrates that the relatively “weak” BCG Tokyo 172 vaccine confers 50–60% protection against pulmonary TB and is more effective in man than the “strong” Pasteur 1173P2 and Danish 1331 vaccines [77,78,79]. A recent cohort study in Kazakhstan reported similar findings with BCG Tokyo 172 exhibiting greater protection effectiveness than BCG Russia and a Serbian formulation of BCG Pasteur 1173P2 [80]. Correlating these results in man with the results from an earlier report of the persistence of the same BCG substrains in mice [37] answers the question posed at the beginning of this paragraph. In effect, there is an inverse correlation between the protection effectiveness of a BCG substrain in man and its persistence in the spleens of mice (Figure 8). This important observation further justifies efforts to improve BCG by reducing its capacity for immune suppression. It also invites a reevaluation of the historical criteria used to evaluate the potency of BCG daughter strains and to designate them as “strong” or “weak”.Finally, the inverse correlation noted above raises a fifth issue about our study. In essence, Figure 8 suggests that the BCG substrain used for genetic modifications will be an extremely important variable in vaccine effectiveness. We used BCG Tice, a vaccine strain that historically was considered to be of relatively low virulence [36] but was reformulated with a subculture of BCG obtained from the Institut Pasteur in 1951 and subsequently has exhibited in vivo persistence in small animal models similar to that observed with BCG Pasteur 1173P2 [81,82,83]. Our modifications to BCG Tice reduced in vivo persistence and per the inverse correlation shown in Figure 8 we expect that 4dBCG will exhibit enhanced protection effectiveness in man. However, we can only speculate as to the magnitude of the improvement and whether 4dBCG will be more or less protective in man than BCG Tokyo 172. Although our modifications were a step in the right direction, ideally the starting strain used for genetic modifications should be a BCG substrain with high protection effectiveness in man. Some progress towards this goal has been achieved; in 2009, while the Aeras Global TB Vaccine Foundation held an exclusive license for Vanderbilt University’s pro-apoptotic bacterial vaccine technology [84], and based in part on our concern about the potential triplication of sigH and other virulence genes in recombinant vaccines derived from BCG Danish 1331 [85] that Aeras was using for vaccine construction [15], we encouraged Aeras to instead introduce the genetic modifications described in the current study and our earlier work [12] into BCG Tokyo 172. Two of these modifications, inactivation of SecA2 and the chromosomal insertion of a dominant-negative ΔH28ΔH76 sodA allele, were introduced into a GMP (good manufacturing practices) stock of BCG Tokyo 172 (Final Progress Report from Aeras to Vanderbilt University, May 2011). Although Aeras decided not to develop this vaccine candidate or modify it further, based on the findings of this study and the experience with “weak” and “strong” BCG vaccines in man (Figure 8), we expect that it would likely exhibit enhanced protection against pulmonary TB.Inverse correlation between protection effectiveness in man and spleen titer in mice of three BCG daughter strains. The protection effectiveness values were taken from a cohort study in Kazakhstan [80] and involved BCG Russia (Microgen), Pasteur 1173P2 (Serbian “Torlak” vaccine), and BCG Tokyo 172 (Japan BCG Laboratory). The log10 spleen titer values were taken from a comparison of 5 BCG strains in Balb/C mice [37] and represent the values at 12 weeks after intravenous inoculation of mice with 106 cfu of the vaccine strain.In summary, we have described an alternative strategy for modifying BCG based on reducing the activity and secretion of multiple immune suppressive microbial factors. Our vaccine strategy challenges the traditional prioritization of BCG vaccines based on their performance in small animal models. Indeed, we consider the virulence attributes of the “strong” BCG vaccines that have previously been regarded as indicators of potency to instead reflect their immune suppressive capacity. In the context of the repeated failure of “strong” BCG vaccines to exhibit protection against pulmonary TB in man and their associated adverse effects, it is time to consider a different direction in TB vaccine development based on the construction and evaluation of modified BCG vaccines with diminished immune suppressive capacity.Genetic tools and bacterial isolates are listed in Table 1. E. coli strains were grown in LB media. Genetic modifications were introduced into BCG Tice. To prepare vaccine inocula BCG Tice and 4dBCG were grown in Middlebrook 7H9 media with 10% oleic acid-dextrose-catalase (OADC) enrichment supplemented with 0.2% glycerol, and 0.05% Tween80. Kanamycin (50 μg/mL or 25 μg/mL), apramycin (50 μg/mL), and hygromycin B (100 μg/mL or 50 μg/mL) were used to select colonies after genetic manipulations in E. coli or BCG, respectively. Erdman S-1 (lot K01) was provided by the Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), USA in accordance with a collaborative agreement between the FDA and the World Health Organization. This challenge strain is a standardized preparation made available to TB vaccine investigators to help reduce variability between laboratories and in two recent studies that compared several M. tuberculosis challenge strains, Erdman S-1 demonstrated relatively high capacity for disseminating to the spleen after aerosol challenge [61,86]. Tools for genetic manipulations and bacterial strains.Plasmid and chromosomal integration vectors were electroporated as previously described [12,88]. After electroporation, Middlebrook 7H9 media was added to the samples, which were incubated in 5% CO2 at 37 °C for 24 h before the suspension was plated on Middlebrook 7H11 agar containing antibiotics as needed. Successful transformation was confirmed by PCR of DNA unique to the vector.To construct dominant-negative (dn) enzyme monomers, glnA1 was PCR-amplified from DNA of M. tuberculosis strain H37RV, ligated into pCR2.1-TOPO, and propagated in E. coli TOP 10. Site-directed mutagenesis was performed using primer overlap extension methods [89]. Codon deletion was verified by DNA sequencing and the sequence data was deposited in GenBank, Accession No. HM217184.The mutant allele encoding ∆D50∆E327 GlnA1 (numbering per homologous enzyme in Salmonella typhimurium [30] but actually representing ∆D54∆E335 deletions in M. tuberculosis GlnA1) was ligated into the shuttle plasmid pHV203 and electroporated into 3dBCG to yield 4dBCG. Immunoblotting was used to compare enzyme quantity. Bacterial lysates were prepared from 25 mL of 108 cfu/mL of each strain grown for 48 h in Middlebrook 7H9 media with 10% oleic acid-dextrose-catalase (OADC) enrichment supplemented with 0.2% glycerol and 0.05% Tween80. Lysates were adjusted to a standard A280 value, applied to a SDS-12% PAGE gel for separation of proteins by electrophoresis and transferred to nitrocellulose membranes and hybridized with a 1:20 dilution of the anti-GlnA1 antibody IT-58 was obtained from Colorado State University as part of NIH, NIAID Contract HHSN26620040091C, “Tuberculosis Vaccine Testing and Research Materials.” To measure glutamine synthetase activity we monitored γ-glutamylhydroxamate formation spectrophotometrically at 540 nm by the γ-glutamyl transfer reaction [90]. As a factor in the cell lysate partially inhibited the assay, we first treated the lysate with 50% ammonium sulfate and then dialyzed the soluble portion with assay buffer, 0.04 M potassium phosphate, pH 7.0, and concentrated to 1 mL before performing the assay. Hydroxylamine, glutamine, and gamma-glutamylhydroxamate were purchased from Sigma.Experiments involving the monitoring of the vaccine strains in the organs of mice were approved by the Vanderbilt Institutional Animal Care and Use Committee (Protocol No. M/06/069). Experiments involving vaccination-challenge were approved by the Syracuse VAMC Subcommittee on Animal Studies (ACORP Protocol No. 005). Female C57BL/6 mice aged 5–6 weeks were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Methods for preparing inocula of the vaccines and challenge strain are described above. Bacterial suspensions were adjusted to a standard absorbance and diluted to achieve the desired inoculum. In studies to compare the clearance of vaccine strains, mice were inoculated by tail vein or retro-orbital injection. In the vaccination-challenge experiments mice were vaccinated subcutaneously and rested until challenge by aerosol with 100 cfu of Erdman S-1. For aerosol infection with Erdman S-1, the organism, supplied frozen, was removed from the freezer, thawed, sonicated and diluted to a final concentration of 1 × 106 cfu/mL and ten mL of the inoculum was aerosolized to deliver ~100 cfu/mouse. Some mice were euthanized and their lungs harvested 24 h post-challenge to verify the challenge dose.To enumerate bacteria in the organs of mice, mice were euthanized with CO2 inhalation and the spleens and left lungs were removed aseptically and placed in a sealed grinding assembly (IdeaWorks! Laboratory Products, Syracuse, NY, USA) attached to a Glas-Col Homogenizer (Terre Haute, IN, USA). Viable bacterial counts were determined by titration on 7H10 agar plates containing 10% OADC supplemented with 2 μg/mL of 2-thiophene carbonylhydrazide. During the course of the 26-week vaccination-challenge experiment one mouse in the 4dBCG vaccination arm was euthanized for humane reasons related to problems with excessive grooming.At the time of harvest, right lungs from each vaccination arm were submersed in formalin. The lungs from each mouse were routinely cut at two levels in antero-posterior sagittal planes. All pieces from a single mouse were embedded flat on the cut surfaces in a single paraffin block. The lungs were cut into 4-μm sections and stained with hematoxylin and eosin (H&E). Occasionally the sections displayed the whole sagittal plane of the right lung at different levels. To illustrate differences in the magnitude of parenchymal lung involvement and loss of alveolar architecture between different vaccination arms, the slides were laid on white paper and photographed with a digital camera. Then three photomicrographs covering 70 to 80% of the visible lung on the slide from three mice in each group were taken using a ×2 microscope objective and digital microscope camera (Olympus America, Inc., Melville, NY, USA). Higher-magnification views were taken as needed to display specific features. To prepare the multi-panel figures, we used Adobe® Illustrator (Adobe Systems, Inc., San Jose, CA, USA) to place photographs within each figure. Then the composite figure was modified within Adobe® Photoshop where autolevel, brightness, and contrast adjustments were applied uniformly to the whole figure to optimize the display.Statistical analyses were performed using Prism 5.0 software (GraphPad) and only significant (p < 0.05) or close to significant (p < 0.1) values are indicated. Unless otherwise indicated, calculations were performed using the two-tailed Mann-Whitney test.The strategy of preferring BCG daughter strains based on in vivo virulence and persistence has failed. The BCG vaccines designated as “strong” on the basis of historical criteria have problems with inadequate safety in immune suppressed persons, adverse reactions in normal hosts, poor memory T-cell responses, and poor protection against pulmonary TB. In this investigation we demonstrate that by targeting microbial enzymes implicated in immune suppression, the in vivo persistence of BCG is reduced while protection in a vaccine-challenge model is enhanced. We believe that the strategy of improving BCG by reducing its immune suppressive capacity addresses the major limitations of current BCG vaccines against TB, modifying BCG in a manner that should make it a safer and more effective vaccine against pulmonary tuberculosis in man. This strategy for improving BCG should also work well in combination with new boosting vaccines against TB, which are more likely to be effective if the initial priming vaccine is highly immunogenic rather than immune suppressive.DSK is a named inventor on issued patents for modified BCG vaccines with reduced activity of anti-apoptotic microbial enzymes including mycobacterial antioxidants. The technology has been assigned to Vanderbilt University and the United States Government as represented by the Department of Veterans Affairs. MB is a named inventor on a patent application for SecA2 deletion filed by the University of North Carolina, Albert Einstein College of Medicine, and the Howard Hughes Medical Institute. None of the other authors have a financial interest related to this work.We thank Gretchen Edwards for assistance with the assays.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Part I. Basic Principles. TB vaccines cannot prevent establishment of the infection. They can only prevent an early pulmonary tubercle from developing into clinical disease. A more effective new vaccine should optimize both cell-mediated immunity (CMI) and delayed-type hypersensitivity (DTH) better than any existing vaccine. The rabbit is the only laboratory animal in which all aspects of the human disease can be reproduced: namely, the prevention of most primary tubercles, the arrestment of most primary tubercles, the formation of the tubercle’s solid caseous center, the liquefaction of this center, the formation of cavities and the bronchial spread of the disease. In liquefied caseum, virulent tubercle bacilli can multiply extracellularly, especially in the liquefied caseum next to the inner wall of a cavity where oxygen is plentiful. The bacilli in liquefied caseum cannot be reached by the increased number of activated macrophages produced by TB vaccines. Therefore, new TB vaccines will have little or no effect on the extracellular bacillary growth within liquefied caseum. TB vaccines can only increase the host’s ability to stop the development of new TB lesions that arise from the bronchial spread of tubercle bacilli from the cavity to other parts of the lung. Therefore, effective TB vaccines do not prevent the reactivation of latent TB. Such vaccines only control (or reduce) the number of metastatic lesions that result after the primary TB lesion was reactivated by the liquefaction process. (Note: the large number of tubercle bacilli growing extracellularly in liquefied caseum gives rise to mutations that enable antimicrobial resistance—which is a major reason why TB still exists today). Part II. Preclinical Testing. The counting of grossly visible tubercles in the lungs of rabbits after the inhalation of virulent human-type tubercle bacilli is the most pertinent preclinical method to assess the efficacy of new TB vaccines (because an effective vaccine will stop the growth of developing tubercles before while they are still microscopic in size). Unfortunately, rabbits are rarely used in preclinical vaccine trials, despite their relative ease of handling and human-like response to this infection. Mice do not generate an effective DTH response, and guinea pigs do not generate an effective CMI response. Only the rabbits and most humans can establish the proper amount of DTH and CMI that is necessary to contain this infection. Therefore, rabbits should be included in all pre-clinical testing of new TB vaccines. New drugs (and/or immunological procedures) to reduce liquefaction and cavity formation are urgently needed. A simple intradermal way to select such drugs or procedures is described herein. Part III. Clinical Testing. Vaccine trials would be much more precise if the variations in human populations (listed herein) were taken into consideration. BCG and successful new TB vaccines should always increase host resistance to TB in naive subjects. This is a basic immunological principle. The efficacies of new and old TB vaccines are often not recognized, because these variations were not identified in the populations evaluated.These Perspectives address both preclinical TB in animal hosts and clinical TB in human populations. It is a guide to how new TB vaccines can be more effectively evaluated in each group. It is not a review of new TB vaccines or the mechanisms in which they may reduce the prevalence or morbidity of TB in the world.A TB vaccine cannot prevent the establishment of a primary pulmonary tuberculous lesion. The establishment of a primary lesion depends on the activation state of the alveolar macrophage (AM) that ingested the inhaled bacillus, i.e., whether the AM was activated nonspecifically (by dust and benign microorganisms) to inhibit the intracellular growth of an inhaled tubercle bacillus. A sufficiently activated AM normally clears the infection, whereas a poorly activated AM allows the bacillus to multiply intracellularly, establishing a primary lesion. The pulmonary AM population contains highly activated AM and poorly activated AM [1]—mostly depending on how long these AM have resided in the alveoli. Many poorly activated AM had arrived there recently.The bacilli (multiplying in poorly activated AM) and these AM themselves release chemotactic factors that cause the migration and accumulation of blood-derived monocyte/macrophages and lymphocytes—establishing a primary tubercle. The initial AM (containing multiplying tubercle bacilli) eventually bursts. The bacilli are then ingested by the accumulating non-activated macrophages from the blood stream [2] in which the bacilli continue to multiply. Lurie called this the stage of symbiosis, because both the number of macrophages and number of tubercle bacilli increase at the site [3]. Bacillary multiplication at this stage of tubercle development is stopped by delayed-type hypersensitivity (DTH) to the tuberculin-like products of the bacillus [4]. The DTH response acts in two ways: (a) by directly killing the infected macrophages by CD8+ T-cells and thrombosis of the macrophages’ blood supply [5] and (b) by enhancing the local accumulation of lymphocytes and macrophages [4,6,7]. Simultaneously, CMI develops and activates the local macrophages by antigen-specific Th1 lymphocytes [8], so that they can prevent the intracellular growth of ingested bacilli and even kill these bacilli. At this stage, the combination of DTH and CMI can arrest the lesion.In individuals vaccinated with effective TB vaccines, lesion progression is stopped earlier, because DTH and CMI are induced more rapidly. This rapid recall would prevent developing TB lesions from reaching X-ray visible size. In other words, effective TB vaccines can prevent clinically apparent disease.Both DTH and CMI are caused by an antigen-specific Th1 lymphocyte population that activates macrophages within tuberculous lesions [1,6,7,9,10,11]. These locally activated macrophages ingest and kill the live tubercle bacilli that they encounter. If many activated macrophages are present, a developing TB lesion is arrested.In brief, good TB vaccines work by more rapidly stopping the progression of small developing pulmonary tubercles. These vaccines do so (a) by DTH, i.e., the more rapid killing of macrophages that contain too many tubercle bacilli for CMI to inhibit—thereby causing solid caseous necrosis in the center of the tubercle in which the growth of tubercle bacilli is inhibited and (b) by CMI, i.e., the more rapid activation of the macrophages that surround the caseous center—so that any ingested bacillus that escapes from the solid caseous center does not grow. Therefore, in the vaccinated host, early developing TB lesions will be arrested sooner (than in the non-immunized host) and never reach clinically overt disease.Both DTH and CMI are cell-mediated immune responses mediated by Th1 antigen-specific lymphocytes [8]. The main difference between them is the concentration of antigen required to elicit the host response [9]. After all, delayed-type hypersensitivity (DTH) is what the name signifies, i.e., an increased sensitivity to the low concentrations of the antigens that elicit it.The classic DTH reaction is the tuberculin reaction, which is an inflammatory reaction—usually measured two days after an intradermal injection of tuberculin, i.e., 5 tuberculin units (0.0001 mg of PPD). At two days, this dose causes a minimal inflammatory reaction (with induration, if any, of less than 10 mm in immunocompetent individuals who never had a pulmonary tubercle). This dose causes a larger induration in individuals who have an arrested a pulmonary tubercle. And, this dose causes a still larger induration (often with a necrotic center) in highly tuberculin-sensitive individuals (probably in individuals whose pulmonary lesion(s) are still more active).CMI seems to be elicited with much higher antigen concentrations than those present in tuberculin preparations. CMI activates macrophages in tuberculous lesions. These activated macrophages kill (or stop the growth of) the tubercle bacilli that they ingest. Yet, very low concentrations of tuberculin can also activate macrophages (i.e., produce CMI) [12], and high concentrations of CMI antigens can probably cause tissue necrosis.In brief, the low concentration at which DTH antigens elicit a reaction seems to be the main difference between DTH and CMI. DTH kills macrophages containing more tubercle bacilli than CMI can inhibit. In doing so, DTH forms the solid caseous center of the tubercle—within which the bacillus cannot multiply. DTH antigens and CMI antigens cause similar tissue reactions. It is only the effective concentration of each antigen that identifies the category to which that antigen belongs. As discussed in Part II below, DTH and CMI must be developed in the proper amount and proper proportion to effectively control the growth of tubercle bacilli in hosts infected with M. tuberculosis (M. tbc). Vaccines that do this will provide the best protection against clinically active disease. In about 95% of the human population, the primary pulmonary tubercle is arrested—often as a minute lesion with a solid caseous center with or without calcification. These individuals have what is called latent TB, because with decrease host resistance, such as in those infected with HIV, the disease becomes active. In tuberculin-positive individuals, latent TB lesions are kept arrested by an active host immune response, i.e., by the macrophages (that surround the solid caseum) becoming activated wherever tubercle bacilli (or their antigens) stimulate local Th1 lymphocytes. The remaining 5% of the human population falls into two categories: (a) the childhood-type of TB (such as miliary TB) in which the disease disseminates by the hematogenous (and lymphogenous) routes and (b) adult-type TB in which liquefaction of the solid caseous center occurs that is frequently followed by cavity formation and the bronchial spread of the disease [3,6,13,14]. In the adult type, the formerly dormant tubercle bacilli multiply in the liquid menstruum (for the first time extracellularly), sometimes reaching a very large number (in which mutations causing antimicrobial resistance may occur). The tuberculin-like products produced by such a large number of tubercle bacilli can destroy the protective surrounding TB granulation tissue (containing activated macrophages) and can erode the wall of a nearby bronchus, forming a pulmonary cavity when the liquefied caseum is discharged into the airway.Increasing host immunity by a TB vaccine increases the number macrophages and their speed of activation, but these host defense cells cannot survive in liquefied (or solid) caseum (probably because of the toxic and allergenic bacillary products present). Therefore, in hosts that have an arrested primary pulmonary tubercle, TB vaccines cannot stop the major clinical cause of reactivation of the adult disease, i.e., the liquefaction of solid caseum. In fact, such liquefaction may even be enhanced by the increased numbers of activated macrophages (and their hydrolytic enzymes) as a result of vaccination (discussed in Item 1.6, below).In adult-type TB, vaccines can only increase the host’s ability to stop the development of new tuberculous lesions that arise from the bronchial spread of tubercle bacilli from the cavity to other parts of the lung. Therefore, effective TB vaccines do not prevent the reactivation of latent TB. Such vaccines only control (or reduce) the number of metastatic lesions that result after the primary TB lesion was reactivated by the liquefaction process.In other words, TB vaccines can reduce (or control) clinically reactivated tuberculosis by arresting the development of secondary pulmonary lesions arising from bacilli spread via the airways from the primary cavity lesion. Vaccines cannot prevent the activation of latent TB (which is due to the liquefaction of the solid caseous center of the primary lesion). In fact, TB vaccines may even enhance the liquefaction process.Chemotherapeutic drug regimens are sometimes ineffective, because of the development of drug resistance in the mycobacterial population. Such drug resistance almost always occurs in the tubercle bacilli that are growing extracellularly in liquefied caseum [15]. The most rapid growth occurs in the inner wall of a pulmonary cavity, where the highest amount of oxygen is present [15], and minimal (if any) growth occurs in solid caseum. Since liquefaction and cavities do not usually occur in tuberculous mice, this species would be less pertinent for evaluating TB drug regimens for their ability to prevent the development of drug-resistant mycobacteria. Rabbits develop cavities more readily than even guinea pigs, and therefore, rabbits would be the best species to use for this purpose [15].To date, the effect TB vaccines have on the development of liquefaction and cavity formation has not been investigated. Such knowledge would be most applicable to vaccines used to boost immunity in individuals who have an arrested primary TB lesion. Drugs (or immunological procedures) to inhibit liquefaction of solid caseum (and therefore cavity formation) would greatly aid the present day antimicrobial treatment of tuberculosis. Such drugs could be evaluated in the skin model of liquefaction presented in reference [15] (see Part 2, Item 2.5, below).TB vaccines (especially BCG) are usually administered to only tuberculin-negative individuals. However, a proportion of these tuberculin-negative individuals would become tuberculin-positive if the skin test was repeated after a few weeks. This response is called the tuberculin “booster reaction” or tuberculin “recall phenomenon” [16,17]. In other words, these tuberculin-negative individuals were really tuberculin-positive with a low expanded antigen-specific T-cell population that was increased by boosting.The identification of such individuals is usually omitted in clinical trials and is one of the reasons why vaccine trials fail to show differences between control and vaccinated groups. (Individuals who produce a booster reaction are already immunized by a natural TB infection that they arrested).A danger exists in vaccinating such individuals. Specifically, many of these individuals have a small arrested tubercle with a solid caseous center. Liquefaction of such caseous centers (and cavity formation) occurs more readily in tuberculin-positive rabbits than in tuberculin-negative ones [15], apparently because more activated macrophages (with high levels of hydrolytic enzymes) surround the solid caseum. Therefore, vaccinating tuberculin-positive individuals might hasten the liquefaction of an arrested (dormant) TB lesion (with subsequent cavity formation). In other words, vaccinating individuals with an arrested TB lesion might sometimes activate this disease. In fact, repeated injections of BCG in tuberculous mice actually caused necrosis in the pulmonary lesions and increased the severity of the disease [18]. Mice are a species in which such necrosis is rare.Note: Tuberculin skin tests do not seem to contain sufficient antigen to stimulate the liquefaction process, but no studies have been made on this possibility. Repeated tuberculin skin-testing has never been shown to produce tuberculin positivity in naive recipients. Yet, BCG vaccination routinely does so. These facts suggest that tuberculin itself is a minimal antigenic stimulus compared to those produced by the intact tubercle bacilli (and some antigenic fractions thereof) that are present in effective TB vaccines.In humans and rabbits, most early pulmonary TB lesions produced by inhaled virulent human-type tubercle bacilli are arrested by the developing immune response [9]. However, in mice and guinea pigs, most of these pulmonary lesions continue to enlarge until the host dies [9]. In other words, humans and rabbits usually develop an effective immune response and mice and guinea pigs usually do not. The immunity of nonhuman primates (to virulent human-type tubercle bacilli) seems to be well below that of rabbits and modern human beings [9]. Probably, the most genetically susceptible humans have long since been killed off by this disease.Rabbits and human beings control the growth of inhaled virulent human-type tubercle bacilli better than any other species. For rabbits, about 300 to 3,000 of these bacilli must be inhaled to establish a single visible primary tubercle [9]. For humans, about 20 to 200 of these bacilli must be inhaled to establish a single visible primary tubercle (Richard L. Riley, personal communication described in reference 9 on pages 224–226). Such tubercles are established in mice, guinea pigs and nonhuman primates with an average inhaled dose of only 10 to 30 of these bacilli [9]. This number for cynomolgus monkeys was not available [9].A good immune response amount requires the host to develop the correct amount of both delayed-type hypersensitivity (DTH) (i.e., tuberculin-like sensitivity) and cell-mediated immunity (CMI) (i.e., local macrophage activation), so that DTH and CMI can work together to inhibit the growth of tubercle bacilli [4,9]. Tuberculous mice develop only weak DTH, but develop good CMI [9]. Tuberculous guinea pigs develop good DTH, but develop only weak CMI [9]. In other words, mice are a poor species to recognize DTH-producing antigens, and guinea pigs are a poor species to recognize CMI-producing antigens. Therefore, each of these laboratory animals will not accurately reflect the DTH and CMI responses to TB vaccines that is found in humans and rabbits (which are species that develop both good DTH and good CMI responses).Several TB antigens promote DTH responses, whilst others induce CMI. Moreover, different animal species respond to the different antigens of the tubercle bacillus in different ways—which is clearly demonstrated when TB in mice, guinea pigs, rabbits and humans are compared [9]. Using both mice and guinea pigs to evaluate new TB vaccines would be much better than using either species alone. However, since the DTH and CMI responses of rabbits more closely resemble those found in human beings, the rabbit is the most relevant laboratory animal for evaluating DTH and CMI responses simultaneously. Mice, guinea pigs, rabbits and humans will each respond to each antigen in TB vaccines in their own manner. Since no animal responds exactly as humans do, new TB vaccines should be evaluated in all three laboratory animal species (and perhaps even in nonhuman primates) before expensive clinical trials are undertaken.The correct balance (or ratio) of DTH and CMI is required for a new vaccine to be better than BCG. Table 1 lists the DTH (i.e., the PPD skin test) and CMI (i.e., the interferon-gamma production by peripheral blood lymphocytes) produced in humans by many candidate TB vaccines now in clinical trials [19]. All of these vaccines increased the IFN-gamma response, and almost all of them converted the PPD skin test.Protective efficacy, delayed-type hypersensitivity (DTH) response and cell-mediated immunity (CMI) response induced by current TB vaccines in preclinical and in clinical trials.Phase I, II and III, early, intermediate and late tests; BCG, Bacille Calmette-Guérin; Y, Better than BCG; EQ, Equal to BCG; ND, Not determined; NA, Not Available; +, positive. In preclinical testing, most of these new TB vaccines protected mice and guinea pig equally or better than BCG. (However, very few of these vaccines have been evaluated in the rabbit model.) In clinical testing, most new TB vaccines produced both Purified Protein Derivative (PPD) skin-test sensitivity (DTH) and antigen-specific peripheral blood IFN-gamma production (CMI) in humans receiving the new vaccines. Adapted from [19].Table 1 also lists the protection (when available) of mice, guinea pigs and rabbits produced by these new TB vaccines when they were compared to the protection produced by BCG vaccine. Several of the new vaccines gave better protection than BCG, and others were just equivalent to BCG in the species tested. However, very few of the vaccine candidates were tested in the rabbit model.DTH and CMI are responses to many antigens. PPD (Seibert’s Purified Protein Derivative of Koch’s Old Tuberculin) contains several antigens that are active in producing the tuberculin skin test [20], and many antigens that produce CMI are being evaluated as improvements or additions to live BCG vaccines [21,22,23]. As discussed above, mice, guinea pigs and rabbits develop different degrees of DTH and CMI when infected with virulent tubercle bacilli. Individual humans also vary in their DTH and CMI responses to tubercle bacilli. Such species and individual differences determine the balance of DTH and CMI produced in the host by new vaccines, and this balance determines how the TB-infected host handles the disease. Insight into the balance of DTH and CMI is best provided by aerosol infection of rabbits with virulent M. tbc. Pulmonary TB in this animal species matches pulmonary TB in humans better than it does in any other animal species. Both rabbits and humans arrest most early developing pulmonary tubercles, and no other laboratory animal species does so.Insight into the balance of DTH and CMI is best provided by aerosol infection of rabbits with virulent M. tbc. Pulmonary TB in this animal species matches pulmonary TB in humans better than it does in any other animal species. Both rabbits and humans arrest most early developing M. tbc pulmonary tubercles, and no other laboratory animal species does so.Quantitation of the tuberculin skin tests (DTH) and IFNγ blood tests (CMI) could be used to characterize the immune response of persons with or without active TB.Pulmonary tubercle counts in rabbits are rarely used to assess the efficacy of new TB vaccines, because most TB investigators do not have facilities to expose rabbits to aerosols containing virulent tubercle bacilli. Existing mouse aerosol exposure chambers can expose 50 mice at a time, but are too small to expose rabbits. Existing guinea pig aerosol chambers can expose 18 guinea pigs at once, but can only house six rabbits for simultaneous exposure.Lurie (using the Wells apparatus) [24] exposed six rabbits at a time. The rabbits were placed loosely in cloth bags (to restrict their activity) and then in metal cylinders (arranged like spokes of a wheel), with the head of each rabbit protruding into the aerosol chamber. Dannenberg [25,26,27] had his rabbits exposed individually at Ft. Detrick (by M. Louise M. Pitt) to 12 aerosols produced from aliquots of a single culture of virulent tubercle bacilli. Each rabbit was again placed in a loose bag and hand-held with its head protruding into the aerosol chamber for exactly 10 minutes. Both Lurie and Pitt collected samples of the aerosol with an impinger. The samples were then cultured in various dilutions to determine the number of viable tubercle bacilli in each aerosol. The dose of the bacilli inhaled by each rabbit was calculated from its respiratory rate. Tubercle counting is currently used by the Gilla Kaplan [28] and the William Bishai groups [25,26,27] for pulmonary TB studies, but they are not (to our knowledge) using the method to evaluate new TB vaccines. The Kaplan group uses a nose-only exposure system [28], and the Bishai group [26,27] currently uses a Madison chamber that is usually used to infect guinea pigs.Different laboratories count tubercles in different ways. Lurie [3] euthanized the rabbits containing five-week old pulmonary tubercles and fixed their lungs by inflating them with 10% formalin (4% formaldehyde) via the trachea. A week or so later, he dissected each tubercle from the lungs and counted them. The number of tubercles can be almost as accurately assessed by palpation of unfixed lungs (so that the unfixed lungs can be cultured for live tubercle bacilli). Surface tubercles can be counted to estimate their total number with about 80% accuracy. Recently, the number of pulmonary tubercles in live rabbits has been estimated by combined computer-assisted tomography (CT) and positron emission tomography (PET) (again with about 80% accuracy) [29,30,31]. This method provides an opportunity and to monitor the progress of TB lesions in the lungs of living rabbits during the whole course of this disease and also provides an estimate of lung inflammation. Since this method detects the amount of inflammation and consolidation in TB lesions in the lungs of living rabbits, it can be used to study the liquefaction and cavity formation as they develop [31]. Use of combined PET-CT imaging has huge potential to improve the evaluation of vaccine efficacy in rabbits, as well as in other animal models. This is the age of molecular microbiology in which the genome of microorganisms is providing clues to their virulence, as well as guides for the development of new antimicrobial agents. The genomic sequence of M. tbc is now known [32] and this sequence enables the identification of products that M. tbc manufactures. The genome of M. tbc transcribes two product categories: (a) those that maintain the life of the bacillus and allow it to reproduce wherever it thrives and (b) those that enable it to withstand the innate and acquired (adaptive) defenses of the host. Here are a few examples of the latter category.(a) Phthiocerol dimycocerosate (PDIM), which makes the mycobacterial cell wall resistant to destruction by macrophages [33], (b) ESAT-6, produced by the RD1 gene region that (among other functions) induces the recruitment of non-activated macrophages in which intracellular bacillary growth readily occurs [34]—the absence of ESAT-6 contributes to the avirulence of both BCG (the widely used TB vaccine) and H37Ra (a common avirulent laboratory strain, (c) cell surface lipids that enable M. tbc to survive within the phagosome and prevent its acidification and maturation [35] and (d) factors that enable the bacillus to survive in solid caseous necrosis in the guinea pig model of tuberculosis [36,37].Antimicrobial drugs and immunological defenses that inhibit the survival and growth of M. tbc may also be effective against a variety of other microorganisms. However, antimicrobial drugs and immunological defenses that reduce the ability of M. tbc to survive for years in solid caseum are probably more specific for this microorganism.Finally, human beings vary considerably in their response to different microbial antigens. A strong immunological defense against specific M. tbc antigens (found in most individuals) is usually able to keep the disease arrested for the person’s lifetime, whereas a weak immunological defense against such antigens (found in some individuals) would allow the M. tbc that escape from an arrested TB lesion to cause clinical disease.The genomic field is still in its infancy, and many more factors involved in host-parasite interactions will be discovered in the next few years.To our knowledge, no laboratory is studying the process by which solid caseous tissue is produced in the center of developing tubercles, and no laboratory is studying the factors that cause this solid caseum to liquefy and forms cavities. Yet, these two processes are responsible, respectively, for the latency and the reactivation of tuberculosis in human populations. The rabbit is the only easily accessible laboratory animal in which both of these processes are readily produced [9]. Mice rarely form caseous tubercles, never form liquefied caseum and never form cavities [9]. Guinea pigs do form solid caseum, but only rarely form liquefied caseum and cavities [9]. (Before cavities develop, guinea pigs usually die of the childhood form of TB that is hematogenously spread rather than bronchial spread).In the rabbit, most early developing tubercles caused by virulent M.tbc (i.e., virulent human-type tubercle bacilli), regress and never reach visible size, because (as stated above in Item 2.1) 300 to 3,000 M. tbc must be inhaled by rabbits to form one tubercle that is visible five weeks afterwards [9]. In rabbits, the majority of the pulmonary tubercles caused by the human-type bacillus would regress when the immune process begins and would be hard to find at necropsy in tissues sections of their lungs.To more easily study the factors involved with the development of solid and liquefied caseum and cavity formation in rabbits, investigators should use virulent bovine-type tubercle bacilli, i.e., virulent M. bovis (e.g., the Ravenel strain), rather than virulent human-type tubercle bacilli [9]. Rabbits develop a visible tubercle when less than 10 virulent M. bovis are inhaled, so that after inhaling a relatively large dose of virulent M. bovis, developing and progressing pulmonary tubercles would be rather easy to find [38,39]. With virulent bovine-type tubercle bacilli, all beginning pulmonary tubercles progress forming a solid caseous center or a liquefying caseous center that is frequently followed by cavity formation. Histological studies and immunological studies, as well as knockout gene and quantitative gene studies, could then be used to elucidate the mechanisms involved in caseation, liquefaction and cavity formation. As stated above, rabbits are the only common laboratory species in which virulent tubercle bacilli frequently liquefy solid caseum and form pulmonary cavities.Liquefied caseum and cavities are produced readily in rabbits inhaling virulent M. bovis [38,39], especially if the rabbits are made tuberculin-positive by vaccines or by a previous exposure to virulent tubercle—probably (as stated above) because increased numbers of activated macrophages (containing increased levels of hydrolytic enzymes) are present. Specific inactivation of such enzymes by pharmaceuticals or by immunological procedures could stop or reduce liquefaction and cavity formation—thereby decreasing the prevalence of tuberculosis in the world.A convenient way to evaluate methods to reduce liquefaction and cavity formation is to produce a surrogate model for these occurrences in the skin of rabbits, specifically, intradermal injections of BCG (or even killed virulent tubercle bacilli) in ascending concentrations (i.e., low to high amounts in 0.1 mL of diluent) [15]. Rabbits receiving effective inhibitor therapy would require a higher concentration of BCG (or dead virulent tubercle bacilli) to produce liquefaction and ulceration of the skin than would rabbits not receiving such therapy. Effective therapies could then be evaluated in the M. bovis-produced pulmonary model of cavity formation, just described.In naive populations (i.e., tuberculin skin-test negative), about 95% of individuals are inherently resistant to active tuberculosis, because they produce an effective immune response to the tubercle bacillus without a vaccine [40,41]. Therefore, an effective TB vaccine could only benefit about 5% of a total population. If exposed, these 5% of individuals would develop active disease and could even die from it. These individuals evidently produce an insufficient immune response and would therefore be an appropriate trial population in which to evaluate TB vaccines.An effective TB vaccine could reduce the number of clinically active tuberculosis cases from 5% to 1% by expanding the appropriate T lymphocyte populations. In other words, the TB vaccine could now protect about 80% of this group [42,43,44,45,46]. Complete protection of every individual may never be achieved.Note that the 95%, 4% and 1% of individuals approximate those found in populations in United States and Europe. Underlying genetic and environmental factors can dramatically shift this ratio [47]. Developing countries with a high percentage of immune-deficient individuals from HIV infection would have a different proportion in each group (see Item 3.2, below). In other words, the efficacy of TB vaccines would vary from one geographic region to another.At present, the only way to select a population that could benefit from a better TB vaccine is to run a preliminary study with a standardize BCG strain and compare the rates of healing between several populations. Populations that healed slowly would have more individuals in the 5% group that could benefit from the vaccine. However, populations that healed quickly would have more individuals in the 95% group in which benefits from the vaccine would be unrecognizable. Several such preliminary trials need to be performed in order to establish the best way to recognize the slow and rapid healers of BCG lesions in clinical populations.A high prevalence of infection with HIV exists in some developing countries, especially in sub-Saharan Africa. HIV infection lowers host acquired (adaptive) immunity to the tubercle bacillus [47,48]. Therefore, HIV-infected persons would respond less well to BCG vaccination and other vaccines than would persons who are not infected with HIV. If the HIV-infected group were identified and separated from the non-HIV group, the beneficial effects of BCG and newer TB vaccines would be more easily recognized.BCG vaccination would protect some M. tbc-infected, HIV-infected individuals from developing clinically active disease when the HIV only partly decreased their immune response. However, BCG vaccination would have no benefit or could even be detrimental if HIV greatly lowered their immune response. In the Karonga/Malawi BCG trial, 57% of cases of clinical tuberculosis were directly attributable to HIV infection [47,48].In developing countries, tuberculosis and intestinal worm infections often occur in the same groups of people [49]. Worm infections may cause some debilitation and also lower host resistance to tuberculosis [49]. Therefore, worm-infested populations would respond less well to BCG vaccination (and to newer TB vaccines) than would non-infested populations [49,50]. Poor nutrition would probably have a similar effect [51,52]. (If identified during these trials, individuals with helminth infections and/or poor nutrition should be given treatment, which would allow their inclusion in future trials).Environmental mycobacteria could have increased immunity in unvaccinated control groups [47,53,54,55], so the beneficial effects of BCG would be hard to detect, as in the Malawi trial [47] and in other inconclusive trials [41,53,54,55]. A comparison of the rates of healing of dermal BCG lesions in Europe and/or USA with those in the developing country would throw light on this possibility.Many individuals in tuberculin-negative groups may show a booster reaction if skin-tested again with tuberculin [16,17,56]. If they did, they would already have increased immunity to M. tbc from a healed natural infection. Therefore, BCG or a new TB vaccine would provide little additional benefit. Groups showing such booster reactions should not be considered tuberculin-negative.Some of these vaccines (including different strains of BCG) will be more effective than others depending on their antigenic composition (discussed in [9]). M. tbc antigens in current clinical trials are in listed in Table 1.BCG vaccination reduced clinical tuberculosis in North American Indians by 50 to 80% [42,43,46]. This indigenous population probably had not been exposed to the tubercle bacillus for as many centuries as had the populations in Europe and Asia and, therefore, might have had more individuals who can benefit from BCG vaccination. In contrast, the trial in Chingleput, South India, showed that BCG vaccination was not beneficial [57]. This may have been because a higher percentage of the population selected belonged to the “95%” group who were naturally resistant to clinical disease.This paper provides perspectives on the immunization of humans, mice, guinea pigs, rabbits and monkeys that have not usually been considered in evaluating new TB vaccines for clinical trials. These perspectives are briefly as follows.Both delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI) must be produced in a host to arrest the progress of tuberculosis. DTH and CMI are similar immunological processes involving Th1 lymphocytes. However, CMI and DTH inhibit the growth of M. tbc by different mechanisms: CMI activates macrophages so that they inhibit the growth of M. tbc that they ingest. DTH kills non-activated macrophages that become overloaded with M. tbc and produces solid caseous necrosis in which the bacillus does not grow. (Non-activated macrophages are present in every active tuberculous lesion and may ingest and become overloaded with them).DTH and CMI are produced by different M. tbc antigens and new vaccines must contain these antigens in the proper amount. Mice (infected with M. tbc) have weak tuberculin sensitivity (DTH) and apparently good CMI, and they usually die of the disease. Guinea pigs (infected with M. tbc) have good tuberculin sensitivity (DTH) and apparently weak CMI, and they also usually die of the disease. However, most humans and rabbits (infected with M. tbc) usually stop the progress if this disease. Therefore, we concluded that mice do not respond well to DTH-producing antigens, and guinea pigs do not apparently respond well to CMI-producing antigens. Yet, humans and rabbits (species that usually arrest the disease produced by M. tbc) evidently respond well to both DTH- and CMI-producing antigens.In human trials, BCG vaccination has not been consistently beneficial. Yet, in laboratory animals, BCG has consistently increased host resistance to challenge with M. tbc. We propose that the rate of healing of the BCG lesions (used as a control for new vaccines in clinical trials) will identify the 95% of humans who arrest infection with M. tbc without the need of vaccination. In the remaining 5%, the benefits of BCG vaccination should be easier to recognize and should be more consistent with those found in laboratory animals.The arrestment of early pulmonary tubercles by the immune process before they become clinically apparent is the very purpose of TB vaccination. Early tubercles in mice and guinea pigs are not as easily arrested, but most early pulmonary tubercles caused by M. tbc in rabbits and humans are arrested.Because of expense, the tubercle counting in rabbits has been not been undertaken before starting much more expensive clinical trials. However, tubercle counting in rabbits could select the most effective new TB vaccines more precisely than any other procedure, because in rabbits (as in humans) the progress of many developing tubercles are arrested by immune forces, whereas in mice and guinea pigs, few, if any, such tubercles are arrested by such forces. Therefore, tubercle counting in rabbits should be performed before clinical trials on new TB vaccines are begun.The antigens recognized by mice and those recognized by guinea pigs together may (or may not) be the same as the antigens recognized by rabbits. And, the antigens recognized by rabbits may (or may not) be the same as the antigens recognized by humans. Such differences and similarities remain to be investigated. Therefore, we urge investigators to always include rabbits along with mice, guinea pigs and, perhaps, monkeys in the preclinical testing of new TB vaccines in order to make preclinical studies more complete.Vaccines containing critical antigens (possibly the Antigen 85 complex, ESAT-6 or hspX) could increase the immunity of the host to a greater extent than that produced by a natural M. tbc infection. Immunization with such critical antigens would increase the host’s ability to neutralize the major components of M. tbc that determine its virulence. Such vaccines could then be used for both TB prophylaxis and TB immunotherapy. However, only some critical antigens have so far been identified.We appreciate the help of Andre Kubler (from the Center for Tuberculosis Research, Johns Hopkins School of Medicine in Baltimore and from the Department of Medicine, Imperial College of London in UK) in formatting our manuscript and in reviewing its contents. Bappaditya Dey was supported by a post-doctoral fellowship from the Howard Hughes Medical Institute.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The Extended Parallel Process Model (EPPM) is an established threat- and efficacy-based behavioral framework for understanding health behaviors in the face of uncertain risk. A growing body of research has applied this model to understand these behaviors among the public health workforce. In this manuscript, we aim to explore the application of this framework to the public health workforce, with a novel focus on their confidence in vaccines and perceptions of vaccine injury compensation mechanisms. We characterize specific connections between EPPM’s threat and efficacy dimensions and relevant vaccine policy frameworks and highlight how these connections can usefully inform training interventions for public health workers to enhance their confidence in these vaccine policy measures.Health providers’ personal decisions not to receive influenza vaccination present a major ongoing public health challenge, with significant implications for morbidity and mortality, medical workforce capacity and healthcare system costs [1]. Low rates of seasonal influenza vaccination among physicians in the U.S. and Israel, for example, reflect the international scope of physicians’ own non-adherence to this critical preventive behavior [1,2].A growing body of research has examined perceptual barriers impeding health providers’ self-adherence to influenza vaccination guidelines. To date, such identified barriers have included a lack of information about novel and seasonal influenza vaccines, the risk of influenza infection, the role of the healthcare worker in transmission and vaccine safety and efficacy [3,4,5,6]. Motivational factors have included the desire to protect self and family through vaccination [5].Of note, modifying factors for providers’ own influenza vaccination behavior appear to be similar for both seasonal and pandemic influenza contexts [6,7,8]. Indeed, these factors resonate with findings from studies that we and other researchers have conducted on pandemic flu response willingness-related attitudes among healthcare providers, including physicians [9,10].Senior public health organizational and regulatory bodies (e.g., Centers for Disease Control and Prevention, World Health Organization, national health ministries) are leading purveyors of influenza vaccination practice guidance for physicians. Accordingly, the effectiveness of these public health authorities in mitigating influenza disease burden hinges substantially on healthcare providers’ adherence to the vaccine guidelines that these agencies deliver—for themselves and their patients.Public health department-based providers represent a critical component of the frontlines of vaccine delivery and are at the heart of the public health emergency preparedness system [11,12]. Despite the importance of public health workers in this context, the research literature has yet to sufficiently examine how this provider cohort’s perceptions toward vaccination and vaccine injury compensation may influence their vaccine-related behaviors for themselves and the communities they serve. Recent research suggests that the Extended Parallel Process Model (EPPM), an established threat- and efficacy-based behavioral framework for understanding health behaviors in the face of uncertain risk, could be usefully applied to understand such vaccine-related perceptions among the public health workforce [13,14,15,16]. In this article, we aim to explore the potential utility of explicitly applying the EPPM to examine public health providers’ confidence in vaccines and vaccine-injury compensation mechanisms.Specifically, we will explore these EPPM-based implications in the following contexts: (1) National Vaccine Injury Compensation Program; (2) Public Readiness and Emergency Preparedness Act (PREP Act) for Pandemic Influenza Medical Countermeasures Utilization Protocol & Decision Tools; and (3) mandatory vaccination policies.Each of these three contexts involves a unique legal infrastructure with specific components that may impact public health workers’ confidence in vaccines. In particular, we focus on the compensation mechanisms that the government has established for individuals and families that experience vaccine-related adverse events. Public health workers’ awareness and understanding of these systems may influence their perceptions of threat and efficacy and, thus, their willingness to receive a vaccination. Introduced by Witte in 1992, the EPPM has the potential to cast light on how the public health workforce can be encouraged to participate in influenza vaccination. The model posits that for a message to induce behavior change, it must simultaneously convey the constructs of threat and efficacy. The construct of threat has two components: severity or the idea that the threat is significant enough to warrant action; and susceptibility or the idea that the person initiating the behavior may be affected by the threat. The construct of efficacy also has two components: self-efficacy or the idea of confidence that the person is able to perform the behavior; and response efficacy or the idea that the behavior change will achieve the intended impact. If the message induces both perceived threat and perceived efficacy in the recipient, the message may be accepted [16]. If the message induces high levels of perceived threat, but does not promote perceived efficacy, fear will be elicited and the recipient will undergo what is known as “fear control”. In this situation, the message may be rejected or defensively avoided [17]. Importantly, self-efficacy or the confidence component of efficacy, has been identified as the component most associated with actual behavior adaptation [18].The EPPM has been both qualitatively and quantitatively applied to or suggested for a variety of preventive health behaviors, including low income mothers’ access to preventive dental care for their children, contraceptive use among women, colonoscopy screening, hearing loss prevention and smoking cessation intentions [19,20,21,22,23,24]. The relative importance of confidence has been affirmed in predicting preventive behavior engagement in some of this work. For example, among smokers with low readiness to quit, threat and efficacy were both seen as important predictors of intention to engage in smoking cessation behavior. However, among those with high readiness to quit, efficacy was most important in this prediction [24].The foundation for the use of this model to understand and promote preventive behaviors toward influenza has recently been developed. Notably, in a study utilizing EPPM-framed forewarning messages to promote preventive behaviors, such as vaccination for H5N1 influenza, perceived threat was associated with fear arousal, which was less positively related to behavioral intention than perceived efficacy [25]. These findings underscore the role of confidence in determining a person’s potential decision to get vaccinated relative to an individual’s perceived threat. In a qualitative study of African American seniors employing the EPPM, issues related to accessing an influenza vaccine dominated the discussion related to efficacy. This population, which receives lower levels of vaccination compared to the general population, identified accessibility, affordability, negative consequences of vaccine (e.g., side effects), physician recommendation and efficacy of vaccination as emergent themes associated with efficacy [26]. In a study of individuals aged 65 and older, while messages developed under the EPPM framework were not shown to significantly increase intention to receive vaccination, the use of the framework in message design did significantly positively influence perceptions of risk and efficacy [27]. Here, we describe in greater detail the three aforementioned law and policy contexts germane to confidence in vaccines among public health providers: (1) National Vaccine Injury Compensation Program; (2) Public Readiness and Emergency Preparedness Act (PREP Act) for Pandemic Influenza Medical Countermeasures Utilization Protocol & Decision Tools; and (3) mandatory vaccination policies.Each year, millions of people in the United States are vaccinated to prevent the morbidity and mortality associated with various infectious diseases. Although the effectiveness and general safety of vaccines have been repeatedly demonstrated, they are not without risk. On rare occasions, some individuals experience side effects, including seizures and anaphylactic shock after receiving a vaccination [28]. Known as vaccine-related adverse events, these responses raise important questions, especially for vaccinations that are recommended by the government. One of the most salient questions to emerge is what, if any, compensation should individuals or families receive after experiencing a vaccine-related adverse event?In the mid-1980s, Congress tackled this issue with the drafting and passage of legislation to establish the National Vaccine Injury Compensation Program (VICP) [29]. The VICP is a government-run program, housed in the U.S. Department of Health and Human Services (HHS). It offers compensation to individuals who qualify, based on the vaccine they received and the nature of the adverse event they experienced. Numerous vaccines are covered by the VICP, including diphtheria, tetanus, pertussis, measles, mumps, rubella and polio. Importantly for public health workers, the VICP covers both Haemophilus influenzae and trivalent influenza vaccines. A publicly available table lists all vaccines and adverse events covered by the program (e.g., anaphylactic shock within four hours of receiving a tetanus vaccination) [30]. To be eligible, claims for compensation must be filed within three years of the initial occurrence of the adverse event. In general, the claim should concern a vaccine and an adverse event that occurred within a timeframe listed on the table. Once a claim is filed, a physician within HHS makes an initial determination about whether the medical information in the claim meets the qualifications for compensation. This recommendation is then shared with a special master tasked with making compensation decisions for the VICP. Compensation may include coverage for medical expenses, attorney’s fees, lost earning capacity and death benefits for survivors [31]. The VICP provides some liability protections for individuals who administer vaccines that are covered by the program.While the VICP’s protections are available to both children and adults, it was intended to cover vaccinations routinely offered to children. The VICP does not offer compensation for vaccine-related adverse events associated with emergency countermeasures, such as vaccines used to treat H1N1 pandemic influenza or smallpox. To address this gap, Congress established the Countermeasures Injury Compensation Program (CICP) with the passage of the Public Readiness and Emergency Preparedness (PREP) Act in 2006 [32]. Like the VICP, the CICP is housed in the U.S. Department of Health and Human Services. It provides compensation to individuals who experienced an adverse event related to a countermeasure they received that was covered by the CICP [33]. Compensation may cover medical expenses, lost income and death benefits for survivors. Unlike the VICP, the CICP only covers countermeasures that are listed in a declaration from the Secretary of the Department of Health and Human Services. This declaration must specify the disease that poses or is likely to pose a public health emergency, the countermeasure that is covered to treat the disease, the period for which the declaration will remain in effect and any population or geographic limitations for administration of the countermeasure [34]. Claims for compensation under the CICP must be filed within one year of the administration of the countermeasure. Since the PREP Act’s inception, the Secretary has issued declarations that covered vaccine countermeasures for smallpox, acute radiation syndrome, anthrax and botulism, as well as pandemic influenza, including the H1N1 and H5N1 strains [35]. The PREP Act includes liability protections for individuals who participate in the development and administration of covered countermeasures [36].Over a century ago, the U.S. Supreme Court established that states can require individuals to receive vaccinations to protect and promote the public’s health [37]. All states, for example, have vaccination requirements that children must meet before they can attend school, with exceptions for individuals with medical contraindications and—in some states—exemptions for those with religious and philosophical objections. These requirements are associated with coverage rates of approximately 95% for children entering kindergarten in the U.S. [38]. In recent years, state and local governments and private employers have grappled with requiring mandatory seasonal influenza vaccinations for healthcare workers who have routine patient contact. These policies, which have been implemented by healthcare institutions in nearly half of the states, contain exemptions for healthcare workers who have medical contraindications [39]. Some also allow healthcare workers to sign a declination statement and take additional precautions (e.g., wearing a mask) if they refuse the vaccination. The policies typically do not mention compensation for vaccine-related adverse events. Should an adverse event occur, compensation would potentially be available from the VICP for seasonal influenza vaccinations. In addition, compensation could be available for pandemic influenza vaccinations from the CICP if the Secretary of Health and Human Services issued a declaration to cover the countermeasure.Mandatory seasonal influenza policies have faced legal challenges from healthcare workers who, in some cases, argue that they contravene collective bargaining agreements. Such lawsuits, which tend to be highly publicized, can undermine confidence in vaccines, as they may raise questions for the general public about the safety and effectiveness of vaccines [40].PREP, VICP and mandatory vaccination policies could potentially serve as effect modifiers toward public health workers’ confidence in vaccines, with attendant implications for vaccine uptake rates among this vital health provider cohort. In that vein, the law has long been recognized as a policy tool with a demonstrated ability to impact behavior. Laws can cause numerous changes—to environments, to product availability, to the legality of certain practices—that may influence individuals’ behaviors. While the implementation of a new law may be associated with behavior change, it may also occur due to the amendment, repeal or expiration of an existing law or to other factors entirely. Two brief examples, from tobacco control and motor vehicle safety, illustrate the law’s potential to influence behavior change.Tobacco control researchers have repeatedly found that the law is a powerful component of smoking cessation and prevention efforts. The implementation of legal measures, such as the presence of tobacco taxes, which raise the price of cigarettes, has been associated with individuals’ increased intention to quit smoking, as well as their decreased purchase of cigarettes. A systematic review of tobacco taxes and smoking behavior concluded that a robust evidence base demonstrates that increasing the price of cigarettes through tobacco taxes is associated with the reduction of smoking, particularly among young people [41]. Several studies have examined clean indoor air laws through the lens of behavior change theories. These studies have similarly concluded that certain theories, such as the theory of planned behavior, can help to explain the interplay between individuals’ intentions to quit smoking and public policies designed to limit tobacco use. For example, in a study grounded in a conceptual model based on the theory of planned behavior, Middlestadt and colleagues found that current smokers who lived in a city with a smoke-free air law and who had high intentions to quit had greater odds of engaging in quitting behaviors than those who had lower intentions to quit and lived in cities without smoke-free air laws [42]. Recently, some researchers have modeled how possible legal approaches—such as a ban on menthol cigarettes—might influence the behavior of current smokers. O’Connor and colleagues found that this type of ban would potentially be associated with quit attempts by approximately one-third of individuals who currently smoke mentholated cigarettes [43].Studies have repeatedly confirmed the important role that laws have played in reducing the number of annual traffic fatalities in the United States, particularly due to their impact on seatbelt use behavior [44]. For example, Chaudhary and colleagues determined that primary enforcement seatbelt laws are associated with increases in both daytime and nighttime seatbelt use [45]. Primary enforcement laws allow police to issue citations for failure to wear a seatbelt; secondary enforcement laws require the presence of another traffic violation before police can give a citation for lack of seat belt use. In an analysis that considered multiple federal and state data sources, Carpenter and Stehr found that laws requiring the use of seatbelts—and particularly those laws that allowed for primary enforcement when seatbelt laws were violated—were associated with statistically significant increases in the use of seatbelts by adolescents [46]. In addition, they found that primary enforcement laws for seatbelts significantly decreased traffic-related deaths within this age group. Laws intended to reduce drinking and driving, especially among young people, have also been associated with statistically significant decreases in motor vehicle fatalities [47,48]. These types of results suggest that laws can influence behavior changes and thus contribute to improved motor vehicle safety.Against this backdrop of law and its relationship to behavior change, EPPM can serve as a useful lens to gauge the extent to which VICP, PREP and mandatory vaccination policies may serve as effect modifiers toward vaccine confidence. In addition, the application of EPPM to this area may assist in the design of relevant training approaches for public health workers’ awareness of and confidence in available protections. Here, the recent public health preparedness literature provides relevant insights. Specifically, EPPM has recently been applied to several cohorts involved in the public health workforce to understand and improve their willingness to respond to a variety of emergencies in the all-hazards disaster spectrum, including pandemic influenza. Notably, the self-efficacy confidence construct was consistently identified as the leading overall predictor of willingness to respond to a pandemic influenza emergency among all cohorts studied [14,49,50,51]. Indeed, EPPM-based research has found that efficacy outweighs threat as a modifier of public health workers’ response willingness toward infectious disease and other crisis scenarios [14]. The implications of these EPPM-related findings are described below.EPPM-based research to date on emergency response willingness points to the applicability of a threat- and efficacy-based behavioral model for future applied research aimed at understanding the interplay between legal protections and confidence in vaccines. A closer examination of how EPPM’s threat and efficacy components apply to the three vaccine contexts discussed above illustrates EPPM’s considerable potential as a conceptual framework for vaccine confidence-boosting initiatives against these policy backdrops. In the case of the VICP, for example, EPPM’s threat dimension applies to public health workers’ concerns about experiencing a vaccine-related adverse event. According to EPPM principles, instilling awareness of this threat dimension (i.e., potential, though rare, adverse events) can motivate public health workers to seek additional information about the protections that the VICP affords and how to access these protections should an adverse event occur. Meanwhile, EPPM’s efficacy dimension in this context applies to public health workers’ confidence in operational implementation of VICP as needed: in the case of self-efficacy, these workers can gain confidence in their knowledge of the VICP protections available to them and they can simultaneously increase their confidence by understanding the requisite protocols to access VICP protections. Under PREP, the elements of EPPM would apply in a similar way to the CICP, with the only difference being that the CICP applies to a more circumscribed set of vaccine-related countermeasures. Finally, in the context of mandatory vaccination, the threat dimension of EPPM reflects public health workers’ potential concerns that they may serve as vectors of vaccine-preventable illness to their own families and to the vulnerable patient populations they serve. With regard to EPPM’s efficacy domain in the case of mandatory vaccination policies, self-efficacy refers to public health workers’ confidence in their comprehensive understanding of the mandatory policy, a thorough knowledge of where to go to receive vaccinations and a clear awareness of whom to contact if they encounter a vaccine-related adverse event. Further, in the context of mandatory vaccination policies, EPPM’s response efficacy dimension would reflect these workers’ confidence that mandatory vaccination actually works to mitigate the likelihood of disease transmission.Importantly, EPPM-centered training is the common operational thread that binds these conceptual understandings with practical implementation strategies that can boost confidence in vaccination amidst the three vaccine-policy backdrops described above. EPPM-centered training efforts could enhance public health workers’ confidence in vaccines through threat- and efficacy-building elements. Specifically, the threat elements of such training should address the respective vaccine policy threat dimensions noted above, as the EPPM indicates that threat awareness can have a positive motivational effect toward adoption of desired behaviors (in this case, vaccination against these respective policy backdrops). Meanwhile, such EPPM-centered training activities should include a particularly strong emphasis on efficacy-building training measures surrounding these vaccine policies, as efficacy has been found to outweigh threat as a modifier of public health workers’ response behaviors in the face of perceived risk [14]. The efficacy component of such EPPM-centered trainings would need to focus on: (1) boosting public health workers’ confidence in their understanding of and knowledge of how to access respective protections accompanying these measures (i.e., self-efficacy per the EPPM); and (2) instilling a clear awareness that the protections afforded are meaningful and substantive and would be fully implemented (i.e., response efficacy per EPPM). The importance of explicit awareness—and confidence in—workplace protective measures is bolstered by the research finding that perceived safety at work is a highly significant modifier of public health workers’ willingness to fulfill role expectations in a variety of disaster contexts [14]. The role of legal protections in boosting confidence in vaccines has become increasingly relevant in an environment where mandatory vaccination for public health workers is a contentious policy issue. Further, EPPM-centered research could assist policy-makers, emergency planners and others to better understand root determinants of the influence of legal protections on vaccine uptake decisions among public health providers. Additionally, as noted above, the EPPM can be used to inform training interventions to enhance public health workers’ awareness of relevant vaccination policies and confidence in them.Future EPPM-based research on policy and practice can thus provide a vital evidence base for enhanced understanding of how public health workers’ confidence in vaccines is modified by relevant protective laws, through the lens of an established threat- and efficacy-centered behavioral model. This type of research can focus not only on public health workers, but also on other health provider cohorts on the frontlines of community vaccination efforts.The authors wish to thank Felicity Marum, MHS, for her literature review assistance that aided in the development of this article.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).An HIV vaccine, once it becomes available, could reduce vulnerability to HIV among African-American women. The purpose of this study was to assess determinants of HIV vaccine acceptability among African-American women across hypothetical levels of vaccine efficacy. Participants were recruited from a hospital-based family planning clinic in Atlanta, GA serving low-income patients (N = 321). Data were collected from audio-computer assisted surveys administered in the clinic waiting room. Psychosocial survey items were guided by Risk Homeostasis Theory (RHT) and Social Cognitive Theory (SCT). Multivariate logistic regression was used to identify determinants of acceptability for two hypothetical HIV vaccines with 50% and 90% efficacy. Overall, 63% of participants would accept a vaccine with 50% efficacy and 85% would accept a vaccine with 90% efficacy. In multivariate analyses, odds of acceptability for a vaccine with 50% efficacy were higher among participants with greater perceived HIV vaccine benefits (AOR = 1.13, p < 0.001) and lower among participants with more than high school education (AOR = 0.47, p = 0.033) and greater perceived costs of HIV vaccination (AOR = 0.95, p = 0.010). Odds of acceptability for a vaccine with 90% efficacy were higher among participants with greater perceived costs of unprotected sex (AOR = 1.08, p = 0.026), HIV vaccine benefits (AOR = 1.23, p < 0.001) and self-efficacy for sex refusal (AOR = 1.11, p = 0.044). HIV vaccine acceptability was high, particularly for a vaccine with 90% efficacy. Findings suggest that demographic and psychosocial factors may impact acceptability of an eventual HIV vaccine. Once an HIV vaccine is available, interventions to maximize uptake may benefit from using RHT and SCT constructs to target relevant psychosocial factors, such as perceived benefits and perceived costs of vaccination.African-American women are disproportionately affected by HIV [1,2,3]. In 2009, an estimated 11,200 women were diagnosed with HIV in the U.S. [4], and the rate of new HIV infections among African-American women was 15 times the rate among White women [4,5]. African-American women are also disproportionately affected by STIs such as gonorrhea, chlamydia, trichomoniasis, and genital herpes (HSV-2), which can increase the risk of contracting HIV and transmitting HIV to others [6,7,8,9]. There is a clear need for effective interventions to reduce the burden of HIV among this population.While several behavioral HIV-prevention interventions have demonstrated efficacy [10], these interventions alone may not be sufficient to control the spread of HIV. The introduction of a safe and effective HIV vaccine provides our best hope for ending the HIV pandemic [11], and would provide women, particularly African-American women, with much needed volitional control over vulnerability to HIV [12]. The promise of an effective vaccine has been supported by a recent vaccine trial in Thailand, which yielded a modest (30%), yet significant reduction in HIV infection, and several promising candidate vaccines are in the pipeline [13,14].Yet an FDA licensed HIV vaccine may be futile if high-risk populations, such as African-American women, are not vaccinated. Little is known about whether African-American women would accept an HIV vaccine. A recent meta-analysis found 30 original studies (21 in the United States) that assessed HIV vaccine acceptability [15]. Among all populations assessed, (high-risk adults, MSM, prison inmates, adolescents, parents, college students, Asian/Pacific Islanders, and military personnel), the range of vaccine acceptability was 37% to 94%, with a mean acceptability of 65.3%. Factors associated with vaccine acceptability across studies included vaccine characteristics, structural factors, HIV vaccine attitudes, ethnicity, and risk group membership [15]. Of note, only three studies have focused exclusively on HIV vaccine acceptability among high-risk minority women [16,17,18], all of which were qualitative. Across studies, findings indicated that barriers to HIV vaccine acceptability among minority women include: concerns about vaccines in general, concerns about HIV vaccine safety and side effects (including reproductive side effects), mistrust of the medical system due to historical ethical controversies (such as the Tuskegee syphilis experiment), concerns about the perceptions of others/stigma, implications for sexual relationships, and affordability/insurance [16,17,18]. Motivators for getting an HIV vaccine included reduced worry about becoming infected with HIV, empowerment to protect oneself against HIV, and getting a recommendation from a health care provider [16,17,18].Several additional studies, although not focused exclusively on women, have assessed HIV vaccine acceptability by gender [19,20,21,22,23]. Across studies, HIV acceptability concerns of particular salience to women included concerns about the HIV vaccine (such as past testing, safety, side effects), sex partner/relationship concerns, negative experiences with healthcare providers, affordability/insurance, and issues related to reproductive/teratogenic effects [19,20,21]. Gender-specific motivators included the ability to conceive a child without worrying about contracting HIV and support from sexual partners [19]. However, other studies did not identify gender-specific differences in HIV vaccine acceptability [22,23].Although these studies provide valuable information, there is a need for larger, quantitative studies assessing anticipated HIV vaccine acceptability among African-American women. The aims of the present study were: (1) to assess anticipated HIV vaccine acceptability depending on varying HIV vaccine characteristics (such as efficacy and cost), and (2) to assess correlates of anticipated HIV vaccine acceptability under varying levels of vaccine efficacy (50% and 90%) among a sample of sexually active African-American women recruited from a hospital-based family planning clinic.Participants comprised African-American women presenting for clinical services at a hospital-based family planning clinic in Atlanta, GA, USA. To be eligible, participants had to self-identify as: African-American; women; aged 18–55 years; HIV-negative or unsure of HIV status; had at least one unprotected sex act (vaginal, anal, or oral) in the past 6 months; and provide verbal informed consent to participate in the study. This study was approved by the institutional review boards at the researchers’ institution and participating hospital.Participants were recruited from the family planning clinic waiting room. Study staff approached women to ask if they were interested in participating in a research study, consisting of an hour-long survey with questions relating to an HIV vaccine. Interested patients were escorted to a private room for eligibility screening. Eligible participants completed audio-computer assisted (ACASI) surveys administered on laptop computers in a private room. The use of ACASI allowed participants to hear survey questions read through headphones, in addition to seeing them written on the computer screen. Study staff monitored survey administration and answered questions as needed. Most patients completed the survey prior to their clinic appointments. If participants were called for clinic appointments prior to survey completion, they were able to pause and resume their surveys after their appointments were finished. All participants who completed surveys received $25 cash as compensation.The survey instrument was designed to investigate multiple factors potentially associated with HIV vaccine acceptability, including demographics, health history, sexual risk behaviors, personality traits, and theory-based psychosocial constructs.HIV vaccine acceptability variables: HIV vaccine acceptability was assessed by asking the following questions: (1) How likely would you be to get an HIV vaccine that would prevent you from getting HIV about half the time (50% effective). (2) How likely would you be to get an HIV vaccine that would prevent you from getting HIV almost all the time (90% effective). Vaccine efficacies of 50% and 90% were chosen based on values used in previously published research [15,24,25]. (3) How likely would you be to get an HIV vaccine if it was available now. (4) How likely would you be to get an HIV vaccine if it required multiple doses (shots). (5) How likely would you be to get an HIV vaccine if it were free or covered by insurance. Participants initially answered questions on 5 point Likert scales ranging from 1 (Very unlikely) to 5 (Very likely). Answers were then dichotomized into “likely/very likely” compared to other answer choices. Additional questions assessed acceptability of out-of-pocket cost for the vaccine as well as who would influence participants’ decision to get the vaccine.Background variables: Demographics included: age, education level, and receipt of public assistance in the past 12 months. Health history included: previous STD diagnosis, previous HIV test, and a brief mental health assessment (5 item Likert scale, higher scores indicate better mental health, alpha = 0.77) [26]. Measures of sexual risk behaviors included: Condom use at last sex, multiple vaginal sex partners in the past 3 months, and at least one casual partner for any sex (vaginal, anal, or oral) in the past 3 months. Personality variables: Personality variables included (1) sexual adventurism [27]; (2) sensation seeking and impulsivity [28]; and (3) positive future orientation [29]. All personality variables were measured on 5 point Likert scales, ranging from 1 (Strongly Disagree) to 5 (Strongly Agree). Scale details are presented in Table 1. Personality and Psychosocial survey scale items.* RHT = Risk Homeostasis Theory; * SCT = Social Cognitive Theory; ** For newly developed measures, all scale items are provided. For existing validated scales, a sample item is given.Psychosocial variables: Psychosocial variables were guided by two key theories: Risk Homeostasis Theory (RHT) [30,31] and Social Cognitive Theory (SCT) [32]. RHT, initially developed by Wilde, posits that humans have a subjective, target level of risk with which they are comfortable. This level of risk depends on (1) the expected benefits of the risky behavior, (2) the expected costs of the risky behavior, (3) the expected benefits of the safe behavior, and (4) the expected costs of the safe behavior [30,33]. People will compare their perceived level of risk with their target level of risk, and adjust their behavior to eliminate discrepancies. In 2007, Eaton and Kalichman proposed an adapted model of RHT to assess risk compensation associated with biomedical HIV prevention, including HIV vaccination. [31]. This study employed an RHT framework, as a person’s target level of risk may impact their decision to accept an HIV vaccine. SCT, developed by Bandura, takes into account the impact of environmental-level factors, (including interpersonal factors) that impact behavior. SCT was founded on the premise of reciprocal determinism, meaning that personal factors (ex. self-efficacy for sex refusal), environmental factors (ex. peer norms supportive of unsafe sex), and behavioral factors (ex. condom use) all influence each other, and thus can impact a person’s behavior. SCT is well-established, and has been shown to predict many heath behaviors, including vaccination [32,34]. This study also employed constructs from SCT to complement the RHT framework, and account for factors influencing HIV vaccine acceptability that may be unexplained by RHT alone. When possible, existing scales validated with African-American females were used. For previously untested constructs, additional scales were created by the authors and reviewed by content experts.Psychosocial scale and index details are presented in Table 1. Psychosocial measures guided by RHT included: (1) perceived benefits of unprotected sex; (2) perceived costs of unprotected sex (3) perceived benefits of HIV vaccination in general; (4) perceived benefits of HIV vaccination in terms of risk compensation; (5) perceived costs of HIV vaccination in general; (6) perceived costs of HIV vaccination in terms of social norms; and (7) perceived HIV transmission risk [35]. All psychosocial variables guided by RHT were measured on 5 point Likert scales, ranging from 1 (Strongly Disagree) to 5 (Strongly Agree), except for perceived HIV transmission risk, which was measured on a 4 point Likert scale, ranging from 1 (Never) to 4 (Always). All RHT variables were scale variables, except for perceived costs of HIV vaccination in general, which was an index variable. Psychosocial measures guided by RHT were all developed for this study, except for HIV transmission risk. Psychosocial measures guided by SCT included: (1) peer norms supportive of unsafe sex, ranging from 1 (None) to 5 (All) [36]; (2) self-efficacy for sex refusal, ranging from 1 (Very hard to say no) to 5 (Very easy to say no) [36]; and (3) barriers to condom negotiation, ranging from 1 (Strongly Disagree) to 5 (Strongly Agree) [37].All analyses were conducted using SPSS version 19.0. Descriptive statistics were used to assess the distributions of all variables. Questions assessing psychosocial constructs were combined into scales, and Cronbach’s alphas were calculated to assess internal consistency. Bivariate analyses were used to assess associations between demographic, health history, behavioral, personality, and psychosocial factors with acceptability for an HIV vaccine with 50% efficacy and an HIV vaccine with 90% efficacy. Only variables that demonstrated significant bivariate associations at the p = 0.10 level were included in multivariate logistic regression analyses. For multivariate analysis, significance was measured at the p = 0.05 level.Of 623 women approached for participation, 508 (81.5%) were interested in screening, 353 (56.7%) were eligible to participate, and valid survey data were obtained from 321 (51.5%). The primary reason for ineligibility was not having unprotected sex (vaginal, anal, or oral) in the past 6 months, and the primary reason for declining to participate was not having enough time. The mean age of participants was 27.4 (SD = 7.7). In terms of education, 68 participants (21.2%) had not completed high school, 139 (43.3%) completed high school only, and 114 (35.5%) completed more than high school. The majority of the sample, (n = 252, 78.5%) received at least one type of public assistance in the past year.More than half of all participants (n = 201, 62.6%) reported that they would accept a vaccine with 50% efficacy, and almost all (n = 274, 85.4%) would accept a vaccine with 90% efficacy. Among participants, 242 (75.3%) would get an HIV vaccine if it was available now, 222 (69.2%) would get an HIV vaccine if it required multiple doses (shots), and 259 (80.7%) would get an HIV vaccine if it were free or covered by insurance. Specifically, 112 participants (34.9%) would pay up to $50 out of pocket, 69 (21.5%) would pay up to $100 out of pocket, 65 (20.2%) would pay more than $100 out of pocket, 56 (17.4%) would only get it for free, and 19 (5.9%) would not get an HIV vaccine. People most likely to influence participants’ HIV vaccine decision-making were family members (n = 161, 50.2%), followed by doctors (n = 155, 48.3%), friends (n = 118, 36.8%), partners (n = 149, 16.4%), media (n = 28, 8.7%), and minister/preacher (n = 25, 7.8%). Further information regarding characteristics of participants willing to accept vaccines with varying levels of efficacy is displayed in Table 2.Characteristics of participants willing to accept vaccines with varying levels of efficacy.* RHT = Risk Homeostasis Theory; * SCT = Social Cognitive Theory.Factors associated with acceptability of an HIV vaccine with 50% efficacy are presented in Table 3. In unadjusted bivariate analysis, the only demographic factor significantly associated with HIV vaccine acceptability at the p = 0.10 level was educational attainment. Participants with more than a high school education were less likely to accept an HIV vaccine with 50% efficacy compared to participants with less than a high school education. In terms of psychosocial variables, participants with greater perceived costs of unprotected sex, greater perceived benefits of HIV vaccination in general, and greater self-efficacy for sex refusal were more likely to accept an HIV vaccine with 50% efficacy. Participants with greater perceived costs of HIV vaccination in general and greater perceived social costs to HIV vaccination were less likely to accept an HIV vaccine with 50% efficacy. No health history, behavioral, or personality variables demonstrated significant associations.Factors associated with acceptability of an HIV vaccine with 50% efficacy.* RHT = Risk Homeostasis Theory; * SCT = Social Cognitive Theory.In adjusted multivariate analysis, the odds of acceptability for an HIV vaccine with 50% efficacy were 1.13 times greater among participants who reported greater perceived benefits to HIV vaccination in general (p < 0.001). Women with more than a high school education and with higher perceived costs to HIV vaccination had a significant reduction in the odds of acceptability (p = 0.033 and p = 0.010, respectively). No other variables remained significant in multivariate analysis.Factors associated with acceptability of an HIV vaccine with 90% efficacy are presented in Table 4. In unadjusted bivariate analysis, health history variables significantly associated with HIV vaccine acceptability at the p = 0.10 level included previous STD diagnosis and higher scores on the mental health scale. The only personality variable associated with acceptability of an HIV vaccine with 90% efficacy was positive future orientation. In terms of psychosocial variables, participants with greater perceived benefits of unprotected sex, greater perceived costs of unprotected sex, greater perceived benefits of HIV vaccination in general, and greater self-efficacy for sex refusal were more likely to accept an HIV vaccine with 90% efficacy. Participants with greater perceived costs of HIV vaccination in general and greater perceived social costs of HIV vaccination were less likely to accept an HIV vaccine with 90% efficacy. No demographic or behavioral variables demonstrated significant associations.In adjusted multivariate analysis, the odds of acceptability for an HIV vaccine with 90% efficacy were 1.08 times larger among participants who reported greater perceived costs to unprotected sex (p = 0.026), 1.23 times larger among participants who reported greater perceived benefits to HIV vaccination in general (p < 0.001), and 1.11 times larger among participants with greater self-efficacy for sex refusal (p = 0.044). No other associations remained significant in multivariate analysis.Factors associated with acceptability of an HIV vaccine with 90% efficacy.* RHT = Risk Homeostasis Theory; * SCT = Social Cognitive Theory.The first aim of this study was to assess anticipated HIV vaccine acceptability depending on varying HIV vaccine characteristics. Previous studies have found that vaccine characteristics, such as efficacy, dose, and cost, are likely to impact HIV vaccine acceptability [38,39]. This study found that HIV vaccine acceptability was relatively high among participants across vaccine characteristics such as efficacy, dosage and cost. A majority of the women in this study would accept an HIV vaccine with 50% efficacy (n = 201, 63%), and a vast majority would accept an HIV vaccine with 90% efficacy (n = 274, 85%). While participants were more likely to accept a vaccine with 90% efficacy compared to 50% efficacy, many participants were likely to accept a vaccine with either scenario. This study also found that a majority of participants would accept an HIV vaccine even if it required multiple doses or out-of-pocket pay. In fact, more than 3 out of 4 participants were willing to pay at least $50 out of pocket, and 1 out of 5 participants were willing pay more than $100 out of pocket. These findings must be considered in the context of the population, the majority of which were unemployed and received some type of public assistance in the past year. There was clearly an interest and willingness to accept an HIV vaccine among the African-American women in this study. HIV vaccine acceptability was higher among the women in this study compared to previous studies conducted with minority women [16], although consistent with levels of acceptability from previous research across diverse populations [15].These results are highly encouraging, particularly given the disproportionate burden of HIV and STIs among African-American women and the opportunity for volitional control over HIV risk that an HIV vaccine would present. Although anticipated vaccine acceptability does not necessarily portend vaccine uptake [40], results of this study indicate that African-American women may be likely to accept an FDA licensed vaccine against HIV.The second aim of this study was to assess correlates of anticipated HIV vaccine acceptability under varying levels of vaccine efficacy (50% and 90%). For an HIV vaccine with 50% efficacy, only one variable remained significantly associated with increased HIV vaccine acceptability in multivariate analyses: perceived benefits of HIV vaccination. This result is consistent with findings from previous qualitative studies indicating that perceived benefits of HIV vaccination, such as reduced worry about contracting HIV or transmitting HIV to other people, will be important motivators for vaccine uptake [16,17,18]. Two variables, higher educational attainment and high perceived costs of HIV vaccination, were associated with reduced HIV vaccine acceptability. It is understandable that participants with higher perceived costs to HIV vaccination (e.g., believing the vaccine might give them HIV, or not wanting a newly developed vaccine) would be less likely to accept an HIV vaccine. In terms of educational attainment, perhaps participants who were more educated were more likely to understand the implications of a vaccine with only 50% efficacy (e.g., they would still have to use condoms to prevent against HIV), and consequently less likely to accept the vaccine. Two of the significant correlates of HIV vaccine acceptability emerged from RHT, which underscores the salience of this model for understanding HIV vaccine acceptability.For a vaccine with 90% efficacy, three variables were significantly associated with increased HIV vaccine acceptability in multivariate analysis: greater perceived costs of unprotected sex, perceived benefits of HIV vaccination, and greater self-efficacy for sex refusal. Two significant variables were psychosocial constructs from RHT (perceived costs of unprotected sex and perceived benefits of HIV vaccination), while one significant variable was a psychosocial construct from SCT (self-efficacy for sex refusal). This finding further supports the use RHT in the context of HIV vaccine acceptability, yet also highlights the potential importance of constructs from additional theories, such as SCT.It is logical that women with greater perceived costs of unprotected sex, including putting themselves and their partners at risk for HIV, would be interested in getting a vaccine that would be highly efficacious. Although research among mixed-race adolescents has failed to find an association between perceived costs of unprotected sex and condom use [41,42], there is little known regarding the salience of this construct in increasing HIV protective behaviors among African-American adult women. Furthermore, getting an HIV vaccine is fundamentally different from condom use in multiple ways. First, getting an HIV vaccine would provide women with more volitional control over vulnerability to HIV compared to condom use. Second, depending on the dosing of the licensed vaccine, getting vaccinated against HIV would likely require a significantly shorter time investment than a potentially lifelong commitment to negotiating condom use. Third, getting vaccinated against HIV would not impact pleasure-related barriers to condom use, such as reduced feeling and ruining the mood. Thus, this construct may be of particular importance for increasing uptake of an HIV vaccine. Perceived benefits of HIV vaccination may also be a critical factor for HIV vaccine promotion interventions to address. It is important to note that perceived benefits of HIV vaccination was a significant predictor of HIV vaccine acceptability for vaccines with both 50% and 90% efficacy, even when controlling for other factors that were significant in bivariate analyses. This finding highlights the potential importance of scientific communication and marketing during an eventual HIV vaccine roll-out. Regardless of vaccine efficacy, a public health emphasis on increasing perceived benefits of vaccination may have a significant impact on vaccine uptake.One additional construct, self-efficacy for sex refusal, was significantly associated with acceptability of an HIV vaccine with 90% efficacy. Previous research among African-American adolescent females has demonstrated an association between positive self-concept (including self-esteem, ethnic identity, and body image) and increased refusal of unprotected sexual intercourse [43]. Although the current study was conducted among African-American adult women, it is likely that positive self-concept continues to be associated with self-efficacy for sex refusal from adolescence into adulthood. It is also likely that women with more positive self-concept are likely to advocate for their sexual health in multiple ways, including refusing unwanted sex and getting vaccinated against HIV.First, this is a cross-sectional study. Thus, we could not assess the causal effects of the predictor variables on HIV vaccine acceptability. Second, the outcome is hypothetical HIV vaccine acceptability, not actual HIV vaccination. It may be difficult for women to predict how they will feel about a vaccine once it becomes available. Third, the data were self-reported, and possibly prone to social desirability bias. Fourth, the study population comprised sexually active African-American women in a low-income clinic setting in Atlanta, GA, USA. Thus, the results of this study may not be generalizable to broader populations. Furthermore, the study population was recruited from a clinical setting. African-American women receiving services from a hospital-based clinic may be more likely to accept biomedical HIV prevention interventions (such as vaccinations) compared to women who do not seek or receive health services. Fifth, although we can speculate reasons why women with higher educational attainment may be less likely to accept an HIV vaccine with 50% efficacy and why women with greater self-efficacy for sex refusal are more likely to accept a vaccine with 90% efficacy, we cannot fully explain these associations. Also, our index measure of perceived costs of HIV vaccination included ten distinct reasons why participants may not want an HIV vaccine. Future research is needed to determine which costs may be the most important barriers to HIV vaccination. Finally, for many of the variables that were significantly associated with HIV vaccine acceptability, the odds ratios were close to one. Thus, although the associations between these variables and HIV vaccine acceptability were statistically significant, it is unclear if these results would be clinically significant or practically meaningful in a public health context.Findings from this study may be useful in identifying priority points to target during future HIV immunization campaigns. Although HIV vaccine acceptability was high overall, particularly for a vaccine with 90% efficacy, there was nonetheless variability regarding vaccine acceptability. Results of multivariate analyses for acceptability of HIV vaccines with 50% and 90% efficacy underscore the importance of vaccine-related perceptions, particularly perceived costs and benefits of vaccination, on HIV vaccine acceptability. Perceived costs of unprotected sex and self-efficacy for sex refusal may also be important determinants of acceptability for an HIV vaccine with 90% efficacy. Given that several psychosocial variables that emerged as significant for vaccines with both 50% and 90% efficacy were guided by RHT, our findings support the salience of this model for understanding HIV vaccine acceptability. Our findings also suggest it may be necessary to supplement the use of RHT with constructs from additional theories, such as SCT, to fully understand HIV vaccine acceptability. Once an HIV vaccine is available, intervention efforts to maximize uptake will need to address negative vaccine-related concerns and sufficiently communicate potential benefits. Interventions may also benefit from taking personal beliefs, such as perceived costs of unprotected sex and self-efficacy for sex refusal, into account. RHT and SCT-based constructs may be useful for guiding intervention efforts. Future studies should investigate factors associated with HIV vaccine acceptability associated with broader samples of African-American women, and women of all ethnicities.The authors wish to thank the Grady Family Planning Clinic staff for their support of the HVARC study. We would also like to thank the HVARC research assistants who assisted with data collection and Dianne Miller for providing administrative support. The project described was supported by Award Number [T32AI074492] from the National Institute of Allergy and Infectious Diseases and Award Number [P30 AI050409] from Emory’s Center for AIDS Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, or Emory’s Center for AIDS Research.The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).To examine changes in seroprevalence of antibodies to hepatitis A virus (HAV) during a period in which universal vaccine recommendations for all U.S. children were implemented, results from serologic testing from the National Health and Nutrition Examination Survey (NHANES) from 2003–2010 were analyzed among 7,989 participants age 6–19 years, born in the U.S. in two birth cohorts (1986–1996 and 1997–2004). Overall prevalence increased over time from 24.4% in 2003–2006 to the highest ever reported (37.6%) in 2007–2010. Specifically, increases reached statistical significance in the birth cohort born in the years after implementation of vaccine recommendations (1997–2004), among those of race/ethnicity other than white, non-Hispanic, and among states where recommendations were implemented later. The greatest increase over time was among the subgroup of persons in states with early implementation who were of race/ethnicity other than white, non-Hispanic. Geographic region and birth cohort based on vaccine recommendations as well as race/ethnicity were the main predictors of seropositivity in 2007–2010. The increase in Hepatitis A seroprevalence occurred during a time of decreasing incidence and increasing vaccination, however race/ethnic disparities persist.Hepatitis A virus (HAV) is transmitted through the fecal-oral route and spread primarily through close personal contact with an HAV-infected person. Hepatitis A was once one of the most frequently reported notifiable diseases in the United States (U.S.) with a reported incidence of 10.7 cases per 100,000 population from 1990–1997, with incidence varying by age, gender, race/ethnicity and geographic region [1]. In 1995, the first Hepatitis A vaccines were licensed in the United States and by 1996 the Advisory Committee on Immunization Practices (ACIP) made recommendations for routine vaccination of children aged 2–18 years living in communities with the highest rates of infection and disease [2]. By 1999, epidemiologic evidence suggested that the strategy had a limited impact on national disease incidence [3]. Therefore in 1999, the ACIP recommended routine vaccination for children living in 11 mostly western states, with mean incidence rates that were at least twice the 1987–1997 national mean (i.e., ≥20 cases per 100,000 population). In addition, the ACIP recommended consideration of routine vaccination of children in an additional six states, where mean incidence rates were higher than the national average, but less than twice that value (i.e., 10–19 cases per 100,000 population [3]. By 2003, acute hepatitis A disease had declined overall by 76%, from a rate of 10.7 per 100,000 population during 1990–1997 to 2.6 per 100,000 population in 2003 [1]. In 2006, ACIP recommended integration of HAV vaccine into the routine childhood vaccination schedule, with HAV vaccine administered for all children at age 12 months [4]. By 2007, the rate of acute Hepatitis A again declined to 1.0 per 100,000 population [5] and by 2009 the rate was the lowest ever reported at 0.6 cases per 100,000 population [6]. The Healthy People 2020 goal is to reduce incident Hepatitis A cases to 0.3 cases per 100,000 population [7].Population-based seroprevalence surveys play a critical role in supplementing data systems for disease incidence, vaccination coverage, and vaccine adverse events in the development of vaccination policy [8]. Before the availability of vaccine, seroprevalence of antibody to HAV (anti-HAV) in the population solely reflected prior infection [9]. Currently, seroprevalence can reflect immunity due to either previous infection or to vaccination. Earlier studies of data from the National Health and Nutrition Examination Surveys described HAV seroprevalence and predictors of seropositivity for the years prior to any vaccination (1988–1994) and prior to the 2006 ACIP recommendation of universal vaccination of all children (1999–2006) [9,10]. Studies of vaccination coverage show increased coverage since 2006, variability in coverage by race/ethnicity, higher coverage in the states where vaccine recommendations were initiated early (1999) but the greatest increase in coverage occurs among states in which vaccination recommendations were initiated later [11]. Our objective was to describe change in seropositivity to HAV in the U.S. among children and adolescents from the pre-vaccine era (2003–2006) to the post-vaccine era (2007–2010) for those age 6–19 years during a time of decreasing HAV incidence. We also evaluated sociodemographic factors associated with seroprevalence in the post vaccine era (2007–2010), and compared these findings with those of previous studies based on data before a vaccine became available.Data for this study came from The National Health and Nutrition Examination Surveys (NHANES), a series of surveys conducted by the U.S. Centers for Disease Control and Prevention’s (CDC) National Center for Health Statistics that obtains nationally representative data on the health and nutritional status of the non-institutionalized, civilian population of the United States. NHANES uses a complex, stratified, multistage probability sample design and collects information using standardized household interviews, physical examinations conducted in mobile examination centers, and testing of biologic samples. The continuous NHANES began in 1999 and data files are released in two-year cycles. We analyzed data from eight years of the continuous NHANES, grouped into two four year cycles (2003–2006 and 2007–2010). For NHANES 2003–2006, non-Hispanic blacks, Mexican Americans, adolescents, and low income persons were sampled at higher frequencies than other persons to provide more precise estimates for these groups. Starting in 2007, adolescents are no longer oversampled and all Hispanic persons were targeted for oversampling rather than just Mexican American persons. More detailed information on survey design for NHANES surveys, including approval from the Ethics Review Board for data collection and analysis, is available from the survey documentation [12].Blood specimens from persons ≥6 years of age (for 2003–2006) and 6–19 years of age (2007–2010) were processed, stored, and shipped to CDC’s Division of Viral Hepatitis Laboratory. A qualitative determination of total anti-HAV in serum or plasma was measured using a solid-phase competitive enzyme immunoassay (HAVAB-EIA, Abbott Laboratories, Abbott Park, IL, USA). Serologic results for those age 6–19 years were used in this analysis.An anti-HAV positive person was considered immune to HAV infection through either vaccination or natural infection. Race and ethnicity were categorized, based on a subjects’ self-reported information, as non-Hispanic white, non-Hispanic black, or Mexican American. Subjects that were not classified into one of these categories were classified as “other”. Country of birth was categorized as U.S. or non-U.S. birth. Poverty index was calculated by dividing family income by a poverty threshold specific for family size. The US Department of Health and Human Services’ poverty guidelines were used as the poverty measure to calculate the poverty index [13]. Education level was measured as last year of school completed using head of household education and grouped into three levels: less than a high school graduate, high school completed, and more than high school completed. Health insurance status was categorized as having any insurance or having none.Analyses were restricted to U.S.-born persons to best reflect U.S.-acquired immunity. Based on restricted use NHANES geographic data, persons were grouped by state of residence into two groups, according to the 1999 ACIP hepatitis A vaccination recommendations and consistent with other analyses [3]: (1) the 17 “early vaccinating states”, where hepatitis A vaccination was recommended (AK, AZ, CA, ID, NM, NV, OK, OR, SD, UT, WA) or where vaccination was considered (AR, CO, MO, MT, TX, WY), and (2) the remaining 33 “later vaccinating states”, where no routine childhood vaccination was recommended in 1999 (AL, CT, DE, FL, GA, HI, KS, KY, IA, IL, IN, LA, MA, MD, ME, MI, MN, MS, NC, ND, NE, NH, NJ, NY, OH, PA, RI, SC, TN, VA, VT, WI, WV). Persons were also categorized into birth cohorts based on restricted data using actual date of birth and into two groups based on initiation of vaccine recommendations; those born between 1987–1996 (before vaccine recommendations) and those born between 1997–2004 (after vaccine recommendations). There were 936 sample persons age 16–19 born before 1987 in survey years 2003–2006 that were not included in either birth cohort and therefore were not included in this analysis. Seroprevalence estimates were weighted to represent the total civilian, non-institutionalized U.S. household population and to account for oversampling and non-response to the household interview and physical examination [14]. Because we utilized a variable based on U.S. geography, we were unable to use the publically released masked stratum and primary sampling units (PSU’s) to designate the complex sample design in our analyses. The true stratum and PSU designations were used instead. Ninety-five percent confidence intervals (95% CI) were estimated by using the exact binary method [15]. Statistical comparisons between subgroups were evaluated using a t-statistic obtained from a linear contrast procedure in SUDAAN (release version 10.0, Research Triangle Institute, Research Triangle Park, NC, USA), a statistical package designed to analyze complex survey data. P-values of less than 0.05, with degrees of freedom equal to the minimum calculated for either subgroup in the comparison, were considered significant. No adjustments for multiple comparisons were made. Because prevalence was very low (less than 10%) in some smaller subgroups as well as very high (approaching 90%) in others, the stability of an estimate was based on both the percent positive and percent negative as well as the number of positive and negative individuals. An estimate was designated as unstable when the relative standard error of the estimate was greater than 30% (RSE = standard error of the percent/percent expressed as a percent) or when the number of negative or positive individuals was <10. Because of small numbers in each survey cycle for several race/ethnic subgroups when stratifying on geographic region, we collapsed race/ethnic categories to white, non-Hispanic and all other race/ethnic groups combined (all others).A logistic modeling procedure in SUDAAN was used to evaluate interactions between change in seroprevalence by survey cycle and either race/ethnic group or birth cohort within each geographic region. Logistic modeling was also used to determine cofactors independently associated with anti-HAV seroprevalence for the data from 2007–2010. Model terms with a Satterthwaite-adjusted F statistic with a p < 0.05 were considered to be significant predictors of HAV seropositivity. There were 4,955 persons aged 6–19 years born in the U.S. between 1987–2004, interviewed in NHANES 2003–2006, and 4,622 in NHANES 2007–2010. Ninety seven percent of those interviewed in both 2003–2006 (n = 4,788) and 2007–2010 (n = 4,488) were examined and 87% of those examined in 2003–2006 (n = 4,185) and 85% in 2007–2010 (n = 3,805) had blood drawn and were tested for Hepatitis A virus antibody. Response to HAV testing varied by many predictors of seropositivity including geographic region and race/ethnic group by ≤5%. Difference in response was greatest for birth cohort (89% for birth cohort 1987–1996 and 79% for birth cohort 1997–2004 in NHANES 2003–2006 and 89% and 80% respectively for birth cohorts 1987–1996 and 1997–2004 in NHANES 2007–2010, p < 0.001). All analyses were repeated with new weights adjusted for the differences in non-response by the three main predictors of seropositivity, geographic region, birth cohort and race/ethnic group. Estimates differed by less than 1% and there were no changes in any results. All results reported were calculated using original weights. Prevalence of anti-HAV among U.S. born individuals age 6–19 years increased over time by 13.1 percentage points from 24.4% (95% CI 16.6–33.9%) in NHANES 2003–2006 to 37.6% (95% CI 32.6–42.7%) in NHANES 2007–2010 (p < 0.05) (Table 1). Prevalence of antibody increased over time in the region with later vaccine recommendations (11.5 percentage points, p < 0.01). A similar effect was found in the region with early recommendations (18.5 percentage points) but it did not reach statistical significance. Overall seropositivity was significantly higher in the region with earlier recommendations as compared to the region with later recommendations in both four year survey cycles (p < 0.001 for both). Prevalence increased significantly over time in the post vaccine birth cohort (1997–2004) (18.9 percentage points p < 0.01) (Table 1). There was not a significant increase in prevalence over time among persons in the pre-vaccine birth cohort (1987–1996). Prevalence was significantly higher in the post vaccine birth cohort as compared to the pre-vaccine birth cohort but only in the later survey cycle (p < 0.001). Prevalence increased significantly over time among non-Hispanic blacks (19.1 percentage points, p < 0.01) and among Mexican Americans (23.3 percentage points, p < 0.001) as well as among all persons combined who were not non-Hispanic white (24.0 percentage points, p < 0.001). There was no increase in prevalence among white, non-Hispanic persons (Table 1). Seroprevalence was lower among white, non-Hispanic persons compared to all others combined and compared to Mexican Americans in both survey cycles (Table 1). Seroprevalence was also lower among white, non-Hispanic persons as compared to black, non-Hispanic persons but only in the 2007–2010 survey cycle (p < 0.01). Black, non-Hispanic persons had lower seroprevalence compared to Mexican Americans in both survey cycles (p < 0.001 for both).Because change in seropositivity over time and initiation of vaccine recommendation policy were both strongly associated with geographic region, additional analyses were conducted stratified on region. Due to small numbers and unstable estimates with the multi-level stratification needed to test for these interactions, differences in race/ethnicity were limited to the two category variable for these analyses—white, non-Hispanics and all others combined. Significance tests for change over time and possible interactions with race/ethnicity or year of birth cohort were conducted using logistic regression stratifying on geographic region and adjusting for the other cofactor. The increase in seropositivity over time, although it did not reach statistical significance, was similar in both birth cohorts in the geographic region with early recommendations (p > 0.05 for both, p > 0.05 for interaction term, Figure 1). This was not true in the region with later recommendations. In the later region, the increase over time was greater in the post-vaccine birth cohort (p < 0.001) as compared to the pre-vaccine cohort (p < 0.05) (p < 0.001 for the interaction term). Note the relative standard error for the estimate for the first survey cycle for those in the post vaccine birth cohort in this region was high (>30%) making the estimate unstable; therefore, results should be interpreted with caution. The increase in seropositivity was similar between white, non-Hispanics (p < 0.05) and all others combined (p < 0.01) in the geographic region with later vaccine recommendations (Figure 2). There was no significant interaction between change in seropositivity (survey cycle) and race/ethnic group in this region (p > 0.10 for interaction term). In contrast, in the region with early vaccine recommendations, the increase in seropositivity over time was much greater among the all others combined race/ethnic group (p < 0.001) as compared to the white, non-Hispanic race/ethnic group (p > 0.05) (p < 0.05 for the interaction term). To determine significant socio-demographic predictors of current HAV antibody seropositivity in the U.S. for the most recent point in time, data from the 2007–2010 survey cycle was analyzed using logistic regression modeling. Possible predictors evaluated were determined from previous analyses published using 1999–2006 data [10]. Significant predictors were defined as those with a p < 0.05 in the full model (Table 2). As expected geographic region based on early vaccine recommendations, year of birth cohort and race/ethnicity were significant predictors of seropositivity. Additional predictors of greater seropositivity included female gender.Because vaccine recommendations were initiated at different times by region of the U.S. and changes in seropositivity over time by race and birth cohort also varied by region, models were run stratified by this variable. Higher seropositivity in 2007–2010 among those in the all others combined race/ethnic group as compared to white, non-Hispanic sample persons was much greater in the geographic region with early vaccine recommendations (odds ratio (OR) 5.1 (95% CI 2.2–11.6) as compared to the region with later recommendations (OR 2.2 (95% CI 1.5–3.2). Other than race/ethnicity, year of birth cohort was the only other significant predictor in the region with early vaccine recommendations. There were no other significant predictors of seropositivity for this region. Possible interactions of each cofactor with year of birth cohort were also examined but estimates were too unstable in many subgroups to stratify on year of birth cohort. Prevalence of HAV antibody among U.S. born 6–19 year old children and adolescents: NHANES 2003–2006 and 2007–2010.HAV = Hepatitis A virus; NHANES = National Health and Nutrition Examination Survey; CI = confidence interval; * Early region includes those states in which the ACIP recommended or considered recommending HAV vaccine in 1999 (Alaska, Arizona, California, Idaho, New Mexico, Nevada, Oklahoma, Oregon, South Dakota, Utah, Washington, Arkansas, Colorado, Missouri, Montana, Texas, and Wyoming). Later region include those states with later HAV vaccine recommendations starting in 2006; (Alabama, Connecticut, Delaware, Florida, Georgia, Hawaii, Kansas, Kentucky, Iowa, Illinois, Indiana, Louisiana, Massachusetts, Maryland, Maine, Michigan, Minnesota, Mississippi, North Carolina, North Dakota, Nebraska, New Hampshire, New Jersey, New York, Ohio, Pennsylvania, Rhode Island, South Carolina, Tennessee, Virginia, Vermont, Wisconsin, and West Virginia); ref = reference group; NS = not statistically significant; a = p < 0.001, b = p < 0.01, c = p < 0.05 from t-test comparing subgroup to reference group within each survey cycle. ^ Pre-vaccine birth cohort were those born from 1987–1996 and post vaccine birth cohort were those born from 1997–2004. + All others refers to the combined race/ethnic group that includes all those other than non-Hispanic white. All others includes non-Hispanic blacks, Mexican Americans, other Hispanics, and other races including multi-racial.HAV seropositivity over time among U.S. born children and adolescents 6–19 years old stratified by birth cohort and region of the U.S.: NHANES 2003–2006 and 2007–2010.HAV seropositivity over time among U.S. born children and adolescents 6–19 years old stratified by race/ethnic group and region of the U.S.: NHANES 2003–2006 and 2007–2010.Predictors of HAV seropositivity from logistic regression models among U.S. born children and adolescents 6–19 years of age by region based on HAV vaccine recommendations and birth cohort: NHANES 2007–2010.HAV = Hepatitis A virus; NHANES = National Health and Nutrition Examination Survey; OR = odds ratio; CI = confidence interval. a Early region includes those states in which the ACIP recommended or considered recommending HAV vaccine in 1999 (Alaska, Arizona, California, Idaho, New Mexico, Nevada, Oklahoma, Oregon, South Dakota, Utah, Washington, Arkansas, Colorado, Missouri, Montana, Texas, and Wyoming). b Later region includes those states with later HAV vaccine recommendations starting in 2006 (Alabama, Connecticut, Delaware, Florida, Georgia, Hawaii, Kansas, Kentucky, Iowa, Illinois, Indiana, Louisiana, Massachusetts, Maryland, Maine, Michigan, Minnesota, Mississippi, North Carolina, North Dakota, Nebraska, New Hampshire, New Jersey, New York, Ohio, Pennsylvania, Rhode Island, South Carolina, Tennessee, Virginia, Vermont, Wisconsin, and West Virginia). NA = not applicable; ref = reference group. * Pre-vaccine birth cohort were those born from 1987–1996 and post-vaccine birth cohort were those born from 1997–2004. ^ All others refers to the combined race/ethnic group that includes all those other than non-Hispanic white. All others includes: non-Hispanic blacks, Mexican Americans, other Hispanics, and other races including multi-racial. c p < 0.001 for comparison of subgroup and reference group in model; d p < 0.05 for comparison of subgroup and reference group in model; e p < 0.01 for comparison of subgroup and reference group in model; f p < 0.05 for interaction of cofactor and year of birth cohort within geographic region; r = RSE > 30% in univariate analysis—estimates may be unstable. In the region with later vaccine recommendations, besides race/ethnicity, post vaccine birth cohort, female gender and having health insurance were associated with greater seropositivity to HAV. In addition, a significant interaction with birth cohort and both race/ethnicity and head of household education was found. Logistic models for this region stratified by birth cohort demonstrated larger race/ethnic differences (higher seropositivity among all others ) in the post-vaccine birth cohort (OR 3.2 (95% CI 2.2–4.8), p < 0.001) as compared to the pre-vaccine birth cohort (OR 1.5 (95% CI 1.0–2.4), p > 0.05). There was no difference between those whose head of household had less than a high school education as compared to those with greater than a high school education in either cohort. Individuals whose head of household educational level was equal to high school had lower seropositivity than those with greater than a high school education but this was only true in the pre-vaccine cohort (p < 0.05).We report here the highest ever prevalence of immunity to hepatitis A among U.S. children (37.6%) through 2010; prevalence was especially high (66%) among children residing in areas with an early recommendation (1999) or consideration of vaccination. This prevalence is almost five times higher than the 8% level found in children during the 1988–1994 NHANES [9], i.e., before the vaccine became available. Given the steady decline in disease incidence [16] our findings of an increase in seroprevalence are consistent with the observed increase in vaccination coverage [17]. In a previous NHANES study period, pre-dating vaccination, children who lived in poverty, in households of less educated adults, or in crowded homes, were more frequently immune because of infection [9]. In this study of U.S. born children in NHANES 2007–2010, we document that socio-economic factors were not associated with immunity. This might be related to the Vaccines for Children Program (VFC), which has been providing vaccines at no cost to children in families unable to pay, since 1994 [18]. The increase in seroprevalence between 2003–2006 and 2007–2010 was similar in both the region with early vaccine recommendations as compared to the region with later vaccine recommendations although it only reached statistical significance in the latter region. Although not statistically significant, the change in seroprevalence in both birth cohorts in the region with early recommendations were similar in magnitude to the significant increase found in the post vaccine birth cohort in the region with later vaccine recommendations. The lack of statistical significance in the early region was partly due to the fact that there were fewer sample persons and fewer stratum and PSU’s in this region thereby resulting in larger standard errors for these estimates and less power to detect statistical significance. The increase in seroprevalence was similar between the two birth cohorts in the region with early recommendations. In contrast, in the region with later recommendations, the increase in seroprevalence was greater in the post-vaccine birth cohort. There appeared to be no difference in prevalence of antibody by birth cohort in this region in 2003–2006 but because of the greater increase in seroprevalence in the post-vaccine cohort, differences between birth cohorts were seen in 2007–2010. The smaller change in seroprevalence in the cohort born before implementation in the region with later recommendations may be due to a variety of factors including: older age of this cohort at the time vaccine recommendations were implemented and the fact that initial recommendations were risk-based and this group may not have been perceived to be at risk.We did observe one disparity: children residing in areas with early recommendations in the all other combined race/ethnic group had a significantly higher prevalence of immunity (82% by 2007–2010) and a significantly greater increase in seroprevalence compared to white, non-Hispanic children. In 2007–2010, controlling for other variables, children in the all others combined race/ethnic group in states with an early vaccination recommendation were 5 times more likely to be protected from hepatitis A than white, non-Hispanic children. In regions with later recommendations, these children were 2 times more likely to be immune to hepatitis A. These findings are consistent with a survey of vaccination coverage among teenagers in 2009 [19] that found that coverage was low overall (two doses 30%) and that white, non-Hispanic teens had the lowest coverage of all racial/ethnic groups (66% in states with early recommendations and 25% in states with no recommendation in 1999). No such disparity has been observed among infants. Vaccination coverage with 2 doses was not significantly different between white and black, non-Hispanic infants; however, coverage among Hispanic and Asian infants was significantly higher than among white, non-Hispanic children [17]. Self-reported vaccination with hepatitis A vaccine was similar among white, black, and Hispanic adults aged 19–49 years in the 2010 National Health Interview Survey [20]. It is possible that the perception of risk for hepatitis A among white children is lower than among children in other race/ethnic groups. The United States is not alone in adopting hepatitis A vaccination with success. Israel was the first country to initiate routine vaccination with a two-dose regimen of hepatitis A vaccine in 1999. There, the incidence of hepatitis A dropped over 95% in children and adolescents by 2007 [21]. In Argentina, the introduction of a single dose regimen for children 12 months of age was followed by an 83% reduction in the average incidence rate by 2007 [22], In Puglia (Southern Italy), vaccination of toddlers and preadolescents was initiated in 1998 after an HAV epidemic in the years 1996–1997. Incidence of HAV infection declined by 95% by 2009 due to both vaccination and reduced circulation although presence of antibody to HAV remained low in some age groups [23].A strength of this study was that there were sufficient data to allow a more in-depth analysis of immunity within geographic areas with and without early vaccination recommendations (i.e., to vaccinate in 1999) as well as by the birth cohorts effected by these recommendations. A limitation of all NHANES data are that findings are generalizable to the U.S. non-institutionalized civilian population of the United States, and do not represent persons residing in institutions (e.g., illicit drug users in prisons). A limitation of the assay used to detect antibodies to HAV is that they are unable to distinguish immunity related to infection or to vaccination.In summary, U.S. children aged 6–19 years through 2010 had the highest ever prevalence of immunity to hepatitis A (37.6%) and immunity reached 66% among children in the region with early recommendations. Prevalence increased over time and this increase was greatest among those of the all other race/ethnic group other than non-Hispanic white. Continued monitoring of immunity, vaccination coverage, and incidence of disease will help guide next steps in the U.S. hepatitis A vaccination policy.The findings and conclusions in this paper are those of the author(s) and do not necessarily represent the official position of the National Center for Health Statistics, Centers for Disease Control and Prevention. The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The failure of current Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccines, given to neonates to protect against adult tuberculosis and the risk of using these live vaccines in HIV-infected infants, has emphasized the need for generating new, more efficacious and safer replacement vaccines. With the availability of genetic techniques for constructing recombinant BCG (rBCG) strains containing well-defined gene deletions or insertions, new vaccine candidates are under evaluation at both the preclinical and clinical stages of development. Since most BCG vaccines in use today were evaluated in clinical trials decades ago and are produced by outdated processes, the development of new BCG vaccines offers a number of advantages that include a modern well-defined manufacturing process along with state-of-the-art evaluation of safety and efficacy in target populations. We provide a description of the preclinical development of two novel rBCGs, VPM1002 that was constructed by adding a modified hly gene coding for the protein listeriolysin O (LLO) from Listeria monocytogenes and AERAS-422, which carries a modified pfoA gene coding for the protein perfringolysin O (PFO) from Clostridium perfringens, and three genes from Mycobacterium tuberculosis. Novel approaches like these should be helpful in generating stable and effective rBCG vaccine candidates that can be better characterized than traditional BCG vaccines. Although still one of the most widely used vaccines in the world, BCG originally developed by Calmette and Guérin more than 90 years ago and first administered to infants in 1921 [1], has not been effective in reducing the global burden of tuberculosis (TB). Further, HIV-infected infants are at greater risk of developing disseminated BCG infections (BCGosis) [2] following immunization, which is a risk in countries like South Africa with high levels of both diseases. BCG vaccines still remain in use because they are associated with protection against childhood TB and reduce the incidence of extrapulmonary (meningeal and miliary) forms of TB in infants. Therefore, a safer and more effective BCG replacement vaccine will reduce the global burden of TB with minimized risk to HIV-TB co-infected individuals. Of the several new TB vaccine candidates that have been studied in human clinical trials over the past decade, three have been recombinant BCG vaccines altered by modern molecular technology to potentially be safer, more immunogenic and efficacious [3]. Two of these rBCG vaccines are described in more detail in this report. The rBCG vaccine, VPM1002 was originally developed by Stefan H.E. Kaufmann and his team at the Max Planck Institute for Infection Biology in Berlin [4,5]. Two genetic modifications were introduced simultaneously into the BCG Danish (Sub type Prague) genome; these included integration of a gene hly from Listeria monocytogenes coding for the protein LLO, and inactivation of BCG’s gene for urease subunit C (ureC) to allow the acidification of the phagosomal compartment. Vakzine Projekt Management, GmbH (VPM) has facilitated the further development and acts as a sponsor for the clinical testing of this novel rBCG vaccine. The non-profit product development organization, Aeras, has sponsored the development of two rBCGs that have entered clinical trials. The first rBCG vaccine, rBCG30, overexpressed a single Mycobacterium tuberculosis (Mtb) antigen (Ag85B) in BCG Tice. This vaccine was shown to demonstrate substantially better protection vs. Mtb challenge in an animal model compared to its parental BCG strain, and elicited Ag85B specific CD4 T cell responses in humans [6]. Although this vaccine was found to be safe and immunogenic in a phase I clinical trial [7], it was not pursued further due to the presence of an antibiotic resistance marker. In a separate development rBCG strain, AERAS-422 was constructed using BCG Danish-SSI 1331 as a platform, to express a mutated pfoA gene coding for the protein perfringolysin O (PFO) from Clostridium perfringens, with a mode of action similar to listeriolysin [8]. It also overexpressed three Mtb antigens (Ag85A, Ag85B and Rv3407).In this review, we provide the details on the developmental strategies used to design and characterize these two novel rBCG vaccines and in addition, summarize the methods and tests used to manufacture and evaluate the candidate products to meet regulatory requirements and describe the pre-clinical assays developed to characterize the critical parameters of these live TB vaccine candidates. Although pharmacopeial guidelines exist for the testing and evaluation of traditional BCG vaccines [9], differences in rBCGs, manufacturing procedures and nonclinical testing of these genetically modified constructs demand new approaches for preclinical evaluation and manufacturing. This article provides a comparison between the modern methods being used to generate and test these new rBCG vaccines and the traditional methods used to generate BCG vaccines. The development of safer BCG replacement vaccines could provide a vaccine that might operate through a mechanism supported by rational design, manufactured by modern cGMP methods and evaluated according to modern cGCP clinical trial designs. VPM1002 was constructed by a single step that disrupted the ureC locus by insertion of a hly gene coding for an LLO expression cassette. The leader sequence of Ag85B was included in front of LLO to allow for secretion of the protein [4]. For construction of the vector used to generate VPM1002, flanking regions of the ureC gene were introduced upstream and downstream of the hygromycin B resistance cassette of the mycobacterial recombination vector. The LLO expression cassette was then inserted between the 3'-region of ureC and the antibiotic marker to result in the final construct, which was electroporated into the parental strain to allow for homologous recombination. Individual hygromycin resistant clones were selected from Middlebrook 7H11 agar containing 80 µg/mL hygromycin B. Genetic modifications were confirmed by PCR, Southern blotting and deep sequencing.VPM1002 was adapted to animal-free Sauton’s medium to create a Master Seed Bank (MSB) and used to produce the Working Seed Bank (WSB). Bulk Drug Substance (BDS) was produced using three 200 mL cultures grown in shake flasks containing Sauton’s medium. The contents of the flasks were used to inoculate a 30-L fermenter containing Sauton’s medium. After meeting the desired growth OD600, contents were harvested, cross filtered, and the concentrated bulk was combined with formulation buffer. The material was then filled into 2 mL amber serum vials and lyophilized, stoppered, crimp-sealed, labeled and stored at 2–8 °C and used for the following nonclinical studies. Female BALB/c mice were vaccinated subcutaneously with 1 × 106 viable bacilli of BCG Danish-SSI 1331, VPM1002 or saline. After 90 days mice were infected with a low dose (200 CFUs) of Mtb H37Rv per animal using a Glas-Col inhalation exposure system. At 30, 60 and 90 days post-challenge, six mice per group were sacrificed, and lungs and spleens homogenized in saline with 0.05% Tween 80 and plated in serial dilutions onto Middlebrook 7H11 agar. Plates were incubated for 3–4 weeks at 37 °C prior to enumerating viable bacterial colony forming units (CFU). Experiments had ethical clearance by the Landesamt fuer Gesundheit und Soziales Berlin under permit number G0307/11.The purpose of these studies was to obtain information on the toxicity of VPM1002 in male and female guinea pigs after single subcutaneous application of the test item followed by periods of 6 to 26 weeks and dosages up to 50-fold human target dose (HTD). The HTD was considered 1–4 × 105 CFU and a total of 6 female and 6 male guinea pigs per experiment were randomly allocated to two treatment groups of 6 animals each (3 female and 3 male animals per group). Body weight was determined three times a week during the observation period, animals were checked three times a week for clinical findings and mortality was recorded daily. At the end of the observation period all animals were necropsied and macroscopically altered organs were evaluated histopathologically.Information derived from this study serves to indicate test-item-related local intolerance reactions, signs of systemic toxicity, mortality and mycobacterial load in target organs. Newborn rabbits were vaccinated subcutaneously within the first 2 days after colostrum feed with either one human target dose (1–4 × 105) of VPM1002, BCG Danish-SSI 1331 or saline followed by an observation period of 90 days, with samples collected 10, 21 and 90 days post-vaccination for gross necropsy and histopathology. Special attention was paid to the local tolerance at the injection site. Target organs were investigated for their mycobacterial load by evaluating the number of CFUs in tissue samples. VPM1002 colonization in target organs was stained and assessed by microscopy. The body weight of each newborn rabbit was monitored on the day of administration, twice weekly until the end of week 2, and weekly from week 3 onwardsThe plasmid vector, pRC131, containing TB antigens Ag85A, Ag85B and Rv3407, was created using the oriM and panCD genes from plasmid pRC128 and the antigen cassette from plasmid pRC102. pRC128 was first digested with BamHI & partially digested with XbaI. The 7.8 kb fragment (plasmid backbone) was ligated with the 2.7 kb antigen cassette generated by PCR from pRC102. pRC131 was digested with PacI to remove the antibiotic marker and self-ligated. The self-ligated, purified plasmid was electroporated into AERAS-413 (a pantothenate auxotroph of AERAS-401 [8]) and selected on antibiotic-free, 7H10 agar plates without pantothenate supplementation. AERAS-422 was adapted to animal-free medium to create an Accession Cell Bank (ACB) and used to produce the Master Cell Bank (MCB). The Bulk Drug Substance (BDS) was produced using three MCB vials grown in shake flasks containing 7H9 medium. The contents of the flasks were used to inoculate a 20-L fermenter containing 7H9 medium. After meeting the desired growth of OD600 5 ± 1, contents were harvested, centrifuged, and the cell pellet was resuspended and stored in vapor phase liquid nitrogen. To produce a final fill lot, vials of concentrated BDS were thawed, combined with formulation buffer, and dispensed into 2-mL amber serum vials. The contents of the vials were lyophilized, stoppered, crimp sealed, labeled and stored at ≤−65 °C and used for the following nonclinical studies. Female Chinese rhesus macaques, between 2–5 years of age, were used in this study. Animals were housed at Advanced Biosciences Laboratory, Inc (Rockville, MD, USA), in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care. Animals were randomized into groups including saline (n = 4), BCG Danish-SSI 1331 (n = 6) and AERAS-422 (n = 6) and were immunized intradermally with 100 µL of saline, BCG Vaccine SSI (1 × 106 CFUs) or AERAS-422 (1 × 106 CFUs). Animals were bled at weeks 0, 6, 8 and 12 and PBMCs were isolated by standard Ficoll-Hypaque method [10]. Intracellular cytokine stimulation and staining was then performed as previously described [11] with LIVE/DEAD viability dye (Violet, Invitrogen) and the following antibodies: CD3-APC-Cy7, CD4-PerCP-Cy5.5, CD8-APC, IFNγ-FITC, IL2-PE, TNFα-PE-Cy7, CD14-Pacific Blue, CD16-Pacific Blue (all from BD). Samples were acquired in a BD LSRII flow cytometer and analyzed using FlowJo Software. Cells were gated on FSC-A vs. FSC-H to exclude doublets. CD3 T cells were then gated against V450 (a dump gate including viability dye, CD14 and CD16), followed by gating on CD4 and CD8 T cells. In a mouse protection study approved by the institutional animal care and use committee of the Center for Biologics Evaluation and Research, C57BL/6 mice were vaccinated subcutaneously with saline, 5 × 106 CFUs of BCG Danish-SSI 1331 or AERAS-422 as described previously [12]. After 8 weeks, the vaccinated mice were challenged by aerosol with 100–200 CFUs of the HN878 strain of Mtb using a Glas-Col inhalation chamber. Mice were sacrificed at 1, 3 and 5 months post-challenge. Lungs and spleens of individual mice were homogenized separately in 5 mL normal saline plus 0.05% Tween-80 using a Seward Stomacher 80 blender (Tekmar). The homogenates were diluted serially and plated on Middlebrook 7H10 agar containing thiophene-2-carboxylic acid (2 µg/mL) to prevent growth of BCG. Lung tissues were processed for histopathology using standard paraffin fixation, sectioning, and H&E staining. The H&E stained lung sections were evaluated using computer-based histopathology analysis as described previously [13].Six- to 8-week-old female SCID mice (Charles River, Wilmington, MA, USA) were immunized with 100 µL (5 × 106) of BCG Danish-SSI 1331(Staten Serum Institute, Denmark; diluted in Sauton’s medium), AERAS-422 and AERAS-401 (Aeras, Rockville, MD, USA; diluted in saline via tail vein injection). Mice were sacrificed when moribund; defined by severe lethargy, hunched with ruffled fur, loose skin or other signs of severe distress in line with FELASA recommendation on limiting clinical signs.For repeat dose toxicity study, guinea pigs were injected intradermally with approximately 10 times greater than the current recommended BCG dose given to humans (~2–8 × 105 CFUs/0.1 mL adult dose). To test excessive dermal reactivity in line with European pharmacopeia and WHO recommendations [14,15] for assessing BCG vaccines, guinea pigs were injected intradermally with BCG Danish-SSI 1331 (8 × 104) or AERAS-422 (5 × 106) and observed for adverse effects. Testing for freedom from virulent mycobacteria was performed to detect the presence of virulent mycobacteria in the lyophilized vials of AERAS-422 to be used in human clinical trials. Guinea pigs were injected subcutaneously with two doses of AERAS-422, low (2.5 × 107) and high (1 × 108), to achieve a target dose of 10 and 50 times the highest anticipated clinical dose. General safety of AERAS-422 was evaluated in both mice and guinea pigs with a five times higher dose (mice; 5 × 106) and a 50 times higher dose (guinea pigs; 2.5 × 106) than the highest anticipated human dose.The rBCG currently in the most advanced clinical studies is VPM1002. VPM licensed the vaccine candidate from the Max Planck Society and has sponsored the preclinical-clinical development to the current clinical phase IIa studies in neonates. This novel construct was produced by integrating the hly gene from L. monocytogenes coding for LLO expression into the genome of BCG [4]. LLO is a non-enzymatic toxin with pore forming functionality. In contrast to other pore-forming bacterial toxins, LLO provides two safety features intended to prevent damage or death of host cells: (1) The pore forming functions of the protein is restricted to acidic pH with optimal activity at pH 5.5 [16]; (2) LLO also carries the PEST amino acid sequence that directs the protein to phagosomal degradation upon appearance in the host cell’s cytosol [17,18,19]. In contrast to BCG, which is restricted to the phagosome and thereby considered to be limited to MHC II antigen presentation, the LLO activity of VPM1002 allows bacterial antigens to enter the cytosol and gain access to the MHC I pathway [4,5]. MHC I antigen presentation and subsequent stimulation of CD8 T cells is considered to more closely resemble the natural mechanism of protection against TB which, through the esx1 region, allows for phagosomal escape [20], and is thus believed to be the appropriate means for improving the induction of immunity by this modified BCG [21]. The targeted integration of this gene into ureC also serves to inactivate BCG’s gene for urease (ureC). Urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia, thus creating a basic environment. Inactivation of this gene is necessary since listeriolysin is optimally active under acidic conditions, as previously mentioned. Both modifications were engineered in one approach by the insertion of hly into the ureC locus, thus disrupting the urease sequence and deleting its activity [5]. Studies have demonstrated that these modifications lead to enhanced apoptosis and enhanced MHC class I antigen presentation [5]. The final VPM1002 construct is sensitive to antibiotics commonly used in the treatment of mycobacterial infection, including isoniazid, rifampicin and ethambutol. The correct genetic nomenclature of VPM1002 is recombinant M. bovis BCG ∆ureC::hly (genetic background Danish, subtype Prague).VPM1002’s preclinical proof of concept for efficacy and safety has been analyzed in 10 studies comprising approximately 600 animals. In general, all studies were performed using BCG Danish, sub type Prague vaccine as a baseline for vaccine induced responses. For efficacy, mice were vaccinated with VPM1002 or BCG and subsequently challenged by aerosol, using either the laboratory strain Mtb H37Rv or a clinical isolate of Mtb. Efficacy was determined by enumerating viable Mtb colony forming units (CFUs) in the lung and spleen. Preclinical testing of VPM1002 in mice demonstrated better protection from Mtb infection compared to BCG SSI (Figure 1). The immune response induced was sustained with bacterial burdens in lungs and spleens of VPM1002-vaccinated mice consistently below the BCG Danish-SSI 1331 control and the difference became statistically significant at 90 days post-infection. In all 10 studies, VPM1002 demonstrated better protection against the TB challenge when compared to the BCG positive control.The safety of VPM1002 was evaluated in a number of animal models and in three different species. Interferon-gamma (IFNγ) knock-out mice served as a model for evaluating the risk of disseminated infection with BCG and VPM1002 vaccines in severely immunocompromised humans, since BCG infection has been related to deficiencies in the IFNγ signaling pathway. No VPM1002-vaccinated animal died during the observation period of 105 days in contrast to BCG (one animal died), and no adverse effects on animal health were observed. However, both VPM1002 and BCG induced local reactions at the site of vaccination.Efficacy study in mice comparing VPM1002 with BCG Danish-SSI 1331. Balb/c mice were immunized subcutaneously with 106 BCG SSI (■), VPM1002 (▲) or with PBS (●). After 90 days, animals were infected via aerosol with 200 CFUs of Mtb (H37Rv) per mouse. Bacterial burden of lung (A) and spleen (B) was determined at 30, 60 and 90 days post-challenge. Data is shown as mean and standard deviation (n = 6 per group) of one representative experiment out of three. One-way ANOVA and Tukey’s multiple comparison was used to determine statistical significance of VPM1002 as compared to BCG SSI (p < 0.05).The target product profile for clinical development of VPM1002 considered HIV-infected individuals, therefore further safety studies were performed in SCID mice which lack an adaptive immune system. As described in Grode et al., 2005 [5], following administration of a 10-fold human target dose (i.e., 5 × 106 CFU per animal), the severity and incidence of findings were similar or less severe than the single human dose of the reference BCG strain (given at 5 × 105 CFU). Bridging studies with cGMP-manufactured vaccine showed comparable levels of safety in SCID mice.Three single-dose toxicity studies were performed in guinea pigs with follow-up periods of 6 to 26 weeks and dosages up to 50-fold human target dose (HTD). In general, weight gain (a highly sensitive marker for TB in guinea pigs) was similar in all treatment groups and in the range of normal variation known for this species. No guinea pig died prior to study end and no lesions typical for TB or related mycobacterial infections were observed during necropsy or histopathological evaluation. Small white spots (approx. 1 mm) were seen on the livers of some animals from all groups (VPM1002, BCG and saline). Histopathology of those spots revealed cell necrosis and hydropic degeneration in both BCG and VPM1002. The pathology seen by microscopy and the naked eye following vaccination with VPM1002 was less than that seen with BCG and no viable bacilli were detected in lung, liver or lymph nodes as observed by culture, although PCR revealed a similar systemic spread of both VPM1002 and BCG nucleic acid in the above mentioned organs. In a safety model using new born rabbits and comparing VPM1002 to BCG (both human target/standard dose), no mortality was observed. Macroscopically and histologically, all animals showed clear infectious reactions, which are expected at the 2-week time point post-immunization with a live BCG vaccine. A preclinical study in newborn rabbits was also performed to compare VPM1002 with BCG for safety, including bio distribution of the bacteria 90 days post-vaccination. The body weight of male and female animals vaccinated with BCG (control) did not gain the same amount of weight compared to animals treated with 0.9% saline. No influence on body weight was noted for the animals treated with VPM1002 (Figure 2). Also there were no local adverse reactions or changes in behavior, external appearance was noted. In addition, all critical assays testing the virulence of VPM1002 met all the requirements of the European Pharmacopeia recommendations for BCG [14]. VPM1002 vaccine safety study in newborn rabbits. Weight development of male (A) and female (B) new born rabbits was evaluated up to 90 days post-vaccination in three treatment groups (Group 1: control 0.9% saline (△), group 2: 1–4 × 105 CFUs BCG (▲) and group 3: 1–4 × 105 CFUs VPM1002 (●) per animal).A second example of a novel rBCG vaccine that has progressed into clinical studies is the AERAS-422 vaccine. The parent strain (BCG Danish-SSI 1331) modified to express PFO, AERAS-401, has been described previously [8]. Using specialized transduction, the panCD genes coding for pantothenate synthase were deleted [22], rendering AERAS-401 auxotrophic for pantothenic acid (vitamin B-5). This allowed for panCD complementation, which helped to retain the episomal plasmid containing two classical Mtb antigens (Ag85A and Ag85B) and one latency-associated antigen (Rv3407), without the need for an antibiotic marker. The overexpression of these antigens by this new rBCG strain, AERAS-422, was confirmed by Western blot analysis using specific antibodies to the Mtb antigens. Antigen expression was also stable after infecting cells from the human macrophage-like cell line, THP1, and plating at different time points and checking colonies for the presence of genes coding for the above mentioned antigens using PCR (Figure 3). Genetic analysis and stability of AERAS-422 plasmid. (A) Schematic representation of the complementation plasmid in AERAS-422 expressing Ag85A, Ag85B and Rv3407c, as well as the complementing panCD gene under its own promoter. (B) The stability of the plasmid was tested by plating a sample of AERAS-422 on 7H10 plates without pantothenate supplement and screening 40 colonies using PCR for the presence of the antigen cassette.Both NHPs [23,24] and mice [25] provide a useful model for immunological testing of vaccines due to their homology with humans, and the availability of a broad range of immunological reagents for the examination of immune responses. AERAS-422 was tested for immunogenicity in NHPs and protective efficacy in the mouse model of TB. As shown in Figure 4, AERAS-422 induced considerably higher CD4 responses against each of the antigens and greater CD8 responses against Ag85B only when compared to parental BCG in NHPs. While these increases did not reach statistical significance, the data present a clear trend toward increased immunogenicity. Responses against Rv3407 remained below the limit of detection for this assay.The protective efficacy of AERAS-422 was evaluated in a mouse challenge model. AERAS-422 showed significantly better protection than animals injected with saline (naïve) or BCG in lungs 12 weeks (p < 0.05) post-challenge and in spleens at both 12 and 20 weeks (p < 0.05) post-Mtb challenge (Figure 5A,B). Using computer-based histopathology analyses, lung sections showed significantly less granuloma-like lesions in both BCG Danish-SSI 1331 (17.6 ± 1.4) and AERAS-422 (20.3 ± 2.5) when compared to naïve mice (48.0 ± 2.5) at 20 weeks post-challenge (Figure 5C).Immune responses to AERAS-422 following immunization of non-human primates. PBMCs from Rhesus macaques vaccinated with either (5 × 106) BCG Danish-SSI 1331 or AERAS-422 were isolated and stimulated with Ag85A, Ag85B or Rv3407c peptides. Intracellular cytokines were examined at weeks 0, 6, 8 and 12. The data is the percent of total CD4+ or CD8+ T cell responses making IFN-γ, TNF-α, or IL-2 alone or in combination following background (DMSO stimulation) subtraction. Groups shown are: ●—Saline —BCG SSI and —AERAS-422. Bars show the Mean ± SD.Bacterial load and lung histopathology following immunization with AERAS-422 and challenge of C57BL6 mice with Mtb HN878. Bacterial load in lungs (A) and spleen (B) were determined 12 (■) weeks and 20 () weeks post-challenge. Statistical analysis was performed using the unpaired t test and the symbols represent the following: * = significantly better than naive at p < 0.05 and # = significantly better than both naive and BCG at p < 0.05. (C) Histopathology at 12 weeks post-challenge demonstrating fewer granuloma-like lesions and more open alveolar space in lungs of mice vaccinated with both BCG Danish-SSI 1331 and AERAS-422.Preclinical safety of AERAS-422 was investigated using a SCID mouse model of infection. In this study, 100% (10/10) of mice immunized subcutaneously with BCG SSI died within the 300 day observation period, while 9 out of 10 mice immunized with AERAS-422 survived as did the naïve control group (Figure 6). As mentioned above, AERAS 422 is derived from AERAS-401 [8] a strain which contains PFO but not the Mtb antigen cassette and AERAS-422 immunized mice also survived significantly better than mice immunized with this parent strain where 5 of 10 mice died. Log-rank analysis of the survival curves comparing BCG Danish-SSI and AERAS-422 yielded a p value = 0.0002. It may be that the high level of antigen overexpression from the plasmid, in addition to expression of the foreign PFO protein, alters the response to the rBCG such that it was contained by the innate immune system present in SCID mice. Survival study in SCID mice comparing AERAS-422 strain with BCG parent strains. SCID mice (10 mice/group) were immunized subcutaneously with high doses (5 × 106) of BCG Danish-SSI 1331 (■), AERAS-401 (●) and AERAS-422 (▼) and compared with a naïve group (▲).AERAS-422 was produced as cGMP compliant material using qualified manufacturing procedures. For toxicological evaluation and release of the cGMP material, acute toxicity, repeat dose toxicity, skin test reactivity (PPD) and freedom from virulent mycobacteria studies were performed. Guinea pigs were used for all of these studies due to their sensitivity to TB [26,27]. In repeat dose toxicity no adverse effects were observed and all animals survived to the scheduled termination (day 7 post-immunization) with no changes in appearance, behavior or body weight. The potency release assay, which was done by tuberculin skin testing with PPD after immunization, demonstrated the biological activity of the vaccines. Finally, freedom from virulent mycobacteria also showed no evidence of tuberculosis following immunization.The World Health Organization (WHO) Expert Committee on Biological Standardization has recently revised the “Recommendations to Assure the Quality, Safety and Efficacy of BCG Vaccines”, which is used by global manufacturers of BCG vaccines [15]. Although important for evaluating current BCG vaccines, new vaccine strains have been constructed using novel molecular techniques and have, in some cases, been manufactured using methods that differ from the traditional methods, for example VPM1002 and AERAS-422 vaccine candidates. New recommendations will therefore be required for general manufacturing and vaccine testing methods, as well as for preclinical and clinical testing. Unlike current BCG vaccines, certain new rBCG vaccines are being produced in facilities not dedicated to BCG production alone. Recombinant BCG vaccines generated by novel molecular techniques require tests for attenuation and possible reversion, persistence, plasmid retention and genetic stability, along with antibiotic sensitivity to frontline treatments. Preclinical safety tests use models of immunosuppression such as SCID and knock-out mouse models to predict the safety of these vaccine candidates intended for use in humans with HIV and other immunocompromised conditions. Clinical trials will need to address issues of safety and efficacy in healthy volunteers and in patients infected with Mtb and HIV. In addition, studies will be carried out using live vaccines as a “prime” and potentially use subunit vaccines as a “boost” to the prime. Although not a regulatory requirement, before proceeding past phase I human safety studies it may prove useful to perform efficacy studies in non-human primate TB challenge studies to investigate prime-boost regimens and, if possible, to compare different recombinant BCG vaccines. For all these reasons, additional WHO recommendations could provide an authoritative framework indicating new standards for assessing the quality, safety and efficacy of new live vaccines for TB. Table 1 lists new tests that may need to be implemented for new live TB vaccines compared to the current tests used for traditional BCG vaccines. Many of these tests may also be used for live attenuated Mtb vaccines such as MTBVAC [28] which has recently entered human phase I studies although additional safety testing such as in non-human primates may need to be performed.Testing strategies for new live TB vaccines compared with traditional tests.* Many of these assays are still under development and have not been standardized; ** Traditional tests as described here may be used in the characterization of new TB vaccines.As previously described [29], investigators face a number of challenges in the rational development of new BCG vaccines to demonstrate that they are safer and more efficacious than the current BCG vaccine. New rBCG approaches should include a novel formulation of BCG which impacts its potential to elicit more protective immunity directed against antigens found in Mtb while not compromising safety. In the two examples presented here, this includes the addition of a heterologous bacterial lytic gene which may promote antigen presentation to the host immune system [5,30] and the expression of Mtb antigens known to elicit protective immune responses in animal models [8]. As rBCG vaccines will be considered a genetically modified organism (GMO), studies to characterize them should meet regulatory requirements relevant to live vaccines such as attenuation, non-reversion, persistence, and shedding. The greatest challenge in rBCG development is demonstrating superior immunogenicity and protection that may be relevant to humans using preclinical animal models. Since there is no known “correlate of protection”, investigators are left measuring immune responses that are only thought to be related to TB protection [31]. In animal protection vs. challenge experiments, which are commonly the most important factor in deciding whether to move a vaccine candidate forward, investigators have used various BCGs as comparators and various immunization and Mtb strains and challenge strategies [32]. As shown in this article and elsewhere [6], rBCGs can be demonstrated to be superior to BCG comparators in animal experiments but results can differ depending upon experimental variables. It is clear from the experience of a number of investigations [33] that time and effort invested in the development of a novel rBCG should be balanced against potential benefits.A new rBCG vaccine may be more acceptable (marketable) to the global community, as long as it can be shown to be safer and more efficacious than BCG, and if the cost is comparable or lower than the currently available BCG. Unlike traditional BCG vaccines, novel vaccines offer several advantages including:
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+            new well-characterized products manufactured by state-of-the-art technologies
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+            shown to be safe and effective in contemporary clinical trials compared to currently used traditional BCG vaccines
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+            may serve as excellent prime vaccines for novel booster TB vaccines currently under development.
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+        new well-characterized products manufactured by state-of-the-art technologiesshown to be safe and effective in contemporary clinical trials compared to currently used traditional BCG vaccinesmay serve as excellent prime vaccines for novel booster TB vaccines currently under development.A robust process for developing candidate products that have undergone extensive preclinical studies such as those described in this article would facilitate acceptance with regulatory agencies for clinical development. In general, it would provide a better-defined product than BCG licensed decades ago by traditional methods. Table 1 outlines testing strategies that could differ between new live BCG vaccines and traditional BCG vaccines. The possibility for an updated regulatory review and product approval of new BCG based vaccines offers an opportunity to develop more rigorous characterization of BCG vaccines and to apply new tests for characterizing purity, safety and, particularly, potency. In historical studies, BCG vaccines have shown variable efficacy in different populations and age groups and the accepted efficacy of currently used BCG vaccines is estimated from meta-analyses of previous clinical trials and case control studies [34,35]. PPD/TST conversion correlates with BCG vaccination with respect to vaccine uptake, but does not appear to correlate well with clinical efficacy [36], although this test has been used for many years by regulatory agencies for the licensure and quality control of BCG. Currently, the identification of relevant biomarkers for immunogenicity and protection by TB vaccines is a high priority [37,38] and it is hoped that some biomarkers may become available for testing in rBCG clinical phase II studies, and may also be tested as correlates of vaccine efficacy or protection in clinical phase III studies. These biomarkers may therefore provide a pathway for regulatory agencies to consider alternative data as supportive evidence for more efficient clinical development towards licensure of rBCG candidate vaccines.Current BCG vaccines can be relatively reactive vaccines that cause ulceration, scarring and lymphadenopathy, as well as serious adverse effects from dissemination in immunocompromised infants [39,40,41]. Robust preclinical safety and human safety studies in the target populations will be critical for licensing a new BCG vaccine. Current WHO/EPI policy calls for immunization of infants at birth who are not HIV-infected, and there is concern related to immunizing infants with mothers who are HIV+ [2]. Safety studies are a major focus of the preclinical studies described here, and together with robust clinical data, rBCGs may be shown to be safer to use in a HIV+ high risk population. A thorough set of safety data demonstrating a lower incidence of fevers, anemia or lymphadenopathy could establish the basis for licensure of an rBCG vaccine if accompanied by robust post-licensure surveillance. Also, since rBCG is similar to BCGs that have a long history of acceptable safety and use in humans, it may be possible to consider a licensure strategy with smaller or fewer efficacy studies, particularly if preclinical immunological data and clinical safety study data is robust and the sponsor commits to large clinical phase IV post-marketing studies. A strategy presently employed by sponsors of TB vaccine candidates in clinical trials is the use of TB subunit vaccines to boost subjects previously vaccinated with BCG. However, several of these antigens are expressed by BCG at low levels or not at all [38]. Development of an rBCG expressing specific Mtb antigens may provide the opportunity to match a prime immunization with the same antigens expressed in the subunit boost vaccine [32]. This offers the opportunity to generate rBCGs overexpressing Mtb antigens that are found in potential boosting vaccines such as MVA85A which is currently being evaluated in a phase IIB efficacy study in BCG vaccinated infants [42]. An NHP study using AERAS-422 boosted by a viral-vectored TB vaccine [43] is an example of an immunogenicity proof of concept preclinical study performed to address the potential of the matched BCG prime-boost concept. However, the ability of an rBCG prime-boost strategy to enhance efficacy remains unclear since some TB vaccine studies have shown little or no booster effects following a BCG prime [44].In this report, we have provided examples of testing strategies that have been successfully applied to two new rBCG vaccines that have entered human clinical trials [45]. Although not without risks, there are a number of potential benefits that may result from introducing new rBCG vaccines. These include a live replacement TB vaccine that is safer to use in all populations, including HIV+ populations, and be more effective either alone or as part of a prime-boost strategy in controlling adult pulmonary TB disease. The TB community continues to assess new live mycobacterial constructs that will be evaluated over the next decade with the goal of contributing to a meaningful reduction in the global TB epidemic.Clinical study of one of the rBCG vaccines discussed in this article, AERAS-422, has been discontinued due to a safety signal observed in the first phase 1 study of this vaccine. A manuscript describing this trial is currently under preparation.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The Kaiser Permanente Vaccine Study Center is a specialized research organization in Oakland, California. They have been an active vaccine research group for many years, and have participated in and led a multitude of vaccine studies. This article will review the last three years of research activities.High vaccine coverage is dependent of public perception of vaccine safety. To this end, it is of utmost importance that ongoing research into the potential association of adverse events with immunization be given priority. This article will focus on the activities of a research group devoted to the study of vaccines and vaccine safety.Founded in 1985, The Kaiser Permanente Vaccine Study Center (KPVSC), part of the Kaiser Permanente Northern California (KPNC) Division of Research (DOR), is a specialized research organization in Oakland, California, which is active in all areas of clinical vaccine research. KPNC is an integrated medical care organization which provides all medical care services to its over 3.2 million members. As part of its core research activities, KPVSC utilizes KPNC member data to conduct a wide range of vaccine effectiveness and safety studies. This article will highlight some of KPVSC’s activities and publications within the past 3 years.The types of vaccines studies that KPVSC design and carry out can be characterized into six broad categories: vaccine clinical trials; observational vaccine effectiveness studies; active surveillance for vaccine safety, including studies with the CDC-sponsored Vaccine Safety Datalink (VSD); observational post-licensure vaccine safety studies; evaluating vaccine safety in special populations and genetic influences on vaccine adverse events, including studies with the CDC-sponsored and Clinical Immunization Safety Assessment (CISA) Network; and studies evaluating the epidemiology of infectious diseases, vaccine-preventable diseases, and diseases related to vaccine adverse events. KPVSC has a long history of conducting Phase 2 and 3 vaccine clinical trials in infants, children and adults. KPVSC centralizes all research activities in our main Oakland office, with trained KPVSC research nurse and physician sub-investigators located at KPNC clinics throughout the Northern California region. Under the direction of the Principal Investigators and the clinical nurse manager Kathy Ensor, RN, the KPVSC RN and physician sub-investigators have been major contributors or, in some cases, the only site for a number of clinical trials. Since 2009, such trials have included:GlaxoSmithKline: 
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+            Evaluated the safety and immunogenicity of a investigational quadrivalent meningococcal vaccine conjugated to tetanus toxoid (MenACWY-TT) in adolescents [1] and infant/toddlers [2]. The United States Food and Drug Administration (FDA) is currently reviewing the adolescent study.
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+            Evaluated the safety and immunogenicity of the combination vaccine DTaP-IPV with MMR with and without varicella vaccine in 4–6 year olds [3].
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+            Evaluated the safety and immunogenicity of an investigational combination measles, mumps, rubella, varicella (MMRV) vaccine with hepatitis A and 7-valent pneumococcal conjugate vaccine in 1–2 year olds [4].
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+            Evaluated the safety and immunogenicity of inactivated hepatitis A vaccine concomitantly with diphtheria-tetanus-acellular pertussis (DTaP) and Haemophilus influenzae type b (HiB) vaccines [5].
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+        Evaluated the safety and immunogenicity of a investigational quadrivalent meningococcal vaccine conjugated to tetanus toxoid (MenACWY-TT) in adolescents [1] and infant/toddlers [2]. The United States Food and Drug Administration (FDA) is currently reviewing the adolescent study.Evaluated the safety and immunogenicity of the combination vaccine DTaP-IPV with MMR with and without varicella vaccine in 4–6 year olds [3].Evaluated the safety and immunogenicity of an investigational combination measles, mumps, rubella, varicella (MMRV) vaccine with hepatitis A and 7-valent pneumococcal conjugate vaccine in 1–2 year olds [4].Evaluated the safety and immunogenicity of inactivated hepatitis A vaccine concomitantly with diphtheria-tetanus-acellular pertussis (DTaP) and Haemophilus influenzae type b (HiB) vaccines [5].Novartis: 
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+            Safety and immunogenicity of MenACWY—CRM in infants [6], toddlers (with and without MMRV vaccine) [7], children aged 2–10 years [8] and adolescents [9,10]. Novartis’ quadrivalent meningitis vaccine uses a mutant diphtheria toxoid as the conjugate. The product is now licensed for ages 2–55 years in the U.S.
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+            Long term follow up studies designed to evaluate the 5 year antibody titer persistence following receipt of MenACWY-CRM in adolescents (P13E1) and infants (P14E1).
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+        Safety and immunogenicity of MenACWY—CRM in infants [6], toddlers (with and without MMRV vaccine) [7], children aged 2–10 years [8] and adolescents [9,10]. Novartis’ quadrivalent meningitis vaccine uses a mutant diphtheria toxoid as the conjugate. The product is now licensed for ages 2–55 years in the U.S.Long term follow up studies designed to evaluate the 5 year antibody titer persistence following receipt of MenACWY-CRM in adolescents (P13E1) and infants (P14E1).Massachusetts Biologics Laboratory:Study demonstrated the efficacy of one dose of a monoclonal antibody at preventing recurrences of Clostridium difficile diarrhea in adults [11,12]. Protein Sciences: Evaluation of the safety and immunogenicity of a baculovirus derived influenza vaccine (FluBlǾk) in adults aged 50–64 years [13]. This recombinant influenza vaccine has recently been approved by the FDA. Pfizer: Immune response to Prevnar 13 in adolescents who had previously received Prevnar 7 as infants [14].Sanofi Pasteur:Safety and immunogenicity of different multivalent pertussis vaccines in infants and toddlers [15].KPVSC is active in conducting Phase IV safety surveillance studies following vaccine licensure, which are typically mandated by the FDA. The size of the studies varies depending on the vaccine, but often range from 10,000 to 100,000 persons vaccinated within KPNC. The studies are observational and evaluate all individuals who received the vaccine as part of routine clinical care. As such, such studies do not involve informed consent; rather, they reflect “real-life” use of vaccines administered to KPNC’s large and diverse population. Using KPNC’s integrated electronic medical record which captures all medical utilization (including vaccinations, laboratory tests, procedures, inpatient, emergency room, and outpatient diagnoses), these studies evaluate post-vaccination outcomes to determine whether such adverse events following immunization (AEFI) are related to the vaccine or whether they are coincidental and unrelated to immunization. In general, our approach to this challenge is to designate a comparison group with which we can compare observed rates of AEFI in the vaccinated group of interest. Comparison groups may include unvaccinated individuals, but vaccinated people can differ in fundamental demographic, health care seeking and other ways from vaccinated individuals [16,17]. It is usually not possible to identify, measure and control for such confounding using data available in electronic medical records, therefore, the results of such studies which rely on unvaccinated controls are difficult to interpret and conclusions drawn may be spurious.For this reason, we favor utilizing a comparable vaccinated comparison group, such as a group of similarly aged individuals receiving a very similar vaccine during a similar time period. However, if such a comparison group is not readily apparent or available (as is often the case), an alternate approach is to study only individuals who were immunized with the vaccine of interest and to compare the rates of AEFIs during a period of time shortly after vaccination (known as the risk interval) with another period of time either before or after vaccination (known as the control interval).This study design is called the “risk interval” approach. We have utilized this study design in the majority of KPVSC’s ongoing or recently completed post-licensure studies listed below.GlaxoSmithKline: Tetanus, reduced content diphtheria and acellular pertussis vaccine (Tdap, Boostrix). This study evaluated the safety among 10,000 10–18 year olds for selected medical events during the 30 days after vaccination using a risk interval design. It also well as compared outcomes of interest using a historical comparison groups of Td vaccinated teenagers. This study did not detect any safety concerns [18]. MedImmune:Live attenuated intranasal influenza vaccine: LAIV or FluMist® in 5–49 and 2–4 [19,20]. A third publication describes our experience with this live nasal vaccine in children 2–4 years of age [21]. Over multiple years we examined the time periods after immunization, to see if visits to medical clinics, emergency departments, and hospitals were more frequent soon after immunization than at later points in time. We also compared outcomes in those vaccinated with LAIV outcomes in killed injectable influenza vaccine (TIV) and no vaccine. One thing we found is that LAIV recipients are very different from TIV recipients and unvaccinated, and these differences were reflected in utilization patterns. This made interpretation of the results somewhat problematic, and many outcomes needed intense investigation to discern any abnormal pattern. After full investigation, the vaccine appears to be safe for all studied ages, and we found no new areas of concern.Merck and Co:
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+            Human Papilloma Virus (HPV4, Gardasil) vaccine [22,23]: This study evaluated the general safety of HPV4 administered routinely to girls using a risk interval design. Rates for all emergency room and hospitalization events in females who received HPV4 (n = 189,629) were evaluated during the post-vaccination risk interval and compared with rates for the same events during the control period which was a post-vaccination interval distant in time from vaccination. This study found that HPV4 was associated with same-day syncope (OR, 6.0; 95% CI, 3.9–9.2) and skin infections in the 2 weeks after vaccination (OR, 1.8; 95% CI, 1.3–2.4). This study did not detect evidence of new safety concerns among females 9 to 26 years of age secondary to vaccination with HPV [22,23].
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+            Zoster vaccine safety [24]: The zoster (shingles) vaccine was approved in 2006 for adults 60 years and older. This study was conducted to monitor the safety of this vaccine when routinely administered to adults and found the vaccine to be very safe. Similar to many post-licensure studies, this study assessed numerous post-vaccination many outcomes, each of which that found a statistical association required follow up and additional investigation. While the study did not identify any safety concerns, interestingly this study found that persons vaccinated with zoster vaccine appeared to be protected against multiple outcomes, including death. Additional analyses revealed that this observation was actually due individuals receiving the vaccine at times when their health was optimized, making it appear (falsely) that the vaccine was protective. In addition, people who were vaccinated appeared to be healthier than the general population of the same age [25].
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+            Varicella long term effectiveness and epidemiology after introduction of the vaccine—a 14 year study [26]. Over the 14-year period, we found that Varicella vaccine was very protective (around 90%) against varicella disease without evidence of waning protection. No cases were seen after a second dose. There was no increase of chicken pox in children as they aged, and no increase in zoster (shingles), both of which had been worries prior to the vaccine.
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+        Human Papilloma Virus (HPV4, Gardasil) vaccine [22,23]: This study evaluated the general safety of HPV4 administered routinely to girls using a risk interval design. Rates for all emergency room and hospitalization events in females who received HPV4 (n = 189,629) were evaluated during the post-vaccination risk interval and compared with rates for the same events during the control period which was a post-vaccination interval distant in time from vaccination. This study found that HPV4 was associated with same-day syncope (OR, 6.0; 95% CI, 3.9–9.2) and skin infections in the 2 weeks after vaccination (OR, 1.8; 95% CI, 1.3–2.4). This study did not detect evidence of new safety concerns among females 9 to 26 years of age secondary to vaccination with HPV [22,23].Zoster vaccine safety [24]: The zoster (shingles) vaccine was approved in 2006 for adults 60 years and older. This study was conducted to monitor the safety of this vaccine when routinely administered to adults and found the vaccine to be very safe. Similar to many post-licensure studies, this study assessed numerous post-vaccination many outcomes, each of which that found a statistical association required follow up and additional investigation. While the study did not identify any safety concerns, interestingly this study found that persons vaccinated with zoster vaccine appeared to be protected against multiple outcomes, including death. Additional analyses revealed that this observation was actually due individuals receiving the vaccine at times when their health was optimized, making it appear (falsely) that the vaccine was protective. In addition, people who were vaccinated appeared to be healthier than the general population of the same age [25].Varicella long term effectiveness and epidemiology after introduction of the vaccine—a 14 year study [26]. Over the 14-year period, we found that Varicella vaccine was very protective (around 90%) against varicella disease without evidence of waning protection. No cases were seen after a second dose. There was no increase of chicken pox in children as they aged, and no increase in zoster (shingles), both of which had been worries prior to the vaccine.Pfizer:Prevnar 13 safety: This is an ongoing FDA-mandated study to follow recipients of Prevnar13 in childhood for possible side effects or unexpected adverse outcomes. To date, no significant safety issues have been raised. Sanofi Pasteur
+        
+          
+            Tdap (Adacel), in adolescents and adults: The study evaluated the safety of Tdap by evaluating all medical events following immunization of more than 120,000 persons who received Tdap as part of routine clinical care. Final analysis is currently underway.
+          
+          
+            Quadrivalent Neisseria meningitidis (ACW135Y) conjugate vaccine (MCV4, Menactra). This study evaluated the safety of this vaccine administered to more than 31,000 individuals ages 11–55 using both observational data in KPNC database (i.e., medical events) and active telephone calls to vaccine recipients. Preliminary analyses using the risk interval method have not detected any safety concerns [27].
+          
+          
+            Menactra in 2–10 year old children. This study will be assessing the safety of this vaccine among a population of 2–10 year old children. Analyses are currently underway.
+          
+          
+            Menactra in 9 and 12 month old children. This study will be assessing the safety of this vaccine among a population of infants and toddlers. Study accrual is underway.
+          
+          
+            Tetanus, diphtheria and acellular pertussis vaccine (DTaP, Daptacel). The primary aim of the study was to assess the general safety of this vaccine in infants. A secondary specific aim focused on the risk of Hypotonic Hyporesponsive Episodes (HHE), a syndrome where infants become transiently less responsive and lose muscle tone. Final analyses are completed. This study identified no safety concerns and no increased risk of HHE related to vaccination.
+          
+          
+            DTaP-inactivated polio-Hemophilus influenza type B vaccines (DTaP-IPV-Hib, Pentacel) safety study. This study is evaluating the general safety of this combination vaccine by evaluating all events in emergency department and inpatient setting and selected outpatient events following immunization of infants and toddlers. Subject accrual has been completed and analyses will begin shortly.
+          
+        Tdap (Adacel), in adolescents and adults: The study evaluated the safety of Tdap by evaluating all medical events following immunization of more than 120,000 persons who received Tdap as part of routine clinical care. Final analysis is currently underway.Quadrivalent Neisseria meningitidis (ACW135Y) conjugate vaccine (MCV4, Menactra). This study evaluated the safety of this vaccine administered to more than 31,000 individuals ages 11–55 using both observational data in KPNC database (i.e., medical events) and active telephone calls to vaccine recipients. Preliminary analyses using the risk interval method have not detected any safety concerns [27].Menactra in 2–10 year old children. This study will be assessing the safety of this vaccine among a population of 2–10 year old children. Analyses are currently underway.Menactra in 9 and 12 month old children. This study will be assessing the safety of this vaccine among a population of infants and toddlers. Study accrual is underway.Tetanus, diphtheria and acellular pertussis vaccine (DTaP, Daptacel). The primary aim of the study was to assess the general safety of this vaccine in infants. A secondary specific aim focused on the risk of Hypotonic Hyporesponsive Episodes (HHE), a syndrome where infants become transiently less responsive and lose muscle tone. Final analyses are completed. This study identified no safety concerns and no increased risk of HHE related to vaccination.DTaP-inactivated polio-Hemophilus influenza type B vaccines (DTaP-IPV-Hib, Pentacel) safety study. This study is evaluating the general safety of this combination vaccine by evaluating all events in emergency department and inpatient setting and selected outpatient events following immunization of infants and toddlers. Subject accrual has been completed and analyses will begin shortly.The Vaccine Safety Datalink (VSD) is a CDC-sponsored collaboration of 9 medical care organizations throughout the United States (U.S.). VSD’s purpose is to monitor the safety of U.S. licensed vaccines for possible AEFI. Safety evaluations include sequentially monitoring in near real-time either newly licensed vaccines for potential new or unanticipated safety issues following vaccine introduction and widespread use in the U.S. population or if there are changes in the recommendations for already licensed vaccines. Investigators at VSD sites have led or participated in numerous important vaccine safety studies in this collaborative project [28]. KPVSC has led numerous studies in VSD, a number of which are detailed below. Methodologies: Case centered method, OBS, Exact sequential analyses, and “difference-in-differences”.Case Centered Method: Many vaccine safety studies are designed to answer the question “what adverse events occur after vaccination?” In this method, we reversed this question by and asked “for all individuals who had a specific outcome, did a larger than expected proportion of them receive a vaccine during specific times prior to developing the outcome?” More clustering of vaccines than expected prior to the outcome may suggest that receipt of a vaccine is causally related to the outcome. We look retrospectively at vaccinated people with specific adverse events, and determine what proportion of vaccinations fall in or outside of a risk interval prior to the event. To find our expected odds of vaccination, we determine the proportion of vaccinated people during the same time intervals, for our entire KPNC population, matched by age and sex to the index cases. We have used this approach in multiple studies [16,29,30,31]. Outcome-Based Surveillance (OBS): This methodological approach extends the principles of the case centered methodology a step further with the goal of monitoring a very large number of vaccine-outcome pairs to preliminarily assess whether a potential relationship exists between the vaccine and the outcome. In order to apply this method, we constructed summary tables of all our members, and eventually of all VSD members, with vaccination, age and sex. This made finding our expected rate of vaccination very efficient, and so we are planning on applying the method to many types of possible adverse events and vaccines, in a project we call Outcome-based surveillance. We expect this project to provide data on various vaccine-AE pairs over years, to be a reference for those looking for potential problems, and to serve as a starting point for vaccine safety research in the years to come. Implementation of this method is ongoing,Exact Sequential Analyses (ESA): This method has been utilized in a number of VSD studies, such as Rapid Cycle Analysis (RCA), where data is analyzed repeatedly over time. ESA is used to adjust for the statistical problem of multiple sequential looks at data. Using an exact permutation test for studies using a binomial outcome, this method improves accuracy, particularly when there are a small number of outcome events. It allows variation in the ratio of the size of comparison groups, and flexibility in controlling for type 1 error over time [32]. Work on a manuscript is ongoing.Specific Vaccine Safety VSD Studies:
+        
+          
+            Measles-mumps-rubella-varicella combination vaccine and the risk of febrile seizures. This study demonstrated that in 1–2 year old children receiving a first dose, receipt of the measles, mumps, rubella and varicella (MMRV) combination vaccine is associated with a twofold increased risk of febrile seizures 7–10 days after immunization in when compared with separately administered same-day MMR and varicella vaccines [31].
+          
+          
+            Risk of Febrile Seizures Following Measles-Containing Vaccine in 4–6 year old Children. This study evaluated risk of febrile seizures after measles-containing vaccine in 4–6 year old children and no evidence that either MMRV or separately administered MMR and varicella vaccines are associated with an increased risk of febrile seizures in children receiving a second dose of vaccine [33].
+          
+          
+            Effect of age on risk of fever and seizures from measles vaccines: We found that the rate of seizures after measles-containing vaccines is modified by the age of the child receiving the vaccine. Children receiving vaccines on schedule, at one year of age, had a lower rate of seizure compared to those receiving the vaccines later in the second year of life [34].
+          
+          
+            Risk of Rheumatoid Arthritis following vaccination with Tetanus, Influenza and Hepatitis B vaccines among persons 15–59 years of age. This VSD retrospective cohort study assessed for an association between rheumatoid arthritis and 3 different vaccines over a 13 year period. This study did not find any conclusive association between immunization and rheumatoid arthritis [35].
+          
+          
+            Immunization and Bell’s Palsy in Children: A Case-Centered Analysis: Using the Case Centered method described above, this study assessed for an association between vaccinations and the occurrence of Bell’s palsy in children and found no association [29].
+          
+          
+            Lack of association of Guillain Barré Syndrome (GBS) with vaccines [30]: As with the Bell’s palsy study, this study also used the case centered method to examine the risk of GBS after vaccination with any type of vaccine. Evaluating data collected over 13 years which included more than 30 million person-years, we found no association between vaccination and GBS.
+          
+        Measles-mumps-rubella-varicella combination vaccine and the risk of febrile seizures. This study demonstrated that in 1–2 year old children receiving a first dose, receipt of the measles, mumps, rubella and varicella (MMRV) combination vaccine is associated with a twofold increased risk of febrile seizures 7–10 days after immunization in when compared with separately administered same-day MMR and varicella vaccines [31].Risk of Febrile Seizures Following Measles-Containing Vaccine in 4–6 year old Children. This study evaluated risk of febrile seizures after measles-containing vaccine in 4–6 year old children and no evidence that either MMRV or separately administered MMR and varicella vaccines are associated with an increased risk of febrile seizures in children receiving a second dose of vaccine [33].Effect of age on risk of fever and seizures from measles vaccines: We found that the rate of seizures after measles-containing vaccines is modified by the age of the child receiving the vaccine. Children receiving vaccines on schedule, at one year of age, had a lower rate of seizure compared to those receiving the vaccines later in the second year of life [34].Risk of Rheumatoid Arthritis following vaccination with Tetanus, Influenza and Hepatitis B vaccines among persons 15–59 years of age. This VSD retrospective cohort study assessed for an association between rheumatoid arthritis and 3 different vaccines over a 13 year period. This study did not find any conclusive association between immunization and rheumatoid arthritis [35].Immunization and Bell’s Palsy in Children: A Case-Centered Analysis: Using the Case Centered method described above, this study assessed for an association between vaccinations and the occurrence of Bell’s palsy in children and found no association [29].Lack of association of Guillain Barré Syndrome (GBS) with vaccines [30]: As with the Bell’s palsy study, this study also used the case centered method to examine the risk of GBS after vaccination with any type of vaccine. Evaluating data collected over 13 years which included more than 30 million person-years, we found no association between vaccination and GBS.The CDC-sponsored Clinical Immunization Safety Assessment (CISA) Network is a collaboration between the CDC and multiple academic organizations. The mission of CISA is to assist national efforts to understand the underlying pathophysiology of adverse effects from immunizations, and to provide guidance in diagnosing and managing difficult individual cases where there is a concern about harm from vaccines. KPVSC has been a part of CISA since its inception in 2001 and has been closely involved in a wide variety of CISA activities, including active participation in working groups which provide clinical guidance and expertise to vaccine adverse event clinic consult expertise to address specific patient and medical provider concerns. KPVSC has both led and collaborated on a wide range of CISA studies. Below are a number of KPVSC-led CISA studies.Immunization rates and safety of vaccines administered to children with inborn errors of metabolism (IEM): Using KPNC’s electronic medical record, we identified children with IEM from 1990 to 2007 and assessed immunization rates and AEFI in these children. This study found that children less than 2 years old with IEM in KPNC were not delayed in their receipt of recommended vaccines when compared with healthy infants. This study also did not detect an increased risk for serious adverse event following immunizations among all children 18 years and younger with IEM during 30 days after vaccination [36].Evidence-based approach to defining post-vaccination risk intervals: KPVSC led a CISA working group with the aims of examining how the choice of specific post-vaccination risk intervals (i.e., number of days in the interval, placement of the interval) affect the results of vaccine safety studies. The WG also developed a method by which the best available evidence would be used to determine the “best” risk interval for various adverse events and vaccines. The WG then applied this approach to assess the “best” risk intervals for (1) fever after influenza vaccines and (2) acute disseminated encephalomyelitis after various vaccines [37].Recurrent Guillain-Barré Syndrome Following Vaccination [38]: This study examined all persons with a history of Guillain-Barré syndrome and determined whether receipt of vaccines after GBS diagnosis was associated with a relapse of GBS. This study was reassuring in that in over more than 30 million person-years, there no relapses of GBS after vaccination identified.Recurrent sterile abscesses after vaccination: In this case report, we described two cases of recurrent sterile abscesses which occurred following vaccines containing aluminum adjuvant and discussed a possible association between receipt of vaccines containing higher levels of aluminum adjuvant and development of sterile abscesses [39].Preterm infant responses to Polio vaccine: In this study, we prospectively enrolled 2 month old preterm and term infants and compared their T cell responses to inactivated poliovirus vaccine. The study found that preterm infants develop poliovirus-specific T cell responses that are comparable to those of term infants. However, preterm infants also demonstrated nonspecific and poliovirus-specific functional T cell limitations. Additional studies will be needed to assess whether these deficiencies have clinical implications [40].Risk of fever and sepsis evaluations after immunization in NICU: In this study, we evaluated whether immunization in the neonatal intensive care unit (NICU) were increased after immunizations due to potential post-immunization changes in clinical status. This study did find that there was an increase in fever and cardiorespiratory events after immunization in the NICU, but routine vaccination was not associated with increased risk of receiving sepsis evaluations [41].Vaccination rates at discharge from the NICU: This study determined immunization rates at discharge from the NICU among infants 2 months of age and found that significant proportion of infants discharged on or after 2 months of age in the NICU was unimmunized or underimmunized at discharge [42].Influenza vaccine and mortality in the elderly [16]: Earlier observational studies had shown that influenza vaccine was more effective at preventing death in the elderly than was plausible. We determined that the differences in persons vaccinated with influenza vaccines compared to those not vaccinated were significant, and that many of these differences are not measured by the medical system, so it is not possible to account for their confounding in studies of Influenza vaccine effectiveness (VE). Therefore, in order to bypass this severe confounding, we used a “difference-in-differences” approach, subtracting out the “effects” of vaccination that seemed to occur outside of the influenza season, when no virus is circulating. Although we verified that previous studies had overestimated VE, we demonstrated that the vaccine can still prevent almost half of all deaths attributable to Influenza infection.Flu and hospitalization [43]: Utilizing our same methodology as in the previous study on Flu vaccine and mortality, we determined the effectiveness of influenza vaccination at preventing hospitalization in persons 50 years and older.Pertussis vaccine effectiveness (DTaP waning, Tdap effectiveness, DTaP vs. whole cell): In 2010–2011, California experienced the largest outbreak of pertussis (whooping cough) in over 50 years. During the epidemic, pertussis rates markedly increased beginning at age 8 years, peaking at ages 10–11 years, decreasing to age 15 year and were low in adults. We hypothesized that the pattern derived from waning immunity to diphtheria tetanus, acellular pertussis (DTaP) vaccines and undertook several case-control studies in KPNC in which we identified person who tested positive for pertussis and compared them with two different controls: one control group consisted PCR test negative individuals and the other controls were matched individuals identified from the entire KPNC population. Using this approach, we found that (1) the 5th DTaP dose wanes by more than 40% each year after vaccination [44]; (2) Tdap is only about 60% effective at preventing pertussis [45]; (3) the risk of pertussis among teenagers who received 4 doses of whole cell pertussis vaccines during the first 2 years of life was much lower than among teenager who received 4 doses of DTaP [46].Invasive Pneumococcal disease following introduction of Prevnar 7 and Prevnar 13 into the KPNC population: We have been collecting specimens from patients with invasive pneumococcal infections since 2000 and serotyping them to see whether the serotypes are those that are included in either the 7- or 13-valent conjugate pneumococcal vaccines. Multiple studies are ongoing. Our system allows us to capture data on the entire underlying population, so that incidence rates of disease can be calculated.Bias and flu shots [17]: Studies of influenza vaccine effectiveness are often confounded by unmeasured variables. We showed this in a study looking at multiple years of influenza vaccines. If a person had been vaccinated for all of the preceding 5 years, and then did not get a vaccination in the current year, mortality rose dramatically. However, if another person did NOT get the vaccine for 5 years, and then got vaccinated in the current year, mortality also rose significantly, making it appear that the vaccine both prevents and causes death. This clearly illustrated the problems inherent in retrospective flu studies, and highlighted differences between vaccinated and unvaccinated individuals.Lopsided Windows: Early on in the course of one of our studies noted above [24], we noted that shorter risk time intervals (compared to comparison intervals) and rare rates of events resulted statistically significant differences weighted against the shorter interval. We showed that this phenomenon is predictable, and can happen in any type of study with comparison intervals that vary in size. This finding has been helpful in assessing and interpreting study results [47].Rates of autoimmune disease [48]: This study assessed baseline rates of various autoimmune diseases in KPNC, which is extremely valuable information in both the design and interpretation of vaccine safety studies.Genetics of Staph epidemiology [49]: This study showed that for KPNC, the USA300 clone of MRSA is found both in outpatients and in hospital- and healthcare-acquired settings.Microbiology and epidemiology of skin and soft tissue infections (SSTIs) [50,51,52]: Within our health plan population, we examined the epidemiology of SSTIs, focusing on trends of staphylococcal and methicillin-resistant staphylococcal (MRSA) infections over time. In our health plan, MRSA rates are decreasing over time, though they are still a very important cause of SSTEpidemiology of Bell’s palsy [53]: This paper provides a descriptive analysis of the largest population of children with Bell’s palsy to date.Incidence of genital warts among adolescents and young adults prior to HPV vaccination [54].Flu surveillance [55]: We have been monitoring Influenza and Respiratory Syncytial Virus (RSV) infections over many years. We provide a service to the KPNC medical providers and administrators, with weekly updates showing trends and predictions, and including guidelines on testing and treatment. Our publication was on how the H1N1 pandemic threw confusion into the surveillance, because the media releases were very early, before disease really hit our area. We found that fear drove utilization for medical services, even more than real disease. Another study analyzed patterns of flu seasons, and found a recurring pattern of latter seasons each year over 9 seasons [56]. We think that peaks of influenza season are more susceptible to the immunity of the underlying population than to weather, humidity, or other fluctuations in natural conditions.Vaccines are one of the most important developments in medical history, providing protection from numerous infections, which in the past resulted in considerable morbidity and mortality. Today, those infections have been nearly eradicated in many parts of the world. Predictably, as vaccine-preventable illnesses wane, and parents have little or no knowledge of their severity and impact, concerns of harm from disease turn to fear of harm from vaccines. Many parents are no longer comfortable accepting that vaccines are without or have minimal risk when they are exposed to many other sources (particularly from the internet) that that tell them otherwise. When people go to the doctor for medical care, they are generally looking for something that helps them and their immediate family; appeals to accept vaccines for the benefit of the community are unlikely to motivate them. Although the risk from vaccines is low, they are not completely risk free: there are known adverse effects associated with vaccines, and new associations may yet be discovered. Real or potential risks must be balanced with the benefits of a vaccine, to both the individual and the public at large. The best way to continue benefiting from widespread vaccine coverage is to assure the public that vaccine safety is a high priority for healthcare providers, for the industry that makes vaccines, and for governmental organizations. This reassurance can best be achieved by ongoing surveillance and research into both the safety and the effectiveness of existing and new vaccines. It is crucial for researchers to actively seek out potential problems with vaccines, to quantify adverse events, even when rates are very small, and to determine how well vaccines actually protect against disease, outside of a clinical trial context. In addition, it is important that the medical and scientific literature publish papers on vaccine safety, whether they point out a new issue, or just show once again that vaccines are relatively safe. Research organizations like the Kaiser Permanente Vaccine Study Center are crucial to the study of vaccine safety, and offer a service to vaccine recipients both here in the United States and also the rest of the world. The Kaiser Permanente Vaccine Study Center has been a leader in vaccine studies over many years, and their research has provided answers to a number of questions about vaccine safety and effectiveness. Studies such as these are essential to national efforts to maintain high rates of vaccine coverage. Both Baxter and Klein have received research grant support from Sanofi Pasteur, Novartis, GSK, Merck, MedImmune, Pfizer, and Protein Sciences.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Rates of delay and refusal of recommended childhood vaccines are increasing in many U.S. communities. Children’s health care providers have a strong influence on parents’ knowledge, attitudes, and beliefs about vaccines. Provider attitudes towards immunizations vary and affect their immunization advocacy. One factor that may contribute to this variability is their familiarity with vaccine-preventable diseases and their sequelae. The purpose of this study was to investigate the association of health care provider year of graduation with vaccines and vaccine-preventable disease beliefs. We conducted a cross sectional survey in 2005 of primary care providers identified by parents of children whose children were fully vaccinated or exempt from one or more school immunization requirements. We examined the association of provider graduation cohort (5 years) with beliefs on immunization, disease susceptibility, disease severity, vaccine safety, and vaccine efficacy. Surveys were completed by 551 providers (84.3% response rate). More recent health care provider graduates had 15% decreased odds of believing vaccines are efficacious compared to graduates from a previous 5 year period; had lower odds of believing that many commonly used childhood vaccines were safe; and 3.7% of recent graduates believed that immunizations do more harm than good. Recent health care provider graduates have a perception of the risk-benefit balance of immunization, which differs from that of their older counterparts. This change has the potential to be reflected in their immunization advocacy and affect parental attitudes.Despite the unparalleled success in improving both individual and population health through vaccination, there is evidence of increasing delay or refusal of some or all childhood vaccines with safety concerns commonly cited as a contributing factor [1,2,3,4,5,6].As vaccination programs succeed in achieving high coverage, the visibility of the disease itself is dramatically reduced [7]. As a result of lower disease prevalence, parents have more familiarity through both experience and media with real or perceived potential adverse events following immunizations than from vaccine preventable diseases. Parental benefit-risk assessments change as both cultural perception of the threat of disease and personal experience with disease decline and some parents come to see the risk of vaccines outweighing the benefits. As cohorts who have forgotten or not experienced vaccine-preventable disease come of childbearing age, their support of vaccines may be less than cohorts who grew up experiencing the effects of polio, measles, rubella, and other infectious diseases. Older mothers are more likely to have children with up-to-date immunizations compared with younger mothers [4,8]. Although there are a variety of individual, geographic, socioeconomic and other factors associated with vaccine uptake, there is evidence that maternal age (a potential proxy for changing parental perceptions) may be the most important factor determining whether a child is fully immunized. This “cohort effect” on vaccine related perceptions, may also affect health care providers’ vaccination beliefs and practices. Younger health care providers who have lived and trained in developed nations are likely to have had little or no personal experience with many vaccine preventable diseases and have been exposed to extensive public discussion of vaccine safety and alleged adverse events [9].Since health care providers are the most frequently used and trusted source for vaccine information, it is important to investigate a potential cohort effect. If health care providers have concerns regarding the safety or importance of immunizations for both individual and public health then they may affect their immunization practice and the counseling provided to parents and patients [10,11,12,13,14,15].We investigated the association of health care provider year of graduation from school with key beliefs about immunization and disease susceptibility, disease severity, vaccine safety, and vaccine efficacy.In a previous study [10], 1630 parents of fully immunized children and 815 parents of children who were exempt from at least one school immunization requirement were mailed surveys in 2004 to examine factors associated with parental vaccine refusal. The children were enrolled in elementary schools in Colorado, Massachusetts, Missouri, and Washington. Parents identified 806 unique primary health care providers who cared for their children at 2 and/or 5–6 years of age. Contact information could not be found for 94 of these providers (8%). Surveys covering vaccine knowledge, attitudes, and beliefs were mailed in 2005 to 712 of these parent-identified providers [16]. The Committees on Human Research at Johns Hopkins University approved this study.Providers answered questions on a 5-point Likert scale regarding vaccine-preventable disease susceptibility and severity and vaccine efficacy and safety (Table 1). Diseases queried included diphtheria, pertussis, tetanus, measles, mumps, rubella, polio, Haemophilus influenzae type b (Hib), varicella, hepatitis B, invasive pneumococcal disease and influenza. The survey also included questions about key immunization beliefs outlined in Table 1 with responses on a 5-point Likert scale and providers were asked, “In what year were you awarded your primary clinical degree?”Questions asked of providers for each of the diseases and vaccines listed.Beliefs regarding disease susceptibility, disease severity, vaccine safety, and vaccine efficacy were analyzed by individual diseases and vaccines and, from these, overall constructs were created. For each category, the responses were averaged across vaccines or diseases (each disease or vaccine weighted equally) to create four overall constructs on the same 5-point Likert-scale. These scores and values for key immunization beliefs were dichotomized 1 to <4 vs. ≥4. Responses of “don’t know” were counted as missing data and excluded from the analysis. Provider graduation year was categorized into 10 approximately five-year intervals: 1954–1959, 1960–1964, 1965–1969, 1970–1974, 1975–1979, 1980–1984, 1985–1989, 1990–1994, 1995–1999, 2000–2002. Due to low numbers in the boundary cohorts, the 1954–1959 cohort (eight health care providers) was combined with the 1960–1964 cohort and the 2000–2002 (two health care providers) was combined with the 1995–1999 cohort. We modeled graduation year in a multiple ways (continuous, quartiles, z-scores, decades, and 5-year intervals) with similar results.Providers were also categorized by patient vaccination status to explore this variable as a potential confounder or effect modifier as the provider sample was not representative of U.S. medical providers. Providers were classified as “non-exempt” if the only parents who identified them were parents of fully vaccinated children. Providers were classified as “exempt” if the only parents who identified them were parents of children exempt from at least one immunization requirement. Mixed providers were identified by at least one parent of an exempt child and one parent of a fully vaccinated child.Associations between provider graduation year and provider vaccine beliefs were explored using logistic regression. Provider vaccine beliefs were set as the dependent variable and provider graduation year interval as the independent variables to test for association between provider vaccine beliefs and graduation year. Analyses presented include the odds ratios adjusted by provider exemption status. Odds ratios are interpreted as the change in odds of a particular vaccine belief for each five-year increase in the year a provider graduated from clinical training (younger vs. older), adjusted for exemption status.Results were considered to be statistically significant if the p-values were ≤0.05. All analyses were conducted using Stata, version 10.Of the 712 provider surveys sent, 44 did not reach the provider due to death, retirement, or a closed practice, and 14 were sent to a non-health care provider resulting in 654 received surveys. Of the received surveys 103 providers declined to participate resulting in 551 valid surveys for an overall response rate of 84.3%. Nine providers who did not provide clinical training graduation year were excluded from the analysis leaving 542 unique providers. The mean and median provider graduation year was 1982 (standard deviation: 9.2 years; range: 1954–2002). There were 380 non-exempt providers, 86 exempt, 74 mixed, and 2 unidentified. Health care providers primarily were medical doctors (MD) (86%) but also included osteopathic doctors (OD) (7%), naturopathic doctors (ND) 2%, nurse practitioners (NP) (3%), registered nurses (RN) (1%), and a licensed practical nurse (<1%).Overall most health care providers had perceptions of high vaccine efficacy (89.4%) and high vaccine safety (92.7%). Lower percentages of providers believed in high disease susceptibility and severity (28.5% and 6.2%, respectively). Beginning with the cohort of providers graduating in the late 1980s there has been a slight downward trend among subsequent cohorts in beliefs in high vaccine safety (Figure 1) and high vaccine efficacy (Figure 2).Percentage of providers reporting high belief in vaccine efficacy by year of health professional graduation.Percentage of providers reporting high belief in vaccine safety by year of health professional graduation.Provider belief in overall vaccine efficacy was significantly associated with provider graduation year with graduates from a more recent 5-year interval having 15% decreased odds of believing vaccines are efficacious compared to providers from the preceding 5-year interval, adjusted for exemption status (OR: 0.85, 95% CI: 0.73–0.99) (Figure 3). Odds ratio of perceived belief for every 5-year interval increase in provider graduation year.Looking at each vaccine individually, there was not a strong effect on belief in vaccine safety by graduation year, although there was a trend towards younger providers having lower belief in vaccine safety. For both types of polio vaccines (IPV and OPV) there were significant decreased odds of believing the vaccine was safe for recent compared to older graduates. Provider belief in vaccine-preventable disease susceptibility was not associated with provider graduation year both overall (OR: 0.94, 95% CI: 0.78–1.13) and for specific diseases (Figure 3). For overall perceived disease severity there was also no association with provider graduation year (OR: 1.05, 95% CI: 0.95–1.17). The direction of association was not consistent among individual diseases. Diphtheria, tetanus, and pertussis were perceived as having lower severity for more recent compared to more experienced health care providers. Conversely, mumps and rubella were perceived as having higher severity for more recent vs. more senior health care providers. In the analysis of specific key immunization beliefs, overall, more recent graduates had greater odds of holding beliefs less favorable to immunization compared to more distant graduates (Table 2). For every 5-year increase in graduation year, there was 24% reduced odds of believing that immunizations are one of the safest forms of medicine ever developed. Recent graduates also had 1.4 times higher odds of believing that immunizations do more harm than good compared to older providers. The one exception to this trend was for the belief that vaccines strengthen the immune system. While the association was small, there was a statistically significant 10% increased odds that more recent graduates will believe that vaccines strengthen the immune system compared to more experienced graduates in the previous 5-year cohort. In our study, a strong majority of health care providers believe that vaccines are highly safe and efficacious, similar to findings from other studies [17,18,19]. However, the proportion believing vaccines are safe and efficacious is lower for those clinicians who have graduated in recent decades. Only a small proportion of health care providers held beliefs of high disease severity and disease susceptibility, and a small minority (3.7%) of recent provider graduates believed that immunizations do more harm than good, likely reflecting the overall downward trend in prevalence of and mortality from vaccine preventable disease in the U.S. [17,18,19]. This changing perception of the risk-benefit balance of immunization may signal a critical change in immunization beliefs in the new generation of providers compared to their older counterparts. Previous studies have shown that parents and providers may be hesitant to vaccinate primarily due to safety concerns. Our original hypothesis was that vaccine safety beliefs would have the strongest association with provider graduation year [10,20,21]. However, vaccine efficacy beliefs exhibited the strongest association with graduation year followed by beliefs about vaccine safety. Our overall analysis showed this skepticism in more recent health care provider graduates for the majority of our key immunization beliefs. In the stratified analysis we saw the strongest effect of graduation year among the non-exempt providers, a subtle or non-effect among mixed providers and no association for exempt providers. One explanation for this finding is that providers who are caring for exempt children are fundamentally different than providers caring for fully vaccinated children. As a result, graduation year does not play a significant role because these providers have already come to a conclusion on the benefits and risks of immunizations.Relationship between provider graduation year and key immunization beliefs (Adjusted for Exemption Status). a Odds Ratio; b 95% Confidence Interval; c Interpretation: For each five year increase in the year a provider graduates from health professional school there is a 24 percent decreased odds that he or she believes that immunizations are one of the safest forms of medicine ever developed, adjusted for exemption status; d Statistically significant results indicated in bold.Previous findings on a cohort effect among providers are mixed. Gust and colleagues showed no difference in proportion of physicians who recommend all vaccines by age category (≤36, 37–42, ≥42 years) [22]. However, by using provider age rather than graduation date differences in educational cohorts may have been blurred. Taylor et al. found no correlation between practice immunization rates for children at 8 and 19 months of age and percentage of practitioners born before 1950 but this is a rough division that is now likely outdated [23]. Koepke et al. grouped children by years their provider had been in practice (<11 vs. ≥11 years) and then looked at the percentage who were up-to-date on vaccines [24]. While not statistically significant, providers who had been in practice greater than 10 years had a higher percentage of children with up-to-date vaccinations compared to those who had been in practice for 10 years or less. The above studies demonstrated interesting trends towards association of provider age and medical cohort with vaccination beliefs, however the analysis done in these studies was not intended to focus specifically on a cohort effect. Our study adds to this body of literature by examining immunization beliefs for specific vaccines and diseases and by more specific provider graduation year intervals.An interesting finding of the study was the differential effect of provider graduation year on perceptions of vaccine vs. disease constructs. While we found a consistent effect of graduation year on vaccine safety and efficacy beliefs, we found a smaller and more nuanced effect on disease susceptibility and severity beliefs. This may indicate that the changing assessment of vaccine benefit and risk by graduation cohort directly related to perceptions of the vaccines themselves rather than to a changing perception of the risk of severity of disease. Similarly it may indicate that with such a low burden of disease, the efficacy and real or perceived side effect of vaccines may be the most significant factors contributing to vaccination beliefs [25].One policy implication of these findings is that provider’s knowledge of disease risk, complications, vaccine efficacy, and safety should be explored to understand if the new provider assessment reflects knowledge gaps or different weighting of the risks of disease and the benefits and risks of vaccines. Similarly parent’s perception of the risks of vaccine preventable disease relative to the other perceived health risks of their children warrants exploration.In addition, the values of providers with respect to the balance between individual autonomy in medical decision making and protecting public health should be explored to fully understand the nuances in immunization related beliefs revealed by this study. Health care providers are one of the most trusted sources for vaccine information and if they have concerns about vaccines it may influence the vaccination status of their patients [10,22,26]. Continued efforts are need to ensure that health care providers fully understand the risks of vaccine preventable disease, the benefits and risks of immunization for the individual child and benefits of immunization to public health. A limitation of this study was that we did not have information on health care provider’s precise age but instead used health professional graduation year as a proxy for age. However, it is likely that providers within each 5-year graduation cohort will have roughly approximate ages compared to providers in other graduation cohorts. Moreover, the expected cohort effect is reflected more by healthcare experience and time period of health profession education than by the provider’s age. Another limitation was lack of sufficient sample size so non-significant findings may be the result of actual non-significance or of a lack of sample size to detect the true association. This study had a cross-sectional design and therefore we cannot assume causal directional relationships between provider’s graduation year and vaccine-related beliefs. However, it is unlikely that vaccine-related beliefs influence a provider’s graduation year. The provider survey was nested within a larger case-control study design and therefore the providers were not representative of all U.S. health care providers but rather provide a spectrum of providers caring for both vaccine exempt and non-exempt children. Although other data supports the overlap of attitudes and exemptions, we do not have data on differences in vaccination coverage by health care provider age and therefore more in-depth research is warranted. Further, we adjusted for provider exemption status based on categorization using a small subset of their patients. This limited provider patient sample size may provide an inaccurate representation of their entire practice. Finally, the study was conducted in 2005 so the association between provider vaccine beliefs and graduation year may have changed for younger health care providers. These findings demonstrated that the most salient difference between recently graduated vs. senior health care providers was that younger providers have lower belief in vaccine efficacy and safety although association was most pronounced for efficacy. More recently graduated health care providers also had higher odds compared to older providers of holding key immunization beliefs supporting the idea that vaccines do more harm than good. These results likely reflect both decreasing prevalence of vaccine preventable diseases and increasing awareness of vaccine adverse effects. Vaccine-related medical curricula should emphasize the importance, effectiveness, and safety of childhood immunizations. Further investigation is needed to determine the impact of this cohort effect on immunization rates overall.This study received support from CDC grant #U01IP000032-02.The authors have no financial relationships or conflicts of interest to disclose with the exception of Neal Halsey and James Taylor. Neal Halsey has received a research grant without salary support from Merck through Johns Hopkins University for the study of human papillomavirus vaccine in sex workers in Peru. He received research grants from Crucell and Intercell for studies of vaccines in Guatemala and he serves on safety monitoring boards for Merck and Novartis. James Taylor was a paid consultant by Pfizer for a one-time meeting to discuss methods for improving immunizations rates.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Hepatitis B remains a significant health issue worldwide, and contributes significantly to the incidence of cirrhosis and hepatocellular carcinoma. Widespread adoption of hepatitis B vaccination strategies has lead to significant declines in acute hepatitis B infections. Current recommendations for vaccination in the non-pregnant population include vaccinating all persons found to have risk-factors for disease acquisition. Hepatitis B virus is known to occur through vertical transmission or early childhood transmission, and strategies to decrease transmission include avoidance of exposure, avoidance of high-risk behaviors, universal screening of women during pregnancy, and active and passive immunization. It is currently recommended that all pregnant women undergo screening for hepatitis B virus at presentation for prenatal care. Those who engage in high-risk behavior should be re-screened at presentation for delivery. Studies have demonstrated the safety and efficacy of the hepatitis B vaccine in pregnancy, and its use is an important component in prevention of disease acquisition. Pregnant women in the United States who are found to be at risk for disease acquisition should be specifically targeted for vaccination.Hepatitis B virus (HBV) is a double-stranded DNA virus of the Hepadnaviridae family. HBV causes infection worldwide and is endemic in Eastern Europe, the Middle East, Africa, Central and Southeast Asia, China and certain areas of South America with prevalence rates as high as 5–20%. The World Health Organization estimates that greater than two billion people worldwide are infected with HBV; 360 million have chronic infection and are at high risk for hepatocellular carcinoma and cirrhosis of the liver [1]. Approximately 600,000 deaths occur worldwide from HBV-related diseases [2]. In 2010, the Centers for Disease Control and Prevention estimated that 35,000 acute hepatitis B infections occurred in the United States, a 29 percent decrease from 2006 [3]. This decline, mirrored in most developed countries, is attributed to the widespread adoption of hepatitis B vaccination strategies.Hepatitis B virus is transmitted by percutaneous or mucosal exposure to the blood or body fluids of infected individuals. In countries where HBV is endemic, vertical transmission from an infected mother to child peripartum or person-to-person transmission in early childhood is most common. In low prevalence countries, HBV transmission occurs more commonly via sexual transmission or sharing contaminated needles, though vertical transmission does occur. The majority of adults who become infected with HBV will eliminate the virus—only 2–8% will develop chronic hepatitis B. The converse is true for younger age groups. Chronic HBV infection develops in 80–90% of infants infected perinatally and 30–50% of children infected before the age of six [4,5].Maternal hepatitis B infection during pregnancy does not increase maternal morbidity and mortality; in fact, it is often asymptomatic and found only on routine prenatal screening. In the absence of HBV immunoprophylaxis, 10–20% of women positive for hepatitis B surface antigen (HBsAg) transmit to their infant—this increases to almost 90% if the mother is seropositive for HBsAg and hepatitis Be antigen (HBeAg) or if she develops acute HBV in the third trimester. This transmission risk decreases dramatically in the setting of universal HBV screening prenatally, immunoprophylaxis given to infants born to HBV infected mothers and, finally, hepatitis B vaccine administered both to high risk mothers and to all newborn infants.In 1970, Krugman, Giles, and Hammond first performed active immunization against HBV by using heated infective human serum. This serum was found to prevent or modify hepatitis B in 69% of children who were challenged [6]. Following this, several vaccines were developed and extensively tested. Initial placebo-controlled studies of an inactivated hepatitis B vaccine were conducted in men who have sex with men (MSM), due to the historically high attack rates of hepatitis B in this group. These studies demonstrated that immunization reduced the incidence of hepatitis B among this high-risk group by 90–95% [7]. This inactivated vaccine was similarly found to protect health care workers with frequent exposure to blood [8]. In 1982, the first hepatitis B vaccine was licensed in the United States. It was a subunit vaccine that contained 22-nm HBsAg particles that were made from the plasma of chronic HBsAg carriers. Contemporary vaccines use HBsAg that is produced by recombinant DNA technology [9]. Currently two single antigen recombinant vaccines are available in the United States: Recombivax HB (Merck) and Engerix-B (GlaxoSmithKline Biologicals). A combination vaccine, Twinrix (GlaxoSmithKline), containing antigens to both hepatitis A and hepatitis B is also available, and is recommended for persons ≥18 years of age who are at risk for both hepatitis A and hepatitis B infections. Two combination vaccines are also available for use in children: Comvax (Merck), a combined hepatitis B and Haemophilus influenzae type b (Hib) conjugate vaccine, and Pediarix (GlaxoSmithKline), a combined hepatitis B, diphtheria, tetanus, acellular pertussis (DTaP), and inactivated poliovirus (IPV) vaccine.Since 1982, a comprehensive strategy for the elimination of hepatitis B in the United States has evolved. The original strategy simply targeted high-risk groups for transmission. This was subsequently shown to have a suboptimal impact on the incidence of the disease [10]. The current strategy against HBV infection includes universal vaccination of infants beginning at birth, prevention of perinatal HBV infection, routine vaccination of previously unvaccinated children and adolescents, and vaccination of previously unvaccinated adults at risk for disease acquisition [9]. In 2006, the Advisory Committee on Immunization Practices updated recommendations to increase hepatitis B vaccination coverage among adults [9,11]. Current recommendations include vaccination of: all infants beginning at birth, all children <19 years of age who have not previously been vaccinated, sexual partners of HBsAg positive persons, sexually active persons who are not in a long-term, mutually monogamous relationship, persons seeking evaluation or treatment for sexually transmitted diseases, men who have sex with men, injection drug users, susceptible household contacts of HBsAg positive persons, health care and public safety workers at risk for exposure to blood or blood-contaminated body fluids, persons with end-stage renal disease, residents and staff of facilities for developmentally disabled persons, travelers to regions with intermediate or high rates of endemic HBV, persons with chronic liver disease, persons with HIV infection, unvaccinated persons with diabetes mellitus age 19 to 59, and all other persons seeking protection from HBV [11]. Considering the last indication to receive vaccination, no specific risk factor must be identified for a person to receive vaccination. Current recommendations also include guidelines for universal vaccination in certain health care facilities known to care for a high proportion of hepatitis B infected persons. These facilities include: sexually transmitted disease treatment facilities, HIV testing and treatment facilities, drug abuse treatment and prevention facilities, health care settings targeting injection drug users, correctional facilities, facilities targeting services to men who have sex with men, chronic hemodialysis facilities, and institutions and nonresidential day care facilities for developmentally disabled persons [11]. Hepatitis B vaccination consists of three intramuscular injections in the deltoid muscle. The three-dose vaccine is administered at 0, 1, and 6 months. According to the ACIP, the minimum interval between the first and second dose is four weeks, and between the first and third dose of 16 weeks, in order to obtain an optimal immune response [11]. Approximately 30–55% percent of healthy adults ≤40 years of age will have a protective antibody response after the first dose, 75% after the second dose, and >90% after the third dose [11]. Accelerated vaccine schedules at 0, 1, and 4 months or 0, 2, and four months, have also been shown to produce similar rates of seroprotection [12]. Serologic testing for immunity, regardless of the vaccination schedule chosen, is not required after routine vaccination. However, it may be recommended in situations in which knowledge of immune status may affect medical management [11].Pregnancy is a unique circumstance in which screening and intervention can be utilized to impact newborn health. Similarly, pregnancy may be a time in which previously unvaccinated women present to the healthcare system, and therefore provides an opportunity for intervention on behalf of the health of the mother. To this end, the American College of Obstetricians and Gynecologists (ACOG) as well as the Centers for Disease Control and Prevention (CDC) recommend prenatal screening of all pregnant women for HBV [9,13]. Pregnant women should have a HBsAg drawn at presentation for prenatal care. Furthermore, pregnant women who undertake high-risk behavior for disease acquisition or who are not previously screened should undergo HBsAg screening when she presents for delivery. According to ACOG, pregnant women who are HBsAg negative and who are at risk for HBV infection should be specifically targeted for vaccination [9,13]. High risk women include having more than one sex partner during the previous six months, been evaluated or treated for a sexually transmitted infection, recent or recurrent injection drug use, or having an HBsAg positive sexual partner [9]. Women who are found to require vaccination for hepatitis A, based on current recommendations, as well as hepatitis B may receive a combination vaccine (Twinrix), which contains antigens to both hepatitis A and hepatitis B [13].There is currently limited data on the use of hepatitis B vaccine in pregnancy. That being said, the American College of Obstetricians and Gynecologists, as well as the Centers for Disease Control and Prevention do not consider pregnancy a contraindication [9,13]. In fact, as mentioned above, vaccination is recommended in pregnancy in specific circumstances. The efficacy of the hepatitis B vaccine in pregnancy has been shown to be similar to the non-pregnant population [14,15]. Grosheide and colleagues examined the seroprotection conversion rates of 16 pregnant women compared to 57 non-pregnant women when the hepatitis B vaccine was given for post-exposure prophylaxis. After six months, all women who received the vaccine had protective anti-HBs levels [14]. Overall, seroconversion rates of 92–94% have been demonstrated in pregnant women [15]. Factors shown to decrease the efficacy of the hepatitis B vaccine in pregnancy include maternal obesity, advancing age, and tobacco smoking [16,17]. Despite this, the vaccine should be provided to all pregnant women at risk for disease acquisition during pregnancy.The safety of the hepatitis B vaccine has been demonstrated in multiple studies [14,15,18,19]. In one cohort, the infants of ten women who received the plasma derived hepatitis B vaccine during the first trimester of pregnancy were followed. No congenital abnormalities were identified at delivery, and all children were physically and developmentally normal at two and 12 months [19]. Studies that have evaluated the safety of the recombinant vaccine have similarly determined that the vaccine is safe in pregnancy [14,15]. In a cohort of 16 women exposed to recombinant vaccine after in vitro fertilization, one woman had a miscarriage two days after vaccination, and one was lost to follow-up. The remaining 14 women subsequently delivered 19 healthy infants, all of who were developmentally normal at 22 months [15]. A critical component of hepatitis B management in pregnancy is proper immunoprophylaxis being provided to the infant after birth. Currently, both ACOG and the CDC recommend that all infants receive the hepatitis B vaccine series as part of the recommended childhood immunization schedule [9,13]. Infants born to mothers who are known to be carriers of hepatitis B, or whose status is unknown, should receive both the hepatitis B vaccine series, as well as passive prophylaxis with hepatitis B immune globulin (HBIG) [9,13]. The current recommended vaccination schedule in the non-pregnant population includes vaccination with the three doses of the recombinant hepatitis B vaccine at 0, 1, and 6 months. Recent studies have demonstrated that accelerated vaccination schedules of 0, 1, and 4 months are as immunogenic as the standard dosing schedule [12]. Within the pregnant population, the traditional vaccination schedule of 0, 1, and 6 months is difficult to complete in the limited time of gestation prior to delivery. After delivery, it is expected that compliance with completion of the vaccination schedule will decrease. It would therefore be ideal to complete the vaccination schedule prior to delivery, while the woman is continuing to receive regular prenatal care. In a cohort of 200 pregnant women, the recombinant hepatitis B vaccine was given at an accelerated schedule of 0, 1, and 4 months [17]. Of the 200 women enrolled, 84% completed the 3-dose vaccine schedule. After three doses, 90% of women demonstrated seroconversion, rates similar to those demonstrated in the non-pregnant population undergoing the traditional schedule of vaccination. This accelerated vaccination schedule has been shown to be an effective means of completing hepatitis B vaccination during the course of pregnancy [17]. Serologic testing for immunity is not required following this accelerated schedule of vaccination. This provides another effective tool in decreasing HBV infection.When discussing the issue of hepatitis B vaccination in pregnancy, maternal acceptance of the vaccine series must be addressed, regardless of the vaccine schedule recommended. In a recent Chinese survey of pregnant women, maternal uptake of hepatitis B vaccine was found to be 33% [20]. The factors associated with maternal acceptance of the vaccine included employment as a healthcare worker, higher education, higher family income, routine medical checkups, and premarital checkups. Based on this, they concluded that the public lacked sufficient knowledge of hepatitis B infection. This was corroborated in a follow-up study in which a questionnaire regarding hepatitis B infection was administered to pregnant Chinese women, and indicated that misconceptions regarding the virus remained prevalent among that population [21]. Interestingly, 87.4% of the 1,623 respondents to this survey correctly answered that hepatitis B infection can be prevented by screening and vaccination, implying that confidence in the vaccine is not a major deterrent to receiving the vaccine. It is clear that maternal knowledge regarding the virus itself is an essential component of vaccine uptake. According to the CDC, implementation strategies for the hepatitis B vaccine include providing information to all adults regarding the health benefits of the hepatitis B vaccination [11]. Pregnant women for whom the hepatitis B vaccine is recommended should equally be educated on the importance of receiving the vaccination, including the benefits to both maternal and infant health.Hepatitis B virus infection is a world-wide public health crisis, both acutely and more importantly, from sequelae in chronically infected individuals. Transmission occurs via vertical transmission or early childhood infection in the majority of cases in countries where HBV is endemic. Effective strategies to decrease this transmission risk include prevention of blood and body fluid exposure, avoidance of high-risk behaviors, universal screening of women during pregnancy, and active and passive immunization. The hepatitis B vaccine has been shown to be both safe and effective in pregnant women, yet is not routinely given to high-risk women in most countries, including developed nations. Education and vaccine availability need to be improved and further investigation into booster dosing and the effects of different vaccine formulations and intervals needs to be encouraged.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Changes in cell surface glycosylation are a hallmark of the transition from normal to inflamed and neoplastic tissue. Tumor-associated carbohydrate antigens (TACAs) challenge our understanding of immune tolerance, while functioning as immune targets that bridge innate immune surveillance and adaptive antitumor immunity in clinical applications. T-cells, being a part of the adaptive immune response, are the most popular component of the immune system considered for targeting tumor cells. However, for TACAs, T-cells take a back seat to antibodies and natural killer cells as first-line innate defense mechanisms. Here, we briefly highlight the rationale associated with the relative importance of the immune surveillance machinery that might be applicable for developing therapeutics. A basic premise underlying immune modalities for cancer is that the immune system can mount a rejection strength response against neoplastically transformed cells [1]. Tumor targeting draws upon two immunological mediated paradigms. The first draws upon concepts of immune surveillance that bridges both innate and adaptive immunity. According to the immune surveillance hypothesis, tumor associated antigens are regarded as “non-self” by the immune system, and a major function of the immune system is to survey the body for the development of malignancy and to eliminate tumor cells as they arise [2]. Innate immunity relies on biochemical and cellular defense mechanisms often observed in the early phases of encounter with microbes. The cellular players include natural killer (NK) cells, dendritic cells (DCs), macrophages, monocytes, γδ T-cells and natural killer T (NKT)-cells. Adaptive immunity involves the expansion of T-cells and B-cells and their humoral and cellular mediators, cytokines and antibodies. In particular, antibodies and NK cells are early participants in the immune response and are particularly effective in eliminating blood-borne metastases [3]. In contrast, T-cells are the effector cells responsible for specific, long-lasting immunity.The second draws upon concepts associated with tissue-specific destruction in the context of acute allograft (acute) rejection, flares of autoimmunity and response to acute infection. This second paradigm requires an understanding of the distinct difference between an anti-tumor immune response and outright tumor rejection. In this context, immune-mediated cancer rejection is a facet of autoimmunity, where the target tissue is the cancer itself. The induction of immune-mediated tumor tissue rejection represents an important conceptual approach to cancer immunotherapy and also remains an important goal in tumor immunology [4,5]. Antigens that function as tumor rejection antigens are considered self, nearly self or non-self [6]. The fact that a tumor antigen elicits a tumor-specific immune response does not necessarily mean that the immune response will cause the rejection of the tumor in vivo. The question remains as to which tumor antigen can or is better at inducing a clinically beneficial response [7]. Tumor-rejection antigen is therefore an operational term describing how well an immune response elicited against a tumor antigen will impact on tumor growth. Tumor antigens can be poor, intermediate or strong tumor rejection antigens, describing quantitatively the impact of the immune response on tumor growth [6].Among potential tumor rejection antigens are glycans expressed on glycoproteins and glycolipids. Aberrant glycosylation is a universal feature of cancer cells with some tumor-associated carbohydrate antigens (TACAs) considered tumor progression markers. A considerable body of evidence put TACAs amongst the most challenging of clinical targets for cancer immunotherapy [8,9], yet immune responses to glycans are noted that could lend to therapeutic strategies and approaches (Figure 1). TACA expression on cancer cells is associated with organ tropism underlying extravasation and metastases, because of glycan receptors on organ tissues [10] or their role in survival. A requisite for metastases is cell survival. Anoikis resistance or survival in the absence of attachment to extracellular matrix (ECM) is a prerequisite for the development of tumor metastases [11,12]. Anoikis resistance has evoked special attention in cancer research because circulating tumor cells in the blood stream are resistant to it. Signaling cascades are intimately interconnected with TACA expression and interaction with the microenvironment. TACAs can regulate the interaction between integrin and Focal Adhesion Kinase (FAK), for example, which, in turn, regulates cancer cell adhesion and invasion [13,14,15,16,17,18,19,20]. Many of the targeted TACAs are found on structures upstream of FAK that can modulate the signaling through FAK [14,17,19,20,21], whereby anti-TACA antibodies might reset anoikis of tumor cells. Glycans are considered as priming agents for T-cells and for B-cells working in concert [22,23,24]. Natural antibodies and induced antibodies can mediate tumor cell killing and tissue destruction by several mechanisms that include complement-dependent cytotoxicity (CDC) [25], antibody-dependent cellular cytotoxicity (ADCC) [26] and through signal transduction pathways, leading to anti-proliferative activity or apoptosis [27]. Antibodies to TACAs have other attributes, such as negating negative signals to immune cells by forming immune complexes with shed TACAs or by blocking attachment of tumor cells to microenvironment constituents. Remodeling the glycan surface of tumor cells either by bio-engineering approaches to facilitate antigen uptake to improve tumor cell immunogenicity [28,29,30] or through inhibitors that affect glycosylation in general may exacerbate the action of antibodies and NK cells reactive with glycan signatures.Immune surveillance targeting of tumor-associated carbohydrate antigens (TACA) allows both attack on tumor cells and interference with the tumor-generated immunosuppressive factors. Differentially expressed glycans mediate tumor cell dissemination and organ tropism. Carbohydrate antigens are bound by natural antibodies, thymus independent B-cell response generated antibodies and, rarely, by thymus-dependent responses. NK cells and CD1-dependent T-cells are also involved. A wide variety of antibody mediated effector mechanisms are at play—complement- and antibody-dependent cytotoxicity, direct proapoptotic effect, interrupting immunosuppressive signaling, migration, extravasation and organ tropism.Much of what we know about immune responses to carbohydrates stem from examining immune responses to microbes and developing pathogen-based vaccines. The success of carbohydrate-conjugate vaccines in anti-microbial strategies has fueled expectations for their success as cancer vaccines, because the pathophysiological process of infection and neoplasia are profoundly affected by similar or the same carbohydrate forms. Some worm glycan antigens, for example, share structural features with host-like glycans and TACAs, including Le(X) (Galbeta1-4[Fucalpha1-3]GlcNAc-), LDNF (GalNAcbeta1-4[Fucalpha1-3]GlcNAc-), LDN (GalNAcbeta1-4GlcNAc-) and Tn (GalNAcalpha1-O-Thr/Ser). Anti-glycan antibody responses are a prominent feature of the immune response, for example, in patients infected with schistosomiasis that display the LeX, LDNF and LDN glycans. It is well known that helminths have immunomodulatory effects on their hosts. They characteristically cause a skew toward TH2 immunity and stimulate Treg cells, while simultaneously inhibiting TH1 and TH17 responses. Carbohydrate antigens can directly stimulate NK cells, without previous antigen sensitization or MHC restriction, to initiate lysis and to produce IFN-γ. Costimulatory signals provided by NK cells, together with the effects of NK cell-derived IFN-γ on B-cell differentiation, isotype switching and immunoglobulin secretion, ultimately result in augmentation of the IgG humoral response against T-cell-independent antigens. In this mini-review, we place into context the selected roles of TACAs reactive immune surveillance. In particular, we focus on glycan-mediated phenomena associated with tissue rejection as a model to understand the rationale of controlling of tumor cell growth by some immune modalities that target TACAs. The rationale for targeting TACAs was elegantly discussed in terms of tissue distribution and therapeutic importance [31]. The transition in glycosylation patterns of cancer cells reflect a myriad of processes that correlate with poor prognosis of cancer, affecting cell signaling and communication, cell motility and adhesion, angiogenesis and organ tropism. Both simple glycan structures and more complex TACAs play a role in these processes. Glycan structures on the tumor cell surface result from the combined action of glycotransferases and glycosidases. The carbohydrate antigens that have been found to be tumor-associated (Table 1) include the mucin related Tn, sialyl Tn and Thomsen-Friedenreich (TF/T) antigens, the blood group Lewis-related Lewis(Y), Sialyl Lewis(X) (SLeX) and Sialyl Lewis(A) (SLeA), and Lewis(X) (also known as stage-specific embryonic antigen-1, SSEA-1), the glycosphingolipids, Globo H, and stage-specific embryonic antigen-3 (SSEA-3), the sialic acid containing glycosphingolipids, the gangliosides, GD2, GD3, GM2, fucosyl GM1 and Neu5GcGM3 and polysialic acid. SLeX and SLeA, in particular, are carbohydrate molecules that mediate the adhesion between tumor cells and the endothelium. Overexpression of SLeX and SLeA is combined with poor prognosis and malignant relapse [32]. The interaction of the antigen SLeX on tumor cells and E-selectin on endothelial cells was shown to mediate adhesion of tumor cells to endothelial cells [33], possibly facilitating tumor cell invasion in blood microvessels, extravasation and migration into tissue. Additionally, colorectal tumor cells expressing SLeX might prefer the liver to form clinically evident metastases, due to interaction with local E-selectin [34]. SLeX expression and lymphatic microvessel density in primary tumors might predict disease recurrence, suggesting that for some cancer, both lymphatic and hematogenous metastasis is mediated by SLeX interactions [35,36].Common carbohydrate antigens targets from tumor biopsy specimens. Many cell-cell contacts are mediated by cell-surface glycans that are redundant on membrane constituents that effect signaling pathways associated with anchorage independent growth and anoikis. Upregulation of the N-glycan branching enzyme β-1,6-N-acetylglucosaminyltransferase V reduces cell-cell interactions within a tumor, promoting cell detachment and invasion of tumor cells [37]. Also, the expression of O-glycans containing an N-acetylglucosamine branch connected to N-acetylgalactosamine (GlcNAcβ1-6GalNAc), which is designated the core 2 branch, is closely correlated with highly metastatic phenotypes of several tumor types [36,38,39]. Core 2 O-glycans expressed on the cell surface can reduce cell-cell interactions [40]. Mucin-type O-glycans containing Core2 branches have distinctly different functions from those O-glycans that contain Core1 structures. Core2 branched O-glycans can have terminal structures that function as ligands for carbohydrate binding proteins [41]. Sialylated Core2 branched O-glycans without additional modifications exhibit anti-adhesive properties, which might be related to anoikis resistance. These results demonstrate that certain mucin-type O-glycans can either facilitate or attenuate cell adhesion to ECM components and lectin proteins, depending on the core structures and the structures of the non-reducing termini. Several studies revealing the role of core 2 O-glycans in immune responses in general show that core 2 expressions is a biologically significant change [42]. Furthermore, the core 2 O-glycan is a key backbone structure in forming some selectin ligands. β-1,6-N-acetylglucosaminyltransferase (C2GnT), expressed in cancer cells, may play important roles in tumor progression through circulatory system or direct invasion [36], since some of these structures inhibit NK cell activation [43,44,45,46]. Thus, O-linked oligosaccharides, in particular those containing core 2 branches, play vital roles in immune responses and may play dual roles in certain situations [40,47]. Although the effectiveness of some of vaccines targeting TACA has been demonstrated in a number of experimental model systems and suggested in several clinical trials, the mechanism underlying their mode of action is uncertain. The distinction between glycans expressed on glycoproteins or glycolipids can translate to differences in how the immune system generates responses [25]. Previous studies showed that the targets for effective CDC were glycolipids (e.g., GM2, GD2, GD3, fucosyl GM1, Globo H or LeY), whereas those in which no lysis was observed were carbohydrate (e.g., TF, Tn, sTn) or peptide (e.g., MUC1) epitopes carried by mucin molecules [25]. It is observed that some TACAs responsible for CDC are expressed on glycoproteins and glycolipids, such as Globo H and LeY. MUC1, Tn, sTn and TF, in contrast, are not expressed on glycolipids, making the distinction clear. It may be that other antibody-mediated mechanisms, such as ADCC, opsonization of tumor cells by leukocytes, induction of apoptosis and blocking of tumor cell invasion or metastasis also differ, depending on the biochemical and biophysical nature of the targeted antigen. Monoclonal antibodies [26] and human serum antibodies from MUC1 immunized subjects [48] can mediate ADCC of human cancer cells. Since tumor tissue rejection is the goal of cancer immunotherapies, broad-spectrum tumor associated antigens, like TACAs, are plausible targets once the problem of their low immunogenicity is solved. The fact that multiple proteins and lipids on the cancer cell are modified with the same carbohydrate structure creates a powerful advantage for TACAs as cancer targets in immunotherapy strategies. Thus, targeting TACAs has the potential to broaden the spectrum of target pathways recognized by the immune response, thereby lowering the risk of developing escape variants, due to the loss of a given protein or carbohydrate antigen. There is an emerging awareness that immune surveillance mechanisms that include antibodies and effector cells are intimately related to TACA reactivity that provides a template for developing strategies for cancer immunotherapy, because of the display of glycans in the context of pattern recognition [49,50,51,52]. Glycans can be clustered representing danger signals to the immune system. Pattern recognition receptors (PRRs) are sentinels of innate immunity that instruct adaptive immunity mechanisms by which long-lived lymphocyte responses are targeted to appropriate antigens [53,54]. Innate immune cells have evolved to sense microbial pathogens through PRRs, coupling pathogen recognition to innate immunity through glycan-dependent mechanisms [55]. The same mechanisms might be operative for glycans expressed on cancer cells. Natural carbohydrate reactive antibodies have been described that mediate tumor cell apoptosis in addition to modulating complement associated cell killing [27,56,57]. Preclinical studies support the hypothesis that antibody-induced responses against TACAs might have their greatest impact in the adjuvant setting, as antibody responses inhibit tumor outgrowth in metastatic models [58]. Such observations suggest that sustained immunity against TACAs should be beneficial to prevent the recurrence of disease, much like the natural ways of immune surveillance. Therefore, maximizing sustained TACA-specific humoral immunity is considered an important goal in developing effective antibody-based immunotherapies against cancer. Like antibodies, NK cells are partners in immune surveillance. Three predominant superfamilies of NK cell receptors (NKR) have been identified that can either inhibit or activate NK cell function: (i) killer immunoglobulin (Ig)-like receptors (KIR) that bind to classical class I MHC molecules; (ii) C-type lectin receptors that bind to non-classical class I MHC molecules or “class I-like” molecules; and (iii) natural cytotoxicity receptors for which ligands are currently not well defined (except for NKp30 binding to B7-H6 and BAG6) [59]. Interestingly, it is possible that some of the natural cytotoxicity receptors may be binding to glycolipids [60]. NK cells can directly lyse virally infected cells and tumor cells without prior sensitization and provide immunoregulatory cytokines that shape the adaptive immune response. Cytolytic signals, triggered by inhibitory and activation receptors on the cancer cell surface, regulate NK cell-mediated cytotoxicity and the production of chemokines and inflammatory cytokines that mediate the immune response. The expression of some TACAs lend to the evasion of NK cell immunity [43,44], while others activate NK cells [61]. Therefore, aberrant glycosylation, while a target for immune surveillance, can regulate negative signaling of NK cells. The eradication of xenografts has been suggested as a model to provide important insights about the role played by immunity in mediating tumor tissue rejection [5]. Tissue destruction occurs with resolution of pathogenic processes (cancer, infection) or tissue damage and organ failure (autoimmunity, allograft rejection) [5]. While xenograft rejection is highly mediated through innate immune mechanisms, in tumor immunology, the primary focus for tumor tissue-rejection is focused on effector cell-mediated tumor rejection and, particularly, the definition of cognate T-cell subsets that define signatures for tumor cell rejection. Nevertheless, underlying the various mechanisms associated with the biology of tissue damage or rejection emerges as a common pattern in tumor tissue-specific destruction relevant to TACA targeting [5]. These patterns were elegantly reviewed by Marincola and colleagues [5], which include the postulates that: (1) Tissue-specific destruction does not necessarily only occur after non-self-recognition, but can also occur against self-or quasi-self-antigens. In the context of tumor targets, TACA reactive antibodies are constantly produced, being inherent in the innate and adaptive immunity. (2) The requirements for the induction of a cognate immune response differ from those associated with the development of its effector phase. Natural circulating anti-TACA antibodies are present and are known to be apoptotic to tumor cells. Therefore, antibodies can function as both judge and jury. (3) Although the mechanisms prompting tissue-specific destruction differ among immune pathologies, the effector phase converges into a common activation of adaptive and innate cytotoxic mechanisms. In this context, glycan-reactive T-cells might work in unison with NK cells and antibodies to target tumor cells. Furthermore, (4) adaptive immunity triggers a tissue-specific reaction, but it is not always sufficient or necessary for tissue destruction. Carbohydrate-reactive antibodies bind to both normal tissue and cancer cells. The binding to normal tissues does not necessarily lend to normal tissue destruction, but may facilitate microenvironment interactions that lend to tumor tissue rejection. Indeed, immune-based therapies have the potential to modulate the tumor microenvironment by eliciting immune system cells that will initiate acute inflammation that leads to tissue destruction [62]. Antibodies can mediate tissue rejection that validates targeting TACAs. A model for glycans as tissue rejection antigens includes the response to the xeno-carbohydrate antigen Galα1-3Galβ1-4GlcNAc-R (alpha-Gal) epitope. The majority of alpha-Gal antigens are built upon the Gal1, 4GlcNAc (type 2) chain, but other inner-core saccharide chains also exist, especially on glycolipids [63,64,65]. Naturally occurring anti-Gal antibody is produced as the most abundant antibody (1% of immunoglobulins) throughout the life of all individuals [66]. Natural antibodies, such as anti-Gal or anti-blood groups A/B antibodies, mediate hyperacute graft rejection and, thus, represent a major hurdle in xenotransplantation [67] and blood transfusions, respectively. In the initial stage of the rejection, anti-Gal IgG binds to R-Gal epitopes expressed on the surface of xenograft cells, triggering antibody-dependent cell-mediated cytotoxicity by human blood monocytes and macrophages. The IgM isotype of anti-Gal is believed to be responsible for the complement activation that leads to complement-mediated lysis of the xenograft cells. While early studies suggested an increased risk of cancer and poor prognosis associated with ABO blood groups, such assertions have not been verified in breast cancer patients [68], but in ovarian cancer, the presence of the B antigen was positively associated with ovarian cancer incidence, whereas blood group A was not associated with risk [68]. One widely-occurring change observed in a large variety of human cancers is deletion of the A or B epitope on tumor cells, associated with accumulation of their precursor H (LeY, LeB), which causes enhanced malignancy [69]. The blood group reactive lectin Griffonia simplicifolia (GS-I), which recognizes alpha-galactosyl moieties is recognized as a surrogate marker to identify tumor expressed antigens reactive with anti-Gal antibodies [70], and GS-I lectin is of utility to interrogate terminal α-GalNAc/Gal expression on human tissues [71]. The antibody-mediated tissue rejection model supports a rationale for targeting TACAs as tumor-induced antibody responses resemble autoimmune responses [72]. Hyperacute rejection is a complement-mediated response in recipients with pre-existing antibodies to the donor (for example, ABO blood type antibodies). Tolerance to autologous ABO blood group antigens seems to depend in part on peripheral control of antibody autoreactivity. However, normal human serum does contain “hidden” natural antibodies reactive with autologous ABO blood group antigens [73]. These naturally occurring antibodies, especially the anti-Gal response, might also have other clinical consequences for immunotherapy [74] in the context of tolerance [75,76], cross-presentation of tumor antigens [77] and increased immunogenicity of cell-based and protein-based vaccines [66]. Consequently, further research is required to develop the translational and clinical applications.As T-cell-dependent antigens, proteins have long been seen as the primary target of adaptive immune responses. In contrast, carbohydrates are characterized as T-cell-independent (either Type 1 or Type 2) antigens [78]; yet, early studies demonstrated that T-cells could recognize carbohydrate antigens [79]. Post-translationally modified T-cell epitopes constitute a small fraction of both MHC-I- and MHC-II-bound peptides, and a number of modifications are identified as natural MHC ligands in vivo [80]. Computer-based sequence analysis suggests that only a minimal portion of experimentally verified T-cell epitopes are potentially N- or O-glycosylated (2.26% and 1.22%, respectively) [81] and T cells are demonstrated to react with processed glycopeptides and glycolipids often representing TACA [82]. Some types of carbohydrates seem to be processed and presented to T-cells by MHC-II [83,84] while others associate with the MHC-I groove [85,86,87]. The demonstration that T-cells can recognize non-protein antigens has modified ideas on the breadth of antigens capable of interacting with T-cells [88]. The size of the carbohydrate chains, as well as O- versus N-glycosylation varies depending on tumor histotypes. However, recent studies suggest that O-glycosylation (GalNAc) presentation on a peptide backbone, while inducing CD4+ T-cells can impact negatively on CD8+ T-cell stimulation [85,86,87]. Structures of MHC Class II/peptide complexes suggest analogies with helical carbohydrate structures that could fit the MHC Class II antigen-binding groove [84]. In some cases, carbohydrate directly stimulates T-cells. Specific T-cell clones have been generated from mice immunized with a meningococcal group C (alpha-2→9-sialic acid) polysaccharide-tetanus toxoid conjugate [90]. These clones were MHC-independent, but still needed contact with antigen presenting cells for optimal activation [90]. Crystal structure analysis of TCR-glycopeptide interactions validate that TCR can recognize glycans presented on a peptide backbone [91,92]. Existing structures display the key interaction of the core of the peptide ligand, with the TCR CDR3 region shaping a “cavity” often accommodating aromatic amino acid residues. The latter are successfully mimicked in size and conformation by short glycans, like TF or the monomer, Tn. The ability of T-cells to recognize mono- and di-saccharides attached to peptides with Ser or Thr might indicate that T-cells might be degenerate in recognizing glycopeptides [51]. It should not be surprising that sometimes glycopeptides offer no significant benefit as targets for cytotoxic immune response. In some cases, CTL, generated upon immunization with glycopeptide, preferentially kills target cells treated with glycopeptide compared to those treated with the core peptide. In other cases, it does not matter [93], and in some cases, it has been suggested that other glycan receptors are involved in T-cell targeting [94]. This is particularly evident in the work of Madsen et al. [89] that clearly suggest that natural processing of GalNAc on MUC1 might not be a suitable for activating CTLs against MUC1. In general, this may or may not matter, because (a) some activated CTLs are cross-reactive with both the glycosylated and non-glycosylated forms of the same peptide and (b) glycopeptides are of low abundance on tumor target cells [93]. Polyclonal CTL have been observed to kill target cells expressing glycolipid [82]. It has been suggested that glycopeptide-specific-restricted CTL and unrestricted glycan-specific CTL belong to different T-cell populations with regard to TCR expression [95]. Such results demonstrate that hapten-specific unrestricted CTL responses can be generated with MHC Class I-binding carrier peptides. It is possible that CTLs activated with non-glycosylated peptides can cross-react with glycopeptides and carbohydrate themselves. Such peptides have been referred to as carbohydrate mimetic peptides (CMPs) or mimotopes. Sequences and structural properties of CMPs have been discussed previously [96,97,98,99]. CMPs are known to generate T-cells cross-reactive with carbohydrates [100] and to tumor cells [76,100,101,102,103]. The similarity of extended peptide structure and carbohydrates that can fit within Class I or Class II groves has also been noted [97]. In addition, select amino acid residues can spatially overlap glycans attached to peptides in the Class I grove [99]. T-lymphocytes from CMP-immunized animals were shown to be activated in vitro by SLeX, triggering IFN-gamma production in a MHC-dependent manner. Stimulation by peptide or carbohydrate resulted in loss of L-selectin on CD4+ T-cells, confirming a Th1 phenotype. An enhancement in CTL activity in vitro against SLeX-expressing Meth A cells using effector cells from Meth A-primed/peptide-boosted animals was observed. CTL activity was inhibited by both anti-MHC class I and anti-L-selectin antibodies. These results further support a role for L-selectin in tumor rejection, along with the engagement by the TCR for most likely processed tumor-associated glycopeptides, focusing on peptide mimetics as a means to induce carbohydrate reactive cellular responses. Immunization of mice with this CMP reduced tumor cell growth in a transplanted mammary tumor model mediated, to a large extent, by CD8+ T-cells [58], but without any damage to normal tissue after vaccination with the CMP [104]. These observations are very important in understanding the complexity of the antitumor response, especially in terms of abnormal glycan expression patterns and developing strategies in vaccine design.Cell-mediated cytotoxicity is a primary effector function of NK cells. It has been known for a long time that NK cells play a major role in tumor immune surveillance by serving as the first line of antitumor immune defense [105,106]. The multifaceted steps early in NK immune surveillance include an orchestrated activation and recruitment to the tissue sites where they, perform effector functions, which may be associated with tumor reactive antibodies. Receptor diversity is crucial in allowing NK cells to respond effectively, mediating their effects through direct cytolysis, release of cytokines and regulation of subsequent adaptive immune responses [107,108,109,110,111]. NK cell lysis is regulated by a balance of intracellular signals transmitted via stimulatory and inhibitory cell surface receptors after specific binding to their respective target cell ligands [112,113,114]. Activation of endogenous NK cells bears limited clinical benefit, as most cancer patients are treated with chemotherapy, and their immune system is compromised. Consequently focus has been directed in recent years to first understand NK suppression mechanisms and how better to exploit NK cell functionality.Antibodies promote NK cell activation through antibody-dependent cell-mediated cytotoxicity. The best example of combining an anti-GD2 antibody with NK cells is in neuroblastoma (NB) [115]. Treatment of patients with high-risk NB with monoclonal antibodies targeting the disialoganglioside surface antigen GD2 has resulted in lower recurrence rates and improved overall survival [116,117,118,119]. In addition to complement-dependent cytotoxicity, the anti-GD2 monoclonal antibody 3F8 achieves NB killing through antibody-dependent cell-mediated cytotoxicity mediated by myeloid and NK cells [117]. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD2-specific chimeric antigen receptor (CAR) comprising an anti-GD2 ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signaling moiety has been described [120]. CAR development in general is a hot topic area in immunotherapeutics, but mostly in developing T-cells for adoptive therapy [121]. The therapeutic efficacy of endogenous NK cells depends on the effectiveness of NK-activating agents to mobilize sufficient numbers of these cells to tumor sites [122]. The clinical utilization of NK cells is considered at the forefront of cancer therapy. It should be clear that adoptive transfer of NK cells should lead to high levels of circulating NK cells, but that does not necessarily translate into mediating tumor regression [123]. This may result from expression of glycans on the tumor cells in addition to glycans shed from the tumor cell surface. A variety of studies have linked the nature of signaling with the glycan ligand NK receptor paring. In this context, interest has focused on the N-glycan biosynthesis of glycoproteins and, in particular, branching enzymes, such as N-acetylglucosaminyltransferase III (GnT-III), GnT-IV, GnT-V and a1-6 fucosyl- transferase (a1-6FucT) [124,125], that can regulate the further processing of the N-glycan structures, which play a pivotal role in tumor development, metastasis and invasion. Heparan sulfate proteoglycans play a role in NK cell initial recognition and activation [126,127]. The interaction of SLeX antigen with lectin-like receptors on NK cells also triggers cytotoxicity [128,129]. Clustered glycoconjugates sharing the common structure motif trisaccharide Le(x) [130] can enhance cytotoxicity specifically by CD16+ NK cells. GlcNAc-terminated glycoclusters are found to be potent inhibitors of receptors on natural killer cells [131]. N-acetyl-d-glucosamine (GlcNAc) transferases, MGAT3 and MGAT5, have major involvement in linking terminating residues on glycans. MGAT5 is responsible for adding β1-6 GlcNAc residues and forming branched structures, which are especially abundant in cancer tissues with high metastatic potential. MGAT3 catalyzes the addition of β1-4 GlcNAc residues and forms a bisecting structure that disables further addition of GlcNAc by other glycosyltransferases, like MGAT5. Expression of terminal GlcNAc is perceived to inhibit NK function supported by experiments in which siRNA targeting these glycosyltransferases in tumor cells are observed to increase NK cell activity towards tumors [132]. Some transformed cells evade immune surveillance and become resistant to NK cell cytotoxicity, mainly because some shed TACA inhibits NK cell activation [45], leading to established primary tumors [133,134,135,136]. In renal cell carcinoma, the presence of higher gangliosides correlates with systematic metastasis. Disialosyl globopentaosylceramide (DSGb5) was identified previously as one of the major gangliosides from renal cell carcinoma (RCC). Siglec-7 (sialic acid-binding Ig-like lectin-7), expressed on NK cells as an inhibitory receptor, has a striking preference for internally branched α2,6-linked disialic gangliosides, such as DSGb5 [135]. These results suggest that DSGb5 expressed on RCC cells can downregulate NK cell cytotoxicity in a DSGb5-Siglec-7-dependent manner and that RCC cells with DSGb5 create a favorable circumstance for their own survival and metastases [135]. Consequently, despite the enthusiasm of using NK cells in adoptive transfer protocols, in most cases, NK functionality needs to be reset by remodeling the tumor glycan surface. The remodeling can lead to activation of endogenous NK cells with anti-tumoral function. Studies exploring such possibilities are warranted and under research.Strategies for cell surface “glycoform remodeling” promise to facilitate the investigation of carbohydrate mediated cell-cell interactions [137] and as cancer vaccines [138]. Expression of the human α1,2-fucosyltransferase, for example, in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis [139]. The T-cell-independent process of delayed xenograft rejection is suggested as a model for glycan remodeling, which augments NK cell activity [140]. While natural antibodies against alpha-Gal epitope cause hyperacute rejection of pig organs in primates, evidence for the role of alpha-Gal in the NK cell-mediated xeno-response has been contradictory [141]. Therefore, while logic would dictate that glycan remodeling facilitates an improved immune response [138], the nature of these responses might be limited to antibodies and T-cells. Nevertheless it was argued early on that inhibition of N-linked oligosaccharide processing in malignant cells is associated with increased susceptibility to natural immunity [142]. Interference with N-glycosylation has been shown both to reduce the membrane expression of MHC class I and to increase the in vitro sensitivity of tumor cells to NK cell killing. It was long recognized that compounds that inhibit glycosylation pathways could affect the growth of tumor cells in tumor bearing animals. Castanospermine, swainsonine and tunicamycin block different steps in the pathways of glycoprotein processing that affect tumor cell dissemination and tumor colonization. This suggested blocking at one of at least two steps could have beneficial effects on tumor cell growth. The antimetastatic effect of tunicamycin may be related to interference in tumor cell-extracellular matrix interactions, whereas treatment with castanospermine or swainsonine appears to block at a stage distal to initial tumor cell arrest [143]. Swainsonine, in particular, is interesting, as it inhibits the formation of N-linked complex oligosaccharides with this inhibition correlative with enhancement with NK cell function. Consequently, inhibitors of N-, as well as O-linked glycosylation need to be expanded, because they should be useful for the treatment of cancer by effectively resetting NK functional activity by disruption of negative signals; given that inhibitors can be specifically targeted to tumor tissue [144]. More recently, it was shown that glycosylation regulates NK cell-mediated effector function through the PI3K pathway [132].Antibodies might also regulate glycan expression patterns in an undefined way that enhances NK activity. The orchestration of glycan remodeling and galectin-1 upregulation by the tumor suppressor p16INK4a in pancreatic carcinoma cells to reconstitute susceptibility to anoikis underscores the potential and tight control of this lectin [145]. Anti-glycan antibodies can function like lectins, mediating cell death signals [58] and cell growth signals [146]. Other galectins can promote NK cell-mediated anti-tumor activity by expanding unique phenotypes [147]. Co-culture of naive NK cells with macrophages from Gal-9-treated mice resulted in enhanced NK activity, although Gal-9 itself did not enhance the NK activity [147]. Antibodies can do the same. Clinical studies have indicated a role for anti-ganglioside IgM antibodies (including anti-GD2) in passive and active immunity against some cancers [148,149,150]. Their mechanism(s) of action is not clear, but a study in which mice transgenic for anti-GD2 IgM antibody were protected from EL4 metastasis and death indicated a role for IgM, complement and NK cells [151]. In these studies depletion of NK cells with anti-asialo GM1 rabbit serum reduced or abrogated the observed anti-tumor effects, suggesting that NK cells play a major role in tumor eradication or suppression [151]. It is possible that the GD2 model actually opens a window on a more general innate circuitry, which has just been further elucidated. Macrophages activated by Toll-like receptor (TLR) ligands appear to stimulate B cells, including through CD40-CD40L interactions, to a state of activation (CD69+ CD25hi, CD317+) in which they produce IFN alpha and stimulate NK cells nonspecifically [152]. The clinical importance of targeting TACAs is highlighted by the success of carbohydrate-based vaccines against infectious diseases, by the role of TACAs in autoimmune phenomena and by the observed anti-TACA antibodies as clinical correlates of positive outcome seen in patients with cancer. Carbohydrate-based vaccines against Haemophilus influenzae Type b, Neisseria meningitidis, Streptococcus pneumoniae and Salmonella enterica serotype Typhi (S. Typhi) are already licensed, and many similar products are in various stages of development. Therefore, factors contributing to the successes and failures of these bacterial vaccines serve as guides to developing carbohydrate-targeting cancer vaccines. The practical benefits of inducing TACA-reactive antibodies in patients with cancer are further demonstrated by observations that patient survival significantly correlates with ganglioside-reactive IgM levels [149,153]. Low affinity natural IgM antibodies have been found indispensable for anti-viral responses [154,155]. An analogous role for natural antibodies as an innate anti-cancer surveillance mechanism has been suggested, but has been underappreciated, so far [156,157]. The fact that survival rates of cancer patients are correlated with low (intrinsic) affinity and low-titer TACA-reactive antibodies argues that more robust antibody responses may not be necessary.The successful development of anti-microbial vaccines has proven that antibodies—particularly those targeting carbohydrate antigens—are ideally suited for eradicating pathogens from the blood stream and from early tissue invasion. Similarly, vaccines targeting TACAs may also prove beneficial in treating micrometastases. This may be the case since anti-TACA antibodies correlate with beneficial effects on the course of malignant disease and long-term patient survival [149]. However, N-acetylglucosamine branch in O-glycans (core 2 O-glycans) expressing cancer cells acquire highly metastatic phenotypes by surviving longer in host blood circulation [43,44,45]. The induction of TACA reactive antibodies and NK cells to leukemic cells might prove specially beneficial, since simple glycan profiles and commonly contained sialyl-T (NeuAcalpha2-3Galbeta1-3GalNAc) and disialyl-T (NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAc) antigens as major O-glycans are observed on these cells [158] and receptors on NK cells bind to alpha2,3-NeuAc-containing glycoproteins [159]. Therefore, maximizing sustained antibody immunity against TACAs that express simple Core 1 and Core 2 (C2GNT-1) structures is an important goal in developing effective cancer vaccines to combat recurrent disease. The ability of the immune system to identify and destroy nascent cancer cells and, thereby, function as a primary defense against cancer, is a long-standing debate. This raises expectations of therapeutic development of antibodies derived from the promise of multifaceted biological potency compared to stoichiometrically binding antibodies that just interfere with receptor binding. It is postulated that anti-carbohydrate antibodies are part of immune surveillance, just as they are a first line of defense against infectious agents. Natural antibodies may not only have a direct cytotoxic effect on intact tumor cells, but also bystander effects. In addition, they are known to contain low affinity self-reactive fraction representing a “grey area” of the tolerance to self. It has been proposed that this “grey” self-reactivity actually detects quantitative rather than qualitative changes of the antigenic landscape—a function especially suited to detecting unnaturally increased expression of TACA [160].Antibodies that bind to a broad spectrum of TACA can reduce tumor cell dissemination by multiple mechanisms, including blocking the adhesion of metastatic cells to adhesion molecules and generally functioning as regulatory molecules to thwart signaling processes that underlie migration and autocrine and paracrine activities that grant immune privilege to cancer. FAK is a non-receptor tyrosine kinase that plays an important role in signal transduction pathways that are initiated at sites of integrin-mediated cell adhesions and by growth factor receptors. FAK is a key regulator of survival, proliferation, migration and invasion: processes that are all involved in the development and progression of cancer. FAK is also linked to oncogenes at both a biochemical and functional level. Moreover, overexpression and/or increased activity of FAK is common in a wide variety of human cancers, implicating a role for FAK in carcinogenesis. Given the important role of FAK in a large number of processes involved in tumorigenesis, metastasis and survival signaling, FAK should be regarded as a potential pathway target in the development of antibodies targeting TACAs that are associated with anoikis [21] and blocking adhesion. Determining populations of glycan reactive antibodies in the repertoire of natural autoantibodies could lead to developing immunotherapies targeting cancer without affecting normal tissues or resulting in adverse side-effects. Thus, the application of natural antibodies, like IVIg, has the potential to be a supportive therapy for the treatment of cancer metastases and provide an opportunity to probe yet undefined roles of natural antibodies relating broad-spectrum reactivity with anti-cancer functional properties. Most anti-glycan antibodies recognize epitopes of two or three sugars. Consequently, antibodies can cross-react with similar terminal structures. This property of recognizing epitopes “shared” by different molecules is characteristic of anti-glycan antibodies and can be considered an example of “antigen mimicry”. In this context, it would seem that anti-Gal antibodies should be reactive with the histo-blood group antigens, LeB and LeY. Blood group B individuals show reactivity to Tn antigen [161], and some anti-Gal antibodies are cross-reactive with the blood group B antigen [162]. Anti-Gal alpha(1,3)Gal antibodies are observed to react with mucin 1 (MUC1) found on the surface of human breast cancer cells [163]. Thus, natural occurring anti-Gal alpha (1,3)Gal antibodies found in all human serum can react with self (MUC1) peptides expressed in large amounts on the surface of tumor cells, but not on normal cells. These findings are of interest and serve to explain reported findings that human cells can, at times, express Gal alpha(1,3)Gal; such expression is suggested as an artifact in that anti-Gal alpha(1,3)Gal antibodies react with mucin peptides [163]. The cross-reactivity of anti-Gal antibodies has been exploited in cell therapy, where autologous cells processed to express alpha-Gal epitopes result in anti-Gal-mediated, in vivo targeting of autologous tumor vaccine to antigen presenting cells (APC) [77,164]. Transfection of cells with the enzyme 1,3galactosyltransferase (1,3GT) with concomitant expression of the Gal epitope followed by immune complex formation by anti-Gal antibodies should increase transport to lymph nodes and processing of anti-Gal complexed vaccines internalized by APC. Anticipated results include an effective activation of vaccine-specific CD4(+) and CD8(+) T-cells and high cellular and humoral immune response [77]. While manipulating the pre-existing anti-Gal response may facilitate an efficacious vaccine response through antigen spreading to antitumor T-cell response, truly tumor-specific antigens are needed to contribute decisively to tumor regression [165].However, some antibodies display exquisite specificity, like those directed toward the TF antigen [166]. Postpartum, carbohydrate structures on the cell walls of the gastrointestinal flora evoke natural antibodies of presumed TF specificity. These antibodies may provide an early barrier against TF-carrying tumor cells. The widely used regimen of neoadjuvant chemotherapy is demonstrated to stimulate the immune response to TACA in some patients, as reviewed by Andre et al. [167]. Small retrospective studies have suggested that post-chemotherapy lymphocyte infiltrates could be associated with better outcome in patients who did not reach pathologic complete response [167]. The high levels of anti-TF antibody before surgery is another example in which antibody targeting is associated with a better survival of stage II breast cancer patients [168]. This may indicate that the selection of immunopotentiating regimens of neoadjuvant chemotherapy might be beneficial for the host in conjunction with the functional activity of natural anti-cancer antibodies. On the other hand, the detectable spontaneous immune responses to T and Tn antigens are not necessarily efficient, since the expression of these antigens correlates with worse prognosis, mostly because of increased metastasis. The reason may be an escape of some cancer cells from the control by immune responses to TACA, like T, Tn and sialyl-Tn. It is also possible that the correlation with higher grade and metastasis is due to the observation that some tumors are resistant to immunoediting. It would be interesting to differentiate between primarily TACA-negative tumors and secondarily negative tumors that arise due to immunoediting. It is likely that specific suppressive influence of the tumor on the production of TF antibodies is associated with the stage and grade of the tumor. Postoperatively, these antibodies rebound, as do lymphocyte counts [169]. The observation of positive correlation between the level of TF antibodies and the count of lymphocytes in TF-responders appears to reflect the adaptive immune response and provides a further explanation for the involvement of anti-TF IgG in cancer-associated immunosuppression. However, the possible protective mechanism of TF antibodies in cancer has yet remained unclear, as is the role antibodies play in the natural anti-cancer defense system. The signs of tumor-immune system interaction, together with the ambivalence of the results, draw attention to the hypothesis that immune surveillance may be just an epiphenomenon of the “knowledge of self” or, at least, still very early in the process of evolutionary optimization. The tools are there, but maybe they are yet to be tuned.Because of their characteristic immunogenicity and/or immunotolerance, most TACAs fail to induce T-cell-mediated immunity that is critical for cancer therapy. Approaches to overcome this limitation or improve their immunogenicity include coupling covalently TACA to proper carrier molecules to form clustered or multi-epitopic conjugate vaccines, coupling TACAs to a T-cell peptide epitope and/or an immunostimulant epitope to form fully synthetic multi-component glycoconjugate vaccines [138]. Polyvalent vaccines containing a variety of tumor-associated antigens are being tested under the hypothesis that a greater number of antigens in a vaccine will increase the probability of containing the correct antigen(s) to stimulate an effective anti-tumor response. The case for a polyvalent cancer vaccine to induce antibodies to TACAs has been made [170], although, in general, there may be more heterogeneity in antibody responses to polyvalent vaccines than that anticipated with monovalent vaccines [171]. More recent studies on carbohydrate-based vaccines are essentially modifications to the basic premise of conjugate formulations [172,173,174,175].The recognition that T-cell receptors can interact with glycopeptides has facilitated concepts for new antigens being developed to activate anti-tumor responses. The feasibility of T-cell antigens design based on carbohydrate structures is strongly supported by crystallography of several HLA/peptide complexes. These include designer glycopeptides to facilitate CTL activation [176], glycan modification of antigens to target to APC to enhance both CD4+ and CD8+ T-cell responses [177,178,179]. One of the more important glycan decorated tumor antigens is human mucin 1 protein (MUC1). Attempts to develop MUC1-targeting cancer vaccines based on carrier-conjugated unglycosylated MUC1 tandem repeat peptides or carrier-conjugated glycosylated epitopes have been largely unsuccessful. Problems here partly relate to the conformational differences between non-glycosylated vaccine sequences and tumor-expressed, aberrantly glycosylated MUC1. Moreover, densely glycosylated MUC1 glycopeptide might be inefficiently processed by antigen-presenting cells, which ultimately means T-helper cells and CTLS aren’t highly activated. More promising results in tumor models have been reported using a two-component vaccine approach based on an MHC I glycopeptide and a T-helper epitope [180]. A multicomponent vaccine comprising a glycosylated MUC1-derived glycopeptide covalently linked to a T-helper epitope and TLR immunoadjuvant elicited potent humoral and cellular immune responses, effectively reversing tolerance and demonstrated potent anticancer effects. The vaccine candidate comprises the thiobenzyl ester of Pam3CysSK4 as a TLR2 ligand adjuvant, together with the composite T-helper epitope and aberrantly glycosylated MUC1 peptide, CKLFAVWKITYKDTGTSAPDT(αGalNAc)RPAP, formulated into phospholipid-based small unilamellar vesicles. To test its effects in vivo, the tripart vaccine was administered to experimental mice and the animals challenged with MUC1-expressing mammary tumor cells after 35 days. A week after the cancer challenge, the mice were given another vaccine boost. Control mice were administered with vaccine constructs comprising either the unglycosylated vaccine or subunits of the overall vaccine structure, i.e., just the glycopeptide or the adjuvant. Immunization with the multicomponent vaccine led to significant reductions in tumor burden and weight when compared with treatment using either empty liposomes or immunization with a control vaccine that didn’t contain the MUC1 glycopeptide epitope or an unglycosylated multicomponent candidate. Immunization with the primary tripartite candidate also elicited robust IgG antibody responses against the MUC1 glycopeptide, including a mixed Th1/Th2 response.However, there are aspects of MUC1 that are largely ignored in the literature that might impact on its utility as an immunogen. Recently it was found that several of the tumor-related glycoforms of carcinoembryonic antigen, and MUC1 might affect CLR signaling and DC differentiation. These are specific ligands for the pattern recognition receptors DC-SIGN [181] and macrophage galactose-type C-type lectin (MGL) [182], expressed on DCs. MGL1/2-positive cells are interesting, as they represent a distinct sub-population of macrophages, having unique functions in the generation and maintenance of granulation tissue induced by antigenic stimuli [183]. MGL1 is postulated to be actively involved in inflammatory processes [184]. Consequently, Tn glycans on MUC1 that bind MGL might instruct DC to drive Th2-mediated responses, which, unlike those of Th1 effector cells, are thought not to contribute to tumor cell eradication. This has several ramifications. Cancer patients with MUC1 expression profiles may exhibit a Th2-skewed cytokine profile within blood and tumor-infiltrating lymphocytes. This Th1/Th2 imbalance would coincide with disease progression and immunotherapy response. Various lines of evidence suggest that in vivo skewing of T-cell responses toward a Th2 type is an important mechanism of immune evasion in cancer patients [185,186,187]. Terminal glycan structures shared by both host and parasitic helminths include LeX, LDN and LDNF and the truncated O-glycans known as the T (Galβ1-3GalNAcα1-O-Thr/Ser) and Tn antigens, all glycan antigens that may interact with host lectins that skew the immune response to Th2 profiles [188]. This skewing may limit the efficacy of immunotherapeutic approaches [189]. Immunization with formulations that reflect a Th2 bias of the native antigen might only exacerbate the Th2 response. Ensuring induction of a strong type 1 response may be critical to the development of effective cancer vaccines. MUC1-derived non-glycosylated peptides are also demonstrated to mimic carbohydrate antigens that include the Gal epitope [76]. Non-glycosylated peptides that mimic TACAs are noted to induce both humoral and cellular responses to tumors. CMPs can induce cellular responses, including CMP- and TACA-reactive Th1 CD4+ and tumor-specific CD8+ cells [100,101], and CMPs can prime for memory responses to TACAs [190]. We have demonstrated that a single CMP can bind to antibodies with differing TACA specificities that, upon immunization, can induce divergent antibody responses that recognize a range of TACAs [191]. Thus, this important and novel feature of CMPs effectively broadens the repertoire of reactive antibodies without inducing autoimmunity in animal models. The capacity to induce a carbohydrate-cross-reactive humoral, a Th and a CTL response with one single CMP is clearly a unique property of this approach. The observations that CMPs can induce both antibody and cellular responses in the absence of autoimmunity emphasize the feasibility of CMP-based vaccination strategies and the potential benefits of maximizing their effectiveness. Furthermore, CMPs can be encoded into DNA and viral vectors to enhance long-term immunity, which precludes the need for repeated TACA-based vaccination to maintain immune surveillance. This approach has led to a phase I study of a carbohydrate mimetic peptide (manuscript in preparation) in stage IV breast cancer subjects. This CMP shares homology with a region of MUC1, but involved reverse engineering using antibody and lectin templates as the basis for CMP development [104]. The mimicking of MUC1 non-glycosylated peptides with the Gal epitope might also have unintended consequences. For example, mimicry might lend to confusion in deciphering the difference in natural antibody levels to MUC1 and clinical outcomes to MUC1-based vaccines if anti-Gal antibodies cross-reactive with MUC1 are not considered [192]. In addition, this mimicry might also skew Th2 type responses to MUC1 vaccines, which is contradictory to the present paradigm that stresses Th1 responses to MUC1 and other tumor associated antigens. In fact, it is easy to see that as MUC1 expressing cancer cells emerge, the Th2 response becomes set. Vaccines that are MUC1-based might only stimulate B-cells and T-cells that are already primed as the Th2 type, exacerbating what might be akin to “original antigen sin” or an amnestic response to MUC1 of Th2 type [193]. In addition, while transgenic mice expressing human MUC1 are perceived to be of importance to understand the immune response to MUC1 in humans, it is often overlooked that these transgenics also express murine MUC1 in which T cells generated to human MUC1 peptides cross-react with naturally expressed murine MUC1 peptides. This cross-reactivity is seldom discussed and has the potential to confound results.Glycans or TACAs are important targets for cancer immunotherapy, as suggested by immune surveillance mechanisms. TACAs display important biological effects in tumor biology and tumor immunology. Most importantly, the recognition properties of glycans by immune effector cells have suggested translational strategies in immune therapy. The diversity of regulatory mechanisms involving glycans expands the range of possible effects of TACA targeting immunotherapeutic approaches. Anti-TACA antibodies, thus, may be involved in more than direct tumor cytotoxicity. Although the exact mechanism may represent a cascade of steps that are still to be established, immunization targeting TACAs has already been shown to yield antitumor effects mediated by NK cells or through neutralization of tumor immunosuppressive factors in the form of soluble gangliosides. Future work should clarify the points of involvement of antibody/carbohydrate interactions in modulating tumor growth and facilitating innate surveillance mechanisms.The abrogation of negative regulatory signals imposed by glycans and the maintenance of the activated phenotype of NK cells can significantly enhance NK cell activity against solid tumors. Manipulating the balance between inhibitory and activating NK receptor signals, the sensitivity of target cells to NK cell-mediated apoptosis and NK cell cross-talk with other immune effector cells might hold therapeutic promise [194,195]. Efforts to modulate NK cell trafficking into inflamed tissues and/or lymph nodes and to counteract NK cell suppressors, might prove fruitful in the clinic. However, a greater understanding of how to downregulate negative signaling, the benefits of combination therapy, characterization of the functional distinctions between NK cell subsets and the design of new tools to monitor NK cell activity are needed to strengthen our ability to harness the power of NK cells for therapeutic aims. The authors declare no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Vaccination provides many health and economic benefits to individuals and society, and public support for immunization programs is generally high. However, the benefits of vaccines are often not fully valued when public discussions on vaccine safety, quality or efficacy arise, and the spread of misinformation via the internet and other media has the potential to undermine immunization programs. Factors associated with improved public confidence in vaccines include evidence-based decision-making procedures and recommendations, controlled processes for licensing and monitoring vaccine safety and effectiveness and disease surveillance. Community engagement with appropriate communication approaches for each audience is a key factor in building trust in vaccines. Vaccine safety/quality issues should be handled rapidly and transparently by informing and involving those most affected and those concerned with public health in effective ways. Openness and transparency in the exchange of information between industry and other stakeholders is also important. To maximize the safety of vaccines, and thus sustain trust in vaccines, partnerships are needed between public health sector stakeholders. Vaccine confidence can be improved through collaborations that ensure high vaccine uptake rates and that inform the public and other stakeholders of the benefits of vaccines and how vaccine safety is constantly assessed, assured and communicated.Vaccines have made enormous contributions to public health allowing, for example, for the global eradication of small pox and elimination of poliomyelitis from most countries [1]. Levels of support for childhood vaccinations have improved, as demonstrated by worldwide coverage in 2010 with the third dose of diphtheria-tetanus-pertussis (DTP) vaccine, Bacille Calmette–Guérin (BCG) vaccine, the third dose of poliovirus vaccine and the first dose of measles-containing vaccine, which was estimated to be 85% or higher among young children, representing at least 109.4 million immunized children on an annual basis [2]. Table 1 summarizes the impact of vaccines in the USA [3]; worldwide, with childhood vaccination, approximately three million lives are saved annually [1] and millions of disease episodes and disabilities are avoided each year [4]. Established immunization programs have provided many economic benefits for individuals, their families and society [5,6]. Impact of vaccines in the USA in terms of numbers of reported cases and deaths associated with disease before and after the introduction of vaccination (reprinted and adapted from Bonanni and Santos 2011 [7] and Roush and Murphy 2007 [3].Hib = Haemophilus influenzae type b; IPD = invasive pneumococcal disease.Despite these benefits, many children and adults are not vaccinated [4,8]. Annually, 19.3 million children from the world’s poorest settings do not receive vaccines, such as DTP [9]. This is recognized by a collaboration of supranational organizations that have named the period 2011 to 2020 the ‘Decade of Vaccines’, with the mission of extending, by 2020 and beyond, the full benefit of immunization to all people, regardless of where they are born, who they are or where they live [10,11]. Significantly scaling up the delivery of vaccines through the introduction of new vaccines and encouraging countries to reach 90% coverage might save the lives of 8.7 million children aged under five years during this decade [12]. Suboptimal vaccination rates are observed not only in developing countries, but also in industrialized regions [4,13,14,15]. This has consequences not only for direct protection of the vaccinated individual, but also for population herd protection, whereby a majority of immunized subjects prevents circulation of infectious agents in the remaining unvaccinated susceptible population [16]. Low vaccination rates may result from a lack of infrastructure or resources, but also from low vaccine confidence. Reasons for the latter include concerns from parents or guardians and healthcare providers about vaccines, most frequently vaccine safety. Vaccines are usually given to large numbers of healthy people in order to prevent disease, which is different from the use of most medicines, which are generally used to treat or control diseases [17]. Since vaccine recipients are healthy and often young children, there is a lower level of tolerance for the risk of adverse events than with other medicines. Vaccine-related adverse events are mostly time-limited and mild [17], most commonly local reactions at the injection site (pain, swelling or reddening), fever and irritability [18]. Rare reactions to vaccination, such as convulsions, thrombocytopenia, episodes of hypotonia and hyporeactivity and inconsolable persistent crying, are usually characterized by spontaneous remission with no sequelae, but can also have a significant impact on health. Anaphylaxis is another rare severe vaccine-related event that can be fatal unless treated in a timely manner [18]. Fear of such reactions can deter people from having themselves or their children vaccinated. Furthermore, as most of the diseases against which vaccines protect are no longer visible, the risks associated with the diseases are often forgotten and the need for immunization programs to control the diseases may be underappreciated [13]. Parents, when considering vaccination, may therefore worry more about possible adverse events than they do about the risks associated with exposure to disease [19]. Other reasons for questioning vaccines are driven by a variety of social and behavioral factors related to complex cultural issues and belief systems [20,21,22,23,24,25,26]. This may include an influence of religious or ethnic affiliation on the perceptions of disease, vaccines and authority or local practices for medical decision-making and vaccine delivery [20,22]. Other factors are related to individuals’ need for control in making thoughtful vaccination decisions for themselves and their dependents [23,24]. Mistrust in the information provided by the pharmaceutical industry and a lack of trust in the scientific research community or in government [13,14,27] may also lead to vaccine refusals. This has fostered misconceptions about vaccination, such as the belief that diseases had already begun to disappear before vaccines were introduced, because of better hygiene and sanitation or that giving a child multiple vaccines for different diseases at the same time increases the risk of harmful side effects and can overload the immune system [28]. The Internet and social media have allowed such concerns to spread rapidly and indiscriminately [29,30], and websites opposing vaccination are now prevalent, publicizing the beliefs of people with negative attitudes to vaccines to a global audience [31,32]. When a decrease in confidence in vaccines results in reduced coverage, the risk of disease outbreaks rises. For example, suboptimal immunization levels with measles vaccines have led to the re-emergence of measles in Europe [33,34], while a boycott of the polio vaccination campaign in Nigeria in 2003 due to public mistrust of mass immunization programs led to fresh outbreaks of polio in the region and to re-introduction of the virus into previously polio-free countries [35,36].It is important to acknowledge and act upon vaccine-related concerns as part of the strategy to achieve high vaccine uptake rates [37]. This review examines how confidence in vaccines is attained by building on trust and by having effective vaccine safety evaluation and monitoring systems that support immunization programs. The partnerships and collaborations that are needed for sustained vaccine confidence in the 21st century are also explored. In a USA survey carried out in 2010, of 376 parents of children aged six years or less, 26% believed that ingredients in vaccines are unsafe and 17% felt that vaccines are not tested enough for safety; only 23% had no concerns about childhood vaccines [38]. This suggests that many people are unaware of the stringent regulatory quality and safety processes involved not only during the vaccine research, development and manufacturing phases, but also post-licensure in order to monitor and respond to safety signals that may appear during vaccine use in large populations. However, knowledge of these safeguards is not sufficient to maintain the long-term success of immunization programs. The development of effective benefit-risk communication messages to build public trust is not straightforward, requiring input from many vaccination stakeholders. In this review, we describe the systems designed to guide and regulate vaccine development and to monitor safety and efficacy. We explore the factors involved in informing the public and others of the benefits and risks of vaccines to sustain trust in immunization programs, as well as the collaboration of different public health sector partners needed to fulfil the various roles outlined in Figure 1.Transparency in the decision-making processes for vaccine policy matters is crucial to counter the conspiracy allegations made on anti-vaccine websites regarding issues, such as government or institutional decisions about vaccine approval for licensure, public funding and safety assessments after licensing [24]. Each country has health policies and develops vaccine recommendations that drive national vaccination programs [39,40]. The development of national and supranational public health policies involves different levels of governments or institutions and numerous stakeholders with diverse needs and interests. Political commitment is also critical to support functional policy-making and regulatory bodies at a national level. Many countries have installed a national immunization technical advisory group, a body of national experts who advise on all technical and scientific topics related to vaccines and immunization [41] and who may elaborate recommendations on which vaccinations are appropriate in which schedule and for which population in order to help protect those at risk. In the USA, the Centers for Disease Control and Prevention (CDC) sets immunization schedules based on recommendations from the Advisory Committee on Immunization Practices (ACIP) [42,43].Factors that promote (outer green boxes) or undermine (outer orange boxes) vaccine confidence and collaborations (central box) associated with improved public confidence in vaccines.Providing evidence-based vaccine recommendations and health policies that meet the needs of parents, healthcare providers and society and ensure that those working in primary care are provided with the support required to implement vaccination programs effectively should be part of immunization implementation programs.Vaccines are biological preparations made in, composed of and/or tested through living systems, with a mode of action that is via the immune system. They are generally administered to large numbers of healthy people to prevent disease and need thorough assessments to ensure that benefits outweigh the risks when used in the target populations [44]. As no medical intervention, including vaccines, is completely safe and without risk, this necessitates ongoing surveillance to identify safety concerns. Then, serious adverse events and associated risks can be compared to the benefits of vaccination in a benefit-risk analysis. Consequently, the processes involved in vaccine development are often complex, specific and stringent. Selection of a candidate vaccine is usually based on public health need (disease burden), scientific feasibility, suitable technologies and manufacturability. The development process (Figure 2) aims to deliver an efficacious vaccine with a strong and long-lasting immune response and minimal adverse effects. Once a candidate vaccine is selected, preclinical studies (in vitro and in animals) are conducted to provide important safety data and evaluate vaccine quality and potency [45]. This is followed by, typically, three phases in clinical development, all of which include vaccine safety assessment in their study protocols. Pre- and post-licensure vaccine development activities.Post-licensure activities are designed to monitor the impact of the vaccine in terms of immunization coverage and effectiveness in protecting against disease and safety surveillance. Manufacturers are usually required to submit risk management plans to the licensing authority that detail a set of pharmacovigilance activities and interventions to identify, characterize, prevent or minimize risks related to the vaccine they intend to market [46]. Phase IV trials are often conducted after the vaccine has been licensed to broaden the vaccine indication (e.g., altered schedule) or assess the vaccine in specific populations, such as immunosuppressed persons, low birth weight infants, chronic disease populations or pregnant women [47,48,49]. In addition, most countries have ongoing vaccine safety assessment through passive or active surveillance systems.The vaccine manufacturing processes that ensure the end product is safe for use are complex, as they require, for instance, sterile manufacturing conditions and involve challenges that differ from those associated with non-biological drug manufacturing [50]. Each type of vaccine (e.g., live attenuated, inactivated or killed whole viruses or bacteria or subunit antigens that are naturally derived or generated using recombinant DNA technology) presents separate specificities in terms of manufacturing, analytic characterization and safety evaluation [47,51], and vaccine development and manufacturing must adhere to stringent rules and regulations, as described below.Currently, extensive scientific and regulatory processes are in place to ensure product quality, safety and efficacy before vaccines are licensed and made available to the public [47]. These processes are mandated by guidelines and rules from organizations, including the World Health Organization (WHO), the Food and Drug Administration (FDA) in the USA, the European Medicines Agency (EMA) and the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), which brings together the regulatory authorities and pharmaceutical industry of Europe, Japan and the USA [44]. Legally-binding quality standards are also laid down in compendial monographs, the European Pharmacopoeia, the United States Pharmacopoeia and other country-specific compendia.Once a vaccine has been approved for marketing, the manufacturer must adhere to quality assurance programs to evaluate various steps of the manufacturing process: from raw materials, through each stage of component preparation to the final formulated, filled and packaged product. Characterization and testing of raw materials are required, and the production procedure must be validated, documented and meet good manufacturing practice (GMP) requirements [52]. GMP covers the methods to be used in and the facilities or controls to be used for the manufacture, processing or packing of a drug to ensure its safety, quality and purity [44]. Final product testing covers aspects such as sterility, general safety, purity, identity and potency. The vaccine manufacturer must also demonstrate production consistency. For each lot of vaccine that is produced, release by national regulatory authorities provides a final check on the manufacturer’s performance in the control of the production process and may include additional, independent testing of sampled lots before approval for release and distribution by the manufacturer [53]. Inevitably, there are occasional disruptions of supply when these multiple steps of control of the production process do not permit a component to be used in a next step. Significant supply disruptions should be notified to authorities and can provide an indication of the reliability of the manufacturing processes.A further oversight to industry processes is delivered through facility inspections by regulatory authorities. Manufacturers that fail to meet product standards or do not comply with GMP may be subject to product recalls, license suspension or withdrawal or even, in exceptional circumstances, closure of the production plant [54,55,56,57]. Safety is monitored throughout the vaccine’s lifecycle, starting at the time of preclinical evaluation and continued during clinical development and, following licensure, is monitored indefinitely while in use in immunization programs. The benefit-risk profile of the vaccine is therefore re-assessed constantly. Safety surveillance is the responsibility of not only those who develop or manufacture the products, but also those who are involved in vaccine distribution and administration [16,58]. For instance, the role of healthcare providers is essential in observing and reporting adverse events. Safety surveillance also requires close collaboration between regulators and industry [59].Similar to the vaccine development and manufacturing processes, safety reporting requirements are guided by rules and regulations. For example, the EMA requires public release of vaccine safety information via Periodic Safety Update Reports (PSURs), which present the vaccine manufacturer’s integrated assessment of benefit-risk and exposure and are provided to the EMA for their assessment. From 2013, PSUR-related documents will be published on the EMA website, increasing the transparency of available safety information [60]. National and supranational vaccine pharmacovigilance programs involve, at the most basic level, passive surveillance of adverse events following immunization (AEFI), in which spontaneous AEFI reports are made to regulatory monitoring organizations, such as the Vaccine Adverse Event Reporting System (VAERS) in the USA and EudraVigilance in Europe [48,61,62]. Passive surveillance, therefore, relies on the detection and reporting of cases by healthcare providers. Where a safety signal is detected or when a new vaccine is introduced, passive surveillance of AEFI is not sufficient, and an “active” vaccine pharmacovigilance is established that involves prospective case finding and data collection [16]. Epidemiological studies may be required to assess whether an AEFI is causally related to a vaccine, as well as pathological or laboratory studies. In the USA, the CDC-sponsored Vaccine Safety Datalink (VSD) system enables active vaccine pharmacovigilance with a near real-time vaccine safety surveillance system via weekly data and sequential statistical analysis [63,64]. In Europe, the Vaccine Adverse Event Surveillance and Communication (VAESCO) project conducts similar vaccine safety studies to complement routine reporting of AEFI to EudraVigilance [65]. The Innovative Medicines Initiative is developing a public-private collaborative framework for rapid assessment of the benefit-risk profile of vaccines [66]. For countries that lack the infrastructure or resources necessary to carry out appropriate pharmacovigilance studies, the WHO Global Advisory Committee on Vaccine Safety (GACVS) provides independent, scientifically rigorous advice on vaccine safety issues of potential global importance [67]. Access to a global network for safety data exchange would also be beneficial [16,68]. National or supranational expert committees often advise local authorities on the nature of the observed events and, where a causal link is established, recommend actions to treat or manage the AEFI at a local level, where appropriate. The national or supranational regulatory authority also decides if the vaccine should be withdrawn from the market or if changes should be made to its licensed indications and safety warnings added to its prescribing information.Active surveillance is important to evaluate the background incidence of rare conditions and autoimmune disorders to determine whether events that are temporally associated with vaccination are occurring at a higher rate than would be expected based upon the background incidence rate for that event [47]. This is essential to address public concerns and to provide accurate and reliable information on vaccines. For example, a tetravalent rhesus-human reassortant rotavirus vaccine licensed in the USA, Rotashield™* (Wyeth-Lederle Vaccines, Madison, NJ, USA; now owned by Pfizer, New York, NY, USA), was withdrawn from the market because of an association with intussusception that was identified through passive surveillance [69] and confirmed following collaboration of the ACIP, industry and managed care organizations [70]. This triggered active surveillance of the two currently available rotavirus vaccines, Rotarix™* (GlaxoSmithKline Vaccines, Wavre, Belgium) and RotaTeq®* (Merck and Co., Inc., Whitehouse Station, NJ, USA) and results have been published from large post-marketing studies conducted in Mexico, Brazil, Australia and the U.S. [71,72,73,74,75]. In its 2013 position statement, the WHO concluded from these data that in some, but not all, settings, a small increased risk of intussusception (about one to two per 100,000 infants vaccinated) was detected shortly after the first dose of both vaccines [76]. Where present, this risk is five to 10 times lower than that associated with the previous rotavirus vaccine that was withdrawn. This AEFI is mentioned as a precaution in the summary of product characteristics for both vaccines, and surveillance for intussusception continues. Current evidence, however, suggests that the benefits of the rotavirus vaccines, in terms of averted deaths and hospitalizations [76,77,78,79], outweigh the risk for intussusception. Data on the efficacy of the vaccine in preventing disease in immunized populations are obtained from controlled studies. Vaccine effectiveness describes protection under programmatic implementation, reflecting the performance of the vaccine in the actual target population, and is generally monitored as part of post-marketing disease surveillance activities [49]. National and international disease surveillance, together with data on vaccination uptake, serve to document the impact of immunization programs. The effectiveness of current vaccines and vaccination policies, such as for a child immunization program, is mainly evaluated through continued passive surveillance, i.e., by routinely reported data from an existing health system. These data may highlight the need for changes in program strategies, for example, from observations of disease trends over time or outbreak patterns from vaccine-preventable diseases. Passive surveillance data are normally evaluated and reported annually in national reports and at a supranational level [80].An additional active surveillance approach, which is more specific and sensitive and also more resource-demanding, may be implemented and is the tool of choice to monitor and evaluate the impact on a vaccine-preventable disease that is targeted for eradication. An example is the active surveillance of acute flaccid paralysis in children aged under 15 years, as recommended by the WHO, to document progress towards reaching the target of polio eradication [81].Information on vaccine uptake is crucial to evaluate the effectiveness of a vaccine or an immunization program. The best quality data are obtained on a real-time and case-based level, but as vaccines are distributed daily and in large numbers, it is highly resource-demanding to obtain data of this quality in all settings. Hence, vaccination coverage assessment may differ from one country to another in terms of the information system used, timeliness of reporting and data analysis methodology [82]. Therefore, to fully understand reports on vaccine effectiveness and impact, taking the heterogeneity of national systems and outputs into consideration, it is important to understand the origin of the data, such as type and timeliness of surveillance, data specifications and analysis methodology, especially when comparisons are made among national reports. To achieve high uptake, there must be broad acceptance of the medical need and safety of immunization, as well as the availability of acceptable health systems that support vaccine delivery. However, the process of developing benefit-risk communication messages to instill public trust is complicated, requiring different types of research at a local community level for each new vaccine introduced. In particular, advocacy and communication strategies must be tailored to the population concerned, as has been demonstrated with the introduction of vaccines against human papillomavirus (HPV). Before licensing, information-gathering exercises showed the importance of raising awareness of HPV as a cause of cervical cancer before introducing an immunization program, the success of which was dependent on targeting primarily young adolescent girls before HPV exposure [83,84]. For example, interviews held with parents of children aged eight to 10 years in the UK before the introduction of the HPV vaccination program showed that most had not heard of HPV and were not aware of its role in cervical cancer [85]. There were also concerns about offering a vaccine that protects against a sexually transmitted infection to children and that the vaccine should be offered at an older age in conjunction with a sex education program. This perceived lack of need and misunderstanding about the optimal time for vaccination, along with some safety concerns, have contributed to low vaccine uptake in some countries [86,87,88]. For vaccine programs to be successful, it is important to present facts about disease burden and vaccine prevention in an accurate, appropriate and easy to understand way, including clear explanation of the risks of disease versus the risks of vaccination (Table 2). Healthcare providers have a central role in maintaining public trust in vaccination through direct communication with the vaccine or the vaccinated child’s parent [13,89,90]. Gaps in knowledge and poor communication from healthcare providers are detrimental to high immunization rates [90], and it is important that healthcare providers have confidence in the information they provide. Stakeholders need to be engaged in a manner that takes into account the knowledge, attitudes, behaviors and values of the local population [12,91]. An example vaccine information statement (VIS) for the measles, mumps and rubella (MMR) vaccine (adapted from VIS produced by the CDC [92] and data in the CDC “Pink Book” [93]). Ear infection (7 persons out of 100) *Pneumonia (6 persons out of 100) *Seizures (jerking and staring) (6 to 7 persons out of 1,000) *Death (2 persons out of 1,000) *Fever (up to 1 person out of 6) Mild rash (about 1 person out of 20) Swelling of glands in the cheeks or neck (about 1 person out of 75) Seizure (jerking or staring) caused by fever (about 1 out of 3,000 doses)Temporary pain and stiffness in the joints, mostly in teenage or adult women (up to 1 out of 4)Temporary low platelet count, which can cause a bleeding disorder (about 1 out of 30,000 doses)Serious allergic reaction (less than 1 out of a million doses)Several other severe problems have been reported after a child gets MMR vaccine, including deafness, long-term seizures, coma or lowered consciousness, permanent brain damageThese are so rare that it is hard to tell whether they are caused by the vaccineDeafness (1 person out of 20,000) *Meningitis (infection of the brain and spinal cord covering) (15 persons out of 100) *Painful swelling of the testicles or ovaries (adults: 1 person out of 2 or 3); rarely sterilityEncephalitis (1 person out of 6,000) **Hemorrhagic manifestations (1 person out of 3,000) **If a woman gets rubella while she is pregnant, she could have a miscarriage or her baby could be born with serious birth defects * Rates reported for measles and mumps complications are from CDC Pink Book; ** complications for rubella are not presented in the VIS and have been sourced from the CDC Pink Book.Therefore, the confidence of healthcare providers in vaccines should be reinforced by better and deeper scientific and public health training on vaccines during their medical studies and through postgraduate education. The WHO has identified several areas that need to be addressed to ensure healthcare providers are well prepared for immunization sessions [94]. These include strengthening of pre-service training within the faculties of medicine, nursing, pharmacy and public health, as well as in-service training through specific support and regular refresher courses, such as vaccinology courses. Moreover, delivery of effective benefit-risk communication messages is highly dependent on a strong immunization program in which policies are driven by processes for developing, manufacturing and monitoring the safety and effectiveness of vaccines that are transparent, i.e., are performed with openness, so that people can trust that they are fair and honest.Although vaccine safety crises are uncommon, they have the potential to disrupt immunization activities and, thereby, affect public health. It is, therefore, important to ensure that vaccine safety communication plans are in place at an early stage, as recommended in the Global Vaccine Safety Initiative developed by the WHO and a group of partners [16,58]. This should include a mechanism for providing feedback to vaccines or parents and healthcare providers on specific concerns raised about vaccines, such as a reported AEFI; not having such feedback could reinforce the subconscious sense that vaccines are dangerous [24].A number of false vaccine concerns have been reported that have fuelled the effectiveness of anti-vaccine advocates. One of the most notable vaccination controversies was related to the presumed link between measles, mumps and rubella (MMR) vaccination and autism in children, which was initiated by an article in The Lancet by Wakefield et al. in 1998 (article retracted) proposing an association between MMR and autism [95]. Despite numerous studies that failed to show a link between the MMR vaccine and autism, media coverage of the allegations was vast, and a significant decrease in vaccination trust and vaccine coverage occurred and still remains in various countries [31,32]. In other cases, immunization programs have been suspended in response to false vaccine concerns, such as the reported link between multiple sclerosis and hepatitis B vaccination that led to the French government suspending, temporarily, the school-based hepatitis B immunization program in 1998 [96]; and disrupted immunization in various countries due to a loss of confidence in pertussis vaccination in the 1970s and 1980s [97]. In both cases, vaccine hesitancy was proven to be unfounded [96,97,98]. Vaccine beliefs should be based on accurate, factual information regarding both the benefits and risks associated with vaccines [24], delivered with tailored advocacy and communication to explain medical need and improve knowledge of vaccine safety systems. This may require social mobilization, where people with influence, such as healthcare providers, local authorities, religious leaders, teachers and community leaders, are involved in addressing local social and behavioral aspects of vaccine hesitancy [99,100]. In the future, the Internet and social media are likely to be used more frequently during the launch of immunization campaigns to prevent misinformation [29,30,31]. This might include web-based decision aids, which have been shown to improve attitudes to vaccination [101]. It has been suggested that the interactive potential of the Internet could be harnessed even further by integrating factual information with people’s own values and preferences, allowing users to receive personalized vaccination recommendations [23]. Strategies for identifying early warning signals of areas of decreased confidence would also encourage concerns to be addressed efficiently, such as the Vaccine Confidence Index developed by the London School of Hygiene and Tropical Medicine [102].According to a survey of over 1,200 parents in the USA, vaccine manufacturers were considered a good or excellent source of information by only 29% of those who did not use the Internet as a source of vaccine information and by 23% of those who did use the Internet [26]. Healthcare providers were regarded as a good source of information by 85% and 69% of those surveyed, respectively. This lack of trust in vaccine manufacturers is possibly linked to the fact that the vaccine industry profits from selling vaccines and that the scientists who develop and test vaccines, as well as the doctors who promote them, are perceived to have a vested interest in highlighting their benefits [13]. Consequently, the public may feel that vaccine manufacturer employees, including research scientists, have a conflict of interest in providing the “full picture” in regards to their vaccines’ benefit-risk profiles and knowledge gaps [103]. Moreover, concerns raised in the past, regarding issues, such as bias in the publication of trial results [104,105,106,107,108], the relationship between industry and the medical profession and patients’ organizations [109,110,111,112] and the regulation of promotional activities and materials [113], have had a negative impact on information provided by the pharmaceutical industry. Openness and honesty attained with bi-directional communication with healthcare providers and the general population is one of the factors that determines trust in industry [114]. It therefore follows that manufacturers of vaccines should provide product information that is accessible to all those interested in vaccine implementation, including the general population. Vaccine manufacturers now post information on the protocols and results of vaccine clinical trials online to meet International Committee of Medical Journal Editors’ requirements [115]. ClinicalTrials.gov, run by the USA National Institutes of Health [116], was the first online registry for clinical trials and is the largest and most widely used. There have been calls to go further and for relevant anonymized patient-level data to be made available upon request for all clinical trials of drugs and devices; since January 2013, the British Medical Journal requires this commitment before publication can be considered [117]. Several pharmaceutical companies have also committed to seek publication of results from all sponsored clinical trials that evaluate their medicines in peer-reviewed scientific journals. In October 2012, GlaxoSmithKline announced that it will make detailed anonymized patient-level data from their clinical trials available to qualified researchers through SHARE (Sharing Anonymized Research Data) [118,119]. Disclosure of comprehensive clinical study results by the vaccine industry would also be desirable [120].Openness and transparency in the exchange of information between the vaccine industry and other stakeholders in vaccine implementation should help to re-build trust and, hence, maintain confidence in immunization programs. Also, increased access to data by outside experts will allow independent trial analysis, offering the potential for different perspectives on the results, which could lead to improvements in trial design or aspects of the immunization program or even the development of novel medicines [103,118].In this Decade of Vaccines, the expansion of immunization programs is necessary to meet the goals of healthcare organizations worldwide and to improve health at a country level. In a world of networks, the vaccination debate has moved from the classical top-down model of information provision to one of social media debate, where each contributor is seen as an equal player. However, websites often present suboptimal and inaccurate information, and as a result, public concerns surrounding vaccinations (most frequently, vaccine safety) have the potential to spread rapidly. Misconceptions and misunderstandings surrounding vaccines have been fuelled by the difficulty of many stakeholders in communicating and informing others in effective ways, be it parents, vaccine recipients or healthcare providers. No medical intervention, including vaccines, is completely safe and without adverse events. The most common AEFIs are time-limited and mild and continuous benefit-risk evaluations are performed by regulatory authorities, using the vaccine safety evaluation and monitoring systems that are in place to identify safety concerns. Serious adverse events and associated risks are assessed and compared to the benefits of vaccination. Even so, reassurances on vaccine safety and of the necessity for vaccination via evidence-based recommendations and policies, as well as stringent safety and disease surveillance procedures, are not sufficient to counter all negative beliefs surrounding vaccines. Further social and behavior science research is needed to determine how to address beliefs appropriately via effective benefit-risk communication messages. Reactions to issues related to vaccine production, safety or implementation programs must be handled with speed and must also be fair, balanced and accurate in the assessment and in all communications. In particular, unexpected and/or serious AEFIs require rapid and comprehensive investigation to provide effective and transparent information regarding causality and management. This necessitates ongoing education and a commitment to communication and dialogue among all involved stakeholders. Importantly, in view of their central role in maintaining public trust in vaccines, healthcare providers need to feel confident in providing advice and need to be updated in a timely manner when scares arise. This might require an increased amount of time spent on the topic of immunization in medical and nursing schools and an increased focus on continued medical education in this area. Better tailored communication materials shared through appropriate channels would further support community engagement. Health authorities need to play an important role in implementing vaccine communication plans and in the wider field of transparency and accountability in vaccine decision-making. Confidence in industry requires a commitment to bring the same high quality and efficacious vaccines to all countries and a major role in the transparent infrastructure for developing, manufacturing and monitoring the safety and effectiveness of vaccines. The vaccines industry must ensure that accurate and reliable information is provided on its products in order to address vaccine hesitancy associated with social and behavioral issues. The industry should also have strategies for providing accurate and reliable information on the benefit-risk profile of vaccines, which are appropriate for each audience concerned and supported by evidence-based and transparent recommendations. This would help other stakeholders to view the pharmaceutical industry as an integral partner in public health issues. Moreover, interactions among vaccination stakeholders should be guided by the highest standards and codes of conduct in order to address any concerns surrounding the impartiality of vaccine manufacturers in terms of benefit-risk communication. This review provides a broad overview of the systems that contribute to building confidence in vaccines and is therefore limited in terms of detail on specific topics. It is clear, however, that to sustain trust in vaccines, partnerships between all stakeholders in the public health sector, such as health authorities, policy makers, national and supranational organizations, healthcare providers, vaccine manufacturers and others, are needed to ensure high vaccine uptake rates, identify and allow the introduction of new vaccines and inform the public and others of the benefits of vaccines and how vaccine safety is constantly assessed, assured and communicated. The development of effective benefit-risk communication messages to instill public trust in vaccines is complex, but can be achieved with collaborative and transparent approaches, thereby encouraging the success of immunization programs.Writing support services were provided by Joanne Knowles (freelance, UK); editing and publication co-ordination services were provided by Véronique Delpire and Mandy Payne (Words and Science, Brussels, Belgium). The authors thank Jane Wynen, Alberta Di Pasquale and Vincent Bauchau (GlaxoSmithKline Vaccines, Wavre, Belgium) for critically reviewing the manuscript. All costs related to the development of this manuscript were met by GlaxoSmithKline Biologicals SA. All authors are employed by GlaxoSmithKline Vaccines. K.H. declares ownership of GlaxoSmithKline stock options and R.S.O., R.A.A. and F.P.M. declare ownership of GlaxoSmithKline shares and stock options.Rotarix is a trademark of the GlaxoSmithKline group of companies. RotaShield is a trademark of Wyeth-Lederle Vaccines, and RotaTeq is a trademark of Merck and Co., Inc.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).DNA vaccination is a disruptive technology that offers the promise of a new rapidly deployed vaccination platform to treat human and animal disease with gene-based materials. Innovations such as electroporation, needle free jet delivery and lipid-based carriers increase transgene expression and immunogenicity through more effective gene delivery. This review summarizes complementary vector design innovations that, when combined with leading delivery platforms, further enhance DNA vaccine performance. These next generation vectors also address potential safety issues such as antibiotic selection, and increase plasmid manufacturing quality and yield in exemplary fermentation production processes. Application of optimized constructs in combination with improved delivery platforms tangibly improves the prospect of successful application of DNA vaccination as prophylactic vaccines for diverse human infectious disease targets or as therapeutic vaccines for cancer and allergy.DNA vaccines are plasmids that combine sequences required for replication and selection in Escherichia coli (bacterial region) with sequences necessary to express an encoded transgene in vertebrate cells (eukaryotic region) after delivery to an organism and transfection of target tissue cells. A great deal has been learned about the mechanism of action of DNA vaccination since the first publication in 1992. After delivery to the patient, the vector encoded transgene antigen is transcribed after entering the cell nucleus. The mRNA is exported to the cytoplasm and subsequently translated. The host-expressed antigen is presented to the immune system by either major histocompatibility complex (MHC) class I or II. Transfected DNA also activates innate immunity which is critical to promote an immune response against the MHC presented antigen [1,2,3]. DNA vaccines are inherently safe since the vectors are non-replicating, encode and express only the target antigen, and are not live and therefore cannot revert to a disease causing form as with viral vectors. A key advantage of DNA vaccination is that, unlike viral vector particles, DNA vaccines do not induce anti-vector immunity, and therefore may be utilized in prime and boost regiments and with multiple products intended for the same patient. DNA vaccination is also effective in neonates even in the presence of maternal neutralizing antibodies [4]. Additionally, DNA vaccine manufacture is much easier and faster than alternative vaccine platforms and the DNA product is highly stable. DNA vaccines are well tolerated and have an excellent safety profile in human clinical investigations with no reported safety concerns such as DNA integration into the host genome, antigen tolerance or autoimmunity (reviewed in [1,2,3]). The licensure of four animal health DNA vaccine products demonstrates the utility of DNA vaccination in large animals including horses and pigs. These licensed products include preventative vaccines for West Nile virus in horses and infectious haematopoietic necrosis virus in fish, a therapeutic cancer vaccine for dogs, and a growth hormone gene therapy to increase litter survival in breeding pig sows [1]. DNA prime-heterologous boost vaccination with influenza hemagglutinin antigen has demonstrated utility to induce broadly cross neutralizing antibodies [5,6,7]. However, efficient plasmid delivery is often required to generate protective responses in large animals and humans compared to mice. Various DNA delivery platforms have been developed that have demonstrated promising results in large animals and humans, including electroporation (EP) [8], needle free jet-injection [9,10] and lipid [11] deliveries. Interestingly, human serum amyloid P binds and inhibits plasmid transfection and DNA vaccine induced adaptive immune responses much more strongly than the murine counterpart [12,13]. This and other species specific differences may collectively account for the greater difficulty in obtaining acceptable efficacy in humans.Most delivery platforms such as electroporation greatly increase plasmid transfer across the cell plasma membrane barrier to directly or indirectly transfect plasmid into the cell cytoplasm but do not deliver DNA to the nucleus [14]. Plasmid transfection into the cell, and vector diffusion through the cytoplasm and nuclear uptake may be enhanced using smaller more compact vectors or nuclear targeting sequences [15]. Within the nucleus, transgene expression levels may be dramatically increased by optimization of the bacterial and eukaryotic regions. In this review, vector innovations that improve DNA vaccine performance are discussed. Critical issues for plasmid manufacturing are also discussed, and exemplary plasmid production processes highlighted.DNA vaccine vectors combine a eukaryotic region that directs expression of the transgene in the target organism with a bacterial region that provides selection and propagation in the Escherichia coli (E. coli) host. The eukaryotic region contains a promoter upstream, and a polyadenylation signal (polyA) downstream, of the gene of interest. Upon transfection into the cell nucleus, the promoter directs transcription of an mRNA that includes the transgene. The polyadenylation signal mediates mRNA cleavage and polyadenylation, which leads to efficient mRNA export to the cytoplasm. A Kozak sequence (gccgccRccATGG consensus, transgene ATG start codon within the Kozak sequence is underlined, critical residues in caps, R = A or G) is included. The Kozak sequence is recognized in the cytoplasm by ribosomes and directs efficient transgene translation. The constitutive human Cytomegalovirus (CMV) promoter is the most common promoter used in DNA vaccines since it is highly active in most mammalian cells transcribing higher levels of mRNA than alternative viral or cellular promoters. PolyA signals derived from the rabbit β-globin or bovine growth hormone genes are typically used. These signals contain accessory sequences upstream and downstream of the polyadenylation site (AATAAA) that increase polyadenylation efficiency resulting in increased mRNA levels, and improved transgene expression. The transcribed 3' and 5' untranslated regions (UTRs) flanking the transgene should not contain open reading frames (ORFs) since ORFs in these regions have been shown to be translated into immunogenic peptides [16]. The bacterial region combines a high copy replication origin, most usually the pUC origin, with a selectable marker. Perhaps surprisingly, certain orientations and compositions of bacterial region sequences can dramatically reduce eukaryotic region directed transgene expression, manufacturing yields, and plasmid quality in the E. coli host [17,18]. Reduced expression with certain backbones may in part be due to production, from cryptic promoters in the vector backbone, of double stranded RNA (dsRNA) that triggers protein kinase R (PKR) mediated translational shutdown or RNA interference [19,20]. Thus, since both bacterial production and expression in the target organism are very sensitive to vector changes (Figure 1a), a critical part of vector design is careful selection and assembly of bacterial region selection and replication sequences.(a) DNA vaccine vector production and application flowchart. Stages 1 and 4 are very sensitive to vector changes and must be optimized coordinately since vector modification to enhance one parameter can have multiple undesired effects on other parameters. Stages 2 and 3 are largely generic; (b) Insert design flowchart.First generation DNA vaccine vectors such as pVAX1 (Invitrogen; Figure 2b) and gWIZ (Genlantis, Figure 2a) contain the kanamycin resistance (kanR) gene as a selectable marker. pVAX1 is a basic vector that contains no eukaryotic or bacterial region optimizations, and consequently has relatively low manufacturing yield and expression in vitro and in vivo (mice) [21]. pVAX1 expression is reduced, compared to alternative CMV promoter vectors, by inhibitory sequences in the bacterial region (see Section 5.1). The pUC origin is oriented such that the pUC origin encoded cryptic eukaryotic promoter [19] will transcribe RNA antisense to the transgene (Figure 2b); this may produce dsRNA and reduce expression by RNA interference or PKR mediated translational inhibition. The gWIZ vector has 5-fold improved expression and 2-fold increased manufacturing yields relative to pVAX1 [21] due to extensive optimization of the orientation and composition of the bacterial region [17] and addition of an intron upstream of the transgene.DNA vaccine vectors. (a,b) 1st; (c) 2nd; and (d) 3rd; generation DNA Vaccine vectors; (e) 2nd and 3rd generation vectors increase in vivo expression compared to first generation vector gWIZ. 5 µg muSEAP vectors delivered intramuscularly with EP to mice on day 0, serum muSEAP assayed on indicated days. 3rd generation vector NTC9385R has significantly higher expression than gWIZ or 2nd generation vector NTC8385 (p-value = 0.05; Mann-Whitney rank-sum test); (f) 3rd generation vectors dramatically increase in vivo expression, compared to 2nd generation. 50 µg muSEAP vectors in 50 µL saline delivered intradermally to mice with EP on day 0, muSEAP assayed on indicated days. 3rd generation vector NTC9385R has significantly higher expression than 2nd generation vector NTC8685 (p-value = 0.05; Mann-Whitney rank-sum test). NTC8685 is a 2nd generation vector similar to NTC8385. The NTC8385 1,518 basepair (bp) bacterial region (spacer region) is reduced to 855 bp in NTC8385-min and 454 bp in NTC9385R. This compares to 2,678 bp for gWIZ, and 1,970 bp for pVAX1.New vectors have been constructed that combine improved transgene expression with superior manufacturing yields and regulatory compliance compared to first generation vectors. Design criteria are outlined below and example vectors described in Section 2.2. See [22,23,24] for comprehensive reviews on DNA vaccine vector and insert design.Regulatory: The FDA and European Union (EU) have issued guidance documents that include vector design considerations for plasmid DNA vectors intended for human use [25,26,27]. Vectors should be minimalized to remove extra nonfunctional sequences, especially ones that encode cryptic ORFs that may be expressed in the target organism. This is especially critical within the transcribed UTRs to prevent production of vector encoded cryptic peptides in the target organism [28] that may induce inappropriate adaptive immune responses [16,29]. For example, the mRNA nuclear export enhancing Hepatitis virus derived posttranslational regulatory element (PRE) included within the transcription unit downstream of the stop codon in some vectors encodes a 178 bp amino acid fragment of the viral polymerase gene [22]; immune response against this viral protein may alter immune responses in individuals with prior exposure and circulating T-cells to Hepatitis. In addition to general concerns regarding use of antibiotic selection markers, the European Union has specifically recommended the elimination of kanR selection markers [27] (see Section 2.2). Vector retrofit to replace the kanR marker with short RNA antibiotic-free markers has the unexpected benefit of improved expression (see Section 5.1).Expression: The 5' UTR upstream of the transgene is typically 50–150 bp and contains a Kozak sequence with no additional ATG motifs upstream of the Kozak sequence that could function as unintended start codons. As well, stable mRNA secondary structures that include the authentic ATG containing Kozak sequence are eliminated since they may reduce translation by prevention of Kozak sequence mediated ribosome recruitment. An intron within the eukaryotic region 5' UTR improves transgene expression [2]. Intron splicing and ultimately transgene expression may be further improved by intron optimization through the addition of splicing enhancers within and flanking the intron [30,31,32]. Insertion of the human T-cell leukemia virus type I R region (HTLV-I R) 5' UTR downstream of the CMV promoter enhances mRNA translation efficiency and further increases transgene expression in mice and nonhuman primates [28,33]. HTLV-I R encoding DNA vaccines have an excellent safety profile established in multiple human clinical trials [7]. To prevent transgene-directed dsRNA formation that may result in RNA interference mediated transgene silencing, bacterial region sequences should not contain cryptic eukaryotic promoters oriented antisense to the transgene [20]. Comparing expression between different constructs in mammalian cells to select an optimal vector must be done carefully since transgene mRNA levels can easily saturate protein production capacity in vitro [34]. Interestingly, minimalization of the bacterial region has recently been demonstrated to improve transgene expression (see Section 5.2). Manufacture/quality: First generation vectors were not optimized for production yield and quality, which can impose significant cost post-licensure. Ideally a plasmid is predominantly monomer with a low propensity for nicking or rearrangement during fermentation, or nicking or denaturation during extraction and downstream purification. Unusual DNA sequences such as runs of homopurine-homopyrimidine tracts, inverted or direct repeats may be prone to instability. Palindromes are unstable and reduce plasmid copy number. AT-rich sequences and cruciforms increase the frequency of plasmid nicking, while Chi sites mediate plasmid multimerization [22,23,35]. Cryptic bacterial promoters within the eukaryotic promoter region may lead to inappropriate expression of the transgene in the bacterial host. In many cases this will be toxic [22], reducing plasmid stability and production yields and will require the creation of designer strains that express transgene-complementary RNA from the bacterial chromosome to prevent translation of the toxic protein [36]. The presence of such undesired sequences in a vector (or gene insert, see Section 2.3) may be identified using bioinformatics [22,37]. The use of antibiotic resistance markers in DNA vaccines has potential regulatory safety concerns. These include production mediated environmental contamination with either antibiotics used in fermentation culture or the plasmid borne antibiotic resistance markers [38], treatment associated transfer of antibiotic resistance to a patient’s endogenous microbial flora (e.g., transfection of skin resident microorganisms with topically applied plasmid DNA), or activation and transcription of the marker from host cell promoters after spurious incorporation into the cellular genome after transfection of the patient’s cells. Selection using ampicillin during production is generally not acceptable due to potential hyper reactivity to residual trace β lactam antibiotics in the product. The European Pharmacopoeia indicates that “Unless otherwise justified and authorised, antibiotic-resistance genes used as selectable genetic markers, particularly for clinically useful antibiotics, are not included in the vector construct. Other selection techniques for the recombinant plasmid are preferred” [39]. The European Medicines Agency (EMA) has further concluded that kanamycin and neomycin are of importance for veterinary and human use and cannot be classified as having minor therapeutic relevance due to current use in critical clinical settings [27]. To address these regulatory concerns, alternative non-antibiotic selection methods are needed. The use of any protein-based selection marker raises the concern that it may be unintentionally expressed and translated in the vaccinated organism. While a number of antibiotic-free (AF) plasmid retention systems have been developed in which the vector-encoded selection marker is not protein based [22,40] superior expression and manufacture has been observed with DNA vaccine vectors that incorporate RNA based antibiotic-free selection markers. For example, the NTC8385 sucrose selection vector (Figure 2c) encodes RNA-OUT, a small 70 bp antisense RNA (Figure 3a) [41]; pFAR4 and pCOR vectors encode a nonsense suppressor tRNA marker (Figure 3c) [42,43], while the pMINI vector utilizes the ColE1 origin-encoded RNAI antisense RNA (Figure 3b) [44,45]. These plasmid borne RNAs regulate the translation of a host chromosome encoded selectable marker allowing plasmid selection (Figure 3). Of these, high yield fermentation processes (>500 mg/L) have been developed for RNA-OUT vectors (1,800 mg/L; [46]) and pMINI (900 mg/L; [47]). In all these vectors, replacement of the kanR antibiotic selection marker resulted in increased transgene expression in the target organism (see Section 5) demonstrating elimination of antibiotic selection to meet regulatory criteria may unexpectedly also improve product performance (reviewed in [48]). RNA selectable marker DNA vaccine plasmids. Purple arrow in bacterial region is pUC replication origin, brown arrow in panels (a) and (c) is the RNA selection marker. Eukaryotic region promoter, transgene and polyA are depicted with orange arrow, blue arrow and green box, respectively. (a) NTC8385 plasmid borne RNA-OUT RNA binds a chromosomally encoded constitutively expressed mRNA that contains the RNA-IN target sequence in the leader. This prevents translation of the downstream levansucrase (sacB), allowing growth on sucrose media; (b) pMINI pUC origin encoded RNAI binds a chromosomally encoded constitutively expressed mRNA that contains the RNAII target sequence in the leader. In the murselect-system, an essential gene (murA) is modified to contain a repressor binding site in the promoter and the RNAII target sequence is incorporated into the repressor mRNA leader. RNAI binding to RNAII prevents repressor translation, allowing expression of the essential gene; (c) pFAR4/pCOR plasmid borne suppressor tRNA allows read-through translation of an amber nonsense codon in a chromosome encoded essential gene. Adapted from Oliveira and Mairhofer, 2013 [48].DNA vaccines, due to in vivo antigen expression, have the advantage that vaccinologists may easily customize encoded antigens through rational transgene design. Commercial gene synthesis has become rapid and inexpensive, thus enabling DNA vaccine antigen design, synthetic codon optimized antigen gene synthesis and vaccine manufacture on a highly compressed timeline. Antigen transgenes for inclusion in DNA vaccines may be an exact copy of the original antigen or a modification to improve efficacy or safety. Antigens may be altered to inactivate enzymatic activity, remove potentially oncogenic sequences or attenuate virulence. Mutations to reduce DNA binding may mitigate concern that immune responses against DNA will be induced by protein/nucleic acid complexes. Alternatively, the antigen may be extensively engineered for immunogenicity using structure based antigen design [49]. For pathogens that contain multiple serotypes, rather than using multiple plasmids, engineering a single broadly cross neutralizing antigen is a possibility. Two technologies that may be used to accomplish this are a bioinformatics approach that generates a consensus immunogen [50] or a directed molecular evolution approach that uses molecular breeding to evolve genes through an iterative process consisting of recombinant generation in vitro followed by selection of cross neutralizing recombinants [51]. Adaptive immune responses may be improved by enhancing antigen processing and MHC class I and/or class II presentation [1,2]. This can be accomplished by the addition of a targeting peptide that routes antigens to various intracellular destinations. DNA vaccine antigens are most commonly targeted to the secretion pathway using a signal peptide [52]. This may use a heterologous secretion signal, or, in the case of a secreted protein, the native secretion signal. Use of an optimized signal sequence may dramatically improve expression over the native sequence. Improvement has been observed using an optimized tissue plasminogen activator (TPA) signal peptide [52,53,54] or IgE gene leader [2]. An optimized heterologous secretion tag is often included in DNA vaccine vectors and the transgene is cloned downstream and in frame with the signal peptide [18]. Alternatively the signal peptide may be included when designing the synthetic gene. In some DNA vaccines, proteosomal targeting using an N-terminal ubiquitin tag (terminal ubiquitin G76 residue altered to A76 to destabilize the fusion protein) is used to promote MHC class I antigen presentation [55] while endosomal targeting by transgene insertion within the LAMP protein is used to promote MHC class II antigen presentation [56]. To experimentally determine optimal antigen targeting, a family of antigen targeting, RNA selectable marker (RNA-OUT), optimized DNA vaccine plasmids with compatible cloning into vectors encoding either N-terminal TPA signal peptide (secretion targeting), N-terminal and C-terminal LAMP1 (endosomal targeting) or N-terminal destabilizing Ubiquitin A76 (proteosome targeting) are commercially available (Nature Technology Corporation, Lincoln, NE, USA). Many other targeting tags have also been described [1,22] including transgene fusion N-terminal to strong immunogens that contain MHC class I and/or MHC class II binding peptides. Fusion to MHC class II peptides that induce CD4+ T-cell help may improve antibody or cytotoxic CD8+ T cell responses [57].Once the antigen protein sequence is finalized, a synthetic gene sequence is designed (Figure 1b), synthesized and cloned into a vector downstream of a consensus Kozak sequence to ensure efficient translation. The optimized protein sequence is reverse translated into a gene sequence, selecting optimal codon usage for the target species. Codon optimization to match high use codons for the target species has been shown to dramatically increase transgene expression [53,58]. Elimination of extensive RNA secondary structure is also important. Some codon optimization programs such as the GeneArt GeneOptimizer® Process combine RNA and codon optimization [58]. An important consideration is that RNA secondary structure between the synthetic gene and the vector 5' UTR is not screened by gene synthesis companies. Secondary structure between the synthetic gene and the 5' UTR encoded Kozak sequence may interfere with ribosome recruitment and reduce transgene expression. Such hybrids may be detected using a program such as mfold [59]. The gene sequence is synthesized and cloned into the DNA vaccine vector backbone.Synthetic gene design is critical. FDA guidance indicates “biodistribution studies may be waived for DNA vaccines produced by inserting a novel gene into a plasmid vector previously documented to have an acceptable biodistribution/integration profile” [25]. Thus a new gene to be inserted into a previously validated vector must not create regulatory concerns due to trivial design issues. New synthetic genes should be screened using the same criteria as described above for vector design to eliminate unusual DNA sequences (e.g., G quadruplex), inverted or direct repeats, palindromes, AT-rich sequences and cruciforms, Chi sites and cryptic bacterial promoters that could affect plasmid quality. Additionally, it is critical to ensure no cryptic splice acceptor or donor sites (sense orientation), polyadenylation sites (AATAAA or ATTAAA), or eukaryotic promoters (both orientations) are present within the insert, since this could result in the generation of aberrant peptides causing regulatory concern [2,22]. Complementary strand promoters would transcribe mRNA that would anneal to the transgene mRNA to create dsRNA that may silence transgene expression. Additionally, as a precaution to reduce potential regulatory agency concern regarding the theoretical risk of insertional mutagenesis of the host genome, large tracks of sequence homology to the target organism identified using the National Center for Biotechnology Information (NCBI) BLASTN program should be removed. A codon optimized synthetic gene typically contains regions with only short tract homology of less than 20 bps of perfect identity to a target genome. These short tracts of homology should not be an issue since characterization of plasmid DNA integration into the genome using repeat-anchored integration capture (RAIC) PCR has demonstrated short homology driven integration events are extremely rare [60]. See Figure 1b and [22] for a detailed insert design flowchart. Several critical factors should be considered prior to large scale cGMP manufacturing for clinical investigations, including product purity and homogeneity (i.e., percent covalently closed monomeric plasmid DNA) specifications, product concentration and formulation, and projected quantities needed for clinical trials and commercialization. Unfortunately, most first generation DNA vaccine vectors are not optimized for fermentation yield and homogeneity and are nicking or dimerization prone [22]. Poor quality is a critical problem, since vector redesign and sequence modification to improve quality to meet clinical specifications may necessitate additional expensive non-clinical toxicology testing to be performed which would delay clinical evaluation. Poor production yield is problematic down the road since it will impose significant cost burden post-licensure. In general, plasmid quality and yield is higher from fed-batch rather than batch fermentation. A few high yield fed-batch plasmid fermentation processes (500–2,600 mg/L) have been described. These processes all couple reduced growth rate (which generally increases copy number) with high copy replication origins [61]. One of these, the patented HyperGROTM inducible fed-batch fermentation process [62], has been utilized to manufacture clinical grade DNA for various plasmids and is generally available for commercial production of research grade (Nature Technology Corporation, Lincoln, NE, USA) or clinical grade plasmid DNA through licenses to several cGMP plasmid manufacturers, including Aldevron, Eurogentec and VGXI. HyperGROTM incorporates novel cell bank and fermentation process innovations that reduce plasmid mediated metabolic burden allowing generic production of a wide range of plasmids with low levels of dimerization or nicking and high fermentation productivity up to 2,600 mg/L [63]. High plasmid homogeneity in the fermentation harvest is critical, since removal of nicked plasmid and dimers is extremely difficult due to similar properties to the desired supercoiled plasmid monomer product. Likewise, high yield is important since increased plasmid yield per gram of bacteria results in improved final product purity [61]. An alternative commercially available high yield fermentation process has been developed by Boehringer Ingelheim and is available for clinical production of plasmid DNA vaccines at their facilities [64].Following fermentation, plasmid DNA is typically extracted using alkaline lysis. Alkaline lysis is difficult to scale, but a number of companies have developed mixing methodologies that remove host cell DNA fragments without denaturing or nicking plasmid DNA. Most commercial manufacturers have developed downstream purification processes that maintain plasmid quality while removing impurities such as endotoxin, genomic DNA, bacterial RNA, and nonsupercoiled plasmid isoforms, for example, anion exchange chromatography followed by hydrophobic interaction chromatography. The reader is directed to several detailed reviews of downstream plasmid purification [61,65,66,67,68]. Combining a fermentation process such as HyperGROTM, that generates high quality supercoiled monomer plasmid with low dimerization and nicking, with an alkaline lysis extraction-downstream purification process optimized to not denature or nick plasmid DNA will provide a plasmid product that will meet stringent plasmid homogeneity specifications [46]. The downstream purification process must remove impurities such as protein, RNA, chromosomal DNA, and endotoxins to acceptable levels. Of these, chromosomal DNA is the most difficult to remove due to similar properties to the plasmid product; thus optimization of alkaline lysis to prevent chromosomal DNA extraction is critical since poor alkaline lysis can result in elevated levels of chromosomal DNA in the final product. While impurity levels for clinical investigation may be relatively easy to achieve (typically <1% protein, RNA, chromosomal DNA impurities, <10 endotoxin units/mg plasmid [61]) the final specifications for protein and chromosomal DNA for commercial use may be tighter since licensed protein products have much lower residual host protein (typically <100 ppm) and gDNA (typically <100 pg/dose) limits [61]. The final commercial specifications may depend on dose, delivery and regulatory agency input. E. coli derived impurities may also detrimentally affect vaccine performance. For example, genomic DNA has been shown to cause skeletal muscle damage after hydrodynamic limb vein delivery [69] and inflammation after lipoplex gene delivery to the lung [70]. Colanic acid polysaccharide impurities in plasmid DNA cause acute toxicity after intravenous injection of plasmid liposome complexes [71]. Critically, impurities such as genomic DNA, ribosomal RNA or endotoxin are ligands of various innate immune receptors. Thus the presence of these impurities may activate innate immunity, inflammation responses, and alter adaptive immunogenicity in a lot to lot, or species-specific fashion. For example, the orphan murine receptor TLR13 triggers cytokine secretion in response to bacterial ribosomal RNA [72]). Potency assays (in vitro and/or in vivo) are product specific and are designed to measure the biological activity of each DNA vaccine lot versus a reference standard to ensure lot to lot vaccination consistency. Typically for early clinical development, an in vitro assay measuring transgene expression after transfection is proposed as a surrogate for immunogenicity [73,74]. However, evidence to support correlation of in vitro expression with in vivo immunogenicity may be required.Plasmid DNA production is typically performed in endA (DNA-specific endonuclease I), recA (DNA recombination) deficient E. coli K12 strains such as DH5α, DH5, DH1, XL1Blue, GT115, JM108, DH10B, or endA, recA engineered derivatives of alternative strains such as MG1655 [75] or BL21 [22,63,76]. Replication of pUC origin plasmids is dependent entirely on multiple E. coli host strain encoded factors [77]. Host encoded replication protein expression level variations between strains likely accounts for observable differences in plasmid properties such as percent open circular plasmid, steady state supercoiling density, catenation, multimerization and yield [78]. For example, high levels of open circle plasmid may be indicative of incomplete replication since plasmids retaining the RNA primer are nicked during alkaline lysis. Variations in open circular plasmid levels between strains may reflect altered levels of DNA Pol I and DNA ligase, since these enzymes are required to remove the replication initiating RNA primer, and create a covalently closed circular (CCC) plasmid, respectively [77]. Different plasmid isoforms may have altered transfection efficiency, intracellular stability, nuclear transfer rate, or promoter activity [22,63] that may dramatically affect transgene expression in vivo so it is critical to control DNA vaccine plasmid production conditions to ensure consistent product quality and in vivo performance during preclinical and clinical development.Significantly, varying production conditions may affect host replication protein expression levels and/or activity which may alter plasmid properties [63]. Negative supercoiling, the under-winding of a DNA strand, is an epigenetic modification that may affect plasmid manufacture and transgene expression. The actions of DNA gyrase (gyrA, gyrB), which increases negative supercoiling, along with relaxing enzymes Topoisomerase I (topA) and Topoisomerase IV (parC, parE; also essential for unknotting plasmid catenates [79]) sets the steady state supercoiling density (σ) [80,81]. σ varies between strains and growth conditions such as growth temperature [82], growth phase [83] and can be perturbed by environmental stress such as nutrient limitation [84] or high temperature spikes during production [85]. Rapidly replicating pUC plasmids in fermentation cultures may not complete replication or reach steady state σ; introduction of a post plasmid production hold step at low temperature to reduce pUC plasmid replication initiation allows completion of initiated replication cycles and plasmid supercoiling to physiological levels [77]. Altering σ may alter transgene expression due to changed susceptibility to stress-induced duplex destabilization (SIDD). SIDD sites are found within transcriptional regulatory regions such as promoters [86] and origins of replication. The activity of many promoters is affected by σ alterations that change the susceptibility to SIDD [87]. Different steady state σ levels may also alter plasmid manufacturing yields between strains. Small plasmids less than 3 kb in size often have poor fermentation yields in standard strains such as DH5α, but can be produced to high yield in XL1Blue [21]: the increased negative supercoiling in XL1Blue relative to DH5α may alter the pUC origin susceptibility to SIDD, facilitating replication of small plasmids [21]. It is critical therefore that the plasmid supercoiling density be maintained during fermentation scaleup and clinical development by tight manufacturing control. Altering epigenetic DNA methylation may also affect transgene expression [63]. While all standard plasmid production strains encode epigenetic dam nucleotide methylation at GATC residues, plasmid from different strains may differ in: (1) strain-specific epigenetic dcm nucleotide methylation (at CCWGG; BL21 and GT115 are dcm-) and (2) negative supercoiling density as described above. From a regulatory perspective, a plasmid with modified epigenetic methylation is a distinct chemical entity and therefore a different product. It is critical therefore that the optimal strain/methylation for plasmid manufacture and performance be identified prior to product definition and subsequent clinical development. In summary, methylation and supercoiling should be monitored during production scaleup since plasmids may have altered biological properties (potency) due to incomplete methylation [63] or nonphysiological supercoiling [21]. Incomplete dam or dcm methylation may be detected by restriction endonuclease digestion (dam: Sau3A cleaves all sites, MboI cleaves unmethylated sites, DpnI cleaves methylated sites; dcm: BstNI cleaves all sites, EcoRII cleaves unmethylated sites) [63] and supercoiling linking number by chloroquine agarose gel electrophoresis [21].Different strains have host chromosome encoded transposons that under stress conditions may transpose into plasmids, for example, IS1 into the neomycin resistance marker promoter during strain adaptation to defined media [88]. This generates a heterogeneous product of plasmid with and without insertion elements which is unacceptable for clinical use [89]. The HyperGROTM cell banking and fermentation process is designed to reduce metabolic stress, and has been shown to not induce IS1 transposon mobilization during cell banking or fermentation unit operations [63]. Extensive research over the last two decades has identified intracellular DNA sensing pathways and mechanisms by which DNA vaccines activate these pathways to induce adaptive immunity (reviewed in [90,91,92]). The application of this knowledge to create strategies to improve DNA vaccine immunogenicity is discussed below.Studies using knock-out mice deficient in various innate immune receptors and signaling molecules have determined that most of the “adjuvant effect” of DNA vaccination is mediated by activation of the cytoplasmic double stranded DNA sensing stimulator of interferon genes/TANK-binding kinase 1 (STING/TBK1) dependent innate immune signaling pathway (Figure 4; reviewed in [93]). This is the primary pathway necessary to induce antigen specific B cells and CD4+ T-cells in response to DNA vaccination. However, several studies have demonstrated a role of endosomal sequence specific CpG DNA sensing Toll-like receptor 9 (TLR9) signaling in priming CD8+ T cell responses [94,95]. Cationic liposome delivered plasmid DNA clearly activates a CpG dependent inflammation response in the lung [96], so the contribution of TLR9 to DNA vaccination induced adaptive immunity may be tissue and delivery specific. Cytoplasmic DNA may also activate the absence in melanoma 2 (AIM2) inflammasome [97], but a role of inflammasome activation and the resultant caspase 1 mediated interleukin-1β production in DNA vaccine immunology has not been established.Molecular mechanisms of DNA vaccines. Transfected B DNA (the most common double helical DNA structure) is sensed in the cytoplasm (cyto) by DNA receptors interferon-inducible protein 16 (IFI16) and DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41) activating the cGAMP synthase (cGAS) [98] /STING/TBK1 pathway to induce type 1 interferon production and NF-κB. An additional cytoplasmic innate immune pathway activated by transfected DNA is the cytoplasmic AIM2 inflammasome. IFI16, DDX41 and AIM2 detect DNA generically and are not sequence specific although IFI16 may preferentially recognize DNA that forms cruciforms or is negatively supercoiled [99]. By contrast, specific CpG motifs in DNA vaccines are sensed by the endosomal (endo) TLR9 innate immune receptor. To improve innate immune activation, addition of optimized immunostimulatory CpG motifs in the vector backbone may be used to increase TLR9 activation while immunostimulatory RNA expressed from the vector may be utilized to activate alterative RNA sensing innate immune receptors such as RIG-I (plasmid backbone adjuvant). Due to limited transgene expression after DNA vaccination in large animals, vector modifications and deliveries that improve transgene expression also improve adaptive immunity. Certain delivery modalities such as EP that improve gene transfer efficiency also activate innate immunity through tissue damage [100,101,102]. EP conditions need to be carefully optimized, since the optimal EP conditions for DNA vaccination are not necessarily those with the highest gene expression [103] and optimal delivery parameters vary between strains [100]. DNA vaccination efficacy may be improved by codelivery by a plasmid encoding adjuvant proteins (Figure 4). Numerous adjuvant plasmids have been developed, including those that express cytokines (e.g., interleukin-12), chemokines (e.g., RANTES), costimulatory molecules (e.g., CD40), or signaling molecules [e.g., interferon regulatory factor-3 (IRF3)] (reviewed in [93,104,105]). An alternative approach is to modify the vector backbone to encode DNA or RNA based adjuvants (plasmid backbone adjuvant; Figure 4). Such modifications avoid the autoimmunity concerns from expressing a human protein and do not limit boosting or multiple product development since the backbone encoded DNA or RNA adjuvant will not be the target of adaptive immunity. As well, this antigen expressing cell-targeted limited immunostimulation approach using backbone modified vectors is safer than nonspecific global stimulation by coadministering a large adjuvant dose such as TLR9 CpG agonist or Melanoma Differentiation-Associated protein 5 (MDA5)/TLR3 agonist poly I:C. Since the ligands for most characterized DNA sensing pathways are not sequence specific, research to improve DNA vaccine immunogenicity by adding DNA motifs has focused on addition of CpG TLR9 agonists. This is complicated by the fact that the flanking sequence determines if a CpG motif is immunostimulatory or immunosuppressive, and that optimal CpG agonists are species specific [106]. Results to date have been variable, but improved immune responses with DNA vaccines incorporating additional CpG motifs have been obtained [107,108]. Some of the variability in response is probably due to unintended alterations of transgene expression from the CpG motif modified vector backbone (see Section 2) as well as differences between delivery modalities in efficiency of endosomal trafficking of CpG motif containing DNA vaccines for TLR9 activation. An alternative approach is to engineer the vectors to coexpress immunostimulatory RNA (isRNA) with antigen. The isRNA is transcribed by either RNA Pol II (isRNA encoded either downstream of transgene in the 3' UTR or in a second transcription unit) or RNA Pol III (isRNA transcribed independently from transgene in the vector backbone). Both RNA Pol II and RNA Pol III expressed isRNA have been shown to improve DNA vaccination induced antigen-specific humoral and/or cellular response [109,110,111]. Increasing DNA vaccine-mediated transgene expression improves immune response in large animals and humans [2]. Recently, as highlighted below, dramatically improved vector expression has been obtained by bacterial region minimalization.A number of bacterial sequences have been shown to inhibit transgene expression in eukaryotic cells [22] (see Section 2). For example, the TN5 encoded kanamycin/neomycin resistance marker is a potent transcriptional silencer that decreases expression from linked eukaryotic promoters [112]. The pVAX1 vector (Figure 2b) has a 1,970 bp bacterial region (spacer between the eukaryotic region polyA and CMV promoter) including this TN5 kanR marker. Dramatically increased expression has been observed with antibiotic-free RNA selection marker pVAX1 derivative vectors, in which the kanR marker is replaced with RNA-OUT (pVAX1-AF, 1,195 bp spacer region) [21], amber suppressor t-RNA (pFAR4, 1,040 bp spacer region) [43] or removed (utilizing the pUC origin RNAII marker for selection; pMINI, 734 bp spacer region) [113] (see Section 2.2 and Figure 3). An alternative interpretation of the improved expression with RNA selection marker retrofitted pVAX1 vectors is that improved expression is due to the reduced vector size. All these vectors encoding transgene are >2,000 bp, which should not have significantly improved cytoplasmic mobility compared to larger plasmids [114]. However, smaller vectors are more effectively transfected into the cell, leading to higher transgene expression [115,116]. Bacterial regions of approximately 1,000 bp or larger, mediate transgene silencing in certain tissues (e.g., liver), while minicircle vectors, containing shorter spacers ≤500 bp, have sustained transgene expression [117]. Silencing may be mediated by the formation of inhibitory chromatin on nontranscribed spacer region sequences [118]. Transcription of a bacterial region in eukaryotic cells using a heterologous promoter improved transgene expression duration [119]. Short bacterial region DNA vaccine vectors may therefore have application to increase antigen expression duration. Persistent antigen expression may improve memory CD8+ T-cell maintenance [120,121]. Consistent with that, sustained expression minicircle vectors elicit superior CD8+ T cell responses compared to plasmid vectors [122]. Minicircle vectors are manufactured from plasmid vectors in the E. coli host via the action of phage recombinases on recognition sequences in the plasmid to create circularized bacterial and eukaryotic regions (minicircle) which are then separated. Minicircle vectors are not practical for DNA vaccine applications since production procedures are very inefficient with optimal reported yields of only 5 mg minicircle per liter culture [123].The pMINI, pFAR4 and NTC8385 RNA selection marker vectors all utilize the pUC origin for selection which can be minimalized to 700 bp without compromising high copy number replication. Replacement of the pUC origin in NTC8385 (Figure 2c) with a minimalized pUC origin (NTC8385-min) reduced the origin-RNA-OUT bacterial region from 1,518 bp to 855 bp. Further reduction of bacterial region size to 454 bp was obtained by replacing the pUC origin with a 300 bp mini-origin (NTC9385R; Figure 2d). The NTC9385R vector bacterial region is below the size limit that mediates transgene silencing in minicircle vectors [117]. Surprisingly, transgene expression level is also dramatically improved with this short spacer region vector (Figure 2e,f) [124]. Similarly improved transgene expression, 2 to 10 fold higher than conventional vectors, have also been observed with novel vectors that contain no spacer region, in which an RNA-OUT-replication origin bacterial region is encoded within an intron of the eukaryotic transcription unit [125,126]. While the mechanism to explain transgene expression enhancement with short spacer region vectors is not clear, these novel vectors have exciting application to improve DNA vaccine performance through improved expression level and duration.DNA vaccines are a new generation biotechnology product that is beginning to enter the marketplace. While not critical in murine models, increased antigen expression correlates with improved immunogenicity in humans and large animals [2]. As reviewed herein, next generation vector designs have been developed that improve antigen expression, manufacturing yield and quality, and regulatory compliance. Application of these improved vectors and high yield manufacturing methodologies will be critical to ensure efficacy, safety and cost effective manufacture of future DNA vaccine products.The author thanks Justin Vincent for preparing the Figures and Aaron Carnes for reviewing the manuscript. James Williams has an equity interest in Nature Technology Corporation. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Since introduction of the human papillomavirus (HPV) vaccine, there remains low uptake compared to other adolescent vaccines. There is limited information postapproval about parental attitudes and barriers when presenting for routine care. This study evaluates HPV vaccine uptake and assesses demographics and attitudes correlating with vaccination for girls aged 11–12 years. A prospective cohort study was performed utilizing the University of Virginia (UVA) Clinical Data Repository (CDR). The CDR was used to identify girls aged 11–12 presenting to any UVA practice for a well-child visit between May 2008 and April 2009. Billing data were searched to determine rates of HPV vaccine uptake. The parents of all identified girls were contacted four to seven months after the visit to complete a telephone questionnaire including insurance information, child’s vaccination status, HPV vaccine attitudes, and demographics. Five hundred and fifty girls were identified, 48.2% of whom received at least one HPV vaccine dose. White race and private insurance were negatively associated with HPV vaccine initiation (RR 0.72, 95% CI 0.61–0.85 and RR 0.85, 95% CI 0.72–1.01, respectively). In the follow-up questionnaire, 242 interviews were conducted and included in the final cohort. In the sample, 183 (75.6%) parents reported white race, 38 (15.7%) black race, and 27 (11.2%) reported other race. Overall 85% of parents understood that the HPV vaccine was recommended and 58.9% of parents believed the HPV vaccine was safe. In multivariate logistic regression, patients of black and other minority races were 4.9 and 4.2 times more likely to receive the HPV vaccine compared to their white counterparts. Safety concerns were the strongest barrier to vaccination. To conclude, HPV vaccine uptake was higher among minority girls and girls with public insurance in this cohort.Human papillomavirus (HPV) is the most common sexually transmitted disease in the United States and is associated with 99% of all cervical cancers [1,2]. In the United States in 2012, approximately 12,170 women were expected to be diagnosed with cervical cancer, and 4,220 died of their disease [3]. Infection with HPV starts at a young age with estimates of cervical HPV prevalence in women aged 18–25 ranging from 27% to 60% [4,5]. Racial disparities result in differential rates of cervical cancer and mortality from cervical cancer. In Virginia, black women have a higher risk of cervical cancer than white women and are twice as likely to die of the disease [6].A vaccine to protect against human papillomavirus was first introduced in 2006 in the United States (Gardasil®, Merck & Co, Inc., Whitehouse Station, NJ, USA). This quadrivalent HPV vaccine protects against viral types 6, 11, 16, and 18, which are known to cause approximately 90% of genital warts and 70% of cervical cancer [7,8]. Cervarix® was FDA approved in 2009 and protects against HPV 16 and 18 (GlaxoSmithKline, Brentford, UK). In March 2007 the Center for Disease Control (CDC) recommended that all girls aged 11–12 years old receive the HPV vaccine as part of their routine adolescent vaccinations [9]. Other vaccines recommended for this adolescent age group include the meningococcal, tetanus, diphtheria and pertussis booster (Tdap), and varicella (if the child has not been infected with chickenpox).It is imperative that the HPV vaccine reach all populations in order to significantly impact rates of cervical cancer. The National Immunization Survey for Adolescents (NIS-Teen) documents adolescent immunization for ages 13–17. These data show national HPV vaccination rates fall below recommended levels and are significantly lower than other adolescent vaccinations [10]. Previous studies have demonstrated that parental decisions to have their children vaccinated are associated with knowledge about HPV, physician recommendation of the vaccine, and belief in the safety and efficacy of the HPV vaccine [11]. A recent in depth analysis of the NIS 2008 regarding HPV vaccination, the first year of HPV vaccination reporting, demonstrated differential rates of vaccination completion by race with minority girls less likely to complete the series [12]. Further, well child visits, insurance status, and provider recommendation were noted to be associated with vaccination [13]. The most recent NIS-Teen data shows differential rates of HPV vaccine uptake by race and socioeconomic status with minority races and low SES having statistically higher rates of HPV vaccine initiation [10]. However, further analysis is still pending regarding the reason behind these trends. In Virginia, doctors reported that cost and reimbursement were the most frequently encountered barriers preventing vaccination [14].In March 2007, the Virginia governor signed into law House Bill 2035 (identical to Senate Bill 1230) requiring the HPV vaccine for girls on or after their 11th birthday but allowing parents to exempt their child via a verbal opt out effective 1 October 2008 [15]. Given that the mandate did not go into effect until October 2008, it did not change school admission requirements until the academic year beginning August 2009. This is the only school mandate in the country and it remains to be seen if it will achieve higher rates of vaccine uptake and decreased racial differences seen with other vaccine mandates [16,17].This study was conducted to determine whether insurance status and other demographic factors play a role in HPV vaccine uptake and completion among girls presenting for a well-child visit at age 11–12 years old. This study also documents baseline vaccination rates for the year prior to enactment of the Virginia school mandate.This prospective cohort was developed by searching the University of Virginia Clinical Data Repository (CDR) at 3 month intervals for all girls aged 11–12 years presenting for a well-child visit between May 2008–April 2009. These dates were chosen to capture the vaccination uptake for the school year prior to enactment of the Virginia HPV vaccine mandate. This included all patients seen at UVA-supported family medicine practices and pediatric practices in a 50 mile radius including urban, suburban, and rural populations. Clinical and demographic data were also collected, including age, race, insurance status, and vaccination status for HPV, Meningitis, Tdap, and Varicella. The primary outcome was HPV vaccine uptake as defined by receipt of ≥1 dose confirmed by billing data. Final collection of HPV vaccine uptake and number of injections received was collected six months after the last identified well-child visit. Of note, this study only evaluated girls as the HPV vaccine had not yet been approved for use in boys at the time of data collection.To provide greater in depth analysis of these findings, the parents of these girls were then contacted 4–7 months after the initial well child visit to allow for time to complete the HPV vaccination series in three batched samples. A cohort of parents/guardians of the original population agreed to participate in the telephone questionnaire.A 50-item telephone questionnaire was developed that took the parents approximately 10 min to complete. The questionnaire included validated questions on insurance status and demographics, previously studied questions on HPV vaccination attitudes and behaviors designed utilizing constructs from the Health Belief Model [18], as well as additional questions regarding other vaccination status, relationship and trust in the provider, and usual health information sources. Lastly, previously studied HPV knowledge questions were utilized. The questionnaire was piloted on a subset of parents who were not included in the final data analysis. All questions referred specifically to the quadrivalent vaccine as the bivalent vaccine was not available in these locations.Parents were contacted first with an advance letter and either called in to the survey center or verbally consented to participate over the telephone. The University of Virginia Center for Survey Research (CSR) trained all interviewers and conducted all interviews and all answers were entered to a database for later data analysis. The parent or guardian who said they were the most familiar with the child’s health history was asked to participate in the survey. To reduce non-response bias CSR made several efforts at “conversion calling” for households where a potentially eligible respondent had refused to participate once or twice.Data analysis included frequency distribution of race, insurance status, and vaccination status in the original population and the cohort participating in the telephone questionnaire using SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA) and SAS, version 9.1 (SAS, Cary, NC, USA). The primary outcome of HPV uptake ≥1 dose received was converted to a binary variable. Associations were determined by chi-square test for categorical variables with p < 0.05 considered statistically significant. Relative risk and 95% confidence intervals were calculated for all cohort data in 2 × 2 tables. Likert scales to assess vaccination attitudes were dichotomized for agree or disagree with the attitude. After preliminary analysis, demographic, knowledge, and attitude data from the telephone questionnaire were utilized in multivariable logistic regression. All factors significantly associated with HPV vaccine uptake or refusal (p < 0.1) were retained in the model. The final model was fit with backwards stepwise elimination of nonsignificant variables. Sensitivity analysis was conducted to verify the relationship between the variable and the outcome of HPV vaccine uptake.Based on demographics of the pediatric population at UVA in 2008, 60% of patients had private insurance. An HPV vaccination rate of 40% based on the National Immunization Survey from 2008 was used as a baseline assumption [19]. In order to detect a difference in vaccination rates of 15% among privately insured patients compared to publically insured or self-pay patients with 80% power, a sample size of 370 patients was calculated.Five hundred and fifty 11–12 year old female patients were identified in the total population of girls seen for a well-child visit between May 2008–April 2009. Of these patients, 72.5% were nonhispanic white race, 53% had private insurance, and 48.2% were vaccinated against HPV during the study period (Table 1).Demographics of total population compared to cohort of telephone interview respondents.In this population, there were noted to be significant associations of HPV vaccination with insurance status and race. Girls with private insurance were less likely to receive the HPV vaccine compared to girls with public insurance or self-pay. Conversely, girls with public insurance were 36% more likely to be vaccinated as compared to girls with private insurance (Table 2).Human papillomavirus (HPV) vaccine uptake (≥1 dose received) by insurance and race in population of 11–12 year old girls presenting for well child visits between May 2008–April 2009.* Compared to publically insured or self-paying patients, Patients with unknown insurance excluded given heterogeneity of the group. † Compared to privately insured patients. § Statistical comparisons not performed given small numbers in groups. Compared to black or other race. ** Compared to nonhispanic white race.Further, nonhispanic white girls presenting for well child visit were overall almost 30% less likely to initiate vaccination compared to all minority girls (RR 0.72, 95% CI 0.61–0.85). Compared to nonhispanic whites, black girls were 36% more likely to have received at least 1 dose of the HPV vaccine. There were no significant differences in HPV vaccination completion rates by race among those who received at least one dose with 84/172 (49%, 95% CI 41%–56%) of nonhispanic whites and 71/154 (46%, 95% CI 38%–54%) of minority girls completing the three injection series.All 550 families from the original population were considered for participation in the telephone questionnaire (Figure 1).Diagram of cohort originating from study population.Four hundred and thirty-two households with a working number were approached for participation in the questionnaire and 242 parents or guardians agreed to participate in the telephone interview for a response rate of 56%. (Of note, 219 of the 242 participants were parents. Thus, the survey respondents are referred to as parents even though 23 individuals were guardians of other familial description.) See Table 1 for demographic comparisons of the cohort participating in the telephone questionnaire compared to the entire population.All parents confirmed the child had a recent visit to the doctor. Eighty-nine percent of participants went with the child to the documented visit, of whom 79% remember the doctor discussing the HPV vaccine. The majority of respondents confirmed that the child had been seeing this physician for 5 years or greater.Overall, 92% of parents were aware of the HPV vaccine, and 85% reported that the HPV vaccine was recommended for their child. In regards to HPV vaccine knowledge, 69% of respondents agreed that the vaccine protects against cervical cancer, but only 20% also identified that the quadrivalent vaccine protects against genital warts. The majority of respondents (59%) agreed that the HPV vaccine is safe. Twenty percent of parents felt that their child did not need the HPV vaccine. There were no statistically significant differences in these attitudes compared by race.After combining billing data with parental report, vaccine uptake rates were compared by race. Blacks and other races were significantly more likely than whites to have received at least one HPV vaccine injection (75.7% and 68.4% vs. 47.5%, respectively; p = 0.003). By comparison, whites were more likely to have received the meningococcal vaccine (74.3% vs. 59.5% and 52.6% respectively; p = 0.043), and rates were not significantly different for Tdap and varicella vaccines (Figure 2).Vaccination rates of recommended adolescent vaccines by race.* Error bars represent 95% confidence interval.Further, rates of HPV vaccine uptake trended toward increased vaccination of children with public insurance or no insurance as compared to those with private insurance (66% vs. 51%; p = 0.06). Again, there were no significant differences for the other recommended childhood vaccines (Figure 3).Vaccination rates of recommended adolescent vaccines by insurance status.* Error bars represent 95% confidence interval.In multivariable logistic regression, patients of black and other minority races were 4.9 and 4.2 times more likely to receive the HPV vaccine compared to their white counterparts. Recommendation by the child’s physician was determined by parents answering yes, no or unsure to the question “has your doctor recommended the HPV vaccination for your daughter?” Daughters of parents who answered yes to this question were twice as likely to initiate the HPV vaccine series. Similarly daughters of parents who disagreed or strongly disagreed with the statement “My daughter does not need the HPV vaccine” (a marker of perceived susceptibility) were also significantly more likely to be vaccinated. Safety concerns were the strongest attitude barrier to vaccination (Table 3). Insurance status was not independently associated with HPV vaccine uptake in the final model.Final regression model showing factors independently associated with initiating the HPV vaccine series.* As compared to nonhispanic white race.In this prospective cohort study, minority girls were more likely to receive the HPV vaccine compared to their nonhispanic white counterparts. In particular, race correlated more with vaccination initiation than insurance status, beliefs about safety and efficacy, recommendation by the pediatrician, and perceived susceptibility although these factors likely also played a role in the parents’ decision to have their child vaccinated. The CDC’s National Immunization Survey-Teen in 2011 documented a similar trend for higher vaccination rates among minorities [10]. However, the NIS-Teen data specifically looks at adolescents aged 13–17 and thus does not specifically address this issue of uptake at the time of the well child visit in younger adolescents. This study demonstrates that these differences may occur at the time of the initial adolescent visit and persistent throughout adolescence. Provider recommendation remains the strongest modifiable event to promote vaccination. However, recent data suggests that provider recommendation is lower for minority races [20]. Conversely, at least one study found that physicians with higher rates of minority patients had higher HPV vaccination rates, suggesting possible targeting of vaccine recommendation [21]. This study cannot provide any information about provider beliefs or attitudes although the strong association between MD recommendation and racial differences in uptake warrant further study in this area. Lastly, it is possible that perceived susceptibility could also play a role in increased uptake among minorities. Racial disparities in cervical cancer incidence and mortality are well documented and have been circulated widely in the lay public.Among girls presenting to the doctor for a well-child visit in this study, privately insured patients were less likely to receive the HPV vaccine than girls with Medicaid or no insurance. This may be related to coverage and reimbursement issues which can be heterogeneous among private payers, possibly resulting in more out-of-pocket costs for parents. By comparison, all children who qualify for Medicaid and/or are underinsured for vaccinations and meet certain financial cutoffs are covered by the Federal Vaccines for Children program for all vaccines recommended by the CDC, including the HPV vaccine. The NIS-Teen survey also noted a difference in vaccination by socioeconomic status with 46.4% of girls below the poverty level initiating vaccination compared to 35.8% of girls at or above the poverty level choosing to be vaccinated [10]. It is possible in our study, that any role insurance status plays is mediated by the difference in race among payer groups. Future studies should assess this in more detail in the questionnaire.This study evaluated HPV vaccination given uniform access to care in a single cohort of girls being seen for well child checks in the same health care system. This is a strength of the study as it controls for regional differences in provider counseling and issues in access to patient care. This research utilized prospective data collection and objective billing data for confirmation of vaccination status allowing for the most accurate determination of vaccination. In addition, utilization of a professional survey group for interviews, validated questionnaire, and a piloted study technique allows for high quality subjective data. This study is limited by its small sample size and evaluation of only a single health care delivery system. For the follow-up questionnaire, we had a relatively low response rate but this is comparable to similar telephone surveys conducted on HPV vaccination [11]. In regards to nonresponder bias, we found that patients with public insurance were less likely to participate in the questionnaire. This may limit our abilities to fully characterize this group’s higher vaccination rates. Further, our research by the nature of cohort selection cannot take into account vaccination barriers related to access to care or determine causation.This study documents HPV vaccination rates, attitudes and barriers prior to initiation of a statewide mandate requiring girls in Virginia to receive the HPV vaccine prior to their 11th birthday for school admission and begins to identify further areas that need elucidation. These data will serve as the baseline for a comparison study of HPV vaccination rates and counseling 5 years after enactment of the mandate. This study is uniquely positioned to address this question given the objective data capture through the billing database combined with a validated parental survey and uniform access to care. Further, documented differences by race and insurance status seen with HPV vaccination but not with other adolescent immunizations will serve as the baseline for post-mandate comparisons. The follow-up study is slated to be conducted starting in August 2013. These data may have implications beyond a single state given similar trends in differential rates of uptake by race seen at a national level.In this study, we found that parents report high rates of understanding that the HPV vaccine is safe, effective, and recommended for adolescent girls. Privately insured patients had lower vaccination rates compared to those with public or no insurance. Racial differences exist with minority girls having higher HPV vaccination rates than their white counterparts. Future studies will be necessary to evaluate whether the verbal opt-out mandate in Virginia will have any effect on these racial differences.This research was funded by the American College of Obstetrics and Gynecology/Merck & Company, Inc. Immunization Award for Residents and Fellows 2008. At no point did Merck or any of Merck’s employees have any input in study design, data analysis, or result reporting and have had no access to data prior to publication. We are very appreciative of the parents and guardians who participated in this study as well as the dedicated staff of the Center for Survey Research, especially Robin Bebel and Tom Guterbock.The authors declare no conflict of interest.HPV vaccine uptake by insurance status and race: supplemental data for Table 2.Supplemental data for Figure 2.* Data missing for 4 patients of white race, 1 patient of black race, and 8 patients of other race.Supplemental data for Figure 3.Insurance status by race.

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+Present address: Metabiota, 1410 Q Street NW, Suite 300, Washington, DC 20009, USA; E-Mail: mguttieri@metabiota.com.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Lassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development.LASV, a member of the family Arenaviridae, is carried by persistently infected multimammate rats (Mastomys natalensis). Humans can become infected by inhalation of aerosolized virus shed in rodent excreta or by person-to-person or nosocomial exposure [1]. LASV is a category A pathogen on the National Institute of Allergy and Infectious Diseases list of biodefense and emerging infectious diseases and is considered a select agent by the U.S. Centers for Disease Control. LASV is endemic throughout western Africa where it is responsible for significant human morbidity and mortality. Among all hemorrhagic fever viruses, LASV is second only to dengue virus in human impact, with an estimated 100,000 to 300,000 LASV infections and 5,000 deaths occurring annually [2]. It is likely the LASV disease burden is greater than estimated as routine surveillance of endemic disease is not performed. Also, approximately 80% of people infected with LASV develop mild symptoms and may not seek medical treatment. Symptoms include fever, malaise, severe headache, and sore throat. Bleeding occurs in about one-third of patients and is a poor prognostic indicator. Pulmonary edema and respiratory distress are common in fatal cases [1]. Mortality among pregnant women is higher than other patients and can reach 30–70% [3]. Hearing loss is observed in about 30% of hospitalized patients with approximately 50% of those patients developing permanent deafness [4]. Although Lassa fever is severe and widespread, there is no evidence of repeat infection in survivors, suggesting an effective vaccine could be developed [2]. Challenges for developing a LASV vaccine include genetic diversity of virus strains [5] and an incomplete understanding of what constitutes a protective or cross-protective immune response. Additional challenges include LASV tropism for dendritic cells and macrophages, which likely interferes with the adaptive immune response, making it difficult to identify appropriate correlates of protection [6,7]. Lassa fever patients and experimentally infected nonhuman primates (NHP) develop strong antibody responses to LASV; however, those antibodies are not neutralizing antibodies and have not been found to correlate with viral clearance. Low levels of LASV neutralizing antibodies, if detected at all, are usually only present after recovery in both humans and nonhuman primates [2]. In contrast, virus clearance has been correlated with the appearance of cytotoxic T cells in LASV-infected nonhuman primates (NHP), with those animals surviving infection displaying stronger and earlier T-cell responses than those that succumbed to LASV infection [6]. Despite these correlations, it is not clear whether or not antibodies are important for protective immunity or recovery if present before LASV infection.There are no FDA-licensed vaccines for Lassa fever, and therapy is generally limited to supportive care. Although intravenous treatment with the antiviral drug ribavirin was found to reduce mortality if given early in the course of Lassa fever, it does not prevent deafness [8]. LASV vaccine development efforts have yet to result in a clear candidate for advanced development due to ineffective protection in animal models or safety concerns. Due to the pathogenicity of LASV, as well as the requirement for handling infectious virus in high-containment laboratories, a recombinant DNA-based vaccine is an attractive alternative to conventional vaccine approaches. To date, several experimental vaccines for LASV derived from recombinant DNA have been tested in guinea pigs and NHP [9,10]. Replication competent viral-vectored candidate vaccines include recombinant vaccinia virus [11], recombinant vesicular stomatitis virus (VSV) [12], and recombinant yellow fever virus [13,14]. Replication-deficient candidate vaccines include a Venezuelan equine encephalitis virus replicon [15] and a virus-like particle (VLP) [16]. Of these candidates, the VSV replicon showed the most promise, in that four vaccinated NHP remained clinically healthy after LASV challenge, although they did develop low-level viremia [12]. Despite these promising results, safety concerns with the VSV live vector remain. With this study, we report the development of a plasmid DNA LASV vaccine delivered via either intramuscular or intradermal electroporation. Additionally, we present immunogenicity and protective efficacy of this vaccine in a lethal guinea pig challenge model. Our data indicate that DNA vaccination offers a safe and potentially effective means to induce protective immunity against LASV.The Lassa virus glycoprotein precursor (GPC) fragment was amplified by PCR using Platinum Taq High Fidelity DNA polymerase (Invitrogen) from a LASV, Josiah strain template using GPC-specific primers. The LASV GPC fragment was then cloned into the NotI site of expression vector pWRG7077 (Powdermed) using T4 DNA ligase (New England Biolabs), generating pLASV-GPC. The gene is flanked by a cytomegalovirus immediate early promoter (CMV IE) and a bovine growth hormone polyadenylation signal (BGH pA). The vector contains a kanamycin antibiotic-resistance gene (KAN). Resulting clones were screened for orientation and sequenced using an ABI 3100 genetic analyzer. Plasmid DNA was purified using Purelink HiPure Mega plasmid purification kit (Invitrogen).The published sequence for LASV GPC gene (Genbank Accession number AY628203.1) was optimized by GeneArt using a proprietary algorithm. In addition to codon usage optimization, negative cis-acting sites (such as splice sites, poly (A) signals, TATA boxes, etc.) which may negatively influence expression, were eliminated where relevant. The GC-content of the LASV GPC gene was adjusted to prolong mRNA half-life. Codon usage was adapted to the bias of Cavia porcellus resulting in an improved codon adaption index (CAI) value of 0.97 (a value of 1.0 being perfect adaption) from a value of 0.68 in the original sequence. For this analysis, any CAI value above 0.9 is considered optimal to ensure robust and stable expression rates in target organisms. The optimized sequence was synthesized and subcloned into the NotI/BglII site of expression vector pWRG7077 (Powdermed) by GeneArt (Germany). The cloned plasmid was sent to Aldevron (Fargo, ND, USA) for scale-up and was provided as a 1 mg/mL solution.To confirm expression, radioimmunoprecipitation assays (RIPA) of the non-optimized vaccine construct were carried out as follows. COS-7 cells at 80% confluency in T25 cell culture flasks were transfected using the FuGene 6 transfection reagent (Roche) with 5 μg of either pWRG7077 or pLASV-GPC plasmid DNA. After 24 hours, monolayers were washed, then treated with 200 μCi Promix ([35S]-methionine and [35S]-cysteine, Amersham) for 4 hours at 37 °C. Once again, monolayers were washed, the cells harvested, lysed, and supernatant collected for immunoprecipitation analysis. A negative control cell lysate was similarly prepared, excluding the Promix incubation. A volume of 5 μL of anti-LASV antibody was preincubated for 2 hours on ice with 500 μL of the negative control lysate. A volume of 10 μL/mL of 10% SDS was added to the radio-labeled lysate, then 200 μL of this mixture was added to the preincubated negative control lysate. The mixture was incubated overnight on ice. The lysate/antisera mixture was then combined with 150 μL protein G sepharose and incubated for 30 minutes on ice. Images were obtained after protein gel electrophoresis and transfer by using a Cyclone phosphorimager (Packard).The intramuscular electroporation device was an ELGEN Twin Injector (Inovio Pharmaceuticals), which consists of an outer housing with an inner wagon carrying two standard 1 mL syringes with needles, 4 mm apart [17]. A gearing system presses the piston of the syringes when the wagon slides forward in the housing to inject DNA during the insertion. The needles subsequently serve as electrodes. The needles penetrate to a depth of approximately 1.6 cm in the targeted muscle, distributing the DNA in a columnbular fashion throughout the muscle and perfectly co-locating the electrical field with the delivered plasmid. The device operates at an applied voltage of 60 and pulses twice, each pulse of 60 ms duration. Upon completion of the electrical pulses, internal motors retract the needle electrodes from the muscle and re-house them in the enclosed wagon. The ELGEN Twin Injector is directly linked to the ELGEN 1000 pulse generator (Inovio Pharmaceuticals), which supplies power to the unit.The ELGEN-MID EP device allows for dermal/subcutaneous DNA delivery at a penetration depth of 5 mm using a four electrode invasive needle array. This minimally invasive intradermal device penetrates the full depth of the skin spanning the epidermis, dermis, and into the subcutaneous space. It operates at standard EP electrical parameters and standard pulse lengths (50 V, 3 pulses, 100 ms). A rectangular electrode configuration (10 mm spacing by 5 mm) and 5 mm depth penetration ensure the distribution of the electric field over a wider skin surface and depth area. Specifically, the EP applicator consists of four gold-plated stainless steel needle electrodes with trocar grinds. The electrodes administer a synchronized pulse. The ELGEN-MID device was built with attachment cord for linkage to the ELGEN 1000 pulse generator (Inovio Pharmaceuticals), which supplies power to the unit.Strain 13 guinea pigs were randomly divided into two groups consisting of six animals each. Group 1 received 100 µg of the mock vaccine (two sites at 50 µg per site) via IMEP. Group 2 was IMEP-vaccinated with 100 µg of plasmid pLASV-GPC, which encodes the GPC gene of LASV, Josiah strain. At each vaccination, approximately 100 mg of plasmid DNA was injected intramuscularly, followed immediately by a two-pulse delivery of 250 mA current. Three vaccinations were administered at 3-week intervals. To collect sera for analysis of virus-specific antibody titers, phlebotomy was performed on all animals before the vaccination series was initiated (pre-bleed) and just before each vaccination session (prime, boost 1, or boost 2). Viral infection was carried out under biosafety level (BSL)-4 conditions. Each animal was administered subcutaneously a single target dose of 1,000 pfu/mL of LASV (strain Josiah) in a total volume of 100 μL physiological saline. After viral infection, phlebotomy was performed at days 7, 14, 21 postinfection and at euthanasia or the study endpoint for survivors. Animals were monitored daily and assigned morbidity scores corresponding to the development of disease signs. Animals were euthanized when moribund, and surviving animals were euthanized on day 28 postinfection. Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the 8th Edition of the Guide for the Care and Use of Laboratory Animals [18].Strain 13 guinea pigs were randomly divided into three groups consisting of eight animals each (IMEP-vaccinated group, ELGEN-MID-vaccinated group, and a virus only group) and two mock-vaccinated control groups consisting of five animals each (IMEP Mock-vaccinated group, ELGEN-MID Mock-vaccinated group). Each animal was implanted with IPTT-300 microchip transponders (BMDS) to measure body temperature. Each group received approximately 100 µg of the mock or authentic vaccine (two sites at 50 µg per site) via IMEP or ELGEN-MID. Three vaccinations were administered at 3-week intervals. To perform the vaccinations, the abdominal fur was shaved, and each animal received two administrations of either an intramuscular injection or shallow dermal injection of DNA-containing solution. For the IMEP group, animals were administered the vaccines via electroporation as described above. To collect sera for analysis of virus-specific antibody titers, phlebotomy was performed on all animals before the vaccination series was initiated (pre-bleed) and just before each vaccination session (prime, boost 1, or boost 2). Viral infection was carried out under BSL-4 conditions. Each animal was administered subcutaneously a single dose of 1,000 pfu of LASV (strain Josiah) in a total volume of 100 μL of physiological saline. After viral infection, phlebotomy was performed at days 7, 14, 21, and 28 postinfection. Animals were monitored daily and assigned morbidity scores corresponding to the development of disease signs. Animals were euthanized when moribund, and surviving animals were euthanized at day 28. Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the 8th Edition of the Guide for the Care and Use of Laboratory Animals [18].Four surviving animals from the ELGEN-MID vaccinated group were held in BSL-4 for 120 days after the vaccination study endpoint to participate in a backchallenge experiment to assess if they could survive re-infection. These animals were re-challenged with a subcutaneous dose of 1,000 pfu LASV 120 days after the end of the vaccination study and monitored for weight, body temperature, and symptom development daily for 30 days postinfection.Serum samples collected pre and after infection were screened for viral titers via a standard plaque assay. Vero cells, seeded in 6-well cell culture plates, were adsorbed with gentle rotation at 37 °C 5% CO2 with 10-fold serial dilutions of serum for 1 hour, then an overlay of 0.8% agarose in EBME fortified with 10% fetal bovine serum and 20 μg/mL gentamicin was applied to each well and allowed to solidify. Cells were incubated at 37 °C, 5% CO2 for 4 days, then stained with 0.001% neutral red solution in PBS. After an overnight incubation, plaques were counted and recorded. Neutralizing capabilities of antibodies in the serum were analyzed by a standard plaque reduction/neutralization test (PRNT), as follows [19]. Twofold serial dilutions of heat-inactivated guinea pig sera were pre-incubated for 1 hour at 37 °C with LASV diluted to approximately 100 pfu. Each serum dilution/virus mixture was then added to confluent Vero cells seeded in 6-well cell culture plates. The remainder of the procedure is as described above for the standard plaque assay. Plaques were counted and compared to control wells containing cells infected with LASV pre-incubated with naïve guinea pig or primate serum. Neutralizing antibody titers (PRNT50, PRNT80) values were identified as the highest dilution of serum yielding a 50% (PRNT50) or 80% (PRNT80) reduction in plaques.Immunohistochemistry was performed on replicate tissue sections for all animals using an Envision-PO kit. A mouse monoclonal antibody against LASV virus (L52-2074-7A) was used at a dilution of 1:15,000. After deparaffinization and peroxidase blocking, tissue sections were incubated with the primary antibody at room temperature for one hour. The sections were then rinsed and incubated for 30 minutes with the peroxidase-labeled polymer (secondary antibody). The sections were rinsed, covered, and incubated with substrate chromogen solution for 5 minutes. The sections were then rinsed, stained with hematoxylin, and rinsed again. Sections were then dehydrated, cleared with Xyless and coverslipped.The LASV GPC product was successfully expressed in COS-7 cells from the pLASV-GPC plasmid. Using guinea pig LASV immune serum, it was possible to immunoprecipitate GPC and GP2, which is released from GPC by post-translational cleavage through the action of a host cell subtilase SKI-1/S1P (Figure 1B, Lane 2) [20]. A plasmid map is provided as Figure 1A. Bands for GPC and GP2 do not appear in the untransfected COS cell lysate Figure 1B, Lane 1). Plasmid Map and Immunoprecipitation and polyacrylamide gel electrophoresis (PAGE) of radiolabeled LASV strain Josiah glycoprotein precursor (GPC, 76 KD). (A) Map of pLASV-GPC cloned into the pWRG7077 vaccine plasmid. (B) Radioimmunoprecipitation and PAGE of LASV GPC and GP2 from COS-7 cell lysate. Expression products from COS-7 cells transfected with (Lane 1) empty vaccine plasmid pWRG7077 or (Lane 2) recombinant pLASV-GPC, and immunoprecipitated with LASV-immune guinea pig serum. The sizes of molecular weight markers M and the location of bands corresponding to GPC and GP2 are indicated.For the pilot study, we produced a candidate LASV DNA vaccine by cloning cDNA encoding the GPC gene of LASV (Josiah strain) into the plasmid vector pWRG7077 [21]. Approximately 4 weeks after the final vaccinations, the guinea pigs were challenged by SC administration of 1,000 pfu of LASV, a standard lethal challenge dose. All of the mock-vaccinated guinea pigs succumbed to LASV infection whereas all but one of the IMEP-vaccinated guinea pigs survived. The IMEP-vaccinated animal that died showed a delayed time to death as compared to controls (Figure 2A). Although the DNA vaccine prevented death in most animals, the challenged guinea pigs developed transient viremia (Figure 2B) and showed mild clinical signs of disease (Figure 2C).Outcomes for IMEP study using the non-optimized LASV DNA construct. (A) Survival curve; (B) Serum viremia as measured by plaque assay; (C) Morbidity score based on observed disease signs.Although neutralizing antibodies were not detected in vaccinated guinea pigs before challenge, they were detected on day 30 after challenge in the vaccinated animals that survived to the study endpoint, indicating that a specific immune response was elicited by the DNA vaccine (Table 1). We next sought to assess whether route of delivery might play a role in the induction of antibody responses, and in particular if we could enhance antibody responses by (a) using an optimized DNA construct and (b) by delivering the optimized LASV vaccine to the dermal tissue [21,22].Plaque-reduction neutralization test (PRNT) titers following vaccination (Day 0) and infection with LASV (Day 30) a.a Neutralizing titers are listed as the reciprocal of the dilution resulting in either 50% or 80% reduction in plaques compared to control.In other studies, we found that optimizing codons of DNA vaccine constructs could improve both their expression and immunogenicity [23]. Subsequently, to determine if we could improve upon the protective efficacy of our DNA vaccine for LASV, we optimized the GPC construct to maximize mammalian codon availability in the guinea pig model and to remove viral elements shown to compromise expression. The optimized GPC sequence went from a codon adaption index (CAI) of 0.67 before adaption to a CAI of 0.97 after adaption, where a CAI value of 1 is considered perfect. Additional changes of note were an increase in the GC content from 43% before adaption to 60% after adaption to prolong mRNA half life and the removal of negative cis-acting sites (such as splice sites, poly A signals and TATA boxes). None of the changes in the GPC gene sequence resulted in changes at the protein level.To evaluate the affect of codon-optimization alone on vaccine efficacy, we vaccinated a group of eight guinea pigs with 100 µg of DNA three times at 3-week intervals, using the same IMEP device as in the pilot study. We also vaccinated groups of guinea pigs with the optimized vaccine using a newly developed ELGEN-minimally invasive intradermal EP device (ELGEN-MID). For this study, we were able to monitor the development of fevers in control animals through the use of the IPTT-300 microchip transponders. These microchip transponders were not available for use in the pilot study. All guinea pigs mock-vaccinated with the empty plasmid or not vaccinated (virus only) became febrile, displayed signs of illness, lost weight, and succumbed to infection between days 15 and 18 postchallenge, whereas all guinea pigs vaccinated with the codon-optimized LASV DNA, regardless of the EP method used, survived challenge (Figure 3A). Unlike the pilot study in which guinea pigs vaccinated with the non-optimized LASV DNA vaccine demonstrated signs of illness, the guinea pigs vaccinated with the codon-optimized vaccine by ELGEN-MID EP did not develop any signs of illness, and remained afebrile (Figure 3B–D). We observed mild signs of disease in some of the guinea pigs that received the optimized LASV DNA vaccine by IMEP (4/8), including low fevers and slight viremias (Figure 3B,C), suggesting that dermal electroporation was more efficacious in this study. Outcomes for dermal versus muscle electroporation using the codon-optimized LASV DNA construct. (A) Survival curve; (B) Serum viremia as measured by plaque assay; (C) Average body temperature changes as a function of time postinfection, and (D) Morbidity score based on observed disease signs. The grey bar indicates the normal body temperature range for guinea pigs. In addition to an improvement in outcome for vaccinated animals surviving challenge, we observed neutralizing antibodies generated as a result of vaccination before virus challenge. Unlike the pilot study in which we were not able to measure neutralizing antibodies by PRNT after three vaccinations, even at the highest serum concentration tested (1:4); detectable neutralizing antibodies were observed in all of the animals receiving the vaccine by IMEP, indicating that codon-optimization alone boosted production of neutralizing antibodies in the absence of virus (Table 2). However, the average neutralizing antibody titer for all animals in this group was just under the PRNT50 level for the highest serum concentration tested (1:8 dilution). Animals vaccinated via ELGEN-MID had consistently higher neutralizing antibody titers before challenge, which we believe contributed the absence of measureable viremia, fever, or other signs of disease in animals in this group. For both the IMEP and ELGEN-MID groups, at least a 20% reduction in plaque formation was maintained for dilutions out to 1:64 before challenge. After challenge, neutralizing antibody titers increased, but not significantly. We believe this is due to the vaccine protecting most of the animals from developing measureable serum viremia postchallenge, thereby mitigating the boosting affect of virus infection. The animals in the IMEP-vaccinated group that developed mild viremia and became transiently febrile (4/8) exhibited the lowest neutralizing antibody titers prechallenge (data not shown), strengthening our hypothesis that the presence of neutralizing antibodies before challenge contributed to preventing viremia in most vaccinated animals. None of the virus only (data not shown) or mock-vaccinated animals (shown in Table 2) developed measureable neutralizing antibodies before virus challenge or at euthanasia. For both the IMEP and ELGEN-MID groups, at least a 20% reduction in plaque formation was maintained for dilutions out to 1:128 at 30 days postchallenge.Plaque-reduction neutralization test (PRNT) titers following vaccination (day 0) and infection with LASV (day 30) a.a Neutralizing Titers are listed as the reciprocal of the dilution resulting in either 50% or 80% reduction in plaques compared to control. b The 1:8 dilution yielded an average 46% reduction in plaque formation, but two of eight animals reached the PRNT50 level at this dilution. Necropsies were performed on animals that met criteria for euthanasia or who survived to the study endpoint with the exception of four of the ELGEN-MID-vaccinated animals. These four animals will be described in the next section. Pathologic findings in virus only or mock-vaccinated animals were consistent with previous reports of the disease process in strain 13 guinea pigs [24]. There was no observed difference in lesion type or severity between the animals in the virus only group and the mock-vaccinated animals. Only mild lymphoid hyperplasia (cervical lymph node or mesenteric lymph node) and/or splenic white pulp hyperplasia was noted in some pLASV-GPC-vaccinated animals at the study endpoint. These findings are consistent with recent viral infection. None of the tissues collected from pLASV-GPC-vaccinated animals, regardless of EP method, were positive for the presence of viral antigen by immunohistochemistry at the study endpoint. Figure 4 illustrates the differences observed in antigen staining for a selection of tissues. As shown, positive LASV antigen staining was present in the lymph node, spleen, adrenal gland, liver, and kidney (Figure 4A,C,E,G,I, respectively) of a mock-vaccinated animal and absent in the corresponding tissues (Figure 4B,D,F,H,J) of a ELGEN-MID-vaccinated animal.Immunohistochemistry staining for LASV antigen in selected tissues of mock-vaccinated or ELGEN-MID-vaccinated guinea pigs. (A) Viral antigen staining of a mock-vaccinated lymph node (40×); (B) lymph node of a ELGEN-MID-vaccinated animal showing lymphoid hyperplasia and a lack of viral staining (20×); (C) Viral antigen staining of a mock-vaccinated spleen (40×); (D) Splenic white pulp hyperplasia in a ELGEN-MID-vaccinated guinea pig (40×); (E) Viral antigen staining of a mock-vaccinated adrenal gland (10×); (F) A lack of viral antigen staining of a ELGEN-MID-vaccinated adrenal gland (10×); (G) Viral antigen staining of a mock-vaccinated liver (20×); (H) A lack of viral antigen staining of a ELGEN-MID-vaccinated liver (10×); (I) Viral antigen staining of a mock-vaccinated kidney (20×); (J) A lack of viral antigen staining of a ELGEN-MID-vaccinated kidney (10×).Four of the surviving ELGEN-MID vaccinated animals were kept at the end of the study in order to assess the ability of the vaccine to protect animals upon secondary exposure to virus after an extended period of time. These animals were maintained in the BSL-4 laboratory for 120 days after the end of the vaccine study, then were re-exposed to 1,000 pfu LASV by SC injection, along with four age/weight-matched control guinea pigs. These animals were observed daily for signs of disease. The vaccinated animals survived to the study endpoint (Figure 5A) and never developed signs of disease compared to the control animals, which lost weight (Figure 4B), were febrile (Figure 4C), and succumbed to disease (Figure 4A).Outcome of backchallenge experiment. (A) Survival curve; (B) Average weights postchallenge; and (C) Average temperatures post-challenge for animals enrolled in the backchallenge experiment.The ability to produce high levels of neutralizing antibodies before challenge is often thought of as a hallmark of a strong vaccine candidate [25,26]. While this is true for many pathogens, protective immunity against LASV in humans is thought to be primarily cell-mediated, and the role of humoral immunity and antibody production in protection is currently unclear [2,6,27,28]. While other vaccination strategies have been undertaken for LASV, to our knowledge, these studies are the first report of a non-replicating LASV vaccine to completely prevent measureable serum viremia in an animal model [12,15,29]. Our data clearly show that a plasmid encoding a codon-optimized GPC gene of LASV, when administered by dermal electroporation, can completely protect guinea pigs from viremia, illness, and death. Although low to modest neutralizing antibody titers were detected in vaccinated animals before virus challenge, we do not believe these antibodies alone account for the protection that we observed.We demonstrated that codon optimization of the vaccine enhanced its efficacy and slightly improved its ability to elicit neutralizing antibodies. All guinea pigs receiving the non-optimized vaccine by IMEP became viremic and were mildly ill, but most survived (5/6). In contrast, guinea pigs receiving the codon-optimized vaccine by IMEP were well, experiencing only transient low-level viremia (4/8) and short-lived fever (4/8), fully resolving by the end of the study. Dermal delivery of the optimized vaccine and EP pulse was the most efficacious, with all animals surviving to the study endpoint with no viremia detected in any of the samples tested and with no observable signs of disease. Our data are consistent with what is currently known about the efficacy of different DNA vaccine delivery technologies. DNA vaccines delivered by needle injection into muscles have been shown to elicit strong cell-mediated responses but historically have not been as effective as other vaccine strategies in eliciting high levels of neutralizing antibodies in animal models [30]. In contrast, delivery of a variety of DNA vaccines to the skin of both animals and humans has been shown to elicit a more balanced humoral and cellular response and to effectively elicit neutralizing antibodies (reviewed in [21,31,32,33,34,35,36]). For example, the ability of gene gun vaccination to stimulate humoral immunity has been hypothesized to correlate with delivery of the DNA to the epidermis and specifically to the ability to target epidermal dendritic cells (Langerhans cells) [37,38,39]. Skin delivery by electroporation, similarly, and probably more efficiently, targets this same highly immunologically active site, as reflected by the improved protective efficacy observed when the LASV DNA vaccine was delivered via dermal electroporation.Although we infer that the protective immunity observed was largely due to cell- mediated immune responses elicited by the LASV DNA vaccine delivered via electroporation, we are currently unable to provide supportive evidence for this in the guinea pig model. Unfortunately, there are few reagents available for testing the cellular immune response in guinea pigs, and the significance of correlative cytokine responses are not well defined for these animals. In ongoing studies, we are addressing this challenge by developing and incorporating gene-based array approaches to measure the guinea pig cellular responses to vaccination and virus challenge. Despite these limitations, our results clearly establish that DNA vaccination accompanied by EP is a viable strategy for inducing immunity to LASV infection and that genetic optimization of the LASV GPC sequence improves its efficacy in the guinea pig model. Further, our data provide preliminary evidence that dermal delivery by electroporation is the optimal method of vaccination with the LASV DNA vaccine. Future studies in the guinea pig model will incorporate dose and schedule refinements in order to establish the minimal protective dose for this vaccine. Additional studies in NHP are in progress, which will allow us to both obtain measurements of cell mediated immunity and to confirm our findings with the LASV DNA vaccine-dermal EP platform.The authors kindly acknowledge the excellent guinea pig phlebotomy assistance of Joan Geisbert, formerly of Virology Division, USAMRIID, Keith Esham, and Michael Winpigler, formerly of Veterinary Medicine Division and Michael Zimmerman, Joshua Moore, and Jimmy Fiallos currently of the Veterinary Medicine Division, USAMRIID. The fine work of Chris Mech of Pathology Division, USAMRIID in preparing the Immunohistochemistry slides is deeply appreciated by the authors.The research presented herein was sponsored by several grants from the Military Infectious Disease Research Program (MIDRP), U.S. Army Medical Research and Materiel Command, and under a collaborative research agreement with Inovio Pharmaceuticals. This research was performed in part while K. Cashman held a National Research Council Research Associateship Award at USAMRIID.Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army or the Department of Defense.KEB and NYS are employees of Inovio Pharmaceuticals and as such receive compensation in the form of salary, stock options and bonuses. All other authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).DNA vaccines can induce both humoral and cellular immune responses. Although some DNA vaccines are already licensed for infectious diseases in animals, they are not licensed for human use because the risk and benefit of DNA vaccines is still controversial. Indeed, in humans, the immunogenicity of DNA vaccines is lower than that of other traditional vaccines. To develop the use of DNA vaccines in the clinic, various approaches are in progress to enhance or improve the immunogenicity of DNA vaccines. Recent studies have shown that immunogenicity of DNA vaccines are regulated by innate immune responses via plasmid DNA recognition through the STING-TBK1 signaling cascade. Similarly, molecules that act as dsDNA sensors that activate innate immune responses through STING-TBK1 have been identified and used as genetic adjuvants to enhance DNA vaccine immunogenicity in mouse models. However, the mechanisms that induce innate immune responses by DNA vaccines are still unclear. In this review, we will discuss innate immune signaling upon DNA vaccination and genetic adjuvants of innate immune signaling molecules.Almost two decades ago, it was reported that plasmid DNA could induce adaptive immune responses against plasmid-encoded antigens [1], indicating it could be used in novel therapeutic applications as a human vaccine for the prevention of various pathogen infections [2], autoimmunity [3], allergy [4], neurological disorders [5], and cancer [6]. In the veterinary field, some DNA vaccines are already licensed for West Nile virus in horse, infectious hematopoietic necrosis virus in salmon, and melanoma in dogs [7]. For human use, DNA vaccines have not been licensed, however, many candidate DNA vaccines are being studied in ongoing clinical trials. The clinical benefits of DNA vaccine are low cost, vaccine stability, high productivity, and easy modification of antigen in comparison with traditional protein vaccines. Conversely, it was reported that the immunogenicity of DNA vaccines was quite low according in clinical trials. Indeed, the immunogenicity of DNA vaccines tended to be weaker than other types of vaccines using live virus, virus vectors, or traditional protein plus adjuvant vaccines. Therefore, the immunogenicity of DNA vaccines was improved by changing promoters, codon usage of antigen sequences, the insertion of genetic adjuvants such as cytokines and innate immune activation molecules, strategies to prime and boost vaccination, and the route of administration [8].Furthermore, elucidation of the molecular mechanisms of DNA vaccines is also important for developing DNA vaccines for human use. TANK-binding kinase 1 (TBK1), and stimulator of interferon genes (STING), was identified as an essential molecule for the induction of adaptive immune responses by DNA vaccination. In addition, double-stranded DNA (dsDNA) is a critical ligand of the STING-TBK1 signaling cascade [9]. These results indicate that dsDNA-induced innate immune signaling lead to induction of DNA-encoded antigen specific adaptive immune responses, like an adjuvant. However, DNA sensing machinery is still controversial. In this review, we will discuss innate immune signaling of DNA vaccines and genetic adjuvants of innate immune signaling molecules.In 1990, Wolf et al. showed that the intramuscular administration of naked DNA led to the induction of DNA-encoded reporter genes in muscle cells [10]. Subsequently, Ulmer et al. demonstrated that the intramuscular administration of plasmid DNA encoding influenza viral protein induced encoded antigen-specific cytotoxic T lymphocyte (CTL) responses, which protected against lethal influenza virus infection [1]. These findings were the first evidence that naked DNA administration alone could induce adaptive immune responses against antigens expressed from plasmid DNA, and suggested that DNA vaccine strategies might be useful for clinical use. Indeed, many researchers evaluated novel DNA vaccines using experimental infectious diseases models [11]. The properties of DNA vaccines represent greater stability, low cost, high productivity, and possibility to improve immunogenicity. In 1998, the first human clinical trial of DNA vaccines against human immunodeficiency virus was reported [12]. This study evaluated the safety and efficacy of DNA vaccines. Importantly, one of the safety concerns for DNA vaccines was the integration of plasmid DNA into the host genome [13]. If integration occurs following DNA vaccination, the integrated-DNA may cause oncogene activation, tumor suppressor gene inactivation, or chromosomal instability. Fortunately, experimental data showed the rate of plasmid DNA integration was lower than the natural rate of mutation in mammalian genomes [14]. Another safety concern is development of anti-DNA antibodies, associated with autoimmune disorders [15]. Anti-dsDNA antibody was increased in mouse after DNA vaccination [16]. In the clinical trials, anti-DNA antibody did not increase in any study subject [17]. However, the improvement of DNA vaccines to enhance immunogenicity may increase the risk of integration and development of anti-DNA antibody. Therefore, evaluation of safety concerns is essential before clinical trials are initiated. Subsequently, research groups have developed novel DNA vaccines against cancer, influenza virus, human papillomavirus, hepatitis, and malaria. However, the early clinical trials showed disappointing results. Although DNA vaccines can induce both humoral and cellular immune responses against plasmid-encoded antigens, the mode of action of DNA vaccines is still unclear. However, when DNA plasmids are administered to muscle, skin, subcutaneous, or the nasal cavity, it is believed that the DNA plasmid enters cells, translocates to the nucleus, and antigen is expressed by the host cellular machinery. In most cases, myocytes and antigen presenting cells (APCs), such as dendritic cells (DCs) or macrophages, appear to capture plasmid DNA. Subsequently, antigen protein is degraded and presented by major histocompatibility complex (MHC)-I in immune cells. Additionally, expressed-antigens can be secreted from cells by active secretion of the protein or released due to apoptosis of the transfected cell. Secreted antigen proteins are taken up, degraded, and presented by APCs on MHC-I and MHC-II molecules. Finally, APCs recruited to the draining lymph nodes activate naïve B cells, CD4+ and CD8+ T cells. In many cases, secreted antigen proteins could induce both IgG1 and IgG2a/c antibody, and cytosolic protein antigens could induce IgG2a/c antibody.Intramuscular electroporation (imEPT) is one method of DNA vaccine administration, which overcomes limitations such as low transfection efficacy and insufficient recruitment of APCs to the injection site, by inducing transient enhancement of cell membrane permeability. Consequently, the increased uptake of DNA into the host cell and induction of low level of inflammation can enhance the influx of APCs to the injection site [18]. This method induces potent immune responses including CTL responses, and is therefore a convenient method for analyzing the intracellular signaling cascade of DNA vaccines. Indeed, for most cases, the contribution of innate immune activation by DNA vaccination is evaluated using imEPT in mouse models. Gene gun [19], needle-free systems [20], and mucosal delivery [21] are studied as other methods for DNA vaccination; however, these methods have not been examined to elucidate the innate immune signaling of DNA vaccination. It is important whether these vaccination methods activate same innate immune signaling cascade. At present, it is known that nucleic acids such as DNA and RNA induce innate immune responses such as type I interferon (IFN) and inflammatory cytokine production. Interestingly, the innate immune activation of DNA is affected by DNA structure and conformation. In 1963, it was reported that rat liver derived-DNA or RNA stimulation could produce type I IFN from chick cells [22]. In 1984, Bacillus Calmette-Guérin-derived DNA was shown to have strong anti-tumor activity [23]. These findings were the first evidence that both host and bacterial DNA induced innate and adaptive immune responses. Subsequently, bacteria-derived unmethylated CpG DNA and synthetic CpG oligonucleotide (ODN) were shown to be direct stimulators of B cells [24]. Additionally, Toll-like receptor 9 (TLR9) was identified as a receptor for CpG motif DNA that activated innate immune responses in immune cells, such as DCs, B cells, and macrophages [25]. Meanwhile, host DNA-induced innate immune activation was forgotten and ignored. In 1999, the independent effects of unmethylated CpG motifs or specific DNA sequences were shown as at least 25 base pairs of synthetic double-stranded (ds), but not single-stranded (ss) DNA up-regulated the expression of genes related to immune responses [26]. Later, the B-form conformation of dsDNA was shown to be more effective at inducing innate immune responses than the Z-form of dsDNA [27]. Stimulation with synthetic B-form dsDNA, poly (dA-dT) poly (dA-dT), resulted in the induction of type I IFN and IFN-inducible chemokines, whereas stimulation with synthetic Z-form dsDNA, brominated poly (dG-dC) poly (dG-dC) only induced CXCL10 release. Studies then focused on adaptive immune responses and demonstrated genomic DNA derived from dead cells induced the maturation of APCs and cellular immune responses, especially CTL responses [28]. In addition, traditional aluminum adjuvant induced cell death and host-derived DNA release, which induced antigen specific IgE production [29]. These results indicate that the immunostimulatory effect of self-DNA could cause the induction of innate immune responses and side-effects in the host. Adverse effects of aberrant DNA have been shown in relation to the function of DNase, an enzyme that digests DNA. DNase II-deficient mice failed to digest DNA from engulfed nuclei of erythroblasts in hepatic macrophages and resulted in the robust production of type I IFN and inflammatory cytokines, which caused severe anemia and rheumatoid arthritis (RA)-like symptoms in a TLR9-independent manner [30,31]. DNase I and DNase III knockout mice developed systemic lupus erythematosis-like symptoms and inflammatory myocarditis, respectively [32,33,34]. The functional mutations of DNase I and DNase III in humans were also shown to cause several autoimmune disorders, such as systemic lupus erythematosis [33,35], Aicardi-Goutieres syndrome [36], familial chilblain lupus [37], or retinal vasculopathy with cerebral leukodystrophy [38]. Thus, DNA-induced immune responses are not only involved in the prevention of microbial infection but also of autoimmune responses. These findings indicate that normal cells are equipped with innate sensing machineries to remove aberrant genomic DNA fragments.In general, DNA vaccines derived from bacterial plasmids contain unmethylated CpG motifs recognized by TLR9, which induce innate immune responses [25]. Therefore, many researchers have attempted to clarify whether TLR9-induced innate immune responses are required for immunogenicity of DNA vaccines. Unexpectedly, some reports suggested that TLR9 was not essential for the induction of immune responses of DNA vaccines in vivo, although plasmid-induced cytokine production from immune cells was completely dependent on TLR9 in vitro [39,40]. Importantly, dsDNA, including plasmid DNA, could activate both immune cells and non-immune cells such as fibroblasts or keratinocytes. Therefore, TLR9-independent DNA sensing machinery might also be involved in the immunogenicity of DNA vaccines [39,40]. TBK1 is noncanonical IκB kinase that directly phosphorylates interferon regulatory factor 3 (IRF3) to produce type I IFN by TLR-dependent and -independent pathways [27,41]. Thus, TBK1 is important for the activation of innate immune responses upon pathogen infection, tumor development, or autoimmune disease. TBK1-deficient mouse embryonic fibroblasts (MEFs) do not induce cytokine production when stimulated with B-form DNA [27]. Interestingly, TBK1-deficient mice were not able to induce either humoral or cellular immune responses upon DNA vaccination [42]. In addition, type I IFN receptor-deficient mice also showed abolished induction of adaptive immune responses. These results strongly suggest that TBK1-dependent but TLR9-independent mechanisms for the type I IFN signaling cascade are critical for the induction of adaptive immune responses following DNA vaccination. Another important molecule is STING (also known as MITA, ERIS, and MYPS) [43,44,45,46] that was firstly reported to be associated with MHC-II-mediated cell death [37]. Subsequently, STING was shown to function as an adaptor molecule that activates innate immune signaling upon cytosolic dsDNA recognition [43]. STING-deficient MEFs did not activate dsDNA-mediated innate immune signaling. Furthermore, STING deficient mice could not induce humoral and cellular immune responses by DNA vaccination [47]. Surprisingly, a recent study showed that STING directly binds to dsDNA to induce innate immune activation [48]. However, it is still unclear whether STING directly binds to plasmid DNA and contributes to DNA vaccine immunogenicity. Other innate immune signaling molecules have been evaluated for their involvement in DNA vaccine immunogenicity and demonstrated that IRF3 is only involved in cellular immune responses but not humoral immune responses [49]. Although STING and TBK1 studies were examined by imEPT to evaluate their contribution to the immunogenicity of the DNA vaccine, IRF3 research has not used the electroporation method. Studies indicate that dsDNA-mediated, but not TLR9-dependent, innate immune signaling regulates the immunogenicity of DNA vaccines [42,47]. Interestingly, our preliminary data showed that other transcription factors are involved in the immunogenicity of DNA vaccines, which are dependent on antigen properties [50].To date, several cellular molecules are reported as DNA sensors that recognize aberrant cytosolic DNA (Figure 1). These sensors are involved in the elimination of invasive pathogens, and induce innate immune signaling. In most cases, recognition of cytosolic DNA by these sensors results in the induction of innate immune responses through the STING-TBK1 signaling cascade [27,43], suggesting that the detection of dsDNA structure of plasmid DNA by cytosolic DNA sensing machinery contributes to the enhanced adaptive immune responses against DNA vaccine-encoded antigens.Z-DNA binding protein 1/DNA-dependent activator of IFN-regulatory factors (ZBP1/DAI) was reported as the first cytosolic dsDNA sensor [51]. Overexpression of ZBP1/DAI increased type I IFN gene expression by dsDNA stimulation such as bacterial and mammalian DNA. Knockdown of ZBP1/DAI resulted in decreased IFN-β production by dsDNA and DNA virus infection but not synthetic dsRNA and RNA virus infection. In addition, ZBP1/DAI directly interacted with B-form DNA in the cytoplasm. Of interest, however, ZBP1/DAI deficient MEFs responded normally to dsDNA, and ZBP1/DAI deficient mice showed normal adaptive immune responses against DNA-encoded antigen [42].Cytosolic DNA sensing machinery.Retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) were identified as cytosolic RNA sensors and activated innate immune responses to protect RNA virus infection [52]. These receptor-mediated signaling pathways are completely regulated by adaptor molecule IFN-β promoter stimulator 1 (IPS-1) (also known as MAVS, VISA, and Cardif) [53,54,55,56]. Although RIG-I acts as a cytosolic RNA receptor, it was shown to be involved in the indirect recognition of cytosolic dsDNA. Knockdown of RIG-I resulted in reduced type I IFN production by both dsDNA and dsRNA stimulation in a human hepatocellular carcinoma cell line, HuH-7. Subsequently, it was shown that RNA polymerase III transcribed 5'-triphosphate RNA from poly(dA·dT)·poly(dT·dA) or pathogen genome DNAs as a template, and facilitated the RIG-I-mediated type I IFN production cascade. Intracellular bacteria-induced type I IFN production was abrogated by inhibitors of specific RNA polymerase III, resulting in the promotion of bacterial growth [57]. Although RIG-I-mediated innate immune signaling is completely regulated by IPS-1, IPS-1-deficient mice had normal adaptive immune responses against plasmid DNA vaccinations [42]. In addition, at least in human cells, knockdown of IPS-1 resulted in decreased type I IFN production after dsDNA stimulation [27]. The involvement of RIG-I-IPS-1 signaling in human DNA vaccination is still controversial.Double stranded DNA induces both innate immune responses and cell death. It was reported that electroporated DNA could induce cell death in murine macrophages [58]. Absence in melanoma 2 (AIM2) was identified as a cytosolic DNA sensor that activated the inflammasome to produce IL-1β and dsDNA-induced cell death. On recognition of cytosolic dsDNA, AIM2 interacts with inflammasome-related molecules to induce pyroptosis, a type of programmed cell death characterized by the activation of caspase-1 and IL-1β production. Deficiency of AIM2 resulted in enhanced susceptibility to bacteria and DNA virus [59,60]. Collectively, electroporation of plasmid DNA might cause aberrant DNA to induce inflammasome activation or cytokine production via AIM2. Histone H2B is a component of chromatin. Recently, we demonstrated that histone H2B recognized dsDNA in the cytosol to induce innate immune responses through IPS-1 and COOH-terminal importin 9-related adaptor organizing histone H2B and IPS-1 (CIAO). In addition, histone H2B sensed host-derived dsDNA after cell damage by electroporation [61]. Taken together, histone H2B might contribute to the recognition of administered plasmid DNA and electroporated-derived DNA to induce adaptive immune responses against DNA vaccines. In addition, interferon gamma inducible protein 16 (IFI16) [62], high mobility group box protein 1 (HMGB1) [63], Ku70 [64], leucine-rich repeat flightless-interacting protein 1 (LRRFIP1) [65], and DDX41 [66] were also identified as cytosolic DNA sensors. Nucleotide second messenger, cyclic-di-GMP, is synthesized by bacteria from two GTP precursors and induced innate immune activation through the STING-TBK1 signaling cascade [67]. Recently, it was reported that after DNA transfection or DNA virus infection cyclic GMP-AMP (cGAMP) was produced by cGAMP synthase (cGAS), a member of the nucleotidyltransferase family. This endogenous nucleotide second messenger induced innate immune responses. Indeed, cGAS binds to DNA in the cytoplasm and catalyzes cGAMP synthesis to act as a cytosolic dsDNA sensor [68]. Furthermore, cGAMP directly interacted with STING to activate IRF3, and knockdown of cGAS suppressed IFN-β production by dsDNA transfection or DNA virus infection. It will be interesting to examine whether DNA vaccination induces cGAMP using plasmid DNA as a template to induce adaptive immune responses.Studies of DNA sensors were performed using different cell types, synthetic DNAs, bacteria, and viruses. However, only limited type of knockout mice have been used for DNA vaccines, although DNA-mediated innate immune signaling is related to the immunogenicity of DNA vaccines. To elucidate which DNA sensors contribute to the immunogenicity of DNA vaccines, the data by using various DNA sensor gene-deficient mice should be accumulated.In general, the immunogenicity of DNA vaccines is lower than for traditional protein vaccines or live vaccines, although DNA vaccines contain a “built-in” adjuvant, the CpG motif. Indeed, addition of several CpG motifs into plasmid DNA resulted in improved immunogenicity of DNA vaccines [69]. Additionally, human specific CpG motifs containing DNA vaccines induced the maturation of human monocytes [70] suggesting that improvements to plasmid DNA for innate immune signaling activation are important for the enhancement of immunogenicity and induction of optimal immune responses. Recently, TLR adaptor molecules, such as myeloid differentiation primary response gene (MyD88) and Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing interferon-β (TRIF) was inserted into plasmid DNA as a genetic adjuvant and enhanced humoral immune responses against plasmid-encoded antigen (Table 1). In contrast, TRIF genetic adjuvant potently enhanced cellular immune responses. Indeed, TRIF genetic adjuvant elicited protection against lethal influenza virus infection and tumor progression [71]. These studies suggest that TLR agonists may act as DNA vaccine adjuvants.Flagellin is a TLR5 agonist that activates innate immune responses. Dermal injection of plasmids encoding flagellin, and influenza A virus nucleoprotein enhanced both humoral and cellular immune responses. Interestingly, the flagellin vaccine adjuvant induced antigen-specific IgA production and enhanced protective immunity to lethal influenza A virus infection [72]. These results demonstrate that expression of DNA-encoded TLR agonists can improve the immunogenicity of DNA vaccines.In addition, IRF1, 3, and 7 were also evaluated as genetic adjuvants for influenza virus DNA vaccines. IRF1 genetic adjuvant strongly enhanced humoral immune responses. In contrast, IRF3 genetic adjuvant induced stronger cellular immune responses. Interestingly, IRF7 genetic adjuvant enhanced both humoral and cellular immune responses [73]. These results suggest that IRF genetic adjuvants can improve both humoral and/or cellular immune responses. In addition, constitutive active forms of IRF3 and IRF7 were evaluated as DNA vaccine adjuvants and elicited both humoral and cellular immune responses to protect against vaccinia virus infection [74]. Furthermore, DNA binding domain-lacked IRF1 (ΔIRF1) was superior to full length IRF1 on HIV TAT DNA vaccines, as ΔIRF1 genetic adjuvant enhanced cellular immune responses [75]. Recently, we showed that TBK1 acts as a genetic DNA vaccine adjuvant. Plasmodium falciparum serine repeat antigen 36 (SERA36)-encoded DNA vaccine administration with TBK1 genetic adjuvant enhanced at least humoral immune responses but not detect any cellular immune responses in this immunization [76]. These results suggest that TBK1 genetic adjuvant improves the immunogenicity of DNA vaccines, at least in anti-malarial immunogenicity. It was reported that ZBP1/DAI interacted with receptor-interacting protein kinase 3 to mediate virus-induced necrosis [77], and electroporated DAI-encoded plasmid DNA facilitated the transcription of type I IFN and proinflammatory cytokines in vivo. In addition, DAI genetic adjuvant enhanced CTL responses by type I IFN and NF-κB-dependent but IRF3-independent mechanisms. Co-administration of DAI-encoded plasmid with melanoma-associated antigen tyrosinase-related protein-2 (TRP2) DNA vaccine resulted in enhanced tumor rejection and protection against B16 melanoma challenge [78]. However, whether the improvement of DNA vaccine immunogenicity involves DAI-mediated cell death is still unclear. These results suggest that at least DAI genetic adjuvant can improve the immunogenicity of DNA vaccines.HMGB1 was also evaluated as a genetic adjuvant for DNA vaccines. Co-immunization with HMGB1 expressing plasmid with HIV-1 Gag and Env expressing DNA vaccines resulted in enhanced humoral and cellular immune responses [79]. In addition, HMGB1 genetic adjuvant also enhanced the immunogenicity of influenza DNA vaccines [80]. Furthermore, chicken (chMDA5) acted as a genetic adjuvant for avian H5N1 influenza virus DNA vaccine. MDA 5 is a RIG-I like receptor that recognizes cytosolic RNAs to induce innate immune responses. In chickens, MDA5 seems to recognize avian influenza virus infection, because chickens lack RIG-I. chMDA5 genetic adjuvant enhanced humoral immune responses and protected against a lethal H5N1 infection [81]. Adjuvant effects of innate immune signaling molecules.*Ab, antibody.About 15 years have passed since the first human clinical trial for DNA vaccines. At present, DNA vaccines are not yet approved for human use. However, many researchers have attempted to improve plasmid DNA, using codon optimization, proper antigen selection, localization changes and addition of antigen signal sequences, appropriate delivery systems and routes, cytokines, and costimulatory molecules as adjuvants, innate immune signaling molecules as adjuvants, targeting for vaccine delivery systems and presentation, and prime boost strategies, amongst others. Indeed, some approaches have succeeded in improving the immunogenicity of DNA vaccines. However, it is important to elucidate the modes of action, such as the cellular and intracellular mechanisms of DNA vaccines. Currently, only dsDNA-mediated STING/TBK1 signaling cascade has been shown to mediate the induction of adaptive immune responses by DNA vaccination. Therefore, it is important to understand how to recognize and induce innate and adaptive immune responses to develop novel, safe, and effective DNA vaccines. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).There is increasingly broad global recognition of the need to better understand determinants of vaccine acceptance. Fifteen social science, communication, health, and medical professionals (the “Motors of Trust in Vaccination” (MOTIV) think tank) explored factors relating to vaccination decision-making as a step to building a multidisciplinary research agenda. One hundred and forty seven factors impacting decisions made by consumers, professionals, and policy makers on vaccine acceptance, delay, or refusal were identified and grouped into three major categories: cognition and decision-making; groups and social norms; and communication and engagement. These factors should help frame a multidisciplinary research agenda to build an evidence base on the determinants of vaccine acceptance to inform the development of interventions and vaccination policies. Immunization has saved millions of lives worldwide since the introduction of the first vaccine more than 200 years ago. However, the sustained success of immunization programs, a cornerstone of public health, is challenged by increasing vaccine questioning, hesitancy, and refusals. These occur for a range of reasons varying from religious and philosophical to concerns about vaccine safety and schedules, or questions about the relevance of some vaccines [1,2]. Moreover, for each vaccine and its target disease(s) there is a unique set of interested “publics” with different positive and negative perceptions and attitudes to vaccination. High profile vaccine scares have brought significant disruption or cessation to entire vaccine programs. For example, despite Andrew Wakefield’s 1998 article in The Lancet [3] being refuted, retracted, and declared fraudulent [4], uptake of measles, mumps and rubella (MMR) vaccination dipped in the UK from 91% in 1998 to 80% by 2004 [5]. There have since been several outbreaks of measles and, 14 years after the local transmission of measles was halted in the UK, the disease was once again reported to be endemic in 2008 [6], and the beginning of 2013 saw the highest rates of measles in two decades. In both 2010 and 2011, there were over 30,000 cases annually of measles in the European region [7]. In another instance, in 2003 five states in Northern Nigeria ordered the boycott of the oral polio vaccine (OPV), alleging that the vaccines were contaminated with anti-fertility substances in a plot by Western governments to reduce the Muslim population [8]. As a result of the boycott, polio reappeared in more than 15 formerly polio-free African countries (and as far afield as Indonesia) [9], and challenges to eradication persist [10]. A more recent example of widespread vaccine refusals was during the 2009–2010 response to the (A)H1N1 pandemic threats, during which populations, including health professionals, around the world had dismally low acceptance of the Influenza A (H1N1) vaccine for a complex mix of reasons from perception of low-to-no disease risk, suspicions around the motives of government and the pharmaceutical industry, and historic memories of reports of an earlier swine flu vaccine causing Guillain–Barré syndrome (GBS). These examples highlight the complex social, historical, political, and power dimensions that influence vaccination uptake [1].These experiences and the research on drivers of vaccination behavior have shown that vaccination decision-making is driven by different factors according to individual or group experiences and contexts, beliefs, and knowledge. Dependent on the viewpoint of the public/s, healthcare professional or government/healthcare system, the spectrum of attitudes toward vaccines ranges from considering them to be life-saving, to viewing them as a danger to health. Research also demonstrates that facts only go so far in determining decision-making; cognitive heuristics are equally important drivers. For example, the regret that people associate with potential adverse events after MMR vaccination has been shown to be a key predictor of MMR uptake [11]. Other studies have shown that many factors that affect behavior are unrelated to facts or awareness, and that traditional modes of health education that are more message-driven rather than dialog-promoting, may have only a small impact on behavior [12,13]. A recent systematic review of the evidence for effective national immunization schedule promotional communications found no evidence that improved knowledge led to increased childhood vaccine uptake, or even intention to vaccinate [14]. To date, much of the literature on vaccination decision-making has identified attitudinal and demographic correlates of complete and incomplete vaccine uptake largely in individuals. The published research has mostly been uni-disciplinary—i.e., drawing from a single specialty (like psychology, or sociology, or public health). While important, the field needs to draw from a range of rich theoretical understandings in other areas of health that can inform more holistic frameworks to understand vaccine decisions and their motivations. Multi-disciplinary approaches to understanding vaccination behavior could also further extend the evidence base, making the most of the tools and frameworks available within the different disciplines. Finally, further development of the field will require the design and evaluation of theory, and evidence-informed interventions at an individual, community, and national level to address the identified influences of vaccination decisions.This paper reports on a two-day workshop with a multidisciplinary group of experts aimed at mapping, firstly, the known and potential drivers and barriers to vaccination at the individual and societal level, and, secondly, a research framework that allows future research to address them. Our ultimate aim was to inform a new research agenda from a rich multidisciplinary evidence base to inform vaccination program design and policy making. In December 2010, an international think tank called “MOTIV” (Motors of Trust in Vaccination) was convened in London. The think tank deliberately assembled professionals with diverse expertise both within, and beyond, vaccinology. The 15 participants were variously expert in medical science, vaccinology, epidemiology, pediatrics, immunization policy and programs, immunization behavior, global health, psychology, anthropology, sociology, decision science, communication science, advocacy, public engagement, and manufacturing. The specific aim was to map the complex web of factors that may influence decision-making about vaccines at all levels, including individuals, peer groups, clinicians, and policy makers. These aims were addressed through a range of interactive sessions. First, a structured brainstorm was carried out where members were asked to spontaneously identify factors affecting vaccination-related behavior—including vaccine acceptance, hesitancy, and refusal—by consumers, professionals, and policy makers. This involved the use of a “reverse brainstorm” technique to help participants look at issues around vaccination uptake “through new eyes”. Here, participants were asked to consider the issue of vaccination from the opposition point of view—and think of ways to make uptake of a vaccine program as poor as possible (in this case a fictitious new vaccine with data from clinical trials showing acceptable levels of safety and efficacy). Following the reverse brainstorm, participants identified factors/determinants of vaccine decision-making. The ideas were captured in an iterative manner and clustered by MOTIV participants into three major domains. Participants were then assigned to three teams that would each explore one of the major domains. Each team reviewed the list of factors/determinants in their assigned domain and then ranked them based on their expert perceptions according to importance, level of evidence, feasibility/actionability, and the need for more research. These key factors/determinants were distilled into research questions that could be taken forward for further investigation. Key factors/determinants for which a research framework could be developed were finally identified and discussed within the entire MOTIV group. The brainstorming identified poor communication, safety concerns, political issues, anti-vaccination activism, and animal rights as the major areas under which a vaccine program might be derailed. Regarding determinants of vaccination decision-making, the MOTIV expert group identified 61 factors that may affect vaccination-related behavior in consumers, professionals and policy makers (Appendix 1). These factors were further iteratively organized into “clusters”, which were grouped under three major domains: cognition and decision-making; groups and social norms; and communications and engagement. Research questions for further investigation were derived across the three major domains of influence on vaccination uptake, as detailed below. Boxes 1 and 2 illustrate two exemplars of such research questions. A range of cognitions influence vaccination decision-making, including heuristics. These are cognitive shortcuts used for making decisions about risk. One well-described heuristic in vaccination decisions is omission bias. This bias occurs if poor outcomes arising from an “action” (e.g., a reaction to a vaccine arising from deliberate acceptance of a vaccine) are viewed more unfavorably than poor outcomes arising from an “inaction” (e.g., disease contraction arising from “taking a chance with fate”), even if those outcomes are objectively identical, or omission is in fact more risky. Psychologically, omission bias has been linked to the emotion of regret: decision-makers tend to experience more regret for an outcome that they perceive as a consequence of their own voluntary “action” than for the very same outcome if that is perceived to be a consequence of luck or fate. Within the context of immunization, typically immunizing is seen as a conscious “action” whereas not immunizing (i.e., “doing nothing”) is seen as “inaction”. Evidence for omission bias has been demonstrated in relation to pertussis [11,12], MMR [13] and H1N1 vaccines [14].Following the identification of drivers and barriers to vaccination and the related decision making processes, we sought to outline a viable research framework. Figure 1 presents an overarching framework aimed at outlining and systematizing a multidisciplinary research approach that is directly linked to evidence-based policy making. The framework is grounded on the types of themes that emerged through the expert brainstorming—namely the cognitive, social/interpersonal, and communication-related influences on attitudes to immunization and vaccination decision-making. A four-step iterative cycle is described. In the first step, descriptive and experimental research offers scientific definitions and illustrations of the issues to be tackled (e.g., omission bias in immunization decisions). In the second step, the findings are translated into interventions—typically including individual decision-makers (e.g., a de-biasing technique to be applied by community nurses or physicians offering vaccinations), the wider public, and also healthcare professionals. In the third step, these interventions are prospectively evaluated for effectiveness and the findings are fed back into the evidence base in the fourth and final step. Which public engagement strategies within the areas of vaccination decision-making and broader healthcare have achieved their goals, and how and why have they achieved their goals?  How does/should communication and engagement change according to culture, geographical region or broadcast channel?Public engagement is an umbrella term for a range of activities that occur at the interface between the specialist and non-specialist. Engagement is defined more by its ethos than by the vehicles of engagement. A key consideration is power: who is driving an engagement process, who owns the conversation, and how far can this process meet different stakeholders’ multiple agendas? The emphasis of engagement is not to get public buy-in for a health program or technology; it is something more collaborative than lobbying or campaigning and goes beyond health promotion. Engagement aims to catalyze a two-way interaction, and well-executed public engagement will ultimately enable more critically aware, insightful decisions for all parties. This may be breaking new and difficult ground for many professionals and scientists and is therefore an important area for future research.A dynamic multidisciplinary research framework to drive evidence-based policy making in vaccination.Importantly, the framework rests on robust multidisciplinary research and development process—which includes key social and behavioral sciences. It also identifies the need for real-life longitudinal tracking not only of coverage and disease outbreaks via epidemiological methods, but also of behavioral and social reactions to immunization. The latter are aimed as explanations for and predictors of the former. The basic premise of the framework is a dynamic approach to the generation and evaluation of new evidence to drive policy-making and program design. Vaccine decision-making is recognized here as a dynamic field of enquiry that can be rapidly affected by new vaccine developments, novel social movements (e.g., newly emerging social networks) and the increasing quest for evidence-based policy. The iterative link described here between multidisciplinary science and real-life coverage/attitudes to vaccines allows this framework to offer insights into policy making. The MOTIV workshop brought together a multidisciplinary group of experts and aimed at mapping drivers and barriers to vaccination to inform future research priorities. The ultimate aim of the workshop was to contribute to a contemporary research agenda, which will in turn inform vaccination policy-making and program design (in the manner outlined in Figure 1). It is clear from the factors identified that public engagement around vaccines needs to be broad and multifactorial, with engagement at multiple levels. These include policy-making (e.g., deliberative democracy), program design (including delivery) and the development of risk communication strategies. Methodological improvements are required for better understanding of vaccine decision-making across populations and contexts and over time. Self-reported vaccine uptake and cross-sectional studies (where we assume causation between a certain attitude and behavior from measurements made only at one time point) limit the robustness of research into vaccination decision-making. In this context, attention to improving research design and data quality is essential, to provide a clear understanding of the relative contribution of factors such as trust, risk perception, online networks, peer networks, and misinformation. Theoretically sound research frameworks and validated methods are also important. Use of recent, robust evidence-based attitude measurement instruments to evaluate the predictors of MMR uptake clearly shows that differences exist in the way vaccine-acceptors and vaccine-decliners think about several key factors regarding vaccination and disease control [15]. The MOTIV approach has some limitations. The faculty consisted of experts across diverse fields but did not exhaust the range of potentially relevant areas of expertise. Moreover, during the group sessions it was agreed that the domain “Communication” is too broad an area and more specific research topics need to be defined within the broader realm of communication. Additionally there was a significant degree of crossover between the domains—for example “Trust” overlaps with “Public engagement”. The question of “What influences policy decision-making?” was identified as missing and was subsequently added to the decision-making category. Further, no formal consensus building methods were applied, as the idea-generation techniques used throughout the workshop were solely qualitative.These limitations notwithstanding, we take the view that the questions outlined, and the proposed framework, are timely. Recent global events have demonstrated a desire for strategic attention to vaccine decision-making. The need to strengthen public support for vaccination efforts is one of the four components that comprise the Global Vaccine Action Plan, endorsed by the World Health Assembly (2012), and catalyzed by the Decade of Vaccines collaboration [13]. Additionally, WHO Strategic Group of Experts (SAGE) on immunization created a working group to specifically address vaccination hesitancy in 2012. Just as vaccine development and testing is informed by science and research, so must our understanding of vaccine decision-making by publics, professionals, and policy makers be informed by robust scientific methods. This understanding will more credibly inform appropriate interventions to support decision-making. This framework provides the groundwork for a more explicitly articulated research agenda on vaccine decision-making. It suggests cross-disciplinary investigations (e.g., applying social networking theories to understanding community influence) and provides a starting point for researchers to identify areas well understood and those needing further enquiry. An approach to researching vaccine decision-making and to translating the research findings into usable building blocks for policy making has been described, involving a range of multidisciplinary factors that cannot be addressed simply with existing health metrics or by one discipline alone. The aim of the MOTIV think tank was to map what we do and do not know about the drivers of vaccination decision-making, and to look beyond the traditional “one-way” approach to health information. In-depth understanding of complex decision-making processes—through appropriate collaborative research across multiple disciplines—is key to better understanding the drivers and barriers of trust in vaccination, and defining how best to engage publics. This provides a first step towards building a dynamic multidisciplinary research network, in both developed and developing countries, that can synthesize research findings within a coordinated research program, develop interventions, and eventually facilitate increasingly evidence-based vaccination policy. This article summarizes the discussions held at the MOTIV think tank meeting in London in December 2010, which was supported by Sanofi Pasteur and the London School of Hygiene & Tropical Medicine and led by Heidi Larson. The authors present these results on behalf of the other MOTIV faculty members: Tim Appenzeller, Nature Journal, London, N1 9XW, UK; Louis Cooper, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA; Pete Cranston, Independent Consultant, Digital Strategy and Knowledge Management, Oxford OX4 1SU, UK; David Heymann, Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, London WC1 7HT, UK; Seth Kalichman, Psychology Department, University of Connecticut, Storrs, CI 06269, USA; Stanley Plotkin, University of Pennsylvania, Doylestown, PA 18902, USA; Claire Topal, Pacific Health Summit, Seattle, WA 98103, USA; Kasisomayajula Viswanath, Department of Society, Human Development and Health, Harvard School of Public Health, Boston, MA, USA; Michael Watson, Vaccination Policy & Advocacy, Sanofi Pasteur, Lyon 69007, France; Jo Yarwood, Department of Health, London SE1 8UG, UK. The authors thank Communigen Ltd. (Oxford, UK) for their assistance in facilitating and summarizing the workshop. The authors take sole responsibility for the article’s content. All authors have completed the Unified Competing Interest form (available on request from the corresponding author) and declare that (1) AT, TA, PC, DH, SK, CM, SP, CT, KV, MW, JY have support from Sanofi Pasteur for the submitted work; JL, HL, and LC have support from The Gates Foundation and London School of Hygiene & Tropical Medicine for the submitted work; SA has support from the Wellcome Trust for the submitted work; NS is a member of the Imperial Center for Patient Safety and Service Quality, which is funded by the National Institute for Health Research (NIHR), UK and is currently funded by an unrestricted research grant by Sanofi Pasteur and also by the National Institute for Health Research (UK), via the Imperial Patient Safety Translational Research Center; JL has previously been an investigator on a study of pediatric influenza vaccination with part funding from Sanofi Pasteur. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health, or Sanofi Pasteur. The funders do not have any editorial control over the work reported in this article; (2) HL, SA, TA, LC, PC, DH, SK, CM, SP, CT, KV, JY have no relationships with Sanofi Pasteur that might have an interest in the submitted work in the previous 3 years; AT and MW have a specified relationship with Sanofi Pasteur that might have an interest in the submitted work in the previous 3 years; (3) HL, SA, JL, NS, TA, LC, PC, DH, SK, CM, SP, CT, KV, JY declare that their spouses, partners, or children have no financial relationships that may be relevant to the submitted work; AT and MW have a specified relationship with Sanofi Pasteur that might have an interest in the submitted work in the previous 3 years; and (4) HL, SA, JL, NS, TA, LC, PC, DH, SK, CM, SP, CT, KV, JY do not have non-financial interests that may be relevant to the submitted work; AT and MW have specified non-financial interests that may be relevant to the submitted work.Factors (Drivers and Barriers) Affecting Vaccination Uptake and Sample Research Questions.Trust in national institutionsTrust in healthcare workersTrust in authorities/expertsTrust in researchTrust in policy makers/accountabilityTrust in mediaTrust in official informationTrust in vaccine industryTrust in vaccinesInterpersonal trustIndustry-policy maker-researcher trustTransparencyCompetencyHeuristics (e.g., confirmatory bias, regret bias, omission bias)Post-hoc rationalisationRisk-benefit assessmentsScientific/medical literacyPhysical sensationsPerceptions about the faith and fear of injecting substancesPerceptions regarding vaccine adverse events, including their likelihood and severityInvisible disease (low perception of risk)Competing priorities; Practical barriers to accessWeighting of the factors may change depending on the contextMulti-factorial equation involving the individual and groups (institution, culture, religion, social network)Experience of adverse events and how they are managedSafety research on vaccines (at an individual levelMandatory vaccination transfers decision-making away from people and leads to opposition and controversiesLevel of education/literacy/ scientific and health literacySocial networksReligious groupsGroups by education-levelAlternative medicine groupsPolitical groupsAnti-vaccination groupsFamily groups/structuresPatient groupsConsumer groupsProfessional groupsPeer pressureHeterogeneous groupsDemographic (socioeconomic) groupsInfluence of alternative healthcare givers such as Chinese medicineSocial and cultural normsVaccination as a routine/normHealthcare workers communication skillsPhysicians poor vaccine educationMediaThe authority of the messenger impacts the credibility of the source of the informationTone in communicationEnsuring that people feel “listened to”Level of how informed and educated (content of training) the media and journalist areHealth care workers level of confidence in providing information to their patientsHealthcare worker temperament

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+Member of the NGIN consortium. Other members of the group are listed in the acknowledgment.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.The ability to elicit HIV-1 neutralizing antibodies (Nabs) is likely to be an essential feature of protective HIV-1 vaccines. The HIV envelope spike is the only glycoprotein presented on the virion and on the surface of infected cells for antibody binding and neutralization, by broadly neutralizing antibodies (bNAbs). Five areas on the HIV trimeric spike have been identified so far as conserved targets for broadly neutralizing monoclonal antibodies cloned from patients including elite neutralizers [1]. Extensive attempts have been undertaken to construct immunogens and use different vaccine strategies to direct antibodies to these areas and to improve functionality, also encompassing antibody-dependent cell-mediated cytotoxicity (ADCC) [2]. However, the growing knowledge of neutralizing epitope structures on the HIV-1 Env does not automatically translate into the generation of improved immunogens, emphasizing the importance of continuing all approaches in the search for HIV-1 vaccine immunogens. Thus, lessons may still be learned from envelopes of rationally selected and/or modified clinical HIV-1 strains e.g. from patients with bNAbs, ADCC and/or a defined favorable clinical course. A stable mimic of the native envelope spike would be an ideal HIV-1 vaccine immunogen, but is technically challenging to construct and produce [3]. Successful attempts to produce in vitro stabilized recombinant glycoproteins include the introduction of SOSIP mutations [4,5] and isoleucine-zipper trimerization signals [6,7], combined with improved gp120/gp41 cleavage site [8]. These modifications were also efficient in inducing neutralizing antibodies [9,10,11]. However, a DNA vaccine expressing selected envelopes intracellularly and in vivo can potentially more closely mimic the native structure and glycosylations, which may differ from in vitro cell line expressed proteins [3]. In addition, a naked DNA vaccine displays the benefits of proven safety, easy manipulation and manufacturing, no anti-vector immunity, and contains in itself an adjuvant effect [12,13]. DNA constructs are also convenient for screening and selection of envelopes which can be rationally modified and tested subsequently to guide protein immunogen production [14]. Despite promising initial studies in small animal models, naked DNA vaccines showed lower immune potency in humans and non-human primates [13]. However, enhanced immunogenicity has now been obtained with several improvements making second generation DNA vaccines ready for trials and use in larger animal models, including humans [15,16,17]. The optimizations of potency include codon-optimized gene sequences [18,19], repeated injection regimens, the inclusion of plasmid adjuvants and various mixed modality (prime-boost) strategies [13,14]. Use of in vivo electroporation as a DNA delivery method has proven very effective in enhancing uptake and immunogenicity of DNA vaccines [20,21,22,23]. SIV/SHIV infection of macaques is the most reliable animal model for preclinical testing of candidate HIV vaccines. However, before such testing, evaluation of potential immunogen candidates needs to be conducted by screening of several immunogens and improved gene versions in smaller animals, such as rabbits or guinea pigs. The rabbit model (Oryctolagus cuniculus) is increasingly used in preclinical HIV-1 vaccine development studies. Firstly, rabbits are large enough to yield sufficient volumes of serum for extensive testing, yet much less challenging to house than non-human primates. Secondly, the rabbit litter size is also large, making it possible to breed for experimental use without endangering the species. The rabbit antibody heavy-chain third complementary-determining region (H3 CDR) is comparable to the length of the VH3 CDRs of human antibodies, whereas the mouse has a shorter VH3 CDR [24,25]. Since length and flexibility in H3 CDRs are structural features necessary for some monoclonal bNAbs [26,27,28,29,30], the rabbit model provides an opportunity for such antibodies to develop. Mice sera are limited in volume and may contain cytostatic factors that down-modulate CD4 receptors on human cells [31], making it less suitable when screening vaccine candidates in HIV NAb assays. Utilizing guinea pigs as models has some of the drawbacks of that with other rodents, but guinea pigs have larger blood volumes than mice and are relatively inexpensive and easy to house and handle.In this study, we have optimized DNA env constructs for immunogenicity, in rabbits and guinea pigs following several steps. The DNA constructs used were based upon the viral reference strain HIV-1Bx08, shown to be commonly recognized by immune sera from a variety of patients [32], and thus, exposing common epitopes for NAbs [32]. We have previously shown that the codon-optimized envBx08 can induce NAbs with limited breadth [18,33,34]. To select potentially better clinical HIV-1 Env immunogens than the EnvBx08, we now hypothesized the opposite, namely that envelope immunogens, which are instead derived from patients with broad neutralizing activity or elite neutralizers, may potentially induce antibodies of broader neutralizing nature. To test this hypothesis, two envelope genes were selected this way and developed into DNA vaccine constructs, and used in a trivalent formulation combined with envBx08. Furthermore, the immunogenicity of env constructs was evaluated with or without the SOSIP-modifications, aiming to stabilize the envelope protein in trimeric conformation. Finally, the optimal vaccine candidate in rabbits and guinea pigs was further tested for immunogenicity in cynomolgus macaques and compared to the immune responses elicited in the smaller animal models.The construction of Bx08 gp140 (Genbank JX473289) plasmid used codons from highly expressed human genes as described earlier [18,33,34] and two other primary Envs from Danish patients, ctl21 (JX473290) and ctl27 (JX473291), were similarly codon optimized. Seven different clade B env constructs were synthesized (syn.) and used (syn.gp140Bx08, syn.gp150Bx08, syn.gp140ctl21, syn.gp140ctl27, syn.gp140Bx08 SOSIP.R6-IZ-H8, syn.gp140ctl21 SOSIP.R6-IZ-H8 or syn.gp140ctl27 SOSIP.R6-IZ-H8). We have previously described the construction of synthetic envBx08 plasmids encoding secreted gp140 and membrane-bound gp150 from HIV-1 Bx08 [18,33,34]. The two primary envctl21 and envctl27 were PCR-amplified from isolated patient virus, cloned, sequenced and then synthesized using only codons from highly expressed human genes (completely codon exchanged) [34,35]. All genes were cloned into the previously described mammalian expression vector pPPI4 [8,10,36]. Plasmids encoding SOSIP.R6-IZ-H8 gp140 variants were constructed as follows: Amino acid substitutions (HxB2 numbering) A501C, T605C and I559P (SOSIP) were introduced as previously described [36]. Additionally, the proteolytic gp120/gp41 cleavage site REKR was substituted with a hexa-arginine motif (R6) to increase cleavage [8]. Together these amino acid substitutions are referred to as SOSIP.R6. The isoleucine zipper (IZ) domain was added to the gp140 C-terminus to facilitate gp140 trimerization [7,37,38]. Also, eight histidine residues (H8) were added to allow downstream protein purification procedures. The vector expressing the Env proteins has been described elsewhere [39,40], but was further modified by mutagenesis to contain a multiple cloning site, including a Hind III site, between the tPA sequences and the env sequences. The env sequences used in this study were then sub-cloned into the resulting vector using Hind III and BamH I. Protein expression was controlled with HEK 293T cells grown in DMEM (Gibco, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin. Transfection was performed with Polyfect transfection agent (Qiagen, Hilden, Germany) and expressed proteins were separated in an 8% tris-glycin gel (Invitrogen, Carlsbad, CA, USA). Env proteins were detected by Western blotting using human anti-HIV polyclonal antisera and visualized with a goat HRP-conjugated antihuman IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). Membranes were developed with SuperSignal West Femto (Pierce, Rockford, IL, USA) and chemiluminiescence was detected with an UVP (AH Diagnostics, Aarhus, Denmark).rgp140 clade C heterotrimer protein mix was produced by transient transfection of HEK 293 T cells. In short, 2 mg plasmid DNA with multiple clade C env expressed constructs, complexed with 3.6 mg PEI, was added to cells. Supernatant was collected after 48 and 96 hours, and after adjusting to pH 8, the media was passed over a cobalt chloride metal-affinity column made from Talon Superflow resin (Clontech, Palo Alto, CA, USA). Protein was eluted with 250 nM imidazole and concentrated and separated by gel filtration chromatography using a Superdex200 26/60 size-exclusion column (GE Healthcare, Buckinghamshire, UK). The gp140 trimer fractions were identified and further purified using a GNA-lectin resin (Vectorlabs, Burlingame, CA, USA).Ten week old female nulliparous New Zealand white rabbits purchased from Charles River Laboratories were housed at Statens Serum Institute Animal Facility (Copenhagen, Denmark). Acclimatization was at least 10 days prior to any experimental procedures. Animal experiments were performed by certified animal handlers and according to the Animal Experimentation Act of Denmark and European Convention ETS 123. Groups of four rabbits were immunized at week 0 (three times during the first week), 4, 8 and 12 with 200 µg DNA injected intradermal (i.d.) and distributed at two injections sites (Figure 1A). The mode of “intensive” priming within week 0 (3 × 200 µg DNA) was initially compared with single DNA immunization and protein immunization. and demonstrated a more rapid and uniform antibody response than both other immunizations (Supplementary Figure 1). Subsequent electroporation using OncoVet™ system (CytoPulse Sciencies/Cellectis, Romainville, France) was done over each injected area. Four groups of rabbits were used, receiving syn.gp140Bx08, syn.gp150Bx08, syn.gp140mix (syn.gp140ctl21 + syn.gp140ctl27 + syn.gp140Bx08) or syn.gp140mix modified (syn.gp140Bx08 SOSIP.R6-IZ-H8 + syn.gp140ctl21 SOSIP.R6-IZ-H8 + syn.gp140ctl27 SOSIP.R6-IZ-H8). The amount of DNA constructs in the mixed formulations was 1:1:1, giving in total a 200 µg/immunization. In all four groups, blood was collected before immunization (w0) and two weeks after last immunization (w14). In addition, rabbits immunized with syn.gp140mix had blood collected at each re-immunization (w4, 8, 12) and rabbits immunized with syn.gp140mixSOSIP.R6-IZ-H8 had blood collected every week until w6 (w1, 2, 3, 4, 5, 6), then every second week until w14 (w8, 10, 12, 14).Dunkin Hartley guinea pigs (HsdPoc:DH) were housed at Statens Serum Institute Animal Facility. Acclimatization was at least one week prior to any experimental procedures. Groups with four 12 week old guinea pigs were immunized at week 0, 4, 8 and 12 with 50 µg DNA injected i.d. and localized to either side of the abdomen area. The vaccination area was subsequently electroporated using the OncoVet™ system. Three groups of guinea pigs were used receiving syn.gp140Bx08, syn.gp140mix or syn.gp140mix modified. Blood samples from a vessel near the eye were taken every second week of the immunization schedule. Adult cynomolgus macaques (Macaca fascicularis), imported from Mauritius, were housed at the CEA facilities (Fontenay aux Roses, Paris, France) and handled in accordance with French national regulations and under veterinary inspectors (Permit number: A 92-032-02). All procedures were carried out under general anesthesia with intramuscular injection of 10 mg/kg ketamine (Rhône-Mérieux, Lyon, France). Four macaques were immunized by the same regimen as the rabbit protocol with intensive priming at week 0, and followed by three subsequent immunizations at w5, 9 and 13, using 800 µg DNA distributed intradermal at four injection sites, and followed by electroporation. All four animals received the DNA construct syn.gp140mix. At w17, the macaques were injected with 80 µg clade C rgp140 heterotrimer protein mixed with 800 µg of the DNA construct syn.gp140mix, and no additional adjuvant. Again the injections were followed by electroporation. Blood was collected before immunization (w0) and at different time points for assessing the immune response. For assessing specific anti-gp120 IgG three different ELISA assays were established for the three different animal models: rabbits, guinea pigs and cynomolgus macaques. The detailed protocol for the rabbit ELISA is summarized here, followed by minor modifications for the guinea pig and macaque protocols. Maxisorp 96-well plates (Nunc) were coated overnight with recombinant gp120IIIB protein (Fitzgerald Industries International, Concord, USA) in carbonate buffer, pH 9.6. Plates were blocked the following day for 1 h at room temperature with blocking buffer containing PBS, 1% BSA, 10% FCS and 1% Triton X-100. Rabbit sera were subsequently added in serial dilutions, diluted in blocking buffer. After an overnight incubation at room temperature, plates were washed five times with washing buffer (PBS, 0.01% Triton X-100). HRP-conjugated mouse anti-rabbit (Sigma, A1949, St. Louis, MO, USA) antibody was added at a 1/2,000 dilution. After 1 h incubation at room temperature, plates were washed and a one-step TMB substrate (Kem-En-Tec Diagnostics, Copenhagen, Denmark) was added. The colorimetric reaction was stopped with 0.2 M H2SO4 and absorbance values were read at 540 nm. Titers were defined as the lowest reciprocal dilution yielding an absorbance value greater than the optical density of twice the background absorbance (wells containing blocking buffer). A mixture of pre-defined high-titer rabbit sera was used as positive control [41]. The ELISA assay for detection of guinea pig and macaque specific anti-gp120 IgG was modified with a prolonged overnight incubation for the coating step to twice overnight. Blocking buffer in the guinea pig assay was PBS, Tween-20, 5% rabbit-normal-serum and in the macaque assay PBS, 1% BSA, 2% skimmed milk powder, 1% Triton X-100. The blocking step was carried out for 1 h on a shaker. Dilution buffer in the guinea pig assay was PBS, 0.05% Tween-20, and in the macaque assay it was the same as blocking buffer. The overnight incubation with diluted animal sera was carried out on a shaker. The guinea pig assay used HRP-conjugated rabbit anti-guinea pig (Sigma, A5545) antibody at a 1/50,000 dilution and the macaque assay used HRP-conjugated mouse anti-human (BD, 555788, Franklin Lakes, NJ, USA) antibody at a 1/500 dilution. The 1 h incubation with conjugated antibody was carried out on a shaker. The colorimetric reaction was terminated with 1 M H2SO4. The guinea pig assay used a mixture of high-titer guinea pig serum as a positive control and the macaque assay used IgG purified from pooled HIV-positive patient serum. Neutralizing activity in sera from immunized animals was analyzed in the pseudovirus-TZMbl assay as described elsewhere [42,43]. Briefly, purified IgG from rabbit sera was used in the TZMbl assay diluted in four 2-fold dilutions, starting at a final concentration of 250 or 400 µg/mL. Rabbit IgG was purified from heat inactivated sera using Protein G HP SpinTrap columns (GE Healthcare). Heat inactivated serial diluted guinea pig and macaque sera were used directly in the TZMbl assay, starting at 1/20 or 1/30 dilution, respectively, and diluted in two-fold steps. Neutralizing activity was expressed as the IgG concentration or reciprocal serum dilution that established 50% inhibition (IC50) of virus infection, as determined by the method described in Fenyö et al. in 2009 [44].The ctl21 and ctl27 envs were selected by screening of patient sera or EDTA plasma for neutralization activity, using PHA-P-stimulated donor peripheral blood mononuclear cells (PBMCs), cultured in RPMI 1640 and Glutamax media (Gibco) supplemented with 10% FCS, recombinant human IL-2, penicillin and streptomycin. Briefly, cells were seeded in 96-well tissue culture plates (105/well) and virus, pre-incubated 1 h with heat-inactivated plasma or serum samples, and added to the wells. Infection was allowed for 24 h, then the plate was washed and new medium added. Culture supernatant was harvested at day 3, 4 and 5 and assayed for p24 production [45]. As a negative control, cells and virus were incubated with serum from a non-infected individual. Differences in neutralizing activity between groups against various pseudotype viruses were evaluated for statistical significance by a Wilcoxon signed rank test. Two-way ANOVA was used to calculate differences in antibody titers between immunization groups. Comparison of neutralization over time in the macaque group was tested using one-way ANOVA with Dunn´s post test. GraphPad Prism v. 5.0 was used for all analyses.To optimize the DNA vaccine to elicit a high and broad immune response in rabbits, the initial evaluation of env DNA concerned the use of gp140 or gp150 genes. The syn.gp150Bx08 or syn.gp140Bx08 DNA plasmids genes are translated into membrane-bound or secreted glycoproteins, respectively [18,34], and induce an antibody response of equal magnitude in guinea pigs [33,34]. The amount of Env-specific binding IgG in rabbit sera 14 weeks after immunization was assessed against recombinant gp120IIIb (Figure 1). Specific IgG antibody titers were induced in the rabbits by both DNA constructs, with a similar increase of >2 logs over the baseline level. Purified serum IgG from week 14 was analyzed for neutralizing activity against a panel of six different HIV-1 viruses (clades A–C). Results are depicted as 50% inhibitory concentration (IC50) of purified serum IgG (Figure 1C and Supplementary Table 1 for individual IC50 values). No significant difference in neutralizing activity was seen in rabbits by the two different constructs. Three viruses (SF162, Bx08 and BaL) were easier to neutralize with IgG from syn.gp140Bx08 rabbit antisera than IgG from syn.gp150Bx08 antisera. Thus, both syn.gp140Bx08 and syn.gp150Bx08 were able to induce a potent antibody response in rabbits demonstrating neutralizing effect on four or three viral strains out of six, respectively, at IgG concentrations between 31 and 400 µg/mL. The viral strains most sensitive to neutralization were all Tier 1 of clade B, which was expected since the DNA construct originated from a clade B virus. Based on this and the possibility of comparing the syn.gp140Bx08 with other gp140 constructs, the results from syn.gp140Bx08 immunization were included in further analysis.To broaden the heterologous neutralization capacity induced by syn.gp140 of Bx08, both rabbits and guinea pigs were immunized with a mix of rationally selected HIV env genes added to the Bx08 and compared to immunization with monovalent Bx08 immunization. Rational selection of the additional env genes was based on screening of neutralizing activity of infected patients’ plasma samples. We hypothesized that envelope immunogens derived from virus of patients with broad neutralizing activity may induce similarly broadly neutralizing antibodies upon immunization in animals or humans if delivered as optimized DNA vaccine constructs. Plasma samples (n = 35) from Danish HIV-1-infected treatment-naïve individuals were collected [46] and screened for neutralization against HIV-1 virus isolates, four clade B and one A1D intersubtype recombinant [47] (Table 1). As expected, the sensitivity to neutralization varied among the virus isolates, with clade B HIV-1BaL being most sensitive to neutralization and recombinant A1D HIV-1DK1 least sensitive. In many samples, neutralization was primarily directed against one or two viruses, but in 17 sera (49%) the neutralizing effect was detected against all five isolates, including the A1D recombinant. Among these, two plasma samples, ctl21 and ctl27, obtained from a male and a female with 9 and 3.5 years of infection, respectively, displayed robust and balanced neutralization titers against all five viruses. To test the hypothesis, the env region including V1-V5 of the clade B virus isolates from ctl21 and ctl27 were cloned, sequenced and synthesized as codon-optimized DNA vaccine constructs, flanked by the N- and C-terminal region of gp120 and the extracellular part of the gp41 region from the HIV-1Bx08 env cassette (see Figure 3A and [18]). The constructs were control sequenced and tested for successful in vitro expression of functional envelope glycoproteins (CD4 binding) (data not shown).Immunization regimen and comparing antibody responses in syn.gp140Bx08 or syn.gp150Bx08. DNA vaccinated rabbits. (A) Schematic immunization schedule with vertical arrows indicating immunizations. Sera were collected before immunization (w0) and two weeks after last immunization (w14). (B) Average IgG response against recombinant gp120IIIb (rgp120IIIb) in immunized rabbits (n = 4). (C) Average neutralizing activity, expressed as IC50, of purified IgG from week 14 rabbit sera against pseudotype virus strains of clade B, C and A (SF162, Bx08, JR-FL, BaL, 92Br025 and 92RW009).HIV-1-specific neutralizing activity in serum from infected individuals (n = 35). Given reciprocal titers correspond to 1/dilution of serum giving 80% inhibitory concentration (IC80) in the PBMC neutralization assay. Color coding: IC80 < 5: no color, 5–25: yellow, 25–125: orange or 125–625: red.§ Four of the HIV-1 panel isolates are R5 clade B. # The DK1 isolate is an A1D intersubtype recombinant form with a clade A envelope gene [47].Guinea pig and rabbit groups were immunized with a trivalent mix encoding syn.gp140Bx08, syn.gp140ctl21 and syn.gp140ctl27 to facilitate heterotrimer formation (referred to as syn.gp140mix). Guinea pigs were also immunized with the same single DNA construct, syn.gp140Bx08, as used in rabbits in Figure 1. Monovalent and trivalent DNA immunizations demonstrated similar immunogenicity in guinea pigs (Figure 2A). In the rabbit model, the syn.gp140mix induced a higher fold increase in IgG response at w14 than syn.gp140Bx08 from Figure 1B (Figure 2C). Immune sera obtained week 14 from guinea pigs and rabbits were analyzed for neutralizing activity (Figure 2B,D, and Supplementary Table 1). Guinea pig sera were diluted and used directly in the TZMbl assay, whereas IgG had to be purified from the rabbit sera because of interference observed in some samples. Guinea pig sera and rabbit IgG were tested for NAbs against a panel of 13 or six different viruses, respectively. In the guinea pig model, syn.gp140mix tended to induce higher NAb titers to most viruses tested (Figure 2B) than monomeric syn.gp140Bx08, although this was not statistically significant (p = 0.054, Wilcoxon signed rank test). In the rabbit model, this tendency was less pronounced (Figure 2D). For both guinea pig sera and rabbit IgG, there was a large variation in neutralizing activity; however, the clade B viruses were the most sensitive to neutralization. Pseudotype virus expressing the unrelated murine leukemia virus (MLV) envelope was included as controls when testing guinea pigs sera and demonstrated no vaccine-induced unspecific effect (Figure 2B). Taken together, these results tended to favor the trivalent mix although broader neutralization could not be demonstrated in the rabbit model. However, since the trivalent mixture induced somewhat higher and broader neutralization in guinea pigs to most viruses and a somewhat higher cross-reacting antibody titer (anti-gp120IIIb) in rabbits, the syn.gp140mix was modified and used in further optimization experiments. In addition, the mixing approach has proven effective in other studies [48,49,50,51].It is believed that vaccine immunogens should closely resemble the native trimer to improve bNAbs. Therefore, several modifications were now introduced in the three DNA constructs included in syn.gp140mix to enrich for stabilized trimeric protein conformations. These are described in the experimental section and have all previously been shown to allow the efficient production of stabilized EnvJR-FL trimeric gene products [4]. A schematic representation of the DNA constructs is shown in Figure 3A,B. The constructs were tested for protein expression (Figure 3C), and a somewhat lower in vitro expression in HEK 293 cells was seen from constructs that included all the modifications (SOSIP.R6-IZ-H8). We also noted that although the IZ domain seemed to enhanced trimerization of SOSIP gp140, it also decreased to gp140 cleavage into gp120 and gp41, despite the presence of an optimal cleavage site (Figure 3C), confirming what others have reported [39,40]. Expressed gp140 with SOSIP.R6 modifications seemed to form monomers, dimers and trimeric proteins as opposed to non-modified gp140 which only appeared as monomers and dimers when analyzed by blue-native PAGE.Both guinea pigs and rabbits were immunized with the modified DNA constructs, syn.gp140mix SOSIP.R6-IZ-H8. The guinea pigs demonstrated specific IgG after the initial immunization which was boosted upon re-immunizations; however, the modified construct, syn.gp140mix SOSIP.R6-IZ-H8, induced significantly lower titers of antibodies when compared to non-modified syn.gp140mix, as per Figure 2A (compared in Figure 4A). Interestingly, vaccination with syn.gp140mix SOSIP.R6-IZ-H8 generated a statistically significant higher neutralizing activity than syn.gp140mix in the guinea pigs (p = 0.021, Wilcoxon signed rank test) despite the lower ELISA titers (Figure 4B, and Supplementary Table 1). However, the more potent neutralizing activity also included non-specific neutralization since a MLV pseudotype virus was also neutralized at high dilutions of guinea pig syn.gp140mix SOSIP.R6-IZ-H8 antisera. This unspecific neutralization was not seen with the non-modified syn.gp140mix in Figure 2B. Immunization of rabbits with the same construct resulted in lower antibody titers for syn.gp140mix SOSIP.R6-IZ-H8, as compared with non-modified construct in Figure 2C (compared in Figure 4C), and similarly, as seen with guinea pig sera. Though, in the rabbit model the two constructs yielded similar neutralizing activity for the six different viruses tested (compared in Figure 4D, and Supplementary Table 1). Only two of the six viruses used could be neutralized to 50% by syn.gp140mix SOSIP.R6-IZ-H8 antisera at the IgG concentrations tested, and they were both clades B pseudotype virus. Comparison of the immune responses in animals vaccinated with monovalent or trivalent DNA. Average IgG responses against rgp120IIIb in immunized. (A) guinea pigs (n = 4) and (C) rabbits (n = 4). Immunization time points are indicated with arrows. Average neutralizing activity, expressed as IC50, of diluted guinea pig serum (B) or purified rabbit IgG (D) from week 14 against pseudotype virus strains of clade A–D and CRF02_AG. Unrelated MLV pseudotype virus was included as non-specific HIV control in the guinea pig setup (red). IgG titers (C) and IC50 values (D) from syn.gp140Bx08 in the rabbit model are derived from Figure 1B,C. Schematic representation of HIV-1 envelope DNA constructs and protein expression. DNA constructs encoding gp140. The tissue plasminogen-activator leader sequence (tPA) and the region encoding the variable regions V1 to V5 are indicated (grey boxes). (A) The gp140ctl21/27 construct with V1-V5 region from ctl21 and ctl27 env flanked by Bx08 env. (B) DNA construct encoding modified gp140 including the SOSIP amino acid substitutions A501C, T605C and I559P (SOSIP), the hexa-arginine cleavage site (R6), the introduced isoleucine-zipper motif (IZ) and the histidine tag (H8). (C) Western blot analysis of protein expression (SDS-PAGE) and oligomerization (Blue-Native PAGE) of EnvBx08 constructs, encoding gp120, gp140, gp140SOSIP.R6 and gp140SOSIP.R6-IZ-H8. These data indicate that neutralizing activity can be improved by use of DNA vaccines encoding for modified Env immunogens, syn.gp140mix SOSIP.R6-IZ-H8, but the increased activity in guinea pigs is non-HIV specific. IgG was purified from a few selected guinea pig serum samples and tested in the TZMbl assay (data not shown). The guinea pig IgG displayed very low neutralizing activity. The unspecific neutralizing activity of modified constructs was only observed in immunized guinea pigs, while rabbit IgG resulting from the modified trivalent vaccine displayed similar neutralizing activity as non-modified. Since the non-modified DNA constructs indeed induced higher cross-reactive IgG titers in both animal models, and the HIV-specific immune response appeared similar for both constructs, we decided to use non-modified syn.gp140mix as the vaccine in the cynomolgus macaques.To evaluate if neutralizing response could be translated from small animal models into non-human primates, cynomolgus macaques were immunized with the same DNA construct, non-modified syn.gp140mix, used in guinea pigs and rabbits, and with the same immunization regimen ranging over four months. Comparison of the immune response in vaccinated guinea pigs (A,B) and rabbits (C,D) with plasmid DNA encoding syn.gp140mix or syn.gp140mix modified. Average IgG response against recombinant gp120IIIb (rgp120IIIb) in immunized (A) guinea pigs (n = 4) and (C) rabbits (n = 4). Immunization time points are indicated with arrows. Asterisk indicates significant difference between the two immunization groups (* p < 0.05, ** p < 0.01, two-way ANOVA). Average neutralizing activity, expressed as IC50, of (B) diluted guinea pig serum or (D) purified rabbit IgG from week 14 animal sera against pseudotype virus strains of clade A–D and CRF02_AG. Amphotropic murine leukemia virus (MLV) pseudotype virus was included as control for the non-specific activity in experiments with guinea pig serum (red). Results from syn.gp140mix immunizations were derived from Figure 2.Evaluation of gp120-specific IgG in immunized cynomolgus macaques demonstrated a response already after the initial priming immunizations; however, the antibody titers did not increase with the same magnitude as in rabbits (Figure 5A). Neutralizing capacity of antisera obtained from the immunized cynomolgus macaques was measured in the TZMbl assay against five different HIV-1 virus strains of clade B and C (Figure 5B). Percent neutralization was compared to guinea pig sera and purified rabbit IgG which had been tested against 10 and six viruses, respectively. Macaque and guinea pig sera were tested at a fixed serum dilution and rabbit IgG at a fixed concentration. Four virus strains, SF162, Bx08, BaL and 92Br025, were tested for NAbs from all three animal species. All four viruses demonstrated lower sensitivity to neutralization by macaque antisera as compared to guinea pig sera or rabbit IgG and could not be inhibited to 50% with macaque serum. The remaining virus tested with macaque sera, MNP.ec3, and was easily neutralized by guinea pig sera, but resistant to neutralization by macaque sera. Kinetics of neutralization of one virus, SF162, was compared between rabbit IgG and macaque sera (Supplementary Figure 2). The rabbits developed neutralizing IgG already after the second immunization at week 4, whereas neutralization in macaques developed more slowly, with a substantial increase in activity after the final immunization at week 13. However, only sera from two out of four animals reached neutralization of the virus at >50% inhibition. Hence, the parallel immunizations using the same DNA construct in three different animal models induced specific antibody responses in all animals, but the neutralization activity was lower in the cynomolgus macaques compared to guinea pigs and rabbits. In order to test if the immune response following the DNA immunizations could be boosted with protein, the macaques were injected with a final immunization including both syn.gp140mix DNA and a clade C rgp140 heterotrimer protein. The final immunization resulted in a fast increase in antibody titers (Figure 5A) and a boost in NAbs (Figure 5C,D).Comparison of immune response in guinea pigs, rabbits and cynomolgus macaques immunized with plasmid DNA encoding syn.gp140mix. (A) Average IgG response against rgp120IIIb in immunized animals (n = 4). Immunization time points are indicated with arrows. IgG titers in rabbits and guinea pigs were derived from Figure 2A,C. (B) Average percent neutralization against pseudotype virus strains of clade A–C, by week 14 rabbit IgG or guinea pig sera and week 17 macaque sera. From rabbit sera, IgG was purified and used in neutralization at one fixed concentration (250 or 400 µg/mL). Sera from guinea pigs and macaques were diluted 20 and 30 times, respectively, and used in neutralization. Neutralization results of rabbit and guinea pigs were derived and recalculated from Figure 2B,D. (C and D) Macaque sera was tested for neutralization at 1/30 dilution against SF162 and MW965 viruses with the addition of a final immunization with DNA and protein at w17 (* p < 0.001, One-way ANOVA, Friedman’s test with Dunn’s Multiple Comparison Test).In this study we have rationally selected, systematically optimized and evaluated HIV-1 env DNA vaccine constructs for immunogenicity in rabbits and guinea pigs. Our evaluation resulted in selection of trivalent gp140 vaccine (syn.gp140mix), encoding no modifications for stabilization of trimer formation. This construct was subsequently used for immunization of cynomolgus macaques and immune responses in the three different animal models that were compared.An optimal DNA vaccine protocol using repeated priming injections during week 0 and i.d. electroporation was established in the rabbit model. The intensive priming resulted in faster, higher and more uniform antibody titers, likely a result of the more frequent or continued presence of the expressed immunogen, as similarly shown for T cell responses [52]. Use of intensive priming DNA vaccination with syn.gp140mix resulted in immunogenicity in the macaques as well. However, compared with the rabbit model, the antibody titers and the neutralizing potency and breadth of the macaque immune response were remarkably low. The guinea pigs demonstrated a very potent immune response, despite the same increase in antibody titers as the macaques. The three different animal sera were diluted slightly differently in the comparative neutralization assay—guinea pigs 1/20 dilution, macaques 1/30, and rabbits 1/25–40 times (according to a total IgG serum level of 10 mg/mL in rabbits [53]). Still, these alterations probably do not influence the large difference seen in neutralization activity, with an average neutralization of 72% for guinea pig sera, 46% for rabbit IgG and 0% for macaque sera.During optimization of the completely codon exchanged synthetic DNA constructs, three different aspects were considered and systematically tested in rabbits and guinea pigs. Firstly, it was evaluated whether a membrane-bound envelope product could induce a higher or broader response than its soluble form. Ideally, the final gene product from the DNA vaccine construct is a mimic of the native Env glycoproteins. Secreted soluble gp140 molecules, containing the gp120 surface glycoprotein and the ectodomain of gp41, exist in several molecular forms from transfected cells e.g. monomers, dimers, trimers, tetramers and higher molecular weight aggregates [54]. The membrane-bound gp150 product has a higher potential to mimic the native trimeric spikes [18] and to induce polyreactive antibodies that are also broadly neutralizing and targeting epitopes in the membrane proximal external region (MPER) of gp41 [55,56,57]. Moreover, expressing membrane-bound protein in the DNA-priming phase before protein boost has been suggested to give a small advantage over soluble gp140 Env proteins in terms of subsequent immune response after protein boost [10]. For these reasons, we hypothesized that the Env membrane-bound product would be superior to the secreted gp140 molecule. However, when rabbits were immunized with syn.gp140Bx08 or syn.gp150Bx08, the membrane-bound gene product did not seem to induce a higher neutralizing activity and three viruses out of six tested were actually easier to neutralize with syn.gp140Bx08 induced antisera. These results are indeed in agreement with our previous publication [34] in which no differences in antibody response were documented when guinea pigs were immunized with syn.gp140Bx08 or syn.gp150Bx08 constructs. The same constructs were also used to immunize rhesus macaques [34] and although syn.gp150Bx08 antisera showed slightly higher in vitro neutralizing activity than syn.gp140Bx08 antisera of homologous HIV-1Bx08, the difference did not reach statistical significance.The second aspect considered the possibility to broaden the neutralizing response by simultaneous immunization with three different env genes. A polyvalent approach of administering multiple Env proteins as opposed to a monovalent Env has proven effective to broaden the Ab response in several studies including rabbits and macaques [48,49,50,51,58,59]. Nevertheless, the antigens need to be selected carefully to maximize the generated immunity. In addition to envBx08 [60], envelope immunogens ctl21 and ctl27 were selected from individuals in whom the neutralizing capacity of serum extended to a panel of clade B R5 HIV-1 strains. We hypothesized that env DNA immunogens from such individuals could induce immunity against several different virus strains. Immunization of rabbits and guinea pigs with the trivalent syn.gp140mix, including syn.gp140Bx08, ctl21, ctl27, did indeed induce a response that could neutralize several different viruses of different clades, but when compared to monovalent vaccine, no increase or broadening of the neutralizing activity was achieved. This could be partly due to all three envs being clade B with not enough differences to induce a broader response. However, it is encouraging that env from only intra-clade B viruses can induce immune response against other clades. Adding more and diverse envelope genes of other clades in the DNA vaccine may further increase the broadness by either focusing the immune response to the shared conserved regions of Env while reducing the dominance of individual hypervariable regions, or simply increase the polyreactivity in an additive manner. The increased immune response observed by boosting with clade C protein/DNA mix in macaques, indicated a recall response to shared epitopes and could thus support a strategy of adding more heterologous env in a more polyvalent mixed vaccine strategy.The final optimization step undertaken in regard to the DNA construct was the use of genetically modified variants of envelope genes, aiming to improve the immune responses by generating more native-like in situ trimers. Immunizations of rabbits with gp140 trimeric proteins with SOSIP modifications have been shown to be superior in eliciting neutralizing antibodies compared to matched monomeric gp120 protein [9,10,11]. In this study, we have engineered plasmids encoding SOSIP.R6-IZ-H8 envelope proteins for all three env genes used, syn.gp140Bx08, syn.gp140ctl21 and syn.gp140ctl27. Modified env constructs were mixed (syn.gp140mix SOSIP.R6-IZ-H8) and used for DNA immunization of rabbits and guinea pigs and compared to a non-modified mix. Immune responses however differed between the animal species. Antibodies from immunized rabbits demonstrated no difference in neutralizing activity when immunized with modified env or non-modified env, whereas sera from guinea pigs immunized with syn.gp140mix SOSIP.R6-IZ-H8 did generate a higher and broader neutralizing activity than syn.gp140mix guinea pig antisera. But this increase in neutralizing activity of syn.gp140mix SOSIP.R6-IZ-H8 in guinea pigs is explained by a non-HIV specific immune response, since MLV control was also neutralized. Thus, the modified constructs seem to have induced a non-specific and broader immune response. This could not be explained by a cross-reactive antibody response, since purified IgG from guinea pig sera only demonstrated low HIV neutralizing activity (data not shown). We can only speculate that there may be a synergistic effect between the specific IgG measured in ELISA and some unspecific serum effects. If this unspecific effect was also present in immunized rabbit sera is not known since only purified rabbit IgG was tested in neutralization assays. However, purified rabbit IgG demonstrated a clear HIV-specific neutralizing effect whereas purified guinea pig IgG did not. Several explanations may be given as to why the immunization experiments described in this study cannot confirm that SOSIP-modifications offer an advantage in the rabbit model in induction of NAbs. Our study differs in many aspects compared to other SOSIP studies using rabbits. Previous studies include SOSIP-modified recombinant glycoproteins [9,10,11], whereas we used SOSIP-modified DNA constructs expressed via DNA vaccination in vivo. When producing SOSIP gp140 recombinant in vitro, it is easier to control and ensure precursor cleavage, an aspect that might contribute to the favorable antigenic and immunogenic properties of SOSIP gp140. Furthermore, it is possibly to purify gp140 trimer proteins out of the mixtures of monomers, dimers, trimers and aggregates that are usually formed. Uncleaved and non-trimeric gp140 forms produced in vivo upon DNA immunization might distract the immune response from cleaved gp140 trimers that better recapitulate the antigenic structure of the native Env spike. Finally, all clinical isolates may not benefit from the same SOSIP mutations deduced from the JRFL strain equally well [4].In order to accelerate the vaccine design process, model systems are important to screen candidate immunogens such as those from selected patient HIV-1 isolates. The model used however is of great importance when assessing immunogenicity, and advantages and drawbacks with each animal model should be considered as well as knowledge of potential antibody gene repertoires and gene usage frequencies [3]. When evaluating a potential human vaccine candidate the most reliable animal model today is the macaque, which shares the pathogenic effects of HIV-1 seen in humans. However, the ethical and financial concerns regarding macaque experiments makes it necessary to assess immunogens in smaller animal models before they can be used in the macaque model. In the present study, optimization of immunogens in rabbits differed somewhat from guinea pigs, and did not automatically translate well into cynomolgus macaques. One conclusion from this is that it is important to select a relevant animal model for optimal selection of immunogens, followed by evaluation of dose, delivery route, method and specific immune response generated in an iterative process. Even with our rationally selected and optimized DNA immunogens, higher antibody potency seemed necessary in the macaques. This could be achieved by using a purified clade C heterologous trimeric protein as a boost in which the adjuvant was in fact the DNA vaccine mixed with the protein that boosted NAb to both clade B and C strains. Among the existing models, mice have not been used extensively for testing of HIV env DNA vaccines due to the Rev dependence and the poor expression of these genes in mice. However, this problem can be overcome by codon-optimization of genes [18,35], which has made it possible to achieve comparable immune responses in mice and macaques [61]. However, rodents, including mice, lack the ability to produce antibodies with long CDR3 loops [24]. Since these loops are important features of several known broadly neutralizing antibodies [3,26,27,28,29,30], the rodent models have a clear disadvantage when screening for immunogenicity. The rabbit model, being a lagomorph and a larger animal, may more closely resemble the macaque model and still maintain the advantages of being less expensive, easy to handle and with large blood volumes to work with. Rabbits may also have an advantage over guinea pigs in generating antibodies, seen after electroporation with an HIV DNA vaccine [21]. This might explain the low potency of guinea pig antibodies we noticed when we purified IgG from a few serum samples with unspecific neutralizing response (data not shown). Thus, the rabbit is a favored model for test of immunogenicity and screening of vaccine candidates, although the model does not guarantee an equal response or protective efficacy in the macaque model. Moreover, even the macaque model may prove not to adequately predict the ability of a vaccine to generate bNAbs and show efficacy in humans. As a consequence, it could prove necessary to actually use macaques or even humans in the screening for optimal HIV-1 Env immunogens eliciting bNAbs and use small animal models primarily to ensure immunogenicity of the DNA constructs.Rational selection of envelope genes and thorough screening concluded that a trivalent mix of non-modified gp140 genes is optimal to induce high and broad NAb in the preferred rabbit model. However, this optimization differed from guinea pigs and did not directly translate into cynomolgus macaques. This suggests species-specific differences in the quality of immune response to HIV-1 env DNA and emphasizes the importance of choosing the correct animal model when screening for future vaccine constructs. The authors gratefully acknowledge Jan Gerstoft for providing the serum samples from Danish HIV patients at the University Hospital of Copenhagen (Rigshospitalet). The project was sponsored by the European Community under EC-FP7-grant “Next Generation HIV-1 Immunogens inducing broadly reactive Neutralizing antibodies” NGIN_201433. Other members of the NGIN Consortium are Mauro Malnati and Lucia Palco, Fondazione Centro San Raffaele del Monte Tabor, Milan, Italy; Hanneke Schuitemaker, AMC at the University of Amsterdam, the Netherlands; Luigi Buonaguro, Istituto Nazionale Tumori “Fond. G. Pascale”, Naples, Italy; Philippe Saudan, Cytos Biotechnology AG, Zurich-Schlieren, Switzerland; Mario Clerici, Milano University Medical School, Italy; Anna-Lena Spetz, Karolinska Institut, Stockholm, Sweden; Jan Albert, Francesca Chiodi and Britta Wahren, SMI/MTC, Stockholm, Sweden; Guido Vanham, Institute of Tropical Medicine, Antwerp, Belgium; Meghna Ramaswamy, National Institute for Biological Standards & Control, Herfordshire, UK; Sylvan Fleury and Anick Chalifour, Mymetics Corporation, Epalinges, Switzerland; Eva-Maria Fenyo, Lund University, Lund, Sweden; and Morgane Bomsel, Institut Cochin, Paris, France. The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The development of vaccines to treat and prevent human immunodeficiency virus (HIV) infection has been hampered by an incomplete understanding of “protective” immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8+ T-cell responses restricted by “protective” HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-α-dependant natural killer (NK) cell responses and plasmacytoid dendritic cell (pDC) responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.The development of human immunodeficiency virus (HIV) vaccines is a global health priority, particularly at a time when therapeutic vaccines are being considered as a component of a strategy for eradicating HIV infection [1]. However, the development of therapeutic HIV vaccines has been hampered by an incomplete understanding of protective immune responses that control HIV infection, as exemplified by the failure of multiple candidate vaccines [2]. Here, we propose the hypothesis that isotype diversification of IgG antibodies against HIV-1 Gag proteins contributes to the control of HIV-1 replication and review the supporting evidence for this hypothesis, including data from our own studies. Furthermore, we discuss how this information might be applied to therapeutic vaccination strategies for HIV-1 infection.Approximately 1% of patients with HIV-1 infection control the infection without antiretroviral therapy (ART) and are referred to as controllers [3]. Intense analysis of controllers is being undertaken to define “protective” immune responses against HIV-1 proteins that might be enhanced by therapeutic vaccines. Studies of HIV-1 controllers suggest that natural immune control of HIV-1 correlates with T-cell responses against viral proteins, particularly CD8+ T-cell responses against proteins of the virus core encoded by Gag that are restricted by “protective” HLA-B alleles [4,5], “helped” by Th1 CD4+ T-cells [6] and are “highly functional” [7]. Similarly, natural control of HIV-2 infection is also associated with high-magnitude polyfunctional Gag-specific CD8+ T-cell responses [8]. Of note, resting CD4+ T-cells “latently” infected with HIV-1 express Gag proteins on the cell surface more than other HIV proteins and are a potential target of immune responses against Gag proteins in patients receiving ART [9]. However, vaccine-induced CD8+ T-cell responses against HIV-1 Gag proteins have not been associated with prevention or control of HIV-1 infection in randomised controlled clinical trials involving large numbers of patients [2,10], though a clinical trial of an Ad5/Gag vaccine as a therapeutic vaccine did demonstrate that vaccine-induced Gag-specific CD4+ T-cells producing IFN-γ correlated with control of HIV-1 replication [11].Approximately one third of HIV-1 controllers do not exhibit evidence of HLA-B-restricted CD8+ T-cell responses against Gag proteins [12], suggesting that other immune responses also contribute to natural control of HIV-1 infection. At least 15 published studies undertaken between 1989 and 2000 in untreated HIV-1-infected adults and children who were not selected on the basis of a controller phenotype, demonstrated that progression of HIV-1 disease was slower in patients with higher serum levels and/or avidity of IgG antibodies to HIV-1 Gag proteins (p17, p24, p55) [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29], suggesting that HIV-1 Gag proteins might be used as vaccine immunogens for eliciting antibodies to control HIV-1 infection. HIV-1 Gag proteins might have the particular advantage of exhibiting high intra-clade and inter-clade epitope conservation, at least for T-cell epitopes [30], and might thereby elicit broadly reactive antibodies. In addition, an increasing amount of evidence indicates that natural control of HIV-1 infection is associated with responses by interferon (IFN)-α-dependant natural killer (NK) cells [31] and plasmacytoid dendritic cells (pDCs) [32,33], which are the major producers of IFN-α [34]. Both NK cells and pDCs mediate innate immune responses against viruses [34,35], but both also function as accessory cells in IgG antibody responses, and therefore, their function might be enhanced by IgG antibodies induced by vaccines. Activation of both cell types induces a diverse anti-viral response that, in particular, includes lysis of virus-infected cells by NK cells and production of type I interferons by pDCs [34,35]. Plasmacytoid dendritic cells also function as antigen-presenting cells for T-cells [36,37,38,39], including cross-presentation to CD8+ T-cells [40,41], and regulate B-cell differentiation [42].Non-neutralising antibodies mediate their effect by activating accessory cells, which also function as antigen-presenting cells and/or elicit innate immune responses. Activation of accessory cells by IgG non-neutralising antibodies is mediated by the Fc region of the antibody binding to Fcγ receptors [43]. Antibody responses of this type elicited against HIV-1 proteins include antibody-dependant NK cell responses (often referred to as antibody-dependant cell-mediated cytotoxicity; ADCC) [44,45], antibody-dependant cell-mediated viral inhibition (ADCVI) [46] and phagocytic antibodies [47,48]. It is currently unclear to what extent these antibody responses are associated with control of HIV-1 infection. Thus, whilst ADCVI responses to whole virus may be associated with prevention of HIV infection after vaccination with recombinant gp120 [46], they are not associated with prevention of HIV-1 superinfection [49]. Similarly, long-term slow progression of HIV-1 infection has been associated with a wide breadth of antibody-dependant NK cell responses to regulatory/accessory proteins of HIV-1 [50], but immune escape from ADCC antibodies to envelope proteins is common [45]. Antibody-induced activation of NK cells (including ADCC) results from ligation of FcγRIIIa and is primarily mediated by monomeric or complexed antibodies of the IgG1 and IgG3 subclass, though complexed IgG2 and IgG4 antibodies can also bind to the 158V genotype of FcγRIIIa, which confers a higher affinity of Fc binding than the 158F genotype [51,52]. Plasmacytoid dendritic cells express the activatory receptor, FcγRIIa, as well as small amounts of the inhibitory receptor, FcγRIIb, in about 10% of healthy individuals, but not the activatory receptors, FcγRI or FcγRIIIa [53,54,55,56,57]. FcγRIIa plays a dominant role in phagocytic antibody responses [58] and has been demonstrated to facilitate the phagocytosis of immune complexes containing “self” or viral nucleic acids by pDCs, resulting in sensing of those nucleic acids by toll-like receptors and pDC activation [54,55,59].Studies in patients with HIV-1 infection have demonstrated that FcγRIIa is the major FcR mediating phagocytosis of IgG antibodies complexed with gp120 [47]. FcγRIIa may be particularly effective in phagocytosis-induced activation of myeloid cells by immune complexes in HIV-1 infection, because, unlike other activatory FcγRs (FcγRI and FcγRIIIa), signal transduction via the immunoreceptor tyrosine-based activation motif (ITAM) of FcγRIIa does not require the FcR common γ-chain adaptor molecule, which is depleted by HIV-1 infection [60]. Support for this is provided by the observation that the 131H genotype of the FcγRIIa gene, which confers higher affinity Fc binding to FcγRIIa than the 131R genotype, is associated with slower progression of HIV-1 disease [61]. In contrast, the “high-affinity” 158V genotype of FcγRIIIa has been associated with an increased risk of acquiring HIV-1 infection [62,63] and also with an increased risk of HIV-1 disease progression [62], though the methods for analysing disease progression in that study are open to criticism. Studies of immune complex binding to FcγRIIa in vitro demonstrate that all four subclasses of IgG are able to ligate the 131H genotype and, to a lesser extent, the 131R genotype of FcγRIIa, especially in the form of large immune complexes [51,52]. Although the affinity of ligation of FcγRIIa by IgG2 and IgG4 is less than that for IgG1 and IgG3, analyses of plasma immune complexes suggest that IgG2 antibodies play a particularly important role in the binding of immune complexes to FcγRIIa. IgG2 is the most abundant IgG isotype in plasma IgG/IgM immune complexes of healthy individuals and a disease-associated increase in the ratio of IgG3 to IgG2 in the immune complexes is associated with decreased binding to Fc receptors on myeloid cells [64]. We have shown that IgG2 is much more abundant than IgG1 in FcγRIIa-binding immune complexes from plasma of healthy individuals and HIV controllers, but that failure to control HIV-1 replication is associated with more abundant IgG1 in the immune complexes [48].It is well-established that IgG2 antibodies and FcγRIIa play an important role in phagocytic antibody responses against polysaccharide antigens of encapsulated bacteria [65,66]. We suggest that IgG2 antibodies also contribute to phagocytic IgG antibody responses against antigens of persistent viruses, such as HIV-1, mediated via immune complexes and FcγRIIa expressed by pDCs. Targeting of viral antigens to FcγRIIa on BDCA-3+ dendritic cells by IgG antibodies has been proposed as a strategy for eliciting T-cell responses against viral antigens [67]. IgG2 is the only IgG subclass capable of covalent dimerization [68], which may enhance the function of this subclass of IgG antibody in phagocytosis and/or immune complex formation. In addition, IgG2 exhibits the highest degree of resistance to proteolytic degradation [69] and may also exhibit greater resistance than IgG1 to the adverse effects of deglycosylation of the Fc region on binding to FcγRIIa [70], though this was not confirmed in another study [52].Support for our hypothesis that an IgG antibody response against HIV Gag proteins that has diversified to include IgG2 antibodies may be beneficial in the control of HIV-1 infection has been provided from studies in HIV-1 controllers or long-term non-progressors (LTNPs). Ngo-Giang-Huong et al. [71] examined plasma samples from 71 LTNPs, who had plasma HIV-1 RNA levels varying from <20 to 860,000 copies/mL and demonstrated that IgG2 antibodies to p55 and p24 were associated with lower plasma HIV-1 RNA levels. In contrast, plasma levels of IgG1 antibodies to these antigens did not correlate with HIV-1 RNA levels. We have examined plasma samples from 32 HIV-1 controllers, of whom 14 were elite controllers (plasma HIV RNA level <50 copies/mL), for IgG1 and IgG2 antibodies to HIV-1 proteins and shown that controllers had higher levels of IgG2 antibodies to Gag proteins than non-controllers and that this association was strongest in patients who did not carry the “protective” HLA-B57 allele [48]. In contrast, Banerjee et al. [72] examined serum from 16 HIV-1 controllers, of whom 13 had a plasma HIV-1 RNA level of <75 copies/mL, and demonstrated that although serum levels of total IgG and IgG1 antibodies to p24 were higher in HIV-1 controllers than patients with progressive HIV-1 disease, serum levels of IgG2 anti-p24 did not differ between HIV-1 controllers and patients with progressive HIV-1 disease. It is unclear why control of HIV-1 replication was associated with IgG2 antibodies against HIV-1 Gag proteins (p55 and/or p24) in two studies [48,71], but only with IgG1 antibodies to HIV-1 p24 in another [72]. Differences might reflect the use of Western blot assays and antigens from virus lysates in the studies by Ngo-Giang-Huong et al. [71] and French et al. [48], as opposed to ELISAs and recombinant HIV-1 proteins in the study by Banerjee et al. [72]. Furthermore, the study of HIV-1 controllers by Banerjee et al. [72] did not subgroup patients according to carriage of “protective” HLA-B alleles.In summary, we suggest that diversification of an IgG antibody response against HIV-1 Gag proteins to include IgG2 antibodies, as well as IgG3 and IgG1 antibodies, may enhance the activation of accessory cell immune responses by NK cells and pDCs via ligation of both FcγRIIIa and FcγRIIa. Further support for our hypothesis that isotype diversification of IgG antibodies against HIV-1 Gag proteins is associated with control of HIV-1 infection is provided by evidence from patients infected by other persistent viruses. Data from patients with acute hepatitis C virus (HCV) infection suggests that IgG2 antibodies to HCV core proteins might be associated with clearance of HCV infection. Zein et al. [73] reported that all of the four patients who spontaneously cleared HCV infection had IgG2 antibodies to HCV core proteins compared with only nine of 23 patients who did not clear the infection. Furthermore, the ratio of IgG2/IgG1 HCV core-specific antibody titres was >1 in three of the four patients. In addition, studies in patients with human papillomavirus (HPV) infection demonstrated that IgG2 antibodies to capsid proteins were associated with protection from HPV disease using an ELISA and virus-like particles as antigens [74], though IgG2 antibodies could not be detected at all in another study when capsid proteins were used as antigens [75]. Isotype diversification of IgG antibody responses occurs during the process of B-cell differentiation and maturation of the antibody response, which occurs in germinal centres of lymphoid tissue follicles following the interaction of naive B-cells with follicular dendritic cells and follicular-helper T-cells (TFH-cells) [76]. Immunoglobulin isotype switching during B-cell differentiation occurs through class switch recombination of immunoglobulin heavy chain genes, with switching to IgG2 and IgG4 occurring “downstream” of IgG3 and IgG1 [77], and results in broadening of IgG antibody function mediated by the Fc region (Table 1). Together, IgG1 and IgG2 comprise about 90% of serum IgG [78] and, therefore, exert the largest functional effect on an IgG antibody response. Regulation of immunoglobulin isotype switching is mediated primarily by molecules expressed on, or produced by, TFH-cells [76]. The most important are the co-stimulatory molecules, CD40 ligand and inducible co-stimulator (ICOS), as exemplified by the association of immunoglobulin deficiency with deficiency of these molecules [79,80], and the cytokines, IL-4, IL-10 and IL-21, as exemplified by the restoration of immunoglobulin production by B-cells from patients with IgA deficiency or common variable immunodeficiency disorder when cultured with these cytokines [81,82,83]. The co-inhibitory molecule programmed death (PD)-1 is also highly expressed by TFH-cells, and ligation by the ligand PD-L1 has been shown to down-regulate ICOS expression and IL-21 production and possibly contribute to TFH-cell dysfunction caused by HIV infection [84]. Pro-inflammatory cytokines, such IL-2, IL-6 and IFN-γ, also contribute to isotype diversification of IgG antibodies, but primarily by enhancing production of IgG subclasses rather than initiating isotype switching [85,86,87,88,89] (Figure 1).Isotype diversification of IgG antibodies leads to broadening of the function of an IgG antibody response. Isotype diversification of an IgG antibody response. IgG antibody isotype switching during B-cell differentiation in germinal centres results from class switch recombination of immunoglobulin heavy chain genes from “downstream” (IgG3 and IgG1) to “upstream” (IgG2 and IgG4) isotypes regulated by co-stimulatory molecules (CD40L and inducible co-stimulator (ICOS)) and cytokines (IL-4, IL-10 and IL-21). Pro-inflammatory cytokines (IL-2, IL-6 and IFN-γ) enhance immunoglobulin production with IFN-γ particularly increasing IgG2 production. CD4+ T-cell production of both IL-21 and IFN-γ is impaired by HIV infection.While B-cell activation and increased production of total IgG is characteristic of HIV infection, driven to a large degree by pro-inflammatory cytokines [90], IgG2 deficiency is common in HIV patients [47,91], and IgG2 and IgA are less abundant in lymph node germinal centres of HIV patients than controls [92]. Indeed, serum levels of the “upstream” isotypes, IgG3 and IgG1, are increased, whereas serum levels of the “downstream” isotypes, IgG2 and IgG4, are decreased in HIV patients [90,93], suggesting an acquired disorder of B-cell differentiation and isotype diversification similar to that in patients with primary antibody deficiency disorders [94]. Data from studies of cytokine regulation of IgG subclass production by B-cells [85,86,88] and of patients with IgG2 deficiency [89] indicate that IFN-γ plays a particularly important role in the production of IgG2. Decreased IgG2 production in HIV patients may therefore be a consequence of both impaired B-cell isotype switching associated with TFH-cell dysfunction [84,95] and impaired IFN-γ production that characterises HIV-induced immunodeficiency, but is preserved in HIV controllers [6]. We provided evidence in support of this proposal from a study of antibody responses to HIV p24 in ART-treated HIV patients enrolled into a clinical trial of a recombinant DNA vaccine encoding a fowlpox virus vector, HIV Gag-Pol and IFN-γ [96]. Although the number of patients was small, this study provided evidence that the vaccine construct containing the gene for IFN-γ increased IgG antibodies to HIV p24, including IgG2 antibodies, which were associated with better control of HIV replication after ART was ceased in patients who possessed the 131H genotype of FcγRIIa, which results in the highest affinity binding of IgG2 antibodies to that receptor. It is notable that lymph node TFH-cells of patients with HIV-1 infection exhibit greater reactivity with Gag proteins than Env proteins [97]. Dysfunction of TFH-cells associated with HIV-1 infection [84,95,97] may therefore contribute to limited isotype diversification of IgG antibodies against HIV-1 Gag proteins.Therapeutic modulation of the isotype of vaccine-induced IgG antibodies is not an established procedure in humans, but has been achieved in dogs with a saponin-adjuvanted Leishmania vaccine [98]. Preliminary data from patients with HIV-1 infection suggest that IFN-γ might enhance vaccine-induced IgG2 antibodies to HIV-1 Gag proteins [96], and this potential approach to therapeutic vaccination should be considered further. Finally, inhibition of immune activation in HIV-1 patients by PD-1 blockade might also have beneficial effects on TFH-cell function [84] and antibody responses [99], and examination of IgG antibody isotype diversification might be examined in clinical trials of therapies that block the PD-1/PD-L1 pathway. We propose that enhancing isotype diversification of IgG antibody responses against HIV-1 Gag proteins during vaccination, to include IgG2, as well as IgG3 and IgG1 antibodies, may result in an IgG antibody response that facilitates the accessory cell responses of NK cells and pDCs to elicit both ADCC responses by NK cells, as well as phagocytosis of complexed antibody by pDCs and a pDC-dependant antiviral response (Figure 2). Further experimental evidence is required to strengthen our hypothesis. In particular, studies are needed to establish that IgG2 antibodies inhibit HIV-1 replication and are not just a marker of Th1 responses. However, at a time when new approaches to the development of HIV vaccines are needed [2], we suggest that consideration should be given to vaccination strategies that will enhance isotype diversification of IgG antibodies against HIV-1 Gag proteins. A diagrammatic representation of how isotype diversification of IgG antibodies against HIV-1 Gag proteins might enhance anti-viral accessory cell responses against HIV-1 infection. It is proposed that IgG antibodies bind to HIV-1 Gag proteins expressed on the surface of cells infected by HIV-1, including resting CD4+ T-cells [9]. Activation of natural killer (NK) cells is elicited by “downstream” IgG isotypes (IgG3 and IgG1) via FcγRIIIa. “Upstream” IgG isotypes (IgG2 and possibly IgG4) may also contribute to NK cell activation by ligating FcγRIIIa, particularly in individuals carrying the 158V genotype. However, it is proposed that multimeric IgG2 antibodies primarily broaden the function of the antibody response by enhancing phagocytic activity against Gag proteins associated with HIV-1 RNA, as a consequence of the functional characteristics of IgG2 (see Table 1), which activates plasmacytoid dendritic cells (pDCs) via FcγRIIa. Activation of pDCs leads to the production of IFN-α, which facilitates NK cell responses and induces the production of interferon-stimulated genes (ISGs) and to antigen presentation and/or stimulation of B- and T-cells (see text). HIV-1 infection impairs diversification of an IgG antibody response to “downstream” isotypes. We acknowledge the financial support of the National Health and Medical Research Council of Australia (grant 510448) and The Medical Research Foundation of Royal Perth Hospital. The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).This Vaccines issue on “Confidence in Vaccines” provides sound evidence through multiple perspectives of life-saving impacts when vaccination programs are effectively implemented in a population. Yet there remain challenges to achieving this impact, including scientific, medical, manufacturing, policy-related and logistical issues. Additionally, socio-cultural, religious and political agendas can come into play, taking public health hostage and sometimes allowing the circulation of myths regarding vaccination. All of these challenges play a role in public confidence in vaccines and vaccination. What we trust, we embrace. What we do not trust, we do not embrace.As a leading vaccine manufacturer, GlaxoSmithKline (GSK) asks: “How do we ensure that we as industry are doing our part in building and maintaining trust in vaccination?” Confronted with a myriad of messages in a world of heightened complexity and high-speed communications, the public is faced with the paradox of having, at the same time, too much and too little information from which to draw informed opinions. The potential vaccinee wants an answer to a basic question: “Do those involved in bringing me (my child) this vaccine have my best interests at heart?”To provide that reassurance, all industry and vaccination players must examine their own ethical behaviours. There is adequate evidence that medical societies, research associations, industry and governments have reflected on standards of ethical behaviour in performing research, in assessing the safety, quality and efficacy of new products, and in defining appropriate ways of working between the medical community and the pharmaceutical industry. Virtually every organisation engaged in vaccine research, development and delivery has, or is developing, a Code of Conduct/Ethics [1,2,3,4,5,6]. At GSK, we recognise the scrutiny to which we are subject given the potential for conflict of interest between commercial objectives and our role as a partner in advancing science through research and development. We firmly hold that there is never a benefit to compromising on the level of quality in vaccine research, development and manufacturing. We know too that it is not enough to state our deeply-held values of “Patient-focus”, “Transparency”, “Integrity” and “Respect for People”; we must demonstrate these in all we do, in our corporate behaviours and in our individual activities as company representatives. To enact our mission and enable trust, it is key to play our role as scientists and physicians and to express science in a manner that is objective, accurate and complete. We define Scientific Engagement as the interaction and exchange of information between GSK and external communities in order to advance scientific and medical understanding, including the appropriate use of our medicines and vaccines, the management of disease and patient care.Our approach to Scientific Engagement is based on five principles for application to our working practices as scientists and physicians, with the aim of earning trust and building confidence in industry:
+	
+	
+	Scientific Engagement with external communities is fundamental to the progress of medical science and to meeting the needs of patients and public health. This principle reminds us of the essential need for partnership with external experts with complementary capabilities, in order to understand healthcare needs and environments, and to advance science and deliver vaccines more rapidly. Interactions between industry and clinicians who acted as clinical trial investigators have facilitated clinical research resulting in registration of new vaccines. Also, a new model of public–private partnerships enables the development of vaccines which may not otherwise be sustainable commercially and demonstrates how external interactions not only promote the advancement of science but can also unblock barriers to new products for diseases of the developing world.
+    
+	GSK physicians and scientists engage in the highest standards of peer-to-peer scientific dialogue to increase understanding of diseases and develop effective prevention and treatment therapies. Industry scientists and physicians come from a vast range of clinical, academic and government institutional experiences and settings. Even with the available staff expertise, research within the company must be subject to challenge and validation by the external community and, as stated in the second principle, these interactions should be of the highest standards in science. Mutual respect builds as shared knowledge and intellectual challenge enables researchers and physicians to advance science for the benefit of health; this industrial science credibility is a cornerstone to building public confidence. Working practices adopted by GSK that realise this principle include the selection of appropriate venues for scientific interactions, involvement of staff members that have the scientific knowledge to engage productively with peers, transparency in how we pay for services from medical consultants, and refraining from practices that could contribute to real or perceived misconduct in external interactions.
+    
+	Scientific Engagement is driven by legitimate scientific need. It is balanced, appropriate and proportionate to the scientific need and intent. Scientific Engagement activities or behaviours cannot be, or be perceived to be, promotional or otherwise designed to influence the prescription, supply, sale or use of GSK products. Every scientist who has ever become passionate about a research project is warned about the potential for loss of objectivity that can accompany that passion. Principle 3 calls on staff to return to scientific and medical need as the baseline from which science must be considered at all times, and to interact and communicate in a manner that is proportionate to that need. We must learn from physicians and scientists, and provide them with appropriate information through appropriate channels in a non-promotional manner. An example relates to the appropriate conduct of advisory boards involving external experts: these are run only when the need for advice is well-defined, the advice has not previously been sought and the company is unable to obtain that advice through internal expert interactions. Such advisory boards will also involve only those staff members needed to contribute to the dialogue to obtain the needed advice, facilitating open discourse; they should never be used as an opportunity to promote for commercial gain.
+    
+	GSK abides by external regulations and internal Policies. Our intentions and actions are driven by our values of patient focus, transparency, respect and integrity. By the company being extremely explicit with staff via this fourth principle, employees are anchored in core values, from which every question and decision can be benchmarked.
+    
+	Scientific Engagement starts in the early stages of development and continues throughout the life cycle of the product and includes all areas of scientific endeavour undertaken by GSK. Accountability and authorisation for Scientific Engagement resides within the Medical Governance framework at GSK. We work in an integrated manner across research, development, manufacturing, medical, regulatory and commercial functions. All are involved in defining strategic directions and operational plans. What this fifth principle tells staff is that accountability in the areas of medical and scientific engagement will sit under a framework of policies and procedures that reinforce our commitment to medical and scientific principles. 
+	
+	Scientific Engagement with external communities is fundamental to the progress of medical science and to meeting the needs of patients and public health. This principle reminds us of the essential need for partnership with external experts with complementary capabilities, in order to understand healthcare needs and environments, and to advance science and deliver vaccines more rapidly. Interactions between industry and clinicians who acted as clinical trial investigators have facilitated clinical research resulting in registration of new vaccines. Also, a new model of public–private partnerships enables the development of vaccines which may not otherwise be sustainable commercially and demonstrates how external interactions not only promote the advancement of science but can also unblock barriers to new products for diseases of the developing world.GSK physicians and scientists engage in the highest standards of peer-to-peer scientific dialogue to increase understanding of diseases and develop effective prevention and treatment therapies. Industry scientists and physicians come from a vast range of clinical, academic and government institutional experiences and settings. Even with the available staff expertise, research within the company must be subject to challenge and validation by the external community and, as stated in the second principle, these interactions should be of the highest standards in science. Mutual respect builds as shared knowledge and intellectual challenge enables researchers and physicians to advance science for the benefit of health; this industrial science credibility is a cornerstone to building public confidence. Working practices adopted by GSK that realise this principle include the selection of appropriate venues for scientific interactions, involvement of staff members that have the scientific knowledge to engage productively with peers, transparency in how we pay for services from medical consultants, and refraining from practices that could contribute to real or perceived misconduct in external interactions.Scientific Engagement is driven by legitimate scientific need. It is balanced, appropriate and proportionate to the scientific need and intent. Scientific Engagement activities or behaviours cannot be, or be perceived to be, promotional or otherwise designed to influence the prescription, supply, sale or use of GSK products. Every scientist who has ever become passionate about a research project is warned about the potential for loss of objectivity that can accompany that passion. Principle 3 calls on staff to return to scientific and medical need as the baseline from which science must be considered at all times, and to interact and communicate in a manner that is proportionate to that need. We must learn from physicians and scientists, and provide them with appropriate information through appropriate channels in a non-promotional manner. An example relates to the appropriate conduct of advisory boards involving external experts: these are run only when the need for advice is well-defined, the advice has not previously been sought and the company is unable to obtain that advice through internal expert interactions. Such advisory boards will also involve only those staff members needed to contribute to the dialogue to obtain the needed advice, facilitating open discourse; they should never be used as an opportunity to promote for commercial gain.GSK abides by external regulations and internal Policies. Our intentions and actions are driven by our values of patient focus, transparency, respect and integrity. By the company being extremely explicit with staff via this fourth principle, employees are anchored in core values, from which every question and decision can be benchmarked.Scientific Engagement starts in the early stages of development and continues throughout the life cycle of the product and includes all areas of scientific endeavour undertaken by GSK. Accountability and authorisation for Scientific Engagement resides within the Medical Governance framework at GSK. We work in an integrated manner across research, development, manufacturing, medical, regulatory and commercial functions. All are involved in defining strategic directions and operational plans. What this fifth principle tells staff is that accountability in the areas of medical and scientific engagement will sit under a framework of policies and procedures that reinforce our commitment to medical and scientific principles. Of course, much more than scientific integrity comes into play to build trust in vaccine manufacturers. Industry Codes of Practice (e.g., EFPIA Code of Practice on the promotion of prescription-only medicines to, and interactions with, healthcare professionals, PhRMA Code on Interactions with Healthcare Professionals, ABPI Code of Practice for the Pharmaceutical Industry) set standards for the best possible practices in the promotion and advertising of medicines. We acknowledge again that words captured in Codes can only come to life through the actions and behaviours of each company employee. Industry standards need to be visible in all ways we engage with the vaccine community, in the way we promote our products, manage safety issues, and interact with regulatory bodies, Ministries of Health and customers—essentially, in all we do. All of this takes leadership, communication and appropriate consequences when standards are breached. Leadership will be taken by industry players becoming actively engaged in defending and promoting the practices that bring credibility to industry. This may mean having more open discussions regarding changes in the ways in which industry and its partners interact. Any practices that result in an expectation, implicit or explicit, of reciprocity can impact the integrity of the dialogue and particularly the perception of impropriety. Long-passed practices of offering t-shirts and gimmicks at industry booths at medical congresses gave a poor impression of the nature of the relationship between industry and healthcare professionals. This is one small example, yet the principle of integrity and transparency in all interactions can apply in many other major and minor ways.In the article, Sustaining Vaccine Confidence in the 21st Century, GSK authors conclude by highlighting the critical importance of communication and collaboration between all parties—researchers, public health organisations, manufacturers, funding bodies, and more—in this vast network that enables vaccination programmes to succeed. Industry efforts—no matter how professionally or passionately undertaken—if uncoordinated and unaligned are sure to result in inefficiencies (manufacturing capacities that do not meet a governmental programme need), gaps in communication (safety information that is not well enough understood by the medical community to address patient questions; myths regarding vaccines’ adverse events are allowed to circulate), and lost opportunities to deliver vaccination (supply shortages). To enable greater collaboration, industry and all those involved in vaccine delivery must increase our competencies in understanding one another—defining vaccination objectives in terms of health needs, and aligning more cohesively industry deliverables to interface with public health needs in a manner that is transparent to all stakeholders and the public.Industry and public health authorities need closer collaboration, to provide accurate and reader-friendly information that appropriately communicates the value and the safety, including risks, of vaccination. Mark Twain is quoted as saying: “A lie can travel halfway round the world while the truth is putting on its shoes.” In a world of “tweets” and viral news through social media, our communication challenge to deliver sound scientific and medical information has never been greater, so that the public can understand vaccination issues as they arise. This means new ways of communicating, which can only be defined in partnership between health authorities, healthcare professionals, and industry. We must consider if we have, today, the proper fora to advance this communication agenda. Clearly, this is an area needing more reflection and action.All of our collaborations, partnerships and interfaces will be judged by the value delivered and the trust engendered. Independent physicians and physician organisations are speaking up about the critical importance of this collaborative spirit based on high standards of interaction between industry and the scientific community [7]. Interactions will remain subject to scrutiny, which in itself is good. It will take all members of the broad coalition of contributors to vaccine advocacy to prove that trust is merited. We must be confident in challenging one another to mutually and continuously keep the bar high. When breaches are encountered, industry has to be proactive and transparent in addressing any short-comings in meeting standards, requirements or legislation through open dialogue, agreed plans to address issues, and timely implementation of corrective action.Having worked with vaccine experts and advocates in industry, governments and academia over the past 30 years, I believe we all share a common objective grounded in public health, and that our ability to deliver to that objective is clearly linked to our ability to create, learn and problem-solve together. We have an incredible opportunity to contribute to the advancement of world health through vaccination to improve and protect quality of life, and it will be through our combined efforts that we will achieve this goal. “Do those involved in bringing me (my child) this vaccine have my best interests at heart?” Industry leadership, communication and collaboration according to highest standards can contribute significantly to making the answer a resounding “Yes”.The author is a full-time employee of GlaxoSmithKline Vaccines.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Recent advances in HIV vaccine development along with a better understanding of the immune correlates of risk have emerged from the RV144 efficacy trial conducted in Thailand. Epidemiological data suggest that CRF01_AE is still predominant in South-East Asia and is spreading in China with a growing number of circulating recombinant forms due to increasing human contact, particularly in large urban centers, tourist locations and in sites of common infrastructure. A vaccine countering CRF01_AE is a priority for the region. An Asia HIV vaccine against expanding B/E or BCE recombinant forms should be actively pursued. A major challenge that remains is the conduct of efficacy trials in heterosexual populations in this region. Men who have sex with men represent the main target population for future efficacy trials in Asia. Coupling HIV vaccines with other prevention modalities in efficacy trials might also be envisaged. These new avenues will only be made possible through the conduct of large-scale efficacy trials, interdisciplinary teams, international collaborations, and strong political and community commitments.South-East Asia (SEA) is home to more than 593 million people. Economic and tourism exchanges within SEA, and between SEA and East Asia, especially China, are thriving and expected to intensify over the next decade [1]. The further development of roads, communications and other major infrastructures promises to intensify the interchange between China and SEA, and the emergence of Myanmar after decades’ long isolation will generate new economic activity and the potential for a geographically continuous market that includes India, China and SEA Asia—constituting nearly 3 billion people.Aggressive HIV prevention measures and expanded access to care and treatment for HIV-infected individuals [2,3] have yielded a 20% decline in new HIV infections from 450,000 in 2001 to 360,000 in 2009, with approximately 4.9 million individuals living with HIV in Asia and the Pacific. Southern Chinese provinces, Cambodia, Indonesia, Malaysia, Myanmar, Papua New Guinea, Thailand and Viet Nam remain the most affected in the region. However, the epidemic still outpaces the response with almost two new HIV infections for every person who starts treatment. Most countries are far from achieving universal access targets for HIV prevention, treatment, care and support [4]. A preventive HIV vaccine as part of a comprehensive prevention package [5,6] remains therefore among the best hopes for controlling the HIV/AIDS epidemic in the region [7,8]. Thailand’s outstanding achievements in HIV prevention [9,10], care and treatment, and HIV vaccine development [11] have paved the way to a regional approach for a preventive HIV vaccine.Considerable HIV vaccine clinical development efforts have been deployed in the region since the mid 1990s [12]. Key studies were conducted in Thailand, which advanced the field considerably. The first Phase III trial in a middle-income country (Vax003) tested a bivalent recombinant gp120 B/E (MN and A244 CRF01_AE) adjuvant in alum (AIDSVAX® B/E) in injecting drug users (IDU) in Bangkok, Thailand [13]. No difference in HIV incidence was detected between the vaccine and placebo arms. HIV-1 CRF01_AE accounted for 77% of infections. No statistically significant effects of the vaccine on plasma HIV-1 load, CD4 cell count, onset of acquired immunodeficiency syndrome-defining conditions, or initiation of antiretroviral therapy secondary end points were observed. A community-based Phase III trial (RV144) provided the first evidence that an HIV-1 vaccine might prevent HIV infection [14,15]. The prime-boost vaccine regimen consisted of a recombinant canarypox vector, ALVAC-HIV (vCP1521) prime, expressing gag, protease subtype B (LAI) and env gp120 TH023 CRF01_AE genes with a gp41 subtype B (LAI) transmembrane anchor, administered at 0, 1, 3, and 6 months and a AIDSVAX® B/E boost given at months 3 and 6. The vaccine regimen was safe and generally well tolerated [16]. The modified intent-to-treat analysis showed 31.2% efficacy after 42 months of follow-up. There was no effect on early post-infection HIV-1 RNA viral load or CD4+ T-cell count. In a post-hoc analysis, vaccine efficacy appeared to be higher (60%) at 12 months post vaccination, suggesting an early, but nondurable, vaccine effect. RV144 was not designed and powered to assess the interaction of vaccine efficacy and risk behavior. HIV risk behavior was assessed with a self-administered questionnaire at the time of initial vaccination in the trial and every six months thereafter for three years. In a post-hoc analysis, participants classified as high or increasing risk at least once during follow-up were compared with those who maintained low-risk or medium-risk behavior as a time-varying covariate; the interaction of risk status and acquisition efficacy showed a greater benefit in low-risk individuals. The authors pointed out that future HIV vaccine trials should recognize potential interactions between challenge intensity and risk heterogeneity in both population and treatment effects [17]. One can speculate that the RV144 regimen was able to protect against infection because the number of sexual contacts were limited in time and could be countered by a marginally efficacious vaccine, which might or might not hold true with communities at higher risk of sexual transmission and increased number of exposures such as MSM or female sex workers. This remains to be demonstrated in future trials using improved immunogens and additional boosts. An analysis of the effect of vaccination on disease progression after infection showed weak evidence of lower viral load and higher CD4+ count in the vaccine group. Vaccination did not affect the clinical course of HIV disease after infection. Interestingly, lower mucosal viral load was observed among vaccine recipients, primarily in semen, suggesting a vaccine-induced effect caused by mucosal immune responses differing from those measured in the peripheral blood. Moreover, a lower RNA viral load in mucosal secretions of vaccine recipients could translate into lower transmission, a potential public health benefit [18].The efficacy observed in the RV144 trial provided the first opportunity to study immune correlations associated with vaccine efficacy against HIV. A case-control study showed that IgG antibodies to the scaffolded V1/V2 region of HIV-1 gp120 correlated with decreased risk of infection while IgA antibodies to the envelope correlated with decreased vaccine efficacy in the vaccine group [19,20,21], but no responses were associated with enhancement of HIV-1 infection risk. The IgG/IgA Env ratio significantly correlated with increased risk of infection (decreased vaccine efficacy) [22]. In the presence of low vaccine-elicited IgA responses, either ADCC or NAb responses correlated with decreased risk of infection. ADCC responses were predominantly directed to the C1 conformational region of gp120. C1 on gp120 is a component of a target epitope for ADCC [23]. IgA antibodies elicited by RV144 could block C1 region-specific IgG-mediated ADCC (via natural killer cells) [22].A sieve analysis identified two signatures of vaccine pressure within the V2 loop corresponding to sites 169 and 181. Intriguingly, vaccine efficacy (VE) against viruses matching the vaccine at position 169 was 48% whereas VE against viruses mismatching the vaccine at position 181 was 78%, supporting the hypothesis that vaccination-induced high V2 binding antibodies were associated with reduced risk of HIV-1 acquisition [24]. The explanation of a greater VE associated against mismatched HIV-1 with the sieve effect at site 181 remains unclear. It is speculated that vaccine-induced responses may have hindered HIV-1 infection with 181 variants, other explanations including involvement of other unidentified sequences near position 181 or inability of this variant to establish infection due to steric hindrance with vaccine-induced antibodies. The assessment of a T-cell based sieve effect in envelope V1/V2 revealed an association between an HLA class I allele and VE, suggesting that VE was restricted to A*02(+) participants and that IgA-C1 antibodies inhibited protective effects of other responses in A*02(−) participants [25].RV305 explores systemic and mucosal immune responses elicited by late boosts (7–8 years post) administered to RV144 vaccine recipients. Another trial (RV306) recapitulating the RV144 regimen is expected to start in 2013 in Thailand and will explore the added value of boosts at 12 months, the immune responses in mucosal compartments and memory B cells. RV328 will evaluate AIDSVAX® B/E alone, recapitulating the Vax003 vaccination regimen to generate samples that will allow immunologic comparisons with RV144-like regimens containing ALVAC-HIV priming.One of the main objectives for future vaccines is to counter HIV-1 variability. Antigenicity studies of the envelope used in RV144 suggest that certain epitopes were better exposed as a result of a non-HIV-1 sequence inserted into the HIV-1 envelope and likely elicited antibody epitope specificities in RV144 [26], in particular higher levels of V2 antibodies. Whether various envelope immunogens eliciting V2 antibodies are functional in a cross-clade manner and universal correlates of risk remains to be demonstrated. Vaccines utilizing a combination of consensus and transmitted-founder envelopes may be able to induce neutralizing responses with greater breadth and potency than single envelope immunogens [27]. In contrast, mosaic HIV antigens expressed by Ad26 vectors markedly augmented both the breadth and depth of antigen-specific CMI responses as compared with consensus or natural sequence HIV antigens in rhesus monkeys [28,29]. Heterologous prime-boost vectored vaccines (Ad26+MVA) encoding mosaic antigens are planned to soon enter clinical trial (RV307) in Thailand. The use of vectors such a ChAdV63 expressing conserved HIV-1 sequences [30] represents another promising approach now tested in humans [8], although not currently envisaged in Asia. Whether vaccine efficacy may prove to be strain-specific, region-specific or universal in populations with various modes of transmission remains to be demonstrated in future efficacy trials. Our current understanding of the immune correlates of protection suggests that no specific vaccine approach should be privileged over the other and that all reasonable vaccine approaches deserve to be pursued. In China, a plasmid DNA and a replication-competent Tiantan vaccinia HIV vaccine vector expressing HIV-1 CN54 CRF07_B’/C gag-pol, env and nef genes [31] is now in Phase II. Preliminary results suggest that both vaccines are safe and immunogenic [32]. An efficacy trial in MSM populations is now envisaged. While in Sub-Saharan African the vast majority of HIV infections occur through heterosexual transmission [33], in SEA and Eastern Asia the epidemic patterns have evolved from heterosexual (female sex workers, new military recruits) now declining to predominantly anal transmission through unprotected intercourse in MSM and transgender (TG) populations [34,35,36,37]. Although previous studies suggested that in Thailand, Indonesia, and Myanmar, there was no significant decline in the prevalence of HIV epidemics in injecting drug users (IDU) [38], recent reports show that harm reduction programs have demonstrated a dramatic and beneficial impact on the epidemic in these populations [39].The majority of HIV strains circulating in South East Asia with growing presence in China are represented by CRF01_AE with an increasing number of recombinant forms with B and C subtypes [40]. CRF01_AE dominates in Indonesia [41], Thailand (developed below), Cambodia [42], Laos, Myanmar, and Viet Nam [43,44]. In Malaysia, co-circulation of CRF01_AE and subtype B [45] has resulted in the emergence of CRF33_01B in approximately 20% of its HIV-1 infected individuals [46], now described in Indonesia [47]. We develop the situation of Thailand and China for their long-standing efforts in HIV vaccine development, and Myanmar, as a recently opened country after decades of isolation and with growing exchanges with China.Approximately 90% of incident infections in RV144 were CRF01_AE infections, a predominant circulating strain in Thailand and much of South East Asia. Among 390 volunteers who were deferred from enrolment in RV144 due to pre-existing HIV-1 infection using a multi-region hybridization assay, full genome sequencing and phylogenetic analyses showed the following subtype distribution: CRF01_AE: 91.7%, subtype B: 3.5%, B/CRF01_AE recombinants: 4.3%, and dual infections: 0.5%. CRF01_AE strains were 31% more diverse than those from the 1990s Thai epidemic that informed vaccine immunogen design. Sixty-nine percent of subtype B clustered with cosmopolitan Western B. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoints analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Forty-three to forty-eight percent of CRF01_AE sequences differed from the vaccine insert in Env V2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. Compared to the molecular picture at the early stages of vaccine development, the analysis of the molecular evolution of the HIV-1 Thai epidemic between the time of RV144 immunogen selection to the execution of the vaccine efficacy trial shows an overall increase in the genetic complexity of the Thai epidemic, increased distance to vaccine immunogens, and represent a clear example of viral evolution that occurred between immunogen design and vaccination for an efficacy trial [48]. Although the level of genetic complexity observed was consistent with the risk levels of a community-based cohort, the changes observed (expansion of the genetic diversity within CRF01_AE, increase in frequency and complexity of B/CRF01_AE recombinants, and shifts in the Western B/B’ balance) may have impacted the efficacy of the vaccine. The molecular epidemic changes that occurred between the time of vaccine design and efficacy trial should be carefully considered at the time of the analysis for future efficacy trials. It also suggests that the evolution of the molecular epidemic, although unavoidable, may be more limited in a region with relatively homogeneous and dominant HIV-1 strains, which may be more propitious for HIV vaccine efficacy testing.In 2007, HIV prevalence among MSM in Bangkok and Chiang Mai was 30.7% and 16.9%, respectively, essentially unchanged from 2005 [35]. The HIV prevalence found in subsequent studies ranged from 5.5–28.3% with an incidence rate of 8.2 per 100 person-years [35,49], and 6% in Bangkok between 2006–2008 [36]. In a recent cohort study conducted in Pattaya, HIV incidence is 5.8 and 6.3 per 100 person-years among MSM and TG sex workers, respectively [50]. New MSM and TG cohort studies are now planned to prepare the conduct of future efficacy prevention trials.Data on the HIV-1 epidemic in Myanmar are still scarce and of questionable representativeness. No HIV incidence data are available. According to the HIV sero-surveillance survey 2011 [51] and the progress report of the National Strategic Plan for HIV/AIDS 2011 [52], the number of female sex workers (FSW) were estimated between 45,000 and 62,000 (12,000–15,000 in Yangon and 7,800–11,000 in Mandalay) with an overall HIV prevalence of 9.4% (12% in direct FSW—defining themselves as sex workers and earn their living by selling sex and 6% in indirect FSW—for whom sex work is not the first source of income; Yangon 18%, high compared to the 2.5% in Bangkok; other cities 5–11%). HIV prevention programs now reach more than 76% of FSW and with more than 95% reported condom use at last sex. HIV-1 prevalence among FSW dropped drastically from >30% in 2006 to current figures. The number of MSM is estimated at 240,000, mostly in Yangon and Mandalay, with an overall HIV prevalence of 7.8% (Mandalay 9%, Yangon 5%), with more than 81% using a condom at last sex. As for FSW, we observe the same decreasing trend over the past five years. Importantly, the current HIV epidemic figures in FSW may offer a unique and perhaps, last opportunity to access high risk heterosexual populations for an HIV vaccine efficacy trial in SEA. However, this window of opportunity may narrow quickly as increased access to prevention services may improve [53]. Recent data on HIV subtypes circulating in Myanmar are limited to the Myanmar-China border, mostly in IDU populations [54,55,56]. However, the predominant circulating recombinant form in Myanmar remains CRF01_AE [40].In the absence of interventions, HIV will spread very quickly in the MSM population [57] with an estimated reproductive ratio of 3.9 [58]. The proportion of MSM in the annually reported HIV cases increased from 12% in 2007 to 33% in 2009 [59]. A recent study showed an overall HIV prevalence of 4.9% with however considerable heterogeneity between provinces, with highest prevalence being up to 18% in southwestern provinces [60]. Approximately 25% of Chinese MSM are married [61,62] and 30% of these individuals have sex with a steady female partner, but with a low rate of condom use [63], constituting a dangerous bridge of HIV transmission from high-risk groups to the general population [64,65]. HIV incidence among MSM populations varies per province: 2.6 per 100 person-years in Beijing [66], 3.9% in Yunnan Province [67], 5.1% in Nanjing, Jiangsu Province [68], and 5.4% in Shenyang, Liaoning Province [69]. As early as 2006, CRF01_AE was found dominant (prevalence of 40.5%, 85.4% being acquired by sexual transmission) in Yunnan Province, in particular at the border with Myanmar [70]. Phylogenetic analysis indicates that the CRF01_AE sequences can be grouped into four clusters, suggesting that at least four genetically independent CRF01_AE descendants were circulating in China, of which two were closely related to the isolates from Thailand and Vietnam. Cluster 1 had the most extensive distribution in China. In North China, CRF01_AE clusters 1 and 4 are rapidly spreading in MSM [71]. In Yunnan, the distribution of HIV-1 strains in MSM was 71.4% CRF01_AE, and 28.6% CRF07_BC [67]. HIV-1 CRF01_AE accounted for 84% of the recent infections among MSM in Liaoning Province of northeastern China [72]. Recent studies give a different breakdown of recombinant forms: In Yunnan, CRF07_BC (18.9%), CRF08_BC (39.1%), CRF01_AE (22.4%), and URFs (subtype C, 5.9% and subtype B, 4.5%) [73]; pol sequences in newly diagnosed HIV-infected individuals from Dehong county, Yunnan, showed that subtype C accounted for 43.1%, unique recombinant forms for 18.4%, CRF01_AE for 17.7%, B for 10.7%, CRF08_BC for 8.4%, and CRF07_BC for 1.7% [74]; in Fujian, CRF01_AE (70.9%), C/CRF07_BC/CRF08_BC (5.8%), B/B’ (15.1%), and unique recombinant forms (8.1%) [75]. Similar trends are observed in Guizhou Province [76], Guangdong, Guangxi [77], Jiangxi and Hunan southern Provinces [78], Hong Kong [79] and Shanghai [80]. The HIV epidemic among MSM in China is expanding to Japan and illustrates the ongoing mixing of CRF01_AE and subtype B lineages unique to HIV-1 circulating in MSM populations in East Asia [81]. New CRF01_AE/B recombinants are now circulating among MSM [82,83]. These constantly evolving patterns illustrate the need to closely monitor the molecular HIV epidemic in potential target populations for HIV vaccine efficacy trials. Unless cross-protection can be demonstrated, the relevance of an HIV vaccine designed to targeting HIV-1 B’/C recombinants in populations mostly infected with CRF01_AE may be questionable, while a CRF01_AE-based vaccine such as the one tested in Thailand would seem more appropriate.An HIV vaccine with VE 50% and with 30% coverage of low-and-middle income country populations could avert between 5.2 and 10.7 million new HIV infections between 2020 and 2030. Based on the WHO 2010 Guidelines for Antiretroviral Therapy and the 2010 antiretroviral drug (ARV) costs for low- and middle-income countries ($155 for first-line and $1,678 for second-line and assuming a second-line ARV decline to $980 by 2015, plus costs of diagnostics and monitoring tests of $180 and service delivery of $176 per patient, per year), an HIV vaccine would save between $46 billion to $95 billion in averted costs of ART provision alone, depending on the characteristics of the vaccine and population coverage levels achieved [84]. In a scenario in which HIV/AIDS programming is scaled up to the UNAIDS Investment Framework targets [85], the number of new HIV infections and costs averted would be between 1.6 and 3.3 million and $14 billion and $29 billion, respectively [86]. A modeling analysis for the province of Sichuan, China, described similar cost-saving patterns [87].A vaccine with rapidly waning protection could have a substantial impact on the epidemic in Thailand [88,89]. Factors influencing the impact of such a vaccine include risk compensation, vaccine efficacy, and duration of protection. Due to the short duration of effect with the RV144 regimen, for example, large numbers of vaccinations would be needed to maintain high population coverage levels.Over the past 15 years, Thailand and China have both considerably invested in HIV vaccine clinical development and (for China) pilot manufacturing [12], and are now spearheading a regional effort that may one day lead an HIV vaccine to licensure. Regional markets are defined epidemiologically and economically in a booming region with genuine regional manufacturing capacity and advanced knowledge-based industry. Vaccine manufacturers may see investment in HIV vaccines as a way to secure access to private vaccine markets for other licensed or developmental products [90]. Infrastructure, know-how, management and leadership and international and regional, in particular through public-private partnerships (PPP) such as the pox-protein PPP (P5) [91] and the AIDS Vaccine for Asia Network (AVAN) [92,93,94,95], collaboration are key elements of success.Because the modest efficacy conferred by the RV144 regimen was observed in a mostly heterosexual Thai population at low risk for HIV infection and with low HIV incidence, follow-up efficacy studies to verify this result in a similar population would necessitate prohibitively large numbers of volunteers. Efficacy trials would be smaller and less costly if implemented in higher risk populations with high HIV incidence. It is argued that vaccine protection might be easier to achieve in high-risk populations with predominantly heterosexual transmission such as those found in Africa [33,96], in contrast to MSM populations with rectal transmission, with the highest risk mode of sexual HIV transmission at 1:20–1:300 infections per exposure [97]. So far, high-risk heterosexual populations suitable for efficacy trials have not been identified in SEA. The rampant epidemic in Sub-Saharan Africa accounting for 69% of people living with HIV worldwide [98] has understandably resulted in a shift in priority for HIV vaccine investments to Sub-Saharan Africa, in particular southern Africa, with less emphasis on Asia. Identification of intermediate- to high-risk populations with predominant heterosexual transmission in Asia deserves greater consideration for future efficacy trials [8,12] as a means of improving trial feasibility and generalizability. For example, the HIV prevalence reported among the female sex workers in Yangon is the highest (18%) in SEA [51]. This opportunity to identify such populations may however rapidly wane with the scale-up of HIV prevention strategies in this country [33]. The success of harm reduction programs has yielded decreasing HIV incidence in IDU [38,39]. For example, only 7 of 1,157 HIV-seronegative IDU acquired HIV over a two-year follow-up in HPTN 058 trial conducted in Xinjiang and Guangxi Provinces, China, and Chiang Mai, Thailand [12,99,100]. Consequently, HIV vaccine efficacy trials in IDU are unlikely to proceed. Moreover, a high proportion of new circulating recombinant forms are now found in IDU, as illustrated by CRF01_AE/B’/C recombinants (42.6%) in northern Myanmar at the border with China [54,55,56] and India [101]. Taken together with the high burden of multiple transmitted/founder variants in IDU [102], it would be unlikely that vaccines eliciting humoral and and/or cell-mediated T-cell responses of limited breadth would be efficacious in IDU populations. This pattern may also become true in other high-risk groups due to increased mixing of populations that may result in increased proportions of HIV-1 circulating recombinant forms, in particular at the borders with China and India.HIV prevention research has shifted to the evaluation of combination prevention programs whereby biomedical, behavioral, and structural interventions are implemented concurrently to maximize synergies among interventions [103]. New prevention strategies to control the epidemic and prevent new infections, including pre-exposure prophylaxis [104], antiviral treatment as prevention [105,106], and topical microbicides [107], are now being actively developed. PrEP reduced the risk of HIV infection by an average of 44% (73% among high adherers) in HIV negative MSM and TG women who participated in the iPrEx, a clinical trial testing a daily oral dose of the antiretroviral emtricitabine/tenofovir drug combination [108]. Following these encouraging results, a recent study suggested that despite multiple challenges, MSM in Thailand would be willing to take PrEP, even if they had to experience inconvenience and expense [109]. Among MSM living in Beijing, despite low awareness of PrEP, 68% were willing to accept PrEP [110]. However, such hypothetical projections may not be matched by the daily reality, as suggested by the cascade of PrEP volunteers in the OLE extension of iPrEx trial [111]. The rationale and theoretical aspects of an efficacy trial of combination prevention modalities such as HIV vaccine and PrEP acting in synergy have been described [112,113]. The conduct of such trials remains however hypothetical and would require high adherence to PrEP, larger sample sizes, be more costly, and complicate the regulatory approval process to licensure.The licensure of HIV vaccines raises several issues that are yet to be addressed. For example, would an HIV vaccine efficacious in MSM be licensed for heterosexual populations if there were no longer the opportunity to test this vaccine in such Asian populations? What would be the requisites of SEA countries for licensure of an efficacious HIV vaccine tested in Thailand only? What would be the requirements for licensure of a vaccine manufactured in Thailand but whose licensure trial lots have been manufactured outside Thailand? What would be the requirements for licensing this vaccine for adolescents? Safety and immunogenicity bridging studies would be required. Efficacy trials in adolescents would provide much needed data in advance of implementing vaccination in this population, however, social and regulatory acceptability represent serious challenges [114]. Public Health Policy, pharmacovigilance, and epidemiological surveillance systems should be put in place before the marketing of the vaccine. A close collaboration from the inception of licensure trial designs between scientists, Public Health Authorities and National Regulatory Agencies is therefore highly desirable and recommended. The use of vaccines expressing multiple HIV proteins, in particular, envelope protein subunits, may increase the risk of vaccine-induced seropositivity (VISP) in some proportion of vaccinated individuals when using routine HIV diagnosis serological tests [115]. This has raised concerns at individual and public health levels [116] as it may seriously complicate the epidemic surveillance of a country if easy-to-perform and cheap tests are not made available [117,118]. Vaccine development has become frustratingly slow. Several factors contribute to this situation including lengthy convoluted approval processes, complicated multi-product vaccine regimens, and timely availability of clinical lots of candidate vaccines, in particular, envelope subunit proteins formulated with potent adjuvants. This situation needs careful consideration and urgent remedies as it may cause communities, donors and scientists experiencing time or budget constraints to waiver in their commitment to HIV-1 vaccine development. The uncertain commitment of pharmaceutical companies and donors for SEA HIV vaccines has led to consideration of sub-optimal strategies including the use of soluble envelope subunit proteins derived from other HIV-1 subtypes (for example, subtype C protein to be used in Africa) that may or may not be suitable for protection against HIV acquisition in SEA, as cross-clade functional reactivity and protection remain unproven. The manufacturing of an envelope subunit protein in SEA, a scenario now being actively pursued, may result in accelerating the availability of this vaccine component for efficacy testing while building vaccine manufacturing capacity for non-HIV vaccines. This will require political and long-term funding commitments. Availability does not guarantee but may impact uptake. Socio-cultural and structural contexts of HIV vaccine acceptability among most-at-risk populations were studied in Thailand. Crosscutting challenges for HIV vaccine uptake such as social stigma, discrimination in healthcare settings and out-of-pocket vaccine costs emerged in addition to population-specific barriers and opportunities [119]. HIV vaccine acceptability and risk compensation among high-risk MSM and TG ranged from 31.6–73.8 on a 100-point scale (mean = 58.3). VISP had the greatest impact on acceptability, followed by efficacy, vaccine-related side effects, duration of protection, out-of-pocket costs and social saturation. Over one-third (34.6%) reported intention to increase post-vaccination risk behaviors in response to a highly efficacious HIV vaccine [120].While challenges are real, epidemiologic patterns and the dynamic of clinical development in Thailand and China represent tremendous opportunities to bring an HIV-1 vaccine to licensure. Better understanding of the immune correlates of protection remains a key element to direct the vaccine effort to counter heterogeneous circulating virus strains among populations with different modes of HIV transmission. The molecular epidemic changes between the time of vaccine design and efficacy trial should be monitored and carefully considered in analysis for future efficacy trials. If emphasis has been recently given to the possible involvement of V2 antibodies in protection against HIV acquisition, other approaches such as immunogens capable of inducing broadly neutralizing antibodies along with cell-mediated immune responses of greater breadth and depth (mosaic and conserved sequences) should be aggressively pursued. While conducting efficacy trials in MSM and TG seems the likely scenario for Asia, genuine efforts to identifying heterosexual populations suitable for such trials, though difficult, should be pursued. Engaging communities, regulatory and public health authorities in a mutually constructive and educational dialogue seems essential to address and help overcome these challenges as well as to prepare for success and access, or failure. A vaccine countering CRF01_AE is a priority for the region. An Asian vaccine against expanding B/E or BCE recombinant forms should be actively pursued. These new avenues will only be made possible through the conduct of large-scale efficacy trials, interdisciplinary teams, international collaborations, and strong political and community commitments.We are extremely grateful to all volunteers and their supporting communities whose willing participation in HIV vaccine clinical trials has greatly advanced the field. We also would like to thank all members of the Thai AIDS Vaccine Evaluation Group for their dedication and achievements and of the AIDS Vaccine for Asia Network for their constant support. This work was supported in part by an Interagency Agreement Y1-AI-2642-12 between U.S. Army Medical Research and Materiel Command (USAMRMC) and the National Institutes of Allergy and Infectious Diseases. In addition this work was supported by a cooperative agreement (W81XWH-07-2-0067) between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense (DOD).The authors declare no conflict of interest. The opinions herein are those of the authors and should not be construed as official or representing the views of the U.S. Department of Defense or Department of the Army.

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+Present address: Kala Pharmaceuticals Inc., Waltham, MA 02452, USA.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA), and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA.In 1990, Wolff et al. [1] demonstrated that direct injection of messenger RNA (mRNA) or plasmid DNA (pDNA) into the skeletal muscle of a mouse resulted in expression of the encoded protein. At the time, the feasibility of development of mRNA vaccines was considered uncertain because of instability in vivo and the technical difficulties in manufacturing RNA at large scale. Hence, much of the subsequent development of nucleic acid vaccines focused on pDNA. Older DNA vaccines have been shown to be immunogenic in a wide variety of animal species and several products are now licensed for commercial veterinary use. These include a West Nile virus vaccine for horses [2], an infectious hematopoietic necrosis virus vaccine for fish [3], a melanoma cancer vaccine for dogs [4], and a growth hormone releasing hormone gene therapy with electroporation delivery for pigs [5]. In humans, proof of principle for induction of both antibody and T cell responses by early pDNA vaccines has been demonstrated for various indications in several clinical trials [6,7,8]. However, the magnitude of these immune responses has been lower than those observed for conventional vaccines consisting of inactivated whole organisms or subunit proteins formulated with adjuvants. The reasons for the shortcomings of pDNA vaccines are not clear, but are likely due, at least in part, to inefficient delivery of pDNA into cells and inadequate stimulation of the immune system. The most promising approaches to overcome these limitations include facilitation of pDNA delivery by electroporation (EP) [9]; and stimulation of the immune system via the use of genetic adjuvants [10,11,12,13].In parallel with the progress being made with pDNA, many of the obstacles to mRNA vaccine development have been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications [14]. Naturally transient and cytosolically active mRNA is now seen by many [15] as a more viable potential vaccine platform. Direct injection of mRNA or “naked” delivery in vivo induces gene expression and generates immune responses [16,17,18,19], with self-amplifying mRNA being more efficient for gene expression in situ [17,20]. The potency of naked mRNA vaccines can be enhanced by cationic molecules [21], or lipid particles [17,22,23]. A recent study has demonstrated that formulated mRNA encoding influenza antigens is immunogenic and protective in animal models [24]. In addition, delivery of mRNA by the gene gun [25,26] or by EP at the site of injection [20,27,28] has been shown to improve immune potency. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These self-amplifying mRNA vaccines differ from conventional mRNA in that they encode an RNA replicon from alphavirus engineered to efficiently undergo a single round of replication and amplify production of subgenomic mRNA encoding an antigen of interest [29]. Here we demonstrate that like EP delivery of pDNA encoding antigens (not replicons), EP delivery of self-amplifying mRNA elicited improved stronger and broader immune responses relative to non-EP delivered RNA. Both of these vaccine technologies have been previously shown to be effective in animal models and human clinical trials [6,18,19,30,31,32,33,34,35,36].After bilateral intramuscular injection of a low dose (1 μg) of naked self-amplifying mRNA encoding for the transgene secreted alkaline phosphatase (SEAP), measureable but low levels of serum SEAP were detectable as early as 3 days after treatment. No significant enhancements in the levels of SEAP expression were observed when the RNA was delivered using EP (RNA + EP) at the same dose, except at the latest time point (Figure 1A). In contrast to the RNA, mice injected with a low dose (1 μg) of naked pDNA showed levels of SEAP expression that were indistinguishable from baseline (Figure 1B), but substantial enhancements in SEAP expression was observed when pDNA was delivered by EP (pDNA + EP) at the same dose. At the 1 μg dose, SEAP expression by pDNA + EP was superior to RNA + EP.In vivo expression of secreted embryonic alkaline phosphatase (SEAP) after intramuscular njection of self-amplifying mRNA (RNA, 1 µg or 10 µg, panels A,C) or pDNA (DNA, 1 µg or 10 µg, panels B,D). The vectors were delivered by bilateral intramuscular injection with (+EP) or without electroporation. Serum SEAP expression was measured on days 1, 3, 10 and 17 after treatment, and represented as the log of mean relative luminescence (RLU)±SEM, n = 5/group. * Statistically significant (student-t test, p < 0.05).After bilateral intramuscular injection of a higher dose (10 μg) of naked self-amplifying mRNA (Figure 1C), measureable levels of serum SEAP were detectable as early as 3 days after treatment and these levels were greater than those observed at the 1 μg dose (Figure 1A). Enhancements in the levels of SEAP expression were observed for RNA + EP by approximately 2-logs (days 3 and 10) compared to naked RNA delivery. High dose (10 μg) naked pDNA showed measurable but low levels of SEAP expression that were lower than those observed for the same dose of naked self-amplifying mRNA. Significant enhancements in SEAP expression were observed for pDNA + EP (Figure 1D). At the 10 μg dose, SEAP expression by RNA + EP was similar to pDNA + EP. Over the course of this 17-day study, it was observed that the kinetics of SEAP expression differed between self-amplifying mRNA (with and without EP) and pDNA. Mice injected with RNA showed measureable levels of expression at day 3, which peaked on day 10, and decreased towards background levels by day 17. On the other hand, mice administered pDNA delivered using EP, displayed high levels of SEAP expression at all time points tested, and these levels remained essentially undiminished for the duration of the study. To assess the effect of EP-enhanced delivery of nucleic acid vaccines in muscle, mice were injected intramuscularly with pDNA or self-amplifying mRNA (5 μg/site) with or without EP. The treated muscles were excised for GFP expression analysis and for hematoxylin and eosin (H&E) staining. Representative images of GFP expression in mouse muscle are shown in Figure 2. Robust GFP expression was detected in both pDNA + EP and RNA + EP treated groups. Low levels of GFP expression were detected in the RNA (no EP) control group, but not in the pDNA (no EP) or 1× PBS control groups. These results are consistent with that observed in the SEAP studies.Histology of muscle tissues (transverse) by hematoxylin and eosin (H&E) staining, and expression of reporter protein (GFP) as delivered by intramuscular injection without EP (No EP) or with EP (+EP). Mice were injected intramuscularly with 50 µL of 1xPBS, pDNA or self-amplifying RNA vectors (pDNA or RNA, 5 µg/site). Images were obtained 2 days following treatment and are shown as representative of 6–8 slides taken per area, n = 4 muscles per group.Potential muscle damage related to pDNA and RNA treatment was assessed in mouse quadriceps 2 days after injection. Mouse muscle injected with 1× PBS only, was used as a treatment control. As shown in Figure 2, the H&E stain for this representative muscle demonstrates typical muscle physiology, with ordered, tightly bundled muscle fibers and some circulatory leukocyte activity. All groups receiving EP treatment demonstrated inflammation at the treatment site, as characterized by an increase in infiltration, consisting mainly of macrophages and neutrophils. There was also a breakdown in the ordered, bundled muscle fibers, typical of an inflammation response. Groups that received either pDNA or RNA (no EP) showed lower levels of leukocyte infiltration and no evidence of muscle fiber dissociation. Hence, injection of pDNA and RNA, with or without EP, induced local inflammation, but the combination of these nucleic acid vectors with EP demonstrated a higher inflammatory response. Self-amplifying mRNA vectors expressing the HIV envelope protein gp140.SF162 (Env) were used to vaccinate mice using a heterologous prime-boost regimen (Figure 3A). Mice were administered 2 doses of self-amplifying mRNA, as the prime, and a third vaccination with recombinant HIV Env protein adjuvanted with MF59 (Env/MF59), as the boost. Viral replicon particles (VRP) and pDNA expressing the same antigen were used as benchmarks. Serum and spleens were collected at the specified time points (Figure 3A). Antibody responses were measured by ELISA for total IgG (Figure 3B,C) and IgG isotypes (Figure 4), and T cell responses were evaluated using an intracellular cytokine immunofluorescence assay (Figure 5). Env-specific IgG responses following RNA + EP or pDNA + EP treatments were only detectable after the 2nd immunization at all the doses tested (1, 10 and 50 µg, Figure 3B). Overall, the results show a dose response for both RNA and pDNA immunized groups, with pDNA producing higher Env-specific IgG titers at 2 weeks after the 2nd immunization (2wp2). At the high dose (50 μg), RNA+EP treatment was significantly more immunogenic than naked RNA (no EP) delivery, as measured by Env-specific IgG levels (GMT of 2,408 vs. GMT of 80), demonstrating the benefits of EP for the delivery for self-amplifying mRNA. EP delivery of pDNA also resulted in higher antigen-specific IgG levels (GMT of 8,910 vs. GMT of 1,936), but this increase was not statistically significant. Vaccination with VRP or Env/MF59 resulted in seroconversion of all the mice after a single dose and significantly boosted after the 2nd dose (Figure 3B). A reduction in titer of 2–3 fold was observed during the 3 weeks following (between 2wp2 and 5wp2) for these groups, which was not seen in case of groups primed with RNA and pDNA vaccines. The highest responding groups at 2 weeks after the 3rd immunization (2wp3) were those vaccinated three times with Env/MF59 (GMT of 99,781, Figure 3C). Immune responses for all groups that received 2 doses of nucleic acid vaccines (RNA, pDNA and VRP) were substantially boosted following administration of 10 µg of Env/MF59. Sera from mice immunized with 50 µg RNA or pDNA, 10 µg Env/MF59 or 1 × 107 IU VRP were also assayed for antigen-specific IgG1 and IgG2a responses before (“-pre”, at 2wp2) or after protein boost (“-post”, at 2wp3). Groups primed with RNA (with or without EP) exhibited a balanced response pre- and post-protein boost, with IgG1 titers being slightly elevated compared to IgG2a (Figure 4). In contrast, the pDNA primed groups (with or without EP) and those that received Env/MF59 only produced a response significantly skewed towards IgG1 production over IgG2a, suggesting that RNA-based vaccines are more effective than plasmid DNA at stimulating Th1-type helper T cell responses.Study timeline for assessment of self-amplifying mRNA vector in an HIV immunogenicity model. (A) Female Balb/C mice received 2 treatments of RNA or pDNA vectors with or without EP (RNA or pDNA, 1, 10 and 50 µg), MF59-adjuvanted gp140.SF162 HIV envelope protein (Env/MF59, 10 µg) or viral replicon particles (VRP, 1 × 107 IU) expressing the HIV Env-protein at weeks 0 and 3. All groups then received a single injection of 10 µg Env/MF59 at week 8. Serum and mucosal samples, and spleens for T-cell quantitation were collected on week 2 (2wp1), 5 (2wp2, pre-protein boost) 8 (5wp2) and 10 (2wp3, post-protein boost). Env-specific total IgG titer for all groups at 2wp1 and 2wp2 (B) and 5wp2 and 3wp2 (C) is plotted as log of mean titer, and the horizontal bar represents geometric mean titer (GMT). Titers < 25 (dotted line) were assigned a value of 5 for calculation of GMT. n = 6 at 2wp1, n = 12 at 2wp2, and n = 8 at 5wp2 and 2wp3. * denotes statistical significance with p < 0.05; ** denotes statistical significance with p < 0.005.Env-specific IgG1 and IgG2a titers in mice sera were measured at 2wp2 (pre-) and 2wp3 (post-protein boost) for nucleic acid vectors with or without EP (RNA and pDNA; 50 µg), MF59-adjuvanted Env (Env/MF59, 10 µg) or viral replicon particles (VRP, 1 × 107 IU) primed groups. Individual titers are plotted and the horizontal bar represents geometric mean titer (GMT) for each group (n = 6 per group). Titers <25 (dotted line) were assigned a value of 5 for calculation of GMT.T-cell responses for groups treated with self-amplifying mRNA (RNA, 10 and 50 µg), pDNA (pDNA, 10 and 50 µg), MF59-adjuvanted Env (Env/MF59, 10 µg) or viral replicon particles (VRP, 1 × 107 IU) at weeks 5 (A,C) and 10 (B,D). Pooled splenocytes (6 spleens/pool) were stimulated with HIV Env-derived antigenic peptides, stained for intra-cellular cytokines, and subjected to flow cytometry (methods). Graphs show the Env-specific (%) frequencies of CD4+ (A,B) and CD8+ (C,D) T-cells with error bars denoting 95% confidence limits.Splenic Env-specific T-cell responses for both the RNA and pDNA vaccinated mice demonstrated a dose response, when assessed at the 2wp2 (pre-boost) time-point (Figure 5). The net frequencies of Env-specific CD4+ and CD8+ T-cells were ~4–5-fold higher for mice treated with 50 µg of RNA+EP compared to those that received RNA without EP. Similar enhancements were observed for pDNA, demonstrating the benefit of EP delivery for both types of nucleic acid vaccines on T-cell responses. The phenotypes of the T cell responses were predominated by Th0/Th1 for nucleic acid vaccines (RNA, pDNA and VRP) and Th0/Th2 for the protein vaccine (Figure 5A,C). Upon boosting with Env/MF59, this relationship was largely maintained. The protein vaccine alone elicited CD4+ T cell responses (Figure 5A), but not CD8+ T cell responses (Figure 5C). Hence, as might be expected, protein boosting of the nucleic acid-primed mice increased CD4+ T cell responses (Figure 5B), but not CD8+ T cell responses (Figure 5D). Overall, EP enhanced the potency of Th0/Th1 CD4+ and CD8+ T cell responses for the self-amplifying mRNA vaccine, as has been previously demonstrated for pDNA [37,38].This report demonstrates that EP is an effective means of increasing the delivery and potency of self-amplifying mRNA vaccines. These enhancements were similar in scope and magnitude to those seen for pDNA vaccines, despite several key differences between the two types of nucleic acid vaccines. First, pDNA vaccines require delivery into the cell nucleus for activity. Achieving a high level of antigen expression from pDNA vector is particularly challenging in the absence of EP in non-dividing cells such as mature myocytes. The delivery of pDNA past the nuclear barrier is considered a rate-limiting step in vaccine effectiveness, but can be overcome by EP. In contrast, RNA vaccines (both mRNA and self-amplifying mRNA replicons) are active in the cell cytoplasm, hence do not require delivery into the nucleus. Second, upon delivery into the cytoplasm, the self-amplifying mRNA vaccine produces many copies of itself and the subgenomic mRNA encoding the transgene. The enhancement of pDNA and RNA vaccine potency by EP, suggests that EP facilitates nucleic acid delivery across both the plasma and nuclear membranes. Third, self-amplifying mRNA produce relatively short-lived expression in situ, possibly due to induction of apoptosis of transfected cells, as occurs in alphavirus infected cells [16,17]. In contrast, pDNA can remain functional in the nucleus of transfected cells for prolonged periods of time, resulting in long-lived expression of reporter genes [34,39,40]. EP delivery of RNA and pDNA did not alter these kinetic profiles. Finally, pDNA and mRNA vaccines likely stimulate the innate immune system in very different ways. E.coli-derived pDNA is known to signal via TLR9, which is believed to play a role in DNA vaccine potency [41]. In contrast, viral RNA has the potential to interact with TLR3, TLR7, TLR8, as well as the intracellular helicases RIG-I and MDA5 [42,43]. Previously, we have shown that effective non-viral delivery of self-amplifying RNA vaccines can be achieved in mice with lipid nanoparticles (LNPs), at low doses of RNA (0.1–1.0 μg) [23]. This novel vaccine technology was found to elicit broad, potent and protective immune responses against RSV F protein that were comparable to a viral delivery technology. This is in contrast to what was observed for EP delivery of self-amplifying RNA in these studies, which required higher doses and two vaccinations to achieve seroconversion, suggesting that there is room for improvement via optimization of EP conditions for RNA. Enhanced delivery of the RNA vaccines by EP was not detrimental to gene expression or vaccine potency. Interestingly, RNA vaccination resulted in a balanced IgG1/IgG2a response, in contrast to pDNA vaccinated groups which exhibited a response skewed towards IgG1. The interaction of non-viral vectors with early and/or late innate immune signaling is very complex and is unique for each types of vector, as demonstrated by our study. A better understanding of the mechanism of action of these vaccines is warranted through further research. We have previously shown that pDNA encoding reporter genes can be successfully delivered into muscle in different animal models such as guinea pig, rabbit, sheep, and mouse by EP [35,44,45,46]. In addition, the delivery of siRNA was facilitated using dermally targeted EP with good tissue tolerability [47]. Others have reported on the EP delivery of self-amplifying mRNA and shown significant enhancement of antibody and T cell responses [20], as well as transient tissue damage [28]. Here, we assessed reporter GFP transgene expression levels and tissue damage in mouse muscle using the Elgen 1000 EP device. GFP expression was found to be robust in groups treated with self-amplifying mRNA and pDNA followed by EP, likely the result of enhanced nucleotide delivery and uptake facilitated by EP. Tissue inflammation was observed in all groups treated with EP, and this damage appeared to be more significant when EP was combined with nucleic acid. In the absence of nucleic acid, the EP-related damage was limited to the immediate vicinity of the electrodes, which is likely due to a localized effect on the muscle fibers caused by the electrical field strength of the EP procedure. The increased effect of EP delivery of nucleic acid may be due to the additional specific innate immune stimulation provided by E. coli-derived pDNA or viral RNA. Qualitative assessment of the treated muscles suggested that groups treated with pDNA + EP induced higher levels of infiltration than the RNA+EP group. The reason for this difference remains unclear and should be investigated further.In summary, EP is an efficient non-viral delivery method for self-amplifying mRNA vaccines and is an effective means of increasing vaccine potency, as has been well documented for pDNA. Substantial enhancements of RNA gene expression in situ, antigen-specific antibody titers and T cell responses (CD4+ and CD8+) were observed. Nucleic acid vaccines (both pDNA and RNA) have several attributes that make them attractive alternatives to protein-based vaccines and viral vectors. Our results show that while some differences exist between RNA and pDNA vaccination, these vectors elicit a broad-based immune response profile including Th1 and CD8+ T cells (unlike protein-based vaccines) and do not induce interfering anti-vector immunity (unlike viral vectors). Enabling delivery technologies, such as EP, are critical to improve potency of nucleic acid vaccines in humans.The gene sequences for the SEAP (GeneBank accession #: U89937) and GFP (GeneBank accession #: KC896842.1) reporter genes and the HIV envelope protein antigen gp140.SF162 [48,49] were cloned into SalI and XbaI sites of a modified VCR construct [29]. Plasmid DNA was purified by standard techniques and the nucleotide sequence of the inserts was confirmed by Sanger sequencing. pDNA was linearized immediately following the 3' end of the replicon by restriction digest and purified by phenol/chloroform extraction and ethanol precipitation. Linearized DNA template was transcribed into RNA using the MEGAscript T7 high-yield transcription kit (LifeTechnologies, Carlsbad, CA, USA). Transcripts were purified by LiCl precipitation, capped using the ScriptCap m7G Capping System (CellScript, Madison WI, USA) and the final product obtained by a second round of LiCl precipitation. RNA was then resuspended in PBS at the appropriate concentration prior to use, reporter or antigen gene expression was confirmed by Western blot analysis of transfected BHK cell lysates.For DNA vaccination, plasmids encoding the reporter gene SEAP or vaccine antigen (gp140.SF162 ) were constructed using standard molecular techniques using the previously described pCMVIII mamalian expression vector[49,50]. Plasmids were grown in E.coli and purified using Qiagen Plasmid Giga Kits (Qiagen, Valencia CA, USA). For histology experiments, gWiz-GFP plasmid DNA (Aldevron, Fargo ND, USA) was used.To compare RNA vaccines to traditional RNA-vectored approaches for achieving in vivo expression of reporter genes or antigens, we used VRPs produced in BHK cells by previously described methods [29]. In this system, the antigen-expressing (or reporter gene-expressing) replicons consisted of alphavirus chimeric replicons (VCR) derived from the genome of Venezuelan equine encephalitis virus (VEEV) engineered to contain the 3' terminal sequences (3' UTR) of Sindbis virus and a Sindbis virus packaging signal (PS) (see Figure 2 of Perri et al.) [29]. These replicons were packaged into VRPs by co-electroporating them into baby hamster kidney (BHK) cells together with defective helper RNAs encoding the Sindbis virus capsid and glycoprotein genes (see Figure 2 of Perri et al.). The VRPs were then harvested and titrated by standard methods and inoculated into animals as solutions in PBS.The subunit vaccine was expressed, purified and formulated as previously described [48,49].Female Balb/c mice at 6–8 weeks old (Charles River Laboratories, Worcester, MA, USA) were housed and handled according to the standards of the Institutional Animals Care and Use Committee. Electroporation was carried out by the Elgen DNA Delivery System (Inovio, San Diego, CA, USA). Prior to immunization, the mice were anaesthetized with isofluorane. A 30 µL volume of DNA or RNA was injected into the quadricep muscle of the hindleg, followed by insertion of 2-needle electrode that flanks the injection site. Two pulses of electricity at 60 ms duration/pulse and 80 V was delivered to the quadriceps muscle. This procedure was repeated on the second hindleg. A total dose of 1 or 10 µg DNA or RNA were delivered per animal.For the histology experiments, 3 week old Balb/c mice were purchased from Charles River (Wilmington, MA, USA) and housed at BioTox Inc (San Diego, CA, USA). Intramuscular injections were performed on mice quadriceps muscles using a 29 gauge syringe delivering either 1× PBS, 5 µg gWiz-GFP plasmid DNA (Aldevron, Fargo, ND, USA) or 5 µg GFP-expressing self-replicating RNA vector (Novartis, Cambridge, UK). The injection volume was maintained at 30 µL across all groups. Immediately following IM injection, EP was performed by inserting the 2-needle array (27-gauge with 4 mm spacing) of the Elgen 1000 EP device (Inovio Pharmaceuticals, San Diego, CA, USA) into the mouse muscle. The parameters for the EP were 2, 80 V pulses of 60 ms same as described above. Mice were humanely sacrificed by cervical dislocation 48 hours post-treatment and muscles were excised post mortem.For immunogenicity experiments, Balb/c mice at 6–8 weeks old were injected with 30 µL of pDNA or RNA at respective doses in the quadriceps muscles of the hindleg and followed by electroporation where required with 2 pulses of 100 V at 60 ms duration. VRP and Env/MF59 were delivered by intramuscular injection to the quadriceps at 50 µL per leg to both legs. A total dose of 1 × 107 IU or 10 µg was delivered per animal, respectively.To assess GFP expression in mouse muscle, excised quadriceps were embedded in OCT (Sakura-Finetek, Torrance, CA, USA) and snap-frozen in liquid nitrogen (LN2)-cooled isopentane (Sigma-Aldrich, St. Louis, MO, USA). The fresh-frozen samples were stored at −80 °C until the cryosectioning procedure. The cryosectioning was performed on a Bright Model OTF cryostat machine. To sample the muscle tissue, two serial 15 µm cross-sections were taken and adhered to separate gelatin-coated slides. Separate slides were prepared for fluorescence imaging and for H&E staining. A 15 µm section was taken every 75 µm through the muscle. Thirty-six sections were prepared for each muscle. Once cryo-sectioned, the tissues were fixed in 4% paraformaldehyde/1× PBS solution for 10 minutes and then washed 3 × 5 minutes with 1× PBS. The H&E staining procedure was carried out using Histo·Perfect™ H&E Staining Kit (BBC Biochemical, Seattle, WA, USA) following the manufacturer’s instructions Briefly, tissues were rehydrated using increasingly dilute ethanol (100%–70%) and DI water and then stained with hematoxylin for five minutes. Slides were rinsed with DI water and submerged into proprietary Acid Wash and Blueing Solutions. The tissues were stained with Eosin for 45 seconds and then washed with proprietary S2 Histo Wash. Finally, slides were dehydrated in xylene for 1 minutes and cover-slipped using xylene-based mounting media. Representative depictions of fluorescence and bright field images were taken using an Olympus BX51 microscope (Olympus of Americas, Center Valley, PA, USA) and MagnaFire SP S camera system (Olympus of Americas, Center Valley, PA, USA) at a 10× magnification. The microscope and camera settings remained constant across all groups.The serum from all groups and time points was sampled for SEAP expression using the Phospha-Light Chemiluminescence Assay Kit (Applied Biosystems, Foster City, CA, USA). The assay was performed according to manufacturer’s protocol for a 96-well plate format, using Microlite* Luminescence Microtiter plates (Thermo Fisher, Waltham, MA, USA). The SEAP chemiluminescence was detected in a luminometer (Berthold Technologies) using 1 seconds duration for signal integration.Detection of gp140.SF162 specific antibodies in serum samples was performed by ELISA using plates coated with 100 µg gp140.SF162 per well, in 96-well flat bottom polystyrene plate (Nunc MaxiSorp, Thermo Fisher, Waltham, MA, USA). The plates were incubated SuperBlock blocking buffer in 1× PBS (Pierce) for 2 hours at 37 °C and then exposed to mouse serum diluted 5-fold serially in assay diluents (5% Goat Serum and 0.1% Tween 20 in 1× PBS). The plates were incubated at 37 °C for 2 hours and washed 3 times with wash buffer at 200 µL/well (1× PBS and 0.1% Tween 20). HRP-conjugated goat anti-mouse IgG, IgG1 or IgG2a (Southern Biotech, Birmingham, AL, USA) prepared in assay diluent was added to the plates at 100 µL/well, followed by 1 hour incubation at 37 °C. The plates were washed 3× with wash buffer, and the amount of HRP bound was quantified using TMB detection system (KPL Labs). Briefly, 100 µL of prepared TMB reagent was added to each well. The plate was immediately transferred to a dark environment for 15 minutes. At the end of this incubation, 1 M H3PO4 was added at 100 µL/well, and chromatic absorbance at OD 450 nm was recorded for each well. Each sample was assayed in duplicate and the average absorbance was used for titer analysis. A standard made of mouse serum with known response to gp140.SF162 was also included in each plate to normalize responses among the plates. The titer each sample was reported as the reciprocal of fold dilution at a pre-determined OD 450 nm, which was 20% of the absorbance of the standard at the highest dilution.Four spleens from identically vaccinated BALB/c mice were pooled and single cell suspensions were prepared. Two antigen-stimulated cultures and two unstimulated cultures were established for each splenocyte pool. Cultures contained 1 × 106 splenocytes, anti-CD28 mAb (BD Pharmingen, #553294; 1 μg/mL), and Brefeldin A (BD Pharmingen, #555029, 1:1,000). HIV-1 SF162 Env-specific T cells were stimulated with a pool of Iad-restricted 20mers (YGVPVWKEATTTLFCASDAK, AYDTEVHNVWATHACVPTDP, ITQACPKVSFEPIPIHYCAP, NVSTVQCTHGIRPVVSTQLL) and H-2Dd restricted 9mer (IGPGRAFYA), each at a final concentration of 2.5 µg/mL. Unstimulated cultures did not contain peptides, and were otherwise identical to the stimulated cultures. After culturing for 6 hours at 37 °C, cells were washed and then stained with Pacific Blue labeled anti-CD4 (BD Pharmingen, #558107) and Alexafluor 700anti-CD8 (BD Pharmingen, #557959) monoclonal antibodies (mAb). Cells were washed again and then fixed with Cytofix/cytoperm (BD Pharmingen, #554714) for 20 minutes. The fixed cells were then washed with Perm-wash buffer (BD Pharmingen, #554714) and then stained with a cocktail of PerCP-Cy5.5-labeled anti-IFN-γ (Ebiosciences, #45-7311-80), Alexafluor 488-labeled anti-TNF-α (BD Pharmingen, #557719), Allophycocyanin-labeled anti-IL-2 (BD Pharmingen, #554429), and Phycoerythin-labeled antiIL-5 (BD Pharmingen, #554395). Cells were washed and then analyzed on an LSR II flow cytometer (BD Pharmingen). FlowJo software (Tree Star, Inc., Ashland, OR, USA) was used to analyze the acquired data. The CD4+8− and CD8+4− T cell subsets were analyzed separately. For each subset in a given sample the % cytokine-positive cells was determined. The net (%) antigen-specific T cells were calculated as the difference between the % cytokine-positive cells in the antigen-stimulated cultures and the % cytokine-positive cells in the unstimulated cultures. The contribution of the various TH subsets to the overall CD4+ T-cell response was plotted using these criteria: TH1: CD4+IFNγ+IL-5neg, TH2: CD4+IFNγnegIL-5+, TH0: CD4+IL-2+/TNFα+and IFNγnegIL-5neg. The 95% confidence limits for the % antigen-specific cells were determined using standard methods [50].All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). For analysis of SEAP expression, student-t test was used with confidence limit of p < 0.05. Self-amplifying RNA vaccines hold promise as next generation nucleic acid vaccines, but may require delivery technologies to enhance potency. Electroporation in situ has been demonstrated to be very efficient for increasing delivery and effectiveness of plasmid DNA vaccines. We show here that electroporation can also be used to markedly enhance the delivery and potency of RNA-based vaccines.We would like to acknowledge the RNA Vaccine Platform Team at Novartis Vaccines and Diagnostics. In particular, Avishek Nandi and Pampi Sarkar for their assistance in producing the vectors for these studies; the Laboratory for Animal Services team for carrying out the animal immunizations and sample collection; Funding for the HIV studies was supported by a HIV Vaccine Research and Design Grant (HIVRAD, 5P01AI066287). The authors are (or were previously) employees of Novartis Vaccines & Diagnostics, Inc or Inovio Biopharmaceuticals.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP) device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21) of enhanced gene delivery with electroporation (EP) was performed to observe the localization of green fluorescent protein (GFP) expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.Delivery of vaccines directly to the skin (intradermal, ID) is an attractive immunization strategy in a clinical setting due to a number of dermal-specific features. Skin is the most accessible organ of the human body, the most easily monitored, as well as being a highly immunocompetent target [1,2]. Indeed, the skin contains a resident population of antigen presenting cells (APCs), specifically a large number of Langerhans cells and dermal dendritic cells, so has the potential for increased immunogenicity through direct transfection and presentation. Human skin is the largest organ of the human body and extends to approximately 2 m2 in area [1]. The most superficial layer of skin is the stratum corneum (SC) which functions as the primary barrier for this organ. The skin has two broad tissue types, the epidermis and the dermis. The epidermis is a continually keratinizing stratified epithelium. Making up approximately 80%–90% of the cellular population of the epidermis, the predominant cell type is the keratinocyte. These cells play both a structural role as well as being immunologically active. Keratinocytes appear to play a role in initiating cell-mediated immune responses in the skin by cytokine release and adhesion-molecule expression [3]. The other three strata of the epidermis (S. granulosum, S. spinosum, S. basale) all contain keratinocytes at different stages of differentiation as well as the immune Langerhans cells and dermal dendritic cells [1,2]. The chief function of the Langerhans cells is to process and present antigens encountered in the epidermal space to naive T cells and to initiate an adaptive immune response [4]. Additional APCs that play a role in the skin immune function and trafficking to regional lymph nodes include veiled cells (resident in the lymphatic system), follicular dendritic cells (resident in the regional lymph nodes), monocytes, macrophages and B cells.The dermis functions primarily as a scaffold for the epidermis, containing a dense collagen matrix, elastic fibers, and extrafibrillar matrix interspersed with fibroblast cells [1]. It is divided into two layers, the superficial area adjacent to the epidermis called the papillary region and a deep thicker area known as the reticular dermis.DNA vaccines are a next generation branch of vaccines which offer major benefits over their conventional counterparts [5,6,7,8]. Unlike conventional vaccines, DNA vaccines are gene based expression plasmids that encode specific antigens and do not require isolated virus for production. Unlike inactivated vaccines, DNA vaccines can mimic the immunological effects of infection since they directly transfect the host’s cells. As a result, gene expression occurs via the host’s own machinery, allowing for antigen presentation through both the MHC class I and II pathways. Such gene-based vaccines also offer the ability to develop, optimize and manufacture large doses of vaccine in a cost-effective, rapid manner. Due to the inherent stability of DNA vaccines, they do not require cold-chain storage which is a major logistical issue with some current conventional vaccines and biologics. This has obvious major implications for their distribution and use in developing countries. Most importantly, DNA vaccines are able to generate both a robust antibody and T-cell response [7,8,9,10]. This ability means that DNA vaccination offers a therapeutic solution against many complex diseases such as HIV/AIDS and cancers.A major obstacle to effective vaccination via gene-based methods is the low efficiency of intracellular delivery. Outside of small rodent models, the delivery of naked DNA through a standard intramuscular (IM) injection is notoriously inefficient. In past studies, this has led to an inability to achieve strong immune responses in large mammals and humans immunized with naked DNA [5,6,7,8]. One physical method to temporarily increase cell permeability is electroporation (EP) and this method has moved to the forefront as the modality of choice for DNA vaccination. EP involves the application of brief electrical pulses that result in the creation of temporary aqueous pathways within the lipid bi-layer membranes of mammalian cells. This allows the passage of DNA and other macromolecules through a cell membrane that was previously impermeable to these molecules. As such, EP increases both the uptake and the extent to which drugs and DNA are delivered to the target tissue of interest [11,12,13,14,15]. Historically, EP has been primarily targeted to muscle tissue and currently multiple clinical trials are being conducted using this route of delivery [16,17,18,19,20]. By the nature of the target tissue, intramuscular EP is an invasive procedure. In an attempt to improve the vaccination experience from the patients’ perspective, recently there has been a significant move towards developing EP devices that target the dermal region. Since the target tissue of skin is considerably shallower from a depth perspective than skeletal muscle, dermal EP devices can be designed to be much less invasive and even completely non-invasive. This has the important implication from a patient tolerability standpoint of not activating deep nerves and muscles. A typical volume for an IM vaccination would be in the range of 1–2 mL whereas ID vaccination injection volumes are generally limited to no more than 100 µL. This raises obvious issues with dose limitation although the dose sparing ability of skin as a target tissue may mitigate this. To be a clinically relevant platform, it is vital that ID EP would still maintain equivalent efficacy in comparison to IM EP procedures. Historically, it had been proposed that IM EP generated robust cellular responses and ID EP humoral responses. However, the current understanding of the platform implies that ID EP can generate both antibody and cellular responses equally well.Devices for ID EP can be classified into different categories depending on their mode of action or application. Examples of non-invasive or surface electrodes are devices such as the caliper [21] and plate electrode platforms [22]. Further skin surface electrodes are the MEA (Multi-Electrode Array) [23,24], and the meander electrodes [25]. In general, these platforms make direct contact with the dermal surface without rupturing the stratum cornea of the skin and require relatively high electrical field strength for efficient transfection. Contactless electrodes can consist of a static spark [26] or a corona charge [27,28] and make no direct contact with the patient’s skin. These modalities also have the obvious benefit of a lack of a disposable device component. Invasive skin EP device configurations generally consist of an array of multiple needles which penetrate into the skin. Roos et al. [29] reported that a device consisting of two parallel rows of 4-needle electrodes (8 in total) using two pulses of 1,125 V/cm and 8 pulses of 275 V/cm field strength resulted in robust immune responses [29,30]. This device was initially assessed in humans to evaluate the safety, effectiveness and relative pain levels of dermal EP [31] and has subsequently been used in several clinical trials to deliver a prostate DNA vaccine (ClinTrials identifier—NCT00859729) and a colorectal cancer DNA vaccine (ClinTrials identifier—NCT00859729).The CELLECTRA®-3P (Inovio Pharmaceuticals, Blue Bell, PA, USA) is a minimally invasive electroporation device which targets dermal and subcutaneous layers of the skin [32,33,34] with mild EP conditions and minimal tissue damage. The device consists of three-needle (3 mm in length) electrodes forming a triangle microarray to cover the DNA injection site. This depth of penetration treats the entire skin thickness and as such targets the dermal cells in the epidermis, dermis and subdermis. Recently, this device has entered the clinic in two studies (ClinTrials identifier—NCT01403155, NCT01405885) sponsored by Inovio Pharmaceuticals (Blue Bell, PA, USA) addressing the delivery of a multi-strain influenza DNA vaccine. The reduced depth of the minimally invasive electrodes has been shown to significantly increase the tolerability of the procedure compared with IM EP [35]. Since these approaches are considered more tolerable, the ability to deliver prophylactic immunizations becomes a reality using this device platform. The electroporation device used for this study is a surface EP device (Inovio Pharmaceuticals, Blue Bell, PA, USA) which features a 4 × 4 array of sharp electrodes that disrupt the stratum corneum, but do not penetrate the epidermis or lower tissue layers [36]. The device design (1.5 mm electrode spacing) and pulse parameters (applied 25 volts) used on this device localize the electrical field to the upper layers of the skin and primarily target the epidermis, rich in APCs, such as Langerhans and dermal dendritic cells.In previous publications, we had detailed the development of this surface EP device (SEP) and demonstrated the utility of the device to induce plasmid expression in the skin which subsequently resulted in robust immune responses [36]. While this publication outlined the proof-of-concept studies with this delivery modality, we were keen to gain a deeper understanding of the mechanism of action and have the ability to specifically identify transfected cells and peak expression times. To achieve this knowledge, a time course assessment of reporter gene expression on both a gross and cellular level was performed. We were able to observe the morphology of transfected cells as well as assess the kinetics of monocyte and granulocyte infiltration at the treatment site. In addition, we demonstrated that ID EP resulted in migration of lymphocytic cells to the treatment site.The results from this study provide insights into expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have future implications when designing efficient ID DNA vaccination strategies for the clinic allowing for peak antigen expression to drive the immunization schedule.Female Hartley guinea pigs (6 months old) weighing ~350–400 grams were used in this study. The guinea pigs were group housed (4 per cage) with ad libitum access to food and water. Animals were quarantined for two weeks prior to experimentation. All animals were housed and handled according to the standards of the Institutional Animal Care and Use Committee.Three guinea pigs were shaved and depilated one day prior to initiating the study for the early time points. All three animals received five separate treatments at each defined time point (1, 2, 4, 6 and 8 h). Therefore there was a total of 15 treatment biopsies generated for each individual time point. To assist with tissue harvesting, animals were treated initially for the 8 h time point and subsequently treated in descending time order. The same experimental set up was applied for the later time points (24 h, 48 h, days 3, 7, 12, 14 and 21) where three animals with five separate treatment sites were used. Again, the treatments were performed in descending order to ensure ease of sacrifice. Each treatment at each time point comprised of a single injection of 50 μg of gWIZ-GFP or gWIZ-RFP (Aldevron LLC, Fargo, ND, USA) in 50 µL of PBS delivered intra-dermally using the Mantoux injection method and immediately followed by electroporation using the surface EP device detailed in the introduction [36]. The Mantoux intradermal injection is a standard clinical technique involving a small gauge needle (usually 29G) inserted parallel to the skin bevel up. The device electrical parameters were three pulses of 100 ms at an applied voltage of 25 volts. The time course spanned 21 days and included early time points of 1, 2, 4, 6 and 8 h. At the relevant times, 8 mm biopsy punches of four of the five treatments from each time point on each animal were taken post-mortem and fixed in 4% paraformaldehyde at 4 °C overnight. The following day, skin biopsies were buffered in a 15% sucrose solution and stored until sectioning at 4 °C. The fifth treatment of each time point on each animal was collected and stored at −20 °C for gross imaging. Biopsies were embedded in OCT Compound and sectioned at a thickness of 15 µm using an OTF Bright Cryostat (Cambridge, UK). Sections from all time points were H&E or DAPI stained and viewed under bright light or fluorescent microscopy. Sections from 1, 2, 4, and 6 h time points were stained with unconjugated primary antibodies against: anti-guinea pig lymphocytes and Langerhans cells (Clone MsGp2, AbD Serotec, Oxford, UK), and anti-keratin 10 (Assay Biotech, Sunnyvale, CA, USA). Sections were then stained with either an anti-mouse Alexa Fluor 555 (MsGp2) or anti-rabbit Alexa Fluor 488 (Keratin) (Life-Technologies, Inc., Grand Island, NY, USA) secondary antibody. An additional stain, Hoechst 33342 (Life Technologies, Inc, Grand Island, NY, USA), was used to visualize nuclei. The slides were then mounted with Fluoromount (Ebioscience, San Diego, CA, USA) and viewed using fluorescent or confocal microscopy.Fluorescent microscopy was carried out using an Olympus BX51 with a Magnafire U-TV1X-2/U-CMAD 3 combo camera for photo acquisition (Olympus, New York, NY, USA). Magnafire software was used to acquire the images.Confocal Images were obtained with a Zeiss LSM 780 laser scanning confocal Microscope (Carl Zeiss, Inc., Jena, Germany) and processed with Zen 2012 Software (Carl Zeiss Inc., Jena, Germany). Z stacks of images (obtained at 0.3 µm intervals) were collected sequentially using a 63× objective and then maximum projected into single flattened stacks for figures. To assess the expression kinetics resulting from gene delivery with a dermal EP device, a plasmid expressing GFP was injected into guinea pig skin at defined time points. Surface EP (SEP) was immediately performed after each injection. The animals were sacrificed post treatments and the skin excised and visualized under a fluorescent microscope (Figure 1). GFP expression appeared at 6 h on the surface of the skin and persisted for seven days. The peak expression was observed at 24 h. During the peak times (24–72 h), the expression was robust and matched in size the diameter of the injection bubble (approximately 5 mm). Within the gross localization pattern of GFP, smaller islands of expression were also noted which coincided in shape and spacing with the direct contact the electrodes make with the skin. The biopsies shown in the figure are representative examples of those seen in multiple treatments on multiple animals.Gene delivery enhanced by dermal electroporation induces sustained expression on the surface of the skin. Time course (1 h post treatment to day 21) of green fluorescent protein (GFP) expression after intradermal (ID) plasmid administration followed by surface electroporation (SEP) in guinea pig skin visualized under natural (top panel) and fluorescent (lower panel) light. An untreated control is also shown. Photos are representative examples of multiple treatments. While the skin surface localization patterns delineate the global expression trends, skin is a squamous epithelial tissue so it was possible that cells below the observable surface (and so not apparent on the surface view) were also being transfected. Through the natural migration of cells in the epidermis, such cells would only later move to the skin surface and so give a falsely slow dynamic. To investigate this further, biopsies were taken of the time points from the original study and fixed sections prepared. Under a fluorescent microscope, sections were observed and scored for GFP positive cells (Figure 2). One hour post treatment, positive cells were identified in skin sections. These cells appeared to be closer to the basement membrane and were likely to be located in the stratum basale level of the epidermis and the mid to upper epidermis (stratum granulosum). As the time course progressed, the transfected cells migrated upwards to the surface of the skin where between day 5 and day 7 the majority of the signal appeared to reside in the stratum corneum. The seemingly brighter GFP signal at day 7 over day 3 is a function of the change in morphology of the reporter gene positive calls as they flatten out in the SC. Post day 7, the GFP positive cells appeared to slough off which is the nature of that barrier layer of the skin. To allow annotation of the structural elements in the skin sections, an enlarged view of the 2 h section is included where the sub-structures and stratum of the skin are noted. In keeping with the shallow electrical field generated by this EP device, all the transfected cells were localized in the epidermis of the skin sections.Histological analysis reveals rapid GFP expression. Histological analysis of GFP expression in a time course (1-hour post treatment to day 21) after ID plasmid administration followed by EP with SEP in guinea pig skin. Skin biopsies were removed, cryosectioned, DAPI stained and visualized under fluorescence microscopy (10×). An injection only control is also shown. Photos are representative examples of multiple treatments. A region of the 2-hour time point section image is enlarged to allow the annotation of skin structures.Magnified DAPI stained images from the 4 h time point plus and minus electroporation (Figure 3A) demonstrate the morphology of the transfected cells as well as the enhancing effect of electroporation. Using a keratinocyte specific antibody, we were able to identify reporter gene positive cells (in this case, red fluorescent protein (RFP)) which also co-stained for K10, the keratinocyte marker (Figure 3B) visualized with an Alexa 488 secondary antibody. In addition to the positive antibody staining, the morphology of the cells depicted through the reporter gene expression is indicative of keratinocytes.Histological analysis reveals reporter gene expression localized to cells in the epidermis. Histological analysis of GFP and red fluorescent protein (RFP) expression after ID plasmid administration followed by SEP in guinea pig skin. (A) GFP treated skin biopsies were removed 4 h post treatment, cryosectioned, DAPI stained and visualized using fluorescence microscopy (20× and 40×). An injection only control (no EP) is also shown; (B) RFP treated skin biopsies were removed, cryosectioned, stained with an antibody against K10 (a keratinocyte cell surface marker), Hoechst stained and visualized using confocal imaging.The biopsied sections from Figure 2 were additionally stained with H&E to observe the dynamics of infiltration at the site following EP treatment (Figure 4). Unlike infiltration in the muscle following EP, which is generally a relatively slow process, monocytes and granulocytes were observed migrating to the skin treatment site within 4 h. The inflammatory context persisted for 14 days and appeared resolved at day 21. No evidence of necrosis or localized tissue damage as a result of the EP was observed at any time point. To allow closer analysis of the infiltration and annotation of the structural elements in the skin sections, an enlarged view of the day 7 section is included where the sub-structures and stratum of the skin are noted.The major APC in the epidermis is the Langerhans cell. Using an antibody against Langerhans cells and lymphocytes, we immunostained skin sections to observe the dynamics of these migratory cells following EP. Although increased numbers of lymphocytic cells were observed following DNA injection alone and EP alone (data not shown), a significant increase in cell numbers was observed following EP enhanced delivery of plasmid expressing GFP at 6 h post treatment (Figure 5). Through analysis of multiple GFP treated sections, lymphocytic cells could be seen migrating towards and congregating in the areas of the skin biopsy where the transfected GFP expressing cells appeared.Rapid and persistent monocyte and granulocyte infiltration is detected at the treatment site following EP-enhanced plasmid delivery. Hematoxylin and eosin (H&E) stained histological analysis of skin sections following ID plasmid administration and EP with SEP in guinea pig skin in a time course. Skin biopsies were removed, cryosectioned, H&E stained and visualized under standard light microscopy (10×). An untreated control (no DNA, no EP) and EP alone post 1 h are also shown. A region of the day 7 time point section is enlarged to indicate skin structures and infiltration of monocyte and granulocyte.Histological analysis reveals infiltration of lymphocytes at the EP treatment site. Guinea pig skin sections were immunohistochemically stained with an antibody recognizing lymphocytes and Langerhans cells (Tetramethylrhodamine isothiocyanate (TRITC) secondary) after ID GFP plasmid administration followed by EP with SEP. Skin biopsies were removed, cryosectioned, antibody and 4',6-diamidino-2-phenylindole (DAPI) stained and visualized under fluorescence microscopy at 20× and 40×. An untreated control (no DNA, no EP) is also shown. Intradermal electroporation is a platform technology which offers a solution to the tolerable delivery of DNA vaccines in the clinic for prophylactic immunization. Many groups have shown the utility of this technology in a range of animal models in preclinical studies as well as recently in human skin in the clinic. Multiple modalities of ID EP devices exist, ranging from contactless to fully penetrating. Since each device has varying modes of action, each will target different compartments in the skin. This will result in the transfection of different resident cell populations and as such, have the capacity to elicit varying immune responses. While a wealth of published literature demonstrates the ability of the platform to elicit robust immune responses in a spectrum of animal models and in the clinic [29,30,31], less is understood about the mechanism of action of dermal EP, especially related to the resulting expression kinetics. Incidences of inflammation at the treatment site following EP has been investigated in guinea pig skin [23] and expression of reporter gene plasmids in skin has been used as a marker by multiple groups. However, these are generally observations at a single time point. Here we investigated the expression of a reporter gene construct following electroporation with a surface EP device that specifically targets the epidermis over a defined time course. This study allowed us insight into peak expression times, duration of expression, and kinetics of infiltration and induced migration of APCs.An elegant study by Roos et al. 2009 [29] investigated the functional properties of invasive EP enhanced intradermal DNA delivery in a mouse model. This group evaluated the kinetics of luciferase transgene expression following DNA injection. Additionally, they identified the location of transfected cells in the skin, the effect on the local tissue environment and the persistence of DNA molecules at the injection site. The work detailed here builds on the Roos study by investigating GFP expression via surface EP in a guinea pig model, histologically identifying transfected cells as well as the kinetics of infiltration at the treatment site.The animal model of choice for many dermatological applications is the guinea pig. This is primarily due to the similarity in skin physiology between these rodents and humans. All studies detailed here were carried out in the Hartley guinea pigs model. A significant difference between the guinea pig model and human skin is the turnover time of cells in the epidermis. The guinea pig has a faster turnover—approximately 2–3 times faster than human skin cell turnover—but is slower than mouse skin cell turnover—approximately seven days. This study allowed us to assess the resulting GFP localization in skin following EP enhanced delivery with a surface device. Here we delivered a 50 µL injection of 50 µg reporter gene plasmid by standard Mantoux ID injection means. While a clear benefit to skin vaccination is the ability to dose-spare, we chose this high dose of reporter plasmid to ensure maximal expression. The resulting injection bubble is approximately 4.5 mm in diameter and so fits appropriately under the electrode array of the SEP device. At the peak expression time point (24 h), the GFP expression pattern corresponds well with the bubble size and array contact. It is also possible to observe small islands of transfection. We believe these islands correspond directly to the contact made between the electrode and the skin.The GFP expression following EP with this device was observed as early as 1 h (microscopically) and persisted through day 7. This timing coincides well with the turnover of cells in the epidermis in guinea pigs. The turnover of cells in humans is considerably longer, more in the range of weeks than days. Analysis of the skin sections suggests that we are directly transfecting cells both in the stratum basale and in the mid to upper epidermis (stratum granulosum) which over the next seven days differentiate as they move towards the upper barrier layer of the stratum corneum. Once trapped in this non-viable but biologically active layer, the GFP disappears as the skin upper layer is sloughed off. Due to the distinct electrode spacing and the low applied voltage of the SEP device, only observing transfection of cells in the epidermis makes sense. The electric field generated by such a modality would be shallow and not penetrate further into the dermis. We believe that this feature of the device will lead to a highly tolerable platform since deep nerves and skeletal muscle will not be activated during the procedure. Higher magnification depictions of the GFP/RFP positive cells in the epidermis reveal distinct cellular morphologies similar to a keratinocyte cell. We confirmed this by additionally staining for a keratinocyte cell surface marker (K10) and observing both antibody positive cells (using an Alexa 488 secondary antibody) and reporter gene positive cells. This is an intuitive finding since keratinocytes make up between 80%–90% of the epidermis cellular population. Since the applied voltage parameter of this device is 25 volts, and the electrode spacing is 1.5 mm, the resulting electrical field is mild and shallow. As such, the finding that there was no obvious cellular damage or treatment associated necrosis was unsurprising. In a previous publication [37], we demonstrated that the lack of skin damage at these low voltages did not compromise the resulting immune responses. Following EP in the muscle, infiltration at the site is not observed until 4 days post treatment [38]. In this H&E skin study; we observed significant monocyte/granulocyte trafficking to the treatment site at the 4 h time point. Clearly there are significant differences between skin and muscle as target tissues but this finding seeks to highlight the benefits of intradermal vaccinations from the perspective of rapid dynamics. Interestingly, increased infiltration is still observed in EP-treated skin 14 days post procedure, significantly longer than the persistence of reporter gene expression (seven days). It is possible that a low number of cells are still expressing the antigen at these later time-points but are below our levels of imaging detection. Antibody staining for lymphocytes/Langerhans cells demonstrated significant increases in detected cells following EP-enhanced plasmid delivery over untreated skin. The majority of positive cells were detected in the dermis region. It is possible that increased numbers of cells would also be detected in the epidermis (alongside the reported gene expression) however more sophisticated imaging equipment may be required to observe this. Ongoing studies are currently underway to further assess the dynamics of this infiltration.When designing clinical protocols involving plasmid transfer, an understanding of the optimal operating parameters of the vaccine delivery device is crucial. The information gained from this study might allow us to design optimal prime/boost regimes from a timing perspective, taking into account expression kinetics and trafficking of immune sensing cells to the treatment site.We would like to thank Maria Yang, Philip Armendi and Jenna Robles for plasmid preparation, and Steve Kemmerrer and Jay McCoy for support of the EP device and Kimberly A. Kraynyak for manuscript assistance. This work was supported in part by a Department of Defense SBIR grant (Phase I and Phase II) number W81XWH-11-C-0051.Authors J.J.M., D.H.A., G.K., C.L.K., T.R.F.S., N.Y.S. and K.E.B. are employees of Inovio Pharmaceuticals and as such receive compensation in the form of salary, stock options and bonuses. Kiosses is an employee of The Scripps Research Institute and declares no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).2013 marks a milestone year for plasmid DNA vaccine development as a first-in-class cytomegalovirus (CMV) DNA vaccine enters pivotal phase 3 testing. This vaccine consists of two plasmids expressing CMV antigens glycoprotein B (gB) and phosphoprotein 65 (pp65) formulated with a CRL1005 poloxamer and benzalkonium chloride (BAK) delivery system designed to enhance plasmid expression. The vaccine’s planned initial indication under investigation is for prevention of CMV reactivation in CMV-seropositive (CMV+) recipients of an allogeneic hematopoietic stem cell transplant (HCT). A randomized, double-blind placebo-controlled phase 2 proof-of-concept study provided initial evidence of the safety of this product in CMV+ HCT recipients who underwent immune ablation conditioning regimens. This study revealed a significant reduction in viral load endpoints and increased frequencies of pp65-specific interferon-γ-producing T cells in vaccine recipients compared to placebo recipients. The results of this endpoint-defining trial provided the basis for defining the primary and secondary endpoints of a global phase 3 trial in HCT recipients. A case study is presented here describing the development history of this vaccine from product concept to initiation of the phase 3 trial.The seminal studies of the DNA vaccine field, published in the early 1990s, demonstrated that administration of plasmid DNA to mice resulted in protein expression of encoded transgenes [1], induction of antibody responses [2], and protection from pathogenic challenge [3]. Following these and other nonclinical proof of concept studies, the first-in-human phase 1 clinical trials were conducted during the mid-to-late 1990s in HIV-1-infected and normal healthy volunteers with DNA vaccines encoding HIV-1 and malaria antigens, respectively [4,5,6]. Since then, DNA vaccines against other infectious disease agents have completed phase 1 and in some cases phase 2 testing including: anthrax, CMV, dengue, Ebola, hepatitis B virus, hepatitis C virus, herpes simplex virus-2, human papillomaviruses, seasonal and pandemic influenza viruses, measles, severe acute respiratory syndrome, and West Nile virus.The results of early clinical trials of DNA vaccine candidates indicated favorable safety and tolerability profiles and evidence of humoral and cell-mediated immune responses [7,8]. However, the perceived need by most investigators to improve the immunogenicity of DNA vaccines resulted in the development of strategies to enhance DNA vaccine performance in humans. These strategies fall into four non-mutually exclusive categories, including: (1) plasmid design (e.g., codon optimization, promoter selection, inclusion of a genetic-adjuvant-encoding plasmid); (2) formulations (e.g., polymer, cationic liposomes, PLGA microspheres); (3) devices (e.g., Biojector® 2000, particle-mediated epidermal delivery, electroporation); and (4) heterologous prime-boost with a viral vector (e.g., NYVAC, MVA, adenovirus) or recombinant protein. Vical Incorporated (hereafter Vical) developed a CMV DNA vaccine candidate utilizing the first two of these strategies. Proof-of-concept testing of the vaccine in a phase 2 trial has been completed and the vaccine is expected to enter a pivotal phase 3 trial sponsored by Astellas Pharma, Incorporated (hereafter Astellas). A case study is presented here of the development pathway of this vaccine.Cytomegalovirus, a β-herpesvirus and the largest virus known to infect humans, initiates a predominantly asymptomatic infection in normal, healthy individuals that persists for life, mostly as a latent infection without evidence of viremia [9,10]. CMV infection rates are high worldwide; the CMV seroprevalence rate in the U.S. is ~60% in ≥6 year olds and increases with age [11]. CMV can cause significant morbidity and mortality in certain high-risk situations including congenital infection of fetuses and infection of recipients of hematopoietic stem cell transplant (HCT) or solid organ transplant (SOT), where treatment-related immunosuppression provides opportunities for CMV replication following viral acquisition or reactivation from latency. Antiviral drugs are licensed for use in transplant recipients either prophylactically or preemptively (upon evidence of viremia as measured by PCR or antigenemia assay). Unfortunately, the use of first line ganciclovir-based drugs or second line foscarnet and cidofovir can result in substantial hematotoxicity and nephrotoxicity [9]. While antiviral drugs have reduced the incidence of CMV end organ disease (EOD) in CMV+ HCT recipients from 25% prior to their licensure to approximately 5% today [9,12], alternative measures for controlling CMV replication after transplantation without attendant drug toxicities are needed. Vaccines represent one such strategy.Numerous phase 1 and phase 2 trials have been conducted with several CMV vaccine candidates but a vaccine has yet to be licensed for any indication [10]. Vaccine candidates include live attenuated vaccines such as Towne strain and Towne/Toledo chimeric strains, subunit vaccines, most notably adjuvanted recombinant gB, and vectored vaccines including recombinant viral vectors using canarypox and alphavirus platforms, and plasmid DNA vaccines [10]. The live attenuated CMV Towne vaccine, derived in 1975, provided proof of concept in SOT recipients for reduced CMV disease severity after transplantation; however, it has not provided efficacy against infection in transplant recipients or in women exposed to CMV in daycare settings, and further development of this vaccine appears to depend upon implementing strategies to improve its immunogenicity [10]. Beginning in the 1990s, a recombinant gB vaccine produced in CHO cells was developed and tested in combination with an MF59 adjuvant in multiple clinical studies over the ensuing decades. Two recent randomized controlled phase 2 studies provided proof of concept for the importance of gB as a protective CMV antigen by demonstrating 50% efficacy in decreasing maternal CMV infection [13] and a significant reduction in the duration of viremia as well as the duration of antiviral ganciclovir treatment in CMV seronegative (CMV−) recipients of kidneys or livers from CMV+ donors [14]. Despite these results it is unclear whether gB adjuvanted by MF59 will continue further development for either indication. Other vaccine approaches such as viral vectored gB [15] and/or pp65 [16,17] have completed phase 1 testing with evidence of safety and immunogenicity. It is conceivable that the recent recognition of the potential importance of creating vaccines that target the pentameric gH/gL/UL128/UL130/UL131 epithelial entry pathway may have contributed to redirected vaccine efforts by some companies, at least for prophylactic vaccines attempting to block both fibroblast (gB-mediated) and epithelial entry pathways [10].Vical built the capabilities for developing plasmid DNA-based products during development of its lead product, an intralesional immunotherapeutic called Allovectin® (velimogene aliplasmid) [18], which is currently being evaluated in a phase 3 metastatic melanoma trial that is nearing completion. In the early 2000s, product development activities began on several infectious disease targets, the first being CMV; by that time all of the key functional areas were in place to design, create, manufacture, release, and clinically test DNA vaccine product candidates. The functional area expertise included vaccine research, molecular biology, pharmaceutics, nonclinical testing, manufacturing, assay development, quality control (QC), quality assurance, clinical research and operations, regulatory affairs, and project planning and management. As described below, the initial CMV vaccine target indication was for CMV+ HCT recipients. A product development timeline with all supporting activities for this vaccine is displayed in Figure 1 and described in the following sections. The vaccine has been referred to by different names throughout its development history including VCL-CB01, TransVax™, and ASP0113, the current designation of this vaccine since its license to Astellas in 2011.Clinical development timeline of ASP0113 (TransVax) from product concept in 2002 to initiation of a phase 3 trial in 2013. Initiation of various activities is shown in blue diamonds; regulatory activities are shown above the timeline and all others below the timeline. Horizontal blue lines and arrows depict the duration of the indicated activities. Development activities that continued and/or were refined during clinical development are shown in rectangular boxes within the large dotted arrow. Abbreviations: IND, investigational new drug application; FDA, U.S. Food and Drug Administration; EMA, European Medicines Agency; GLP, good laboratory practices; QC, quality control; DS, drug substance.CMV-seropositive recipients of an allogeneic HCT have high attack rates of CMV reactivation following transplantation: 50% to 70% will develop detectable CMV viremia within the first 100 days [19] and are at increased risk for developing CMV EOD if antiviral therapy is not initiated. Due to the overall high CMV-seroprevalence rate, CMV+ subjects represent a majority of subjects who undergo HCT. The original intent was to vaccinate normal healthy HCT donors with a 3 dose vaccination series to prime or boost CMV immune responses and then to vaccinate recipients at the time of transplantation to boost the donor’s cells. This dosing regimen was thought to be superior to dosing only the recipient at the time of transplantation because of the potential reduction in vaccine efficacy due to the immunosuppressive medications required post-transplant. After initial testing in CMV+ HCT recipients, Vical planned subsequent evaluation of the same vaccine product in CMV− SOT recipients of organs from CMV+ donors (D+/R−), who represent approximately 20% of all solid organ transplantations and those at highest risk for CMV infection and disease.There are several key advantages that plasmid DNA vaccines can offer relative to other vaccine approaches for application in transplant recipients. First, there is a considerable safety advantage compared to live attenuated virus or viral vectors which may replicate to undesirable levels in an immunosuppressed setting typical of myeloablative or nonmyeloablative conditioning prior to transplantation. Second, there is the ability to stimulate the desired antiviral T-cell responses like live virus vaccines but unlike protein vaccines. Third, repeated vaccine administration would be required for a vaccine in this population, which is readily accomplished with plasmid DNA vaccines, but which may be difficult with viral vector vaccines which typically invoke antivector immune responses during repetitive injections. Fourth, DNA vaccines permit a focusing of immune responses on select antigens in addition to avoidance of immunoevasive CMV proteins coincidentally expressed by live attenuated CMV strains. A DNA vaccine therefore offers the potential for T-cell inducing qualities reminiscent of a live virus vaccine with the safety profile of a protein subunit vaccine—an ideal vaccine profile for transplant recipients.Vical initiated development of a CMV DNA vaccine for transplant recipients in 2002 by first selecting putatively protective CMV antigens and constructing the corresponding plasmids. The control of CMV replication in transplant recipients was predominantly attributed to both CD8+ and CD4+ T-cell responses but a role for a vaccine-induced antibody response in reducing CMV viral loads could not be excluded and could be important in high risk CMV− SOT recipients who receive an organ from a CMV+ donor. Based on the prevailing knowledge at that time, dominant T- and B-cell antigens recognized after natural infection were identified as vaccine candidates including a tegument phosphoprotein, pp65, a dominant T-cell antigen and an envelope glycoprotein, gB, a major target for neutralizing antibodies [20,21,22]. A comprehensive study was subsequently published in 2005 in which the prevalence of both CD4+ and CD8+ T-cell recognition of essentially all (213) CMV proteins was characterized in 33 CMV+ normal, healthy subjects; the results further supported the prevalent recognition of these antigens [23]. Finally, adoptive transfer studies of CMV-specific T cells supported the hypothesis that the antiviral activity of pp65-specific T-cells can control CMV replication in transplant recipients [24,25,26,27]. The DNA vaccine concept was to elicit both humoral and T-cell mediated immune responses to key CMV antigens that could provide immune control early after allogeneic transplantation when CMV+ recipients face the highest risk of CMV reactivation and EOD, namely within the first 100 days.The sequences of the CMV antigens encoded by the plasmids were derived from the laboratory-adapted CMV strain AD169 [28]. Prior to gene synthesis and cloning, the protein sequences for both gB and pp65 were modified in silico. A secreted form of gB was created (713 amino acids in lieu of the full-length 906 amino acids) that could retain conformationally-intact epitopes and provide higher antibody titers than a nonsecreted form [29]. Four amino acids, 435RKRK438, were deleted from the otherwise full-length, 561 amino acid, pp65, thereby eliminating a putative kinase site [30] and mitigating theoretical safety concerns of expressing wild-type pp65. Once the final protein sequences were established, a codon-optimization using an algorithm developed at Vical was performed by changing the codon usage of five amino acids to more frequently used codons (U.S. patent 7,410,795). Each codon-optimized gene was synthesized and cloned into the Vical plasmid backbone VR10051, containing a human CMV IE1 promoter/enhancer and intron A and a modified rabbit beta globin polyA/terminator [28]. The final plasmid nomenclatures were VCL-6365 and VCL-6368 for gB- and pp65-expressing plasmids, respectively.A synthetic, nonionic triblock poloxamer, CRL1005, was selected as the lead formulation candidate for this product. This poloxamer consists of a polyoxypropylene (POP) core flanked on each side by a polyoxyethylene (POE) block. CRL1005, developed by CytRx Research Laboratories (Atlanta, GA), had been evaluated in clinical studies with protein-based vaccines and was found to have a favorable safety profile at doses up to 75 mg/injection [31]. Of direct relevance to DNA vaccines, more recent studies revealed that injection of rhesus macaques with an HIV-1 gag-encoding plasmid formulated with CRL1005 provided the highest T-cell responses compared to other formulations following boosting with an adenovirus-5 expressing gag [32]. Subsequently these investigators incorporated a cationic surfactant, benzalkonium chloride (BAK) capable of binding both CRL1005 and DNA resulting in nanoparticle formation; they demonstrated increased T-cell responses to an HIV-1 gag plasmid with this formulation compared to DNA with CRL1005 or DNA alone [33,34]. BAK, at the concentration used with CRL1005, is an inactive ingredient used in a variety of injectable (e.g., intramuscular, intradermal and other routes) FDA-approved products and as such is not considered a new raw material [35].The combination of CRL1005 and BAK self-assembles into stable nanoparticles. The formulation appearance is a milky white suspension at room temperature that becomes clear as the temperature drops below the CRL1005 cloud point (5 °C–7 °C). The final bivalent vaccine formulation was designed to be a 1-mL intramuscular (IM) injection consisting of a 5-mg total plasmid dose (2.5 mg of each plasmid), 7.5 mg of CRL1005, and 0.1 mg/mL of BAK, all dissolved in phosphate-buffered saline (PBS) and stored frozen [36]. This formulation provided key practical advantages compared to an alternative cationic lipid-based formulation undergoing development during that time: A higher DNA dose could be formulated (up to 5 mg/mL) and a single vial formulation was possible, in contrast to a multivial format (up to 1 mg/mL) with reconstitution of dried lipid film and mixing for the cationic lipid formulation.Because of the species tropism of human CMV, no animal challenge model of efficacy exists for testing human CMV vaccine candidates. Instead, immunogenicity testing was conducted in BALB/c mice to characterize the gB-binding serum IgG levels and the frequency of pp65-specific IFN-γ producing-T cells as measured by ELISPOT assay with overlapping peptides [28]. Two-dose and three-dose regimens of the bivalent plasmids and each monovalent plasmid were tested with and without CRL1005/BAK formulation. This formulation was found to significantly increase the immune responses compared to the bivalent vaccine in PBS only, thereby abrogating the immunological interference that was encountered when the combination was prepared in PBS only [28].Additional in vivo studies were conducted in conjunction with a more detailed physical characterization of this formulation [36]. These studies utilized a plasmid encoding a model antigen, influenza A nucleoprotein (NP), which elicited antibody responses and CD4+ and CD8+ T-cell responses to defined epitopes. Following a three-dose immunization schedule, DNA/CRL1005/BAK-formulations produced significantly higher responses than the same dose of DNA alone; NP antibody responses were 1.6-fold higher (p < 0.001), CD4+ responses to defined class II-restricted peptides were 1.7-fold higher (p < 0.01) and CD8+ T-cell responses to a class I-restricted peptide was 1.9-fold higher (p < 0.01; [36]). In vivo expression studies were also conducted to determine the degree by which this formulation enhanced delivery. Mice received a single injection of plasmid encoding reporter transgenes luciferase (cytoplasmic protein) or erythropoietin (secreted protein). DNA/CRL1005/BAK-formulations compared to DNA alone provided a 3-fold increase in luciferase in muscles and a 5-fold increase in erythropoietin in muscles as well as serum [36]. Collectively these results support the hypothesis that increased immunogenicity can be attributed at least in part to enhanced expression of plasmid with this delivery system.The first of two good laboratory practice (GLP)-compliant nonclinical studies conducted with the vaccine ASP0113 was a repeat dose safety-toxicology study in rabbits that received 4 IM injections at 2-week intervals of 0 mg, 0.5 mg, and 5 mg doses of product. This study was designed to evaluate clinical signs including injection site reactions (Draize scores), body weights, food consumption, ophthalmological exams, and mortality, as well as clinical pathology including hematology, coagulation, and clinical chemistry panels and anti-nuclear antibodies. No product-related changes in these evaluations were found with the exception of increased but reversible creatinine phosphokinase levels after the last injection and minimal to moderate inflammation in muscle and skin encompassing the injection site, which largely resolved during the recovery period. These are expected observations for IM injected vaccines.The second of two GLP-compliant nonclinical studies conducted with ASP0113 was a single dose biodistribution/integration study in mice. Various tissue samples were collected at days 2, 14, 28, and 61 after injection of 100 μg of bivalent product to assess the tissue distribution and clearance kinetics of plasmid over time and compared these results with a control plasmid (formulated in PBS only) previously tested in GLP and clinical studies. Plasmid copies cleared rapidly after injection such that by day 28 the highest copies were found in injection site muscle and with detectable levels only in spleen and bone marrow. There were no significant differences in the plasmid-copy clearance between the two test articles over 61 days. An integration study was conducted and the results supported the conclusion that the risk of plasmid integration was negligible. Pharmacodynamics and pharmacokinetic studies were not required.Prior to the production of ASP0113, Vical had acquired extensive chemistry, manufacturing, and control (CMC) experience in developing plasmid DNA-based products for clinical testing. For clinical testing of ASP0113, Vical produced all bulk drug substance (DS) lots of each of the CMV plasmids according to current good manufacturing practices (cGMP). Each plasmid was produced by bacterial fermentation using E. coli strain DH10B under kanamycin selection. A master cell bank was created for each plasmid with specifications for purity, potency, and identity. A manufacturer’s working cell bank was derived from the master cell bank and used to inoculate an overnight culture which in turn was used to inoculate a fermentor (initially 100 L and later 500 L scale). Bacterial cell paste was collected following fermentation and was processed by alkaline lysis followed by filtration and precipitation procedures. Following downstream chromatography steps, the final purified bulk DS for each plasmid was adjusted to the final DNA concentration and frozen. In-process QC testing was conducted throughout all of the above steps. Aliquots from the final bulk DS lots were placed on a 36-month stability program and other samples were submitted for release testing that incorporates measures of purity, strength/potency, and identity. Vical’s DS release specifications, including tests for the host E. coli macromolecules, genomic DNA, RNA, and protein (all <1%), endotoxin <40 EU/mg DNA, and percent of supercoiled plasmid DNA at >80% follow the acceptance criteria recommended in the 2007 FDA Guidance for Industry document for plasmid DNA vaccines.The final drug product (DP) was manufactured by mixing each monovalent bulk DS at a 1:1 mass ratio to create a bivalent bulk DS that was then formulated with CRL1005 and BAK under aseptic conditions, resulting in the bulk DP. A challenging aspect of the formulation development was the requirement to maintain the temperature of the CRL1005-containing solution below the cloud point during filtration. During early development the bulk DP was sterile filtered below the cloud point. However, significant product losses were incurred, leading to optimization of the process prior to phase 3. Sterile filtration of a premix containing BAK and CRL1005 below the cloud point was implemented, followed by introduction of sterile-filtered bulk DS to form the bulk DP.Bulk DP was aseptically filled into glass vials and stored frozen. Samples of DP were tested for release as well as placed on a 36-month stability program; both DS and DP achieved at least 36 months of stability. The current DP release tests used for purity include appearance, endotoxin, sterility, percent supercoiled plasmid DNA, particle size, general safety test, and particulate matter. The release tests used for identity include pH, total DNA size, and Western blots for the products of each plasmid. The majority of the DS and the DP release assays are standard, i.e., applicable regardless of plasmid. However, one plasmid-specific DP release assay, a relative potency assay, merits discussion for its importance as a robust assay for release and stability testing.A TaqMan®-based reverse-transcriptase, polymerase chain reaction (RT-PCR) assay was developed which measures the mRNA expression of both gB and pp65 following transfection of the bivalent product, as an indication of the product’s in vitro potency [37]. Messenger RNA expression from plasmid DNA is the most immediate biological activity measurable after DNA vaccine delivery. This highly-specific assay was designed to detect expression of each of the plasmid-derived transgenes in comparison to a reference standard to establish the percent relative potency (%RP) of any test sample. The %RP assay was incorporated as a DP release test for phase 2 clinical trial material (CTM) lots as well as for measuring stability of each lot and lot-to-lot consistency. Furthermore, a correlation was established between in vitro potency and in vivo gB antibody responses in mice with samples subjected to forced degradation by heat; the %RP assay proved to be an excellent indicator of in vivo potency [36].By late 2003, Vical had completed all of the appropriate activities and compiled the requisite documentation for filing an investigational new drug application (IND) with the U.S. FDA. IND allowance occurred in early 2004 followed shortly by initiation of a phase 1 trial.The first-in-human clinical testing of ASP0113 (denoted then as VCL-CB01) was a multicenter open label phase 1 trial in 44 normal, healthy adults (22 each CMV+ and CMV−) age 18–45 years to evaluate the safety and immunogenicity of the bivalent vaccine [38]. Subjects received IM injections of 1-mg or 5-mg DNA doses on a 0-, 2-, and 8-week schedule or 5-mg DNA dose on an accelerated 0-, 3-, 7-, and 28-day schedule.In that setting, the vaccine was well tolerated, with no serious adverse events (SAEs) and no discontinuations due to vaccine-related adverse events (AEs). The most frequent AEs were injection site pain, myalgia, headache, and malaise and were of mild to moderate severity. Systemic reactions included mild to moderate malaise and myalgia. Local reactions included injection site pain, induration, swelling, and erythema [38].T-cell responses through Week 16 were assessed in an ex vivo ELISPOT assay using pp65 or gB overlapping peptides [38]. Antibody responses to gB were assessed in an indirect binding ELISA using full-length recombinant gB protein isolated from transfected CHO cells. T-cell and/or antibody responses to vaccine encoded antigens were elicited in 37.5% and 50% of the CMV- subjects in the 1-mg and 5-mg dose groups, respectively, suggesting a possible dose response; however, the number of subjects in each group was not powered to detect a difference in the response rates for the different doses and vaccination schedules. T-cell responses to pp65 were detected in 12.5% to 37.5% of CMV+ subjects, but no CMV+ subject in any group had greater than a 2-fold increase in gB antibody levels, suggesting that the antibody responses were not boosted by the vaccine.To evaluate the duration of the T-cell responses in CMV- subjects, the ex vivo ELISPOT assay was performed with Week 32 specimens from CMV− subjects on the 0-, 2-, 8-week injection schedule. Neither of the 2 subjects in the 1-mg dose group who were responders in the assay at earlier time points had detectable responses at Week 32. Conversely, in the 5-mg dose group, 5 subjects (62.5%) had detectable responses, including all 3 of the subjects who had T-cell responses by Week 16, and an additional 2 subjects who did not have detectable responses by Week 16 (p = 0.0256; Fisher’s exact test, for responses in the 1-mg and 5-mg dose groups at Week 32). These results suggested a dose response for CMV− subjects, even though a dose response was not established in the initial evaluation of the T-cell responses through Week 16.T-cell responses at Week 32 in CMV− subjects on the 0-, 2-, 8-week injection schedule and at Week 16 for those on the 0-, 3-, 7-, 28-day schedule were also evaluated by the cultured ELISPOT assay, which demonstrates the ability of vaccine-primed memory T cells to proliferate and produce IFN-γ on exposure to vaccine encoded antigen. In the assay, peripheral blood mononuclear cells (PBMC) were cultured with pp65 and gB peptides and recombinant human IL-2 for 10 days prior to evaluation in the IFN-γ ELISPOT assay. Altogether, vaccine-primed memory T-cells were detected in 15 of 22 CMV− subjects (68%) [38]. Moreover, memory T-cell responses were detected in 5 CMV− subjects who failed to demonstrate T-cell responses in the ex vivo ELISPOT assay at any time point indicating that priming of memory T-cell responses had occurred even in the absence of a detectable effector T-cell response.The second clinical trial of ASP0113 was a multicenter, randomized, double-blind, placebo-controlled phase 2 trial in CMV+, allogeneic HCT recipients aged 18–65 years with various forms of lymphoma and leukemia [39]. According to the original trial protocol 80 donor-recipient pairs undergoing HCT were planned for enrollment but donor vaccination proved logistically impractical and the protocol was amended to enroll a recipient-only (unpaired) arm. Subjects were randomized 1:1 to receive 5-mg doses of ASP0113 or PBS placebo prior to ablative conditioning and at approximately 1, 3, and 6 months after transplantation and were stratified by clinical site, donor-recipient human leukocyte antigen match, and by donor CMV serostatus. All subjects who received at least one dose were included in the safety analysis and a total of 74 subjects were included in the per protocol (PP) population analysis. The two groups were well-balanced for demographics, conditioning regimens, and donor relatedness and serostatus.All HCT recipients developed at least 1 treatment-emergent adverse event and the majority (>70%) of subjects in both groups developed an SAE during the study, which was not statistically different between groups. The incidence of local reactogenicity was higher in the ASP0113 group than in the placebo group (22.9% and 10.9%, respectively), primarily due to injection site pain. One SAE in the vaccine group, an allergic reaction which resolved after treatment, was deemed possibly related by the Sponsor. Overall, the safety profiles of both groups were similar and the vaccine was considered well tolerated in this trial [39].The primary efficacy endpoint, the rate of initiation of CMV-specific antiviral therapy was lower in the PP population for the vaccine group (19/40, 47.5%) compared to the placebo group (21/34, 61.8%) but the difference did not achieve statistical significance (p = 0.145). Considerable variability existed among the 16 trial sites in the types of local laboratory assays used for detecting CMV and each institute had different treatment algorithms for initiating preemptive antiviral therapy. In contrast, measurement of plasma levels of CMV DNA (also referred to as CMV viremia) using a quantitative PCR assay performed at a single, central laboratory revealed statistically significant differences between the two groups. Compared to the placebo group, the vaccine group had significantly lower occurrence of detectable CMV viremia (p = 0.008), fewer CMV viremic episodes (p = 0.017), longer time to initial viremia (p = 0.003), and shorter duration of viremia when normalized to days on study (p = 0.042).Several secondary endpoints representative of the clinical manifestations attributed directly or indirectly to CMV infection were numerically lower in the vaccine group compared to the placebo group but did not achieve significance; however, this study was not powered to detect differences in these endpoints. These endpoints included CMV EOD (manifest as pneumonia or gastroenteritis), overall mortality, grade 3–4 acute GVHD, and severe chronic GVHD; a post-hoc analysis of a composite of these endpoints revealed a difference in favor of vaccine which did not achieve statistical significance (p = 0.10). Based on the phase 2 findings and as described further in Section 5.3, overall mortality was selected for inclusion into the primary endpoint for a pivotal phase 3 trial, with the prospect for also including several other endpoints as a composite.T-cell responses to pp65 and gB for HCT recipients in the ASP0113 and placebo groups were assessed in the direct ex vivo IFN-γ ELISPOT assay and levels of gB-specific antibody were assessed in the indirect binding ELISA [39]. T-cell responses and antibody levels were similar in the ASP0113 and placebo recipients prior to transplant. However, donor-derived T-cell responses to pp65 or gB were not evaluated, so an impact of imbalances in these T-cell responses cannot be ruled out.T-cell responses to pp65 were numerically higher in the ASP0113 group relative to the placebo group at all of the time points evaluated after transplant. Despite the variability in the magnitude of the responses, there was a trend towards a significant difference in the responses at Day 56, and statistical significance was reached by Day 84 (p = 0.075 and 0.036, respectively; Wilcoxon rank-sum test). In a post-hoc analysis of the T-cell responses to pp65, an ordinal logistic regression model was used because the T-cell responses had a U-shaped distribution in both groups [40]. Three categories, defined as <750, 750–2,999, and ≥3,000 SFU/106 PBMC (with boundaries at ½ and 2 times the mean of approximately 1,500 SFU/106 PBMC in normal CMV+ individuals), were used for analysis of the pp65 responses. The treatment effect p-value of 0.022 for Day 56 through Day 365, suggests that recipients of ASP0113 were significantly more likely to have high levels of pp65 T cells compared with the placebo group. This finding was reflected in the mean values of pp65-specific T cells which were more than twice the mean for normal CMV+ adults (1500 SFU/106 PBMC). The mean T-cell responses to gB were also numerically higher in the ASP0113 group than in the placebo group at all time points after Day 84; however, the differences at any time point were not significant.The pp65 T-cell responses in the ASP0113 group could have been enhanced by ablative conditioning prior to transplant, which may have created “immunological space” for the rapid expansion of CMV antigen-specific T-cells upon exposure to vaccine encoded antigens. Because expansion could also occur in either group as a result of exposure to CMV during viral reactivation, in a second post-hoc analysis using the ordinal logistic regression model, the pp65 T-cell responses were censored after the occurrence of viremia to eliminate the influence of exposure to CMV. The pp65 T-cell responses in the ASP0113 group were still higher than those of the placebo group with a treatment effect of p = 0.005, suggesting that the comparatively higher responses in the ASP0113 group were independent of CMV viremia [40] and likely due to vaccination with ASP0113. Furthermore, for those subjects who did not have CMV viremia by Day 56, the pp65 T-cell responses at Day 56 were significantly higher in the ASP0113 group than in the placebo group (p = 0.030; Wilcoxon rank-sum test), indicating that the divergence of the responses in the two groups begins early in the vaccination regimen, providing protection from CMV reactivation during the time period when most CMV viremia occurs.The geometric mean gB antibody levels were not significantly higher in the ASP0113 group than in the placebo group until after the fourth injection, when the gB antibody levels showed a trend towards significance at Day 210 and reached significance by Day 365 (p = 0.064 and 0.009, respectively; Wilcoxon rank-sum test). The late divergence of the gB antibody levels in the two groups compared with the early divergence of the T-cell responses may reflect differences in both the persistence of antibodies and T cells after ablation and the kinetics of their recoveries after transplantation.The design of a global phase 3 trial of ASP0113, sponsored by Astellas Pharma Global Development, Inc. with Vical as collaborators, has been recently registered at www.clinicaltrials.gov. The official title of this protocol, 0113-CL-1004, is “A Randomized, Double-Blind, Placebo-Controlled, Phase 3 Trial to Evaluate the Protective Efficacy and Safety of a Therapeutic Vaccine, ASP0113, in Cytomegalovirus (CMV)-Seropositive Recipients Undergoing Allogeneic Hematopoietic Cell Transplant (HCT)”. Subjects will be randomized 1:1 to receive ASP0113 or PBS placebo. The primary efficacy endpoint of this trial is overall mortality at one year post-transplantation. The safety of ASP0113 in HCT recipients will also be monitored. This trial is anticipated to enroll 500 subjects and is analytically divided into two parts, with Part 1 enrolling 100 subjects and Part 2 enrolling 400 subjects. Part 1 will be used to evaluate the adequacy of the primary endpoint and, if necessary, to modify the primary endpoint to be specified for Part 2. This trial will enroll subjects at approximately 90 HCT centers in North America, Europe, Asia, and Australia and is anticipated to be completed in September of 2016.During production of DP for testing in phase 1 and phase 2 trials, improvements in CMC-related processes and refinements in analytical testing methods and assay specifications were identified and implemented prior to phase 3 testing. Furthermore, the DP lot sizes sufficient for phase 1 and phase 2 CTMs had to be increased by scaling up the manufacturing procedures to meet the CTM demands for phase 3. Process improvements enhanced manufacturing efficiencies and lot-to-lot consistency by streamlining/simplifying procedures, upgrading equipment, and enriching product yield. Some assays underwent refinements that resulted in increased sensitivity, precision, ease of use, and reagent stability and availability. Each plasmid-specific DS and DP release assay was validated in preparation for use in phase 3. All of the CMC-related changes were submitted to the FDA under the current IND in 2012.The timeline in Figure 1 integrates all of the key activities described above and illustrates both the typical and unanticipated factors that can impact product development timelines. A reasonably representative time frame ensued from product concept to completion of phase 1 testing and accordingly the indication of the favorable safety profile and demonstration of immunogenicity in humans [38,41], a vital first stage for a vaccine program. In contrast, the phase 2 trial in HCT recipients took an extended period of time to enroll, largely due to the novelty of the approach and the inherently-complex nature of conducting a vaccine trial in subjects with such underlying conditions and treatment-associated morbidity; furthermore, as described in Section 2.2, the original premise was to vaccinate donors but due to the limited time donors were available prior to transplant, this proved impractical and this arm was discontinued while leaving the recipient-only arm open. In a strategy to mitigate some of the risks inherent in product development, most optimization and assay validation activities were delayed until late into the phase 2 trial. Ultimate agreement with the FDA, in addition to discussions with other global regulatory agencies in Europe and Asia, required thorough and frequent communications to define acceptable endpoints for licensure. Vical licensed this program to Astellas during this period, with joint participation from both groups for regulatory discussions on phase 3 trial endpoints. While some aspects of this product development history could have been shortened, small biotechnology companies such as Vical typically encounter resource constraints and risk management plays an important role in the timing of activities. Fortunately, the partnership for continued development of ASP0113 provides a tremendous opportunity for advancing this first-in-class therapeutic CMV vaccine towards product licensure.The proof of concept findings for the bivalent CMV vaccine in HCT recipients provides support for testing this product in SOT recipients. A randomized, double-blind, placebo-controlled phase 2 trial is planned with ASP0113 in CMV− recipients receiving a kidney transplant from either a living donor or a deceased donor who is CMV+, the highest risk group among SOT recipients for experiencing CMV disease.Finally, a CMV prophylactic vaccine that can prevent congenital CMV infection would have a tremendous public health impact. However, the development of a vaccine for this indication will require considerable resources due to a longer clinical development pathway and potentially high numbers of subjects for clinical trials. Furthermore, the clinical endpoint required for licensure of such a vaccine has not been finalized [42] and has only recently received discussion between government, academia, and manufacturers [43]. Vical initiated development of a prophylactic congenital CMV vaccine (CyMVectin™) by developing a different product formulation, a cationic lipid adjuvant called Vaxfectin®, combined with gB and pp65-expressing plasmids and recently published nonclinical immunogenicity and GLP safety studies [44]. Given the enormous scope of developing such a vaccine, further development of CyMVectin™ would ideally proceed in partnership with a large pharma company. In parallel, additional research is ongoing to identify one or more additional plasmids expressing other CMV genes (Section 1.2) for potential inclusion in this vaccine. The encouraging proof of concept results of ASP0113 in the difficult HCT recipient population provides optimism for developing CMV vaccines for additional unmet needs. All authors are employees of either Vical Incorporated or Astellas Pharma Global Development and may own company stock or stock options.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.DNA-vaccines are promising tools with great potential for combating infectious disease. Non-living/replicating DNA vaccines have several advantages over living viral delivery vectors, such as lower production costs, increased stability, a higher overall safety profile, and recent evidence indicates that they can provide humans with protective immunity to viral infection [1]. However, living viral vectors used in DNA vaccine settings (such as Adenovirus) can still elicit stronger immune responses in humans than naked DNA. Yet in the case of adenovirus, evidence suggests that they may not promote the desired immune responses to the recombinant antigen. As results from clinical trials show, the use of a viral vector can, possibly as a consequence of the anti-vector immunity, potentially even enhance the risk of infection with certain pathogens [2]. These observations emphasize the critical need to continue research on methods for adjuvanting minimal, non-living/replicating DNA vaccines.There are many approaches to improving the efficacy of plasmid DNA vaccines such as choice of delivery method, modifications of antigen location/stability/presentation, and the use of immunopotentiators [3]. Here, we investigate a formulation-compatible immunopotentiating adjuvant, with the potential to activate innate and adaptive immune responses through Toll-like Receptor 5 (TLR5) and/or possibly Nod-like Receptor (NLR) family members NLRC4 and Naip5 [4]. This approach employs plasmid DNA encoding a secreted form of flagellin (FliC) from Salmonella typhimurium as an adjuvant in DNA vaccinations. This adjuvant allows mammalian cells to create an environment of sterile-inflammation, thus mimicking natural infection in a safe manner and promoting adaptive immune responses to co-delivered DNA-encoded antigens [5]. This approach is unique in that it uses a plasmid-encoded agonist of innate immune receptors to activate a large variety of molecules capable of promoting adaptive immunity, unlike many other approaches which use single cytokines or chemokines [3]. A major benefit of this system is that it works without physically linking the antigen to flagellin. This ensures that the antigen is properly folded and processed and constitutes a major practical advantage as the system is flexible and can be applied with ease to various antigens without the need for time-consuming development of fusion-constructs. Recombinant flagellin produced in bacteria is currently being used by many as an experimental adjuvant to promote humoral and cellular immunity against microbial pathogens [6,7,8]. However, the use of flagellin in protein-form presents formulation and stability issues with non-living/replicating DNA vaccines such as plasmids.In previous work, we vaccinated mice epidermally, using a gene-gun, with a transmembrane-anchored form of flagellin (pFliC-Tm) and secreted ovalbumin (pOVA). We observed significant increases in antigen-specific serum IgG levels compared to pOVA alone as well as strong antigen-specific CD4+/8+ cellular immune responses [5]. Importantly, we also showed that the pFliC-Tm adjuvant delivered with a DNA-encoded nucleoprotein gene from Influenza A resulted in a strong antigen-specific CD4+/8+ cellular immune response which correlated with protection from lethal virus infection [5]. This work demonstrated that pFliC-Tm acts as an adjuvant when delivered dermally however it is not clear whether this is the optimal route for eliciting the broadest or strongest immune responses. Additionally, not all DNA vaccination approaches are applied dermally therefore further studies of adjuvant effects induced by various delivery routes are warranted.The HIV-1 pandemic has been estimated to have according with WHO/UNAIDS reports been spread globally and infected individuals exist in all countries in the world. So far, only a few experimental vaccine studies have shown promising and protective results in clinical trials. Thus there is a continued need to find more efficient vaccination strategies to provide protective immunity against the infection. Since, the main route of infection is via sexual transmission and via mucosal transmission such as breast-feeding, a vaccine that can provide mucosal immunity would be desirable. However, mucosal vaccines against infectious disease are few, and only polio, influenza, rotavirus, S. typhi, and V. cholerae have commercially available vaccines [9]. There are numerous adjuvants now found to promote mucosal immune responses, some of them lipid based, however none of these are in themselves plasmid-DNA based technologies [10].Here, we studied if a plasmid vector expressing secreted FliC (pFliC(-gly)) activates TLR5- and NLRC4/Naip5-specific innate immune responses and acts as an adjuvant to plasmid-encoded antigen by three different routes representative of dermal, systemic, and mucosal locations. Additionally, we performed intranasal mucosal immunizations using plasmid encoding the clinically relevant HIV-1 antigen gp160 followed by recombinant HIV-1 protein booster. The ability of pFliC adjuvant to enhance HIV-1 gp160 envelope immune responses at mucosal and systemic compartments was also investigated.pOVA and pFliC-Tm(-gly) have been described previously [5]. pFliC-Tm(-gly) was subjected to site-directed mutagenesis to insert two translational stop-codons after AA 459 of FliC(-gly) (AA numbering is based on GenBank Accession #D13689). Changes of all constructs were confirmed by DNA sequencing. Variants of FliC(-gly) were created for testing the molecular mechanism of FliC(-gly) detection in vitro. First, to make a cytoplasmically-expressed FliC(-gly) the secretion leader sequence of FliC(-gly) was removed from the pFliC(-gly) vector by PCR amplification using the primers 5'-ccaggttccAATCTTATGTatccatatgatgttccagattatgct-3' and 5'-GCAGCCGCGGATCCCGGGGTACCTATCGCAGTAAAGAGAGGACGTTTTGCGG-3' with the pFliC(-gly) template encoding a starting methionine, HA-tag, and complete FliC(-gly) open-reading frame (ORF) without the secretion leader sequence. Full-length products were digested with Hind III/BamH I and ligated into pcDNA 3.1/Zeo(+) prepared with HindIII/BamHI to make pcFliC(-gly). To remove the COOH-terminal 34 amino-acids of FliC a section of the FliC(-gly) gene encoding AA282 to 461 residing on a BsrG I/Xho I fragment (5'-atgtacaagttgcaaatgctgatttgacagaggctaaagccgcattgacagcagcaggtgttaccggcacagcatctgttgttaagatgtcttatactgataataacggtaaaactattgatggtggtttagcagttaaggtaggcgatgattactattctgcaactcaaaataaagatggttccataagtattgatactacgaaatacactgcagatgacggtacatccaaaactgcactaaacaaactgggtggcgccgacggcaaaaccgaagttgtttctattggtggtaaaacttacgctgcaagtaaagccgaaggtcacaactttaaagcacagcctgatctggcggaagcggctgctacaaccaccgaaaacccgctgcagaaaattgatgctgctttggcacaggttgacacgttacgttctgacctgggtgcggtacagaaccgtttcaactccgctattaccaacctgggcaacaccgtaaacaacctgaattctgcccgtagccgtatcgaagattccgactacgcgacctagtagctcgaga-3') was synthesized (and used to replace the 3' end of both the pFliC(-gly) and pcFliC(-gly) constructs after digestion by BsrG I/Xho I to create pFliC(-gly)Δ34 and pcFliC(-gly)Δ34.pFliC(-gly), pcFliC(-gly), pFliC(-gly)Δ34, or pcFliC(-gly)Δ34 were transiently transfected into 293T cells and 2 days later cell lysates were prepared as described [5], total protein concentration was determined by BCA assay (Pierce Thermo Scientific, Walthman, MA, USA), normalized, and subjected to SDS-PAGE (NuPAGE, Invitrogen Life Technologies, Stockholm, Sweden) followed by western blot analysis (anti-HA tag, Covance Research Products, Brussels, Belgium).The ORF of FliC(-gly) gene and three variants were excised using MfeI and XhoI and inserted into the retroviral expression vector pMSCV-IRES-GFP/neo digested with EcoR I and Xho I. Constructs were transfected into 293T cells and proteins from cell lysates and supernatants were analyzed for the presence and correct molecular weight of FliC(-gly) and variants by Western blotting as described above (data not shown).Plasmid vectors expressing gp160 and p24gag from HIV-1 clade B strain Ba-L have been described previously [11].All plasmid DNAs were prepared using a Qiagen EndoFree Plasmid Maxi Kit (Qiagen, Hamburg, Germany). Macrophage stimulations were performed as follows. Alveolar macrophages were harvested by BAL from C57BL6/N or TLR5−/− mice backcrossed >10 generations onto C57BL6. Cells were seeded 50,000 cells/well in 96 well plates (Costar) in 50 µL RPMI 1640, 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Pierce Thermo Scientific, Walthman, MA, USA) and allowed to settle for 1 h. 50 µL of two day culture supernatants from 293T cells, transiently transfected with 0.8 µg of pFliC(-gly), pFliC(-gly)Δ34, empty vector, recombinant FliC at 100 ng/mL (a gift from A. Gewirtz, Emory, GA, USA) or ultra-pure LPS at 100 ng/mL (InVivogen, San Diego, CA, USA.), was placed on cells and incubated at 37 °C at 10% CO2 for 4 h. Cell supernatants were harvested and subjected to standard ELISA to detect secreted mouse TNFα (BioLegend, San Diego, CA, USA).Retroviral Lethality Screen was performed as follows. pMSCV-IRES-GFP/neo alone or containing FliC(-gly), cFliC(-gly), FliC(-gly)Δ34, or cFliC(-gly)Δ34 ORF were packaged in Phoenix amphotropic virus packaging cells. After transfection media was replaced at day one and viral supernatants were harvested at days two and three. Pooled supernatants were 0.45 µm filtered, concentrated by centrifugation at 2,300 × g for 18 h at +4 °C, and frozen at −80 °C. 2 × 105 J2-virus immortalized mouse bone-marrow derived macrophage cells (BcgR) or 293T cells were pre-treated with BX795 (5 µM) for 30 min at 37 °C to improve transduction efficiency [12] followed by 200 µL virus mixed with polybrene 8 µg/mL (Sigma, St. Louis, MO, USA) and centrifuged for 45 min at 27 °C. 4 days after triplicate transductions cells were subjected to analysis for GFP by flow cytometry using a 4-laser LSRII-Fortessa with standard filter sets (BD Bioscience, Stockholm, Sweden). >40,000 non-debris singlets were analyzed in every sample. FACS data was analyzed using FlowJo v9.2 (Tree Star, Ashland, OR, USA).For experiments using OVA antigen female C57BL6/J-crl sub-strain mice (8–12 weeks at priming) from Charles River Laboratories (Sulzfeld, Germany) were used and housed under standard specific pathogen-free conditions (Swedish Institute for Infectious Disease Control). All procedures were reviewed, approved, and performed under both institutional and national guidelines. Plasmid DNA was prepared using a Qiagen EndoFree Plasmid Maxi Kit (Qiagen, Hamburg, Germany) as described by the manufacturer without exception. Vaccinations were done in the animal facility at approximately 24 °C and 60% relative humidity. Mice receiving intramuscular vaccinations were injected with DNA resuspended in PBS in a total volume of 50 μL in one quadricep. Standard ballistic dermal vaccinations were performed as described [5]. For intra-nasal vaccinations, plasmid DNA was resuspended in 0.1 M Tris-HCL, pH 8.0 and placed on ice and mixed with a 1:1 volume of 2% Eurocine cationic N3 lipid (called N3) to make a final volume of 1% N3 lipid adjuvant (see Table 1 for details). Preparation of N3 was carried out as described [13] and was gently stirred with DNA on ice until homogenous then brought to room temperature. Mice were briefly anesthetized with Isofluran, placed dorsal side up, and 4 μL/nostril of N3/DNA mixture was applied to each nostril using a standard laboratory pipette. Mice were gently supported in this position until the mouse revived and attempted to turn over.Vaccinated groups for ovalbumin (OVA) experiments.a g.g.=gene-gun, i.m.=intra-muscular, i.na.=intra-nasal.For experiments involving gp160/p24gag, eight to ten-week-old female BALB/c mice were purchased from Scanbur BK, Sollentuna, Sweden. Six groups (n = 35) were vaccinated with DNA-plasmids expressing gp160 and p24gag (promoter CMV-IE) as previously described [11] with or without adjuvant (Table 2). For plasmid-DNA priming, mice were given 10 µg/plasmid dose/mouse as 5 µL/nostril/mouse with N3 prepared as described above. HIV-1 recombinant protein-boost antigens were gp160 and p24gag (Protein Sciences Inc., Meriden, CT, USA), containing HIV-1 gp160 LAI and p24gag prepared with anionic L3B as described [14]. Mice were given 5 µL of vaccine in each nostril, corresponding to 1 µg recombinant protein antigen and 0%, or 2% of L3B adjuvant or 1% N3 with 5 µg pFliC(-gly) (see Table 2 for details). For delivery of intranasal vaccinations, the mice were treated as above. Groups studied for longetivity of immune responses were immunized three times at three weeks intervals then mice were sacrificed 4, 8, 12, 24, and 36 weeks after the last immunization for analysis. Mice studied for general immune reactivity were sacrificed 4 weeks after the final immunization.Vaccinated groups for gp160/p24gag experiments.a Plasmid DNA; b Recombinant protein.Anti-OVA humoral responses were performed as follows. Briefly, serum, fecal pellets (100 mg feces solublized in 1 mL PBS with protease inhibitors, Complete Mini, Roche, ) and vaginal washings (50 μL of PBS with protease inhibitors, as above) were subjected to anti-OVA ELISA as described [5]. Assessment of antigen-specific IgA in lungs was done as follows. Isolated lungs were rinsed in cold PBS then minced in PBS with protease inhibitors. Solids were removed by centrifugation and total IgA in washings were determined by ELISA, using a primary monoclonal goat-anti mouse IgA (Sigma 098K4823 clone ISO2-1KT, St. Louis, MO, USA) and secondary rabbit anti-goat IgG HRP (Dako, Stockholm, Sweden). IgA anti-OVA titers were determined by ELISA then normalized for total IgA content. Individual samples were tested in triplicate at a dilution of 1/10. Amino acids were numbered according to the Los Alamos Data Base on Retroviruses (peptide source: AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: from DAIDS, NIAID and J&J, San Diego, CA, USA) [15]. For IgG anti-gp160 measurements, individual mouse sera were diluted in ten-fold steps from a starting dilution of 1:100 in ELISA-buffer (2.5% dry milk and 0.05% Tween-20 (Sigma, St. Louis, MO, USA) in PBS to end-point. Goat-anti-mouse IgG (H+L)-HRP secondary conjugate (Bio-Rad, Hercules, CA, USA) was used, diluted 1:3,000 to detect IgG anti-gp160 immune complexes. Anti-gp160 IgA and IgG isotype subclasses were measured using a mouse monoclonal antibody isotyping reagent (Sigma, St. Louis, MO, USA) according to the manufacturer’s protocol with peroxidase-conjugated anti-Goat IgG (Sigma, St. Louis, MO, USA), diluted 1:2,000. For developing ELISA reactions, O-phenylenediaminedihydrochloride (OPD) (Sigma, St. Louis, MO, USA) was used. Based on earlier studies, an OD of 0.2 was set as the cut-off value for positive samples. Clade A, Uganda 29 (UG29) and C, Brazil (BR25) envelope antigens were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIAID, NIH, Bethesda, MD, USA). Mucosal wash IgA analyses were performed as previously described [16,17,18]. Briefly, IgA was isolated from secretions collected by nasal washing using Kaptive/IgA/IgE reagents (Biotech IgG, Copenhagen, Denmark) as recommended by the manufacturer. IgA quantities were determined using an in-house murine IgA capture ELISA, and commercial murine IgA (1 mg/mL, Sigma, St. Louis, MO, USA) was used to prepare a standard curve. The purified IgA and the standard IgA were diluted in ten-fold serial dilutions. From each dilution, 100 µL was added to each well of a 96-microwell plate pre-coated with rabbit anti-murine IgA (Dakopatts AB, Sollentuna, Sweden). Goat-anti-mouse IgA-HRP secondary conjugate (SouthernBiotech, Birmingham, AL, USA), diluted by 1:3,000, was used to detect IgA anti-gp160. The total amounts of IgA in nasal samples were determined by comparing the OD values of the test samples with the IgA standard and final anti-gp160 values were normalized to total IgA values.T-cell responses to OVA was performed as described [5]. Briefly, spleens were isolated, single-cell suspensions were Ficoll-purified, washed twice with PBS and used in IFNγ ELISPOT analysis according to the manufacturer guidelines (Mabtech, Nacka, Sweden). Ag restimulation was performed using either the H-2Kb binding OVA peptide SIINFEKL (257-264) at 1 μM final concentration or the I-Ab binding OVA peptide ISQAVHAAHAEINEAGR (323-339) at 1 μM final concentration (GenScript, Piscataway, NJ, USA).). Cell reactivity was confirmed by incubation with ConA. Spot-forming cells were quantified after 24 h incubation and counted by AID ELISPOT reader (AutoImmun Diagnostika, Straßberg, Germany).T cell immune responses to gp160 were measured using a cell-in-well murine cytokine capture-ELISA assay as described previously [11]. Briefly, 96-well ELISA plates were coated with capture anti-IFNγ (AN18) or anti-IL-5 (TRFK4) according to the manufacturer’s protocol (Mabtech, Nacka, Sweden) overnight at 4 °C. Following well washing and blocking according to the manufacturer’s protocol 2.5 × 105 ficoll-purified splenocytes from individual mice were added to each well, either with or without recombinant HIV-1 gp160, p24gag (Protein Sciences Inc., Meriden, CT, USA), control antigen (Sf9 cell lysate), the positive control Concanavalin A (2.5 µg/mL, Sigma, St. Louis, MO, USA) or RPMI 1640-medium alone. Plates were incubated at 37 °C, 5% CO2 for 5–6 days. Cells were then removed, plates were washed with PBS, and biotinylated detection antibodies were added, washed, followed by streptavidin-ALP (Mabtech, Nacka, Sweden). The plates were developed with substrate solution (Mabtech, Nacka, Sweden) for 5–10 min until spots became visible, and the color reaction was stopped by 2.5 M H2SO4. Plates were then read in an ELISA reader (BioRad, Hercules, CA, USA) at 405 nm.T-cell proliferation to gp160 was performed as described previously [18] using 1 µg/mL of rgp160 or rGag p24 as specific antigens.Nasal washings were performed trice with 25 µL PBS/nostril on Isofluran sedated mice kept in supine position. Collected washings were frozen until analyzed. Nasal washings were tested individually for the cytokines IL-6, IFNα, and IFNγ according to the manufacturer’s protocols (R&D Systems, Minneapolis, MN, USA).The HIV-1 neutralization assay was performed as described previously [11]. The viral isolates used for the neutralization were the subtype B laboratory strains IIIB LAI (vaccine homologus) and the primary subtype B isolate 6,794. Briefly, the sera from mice were pooled group wise and inactivated at 56 °C for 1 h to prevent complement-mediated neutralization. Sera were diluted in RPMI 1640 medium (Invitrogen Life Technologies, Stockholm, Sweden) in 96-well tissue culture plates (Nunc microwell plates, Nunc, Pierce Thermo Scientific, Walthman, MA, USA). Dilutions were mixed with virus and incubated at 37 °C for 1 h followed by the addition of 1 × 105 human PBMCs (activated by phytohemagglutinine and rIL-2; PeproTech, Rocky Hill, NJ, USA) or Jurkat T cells. The cultures were incubated at 37 °C in 5% CO2 over night, after which the cells were washed twice with RPMI 1640. After 6 days of culture, the presence of HIV-1 p24 antigen in the culture medium was measured by ELISA [19]. The background in the p24 ELISA was determined for each plate and subtracted from all wells before the percentage neutralization was determined as [1-(mean p24 OD in the presence of test serum/mean p24 OD in the absence of test serum)] × 100. Ethical permission for use of huPBMCs was approved by the ethical committee at Linköping University Hospital.Statistical analysis was performed using GraphPad Prism 5 (La Jolla, CA, USA). Comparisons between groups with the HIV-1 antigens were performed by using the non-parametric Mann-Whitney U test with Bonferroni correction, p < 0.05 was considered significant. A secreted variant of flagellin with reduced glycosylation (called pFliC(-gly)), based on the pFliC-Tm(-gly) plasmid [5], was constructed by removing the human transmembrane PDGFR domain from the ORF to eliminate potential immune responses to this region and to prepare a base vector for adjuvant use. Three additional variants of pFliC(-gly) were also constructed to test the ability of FliC(-gly) to activate the two known innate immune receptors capable of sensing flagellin TLR5 and NLRC4/Naip5. These four constructs are depicted in Figure 1a relative to the defined domains of Salmonella typhimurium FliC. To prepare pFliC(-gly) control variants capable of activating cytoplasmically expressed NLRC4/Naip5 we recloned the FliC(-gly) insert sans leader sequence (pcFliC(-gly)). We also prepared additional control versions of pFliC(-gly) and pcFliC(-gly) removing the COOH-34 amino-acids of FliC(-gly) shown to activate NLRC4/Naip5 [20]. These versions were designated pFliC(-gly)Δ34 and pcFliC(-gly)Δ34 respectively. All four vectors were capable of expressing proteins of predicted size with an apparent polypeptide of approximately 52 kDa for pFliC(-gly) and pcFliC(-gly) and approximately 48 kDa for pFliC(-gly)Δ34 and pcFliC(-gly)Δ34 (Figure 1b). To determine if secreted FliC(-gly) protein produced from pFliC vectors could activate TLR5 culture supernatants from pFliC(-gly), pFliC(-gly)Δ34 transfected 293 cells, or recombinant FliC protein were applied to alveolar macrophages from B6 or TLR5-deficient mice. Plasmid vectors produced full-length or Δ34 secreted FliC(-gly) able to activate B6 alveolar macrophages to produce TNFα but not macrophages from TLR5-deficient mice (Figure 1c). To determine if secreted FliC(-gly) has the potential to activate cytoplasmic NLRC4/Naip5 inflammasome responses we performed a retroviral lethality screen using the macrophage cell line BcgR which undergoes pyroptosis in the presence of the COOH-terminal tail of FliC [21]. This assay detects the ability of macrophages virally transduced with genes expressing GFP as well as various flagellin constructs to undergo pyroptotic cell death in response to whole flagellin dependent on the NLRC4/Naip5 35 amino-acid carboxy-terminal activating domain [20,21]. GFP positive BcgR cells are taken as evidence of a lack of NLRC4/Naip5 activation while GFP negative cells, relative to GFP positive identically transduced 293T control cells, are taken as evidence of NLRC4/Naip5 activation. FliC(-gly), cytoplasmic expressed FliC (cFLiC(-gly)), and a variant of each lacking the final 34 amino-acid COOH-tail (Δ34) (Figure 1a) were subcloned into the retroviral vector pMSCV-IRES-GFP which are designed to produce FliC(-gly) and variants as well as GFP upon transduction. Various FliC(-gly) constructs were Amphotropic packaged, and used to transduce BcgR or 293T cells. Using GFP as a reporter for FliC expression we observed that all versions of FliC(-gly) were expressed at nearly equal frequency in 293T cells indicating that all vectors were packaged with equal efficiency and could deliver GFP and FliC genes (Figure 1d). However, when identical vector preparations expressing FliC(-gly) were trandsuced into BcgR cells we observed GFP expression only with Δ34 versions (Figure 1d). These results demonstrate that secreted form of FliC(-gly) we use as an in vivo adjuvant has the ability to activate NLRC4/Naip5 pyroptotic cell death when expressed in a responsive cell type. FliC(-gly) produced from pFliC(-gly) stimulated TNFα production in a TLR5-dependent manner as well as inflammatory cell death (pyroptosis) dependent on a defined FliC region known to be a NLRC4/Naip5 agonist. These results suggest that TLR5 and NLRC4 expressed in vivo could be important factors in the adjuvant effects of pFliC(-gly) in immunized mice. It is interesting that FliC(-gly) destined for secretion has the capacity to activate the cytoplasmic flagellin detectors NLRC4/Naip5. We consider it likely that a portion of secreted FliC(-gly) undergoing translation is retro-translocated from the endoplasmic reticulum back into the cytoplasm where it may detected by NLRC4/Naip5 leading to the induction of pyroptosis.To compare the effectiveness of secreted flagellin (pFliC(-gly)) as a DNA adjuvant by various routes, DNA vaccinations were carried out using plasmid pOVA together with empty vector (pcDNA3.1/Zeo(+)) or with vector expressing pFliC(-gly). Empty vector control was used to include possible adjuvant effects contributed by sensing of B-DNA by innate immune receptors [22,23] but to exclude adjuvant effects contributed by secreted flagellin (Table 1). Vaccinated mice were primed once, boosted once and then sampled 9 and 10 days later (Figure 2a). The amount of total DNA given to the mice varied and was dependent on the limitations of the delivery route (Table 1). Mice were vaccinated by three different routes representative of dermal (gene-gun, g.g.), systemic (intramuscular, i.m.), and mucosal tissues (intra-nasal, i.na.). A constant sub-optimal amount of pOVA was used with each route (0.5 μg/g.g., 10 μg/i.m., 5 μg/i.na.) to allow the study of the adjuvant effects of flagellin.To determine if pFliC(-gly) promotes humoral immune response to co-delivered DNA-encoded antigen (pOVA), we studied antigen-specific antibody responses in dermal, systemic, and mucosal compartments. When anti-OVA antibody responses were examined in the sera of vaccinated mice we observed that the pFliC(-gly) adjuvant increased the antigen-specific total IgG antibodies in the sera of mice vaccinated with pOVA by all three routes (Figure 2b). These responses were dependent on the dose of pFliC(-gly) used as mice given 0.1 or 0.2 μg of pFliC(-gly) by g.g. or mice given 2 or 5 μg of pFliC(-gly) by i.m. did not exhibit any increases in anti-OVA antibody responses (data not shown; Table 1, Groups 2, 3 and 6, 7 respectively). Significant increases were seen when mice were vaccinated by g.g. or i.na., however, we also observed a reproducible trend of pFliC(-gly) to promote increases in anti-OVA total IgG when mice were vaccinated i.m. To see if the adjuvant effects of pFliC(-gly) delivered by various routes affected skewing of anti-OVA IgG isotypes, we analyzed the titers of anti-OVA IgG1, IgG2b, and IgG2c in the sera of vaccinated mice. We observed increases in all three IgG isotypes when pFliC(-gly) adjuvant was used, regardless of the route of delivery (Figure 2c–e).Flagellin (FliC)(-gly) variants for in vivo and in vitro use activate innate immune responses. (a) Depiction of pFliC(-gly) and variants relative to FliC polypeptide and domains produced by S. typhimurium. Grey domains D0/1 indicate conserved regions important for activating innate immune responses. L and HA indicates a leader and HA-epitope tag domains respectively. Names of the four FliC(-gly) constructs used in this study are indicated to the right of the drawings; (b) Western blot analysis of cellular lysates from 293T cells transfected with the indicated constructs. Apparent molecular weights were determined by comparison to the standard depicted to the left of the blot. Signals were not detected from cells transfected with empty vector (data not shown); (c) Release of TNFα from B6 alveolar macrophages but not Toll-like Receptor 5 (TLR5) −/− alveolar macrophages after stimulation with FliC(-gly) and FliC(-gly)Δ34. Supernatant from 293T cells transfected with pFliC(-gly) and pFliC(-gly)Δ34 vectors was incubated with cells for 4 h followed by analysis of secreted TNFα by ELISA. Data are mean ± SEM of triplicate samples representative of two independent experiments; (d) Activation of pyroptotic cell death by retroviral transduction of BcgR macrophages with constructs expressing FliC(-gly) but not FliC(-gly)Δ34 as determined by GFP expression. Upper panels represent representative data from BcgR cells transduced with FliC(-gly), FliC(-gly)Δ34 and controls (as indicated) when comparing GFP and forward-scatter (FS) parameters. Quantitative data of the percentage of GFP positive BcgR cells from each construct after transduction. Lower panel represent representative data from 293T cells transduced in identical fashion. Data are mean ± SEM of GFP positive cells observed during three independent transduction experiments. * Differences of the response relative to the FliC(-gly) construct without Δ34 defined as p ≤ 0.05 calculated using a two-tailed unpaired Student t test.Vaccination schedule and serum antibody responses to OVA. (a) Immunization and sample isolation timeline; (b) Anti-OVA total IgG responses. Anti-OVA IgG1 (c), IgG2b (d), IgG2c (e) responses. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly). Results are representative of two independent experiments (n = 7–8 mice/group). The concentration of OVA-specific Abs are expressed as the reciprocal of the last dilution of samples giving an OD equal to, or higher than, the mean + 3 SDs (the determined cutoff value for the assay) of the values of serum samples from unimmunized mice. Absorbance values equal to or above the cutoff value were considered positive. The error bars represent 95% confidence intervals calculated from the geometric mean titers. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using a two-tailed unpaired Student t test.To assess whether DNA vaccination of mice with pOVA and empty vector or pFliC(-gly) by various routes could elicit antibody responses in mucosal compartments, we collected extracts from fecal pellets, vaginal washes, and extracts from lung homogenates. We observed no significant differences in total amounts of total IgG or IgA immunoglobulins isolated from g.g., i.m. or i.na.-immunized animals (data not shown). Fecal extract samples were assessed for the relative amount of anti-OVA total IgG and IgA. We were able to detect significant increases in fecal anti-OVA IgG and IgA in the groups of mice vaccinated intranasally with pOVA together with the highest amounts of pFliC(-gly), but not when the same plasmids were delivered by g.g. or i.m. (Figure 3a–b). Similarly, only animals receiving the highest doses of pFliC(-gly) and pOVA intranasally developed measurable anti-OVA IgA in the vaginal washes (Figure 3c) and lungs (Figure 3d).We find it interesting that pFliC(-gly) promotes antigen-specific IgG and IgA responses to in mucosal compartments after mucosal delivery but not when it is delivered systemically or dermally. Despite this specificity we observed antigen-specific IgG in the sera by all routes. Other studies using purified flagellin protein and mucosal cell populations however, has revealed that the small intestine lamina propria contains CD103+ dendritic cells which express TLR5 and respond directly to flagellin to promote T cell-independent class switching of naive B cells from IgM+IgD+ to IgA [24]. It may be that a similar phenomenon occurs in vivo when flagellin is present in the compartments of the nasal mucosa and upper airway. However, it is not known why flagellin acts to promote humoral immune responses by all routes explored, but does not elicit mucosal antibodies when delivered systemically or dermally. Differences in the numbers, types, or the immune-skewing potential of flagellin-responsive cells interacting with flagellin after i.m. or g.g. delivery could be responsible for these effects.Mucosal antibody responses to OVA. (a) Fecal anti-OVA IgG and (b) IgA responses; (c) Vaginal anti-OVA IgA responses. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly); (d) Lung anti-OVA IgA responses shown are from mice only vaccinated i.na. and immunizations given are shown below the axis. Results are representative of two independent experiments (n = 7–8 mice/group). The concentration of OVA-specific Abs in samples are expressed as OD equal to, or higher than, the mean OD of the values of samples from unimmunized mice. The error bars represent SEM calculated from the mean OD. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using a two-tailed unpaired Student t test.MHC class I-dependent responses were analyzed by stimulation of splenocytes from immunized mice with peptide representing the immunodominant OVA H2-Kb restricted epitope. We observed significant increases in the numbers of antigen-specific IFNγ-producing cells in mice receiving either g.g. or i.na. immunization with pOVA and pFliC(-gly) when compared to mice receiving pOVA together with empty vector (Figure 4a). We also observed a reproducible trend of pFliC(-gly) to promote antigen-specific increases in IFNγ-producing cells when mice were vaccinated i.m. (Figure 4a). These Class I cellular responses were dependent on the dose of pFliC(-gly) delivered as mice given 0.1 or 0.2 μg of pFliC(-gly) by g.g or mice given 2 or 5 μg of pFliC(-gly) i.m. did not exhibit detectable Class I-dependent responses (data not shown; Table 1, Groups 2, 3 and 6, 7 respectively). When Class II-dependent cellular immune responses were studied by stimulating splenocytes with the immunodominant I-Ab binding OVA peptide we observed significant increases in the numbers of antigen-specific IFNγ-producing cells in mice receiving pOVA intranasally together with the highest amounts of pFliC(-gly), but not with pOVA and empty vector (Figure 4b). We did not observe any OVA-specific class II-restricted responses after i.m or g.g. immunization (Figure 4b).Class I- and Class II-dependent T cell responses to OVA. IFNγ ELISPOT analysis of splenic T cell responses to (a) Class-I and (b) Class-II MHC binding OVA peptides after vaccination. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly). Results are representative of two independent experiments (n = 7–8 mice/group). Data is expressed as the calculated geometric mean of the Ag-stimulated cells minus unstimulated cells. The error bars representSEM calculated from the mean SFC/106 splenocytes. Statistical analyses were conducted using a two-tailed Student t test. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using an two-tailed unpaired student t test.We observed unique Class-II dependent IFNγ-responses in the spleen after mucosal delivery of pFliC(-gly) but not when delivered systemically or dermally. This mirrors our observations of mucosal IgG and IgA with pFliC(-gly) use. Why does pFliC(-gly) promote strong Th1-like CD4+ T cell and IFNγ-producing CD8+ T cell responses when delivered mucosally but only promotes increases in IFNγ-producing CD8+ T cells when applied systemically or dermally? It could be that splenic Th1-like CD4+ cells have trafficked to other locations before analysis or are below the level of detection. Alternatively, there may be a strong ability of FliC to promote CD4+ responses when delivered mucosally. It has been observed that certain mucosal DC populations expressing TLR5 have a special ability to promote flagellin-specific CD4+ T cell responses [25]. However, it remains to be seen if these TLR5-dependent responses can be extended to other antigens encountered in the same environment as flagellin. Nevertheless, as an adjuvant, we find it interesting that flagellin can promote different immune responses to the same antigen encountered in different environments. This may have relevance to immune responses elicited by flagellated pathogens.Adjuvant effects of pFliC(-gly) were dose-dependent. Lower doses delivered intranasally re-capitulated the effects seen after systemic and dermal routes giving increases in anti-OVA IgG in the sera as well as IFNγ-producing Class I-dependent cellular responses. Higher doses of pFliC(-gly) however, were able to induce mucosal anti-OVA IgG and IgA responses. These observations suggest there may be a lower threshold for flagellin to promote systemic responses to an antigen compared to mucosal responses, which might require more of the adjuvant. Whether this could be through the triggering of a threshold of pre-existing cells at the vaccination site, recruitment of new cell populations to the site after vaccination, or differences in triggering TLR5 and NLRC4 responses is not known.Experiments with pOVA and pFliC(-gly) indicated that delivery of plasmids using N3 and the intranasal route was able to promote cellular immune responses as well as humoral mucosal immune responses. To compare the effectiveness of secreted flagellin to promote immune responses to a clinical antigen using a heterologus prime/boost regime, priming intranasal DNA vaccinations were carried out using plasmid pgp160Lfai/pRev [16] with delivery lipid N3 alone or together with pFliC(-gly). Boostings were performed using recombinant gp160 proteins with a protein-delivery lipid L3B alone or together with N3 mixed with pFliC(-gly) (Table 2). In these experiments mice were given doses of antigen and adjuvant believed to maximize detectable responses. Mice were primed, boosted, and analyzed according to the indicated timeline (Figure 5a). Four weeks post-final boost serum total-IgG titers anti-rgp160 indicated that addition of N3 to pgp160 was able to strongly promote anti-gp160 antibody responses (Figure 5b). Similar to responses seen using OVA, addition of N3/pFliC(-gly) to the immunization regime enhanced antigen-specific antibody titers further (Figure 5b). The adjuvant effect was dependent on N3. Likely due to it’s ability to encapsulate plasmid DNA and protect it from the degradative environment of the mucosal compartment. These higher titers of antigen-specific IgG in the sera and the presence of antigen-specific mucosal IgA indicate that the mucosal adjuvant effects of pFliC(-gly) are not limited to experimental antigens. Kinetic analysis of anti-gp160 IgG isotypes revealed that addition of N3 to pgp160 was able to promote anti-gp160 IgG1 at 4 weeks following the final boost. This titer was generally sustained to 24 weeks but fell sharply by week 36 (Figure 5c). A similar trend was seen when anti-gp160 IgG2a was studied (Figure 5d). When pFliC(-gly) was added to N3/pgp160 vaccinations IgG1 and IgG2a anti-gp160 titers were enhanced further as well as sustained to later time points (Figure 5c,d). These results suggest that the adjuvant effects of pFliC(-gly) not only boost antigen-specific antibody but that these effects may also lead to longer persistence of antibody.One should bear in mind that the antigen doses used were chosen to be relatively weak at inducing immune responses when given via the nasal route in small volume once or twice without adjuvant. Therefore, it was not surprising to see a short-lived serum and mucosal antibody response unless adjuvant was used. Longevity of total anti-gp160 IgG was significantly enhanced in groups where the N3 adjuvant was used especially when combined with pFliC-DNA. Since the N3 adjuvant has cationic and surfactant properties one proposed mechanism would be that there is an increased mucosa-penetrating property when HIV-antigen and adjuvant is given in mixture. This would increase the amount of antigen reaching below the mucosal surface, thereby making the antigen available at higher dose for antigen-presenting cells in the mucosa. Equally important, the capacity of the serum immunoglobulins to neutralize HIV-1 virus in vitro, both cell-line adapted (HIV-1IIIB), and primary patient isolate (HIV-1 6794B) remained clearly detectable at 36 weeks in serum from groups 2 and 4 (Figure 5e). Although we observe enhanced titers of anti-viral spike antigen antibodies in the serum of animals immunized with antigen and pFliC(-gly) adjuvant the antibodies in mucosal secretions may be more likely to potentially neutralize viral particles. Studies of vaginal IgA anti-gp160 responses had assay backgrounds that precluded the determination of antigen-specific titers (data not shown). As a surrogate location representative of antigen-specific IgA responses we studied the titers of IgA anti-gp160 harvested from nasal washes. We observed trends similar to those found in the serum. Addition of N3 to pgp160 vaccinations followed by L3B protein boostings lead to clear IgA anti-gp160 titers which could be further enhanced by the addition of pFliC(-gly) (Figure 6a). The ability of nasal IgA anti-gp160 to cross-react against homologous (clade B) as well as heterologus clades (A and C) of HIV-1 gp160 was also tested. We observed nasal wash reactivity to clades A and B (Figure 6b). As with serum IgG, N3 could promote detectable anti-gp160 antibodies while pFliC(-gly) could enhance responses even further (Figure 6b). These results indicate that higher titers of antigen-specific clade B160 IgA also correlate with higher titers of antibody able to cross react with HIV-1 clade A gp160. Increases in cross-clade reactivity may likely be a behavior central to the development of an effective vaccine.Vaccination schedule, serum antibody responses to gp160, and virus neutralization titers. (a) Immunization and sample isolation timeline. Priming (ImmunogenP, plasmids) and boostings (ImmunogenB, rec proteins) are indicated in days while time after the final boost are indicated in weeks. Immunization details are listed in Table 2; (b) Serum IgG titer against rgp160 at 4 weeks post immunization in all seven study groups; (c) Serum titer anti-rgp160IgG1 isotype kinetics in the four first study groups in Table 2; (d) Serum end-point titer anti-rgp160 IgG2a isotype kinetics in the four first study groups. The concentration of rgp160-specific Abs are expressed as the end-point titers giving an OD equal to, or higher than, the mean + 3 SDs (the determined cutoff value for the assay) of the values of serum samples from unimmunized mice. Absorbance values equal to or above the cutoff value were considered positive; (e) Serum neutralization of HIV shown as IC50 in serum samples of the four first study groups in Table 2. The TCID50 (the reciprocal of the virus dilution where 50% of the cultures were infected) of IIIB (LAI) or 6794 was incubated with sample mouse serum (dilutions: 20, 60, 180, 540, 1 620). 5 × 104 cells well were then added, incubated, washed, and incubated for 7 days. Culture supernatants were tested for virus production by HIV-1 p24 capture ELISA. The lowest serum concentration giving a 50% reduction (IC50) of ELISA absorbance value compared with the mean of the negative controls are presented [19]. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups defined as p ≤ 0.05 were considered significant. n.s. = non-significant. Comparisons between groups with the HIV-1 antigens were performed by using the non-parametric Mann-Whitney U test with Bonferroni correction, p < 0.05 was considered significant. Mucosal antibody responses to gp160. (a) Nasal IgA anti-rgp160; (b) Nasal IgA anti-gp160 cross-reactivity against clade A, B, and C envelope antigens. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. ELISA was performed using individual serum from the indicated immunization groups. Absorbance values equal to or above the cutoff value were considered positive. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups defined as p ≤ 0.05 were considered significant.To determine the breadth of antibody responses against hypervariable regions of gp160 within antigen-specific IgG we performed B cell epitope mapping of group-pooled serum against individual 20-mers of gp160 from AA249–499 containing the V3 variable loop region. We observed clear populations of IgG anti-gp160 peptide immune responses in the sera of mice immunized with pgp160 with N3 followed by boosting with rgp160 protein with L3B (Figure 7a). However, the addition of pFliC(-gly) to immunizations expanded the number of detectable populations by five (Figure 7a). Analysis of amino acid identities and similarities between FliC and clade B LAI gp160 within the region encoded by the peptides were performed using NCBI BLASTP analysis (v2.2.26+) with default settings. Two regions were identified containing identity and similarity. Of these two regions only one (containing 36% identities and 55% similarity) overlapped with a region of increased reactivity (peptides AA 439 and 444) and was excluded. There were no regions of alignment identified within the 5 annotated populations that exhibited equal or greater identity and similarity than the 22AA region. B cell epitope mapping to C2-C5 region of gp160 after immunization with gp160 with and without adjuvant. ELISA was performed using group-pooled serum (equal volumes) from immunization group 2 (n = 35) or 4 (n = 35) against individual peptides. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. The concentration of gp160-peptide specific Abs are expressed as the end-point titers giving an OD equal to, or higher than, the mean + 3 SDs (the determined cutoff value for the assay) of the values of serum samples from unimmunized mice. Absorbance values equal to or above the cutoff value were considered positive. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups defined as p ≤ 0.05 were considered significant. These use of pFliC(-gly) appears to promote a broadening of B cell epitope reactivity to rgp160 and/or presentation of additional “masked” epitopes. Similar responses have been seen in response to a TLR-adjuvanted malaria vaccine using advanced techniques [26]. However, how this increased reactivity occurs is not known. It may be that the higher titers of anti-gp160 elicited by use of pFliC(-gly) revealed reactivity normally below threshold when samples from un-adjuvanted groups were studied. Additionally, it may be that pFliC(-gly) is able to promote expansion of B cell populations normally under stimulated or neglected due to competition. However, it may also be possible that there is cross-epitope reactivity between rgp160 and FliC, and new regions of anti-gp160 reactivity are actually due to anti-FliC antibodies. Although this cannot be formally excluded here it may be unlikely due to the low nature of homology between antigen (gp160) and adjuvant (FliC). Nevertheless, these results suggest that detailed study of antibody responses in mice receiving pFliC(-gly) are warranted.To study T cell immune responses to DNA-prime/protein-boost i.na. immunizations we chose to assay standard cytokines associated with Th1-like (IFNγ) or Th2-like (IL-5) populations. Responses to gp160 were assayed following individual splenocytes harvesting at 4, 8, 12, 24, and 36 weeks after final boost and restimulation with rgp160. Observed response trends were similar to those seen when studying antibody responses. Addition of N3 to pgp160 vaccinations followed by L3B protein boostings lead to clear and significant IFNγ, IL-5, and proliferative responses over mice immunized without adjuvant (Figure 8a–c). The IFNγ and proliferative responses could be further enhanced by the addition of pFliC(-gly) but not IL-5 (Figure 8a–c) demonstrating the ability of pFliC(-gly) to act as an adjuvant but with a propensity to strengthen Th1-like responses.To study the T cell immune responses to p24gag individual splenocytes were harvested at 4 weeks after final boost and restimulated with rp24gag. Interestingly, the addition of N3 to p24gag vaccinations followed by L3B protein boostings only lead to clear and significant increases in IL-5 and proliferative responses compared to mice immunized without adjuvant (Figure 9a–c). Increases in IFNγ production were not seen. IFNγ and proliferative responses could be significantly enhanced by the addition of pFliC(-gly) but the IL-5 responses gained by use of N3 were suppressed by the addition of pFliC(-gly) (Figure 9a–c).Interestingly the abilities of N3 and pFliC(-gly) to act as adjuvants did not completely overlap and, in one combination, even counteracted the other. In our immunizations the secreted antigen gp160 with N3 promoted a somewhat Th2-like response (including IFNγ) which was further enhanced by the addition of pFliC(-gly). Similar results were seen with the intracellular antigen p24gag where addition of pFliC(-gly) promoted IFNγ responses [27,28,29,30,31]. However, with p24gag the pFliC(-gly) addition actually suppressed IL-5 responses.Together these results suggest that N3 has the ability to promote Th2-like adjuvant effects (IL-5 and proliferation) to extracellular and intracellular antigens whereas the effects of pFliC(-gly), which were greatly dependent on the presence N3, promoted Th1-like adjuvant effects (IFNγ, proliferation) sometimes at the expense of Th2-like responses (IL-5). Why these effects were dependent on the “location” of the antigen is unknown. However, it does suggest that with complex antigen/adjuvant mixtures that we are unable to predict exactly how they will affect adaptive immune responses.To determine the types of inflammatory factors elicited intranasally by the adjuvants used we performed nasal mucosal washings at various time points after adjuvant delivery. We detected increases in the inflammatory cytokines IL-6, IFNα, and IFNγ at 18 to 48 h post-nasal adjuvant administration compared to nasal saline exposure (Figure 10a–c). Significant increases in IL-6 and IFNγ were elicited by N3 use at 18 hours post exposure while significant increases in IL-6 was elicited using a combination of pFliC(-gly) and N3 only at later time points (Figure 10a). These increases in IL-6 were dependent on the use of N3 with pFliC(-gly) but were not due to N3 or pFliC(-gly) alone.Currently, it is still likely that several alternative prime-boost combinations will need to be tested to identify the most promising vaccine/adjuvant and vaccine design regimes for HIV-1 vaccines [32,33,34].Kinetic analysis of T cell responses to immunizations with gp160 with and without adjuvant combinations. (a) Anti-mIFNγ ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naive mice. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2; (b) Anti-mIL-5 ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naïve mice; (c) Proliferative response to stimulation with rgp160 defined as stimulation index relative to identical stimulations using splenocytes from naïve mice. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups at week 4 after final boost defined as p ≤ 0.05 were considered significant. n.s. = non-significant. Comparisons between groups with the HIV-1 antigens were performed by using the non-parametric Mann-Whitney U test with Bonferroni correction, p < 0.05 was considered significant.Analysis of T cell responses to immunizations with p24gag with and without adjuvant combinations. (a) Anti-mIFNγ ELISA was performed on cells restimulated with p24gag. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naïve mice. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2; (b) Anti-mIL-5 ELISA was performed on cells restimulated with p24gag. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naïve mice; (c) Proliferative response to stimulation with p24gag defined as stimulation index relative to identical stimulations using splenocytes from naïve mice. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups at week 4 after final boost defined as p ≤ 0.05 were considered significant. n.s. = non-significant. Comparisons between groups with the HIV-1 antigens were performed by using the non-parametric Mann-Whitney U test with Bonferroni correction, p < 0.05 was considered significant. Cytokines produced after intranasal adminstration of adjuvant combinations. Kinetic analysis of (a) IL-6, (b) IFNγ, and IFNα2 (c) at 18, 36, and 48 h by ELISA using nasal wash samples. Mock adjuvant shown as White bars, use of pFliC(-gly) as Striped Bars, and N3 as Grey bars (n = 5 mice/group). Data is expressed as the calculated mean ± SEM. Statistical analyses were conducted using a two-tailed Mann-Whitney test. ** Differences relative compared groups defined as p ≤ 0.005 were considered significant.There are several important aspects concerning the flexibility associated with HIV-vaccine antigen development: first, the selection of immunogens and adjuvants. In this study, and in several others, DNA-plasmids should be selected that have long-lasting stability and allow persistence as stable antigen-expressing vectors. Second, production of DNA-plasmids, is today quite efficacious and can easily be performed at large-scale. Third, DNA-plasmids are attractive due to their great adaptability, and modifications in expression efficacy, gene exchange, or other desired modification is today easy to carry out.Recombinant HIV-proteins as vaccine antigens are instead more of a challenge, especially when it comes to production and expression of such delicate proteins like the HIV-1 outer envelope. If they need to structurally mimic the envelope spikes found at the surface of HIV-1 primary isolates they need to be produced and maintained as multimeric, glycosylated envelope proteins. This production is not a trivial matter, and search for the ideal HIV-1 envelope vaccine candidate is still an unsolved issue [32,33]. Finally, the stable storage of sensitive recombinant proteins antigens is likely more of a problem. Thus a potent and safe adjuvant, formulated with obtainable amounts of quality antigen, may be a critical way to use these immunogenic proteins as vaccine candidates. In this study, we chose the recombinant baculovirus expressed HIV-1 gp160 due to its modest immunogenicity, its fair degree of glycosylation and trimeric structure and the content of both gp41 and gp120 envelope proteins [16,18].From an immunological perspective, immunizing with a DNA plasmid with its endogenous in vivo expression of the HIV-antigen and especially of a highly glycosylated, conformation-sensitive antigens as HIV-1 gp120 is an attractive technology. Much of the trouble of production, safe storage and efficient administration of a neutralization antibody-inducing multimeric protein may then be reduced or avoided [35]. The durability of protective immune responses can often be enhanced by broadening antigen recognition ability through enhanced antigen delivery, enhanced antigen uptake, and/or prolonged antigen exposure [36,37]. Furthermore, by triggering several innate immune recognition pathways such as multiple Toll-like receptors (with DNA-plasmid CpG motifs, (TLR9), with FliC expression (TLR5), cytoplasmic DNA sensors [38,39] and increased induction of cell death using surfactant adjuvants in conjunction with antigen presentation as with cationic lipids more potent immune enhancement may be obtained [40]. In this study, we endeavored to optimize the immunological outcome (strength and longevity) of these vaccine technologies by methodologically studying optimal route delivery for adjuvants with DNA-plasmid vaccination followed by the application of this knowledge to a heterologus prime-boost regime using HIV-1 antigens. Importantly, we show that in the HIV-1 study group (group No.4) receiving potentially the broadest HIV-1 antigen variants and the most complex adjuvant combination we obtained the most long-lasting humoral and cell mediated immune responses.Certain studies have described the induction of innate and adaptive immune reactivity when immunogens and adjuvant have been given to mice [41,42,43]. In the current studies we have performed analyses on how the cationic lipid-based N3 adjuvant, the DNA-plasmid-based FliC (-gly) adjuvant, and the combination of both together influence the local and systemic innate and adaptive immune responses. The data indicate that the adjuvants stimulate the production of pro-inflammatory IL-6 and interferon (IFN), two cytokines described promote innate immunity and B cell activation [44,45].Each of the adjuvants are likely to contribute, in their own way, to immune activation, and lipid-based adjuvants (such as the N3) have been observed to induce cell death (as seen with N3 in vitro, data not shown) and local inflammation. This would provide danger signals to attract antigen-presenting cells, stimulate antigen uptake, and induce dendritic cell maturation. For instance, cell death and endogenous DNA release would be able to function as an endogenous adjuvant capable of supporting IL-6 release and a T-helper type 2 response [41,45,46]. Inflammatory cytokine patterns, accompanied higher antibody titers have been reported by Valesi et al., when using the MF59 lipid emulsion adjuvant and influenza vaccine given to mice [47]. Similar results in inflammatory cytokine production with the N3 lipid adjuvant and HIV antigens were also observed in our work.DNA-plasmid vaccination used to express antigen and adjuvant proteins can trigger TLR systems and promote inflammatory cytokine production (such as IL-6) [48]. Intranasal recombinant FliC polypeptide has also been observed to induce inflammatory cytokines including IL-6 [49]. Interestingly, the induction of IL-6 secretion in mucosal stimulation has been reported to improve transepithelial transport over mucosal epithelial barriers [50]. This effect of IL-6 may explain the enhanced mucosal immune responses we observe using mucosally delivered pFliC(-gly). However, the ability of FliC to promote mucosal adaptive immunity is complex and appears to also involve the production of numerous other factors not studied here such as IL-17, IL-22, and IL-23 [51,52].Our observations of mucosal, antigen-specific IgA elicited by intra-nasal immunization indicates presence of a Th2-type response with N3 use while IFNγ production detected in the nasal washes of pFliC(-gly) immunized animals suggest a Th1-type response. Our results also suggest that these responses need not be mutually exclusive. Adjuvant emulsions such as MF59 and similar products applied mucosally have been shown to act as Th2-type adjuvants, and addition of additional stimuli such as CpGs, and other TLR agonists have been observed to skew immune responses towards a Th1-type or a balanced Th1/Th2-type immune pattern [38,53]. Here, we show data that immune responses induced using a Th2-enhancing lipid adjuvant (N3) may be modified by the addition of a TLR-agonist and inflammasome trigger (pDNA, FliC), to also promote a Th1-type (IFNγ) response when administered nasally.Adjuvant choice during DNA vaccine development will depend on formulation relative to method of delivery, the recipient, the protective antigens used, as well as the desired induction of immunity at the portal of infection. As the detection of flagellin by innate immune receptors is evolutionarily conserved, it has the potential to be easily used in many species without the need to isolate and prepare species-specific adjuvants such as cytokines [27,28,29,30,31]. These unique properties as well as its ability to promote both humoral and cellular responses to co-delivered antigens by multiple routes without a need for manipulating the antigen indicate that it works as an easy and efficient adjuvant to improve non-living non-replicating DNA vaccines.Finally, in this work, all immunizations with HIV-antigens were delivered as heterologous prime boost immunizations. Antigens are presented both through endogenous expression and as recombinant soluble proteins to the immune system to provide the antigenic regions or epitopes with greater variation than in a homologous immunization. With this approach we demonstrate that cationic lipids formulated with plasmid FliC-DNA and plasmid HIV-DNA, followed by cationic lipids formulated with plasmid FliC-DNA and recombinant HIV-1 proteins (study group 4) elicited the longest-lived immunity and broadest antigen epitope recognition.Toll-like ReceptorNod-like Receptor family CARD domain-containing protein 4Neuronal apoptosis inhibitory protein 5Human Immunodeficiency Virus-1This work was supported by the Swedish International Development Cooperation Agency (SIDA), the Swedish Research Council (VR), and Läkare mot AIDS research fund. We would like to thank Lech Ignatowitz for fresh N3 preparation, Eurocine Vaccines AB for providing N3 and L3B adjuvants. David Hallengärd for excellent ELISPOT assistance.Jorma Hinkula and Ulf Schröder have IP-rights and economical interests in the N3 adjuvant. All other authors have no conflicts to declare.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).We are witnessing a new era of immune-mediated cancer therapies and vaccine development. As the field of cancer vaccines advances into clinical trials, overcoming low immunogenicity is a limiting step in achieving full success of this therapeutic approach. Recent discoveries in the many biological roles of chemokines in tumor immunology allow their exploitation in enhancing recruitment of antigen presenting cells (APCs) and effector cells to appropriate anatomical sites. This knowledge, combined with advances in gene therapy and virology, allows researchers to employ chemokines as potential vaccine adjuvants. This review will focus on recent murine and human studies that use chemokines as therapeutic anti-cancer vaccine adjuvants. Chemokines are a group of related chemoattractant peptides that are essential regulators of the immune system, both during homeostatic and inflammatory conditions. Over the last few decades, chemokines are found to be involved in almost every aspect of tumorigenesis and antitumor immunity [1]. While a function of chemokines is to regulate lymphocyte trafficking, the view that chemokines act simply as “chemotactic cytokines” has evolved to include the many critical roles they play in regulating innate and adaptive immune responses. For example, in addition to chemotaxis, chemokines modulate lymphocyte development, priming and effector function [2] and play a critical role in immune surveillance. Some inflammatory chemokines have proven essential in memory T cell generation [3]. In the context of cancer, the chemokine-chemokine receptor system plays paradoxical roles. On one hand, the chemokine network is used by tumors to evade immune surveillance, resist apoptosis, and metastasize. On the other hand, the chemokine system also plays a crucial role in the induction of antitumor immune responses and optimal effector function regulation of immune cells [1,4,5].To date, there are more than 50 chemokines and 18 chemokine receptors identified [6]. These molecules are classified into four families (CC, CXC, C, and CX3C) based on the way the first two conserved cysteine residues are arranged, creating a structural motif [6]. Two nomenclature systems are often interchangeably sited in the literature: the name at the time of discovery, and the systematic nomenclature as described in Table 1 [6]. For consistency, this review will henceforth use the systematic nomenclature. Most chemokines bind to more than one receptor, while most receptors also display overlapping ligand specificity [5]. Functionally, chemokines are described as inflammatory (inducible) or homeostasis (constitutive) based on their pathophysiological activities. Inflammatory chemokines are secreted in inflamed tissues by resident and infiltrated cells after stimulation by pro-inflammatory cytokines or during contact with pathogens. They specialize in the recruitment of effector cells, particularly monocytes, granulocytes, and effector T cells to sites of inflammation, tissue destruction, or tumor microenvironment (TME). Homeostatic chemokines are constitutively produced and regulate physiologic trafficking of immune cells during hematopoiesis, antigen sampling in secondary lymphoid tissue and immune surveillance. Some chemokines are also defined as angiogenic or angiostatic based on their role in promoting or suppressing tissue neovascularization, respectively [7].Chemokine nomenclature, corresponding receptors, and category based on function. Adopted from [6,7]. Chemokines used as adjuvants for vaccines in murine and human studies are highlighted in color.Development of an effective antitumor immune response depends upon the unified interaction of immunocompetent cells and their trafficking pattern between the tumor site and secondary lymphoid organs (e.g., lymph nodes (LNs) and spleen). This trafficking pattern is coordinated by chemokines acting through their corresponding receptors [5]. Dendritic cells (DCs) are professional APCs responsible for initiation or inhibition of immune responses by priming or tolerizing T cells [8]. Chemokines play a key role in the migration and recruitment of DCs. DC precursors in the peripheral blood migrate into peripheral tissues and differentiate to become immature DCs (iDCs), characterized by high phagocytic ability and increased levels of MHC molecules, but a lack of costimulatory molecules [9]. iDCs are guided by inflammatory chemokines (CCL2, CCL3, CCL4, CCL5, CCL7, and CCL20) to migrate to sites of inflammation or tissue damage, where they pick up antigen, upregulate costimulatory molecules, and become activated, mature DCs (mDCs). This chemotactic migration of iDCs within tissue is related to their expression of CCR1, CCR2, CCR5, and CCR6, while mDCs downregulate these chemokine receptors and upregulate CCR7 [1,9,10]. It is the constitutive expression of the CCR7 ligands CCL19 and CCL21 by the stromal cells in the T cell zones that guides the mature and antigen-loaded mDCs to secondary lymphoid organs, where they present processed antigens to the CCR7-expressing naïve or central memory T cells [1,9,10,11].To become effective tumor-associated antigen (TAA)-specific killer cells, cytotoxic T lymphocytes (CTLs) require effective priming by DCs, which in turn require licensing by CD4+ T cells [12]. For this purpose, naïve CD8+ and CD4+ T cells, expressing CCR7, continuously scan the surface of DCs in secondary lymphoid organs in search for their rare cognate antigen [13]. Several chemokines are found to be critical to this process. CCL3 and CCL4 secreted by DCs in inflamed lymph nodes help guide naive CD8+ T cells expressing CCR5 to sites where CD4+ and CD8+ T cells are actively interacting with antigen-presenting DCs. The ternary cluster formed by the naïve CD8+ T cell, the CD4+ T cell and the DC enhances memory CD8+ T cell generation [3,14]. Additionally, mDCs secrete CCL19 to increase scanning behavior and antigenic response by naïve CD4+ T cell [15]. Upon TCR-MHC engagement, chemokine receptors also act as co-stimulatory molecules in the immunological synapse to further enhance signal transduction between the T cells and the APCs [16]. Following priming and T cell expansion, a change in the pattern of chemokine receptor expression is required for the redistribution of T cells from the secondary lymphoid organ back towards the target tissue. Once effector T cells have differentiated, they downregulate CCR7 and upregulate receptors specific to chemokines expressed in target tissues, such as CCR1, CCR2, CCR3, CCR5, and CXCR3 [5,17]. Thus, chemokines are critical in regulating the traffic of immune cells between the TME and draining LNs, as well as enhancing differentiation of naïve T cells into TAA-specific CTLs.Effective cancer vaccines are designed to boost host adaptive immunity from a functionally tolerized state against cancer cells to one that can mount a functionally competent, tumor-specific, CD4+ and CD8+ effector and memory T cell-mediated immune response. As such, adjuvants such as Toll-like receptor (TLR) agonists (for example, CpG and PolyI:C) have been used in cancer vaccine to achieve this effect [18,19,20]. Due to their multifaceted roles in tumor immunology, chemokines represent another class of molecules that are attractive candidates for manipulation in cancer immunotherapy. Various chemokine-based tumor immunotherapies have been investigated, most of them in early preclinical models. A challenge to investigators in this research arena is that chemokines have been shown to be pro-tumorigenic in some tumor systems while anti-tumorigenic in others [1,4,8]. Some strategies target the pro-tumorigenic roles of chemokines by inhibiting chemokines and chemokine receptors that promote angiogenesis, tumor growth, and metastasis in certain tumor models [8]. Other strategies that deliver chemokines within the tumor microenvironment (TME) have been associated with enhanced antitumor immune response, increased angiostatic effect, low recurrence rate and increased patient survival [5]. In this light, immune-based cancer vaccines are strategies that can benefit from the addition of chemokines. These strategies vary based on the mode of tumor antigen loading unto professional APCs (e.g., peptide/protein-pulsed DC vaccines and peptide/DNA vaccines) [21]. This review will focus on the current use of chemokines as cancer vaccine enhancers. The main goal of cancer vaccines is to elicit a tumor-specific adaptive immune response by activating CD8+ cytotoxic T lymphocytes for tumor cell lysis and Th1 CD4+ T cells to enhance CTL activity [1,22,23]. Cancer vaccines are likely to be most effective in a setting of minimal residual disease (MRD), once the bulky tumor has been reduced by other therapeutic modalities [1]. Since the FDA has approved the first therapeutic cancer vaccine for metastatic castrate-resistant prostate cancer, a wide range of cancer vaccines are now undergoing evaluation in Phase II and III clinical trials [23]. Various cancer vaccines are currently under investigation in clinical trials, including peptide, viral vector, whole cell/lysate, genetically modified tumor cell, and DC-based vaccines [21,23]. Each of these vaccine groups has their unique properties that create specific advantages and challenges. The common disadvantage in all cancer vaccines is the realization that TAA presentation alone is not sufficient to create the most efficient tumor eradication and memory response. Therefore researchers now focus on various techniques to enhance TAA immunogenicity and vaccine efficacy. As described below, chemokines can be useful adjuvants in different vaccine settings. The choice of chemokines varies from homeostatic (e.g., CCL19 and CCL21) to inflammatory (e.g., CCL3 and CCL5). The major contribution provided by chemokines is more robust recruitment of relevant immune cells towards tumor recognition, immune priming, and killing. These discoveries lead to several murine cancer vaccine studies with chemokines as additives (summarized in Table 2), and provided the scientific rationale for subsequent Phase I and Phase II clinical trials (summarized in Table 3). Vaccine approaches incorporating various chemokines.* pDNA, plasmid DNA; & pCCL21, plasmid DNA encoding CCL21.Clinical trials using chemokines as cancer vaccine adjuvants.* GM.CD40L, genes encoding GM-CSF and CD40L.DCs are potent APCs that are capable of activating naive T cells and generating strong anti-tumor immunity [63,64]. iDCs can efficiently internalize antigen and, subsequently, process and present antigen peptides in conjunction with major histocompatibility complex (MHC) class I and II molecules to T lymphocytes. However, concerns have been raised regarding the use of iDCs in clinical trials since they have been associated with inducing T cell tolerance [64]. However, mDCs have a higher expression of MHC and costimulatory molecules after activation by danger signals in the periphery, and are therefore better equipped to activate antigen-specific T cells in secondary lymphoid organs. For this reason, mDCs loaded with TAAs in vitro have found clinical applications. Phase II studies have been conducted to evaluate the effectiveness of DC-based vaccines using various strategies (protein-pulsed, peptide-pulsed, or viral-vector infected DCs) to treat patients with prostate cancer, colorectal cancer, melanoma, glioma, and other cancers [21,23]. Of these approaches, a major challenge is that these vaccines do not always result in robust T cell activation, tumor killing by effector T cells, or generation of memory T cells. A reason for this is insufficient physical contacts among relevant immune cell types for optimal immune response generation. For these reasons, chemokines have been added to DC vaccines in an effort to improve antigen presentation and immune cell recruitment. In addition, chemokines have also been used to enhance DC recruitment in vivo for subsequent in vitro expansion. For example, He et al. showed that intravenous injection of CCL3 and CCL20 prior to DC collection improved recruitment of DCs. Subsequent transduction of those DCs with the melanoma TAA MAGE-1 gene resulted in improved melanoma tumor rejection ex vivo and in vivo [24]. In another study, CCL3 pre-treatment of mice resulted in the recruitment of more effective DCs in the peripheral blood. These DCs expressed a higher level of CCR7, displayed a more significant chemotactic response towards secondary lymphoid tissue, and generated a stronger CTL responses resulting in enhanced rejection of melanoma [26]. An attractive approach to enhance DC vaccine efficacy is to combine DCs with plasmid DNA (pDNA) encoding specific chemokines. Jiang et al. undertook such an approach by administering DCs pulsed with glioma cell line (GL261) lysate subcutaneously (SQ) into mice bearing glioma tumor [27]. A cohort of mice also received a plasmid encoding CXCL10 (pcDNA3.1-mIP-10) at the same vaccination site. As CXCL10 is a chemokine that has both anti-angiogenic and T cell recruitment properties into the CNS [65], mice receiving combination therapy had significantly improved survival rates (60% vs. 0%). A different group of researchers has attempted retroviral introduction of the CXCL10 gene into DCs and observed improved CD8+ T cell response and tumor rejection [28]. Li et al. pulsed bone marrow-derived DCs with murine prostate tumor lysate and transfected these cells with a plasmid vector encoding for CXCL10 [32]. Tumor rejection and survival was improved compared to mice receiving pulsed DCs or non-pulsed DCs with CXCL10 gene alone. CCL21 has also been implemented in DC vaccine strategies. Although considered to be a homeostatic chemokine, CCL21 influences T cell migration to secondary lymphoid organs during inflammation and enhances the Th1 T cell response [66]. Liang et al. transfected murine iDCs with the CCL21 gene using the recombinant adeno-associated virus serotype 2 (rAAV2) as a gene delivery vector [31]. When CCL21-transfected DCs were injected intratumorally in mice bearing hepatocellular carcinoma (HCC), mice exhibited delayed tumor progression, increased intratumoral T cell infiltration and overall improved survival. Yang et al. took a similar approach by transducing DCs with adenoviral vector encoding the CCL21 gene. Their data again showed better tumor eradication and tumor-protective immunity in the mouse cohort receiving CCL21-expressing DCs intratumorally [34]. Another study not only introduced CCL21 gene-encoding plasmid (pAAV-IRES-hrGFP/SLC) into bone marrow-derived DCs but also pulsed DCs with whole tumor lysate and then injected the construct into tumor-bearing mice with similar efficacy [29].CCL20 was recently shown to direct iDC migration and is postulated to play a role in tumor immunotherapy [67]. SQ injection of irradiated tumor cells expressing CCL20, followed by a second vaccination of DCs pulsed with irradiated tumor cells at the same injection site resulted in significantly more robust tumor rejection than DC vaccine alone [30].XCL1 is a chemokine that has shown the ability to attract effector cells (NK cells and CD8+ cells) and has been tested as a DC vaccine enhancer [68]. Xia et al. immunized mice with DCs co-transfected with XCL1 and melanoma antigen gp100 (XCL1/gp100-DC) using an adenoviral vector. Their results showed enhanced effects of CTL and NK cell activation and increased production of IL-2 and interferon-gamma. The XCL1/gp100-DC immunized mice exhibited resistance to tumor challenge more effectively compared to controls [25]. CCL5 is one of the central chemokines that regulates T cell migration towards sites of tissue injury and inflammation, as well as Th1 differentiation [69]. CCL5 has been tested in murine models as adjuvant therapy for tumor lysate-pulsed DC vaccines. Mice received tumor lysate-pulsed DC vaccine followed two days later by intraperitoneal (IP) injection of CCL5-expressing recombinant vaccinia virus [33] showed a significant reduction in rates of tumor growth and increased survival, which correlated with increased immune cell infiltration into tumor sites.CCL19 is a potent inducer of T cell proliferation [70]. To bypass the labor-intensive process of isolating, expanding and loading DCs from individual patients ex vivo, Kumamoto et al. developed an approach to entrap epidermal Langerhans Cells (LCs) in situ and load them with TAAs [71]. They used subcutaneously (SQ) implanted CCL19-coated polymer rods to create a LC-attracting chemokine gradient during their migration from the epidermis to the draining LN. The entrapped LCs were antigen-loaded in situ by co-implantation of a second polymer rod releasing tumor-associated antigens. Once loaded with TAA in situ, CCL19 administration allowed LCs emigration from the epidermis to the draining LN to activate a strong antigen-specific CTL response [71]. These preclinical investigations lead researchers to successfully transduce CCL21-expressing human DCs [72], setting the ground work for future clinical trial development. A recently closed Phase I clinical trial in melanoma applied intradermal injections of adenovirus-CCL21 transduced class I peptide-pulsed DCs [55]. Dose-escalation studies of intratumoral autologous DC-adenovirus CCL21 vaccine in patients with advanced lung cancer are also currently open [56,57] (Table 3).DNA vaccines encompass DNA constructs that encode TAAs. Once administered SQ or intramuscularly (IM), DNA constructs are taken up by local cells, including APCs, that then express the TAAs on the cell surface in conjunction with MHC class I molecules. This TAA presentation ultimately leads to T cells response against TAA and therefore the tumor cells [73]. The use of DNA vaccines in cancer immunotherapy has many advantages (e.g., less costly, vastly available, safe, lack of autoimmunity, and less potential for rejection) [74]. However, the main challenge of such vaccine approach is their low immunogenicity [75]. As discussed above, CCL19 is a potent inducer of T cell proliferation [70], a feature that prompted trials of its use as an adjuvant for DNA vaccination in murine models [35]. Westermann’s group compared how mice-bearing tumors responded to vaccine with plasmid DNA (pDNA) encoding tumor DNA alone or vaccine with tumor DNA and CCL pDNA. Co-expression of pDNA encoding CCL19 and tumor antigen resulted in enhanced Th1 immune response and increased CD8+ T cell infiltration in the tumor bed. Similar experiments were conducted by injecting tumor-bearing mice IM with pDNA encoding Her2/neu with or without CCL19 pDNA [36]. Again, mice injected with both Her2/neu pDNA and CCL19 pDNA had substantially improved tumor protection (58% versus 22% tumor-free incidence). Similar results were obtained with CCL21 pDNA [39]. As CCL21 is another potent inducer of T cell proliferation, Yamano et al. injected CCL21 into mice at various time points before and after vaccination with TRP vaccine and showed CCL21 enhanced responses best when it was administered into the vaccine bed 24 hours prior to TRP DNA injection [44]. Another study tested CCL21 administration 24 hours before cTERT DNA vaccine [37]. Again, results showed significantly improved anti-TERT cell immunity in mice that received CCL21 chemokine compared to vaccine alone. Incorporating plasmid DNA encoding CCL21 gene (pCCL21) into a DNA vaccine construct containing fused common Her-2/neu and p53 (HP) to the Fc portion of IgG improves MHC II class presentation [41]. Injection of the end-product construct pCCL21-HP-Fc into melanoma-bearing mice resulted in improved tumor free survival (40% vs. 0% at 45days when compared to Fc controls) and better protection against subsequent tumor re-challenge. Similar DNA vaccine constructs encoding a single tumor antigen-E7 (pCCL21-E7-Fc), or multiple epitopes (pCCL21-3P-Fc), also showed improved tumor rejection and memory T cell generation in both cases [42,43]. The chemokine CX3CL1 contains chemoattractant properties for CTLs, NK cells, and macrophages [76], and was evaluated in pre-clinical models as a DNA vaccine adjuvant. DNA vaccine co-expressing HIV-1-RT antigen and CX3CL1-Ig promoted enhanced tumor rejection compared to DNA vaccine without CX3CL1-Ig [40].Dorgham et al. identified a CCL5 analog (super-agonist) that has an increased capacity to engage CCR5 [38]. Aravindaram et al. delivered CCL5 cDNA into the vaccination site before human gpDNA (hgp100) vaccination [45], and continued to augment the antitumor effect by injecting viral vectors expressing mRNA for both CCL5 and hgp100. Their results showed a significant immune cell infiltration at the vaccination site and a strong anti-tumor response [45]. Inoculation of a new CCR5 mutant, 1P7-immunoglobulin (1P7-Ig), along with tumor DNA, resulted in an increased CD8+ T cell presence in the tumor beds and a better protection against tumor growth. These murine studies illustrate the potential benefit of using chemokines in DNA cancer vaccine preparations. Another vaccine strategy is to exploit the fact that chemokines are internalized upon binding to their corresponding receptors on iDCs, thereby facilitating the delivery of accompanying antigens to APC for processing and presentation. Biragyn et al. generated genetically fused proteins consisting of inflammatory chemokines and TAA, where the chemokines serve as a carrier for the previously non-immunogenic TAA [53,54,77,78]. Once internalized along with the chemokine via the chemokine receptor, TAA presentation on DCs increases 100 to 10,000-fold [7,79,80] and results in the generation of protective antitumor immunity. This strategy was tested in murine lymphoma cell lines whose non-immunogenic variable region sequences (sFv) was genetically fused to chemokines CCL7, CXCL10 [54] and CCL20 [53]. Immunization with chemokine-sFv protein elicited a T-cell dependent antigen-specific protective antitumor immunity [54]. This response was dependent on the ability of chemokines to deliver the fused TAA to a chemokine receptor for internalization, whereas the recruitment of DCs alone to the site of antigen immunization by non-fused mixtures of chemokine and antigen was not sufficient to break the non-responsiveness to tumor antigen [54]. Therefore, this strategy can potentially be used in the same manner with any chemokine that binds to chemokine receptors present on iDCs (e.g., CCR1, CCR2, CCR5, and CCR6) [1], facilitating the efficient delivery of tumor antigens to MHC class I processing and cross-presentation pathway [81]. Whole cell/cell lysate vaccines are prepared by irradiating or lysing autologous or allogeneic tumor cells [21]. They can be genetically modified further to express certain TAAs or other molecules [21]. This approach provides another way to include chemokines as adjuvants to increase vaccine immunogenicity. Zibert et al. created genetically modified leukemia/lymphoma vaccine to express CCL3 plus IL-2 or CCL3 plus GM-CSF [48]. Data showed that groups of mice receiving CCL3 plus IL-2 had 46% survival and the CCL3 plus GM-CSF group had 75% survival compared to 0% in the control group. Injection of CCL3 as a single agent showed 29% survival. These results were accompanied by enhanced effector cell infiltration in the tumor beds. Nomura et al. designed mouse fibrosarcoma and ovarian carcinoma cells to encode genes for CCL21, CCL19, or CXCL12 in the presence or absence of co-infection with GM-CSF and IL-2. Chemokine addition alone showed additive anti-tumor effect, while the combination of chemokine plus IL-2/GM-CSF boosted the response even further [52]. As CXCL12 is implicated in tumor pathogenesis [82], its future in immunotherapy is still being debated.B16 melanoma cells engineered to stably express CCL21 chemokine elicited a robust effector T cell infiltration when used as a vaccine [47]. Li et al. used prostate cancer cells to develop a novel fusion gene using three common cancer gene epitopes: hPSM-hPAP-hPSA (“3P”) [49]. Fusing this gene construct with plasmid DNA coding for CCL21 (pCCL21-3P-Fc), the investigators introduced this construct into the B16FO melanoma cell line to create a genetically modified tumor vaccine. Injection of this vaccine into mice bearing melanoma tumors showed efficient tumor rejection and improved survival, with additional therapeutic benefits when the regimen was combined withanti-PD-L1 antibody administration. Inoue et al. evaluated the effect of adding either CCL5 or CCL17 to irradiated GM-CSF producing WEHI3B cells [50]. Addition of both chemokines in this study showed additional benefits in survival and tumor rejection, with significant CD4+ and CD8+ T cell infiltration in TME [50].Based on the above studies, a Phase I clinical trial is evaluating the cell-based vaccine composed of irradiated tumor cells transduced with granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD40-ligand (CD40L) genes, called the GM.CD40L vaccine, in the presence or absence of CCL21 in patients with lung cancer [58]. Another Phase I study of XCL1and IL-12gene-modified autologous neuroblastoma vaccine for relapsed/refractory neuroblastoma has been completed recently [59]. Additionally, a Phase I-II study is open for pediatric patients with advanced neuroblastoma using repeated immunization with gene-modified, IL-2/XCL1-secreting neuroblastoma tumor cell vaccines [58,60] (Table 3).Even though chemokines are a promising adjunct to growing cancer vaccine protocols, some studies have also uncovered deleterious effects of adding chemokines to cancer vaccines. For example, the addition of CCL3 to GM-CSF producing glioma cells nullified the therapeutic effect of GM-CSF [51]. In another trial, the triple gp100 DNA + CCL21 DNA + IL2 vaccines failed to demonstrate a benefit over the dual gp100DNA + CCL21 DNA vaccine combination, while gp100DNA + IL-12 DNA vaccines showed some efficacy [46]. These observation highlights caution when choosing a combination of chemokine-cytokine vaccines. The addition of chemokines into cancer vaccine strategies has the potential to provide great benefits in overcoming tumor tolerance by improving antigen presentation by APCs, enhancing effector cell priming, tumor eradication, and sustaining T cell memory responses (summarized in Scheme 1). Several studies have moved these concepts forward from pre-clinical studies into Phase I and Phase II trials in both adult and pediatric populations. Results of these highly anticipated trials would better inform investigators regarding next phases of new therapeutic development. Clearly, defining the precise disease stages (i.e., bulky disease versus minimal residual disease) and timing during therapy when administering chemokine adjuvant therapy will be important next steps. Furthermore, characterizing which chemokine(s) to employ in various tumor types and the spectra of clinical scenarios in which to employ them will help to optimize the specific biological effects of these molecules for desired therapeutic outcomes. As cell-based immunotherapy (e.g., chimeric antigen receptor (CAR) lymphocyte therapies) [83,84,85] and immune checkpoint blockade (e.g., anti-PD1 and anti-PD-L1 antibodies) [86,87,88] approaches arrive at the forefront of novel cancer therapeutic development, the inclusion of relevant chemokines may further enhance therapeutic effectiveness by providing directed trafficking, accumulation and effector function delivery of therapeutic immune cells to the relevant body sites.Mechanisms of chemokine-enhanced cancer vaccines.Evolving cancer vaccine strategies reflect our growing knowledge of tumor immunology, as classes of molecules (such as TLR agonists and chemokines) that are important in orchestrating effective host immune response find their way into various pre-clinical and clinical cancer vaccine and immunotherapy applications. An in-depth knowledge of the role of chemokines, cytokines and other biological agents will bring about their incorporation into vaccine preparations in the future to further boost therapeutic efficacy. In particular, current use of chemokines in cancer vaccines focuses on these molecules’ effect on migration and recruitment of relevant immune cells for effective antigen delivery and recognition. The potential additional effect of chemokines as direct functional co-stimulatory molecules on responding cells still remain largely unexplored. Future insights regarding the multi-faceted role of chemokines in immune response orchestration will further propel the field of cancer vaccine forward. In addition, it remains to be determined which vaccination strategies, timing and routes of administration involving chemokine adjuvants will be most efficacious in the clinical setting through well-designed clinical trials.The authors gratefully acknowledge the following funding support for this work: the National Cancer Institute R01 CA154656 (AYH), the St. Baldrick’s Foundation (AYH, AG), the Cancer Research Institute (AYH), the Alex’s Lemonade Stand Foundation (AYH), the Hyundai Hope-on-Wheels Program (AYH, AG), the Rainbow Board at the Rainbow Babies & Children’s Hospital (IDB, AG), the Steven G. AYA Foundation (AYH), and Department of Pediatric Pilot Grant at Case Western Reserve University (AG).The authors declare no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).In veterinary medicine, there have been different experiences with the plasmid DNA vaccination. In this area and with the hypothesis to demonstrate the effectiveness of different plasmids encoding porcine respiratory and reproductive syndrome (PRRS), five DNA vaccines against PRRS were evaluated for their innocuity and efficacy in pigs. Eighteen animals were divided into five groups which were injected with five (A, B, C, D, E) different DNA vaccines. Albeit, none of the proposed vaccines were able to protect the animals against PRRS virus. Only vaccines A and B were able to reduce the clinical signs of the infection. ELISA IgM were detected 30 days after the first vaccination in the pigs injected by Vaccine A or B. ELISA IgG were detected 90 days after the first vaccination in the pigs injected by Vaccine B or C. Neutralizing antibody were detected Post Challenge Days 61 (PCD) in all groups. In the pigs inoculated with Vaccine C, IFN-γ were detected 90 days after first vaccination, and after challenge exposure they increased. In the other groups, the IFN-γ were detected after challenge infection. Pigs injected with each of the vaccines A, B, C, D and E showed a significantly higher level of CD4−CD8+ lymphocytes (p < 0.001) after infection in comparison with their controls.Porcine reproductive and respiratory syndrome (PRRS) is the most economically relevant disease in swine herds. It is responsible for respiratory and reproductive clinical signs, but, in recent years, reproductive failure has been more prevalent in swine herds. The continuous circulation of the virus among the pig population causes severe economic loss for the swine industry.The causative agent of PRRS is an enveloped virus which belongs to Arteriviridae family [1]. This virus contains a linear, single-stranded RNA (+) genome of 15 kb composed of 10 open reading frames (ORFs-ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a, ORF5b, ORF6, ORF7) encoding the different functional and structural viral proteins (Figure 1). In particular, the principal non-structural proteins, encoded by ORFs 1a and 1b, have replicase and helicase activities, whereas the three major structural proteins GP5, M, and N are encoded by ORFs 5, 6, and 7, respectively. The products of ORFs 2, 3, and 4 (GP2, GP3 and GP4) represent additional components of the PRRS virion. GP4 contains an immunodominant, neutralizing epitope that shows an extensive degree of variation. This fact indicates that it does not play a direct role in cell-entry or fusion processes, but that it is most probably located in close proximity to that region. Costers et al. indicates that accumulation of amino acids (aa) substitutions in the GP4 neutralizing epitope play a role in the inefficient PRRSV elimination from pigs with a primed anti-PRRSV neutralizing antibody response at the onset of infection [2].The GP5 is a major envelope glycoprotein as a key PRRSV neutralization target. Monoclonal antibodies against GP5 showed neutralizing activity to the homologous strains of PRRSV. The specific sequences of neutralization epitopes in GP5 were further identified as different amino acids of the European strain (Lelystad virus, type I) or North American strain (VR-2332, type II). Also, the neutralization epitopes were defined as linear peptides. Vanhee et al., 2011 have demonstrated that the antibodies specific to GP5 peptides from pigs infected with type I did not neutralize the virus [3]. Li et al. have demonstrated that GP5 ectodomain peptide epitopes are accessible for host antibody recognition, but are not associated with antibody-mediated virus neutralization [4].Recently, based on the bioinformatics analysis of the gene encoding GP5, two gene fragments were amplified by PCR and designed as GP5a and GP5b, respectively. These fragments were then cloned into a plasmid vector for the production of the protein, respectively [5].Current strategies for the control of PRRS infection include live-attenuated and inactivated vaccines. Unfortunately, these strategies of immunization are not fully successful against PRRS because they do not allow the priming of an appropriate immune response. Furthermore, reversion to virulence of the attenuated strains is of high concern as already occurred in the past. Accordingly, a high immunogenic and safe vaccine against PRRS is needed. Previous findings [6,7] demonstrated that the DNA vaccination against PRRS is at least partially successful in mice [8], suggesting that this strategy of immunization may be effective also in pigs.The aim of this study was to evaluate the effectiveness and safety of five DNA vaccines against PRRS. The DNA-based vaccines proposed herein are plasmids encoding for ORF4 or ORF5 of PRRS. In order to increase the immune response elicited by the DNA vaccination, these plasmids were also engineered including immunostimulatory cytidine-phosphate-guanosine (CpG) motifs. Two of the vaccines also include UbiLacI, a sequence that encodes for a strong proteasomal degradation signal and that should be able to enhance the priming of a cell-mediate immunity against PRRS.Schematic genome of porcine reproductive and respiratory syndrome virus (PRRSV) composed of 10 open reading frames (ORFs) encoding the different functional and structural proteins. In particular, ORF4 and ORF5 are used in the plasmid encoding GP4 or GP5 proteins.The strain 2000/BS 114 L of PRRS type I was selected for this study. The virus was used at the third passage on fetal monkey kidney (MARC 145) cell cultures at a titre of 105.50 TCID50/mL. All plasmids derived from pVAX1 (Invitrogen, San Diego, CA, USA). Plasmids were constructed by cloning PRRS genes encoding GP4 and GP5 into different plasmids: pVAX1-48CpG-NeuL-ORF4 (Figure 2); pVAX1-48CpG-NeuL-ORF5 (Figure 3); pVAX1-48CpG-UbilacI-ORF4 (Figure 4); pVAX1-48CpG-UbilacI-ORF5 (Figure 5). NeuL sequence was cloned into EcoRI end Hind III restriction sites and encoded for strong proteic secretion signal. Therefore, NeuL should allow for the protein processing by the endoplasmatic reticulum that is a step required for a new synthesized protein to be trans-located to cellular membranes and/or be secreted. These events allow for the viral antigen produced by the plasmid to be presented to the immune system in order to stimulate an antibody response. The sequence neuL includes Her-2/neu 5'UTR. The secretion signal DNA fragment was obtained by enzymatic amplification of DNA using the pCMV-ECD-TM vector [9,10] as a template, T7 primer (5'-TAATACGACTCACTATATAGGG-3') as a sense oligonucleotide and an oligonucleotide antisense having a terminal EcoR I site, “neuL” antisense EcoR I (5'-CATGGAATTCCGCGATTCCGGGGGGCAGGA-3'). The sequence UbiLacI encodes for a signal that leads to the proteasomal degradation [11]. This sequence was generated by polymerase chain reaction (PCR) from reference sequence [12] and cloned into Hind III and EcoR I restriction sites in line with viral sequence using the following primers: 
+		
+		sense 5'-GCCCAAGCTTCCGGAGCCGCAGCCGCCACCATGCAGATCTTCGTGAAGACCCTGACTGGTAAGACC-3'; antisense 5'-GCCCGAATTCTCGGGAAACCTGTGGTGCCAGCTGCATTAA-3'.
+		
+		
+		sense 5'-GCCCAAGCTTCCGGAGCCGCAGCCGCCACCATGCAGATCTTCGTGAAGACCCTGACTGGTAAGACC-3'; antisense 5'-GCCCGAATTCTCGGGAAACCTGTGGTGCCAGCTGCATTAA-3'.Plasmid pVAX1-48CpG-NeuL-ORF4.Plasmid pVAX1-48CpG-NeuL-ORF5.Plasmid pVAX1-48CpG-UbilacI-ORF4.Plasmid pVAX1-48CpG-UbilacI-ORF5.The oligo sense is designed to add Kozak sequence, necessary for good transcription in mammary cells [13]. Conjugation of antigen with ubiquitin should target the endogenously synthesized antigen to the proteasome, resulting in enhanced MHC-I presentation. Plasmid pVAX1-48CpG (Figure 6) was constructed by introduction in pVAX1 of specific CpG motifs based on the immunostimulatory sequence from ODN 2135 [13]. Briefly, two complementary oligodeoxynucleotides (BstE II forw: 5'-AATTCGGTTACCTCTAGACAAACCAACCAAT-3'; BstE II rew: 5'-CTAGATTGGTTGGTTGGTCTAGAGGTAACCG-3') were annealed to form a duplex containing the restriction site BstE II, and then cloned between EcoR I and Xba I sites in pCDNA3.1 (Invitrogen, San Diego, CA, USA). Another two complementary oligodeoxynucleotides were annealed to form a duplex containing 12 CpG motifs with protruding ends complementary to the restriction site BstE II.CpGBstE II sense: 5'-GTTACGTCGTTTGTCGTTTTGTCGTTTCGTCGTTTGTCGTTTTGTCGTTTCGTCGTTTGTCGTTTTGTCGTTG-3';CpGBstE II antisense: 5'-GTAACCAACGACAAAACGACAAACGACGAAACGACAAAACGACAAACGACGAAACGACAAAACGACAAACGAC-3'.Plasmid pVAX1-48CpG.Since the oligo CpG BstE II sense has a C in spite of a G, it was possible to clone in succession four CpG annealed product into BstEII locus. Finally, the whole sequence was modified by PCR using the primers: sense: 5'-GTGTGGTGGAATTGGGTTACGT-3'; antisense 5'-GTGCGGGCCCACTAGAGGAAACCAACG-3'; and blunt-cloned into Eco721 site of pVAX1. Plasmid DNA for immunization was purified from Escherichia coli strain DH5α using Qiagen Plasmid-Giga kits (Qiagen, Milan, Italy), resuspended at 1 mg/mL in sterile endotoxin water (Gibco BRI) and stored at −20 °C.We cloned into the restriction site Xba I of pVAX1-48CpG-neuL-ORF4 and pVAX1-48CpG-neuL-ORF5 a sequence encoding for the antigenic Myc tag epitope EQKLISEEDL. This modification lead to the expression of ORF4 or ORF5 in fusion with the antigen Myc tag that is recognized by a commercial antibody FITC conjugated F2047 (Sigma-Aldrich, Milan, Italy) and then allowed us to follow the expression of ORF4 and ORF5 in mouse embryonic fibroblast cells (NIH-3T3) by confocal microscopy. Transfections of NIH-3T3 using a lipofectamine established protocol (Invitrogen, San Diego, USA) of either pVAX1-48CpG-NeuL-ORF4-Myc or pVAX1-48CpG-NeuL-ORF5-Myc led to a marked cytosolic expression of the encoded antigens (Figure 7A,B). FITC immunofluorescence of mouse embryonic fibroblast cells (NIH-3T3) transfected with pVAX1-48CpG-NeuL-ORF4-Myc. (A) or pVAX1-48CpG-NeuL-ORF5-Myc; (B) after 48 h from transfection.Twenty-two animals of one month of age, devoid of PRRS ELISA antibodies, were used. The pigs were housed in isolation units at Lombardia and Emilia-Romagna Experimental Zooprophylaxis Institute Brescia (Italy), and fed twice a day with a diet of concentrate and water ad libitum. The maintenance and experimental protocols were established according to the animal care guidelines of International Guiding Principles for Biomedical Research Involving Animals and the European Agency for the Evaluation of Medicinal Products (CVMP/IWP/07/98). The experimental design was performed after the approval of the local ethical committee.The experimental animals were divided into seven groups (Table 1). The pigs in the first four groups each composed of four animals, were injected with the vaccine by subcutaneously (s.c.) route in the retroauricolar region; the fifth and sixth groups were injected with the plasmid with 48-CpG or only plasmid; the animals in the seventh group served as controls. Group 1 received 500 µg of pVAX1-48CpG-NeuL-ORF4 plasmid in 500 μL of 0.1 M phosphate buffer saline, (PBS) (Vaccine A); Group 2 received 500 µg of pVAX1-48CpG-NeuL-ORF5 plasmid in 500 μL of 0.1M PBS (Vaccine B); Group 3 received 500 µg of pVAX1-48CpG-UbilacI-ORF4 plasmid in 500 μL of 0.1 M PBS (Vaccine C); Group 4 received 500 µg of pVAX1-48CpG-UbilacI-ORF5 plasmid in 500 μL of 0.1 M PBS (Vaccine D); Group 5 received 500 µg of pVAX1-48CpG plasmid in 500 μL of 0.1 M PBS (Vaccine E); Pigs in group 6 received 500 µg of pVAX1 in 500 μL of 0.1 M PBS. The animals in Group 7, were used as challenge infection controls. All animals were immunized three times, at 28-day intervals. Ninety days following the first immunization, all animals were challenged with a virulent PRRS. The virus was given by intranasal route (i.n.) at a dose of 4 ml × 105.50 TCID50/mL for each animal. The pigs were observed for 90 days after challenge and temperatures were taken daily for 15 days. Serum samples were taken from each pig on the day of challenge (PCD 0) and on PCD 14, 28, 61, 90. At the end of the study, the animals were killed and the target tissues (lung, mediastinic lymph-node, tonsils) were collected for histological observation.Total RNA was extracted from 200 μL of each serum using RNeasy™ Mini Kit (Qiagen, Milan, Italy) by QIAcube platform (Qiagen, Milan, Italy) according to the instructions of the manufacturer, and eluted in 50 µL of RNAsi-free water. Positive serum control was previously prepared from pigs subjected to challenge infections with the PRRS type I and negative serum control was prepared from pigs free of PRRS infection. The TaqMan® probe Real-Time PCR amplification was performed in the CFX96™ Real-Time System (Bio-Rad, Milan, Italy). The PRRS amplification was performed in accordance of the protocols of Revilla-Fernandez et al. [14].Twenty-five µL of undiluted serum samples and two-fold dilutions of each were mixed with 25 µL of 100 TCID50 of strain 2000/BS 114 L of PRRS type I in 96-well microtitre plates (Corning Inc., Corning, New York, NY, USA). Positive serum control was previously prepared from pigs subjected to challenge infections with the PRRS type I and negative serum control was prepared from pigs free of PRRS infection. Neutralization titres were expressed as log2 of the highest dilution inhibiting cytopathology. The protocols adopted were in accordance with Yoon et al. [15]. Porcine respiratory and reproductive syndrome (PRRS) DNA vaccines used in the experiment.1 All groups of pigs were housed together; 2 Vaccine or plasmid were administered three times, i.e., 84, 56 and 28 days before challenge infection; 3 The vaccine or plasmid were inoculated by subcutaneous route (s.c.); 4 NA, not applicableSerum samples were used for the evaluation of immune response. Immunoglobulin M (IgM) or G IgG were evaluated by commercial ELISA tests (IgM—LSI kit, LSI, Lissieu, France; IDEXX Herdcheck—IgG PRRS kit, IDEXX Corporation, Westbrook, ME, USA). The protocols adopted were used in accordance with the instructions of the tests. IFN-γ was evaluated in serum samples by using a commercial ELISA test (Pierce, Endogen, Rockford, IL, USA). The protocol adopted was in accordance with the instructions of the test.Flow cytometry analysis was carried out according to a previous protocol [16]. Briefly, 50 μL of heparinized blood were mixed with 5 μL of the specific antibodies in a plastic tube. After 15 min of room temperature incubation in the dark, the cells were washed in PBS supplemented with 1% FCS and centrifuged for 5 min at 400 × g. The contaminating red cells were lysed by treatment with NH4Cl solution, pH 7.2, for 15 min at room temperature in the dark. The cell suspension was then washed twice in PBS supplemented with 1% FCS, centrifuged for 5 min at 400 × g, re-suspended in 0.5 mL in PBS supplemented with 1% FCS and finally set aside for the flow cytometry (Epics XL-MCL, Coulter). The antibodies used were as follows: anti-CD8α-FITC (Southern Biotech, Birmingham, AL, USA); anti-CD4α-R-PE (Southern, Biotech, Birmingham, AL, USA); mouse anti-pig CD8β, TCR γ/δ (VMRD Inc., Pullman, WA, USA); mouse anti-pig CD25, CD16-FITC (Serotec, Milan, Italy). Necropsies were performed after euthanasia (Tanax ®) and specimens were collected from target tissues. Specimens were fixed in buffered formalin solution 10% w/w, pH 7.4 and wax embedded (56–58 °C, Bio-Optica, Milan, Italy). Paraffin microtome sections, 5 µm thick, were stained with H&E, Van Gieson and Schiff’s reaction (PAS).Slides were studied with conventional optical microscope (Nikon Eclipse E800) PLAN APO lens. Positive values were statistically compared as cumulative data between treated and control groups at different times by ANOVA-Dunnett’s test. p < 0.05 was considered significant.The test vaccines did not induce any clinical signs in immunized pigs prior to challenge at day 90. Rectal temperatures were within normal values and similar to the control values (39.0–39.5 °C). After challenge, all immunized pigs presented clinical signs which were similar to those observed in controls (Table 2). They had high fever (40.1–41.3 °C) from PCD 2 (Group 6, 7) and PCD 4 (other groups), which lasted for eight days. Inappetence, cough, dyspnoea, lethargy, were detected from PCD 1 (Groups 1 and 2), PCD 2–4 (other groups) from one to eight days. All animals recovered after three weeks following challenge. A significant difference in hyperthermia was detected in groups inoculated with vaccine A (p < 0.017) and vaccine B (p < 0.008), respectively.Clinical response of pigs immunised with experimental PRRS DNA vaccines and challenge infected with virulent PRRSV.1 See Table 1 for the vaccine identification; 2 Vaccine or plasmid only were administered three times, i.e., 84, 56 and 28 days before challenge infection; 3 Day of onset after challenge infection/length of period during which clinical signs were detectable.Viral RNA sequences were detected in pigs of all groups from PCD 2, 9 and 14. On PCD 20, only some animals in Groups 1, 3 and 4 were negative, while on PCD 28, 61 and 90 all pigs resulted to be devoid of virus (Table 3). No statistical significant differences were evidenced among the different groups in viremia as shown by RT-Real time PCR. Porcine reproductive and respiratory syndrome virus (PRRSV) detection in serum samples by RT-Real time PCR from pigs, immunised with experimental DNA vaccines, and challenge infected with virulent PRRSV. 1 See Table 1 for the vaccine identification; 2 Vaccine or plasmid only were administered three times, i.e., 84, 56 and 28 days before challenge infection; 3 Number of pigs from which virus was recovered.No increase in antibody titre to PRRS was detected in the vaccinated pigs (Table 4). No seroconversion was detected in the control group inoculated with the plasmid vector. After challenge infection neutralizing antibodies evaluated on PCD 61, 90 had a titre from 1.50 log2 (Groups 6, 7) to 3.50 log2 (other groups).Serum neutralizing antibody response of pigs immunised with experimental PRRS DNA vaccines and challenge infected with virulent PRRSV.1 See Table 1 for the vaccine identification; 2 Vaccine or plasmid only were administered three times, i.e., 84, 56 and 28 days before challenge infection; 3 Expressed as log2 of the reciprocal of the highest dilution inhibiting cytopathic effects (mean value).IgM were first detected on PVD 30 in pigs injected with vaccine A or B with a mean titre of 2.01 log2. These titres increased to 3.07 log2 and 3.13 log2 on PCD 0, respectively, and decreased until PCD 90 with a mean titres 2.19 log2 and 2.10 log2, respectively. No IgM were detected in the other vaccinated groups as well as in the controls on PCD 0. In these animals as well as in the control group, IgM were detected only following PRRS experimental infection with mean titres of 2.78, 2.88, 2.85, 3.04, 2.91 log2 for pigs vaccinated with products C, D, E, plasmid vector and challenge infection controls on PCD 14. These titres did not vary significantly on PCD 28, 61, 90 (Table 5). ELISA Immunoglobulins M (IgM) response of pigs immunised with experimental PRRS DNA vaccines and challenge infected with virulent PRRSV.1 See Table 1 for the vaccine identification; 2 Vaccine or plasmid only were administered three times, i.e., 84, 56 and 28 days before challenge infection; 3 Expressed as log2 of the reciprocal of the highest serum dilution positive by ELISA (mean value).IgG were first detected on PCD 0 in pigs inoculated with vaccines B or C with a mean titre of 1.45 log2 and 1.59 log2, respectively. These titres increased to 3.02 log2 and 3.03 log2, respectively, on PCD 61. Then, they decreased to a mean titre 2.82 log2 and 2.42 log2, respectively, until PCD 90. No IgG were detected in the other vaccinated groups as well as in the controls on PCD 0. In these animals as well as in the control group, antibodies were detected only following PRRS experimental infection with mean titres of 2.43, 2.12, 2.50, 2.55, 2.12 log2 for pigs vaccinated with products A, D, E, plasmid vector and challenge infection controls, respectively on PCD 14. These titres increased from 2.69 log2 to 3.13 log2 on PCD 61and decreased from 2.34 log2 to 2.82 log2 on PCD 90 (Table 6). IFN-γ were detected only in the groups of pigs inoculated with vaccines B or C on PVD 84 with a mean titre of 13 and 10 pg/mL, respectively. A further increase was detected in these animals on PCD 14 when the mean was 41 and 59 pg/mL, respectively. In the other groups immunized with Vaccines A, D, E, plasmid vector and in the challenge infection controls, IFN-γ were detected only on PCD 14, with a mean titre ranging from 12–36 pg/mL. In all groups, a decreased of IFN-γ was detected on PCD 61 with a mean titres of 2 pg/mL (Figure 8).ELISA Immunoglobulins G (IgG) response of pigs immunised with experimental PRRS DNA vaccines and challenge infected with virulent PRRSV.1 See Table 1 for the vaccine identification; 2 Vaccine or plasmid only were administered three times, i.e., 84, 56 and 28 days before challenge infection; 3 Expressed as log2 of the reciprocal of the highest serum dilution positive by ELISA (mean value).IFN-γ response (pg/mL) in pigs immunised with experimental PRRS DNA vaccines and challenge infected with virulent PRRSV.After challenge, a significant difference was detected in the time-related changes of CD4-CD8+ lymphocytes (p < 0.001) between the pig groups injected with the different plasmids and the controls. A higher difference was observed also on PCD 14 and 28 for γ/δ (p < 0.037) and CD16 (p < 0.0001) (data not shown). Necropsy was conducted on PCD 90. Gross pathology related to PRRS infection was observed in vaccinated and control pigs. Histopathology lymphoid organs were characterized by lymphocytic hyperplasia in infected pigs. Moreover, mononuclear parenchyma infiltrations were constantly observed in lungs. No histological lesions were observed in other target tissues collected in pigs of all groups.Viral glycoproteins of PRRS virus have been identified as the main targets for humoral and cell-mediate immune responses and they have been selected as candidate antigens in novel vaccine strategies such as DNA immunization. The genome of PRRS virus contains 10 open reading frames (ORFs). ORF1 encodes for viral replicase polyproteins that are immediately translated upon viral entry, and proteolytically processed by viral encoded proteinases into different non-structural proteins. ORF2a, ORF2b, ORF3, and ORF 4 encode for the structural proteins GP2a, GP2b (E), GP3 and GP4, respectively. ORFs 5–7 encode for three major structural proteins, respectively, i.e., the envelope glycoprotein GP5, the non-glycosylated membrane protein (M) and the nucleocapsid protein (N). Immunization protocols against GP4, GP5, M, N proteins have already been tested with the aim to evaluate their safety and efficacy in miceand pigs [7,17].Currently, the control of PRRS infections is performed by using two types of commercial PRRS vaccines based on the use of killed-virus (KV) vaccine or modified-live virus (MLV) vaccine. It was proven that KV vaccine can stimulate partial immune protection to PRRSV [18], while PRRS MLV vaccines seems to be more efficacious for protection against clinical signs induced by homologous infection; however, PRRS MLV vaccines have the disadvantage to revert to virulence [19,20,21,22]. With the aim to achieve the above mentioned, a new generation of PRRS vaccines is being explored and in recent years DNA vaccines have been able to induce effective humoral and cell-mediated immune responses in different animal models [4,5,6,7,23,24,25]. However, most viral DNA vaccines are designed to express only one antigen and, thus, their efficacy is lower compared to conventional vaccines [26,27]. Different strategies have been performed to improve DNA vaccines such as the choice of vector and target protein as well as the use of an adjuvant or co-immunogen [27,28,29]. The pVAX1 plasmid used in this study was selected due to its small size that provides a very high level of protein expression, thus minimizing extraneous genetic elements. This strategy appeared to be successful as the expression of proteins from different plasmids in NIH-3T3 cells was confirmed by immunofluorescence, clearly demonstrating the ability of the different genes to be expressed in vitro. In order to increase the immunogenicity of DNA vaccines, we used novel adjuvant approaches i.e., the incorporation on the DNA vaccines of CpG oligodeoxynucleotides (ODN) [23]. In this study, a plasmid (pVAX-48CpG) was constructed containing 48 copies of the CpG hexamer (GTCGTT), organized in the same way as the ODN 2135 [24]. This vector was used to express the GP4 or GP5 of PRRSV (Vaccines A and B).In Vaccines C and D, an additional sequence (UbilacI) was included in order to enhance the cell response to GP4 or GP5. UbilacI encodes for a proteasome-dependent degradation signal that mediates intracellular protein degradation and the production of peptides for antigen presentation via MHC class I. Hence, the proteasomal degradation of GP4 and GP5 should increase the number of peptides available for MHC-I binding, which may enhance the cell-mediated immune response to the vaccine antigens. The experimental infections conducted showed that PRRS DNA vaccines in our study did not protect pigs against infection with virulent PRRS but that they were able only to reduce clinical signs. In particular, pigs treated with Vaccines A and B developed milder clinical signs compared to controls. In contrast, pigs of Group B cleared the virus more slowly than pigs in Groups A, C, D and E.The results obtained in this study are in contrast with those obtained from previous studies carried out with traditional PRRS inactivated vaccines. In particular, commercial KV vaccines were not able to reduce clinical signs in vaccinated pigs and challenge infected with a homologous virus [22]. On the other hand, results obtained by Vaccines A and B are similar to those detected following the vaccination with MLV vaccines which can reduce the duration of clinical signs by up to about one week [30,31,32,33]. A significant increase of IgM was observed only in pigs of Groups A and B (PVD 30). In contrast, IgG were detected only in Groups B and C on the day of the challenge (PCD 0). These results are in accord with data reported by others [7], following DNA vaccination and no antibodies to GP4 and GP5 were detectable by ELISA test for a period of 12 weeks after vaccination. In contrast, both KV and MLV vaccines induce IgM and IgG response due to the presence of complete virus particlesin the vaccine [32,34].In this study, no increase in neutralizing antibody was found in all vaccinated pigs in agreement with other authors [17] but they are in contrast to the findings of Kwang et al. who detected neutralizing antibodies to GP4 and GP5 at a dilution 1:8 on week 12 after vaccination [7]. Similarly, Du et al. demonstrated the presence of neutralizing antibodies, although at a low level, following vaccination with a plasmid which expressed in fusion form GP3 and GP5 and the titres increased by using a plasmid encoding IFN-γ and IFN-α [28]. On the contrary, findings of studies carried out by other authors showed the inability of the GP4 and GP5 to stimulate an immune response from the host [4]. GP4 and GP5 of PRRSV have been able to stimulate neutralizing antibodies, and accordingly, GP5 has been suggested as a candidate for this study [7,25,35,36,37,38]. On the contrary, Li et al. have demonstrated the inability of pig anti-GP5 ectodomain antibodies or GP5/M ectodomain polypeptides to inhibit infection of permissive cells, indicating that GP5 and M surface epitopes are not directly involved in virus interaction with host cells [4].The results of this study showed that plasmid encoding GP4 or GP5 gene was not able to stimulate immune response, in contrast to what was supported by others authors [8] who demonstrated the immune efficacy of GP5 associated with cytokine as adjuvant. In particular, interleukin 15 (IL-15) can activate immunologic system via IL-2 receptor and it plays a role in generating and maintaining high activity of T cell responses to pathogens. Observed inefficacy to stimulate neutralizing antibodies against GP4 might be the result of hypervariability of the region encoding neutralizing epitope of GP4 [2]. Moreover, failure in inducing neutralizing antibodies following DNA vaccination could be the consequence either of a reduced transcription/translation process of the ORFs cloned sequences or of the inability of the commercial ELISA test (IDEXX) to detect antigens encoded by ORF4 and ORF5 genes [39]. Findings from the performed study suggest that vaccine C seems to be more immunogenic than other types as indicated by viremia, lasting for a shorter time and by stimulation of cell-mediated immune response.In contrast, NeuL sequences included in Vaccines A and B, ubiquitin sequences included in vaccine A and D and CpG motifs included in vaccine E were not able to stimulate any immune response. GP4 protein expressed with 48 CpG motifs and ubiquitin in Vaccine C, presumably due to insufficient amounts of antigen, did not stimulate B-cells and that did not produce the humoral immune response. Moreover, it is known that PRRS virus is unable to stimulate an efficient immune response as shown by the delayed appearance of neutralizing antibodies and cell-mediated immunity, following either infection with a virulent strain or vaccination [40]. According to that, a similar behaviour is expected to be detected following DNA vaccination. The unsatisfactory results of the study can be explained by the inability of the plasmid used to activate (priming) the immunological system as well as by the partial expression of virus antigens—only limited to GP4 and GP5 proteins—that can be responsible for the stimulation of an immune response at a lower level induced by a complete virus.No DNA vaccines used in this experiment showed any residual pathogenicity for respiratory apparatus as shown by macroscopic investigations and histology. These findings could be caused by the long interval period between challenge infection and necropsy. To conclude, the results of this study indicate that vaccination of pigs with DNA vaccines expressing GP4 of PRRS combined with CpG motifs and ubiquitin sequences has been able to prime the immune system. However, this response was not able to protect the animal from virulent PRRS challenge infection as shown by the lesser severity of clinical signs and the viremia.No DNA vaccines used in this experiment showed any residual respiratory pathogenicity as shown by anatomo-pathological investigations. These results indicated that PRRS DNA vaccines expressing GP4 combined with CpG oligodeoxynucleotides (ODN) in the plasmid backbone could be used for priming the immune system against PRRS infection.This research was financially supported by the Ministry of Public Health. The authors are grateful to Gigliola Canepa, University of Milan (I) for language revision. A special thanks to Silvia Dotti, Annalisa Guizzardi, and Sabrina Guana for her technical support and for their data elaboration.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Increasing numbers of HIV-infected individuals have access to potent antiretroviral drugs that control viral replication and decrease the risk of transmission. However, there is no cure for HIV and new strategies have to be developed to reach an eradication of the virus or a natural control of viral replication in the absence of drugs (functional cure). Therapeutic vaccines against HIV have been evaluated in many trials over the last 20 years and important knowledge has been gained from these trials. However, the major obstacle to HIV eradication is the persistence of latent proviral reservoirs. Different molecules are currently tested in ART-treated subjects to reactivate these latent reservoirs. Such anti-latency agents should be combined with a vaccination regimen in order to control or eradicate reactivated latently-infected cells. New in vitro assays should also be developed to assess the success of tested therapeutic vaccines by measuring the immune-mediated killing of replication-competent HIV reservoir cells. This review provides an overview of the current strategies to combine HIV vaccines with anti-latency agents that could act as adjuvant on the vaccine-induced immune response as well as new tools to assess the efficacy of these approaches. Despite the undeniable success of antiretroviral therapy (ART) in limiting HIV replication, it has become increasingly evident that ART is not a long-term solution for HIV-infected individuals. Besides the deleterious side effects, ART does not eradicate HIV and does not optimally reconstitute the immune system [1,2]. Novel immunotherapeutic strategies that would induce the immune-mediated control of HIV replication in the absence of ART (also called “functional cure”) are needed. However, tested vaccination strategies have so far shown limited success (reviewed in [3]). Immune mechanisms of HIV control are still unknown and need to be elucidated in order to help develop these therapeutic interventions. In individuals who naturally maintain undetectable viral load without ART, called elite controllers (ECs), the control of HIV replication in the absence of treatment has been primarily attributed to an effective adaptive immune response [4]. However, in most HIV-infected subjects, HIV-mediated immune damages (mainly CD4+ T cell depletion and chronic inflammation) lead to a dysfunctional immune response that is not restored by ART [1,5]. Moreover, viral control is only achieved by ART and treatment interruption results in a rapid return of viremia. The major obstacle to HIV eradication is the persistence of latent proviral reservoirs that are not targeted by antiretroviral regimens [6,7,8,9,10]. Eradication strategy aims at the induction of viral replication in latently-infected cells and at the elimination of these reactivated cells by either direct cytolytic targeting or by immunotherapeutic intervention [11]. HIV-specific CD8+ cytotoxic T cells (CTLs) are important for the control of HIV replication in non-treated individuals but in subjects under ART their number is too low to kill the latent reservoirs. Recently, Dr. Siliciano’s group showed that after in vitro expansion of HIV-specific CD8+ T cells from ART-treated subjects, these cells were able to eliminate HIV-infected CD4+ T cells [12]. This seminal study provided the rationale for new therapeutic strategies that combine agents that reactivate latently-infected CD4+ T cells with immune interventions that increase the numbers and function of HIV-specific CD8+ CTLs to clear HIV reservoirs in individuals on ART. However peptide-stimulated HIV-1-specific CD8+ T cells were used in this study, which might not reflect in vivo approaches. In addition, in this study only one histone deacetylase inhibitor (HDACi), Saha, was used. This molecule was also used in a clinical trial and was able to increase the levels of HIV-DNA in ART-treated donors [13]. Other more potent HDACis may induce stronger cytotoxic effects during HIV-1 reactivation, and cause selective cell death in CD4+ T cells in which successful viral reactivation occurs. Different approaches are currently being tested to reactivate latently-infected cells and restore immune functions. These molecules include HDAC inhibitors, mediators of T cell homeostasis or antibodies to block negative regulators; they all focus on reactivating latently-infected CD4+ T cells to render them susceptible to immune-mediated killing while also potentiating HIV-specific CTLs to kill reactivated CD4+ T cells. This review will describe the current knowledge and advances using these therapeutic strategies.Functional cure from HIV infection was achieved for the first time by Timothy Brown, the Berlin patient, who was given hematopoietic stem cell transplant (HSC) from CCR5 delta 32 donor (mutation of the gene required for HIV entry) [14]. Brown remains HIV free without ART after 6 years. Recently, two subjects from Boston, USA, with Hodgkin’s lymphoma and treated with ART were given a CCR5+/+ haemopoietic stem-cell transplant [15]. The two subjects had undetectable HIV-DNA years after transplantation. These findings suggest that ablative conditioning, immunosuppressive treatment, and post-transplant graft-versus host might be the reasons of the functional cure, more than the CCR5 deletion.The components of an efficient immune response able to control HIV replication after treatment interruption are still to be identified. In ECs, the natural control of HIV replication has been mainly attributed to strong T cell responses directed to dominant epitopes restricted by HLA types associated with viral control [4,16,17]. However, these responses cannot be elicited by a vaccine regimen in subjects that do not carry these HLAs. Recent studies suggested that initiating ART in early HIV infection could lead to control of HIV replication control in 5% to 15% of individuals after analytical treatment interruption (ATI) in individuals missing the known genetic characteristics of ECs [18,19,20,21,22,23,24,25,26,27]. In the VISCONTI cohort, 14 subjects achieved long-term post-treatment control of HIV replication after ART cessation [18,21]. Very early treatment (30 h after birth) was given to the Mississippi baby that was the first case of functional cure of an infant [19]. What cured the baby is still not fully understood but one reason might be prevention of the formation of latent reservoirs by very early treatment. These rare cases initiated ART during the early/acute phase of infection and interrupted ART after some years of ART, suggesting that early treatment initiation in acute HIV infection would lead to specific immune functions that are able to control HIV replication after ART cessation. Early and prolonged ART has been recently shown to be associated with an HIV-specific CD8+ T cell cytokine profile comparable to that of long-term non-progressors [28,29]. However, the mechanisms of viral control in this small number of subjects have not been elucidated yet. Furthermore, the magnitude of HIV-specific CD8+ T cells is low in these very early treated donors, which may limit studies. Moreover, no evidence of HIV-specific CD8+ T cells-mediated control of viral control after ART cessation was demonstrated in the VISCONTI cohort [21]. Other studies are currently testing whether the very early treatment in acute infection could lead to control of viral rebound after ATI [30,31]. The analysis of the characteristics of the immune response directed against HIV before and after ART interruption in these rare cases will provide clues that will guide the development of new vaccine interventions. Several studies suggest the need for efficient HIV-specific CD8+ T cells to control viral replication after treatment cessation. HIV-specific CD8+ T cells are a promising tool to eliminate reactivated, latently-infected CD4+ T cells and cure HIV infection as they are already important for the control of HIV-1 replication in non-treated individuals [16,32]. However, upon initiation of ART, their frequency declines rapidly and few memory cells are maintained. Previous efforts aimed at augmenting HIV-specific CD8+ T cell responses with structured treatment interruptions or vaccine regimens in ART-treated donors and in non-human primates have not been successful to control viral replication after ART cessation. Recently, Hansen et al. described a new RhesusCMV viral vector that induced the viral control of half of the infected macaques after challenge [33]. The wide breadth and non-conventional CD8+ T cell responses induced by the vaccine could contribute to the dramatic control of SIV replication in the vaccinated animals [33]. These results suggest that different and more efficient CD8+ T cell responses targeting HIV epitopes could control HIV replication. HIV-specific CD8+ T cells play a crucial role in mediating antiviral immunity by killing the productively infected CD4+ T cells. The critical role of CD8+ T cells in controlling viral replication has been demonstrated in acute infection in the SIV model, where CD8+ T cells depletion leads to a sharp increase in viremia [34]. Several other observations suggest that HIV-specific CD8+ T cells are important for the control of HIV-1 replication, including the generation and maintenance of viral escape mutations in CTL epitopes or the superior control of viral replication by certain HIV-specific clonotypes restricted by HLA-B57 and B27 [16]. Yang et al. demonstrated a significant association between CD8+ T cell viral inhibition activity in vitro and the rate of CD4+ T cell loss in early HIV infection and CD4+ T cell decline in chronically infected individuals [35]. In a macaque model for HLA-B27 mediated viral control, the control of viral replication was associated with high frequencies of SIV-specific CD8+ T cell responses directed against three epitopes [36]. These data highlight the important role of eliciting efficient CD8+ T cells for the control of viral replication [23]. Previous reports have shown that in vivo induction of high-avidity, high-frequency CD8+ T cell responses were associated with antiviral protective immunity [36,37,38]. The selection and maintenance of high affinity clonotypes early in acute infection could play an important role in the efficient killing of infected CD4+ T cells after treatment interruption [16,39,40]. However, clonal depletion has been observed during primary HIV infection [41]. We have previously shown a marked degree of clonotypic turnover within HIV-specific CD8+ T cell populations as a consequence of antigen decay after the initiation of ART with particular clonotypes selected for their higher functional sensitivity [42]. Therefore, maturation of the TCR repertoire towards these high affinity clonotypes could play an important role in viral control after ATI. However, previous efforts aimed at augmenting CD8+ T cell responses in ART-treated donors with structured treatment interruption or using vaccine regimens have not been as successful as hoped to control viral replication after ART cessation [24,43,44,45,46]. Novel CD8 T cell-based vaccine strategies are therefore needed to increase the number and function of HIV-specific CD8+ T cells that could control viral rebound after ART cessation, with the ultimate goal of achieving spontaneous control of viral replication without treatment [47]. Several molecules currently tested in clinical trials to reactivate HIV from latently-infected CD4+ T cells have demonstrated promising in vitro and ex vivo activities in reactivating latent HIV reservoir (shock) [48,49,50,51]. These molecules include HDAC inhibitors, mediators of T cell homeostasis or antibodies to block negative regulators. These molecules could also differentially influence the selection, expansion, persistence and function of HIV-specific CD8+ T cell responses stimulated contemporaneously by a vaccine strategy (kill).Post-translational modification including phosphorylation, acetylation, methylation and ubiquitination are thought to contribute to transcriptional regulation by inducing an “open” (transcriptionally permissive) vs. “closed” (transcriptionally repressive) state of chromatin. During HIV infection, many transcription factors bind to the LTR and induce silencing of the HIV promoter by recruiting histone deacetylases (HDACs). C-promoter binding factor-1 (CBF-1) and the NF-kappaB homodimer p50/p50 are two of the transcription factors that have been shown to bind the LTR enhancer sequence thus promoting transcriptional silencing during the establishment of HIV-1 latency [52,53]. Thus, one of the promising candidate families of molecules tested in clinical trials to reactivate latent HIV reservoirs are histone deacetylase inhibitors (HDACis). Among the HDACis, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) have been tested in HIV infected subjects under ART. However in the first clinical trial, VPA failed to reactivate the virus from latently infected cells and thus to decrease the reservoir. Most promising results in a recent clinical trial showed that SAHA was able to reactivate viral reservoirs in subjects on ART [13]. There is limited knowledge of the chromatin remodeling and non-epigenetic effects of HDACis on the differentiation of CD8+ T cells in humans. Studies in mice have shown that CD8+ T cells activated without CD4+ help, fail to develop functional, protective memory and remain hypo-acetylated. Treatment with an HDACis increased histone acetylation in unaided CD8+ T cells and restored their ability to differentiate into functional memory cells capable of immediate cytokine production and providing protective immunity [54]. Besides being able to reactivate viral production in latently-infected CD4+ T cells and increase HIV antigen presentation by MHC I, HDACis could also change the fate of HIV-specific CD8+ T cells induced by a vaccine.The combination of systems biology, phenotypic and functional profiles suggests that PD-1 is a good target for therapeutic interventions aimed at restoring CD8+ T cell function in HIV infection. Different groups have already shown that a PD-1 blockade in various diseases such as HIV, hepatitis B, and hepatitis C is able to restore T cell proliferation, cytokine production and thus effector function [55,56,57,58,59,60,61]. While it has been suggested that blocking PD-L1 has a better capacity to restore T cell function than targeting PD-1 itself [55,62], in both cases blocking this interaction resulted in increased HIV-specific CD8+ T cell proliferation [63]. Recent in vivo studies have been conducted in the rhesus macaque SIV infection model [58,64]. Velu et al. showed that PD-1 blockade resulted in increased frequencies of SIV specific CD8+ T cells, increased cytotoxic function and decreased viral load [58]. Many groups have also shown that the exhaustion of HIV specific CD8+ T cells from chronic HIV infected subjects is associated with the co-expression of different negative regulators, such as PD-1, CD160 and 2B4. The most exhausted cells simultaneously express multiple negative regulatory receptors on their cell surface and their expression positively correlates with viral load and decreased cytokine production [65,66]. Blocking the interaction of CD160 and HVEM was able to enhance and rescue HIV-specific CD8+ T cell proliferation and cytokine production. All together, these findings are in agreement with previous studies that provide a strong rationale for initiating human clinical trials targeting PD-1 with blocking antibodies in HIV-infected subjects. Blocking PD-1 or multiple negative regulators might not only increase effector functions of exhausted HIV-specific CD8+ T cells but could also reactivate the viral reservoir from latently infected CD4+ T cells [67]. Thus, combination therapy that combines vaccination under ART with blocking antibodies for PD-1 signaling might prove to be more potent in increasing CD8+ T cell killing of latently infected cells and to preventing reactivated virus from re-infecting new CD4+ T cells. Gamma chain cytokines (IL-2, IL-15, IL-7 and IL-21) that converge on the STAT5A/B signaling pathway have been considered as candidates to regulate reservoir reactivation. These cytokines can also be used as modulators of a vaccine immune therapy [68]. During HIV infection, production of some of these cytokines, such as IL-2 and IL-15, is downregulated, while IL-7 levels are increased as a consequence of lymphopenia. Extensive phase I and II studies were done in the late 1990s with IL-2 as a candidate cytokine for treatment of subjects with HIV infection. These studies demonstrated that this cytokine increases the frequency of naïve and central memory (TCM) CD4+ T cells, as well as CD8+ cytotoxic functions [69,70,71]. However, phase III clinical trials demonstrated that IL-2 increased CD25 and FOXOp3 expression (and thus regulatory T cells), which was associated with increased risk of opportunistic diseases [71,72,73]. Similar to IL-2, IL-15 signals through the IL-2RB (CD122) and γ-chain (CD132) receptors and plays an essential role in T cell survival [74]. Although both IL-2 and IL-15 induce identical signal-transduction pathways and proliferative responses in T cells and NK cells, IL-2 favors maintenance of peripheral regulatory T cells and participates in activation-induced cell death while IL-15 preferentially stimulates expansion of CD8+ T cells, NK, and NKT cells [75]. Stimulation of cells from HIV-infected subjects with IL-15, enhances the frequency of effector memory CD8+ T cell, promotes their survival and cytotoxic functions [76,77,78,79,80]. Stimulation with IL-15 might also relieve HIV-specific CD8+ T cells from their functional and phenotypic block in a transitional memory (TTM) phenotype (CCR7−, CD27+, CD45RA−) and induce their differentiation in functional effector antigen-specific CD8+ T cells [67]. Preclinical studies have shown that IL-15 enhances CTL responses in murine tumor models and the administration of IL-15 in combination with anti-PD-L1and anti-CTLA-4 antibodies showed greater cytolytic and effector functions on the CD8+ T cells of metastatic tumor-bearing animals [81,82,83,84,85]. Studies in non-human primates showed that daily administration of IL-15 was not only safe but increased the frequency of effector memory (TEM) CD8+ T cells of 100-fold [86,87]. However another study showed that administration of IL-15 in chronically SIV infected macaques, in combination with ART, resulted in a delay in viral suppression and failed in the reconstitution of CD4+ T cell numbers after ART interruptions [88]. IL-7 regulates T-cell maturation and supports peripheral T cell homeostasis. Preclinical studies in SIV-infected macaques showed that IL-7 injection was not toxic and was not increasing viremia [89,90]. Phase I/II trials in HIV infected subjects with persistent lymphopenia have demonstrated that the administration of IL-7 was able to restore circulating CD4 T-cell counts as well as the frequency of CD8+ T cells, mainly those with a central memory (TCM) phenotype [91] without any effect on the number of regulatory T cells. IL-21 is another γ-chain cytokine that induces a potent cytotoxic activity of NK and CD8+ T cells from HIV infected subjects and promotes antiviral activity in human CD8+ T cells [92]. HIV-specific IL-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function [93]. The benefic role of this cytokine has been confirmed in a pilot study where IL-21 was injected in SIV infected rhesus macaques in late-stage disease [94]. Administration of IL-21 resulted in increased cytotoxic effector molecules in CD8+ T cells and NK cells and as well as enhanced B cell differentiation.Eradication strategies should rely not only in boosting CTL activity, but also at enhancing HIV reactivation from latently infected CD4+ T cells. Few studies have shown that among the γ-chain cytokines, IL-7 is a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs [50,95]. Vandergeten et al. showed that IL-7 increases reactivation in productively infected cells but has no effect in latently infected CD4+ T cells whereas IL-15 seems to be a more potent inducer of viral production from latently infected CD4+ T cells than IL-7 [96,97]. Administration of IL-15 during the contraction phase (7–14 days) and not during the expansion phase could have a better effect in the enhancement of HIV-specific CD8+ T cells response after vaccination. Further studies should be done to determine the timing of cytokine delivery in combination with a vaccine regimen. All together these findings provide a good rationale for complementing vaccine therapy with γ-chain cytokines in eradication strategies aimed at purging latent HIV from CD4+ T cells and at boosting HIV-specific CD8+ T cell responses.Few parameters including frequency, phenotype, and cytokine production are commonly used to assess T cell responses [98]. The frequency and phenotype of T cells are limited in evaluating their functions. T cell poly-functionality has been suggested to correlate with HIV disease control, however it is still unclear if this feature is sufficient to provide T cell-mediated immune control [99,100]. Therefore, new functional assays have to be developed to measure T cell functions focusing on select immune parameters known to impact the control of HIV replication. The cytolytic function of CD8+ T cells is critical for eradicating HIV-infected CD4+ T cells and control viral replication after ART interruption. New in vitro methods to evaluate the viral replication inhibition by CD8+ T cells have been recently developed [12,101,102] and can be used to recapitulate the in vivo cytotoxic activity of CD8+ T cells after ATI. A study by Yang et al. demonstrated a significant association between CD8+ T cell antiviral activity in vitro and the rate of loss in early HIV-1 infection and CD4+ T cell decline in chronically infected individuals using an in vitro viral inhibition assay [35]. We developed a new assay to quantify the intrinsic killing capacity of HIV-specific CD8+ T cells measured in lytic units [103]. Using this assay, we have observed that HIV-specific CD8+ T cells in primary infection exhibited a significantly higher cytotoxic capacity than HIV-specific CD8+ T cells in chronic infection [104]. These new functional cell-based assays can help determining if an enhanced cytotoxic activity of CD8+ T cells is associated with viral control after ATI. Furthermore, new assays that can recapitulate the killing of primary latently-infected CD4+ T cells by HIV-specific CTLs from ART treated donors would be an important platform to test the immune intervention strategies of reservoir eradication as it would provide the direct readout of elimination of reactivated latently-infected cells. New CD8+ T cell-based vaccination strategies under ART should be evaluated in combination with reactivating agents to achieve control of viral replication upon ART interruption. Immunotherapeutic interventions not only need to increase the number of HIV-specific CD8+ T cells in vivo but also induce efficient cytotoxic CD8+ T cells to kill reactivated latently-infected CD4 T cells. However, the mechanisms that underlie such successful immunotherapies remain unknown. These immune interventions have to be combined with contemporaneous strategies aimed at reactivation of the latent HIV reservoir. Combinations could also include new strategies such as vaccination with a biologically active Tat protein that protected non-human primates against an R5-SHIV challenge by neutralizing antibodies [105]. These combinations have to be judiciously selected to potentiate the vaccine-induced response. The effect of reactivating agents on the immune response induced by therapeutic interventions has to be also evaluated. The accurate assessment of new therapeutic vaccine regimen would accelerate the development of successful therapies for a functional cure.This work was supported by the Office of Tourism, Trade and Economic Development of Florida.The authors declare no conflicts of interest.

Med-MDPI/vaccines/vaccines-01-04-00497.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The HIV-1 envelope glycoprotein spike is the target of neutralizing antibody attack, and hence represents the only relevant viral antigen for antibody-based vaccine design. Various approaches have been attempted to recapitulate Env in membrane-anchored and soluble forms, and these will be discussed here in the context of recent successes and challenges still to be overcome.To fully grasp the potential of the HIV-1 Env trimer for vaccine use, its structure and function need to be understood. HIV-1 carries a single virally-encoded structure on the outer surface of its envelope, the Envelope glycoprotein (Env). Env consists of a non-covalently linked trimer of heterodimers, each heterodimer composed of one surface gp120 subunit and one transmembrane subunit (Figure 1). The env open reading frame codes for a precursor trimer polypeptide (gp160) that is trafficked from the endoplasmic reticulum to the Golgi, within which it is cleaved by a cellular protease into its mature components [1]. Cleavage refolds Env into an activated, fusion-competent state via undefined structural rearrangements within the trimer. The mature trimer is then trafficked to the plasma membrane via a poorly defined pathway [1] that may involve regulated secretion induced by contact of the infected cells with an uninfected, receptor-bearing cell [2]. The cytoplasmic tail of gp41 carries endocytic motifs that drive traffic Env from the plasma membrane into either maturing endosomes leading to degradation or recycling endosomes, meaning that at steady state a large proportion of Env is within intracellular compartments [3,4]. This is suggested to be an immune evasion strategy, reducing cell surface Env recognition by B cells. Env targets lipid rafts via an acylation signal on gp41, meeting Gag at the cell membrane to initiate budding of the nascent virion [1]. The surface location of Env is dependent upon the infected cell type: CD4+ T cells predominantly express Env at the plasma membrane, whereas macrophages express Env principally within an intracellular compartment continuous with the plasma membrane called the virus-containing compartment (VCC) [5,6]. The Env gp120 subunit is the receptor-binding component, and is comprised of a three-domain substructure: the inner and outer domains and the linking bridging sheet [7] (Figure 1). Gp120 engages CD4 and a coreceptor (CR), either one of the chemokine receptors CCR5 or CXCR4, in a two-step process. The CD4 binding surface (CD4bs) on gp120 is constitutively accessible to CD4 on Env, but undergoes a conformational change stabilized by CD4 binding that triggers both high-affinity CD4 binding and structural rearrangement of the trimer to reveal the chemokine-binding surface (CRbs) [8]. Subsequent CR engagement by gp120 leads to further conformational rearrangement of the Env trimer that triggers gp41 activation leading to its refolding and proposed penetration of the target cell membrane [8]. The formation of a coiled-coil gp41 structure brings the viral and target cell membranes into close apposition, driving their fusion and allowing entry of the viral core into the cell [9].HIV-1 Env. (A) Cartoon of gp120 with major features represented. The location of the V1V2 loops, missing from gp120 crystal structures, is predicted from analysis of trimeric Env by electron tomography [10,11] and from the location of the quaternary conformation epitope-specific antibody PG9 by negative stain electron microscopy [12]; (B) Molecular model of gp120 based on crystal structures and obtained with permission from [13]. The gp120 surface is colored grey for inner domain, red for outer domain and blue for the bridging sheet. The initial contact surface for CD4 is shown in yellow cross-hatching, and the recognition surface of broadly neutralizing CD4bs antibody VRC01 is green; (C) Cartoon of the Env trimer with broadly neutralizing antibody epitopes depicted; (D) Molecular model of Env trimer with glycans. Red surface is gp120 density, yellow represents the CD4 binding site, hybrid and complex glycans are represented in blue and the 2G12 epitope high mannose glycans in white (from [14]).Env is essential for viral infectivity, and antibodies targeting functional Env trimers will neutralize virus by preventing receptor engagement and/or virus-cell fusion, inhibiting both cell-free and cell-to-cell spread [15]. Non-neutralizing antibodies may also impact upon HIV-1 replication by binding non-functional Env on HIV-1-infected cells and mediating effector functions such as antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis and IgA-mediated viral aggregation and sequestration in mucous [16,17,18]. Since the correlates of protection from immunodeficiency virus infection by neutralizing antibodies are robust [19,20,21,22,23,24], whereas those for non-neutralizing antibodies are not [25], here I will focus on neutralizing antibodies. HIV-1 has evolved a series of immune evasion strategies to reduce or abrogate the impact of neutralizing antibodies on replication. These have been extensively reviewed elsewhere [26,27,28], therefore the major mechanisms of antibody evasion by Env are only briefly, and non-exhaustively summarized. The primary immune evasion strategy concerns amino acid sequence variation in Env [29]. Although there is relative conservation of the receptor binding surfaces on gp120 and of several other regions of gp120 and gp41, major segments appear to be immunorecessive compared to the more variable portions of the molecule [27]. Thus the gp120 hypervariable regions, particularly the loop structures V1, V2 and V3, are primary antibody targets, but these antibodies can only neutralize a fraction of circulating viruses. Focusing the B cell response away from hypervariable regions and towards more conserved regions is a major challenge to antibody-based vaccine design.Env is very heavily glycosylated, and glycan masking of the underlying protein surface prevents ready access of antibodies to vulnerable conserved Env regions [30]. A specific example of this is the CD4bs, where a “fence” of glycans reduces the ability of antibodies to probe the CD4bs in the same way as CD4 itself [31], resulting in antibodies that bind more weakly to Env and hence have limited neutralization activity [32]. However, recent crystallographic studies have revealed that glycans can also entirely comprise [33], or be a significant part [34,35,36], of neutralizing antibody epitopes, and so may in fact turn out to be part of the solution to the problem. A related accessibility issue concerns steric hindrance of antibody access to conserved neutralizing epitopes by structures other than glycans. The CD4bs forms a shallow valley on gp120 into which domain 1 of CD4 can fit snugly (a single immunoglobulin domain), but which an antibody variable region (two immunoglobulin domains) does not readily access [7]. A second level of steric exclusion is also exercised within the Env trimer since the gp120 protomers are arranged in a configuration that presents a very limited “angle of approach” to the CD4bs [37]. The element of the CRbs that is formed when gp120 adopts the CD4 bound state is also sterically restricted for antibody access when the Env spike is engaged with target cell CD4: there is insufficient space between the target cell membrane and the top of the Env trimer to accommodate the bulk of an IgG molecule [38]. Size restrictions may also apply to the accessibility of MPER antibodies to their epitopes, as they are wedged between the lower surface of gp120 and the viral envelope [39].The trimer structure is reported to be metastable and able to adopt different conformational states, which are hypothesized to relate to an unliganded state, a CD4 bound, CRbs-accessible state, and an intermediate between these. Evidence for this comes from the ensemble of antigenic [40,41], functional [42], biochemical [43] and electron tomographic or single particle [44,45,46] analyses of different conformational states of intact trimers. Oscillation of Env between different conformational states is proposed to be part of a conformational barrier to antibody (and by extension B cell receptor) recognition [47]. An extreme example of conformational change comes from the dissociation of the trimer into subunits, by which gp120 is released in a soluble form [48,49] and gp41 adopts a post-fusion membrane-anchored conformation. This can be CD4-induced or spontaneous. Neither of these non-functional forms is likely to be able to induce potent or broad spectrum neutralizing antibodies. Env expressed at the plasma membrane of cells may be misfolded or uncleaved gp160, which are poor mimics of the functional trimer. Furthermore, HIV-1 infected cells may be lyzed by CTL or die by necrosis, releasing non-functional precursor forms of Env that will be recognized by B cells [50]. A final consideration relates to the number of Env spikes on a virion. This has been estimated to average approximately 10 [51,52] (although this number varies between different viral strains), a surface density that is suboptimal for cross-linking B cell receptors (BCRs) [53]. It has been hypothesized that the low number of spikes may lead B cells to undergo heteroligation (binding to two different epitope structures on an antigen) in order to drive high avidity antibody production [53]. When taken together, these evasion strategies provide a formidable barrier to antibody efficacy during natural infection, and make vaccine antigen design particularly difficult. However, results from diverse studies provide hope that trimeric Env, either native or structurally modified, might nevertheless be a useful antigen to elicit neutralizing antibodies in the appropriate immunization context. Historically, the first attempts at immunization with Env-based antigens used monomeric gp120 [54]. The neutralizing antibody responses obtained were relatively weak and were directed against a very limited spectrum of neutralization sensitive viral strains, termed tier-1 viruses, which do not represent most circulating strains [55]. It became apparent that the hypervariable V3 loop is a particularly immunodominant surface on gp120. However, despite efforts over 2 decades, V3 loop-based immunogens have failed to elicit antibodies that robustly neutralize a substantial proportion of circulating viruses that are intrinsically relatively neutralization resistant (termed tier 2) [56]. The most likely explanation for this is that the V3 loop is not well exposed on the majority of non-CD4-bound HIV-1 Envs, most likely as a result of packing into the surface volume of the trimer [57,58]. On soluble gp120 the V3 loop is liberated from constraints imposed within the trimer and becomes highly accessible to B cell recognition [59]. Analyses of antibody binding in sera from animals immunized with monomeric gp120 revealed strong binding to monomeric gp120 but very weak binding to trimeric Env [60]. This was one of the first indications that the immunogenic surfaces of gp120 that are exposed when the Env subunits dissociate are not represented on the trimer and are neutralization irrelevant. Together with the observed correlation between antibody binding to trimeric Env and neutralization [61,62], these findings prompted two considerations: firstly that antibody binding to functional forms of trimeric Env may be sufficient for neutralization, secondly that trimeric Env or proper mimics thereof may make useful vaccine antigens. The principal determinant of neutralization in these studies was antibody avidity, suggesting that the precise target epitope of the antibody was a less important consideration than the tightness of antibody binding to the functional trimer [61]. Moreover, unlike with simple antigens such as V3 peptides for which the antibody dissociation rate was the kinetic parameter correlating best with neutralization [63], on the Env trimer the association rate may also be a significant factor, implying that antibody accessibility to the epitope on the trimer is an important kinetic barrier [60]. This can now be explained by our current structural understanding of Env immune evasion properties relating to steric barriers to antibody-epitope engagement, including glycans and trimer architecture (Section 2). The rate at which an antibody binds its epitope is an important functional feature under conditions of competition with viral receptor engagement and entry, and requires further investigation.The recent isolation of multiple monoclonal antibodies with potent neutralization activity against a broad range of viral strains (broadly neutralizing antibodies) highlights the fact that Env has conserved, antibody-accessible surfaces on the functional trimer. Some of these antibodies neutralize up to 90% of circulating strains [64], testifying to high conservation of their epitopes. Structural analysis reveals that these antibodies fall into several different epitope-binding clusters, described in more detail elsewhere [29,65,66,67,68,69,70] and summarized in Figure 1. Briefly, these are: (I) the CD4bs; (II) a highly conformational epitope cluster formed from the quaternary folding together of the V1V2 and possibly V3 gp120 loops of at the apex of the trimer; (III) a patch of high mannose glycans alone (2G12 antibody), or together with the underlying protein surface forming glycoprotein epitopes; (IV) a segment of the extramembranal portion of gp41 termed the membrane proximal external region (MPER). Characterization of the atomic structure of the epitopes of these monoclonal antibodies has led to a flurry of activity in the molecular modeling field with the aim of producing conformationally constrained epitope mimetics that could be used to focus B cell responses towards what are otherwise immunorecessive targets. At present this field has produced antigens that share close atomic topology to their native counterparts in the trimer, a spectacular feat of engineering, but these have yet to elicit neutralizing antibodies by immunization [71,72,73,74]. One strategy will therefore be to use such epitope mimetics to prime or boost antibody responses made against native Env trimer structures. This will be discussed in more detail below.There have been numerous attempts to express both membrane-anchored and soluble forms of trimeric Env for immunization. To date none of these attempts have led to induction of potent neutralizing antibodies against highly conserved Env surfaces, but some incremental improvements in neutralization compared to immunization with gp120 have been observed, suggesting that this strategy is worth pursuing [75]. The most straightforward way to produce trimeric Env with a native, functional conformation is to express it in a membrane context. This can be achieved by in vivo expression from DNA or from vectors encoding env. Obvious advantages of this approach are that the glycoprotein is assembled and expressed in the host cells, therefore being post-translationally processed in a similar manner to the virus during an infection. A downside of this approach is that the amount of Env expressed in vivo is unknown, but is likely to be relatively low, generally resulting in weak antibody responses without further protein boosting. Moreover, the antibody evasion mechanisms inbuilt into Env, including endocytosis and the presence of misfolded, uncleaved and subunit dissociated forms will be active in this setting. A more controlled strategy that can potentially deliver greater quantities of antigen is the ex-vivo expression of env in the context of Gag, resulting in production of virus-like particles (VLPs) [76,77]. VLPs contain functional trimers and can be purified to high concentration. Membrane-anchored Env produced in high-level expression systems is not free from non-functional forms and contains uncleaved monomer gp160, gp41 stumps and other membrane-anchored “junk” [78,79] (Figure 2). Of particular interest, treatment of membrane Env with protease eliminated most non-native forms whilst preserving native Env, as probed by native gel electrophoresis and neutralizing antibody binding [79,80]. Moreover, soluble gp160 can be liberated from lipid using mild detergent and can be isolated in an intact form, allowing preparation of relatively pure forms of the antigen. Although these approaches raise the challenge of producing sufficiently large quantities of such native antigen for immunization purposes, they are nevertheless promising and deserve further development.Soluble glycoproteins have obvious advantages over their membrane-anchored counterparts, including ease of manufacture and availability of higher antigen concentrations for immunization. However, the removal of Env from its membrane environment by genetic truncation prior to the transmembrane region (producing a gp140 molecule) results in loss of trimer stability leading to misfolding and gp120-gp41 dissociation (Figure 2). Elimination of the cleavage site between gp120 and gp41 overcomes much of this instability, and many trimeric forms of gp140 have been produced with this feature [75]. Additional stability can be introduced by fusion with an exogenous trimerization motif or by strategic incorporation of a disulfide bond linking gp120 to gp41 in cleaved trimers, termed SOS gp140 [44]. Several studies have compared such trimeric Env antigens with gp120 for immunogenicity and for induction of neutralizing antibodies. The general conclusion from these studies is that first generations of trimeric gp140 yield a small incremental increase in neutralizing antibody elicitation, but to date fail to induce antibodies capable of neutralizing a broad spectrum of HIV-1 circulating strains [81,82,83,84]. Although the field lacks an atomic resolution structure of the assembled functional Env trimer, recent antigenic and structural analyses have nevertheless helped define shortcomings in the first generations of soluble trimers, and may provide a path forward to more faithful Env mimics. First, deletion of the gp120-gp41 cleavage site results in a molecule that predictably fails to fold into the mature functional form, and therefore does not antigenically mimic the target antigen on the virus [85,86,87]. The elimination of the gp120-gp41 cleavage site leads to various abnormalities in glycoprotein folding, including the potential for formation of aberrant disulfide bonds between gp120 and gp41 subunits. Although certain surfaces, such as the CD4bs, are well preserved and presented on soluble uncleaved gp140, others such as the quaternary V1V2V3 mAb epitopes are perturbed [88]. Indeed, these antibodies have become extremely useful probes for the conformational integrity of Env, and allow rapid and clear evaluation of trimer folding. The lack of quaternary broadly neutralizing antibodies binding to soluble uncleaved gp140 appears to correspond to structural rearrangements in the trimer that are represented as an “open” conformation in electron tomographic reconstructions of both membrane-anchored and soluble forms of Env [44] (Figure 2). This open conformation can be stabilized in membrane anchored Env by ligation with soluble CD4 or some neutralizing antibodies, and therefore represents the CD4-bound state of the Env trimer, exposing the CRbs and allowing binding of CRbs-specific monoclonal antibodies [44]. In conclusion, uncleaved gp140 represents a form of Env that fails to present some neutralizing antibody epitopes (quaternary epitopes), may present other broadly neutralizing antibody epitopes such as the CD4bs in a manner that fails to mimic the functional trimer, and exposes non-neutralizing antibody epitopes [87]. Inappropriate presentation of the CD4bs will lead to production of antibodies that recognize the surface with an angle of approach that is inconsistent with binding to the functional trimer [37], hence these antibodies will neutralize weakly or not at all. Non-neutralizing surfaces such as cluster-I and -II gp41 epitopes are likely to dominate immunogenicity, and may therefore be deleterious for elicitation of neutralizing antibodies [87]. It therefore seems unlikely that the use of this type of trimeric antigen will yield appropriate neutralizing antibody responses after immunization. As mentioned above, another approach to producing soluble native trimeric Env has been to genetically add a disulfide bond (SOS) that holds the extramembranal portion of gp41 to gp120 post cleavage without obviously disturbing the trimer conformation [89]. When expressed in a membrane bound form these trimers can drive receptor-mediated fusion in the presence of a reducing agent to break the introduced disulfide bond, and so are functional [90]. More recent iterations have introduced a further stabilizing mutation into gp41, producing a construct called SOSIP [91]. Recently described gp140 SOSIP trimers from the BG505 HIV-1 strain lacking most of the MPER have improved solubility [92], a compact and stable structure similar to that of membrane-anchored gp160 as defined by negative stain single particle EM [12,46,93] (Figure 2), and bind all broadly neutralizing antibodies and very few non-neutralizing antibodies [93]. Of particular importance, this antigen binds quaternary epitope-specific mABs, strongly implying correct gp120 folding within the trimer [12,93]. These antigens therefore appear to be very close mimics of the native membrane anchored trimer and the prototype of a new generation of potential vaccine antigens.HIV-1 Env trimers for experimental vaccine use. (A) Functional, cleaved HIV-1 Env expressed in a viral, infected cell, or VLP membrane; (B) Uncleaved soluble gp140 trimer with “open” structure; (C) Cleaved gp140 is unstable resulting in subunit dissociation; (D) SOSIP gp140 is stabilized by disulfide bonds and maintains a compact “closed” conformation; (E) EM reconstruction of BG505 SOSIP gp140 at 24A resolution showing compact globular morphology, from [93] with permission; (F) HIV-1 gp160 expressed from infected or transfected cells contains a proportion of so-called “junk” forms that may compete with the native trimeric forms for induction of neutralizing antibodies [79]. These may be at least partially removed by protease treatment [79]; (G) Disulfide (SOS)-stabilized membrane-anchored Env trimer [79,90]. Recent advances in the field have increased optimism that pure populations of correctly folded Env trimers can be produced in membrane anchored or soluble forms. This is excellent news as it will, for the first time, allow an evaluation of the structure, antigenicity and immunogenicity of such antigens without contamination from potentially immunodominant, incorrectly folded forms of Env. If immunogenicity could be predicted directly from antigenicity, then these antigens would be expected to elicit antibodies against the surface exposed conserved neutralizing epitopes and not against other non-neutralizing epitopes. However, the relationship between antigenicity immunogenicity is complex and the B cell response to these antigens is unpredictable [27]. In this respect the broadly neutralizing antibodies isolated from HIV-1-infected individuals, which provide proof of principle for the immunogenicity of the target epitopes that elicited them, may not be readily re-elicited by active immunization. Firstly, these antibodies have unusually high levels of somatic hypermutation, most likely driven by reiterative rounds of viral escape by amino acid variation [94]. Such variation will focus B cell responses towards more conserved regions of the antigen, which have unusual epitope structures potentially requiring substantial structural rearrangement of the antibody paratope. Examples of this are: (I) the CD4bs, which as mentioned above imposes stringent steric constraints on the size and shape of the paratope and the angle of approach of the immunoglobulin molecule to the antigen; (II) the MPER that is sandwiched between the lower portion of gp120 and the target cell membrane, driving the selection of antibodies that may bind both protein and lipid [39]. However, lipid binding may not be a prerequisite for MPER antibody function [95], and a recently isolated MPER reactive antibody (10E8) does not require lipid binding, making this epitope a potentially more straightforward target [96]. An epitope cluster that may not impose such stringent steric constraints for BCR and antibody recognition is that recognized by the Env quaternary conformation-specific antibodies. This epitope located at the apex of the trimer [12] (Figure 1) appears, at least in models made at molecular-level resolution, not to be subject to the same angle of approach and steric constraints as some other neutralization epitopes [83]. The virus may rely upon the conformation instability of this epitope on the virion to evade efficient B cell recognition. Thus if this epitope cluster was sufficiently stable on the trimer, it may make an effective immunogen for elicitation of this specificity of neutralizing antibody. An approach to eliciting this quaternary conformation Env antibodes may be to completely “fix” the trimer into the appropriate stable conformational state, either using molecular biology to introduce covalent bonds, or by using targeted chemical crosslinking strategies. In this respect, glutaraldehyde cross-linked membrane-bound Env completely stabilizes trimer structure but conserves binding of quaternary conformation antibodies [97].The immunorecessive nature of particular epitopes may be overcome by priming B cells with epitope mimetics that focus responses to these surfaces. The concept relies upon the presentation of the mimetics by a series of heterologous scaffolds that would each only be used once for immunization thereby focusing B cell responses to the mimetic [74]. This approach is elegant but has its critics, particularly as a stand-alone concept, since re-elicitation of a unique antibody paratope by an isolated epitope is highly improbable [98,99]. However, additional approaches may increase the probability of success. For example, B cell responses initiated by epitope mimetics may be boosted using trimeric Env to drive high-avidity recognition of the native target epitope in its correct molecular environment. If mimetic recognition by naïve BCRs fails, then epitope variants can be engineered to activate naïve B cells and subsequently guide the B cell response towards high-affinity recognition of the target epitope [12,94,100,101]. The degree of somatic mutation observed in neutralizing monoclonal antibodies obtained from infected individuals is likely to be very difficult to elicit by active vaccination using a non-persisting antigen. However, the magnitude of mutation observed may be a consequence of B cells reacting to the relentless variation of the target epitope until a non-variable target is arrived at, potentially through inability of the virus to further mutate that region without loss of function. Thus an invariant vaccine antigen may take a short cut to induction of high affinity antibody binding without requiring such a high degree of somatic mutation. Potent new adjuvants, in the form of extrinsic formulations or as co-stimulatory genetic fusions with the antigen may help to achieve efficient affinity maturation by activating T cell help, particularly from T follicular helper cells. Combinations of vector-delivered Env with protein boosting may also increase antibody titers and avidity. The field of trimer-based vaccine antigen development for eliciting neutralizing antibody responses has come a long way over the past 2 decades, and is currently in its most exciting phase. Both membrane-anchored and soluble forms of trimer that are precise mimics of the in-situ viral spike have been produced and are currently being characterized for their biophysical, structural, antigenic and immunogenic properties. The next few years will deliver critical information on whether this concept can work, alone or combined with other developing vaccine technologies.This work was supported by The International AIDS Vaccine Initiative Neutralizing Antibody Consortium. QJS is a James Martin Senior Fellow and a Jenner Vaccine Institute Investigator.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).When HIV was discovered and established as the cause of AIDS in 1983–1984, many people believed that a vaccine would be rapidly developed. However, 30 years have passed and we are still struggling to develop an elusive vaccine. In trying to achieve that goal, different scientific paradigms have been explored. Although major progress has been made in understanding the scientific basis for HIV vaccine development, efficacy trials have been critical in moving the field forward. Major lessons learned are: the development of an HIV vaccine is an extremely difficult challenge; the temptation of just following the fashion should be avoided; clinical trials are critical, especially large-scale efficacy trials; HIV vaccine research will require long-term commitment; and sustainable collaborations are needed to accelerate the development of an HIV vaccine. Concrete actions must be implemented with the sense of urgency imposed by the severity of the AIDS epidemic.The development of an HIV vaccine has been a long and tortuous process that, thus far, has consumed nearly 30 years of intense laboratory and clinical work. When HIV was discovered and established as the cause of AIDS in 1983–1984 [1], many people believed that a vaccine would be easily developed and rapidly deployed. After all, vaccinologists had been very successful in developing vaccines for a whole range of viral diseases. However, the paradigm that allowed the development of most existing viral vaccines, which is based on the recreation of the protective immunity that develops after natural infection, does not work in the case of HIV. In AIDS, virus-induced immune responses are not capable of preventing re-infection or are very inefficient in slowing progression to disease. The history of HIV vaccine development has been a tour de force in trying to develop protective immune responses that nature has not learned to produce. Although we are closer today to an HIV vaccine than we were in 1983, it is not possible to predict when we will have a vaccine with sufficient efficacy for use in public health programs. Nevertheless, if we learn lessons from the past and, most importantly, have the wisdom to apply them, we may be able to accelerate the development of a much needed HIV preventive vaccine.This article summarizes past efforts made to develop a preventive HIV vaccine. It discusses the insights that have guided those efforts, and identifies lessons that can inform the path forward.This discussion is based on personal experiences after more than 25 years of involvement in the global HIV vaccine effort. First at the World Health Organization (WHO), and the Joint United Nations Program on AIDS (UNAIDS) in Geneva, Switzerland (from 1986 to 2004), and more recently at the Bill & Melinda Gates Foundation in Seattle, WA, United States (since 2004). I recently reviewed the history of HIV vaccine development [2], and this section presents a summary. Table 1 includes some of the key events in the basic science, clinical trials and organizational fronts.Key events on the history of HIV vaccine research and development.The initial efforts to develop an HIV vaccine were based on the concept that neutralizing antibodies would be sufficient to protect against HIV infection. These efforts followed the paradigm established by the licensure in 1986 of the first recombinant vaccine, against hepatitis B. After all, most existing vaccines work through antibodies that block infection or interfere with systemic infections [3]. Different HIV vaccine constructs were developed based on the envelope glycoproteins of the virus (mainly gp120 and gp160), which are responsible for virus binding to the target cells, and serve as the main targets for the neutralizing antibodies. The first HIV vaccine trial conducted in the US started in 1988 and evaluated a recombinant form of gp160 produced in a baculovirus-insect cell system [4]. Other envelope constructs were designed, especially gp120 and gp160 molecules produced in mammalian cell systems. This period of the antibody paradigm was very active, with different lines of research being explored. These included the use of poxvirus vectors to prime the antibody responses [5]; the development of non-human primate (NHP) models for HIV vaccine research; the identification of different genetic subtypes of the virus [6]; the classification of R5 and X4 virus phenotypes [7]; and the finding that primary and cell-cultured isolates of HIV have different sensitivity to neutralizing antibodies in vitro [8]. Other avenues of research that were less productive included: the general agreement that the V3 loop of gp120 constituted the Principal Neutralization Domain (PND) of HIV [9]; the observation that NHP could be protected with whole inactivated SIV, which turned out be mediated by a xenoimmunization mechanism [10]; and the potential use of live attenuated vaccines [11].Nevertheless, this period was characterized by the expectation that a vaccine would be developed within the next few years. That belief led to major efforts to prepare international sites for the conduct of vaccine efficacy trials [12]. This energy was also reflected by the creation in 1995 of the AIDS Vaccine Advocacy Coalition (AVAC), the challenge that President Clinton posed to the scientific community in 1997 to develop an HIV vaccine within 10 years [13]; and the establishment in 1996 of the International AIDS Vaccine Initiative (IAVI).This first period came to an end in 2003, when the negative results of the VaxGen trials were reported. Those were the first two efficacy trials of any candidate vaccine, simultaneously conducted in Thailand (VAX003) and North America (VAX004), to test the protective efficacy of two different preparations of recombinant gp120 vaccines [14,15]. The failure of the VaxGen trials catalyzed a rethinking in the field with a re-examination of the scientific basis for HIV vaccine development. The second wave of HIV vaccine development began with the recognition in the early 2000s of the critical importance of CD8+ T-cell responses in the control of HIV infection [16]. This new paradigm led to the development and refinement of live recombinant viral vectors, especially poxvirus and adenovirus vectors, as well as of DNA vaccines.On the organizational side, the VaxGen results stimulated the search of mechanisms for a more strategic and coordinated approach to solve the HIV vaccine challenge. This led to the eventual establishment of Global HIV Vaccine Enterprise, initially proposed in 2003 [17]. The Enterprise stimulated new investments in the field, including the launching in 2005 of the NIH-funded Center for HIV Vaccine Immunology (CHAVI) and of the Bill & Melinda Gates Foundation supported Collaboration for AIDS Vaccine Discovery (CAVD) in 2006. This period saw much work trying to understand the dynamics of cell mediated immunity (CMI) in natural infection and in animal models. Those studies provided strong evidence that cytotoxic T-lymphocytes (CTLs) were important in controlling virus replication in infected people, although not sufficient to completely eliminate the virus. Moreover, NHP protection experiments repeatedly showed that CTL-based SIV vaccines could not prevent acquisition of infection, although some of them decreased virus load and progression to disease in vaccinated animals that became infected after performing a virus challenge. This, combined with the conviction that gp120 vaccines would not offer significant protection against primary infection, led by 2007 to the conclusion that the best that could be done was to develop disease-modifying vaccines [18]. The field then turned to develop vaccines that stimulate CD8+ T cells in humans. Research done in the early 1990s has shown that recombinant plasmid DNA delivered into the skin or muscle induce viral specific immune responses. This relatively simple technology was seen as a potential modern replacement of live-attenuated vaccines, capable of inducing a whole range of immune responses. Starting in the mid-1990s, DNA technology began to be explored in the SIV/macaque model and in human trials. Very quickly, however, it was found that the robust immunogenicity observed in small animals did not translate to NHP or humans. To address the problem, different technologies were explored to enhance the immunogenicity of DNA vaccines including electroporation, co-administration with cytokines, and the use of prime-boost regimes [19].During this period two viral vectors were preferentially used for the development of HIV vaccines, poxviruses and adenoviruses. Although poxviruses were the first to be used for HIV vaccine development [20], including the first poxvirus-prime/protein-boost trial conducted in the US in 1991 [21], the emphasis during this wave shifted to adenovirus vectors. A major driving force of the adenovirus vector effort was the pharmaceutical company Merck and Co, Inc. (Whitehouse Station, NJ, USA) which in 2001 announced results from their initial NHP protection experiments using a replication-defective adenovirus 5 (Ad5) vector expressing the SIV gag gene [22]. Based on those results, a candidate vaccine using a mixture of recombinant Ad5 vectors expressing the HIV gag, pol and nef genes moved in 2004 to two sequential efficacy trials: STEP, in the US and other countries in the Americas, and Phambili, in South Africa. Both trials were halted in September 2007 due to an interim review of the STEP trial that revealed the vaccine was not protective and that vaccination appeared to be associated with an increased risk of HIV acquisition in vaccinated individuals who had preexisting antibodies against Ad5 [23,24,25].An early concern related to the potential use of Ad5 vectors was that the preexisting immunity to Ad5, which is quite prevalent especially in less developed countries, could impair its immunogenicity. This concern led to the development of alternative adenovirus vectors, either based on less prevalent human adenovirus serotypes or on simian adenoviruses for which no preexisting immunity exists in human populations [26]. It still remains unclear if the enhancement of HIV infectivity observed in the STEP trial is a common characteristic of all adenovirus vectors, or if it is a specific trait of the Ad5 vectors. Since different adenovirus serotypes have different biological properties, the answer to that question deserves additional research and consideration.The negative results from the STEP trial came as a surprise to the scientific community who had high expectations for the cell-mediated immunity approach. In response, the scientific community reacted with a call to reconsider the clinical research strategy and to focus more on a basic research agenda [27].The third wave of HIV vaccine development, aimed at exploring combinations of immune responses, was initiated after the disappointing results from the STEP trial were announced. However, two years after the STEP trial was stopped, surprisingly positive (although modest) results from the RV144 trial were reported, in October 2009. The trial was conducted among 16,402 adults in Thailand to test the protective efficacy of a prime-boost combination of two vaccines: a canarypox-HIV recombinant vector followed by a recombinant gp120 protein, and demonstrated a 31.2% efficacy in preventing HIV infection [28]. An unprecedented scientific collaboration was organized to try to identify potential immune correlates of protection, which generated the hypothesis that V1V2 antibodies may have contributed to protection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of the protective antibodies [29]. The analysis failed to identify neutralizing antibodies as a potential correlate, turning the attention to the potential role of non-neutralizing antibodies, probably those involved in antibody-dependent cell-mediate cytotoxicity (ADCC). Discussions are now underway to confirm and extend results from the RV144 trial to other populations in southern Africa and Thailand (through the so-called Pox-Protein Public Private Partnership, or P5) [30].To some extent, the modest success obtained with the RV144 trial brought new attention to the importance of conducting clinical trials, especially efficacy trials, to complement the basic research effort. This, taken together with the failure of the CTL vaccine tested in the STEP trial, turned the HIV vaccine paradigm pendulum back to the induction of antibodies. The Holy Grail of HIV vaccine research has been the development of immunogens capable of eliciting broadly neutralizing antibodies (bnAb) that can protect against the large number of immunologically different strains of HIV that circulate globally [31]. It is known that roughly 20% of HIV infected individuals develop such bnAb, typically after two years of infection. As early as 1992, the first broadly neutralizing monoclonal antibodies (bnmAb) were isolated and characterized. In recent years there has been an explosion in the discovery of new bnmAb, targeting different epitopes in the HIV envelope glycoproteins. These epitopes are being explored as potential targets for vaccine development, an effort that has been facilitated by new knowledge on the molecular structure of the HIV envelope. However, a major challenge the field is confronting is the dissociation between antigenicity (the ability of a molecule to be recognized by given monoclonal antibodies) and immunogenicity (the ability of those molecules to induce in animals or humans the corresponding antibodies). In this regard, much has been learned in the last two years about the mechanisms for the development of broadly neutralizing antibodies [32]. The success of passive immunization experiments in animals, using different bnmAb, is stimulating research on the potential use of those antibodies for prevention and treatment of HIV infection in humans [33].However, T-cell vaccines received a surprising boost in 2011, with the description of a profound early control of SIV by effector-memory T cells induced by a Rhesus cytomegalovirus (RhCMV) vectored vaccine [34,35]. On the other hand, the latest disappointment in the field was the stopping of the HVTN 505 trial in April 2013, for lack of efficacy [36]. The vaccine tested consisted of a prime-boost regimen involving DNA priming and boosting with Ad5 vectors.Three decades of HIV vaccine research has taken the field through a roller coaster of many failures and a few modest successes [2]. It is important to take stock and to draw lessons from the past to avoid the definition of insanity attributed, among others, to Albert Einstein: “doing the same thing over and over again and expecting different results.”In this regard, I recently discussed several lessons from the 1954–1955 field trial of the Salk inactivated polio vaccine, that could inform the development of an HIV vaccine, as follows [37]:
+      
+        
+          Paradigms change, and “expert” opinion can be wrong;
+        
+        
+          Basic science is essential, but it alone will not be sufficient to develop a vaccine;
+        
+        
+          Human data trump everything we do in vitro or in animal models;
+        
+        
+          Different vaccine concepts need to be tested in parallel;
+        
+        
+          The availability of other preventive interventions may decrease the interest on vaccine development;
+        
+        
+          Sustained support over the long term is needed;
+        
+        
+          Invest in the future by protecting the funding necessary for vaccine development; and
+        
+        
+          Preparation for success can shorten the time between vaccine development and public health impact.
+        
+      Paradigms change, and “expert” opinion can be wrong;Basic science is essential, but it alone will not be sufficient to develop a vaccine;Human data trump everything we do in vitro or in animal models;Different vaccine concepts need to be tested in parallel;The availability of other preventive interventions may decrease the interest on vaccine development;Sustained support over the long term is needed;Invest in the future by protecting the funding necessary for vaccine development; andPreparation for success can shorten the time between vaccine development and public health impact.This article specifically focuses on lessons learned after 30 years of HIV vaccine research. In order to have a more robust and informed discussion, I reached out to a number of colleagues. Thirty-six of whom responded with thoughtful comments (which are italicized in the text, unattributed) and their names are listed in the acknowledgment section. Nevertheless, the author assumes full responsibility for the views expressed. Perhaps, the first lesson is that “we need to be willing to learn from the experiences of the past.” The same advice was voiced by the Spanish-born poet and philosopher George Santayana (1863–1952), who reminded us that “Those who cannot remember the past are condemned to repeat it” [38]. “If it were a simple problem, someone would have solved it by now.” The naivete of the first two decades of HIV vaccine research has now been replaced by the sobering conclusion that developing a HIV preventive vaccine is one of the most difficult challenges that biomedical research is confronting. “The field has to move away from a home run philosophy”, which seems to have equally affected researchers, funders and advocates. The priority over the last few years has been “to win, not to think.” A more systematic and coordinated approach to problem solving needs to be adopted. On the positive side, recent results from the RV144 trial suggest that developing a vaccine that prevents HIV acquisition is possible. However, there are more cautious voices that argue that in reality we do not know if we would be able to solve the problem, or when a practical vaccine will be developed. The reality is that after 30 years of HIV vaccine research “we are mostly in the discovery phase.” It is important to be guided by data and to “resist the temptation of trusting our own beliefs, preconceived ideas and feelings of certainty.” We constantly need to remind ourselves that “good science (rational or empirical) matters!” Preconceived ideas herded the first two waves of HIV vaccine development. As discussed above, the hepatitis B vaccine model guided the antibody paradigm wave of HIV vaccine development, which was followed by a second wave based on the conviction that protective responses observed in elite controllers could be directly translated into the development of a preventive vaccine. Although it made sense to explore those lines of research, the problem was that the entire field followed the fashion with an almost religious fervor. “Everyone was running after the same ball, which sometimes changed direction.” Funding agencies did the same, providing limited funding to explore alternative approaches. “HIV research has provided much knowledge but not a vaccine.” Scientific knowledge needs to be translated into products for clinical trials.Results from three efficacy trials (the two VaxGen trials reported in 2003 and the STEP trial in 2007) were determinant in changing the prevailing vaccine development paradigms. The current paradigm, which is exploring a broader range of immune responses, was reinforced by the 2009 results from the RV144 trial.The current dilemma and a potential problem is that “many scientists have built entire careers in HIV, which has become a research industry.” In some cases there is little motivation to “kill early and kill hard,” with a reluctance to move fast into the clinic, where negative results could have detrimental consequences for grants or professional careers.One critical lesson is that “nothing replaces clinical trials,” whose results are often “unpredictable and surprising.” Clinical trials are “time-consuming, expensive and dependent on appropriate clinical trial infrastructure.” In addition, the conduct of clinical trials, especially large scale trials “can be very controversial” and, often, “decisions need to be made without first achieving consensus.” However, “judicious use of large scale efficacy trials” is essential to advance the field. The recent development of effective non-vaccine prevention intervention for HIV (such as microbicides and pre-exposure prophylaxis) is welcomed. These interventions would surely have a significant public health impact, at least in some populations, especially in developed countries. However, an HIV vaccine is believed to be needed to fully accomplish the goal of an AIDS-free world and we should “avoid complacency because of the existence of those other HIV preventive interventions.” A practical problem that the field needs to solve is how to conduct vaccine clinical trials in the context of other prevention interventions. These interventions would decrease HIV incidence in the trial population, making it more difficult to assess vaccine-induce protection.What we cannot afford is to give-up, this requiring “long term perseverance and dedication of scientists and funders.”“HIV vaccine development is not for the faint of heart.” Younger generations of scientists are needed to continue the effort and “new people need to be attracted to the field.” However, there is a perception among some young scientists that the HIV field is “too crowded for them to have any chance and it would be better to focus on a new research area.” “Unless they can see a career path we will fail to recruit the brightest and the best to this effort.” I believe, however, that the HIV vaccine field presents to the young scientists the incredible challenge and opportunity to work on a major research challenge that for years has resisted solution, also contributing to the solution of one of the major global health problem of our time.Given that “classical vaccinology has not helped developing an HIV vaccine,” the field has to keep an open mind, exploring innovative approaches that have not been explored before. In this regard, experience has shown that “each new vaccine needs a champion.”The development of an HIV vaccine is a complex scientific endeavor which requires multiple collaborations. Unfortunately, “we have not applied all available knowledge because of the silo nature of science,” although both “big science and small science can and should complement each other” [39]. Different modalities of collaborations have been established throughout the years, especially for the conduct of clinical trials where expanded access to trial populations is needed. Examples of these collaborations are the AIDS Vaccine Evaluation Groups (AVEG), the Preparation for AIDS Vaccine Evaluation (PAVE) initiative, and the HIV Vaccine Trials Network (HVTN). Most recently, the P5 partnership has brought together different players from the public, private and philanthropic sectors.Other modalities of collaboration were also established to tackle upstream aspects of HIV vaccine research, including the IAVI’s Neutralizing Antibody Consortium, as well as the Center for HIV Vaccine Immunology (CHAVI) and the Collaboration for AIDS Vaccine Discovery (CAVD). Regional collaborations were also established, including the African AIDS Vaccine Programme/Partnership (AAVP) and the AIDS Vaccine for Asia Network. The 2003 proposal to establish the Global HIV Vaccine Enterprise as a mechanism to accelerate the development of an HIV vaccine was received with great enthusiasm and heralded a new era of intensified collaboration [17]. One of the most successful examples of true partnership has been the multiple collaborations established by different groups of Thai scientists and international collaborators, a commitment that spanned many years culminating with the RV144 trial [28,40,41]. However, an analysis of the “natural history” of many collaborative efforts reveals that after a period of initial excitement and support, the original goals of many of these efforts are forgotten or weakened and many of them fail to thrive. With time, some of these collaborative efforts lose strategic focus and/or financial support, remaining in place just as faint memories of unfulfilled hopes. As support for global health projects declines, the HIV vaccine field will have to be creative and commit to more coordination and collaboration [42].The HIV vaccine field has been “too inward looking” and it is important to learn from other vaccine efforts. Future collaborations should learn from experience, ensuring “clarity of objectives,” “industrial involvement and partnerships,” “full involvement of local investigators and communities” and developing novel approaches to “prioritize, synergize and interconnect.”A major global effort to expand access to the existing prevention and therapeutic interventions has already resulted in a decrease in the number of new HIV infections, from 3.5 million in 2005 to 2.3 million in 2012. Ongoing work by UNAIDS indicates that an intensified effort to provide existing interventions to the appropriate target populations would result in additional reductions in HIV incidence, although it would not be able to bring the epidemic to zero. However, moving forward with that intensified effort would cost up to 23 billion US dollars per year, a financial commitment that would be challenging to maintain. Preliminary modeling work by IAVI, indicates that a 80% effective vaccine introduced in 2025 would be critical in significantly reducing the number of new HIV infections in an effort to achieve the goal of an AIDS free generation [43]. The world has invested almost 9.5 billion US dollars in HIV vaccine research since 2000, and the current annual level of investment dedicated to vaccine development is close to 850 million US dollars. That amount seems relatively modest, considering that a vaccine would be the most cost-effective intervention to control the HIV pandemic. However, additional work is needed to estimate the costs of rolling out a global HIV vaccination campaign.Summarizing the many lessons from the past, I would like to make a set of personal recommendations. These recommendations are intended to stimulate the discussion and intellectual dialogue that the field needs to accelerate the development of an HIV vaccine.Establish and maintain a program of truly innovative research with protected funding to explore out-of-the-paradigm approaches (perhaps not less than 10 percent of the total investment);Continue the basic research effort, exploring novel opportunities to conduct translational research, including the implementation of small experimental medicine trials (small human trials designed to answer critical questions prior to embarking on formal product development activities);Discuss an ambitious goal of initiating a certain number of well-coordinated efficacy trials in the next five years. Planning for these trials would help structure the discussion around scientific questions, vaccine manufacturing capacity, access to and preparation of trial populations, and funding issues;Design appropriate strategies and trials to answer lingering questions in the field, such as the potential protective efficacy of vaccines against different HIV clades and routes of transmission, which differ in different geographic regions of the world;Strengthen the global HIV vaccine architecture by supporting the role of different national, regional and global organizations (including WHO and UNAIDS) which have different audiences and constituencies. In particular, strengthen the role of the Global HIV Vaccine Enterprise as a venue where multiple partners plan their collaborative effort; andBring new partners to the HIV vaccine field and strengthen interactions with other organizations that work in the HIV prevention arena.All of the above would need to be conducted with the necessary sense of urgency that the epidemic is imposing on us. I express my thanks to the many colleagues who generously shared their views on lessons learned which I have incorporated in the article. They are: Dan Barouch, Alan Bernstein, Don Burke, Jon Cohen, Patrice Debré, Max Essex, Mariano Esteban, Pat Fast, Mark Feinberg, Robert Gallo, Marc Girard, Barney Graham, Shiu-lok Hu, Peggy Johnston, Pontiano Kaleebu, Wayne Koff, Thomas Lehner, Shan Lu, Malegapuru Makgoba, Margie McGlynn, Charles Mgone, David Montefiori, Lynn Morris, Rafael Nájera, Stanley Plotkin, Fil Randazzo, Harriet Robinson, Mauro Schechter, Jim Tartaglia, Gerald Voss, Britta Wahren, Bruce Walker, Mark Weinberg, Robin Weiss, Hans Wigzell and Susan Zolla-Pazner. I specially thank Robin Shattock for encouraging me to write this article and for providing excellent comments on the first draft of the manuscript. I also wish to thank Brianna Thompson for editorial support. Parts of this article were presented at a meeting of the WHO-UNAIDS Vaccine Advisory Committee (Beijing, 14 June 2013) and at the closing session of the XIII AIDS Vaccine Conference (Barcelona, 10 October 2013). The author declares no conflict of interest. The views and opinions expressed in this article are those of the author and not necessarily those of the many colleagues who provide comments, neither the official policy nor the position of the Bill & Melinda Gates Foundation.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The success of cellular immunotherapies against cancer requires the generation of activated CD4+ and CD8+ T-cells. The type of T-cell response generated (e.g., Th1 or Th2) will determine the efficacy of the therapy, and it is generally assumed that a type-1 response is needed for optimal cancer treatment. IL-17 producing T-cells (Th17/Tc17) play an important role in autoimmune diseases, but their function in cancer is more controversial. While some studies have shown a pro-cancerous role for IL-17, other studies have shown an anti-tumor function. The induction of polarized T-cell responses can be regulated by dendritic cells (DCs). DCs are key regulators of the immune system with the ability to affect both innate and adaptive immune responses. These properties have led many researchers to study the use of ex vivo manipulated DCs for the treatment of various diseases, such as cancer and autoimmune diseases. While Th1/Tc1 cells are traditionally used for their potent anti-tumor responses, mounting evidence suggests Th17/Tc17 cells should be utilized by themselves or for the induction of optimal Th1 responses. It is therefore important to understand the factors involved in the induction of both type-1 and type-17 T-cell responses by DCs.Dendritic cells (DCs) regulate the activation of naive T-cells by the uptake and processing of antigens and presenting them on their cell surface as peptides bound to major histocompatibility complex molecules (MHC), which is considered antigenic “signal 1”. Together with peptide:MHC complexes, DCs also express co-stimulatory molecules that provide signal 2 [1]. The combination of signal 1 and 2 regulate the specificity and magnitude of the T-cell response. DCs also provide a third signal (signal 3) to the T-cells, which is in the form of cytokines. Signal 3 determines the type of T-cell response that is elicited [2,3].Different CD4+ T-helper cell populations are recognized based on their function and cytokine production [4,5,6,7]. Much of the early work in characterizing these populations and the conditions that favor their differentiation was performed on the T-helper (Th) 1 and Th2 subsets, as these were the first two populations described. Type-1 polarized CD4+ T-cells provide protection against intracellular infections, produce high levels of IFNγ and express the transcription factor, T-bet [8], while Th2 cells express the transcription factor, GATA-3 [9], produce IL-4, IL-5 and IL-13, enhance humoral immunity and protect against helminthes infections [10]. The production of interleukin (IL)-12p70 by DCs during the priming of CD4+ T-cells results in the induction of Th1 cells [11]. In contrast, the secretion of IL-4 during CD4+ T-cell priming results in the generation of Th2 CD4+ T-cells. More recently, regulatory CD4+ T-cells were described, which are recognized by the expression of the transcription factor, Foxp3, are dependent on IL-2 signaling and regulate immune responses by inhibiting T-cell proliferation and function [12]. Recently, a new population of CD4+ T-cells was described based on the expression of the transcription factor, retinoic acid-related orphan receptor (ROR)-γt and the production of IL-17A, IL-17F and IL-21 [7,13]. Th17 cells play a role in the immune response against extracellular bacterial and fungi infections and have been associated with various autoimmune diseases [14,15,16], as well as cancer [17].The conditions that polarize a particular T-cell subset (e.g., IL-12 and IL-4) inhibit the differentiation of the other T-helper cell subsets, further enhancing the desired response. The presence of IL-12 during T-cell priming not only induces Th1 differentiation, it also inhibits Th2 differentiation, while the presence of IL-4 during priming regulates Th2 differentiation while preventing Th1 development. Furthermore, IFN-γ, produced in type-1 responses, downregulates IL-4 expression, thereby minimizing Th2 development [18], while IL-4, produced in type-2 responses, blocks expression of IL-12R, preventing Th1 development [19,20]. Similar to the effect of IL-12 on Th2 development and IL-4 on Th1 development, both IL-12 and IL-4 have been shown to prevent Th17 differentiation [21,22]. Likewise, the expression of transcription factors specific to a T-helper cell subset can also prevent the differentiation of other subsets. Th2 cells transduced to express T-bet developed into Th1 cells that produced IFNγ and lost their IL-4 and IL-5 production [8]. Conversely, the expression of GATA-3 by CD4+ T-cells prevented the production of IFNγ by T-cells primed under Th1-inducing conditions [23]. Furthermore, expression of T-bet prevented the generation of IL-17 secreting T-cells in a mouse model [24].Th1 cell differentiation has been well-described for years and is fairly straightforward: their differentiation is promoted by inflammatory environments that contain intracellular pathogens, such as viruses and some bacteria and protozoans and can also be induced by the presence of cytokines during T-cell priming; IL-2 and IL-12, in particular, synergize to promote IFNγ-secreting Th1 cells [25,26,27].Th17 differentiation is not nearly so simple. Because IL-17 producing CD4+ T-cells are important in inflammatory and autoimmune diseases, many studies have explored the factors required for the differentiation of Th17 cells and the cellular origin of these cells; see Table 1 for a summary of the literature discussing the role of cytokines in human Th17 cell differentiation. The origin of human Th17 cells has received a fair amount of controversy, since initial studies were unable to show the differentiation of Th17 cells from circulating CD45RA+CD45RO− (naive) CD4+ T-cells [28,29]. Cosmi and colleagues examined Th17 differentiation in detail and showed that IL-17-producing cells exclusively originated from CD161+ precursor cells [28]. Since naive circulating T-cells in adults are CD161−, this observation provides an explanation for the inability to detect Th17 development from naive CD4+ T-cells. In contrast, naive CD4+ T-cells derived from umbilical cord blood (UCB) or the thymus express CD161, and these naive CD4+CD161+ T-cells can be induced to differentiate into Th17 cells [28]. It was subsequently shown that all IL-17-producing T-cells, as well as precursors to IL-17 producing cells express CD161, which was, in part, regulated by the transcription factor, ROR variant 2 (RORC2), the human ortholog of mouse RORγt [30,31].Summary of the literature for cytokines involved in human Th17 cell differentiation. Initial studies showed a correlation between IL-23 expression and the presence of Th17 cells, suggesting a prominent role for this cytokine in the differentiation of naive CD4+ T-cells into Th17 cells [40,41]. Subsequent studies have shown that IL-23 is required for Th17 cell effector function, expansion and survival, but not for their differentiation [34,39]. However, there are a number of studies that demonstrate that IL-23 is a critical component of the cytokine milieu supporting Th17 differentiation [29,32,33,34,35,36]. IL-23 by itself has been shown to be capable of supporting the differentiation of naive human CD4+ T-cells into Th17 cells [35]. Furthermore, DCs stimulated with a derivative of bacterial peptidoglycan (PGN) produce IL-23 and IL-1 and stimulate the development of Th17 cells, which could be inhibited by blocking IL-23 [29]. Although the precise role of IL-23 in Th17 cell differentiation remains to be fully defined, the data suggest that IL-23 has a valuable role in the survival and expansion of Th17 cells and might also play an important part in the differentiation of (human) Th17 cells.Mouse studies showed that the activation of CD4+ T-cells under inflammatory conditions in the presence of TGFβ and IL-6 results in the differentiation of Th17 cells [34,42]. In humans, however, the requirement for TGFβ in the differentiation of Th17 cells is still unclear. Various studies using Th17-associated cytokines revealed no role for TGFβ in the differentiation of human CD4+ T-cells into Th17 cells, and some found that TGFβ had a negative effect on Th17 differentiation. These studies showed that the differentiation of Th17 cells was dependent on the cytokines, IL-6, IL-1β and IL-23, alone or in combination [22,35]. However, recent data showed that TGFβ, in combination with IL-23, IL-1β and IL-6, was indispensable for driving the development of IL-17 producing human CD4+ T-cells. [32,36]. The observed contradictory roles for TGFβ in the development of human Th17 could be due to difference in the concentration of TGFβ used by the different research groups, with lower concentrations (1 ng/mL) being favorable for Th17 cell development and higher concentrations (5–10 ng/mL) being unfavorable [22,32,35,36,37,38,39,40,41,42].IL-6, as mentioned above, is critical for mouse Th17 cell differentiation, but, like TGFβ, its role in human Th17 cell differentiation has been less clearly defined. Several of the studies discussed above showed that IL-6 is necessary for Th17 cell differentiation in concert with other cytokines [22,32,33,36]; however, there is considerable data demonstrating that it is not required [29,34,35,37]. Treatment of patients with rheumatoid arthritis (RA) with antibodies against the IL-6 receptor resulted in the skewing of T-cell differentiation towards Foxp3+ Tregs and away from Th17 cells [43]. Interestingly, IL-6 signaling induces the rapid upregulation of the IL-23 receptor on CD4+ T-cells, leading to Th17 cell induction, suggesting that IL-23 might play a role in the differentiation of Th17 cells [44]. IL-1β is frequently cited as one of the “pro-inflammatory” cytokines that either induces or enhances the differentiation of human Th17 cells [22,29,32,33,34,35,36]. This cytokine does not appear to be involved in the differentiation of Th17 cells in mice; however, the literature in humans supports the notion that it is a necessary component in human Th17 cell development.While most studies focus on the development of Th17 cells, it appears that the cytokines required for the differentiation of IL-17-producing CD8+ T-cells (Tc17) are similar to those for Th17 differentiation. Several mouse studies have shown that the stimulation of splenocytes or isolated CD8+ T-cells in the presence of recombinant IL-6 or IL-21 strongly reduced IFNγ production by CD8+ T-cells. The addition of TGFβ further inhibited the Tc1 differentiation by blocking granzyme B expression and upregulating IL-17 production [45,46,47]. Furthermore, CD8+ T-cells activated in the presence of IL-6 and TGFβ expressed elevated levels of the transcription factor, RORγt [47]. In humans, patients with Helicobacter pylori infection show an increased expression of IL-23, which resulted in elevated production of IL-17 by CD8+ T-cells, as well as CD4+ T-cells, suggesting a role for this cytokine in Tc17 development [48]. In a human hepatocellular carcinoma study, it was shown that monocytes/macrophages recently activated in the tumor-microenvironment efficiently induced the development of Tc17 cells and that this development could be blocked by antibodies directed against IL-1β, IL-6 and IL-23 [49], suggesting that TGFβ is not required in humans for Tc17 development. Interestingly, a large percentage of these Tc17 cells also produced IFNγ.Traditionally, autoimmune diseases have been associated with self-reactive hyperactive Th1 cells. However, mice lacking functional IL-12p70, lacking IFNγ or deficient in IFNγR signaling still developed certain autoimmune diseases. These paradoxical observations were resolved by the discovery of a new cytokine, IL-23, which is comprised of an IL-23p19 subunit and the IL-12p40 subunit, which it shares with IL-12p70 [50]. Experiments with mice lacking the IL-23p19 subunit, which are deficient in IL-23, but produce functional IL-12p70, revealed that these mice are resistant to the induction of experimental autoimmune encephalitis or collagen-induced arthritis, demonstrating the role of IL-23 in the pathogenesis of autoimmune diseases [51]. IL-23 is critically involved in maintaining the effector function of Th17 cells, and thus, the evidence linking IL-23 and autoimmune disease led to the association of Th17 cells and autoimmunity. This assertion has been supported by the detection of elevated IL-17 levels in the synovial fluid from rheumatoid arthritis (RA) patients [52], as well as in the serum of patients with inflammatory bowel disease [53]. Furthermore, models using IL-17-deficient mice or in which IL-17 was blocked by antibody treatment showed reduced inflammation and disease severity in rheumatoid arthritis and experimental autoimmune encephalomyelitis (EAE) models, further linking IL-23, IL-17 and autoimmune disease [14,16,54,55,56].T lymphocytes, both CD4+ and CD8+, are critical mediators in the immune system’s elimination of transformed cells. However, most studies have focused on differently polarized CD8+ T-cells, as these cells were considered the effectors, while CD4+ cells were thought to be supporting cells. CD8+ T-cells are infamous for their ability to lyse infected and transformed cells via the perforin/granzyme B pathway and the Fas/FasL pathway, earning them the reputation as the primary anticancer T-cells. CD4+ T-cells, on the other hand, were thought of only as support cells that would prime and sustain CD8+ T-cells and activate macrophages. However, it is now clear that CD4+ T-cell can kill tumor cells through direct cell contact via FasL- and TRAIL-dependent pathways, as well as through the perforin/granzyme B pathway, which is classically associated with cytotoxic CD8+ T-cells [57,58,59]. CD4+ T-cells also regulate the production of chemokines and, thereby, the attraction of cytotoxic CD8+ T-cells and other immune cells. Additionally, while it has been demonstrated that primary cytolytic CD8+ T-cell responses can be generated without CD4+ T-cells, CD4+ T-cells are necessary for the generation of CD8+ memory T-cell responses and the ability to rapidly and effectively extinguish future antigen challenges [60,61]. Taken altogether, T-helper cells have an integral role in the host defense against malignancy, and their incorporation into immunotherapy regimens is critical to the long-term success of such treatments.Th1 cells are considered the primary T-helper cell subset involved in antitumor responses; they have been associated with anti-tumor responses in mouse models, achieved in part by their secretion of IFNγ. IFNγ has a myriad of functions in the immune system’s ability to control the growth of or eliminate tumors, notably the recruitment and activation of cells of the innate immune system and enhancing the production of anti-tumor chemokines [62,63]. IL-12p70, which strongly promotes the differentiation of type-1 T-cells, enhances IFNγ and granzyme B production, prolongs T-cell survival and enhances immune recognition of tumor antigen-expressing cells [64,65,66]. These factors together demonstrate why Th1 cells have been considered the premier cell to include in cancer immunotherapy regimens for most of the last decade. The role of Th17 and IL-17 producing cells in cancer, on the other hand, is controversial. Human cervical tumor cells transfected to express IL-17 were shown to have enhanced growth when transplanted into nude mice [67]. Furthermore, mice lacking IL-17 showed a reduced growth of B16 melanoma tumors and MB49 bladder carcinomas, suggesting a role for IL-17 in promoting tumor growth. Conversely, the growth of the tumors was enhanced in mice lacking IFNγ [68]. High levels of IL-17 producing cells in human breast cancer has been associated with decreased disease-free survival and tumor growth [69,70]. The elevated levels of Th17 cells in breast tumor tissues appears to be a result of increased levels of IL-23, due to tumor-produced prostaglandin E2 (PGE2) [71]. In colorectal tumor tissues, the production of IL-22, one of the cytokines produced by Th17 cells, was enhanced compared to normal tissue and correlated with enhanced IL-23 levels. IL-22-producing tumor-infiltrating lymphocytes (TILs) promote tumor growth and metastasis in a mouse model when co-transplanted with tumor cells [72]. Furthermore, while IL-12 has been shown to promote the infiltration of cytotoxic T-cells into tumors, IL-23 was shown to inhibit the migration of cytotoxic T-cells to tumor tissue and promote angiogenesis [73]. In contrast, some recent studies suggested an anti-tumor role for Th17 cells and IL-17 producing CD8+ T-cells. Muranski and colleagues showed that adoptive transfer of tumor-specific Th17 cells could be an efficient treatment of established tumors [74]. The Th17 cells showed a survival advantage over other transferred T-cells, and the anti-tumor immune response appeared to be dependent on IFNγ production [74]. The Dong group showed that adoptive transfer of tumor-specific Th17 cells resulted in robust activation of tumor-specific cytotoxic T-cells, and the anti-tumor response elicited was dependent on the Th17 cell-mediated production of CCL20 [75]. The increased chemokine secretion resulted in the enhanced attraction of DCs, T-cells and other leukocytes to the lungs and an increased activation of cytotoxic CD8+ T-cells. Interestingly, the transferred Th17 cells showed superior anti-tumor responses compared to transferred Th1 cells, and the Th17 cells retained their phenotype in vivo [75]. Another study evaluating anti-tumor responses in RORγt-deficient mice showed that reduced presence of Th17 cells in the tumor microenvironment resulted in enhanced tumor growth. This effect could be counteracted by adoptive transfer of Th17 cells [76].A study in melanoma patients showed that vaccination with cell lysate-pulsed DCs resulted in an increase in both Th1 and Th17 cells in the peripheral blood. The increase in effector cells resulted in an enhanced immune response to tumor-antigen, as measured by delayed-type hypersensitivity (DTH) reaction and correlated with enhanced survival [77,78]. However, it is unclear whether the Th17 cells have a direct effect on tumor regression or an indirect effect by the attraction of type-1 cells. In ovarian cancer, the presence of Th17 cells in tumor tissue is positively correlated with effector cells and negatively associated with Treg infiltration [38]. IL-17 synergized with IFNγ to enhance the production of the Th1-associated chemokines, CXCL9 and CXCL10, leading to an increase in effector cell infiltration. Together, these data suggest that Th17 cells might play an indirect role in tumor immunity through the attraction and activation of effector (Th1/Tc1) cells. See Table 2 for a summary of the literature discussed above regarding the pro-versus anti-tumor effect of Th17 cells.Highlights of the literature reviewing the pro- versus anti-tumor effect of Th17 cells. Human cervical tumor cells overexpressing IL-17 have enhanced growth in nude mice [67].Mice lacking IL-17 had reduced growth of melanoma and bladder tumors, and the growth of tumors was enhanced when IFNγ was lacking [68].High levels of IL-17-producing cells associated with decreased disease-free survival [69] and increased tumor growth in breast cancer [70].Evidence that tumors produce PGE2, which increases IL-23, which, in turn, enhances presence of Th17 cells in breast cancer [71].In a colorectal cancer mouse model, transfer of IL-22-producing tumor-infiltrating lymphocytes with tumor cells promoted tumor growth and metastasis [72].IL-23 is overexpressed in local tumor environment of human colon cancer patients and in mice has been shown to increase angiogenesis and inhibit migration of cytotoxic T-cells [73].In ovarian cancer, the presence of Th17 cells is positively correlated with effector cells and negatively associated with Treg infiltration [38].Adoptive transfer of tumor-specific Th17 cells can actually eradicate established melanoma tumors; Th17 cells showed survival advantage over other transferred cells, and the anti-tumor effect was dependent upon IFNγ production [74].IL-17-deficient mice more susceptible to lung melanoma, and the adoptive transfer of tumor-specific Th17 cells prevented tumor development; transferred Th17 cells showed superior anti-tumor immunity as compared to transferred Th1 cells; the Th17 cell-mediated anti-tumor response was dependent on Th17 cell-produced CCL20 [75].RORγt-deficient mice had reduced numbers of Th17 cells in the tumor microenvironment, and this led to enhanced tumor growth; adoptive transfer of Th17 cells reversed this effect [76].Several groups have examined the adoptive transfer of IL-17 producing CD8+ T-cells. Similar to Th17 cells, the transferred Tc17 cells showed an enhanced survival compared to other transferred cells, which was associated with enhanced expression of IL-7Rα [45]. Furthermore, the anti-tumor effects of Tc17 cells appeared to be dependent on IFNγ produced in the tumor microenvironment, although it is not clear whether this IFNγ is produced by the Tc17 cells, after in vivo conversion into Tc1-like cells [45], or if it is produced by other cells in response to the Tc17 cells [79]. The mechanism by which Tc1 and Tc17 cells inhibit tumor growth appear to be different [79], and while Tc1 cells are more efficient than Tc17 cells, the latter might be important for the attraction of Th1/Tc1, as well as monocytes and neutrophils to the tumor [80]. On the other hand, while some studies showed that Tc17 cells lack granzyme B expression and cytolytic capacity, Tc17 cells might acquire IFNγ production (IL-17+/IFNγ+ CD8+ T-cells), granzyme B and FasL expression, as well as cytolytic ability after adoptive transfer or under the influence of IL-12 [81,82].Although there is evidence suggesting a pro-tumor role for Th17 cells, accumulating data supports the notion that Th17 cells play an important anti-tumor role, particularly in the context of immune responses with combined Th1/Th17 properties and IFNγ production.Due to the ability of DCs to regulate immune responses, much effort has been put in the generation of DC-based therapies for the treatment of various diseases, such as cancer and autoimmune disease, as well as treatment for organ transplantation [83]. In 2010, the Food and Drug Administration approved the use of a cellular vaccine, Provenge (Sipuleucel-T), for the treatment of hormone refractory prostate cancer. This DC-containing vaccine improved overall survival in patients, but failed to induce tumor regression or to prolong time to disease progression, possibly due to the lack of mature DCs. These results highlight the feasibility of DC-containing cellular vaccines in the treatment of cancer, but also show the need to improve DC-maturation protocols [84,85]. The safety of DC-based therapies for the treatment of cancer has been demonstrated in clinical trials for the treatment of melanoma, lymphoma and renal cell carcinoma and breast cancer [86,87,88,89,90]. DC vaccines have also been proposed for diseases other than cancer, such as auto-immune diseases and tissue transplantation, in particular, based on their ability to induce peripheral tolerance via Treg cells specific for auto-antigens [91,92,93,94]. Several broad requirements can be defined for the generation of suitable DCs for vaccination: first, the source of the DCs should be such that large numbers of clinical-grade DCs can be easily made. Secondly, the DCs should be matured in such a way that they express the desired co-stimulatory receptors and produce the appropriate cytokines and chemokines in order to effectively activate and polarize the T-cell response. Thirdly, the DCs should express the appropriate chemokine receptors and integrins to migrate to lymphoid organs or to specific tissues to interact with T-cells and induce the desired type of T-cell response. Each of these requirements, once a barrier to successful DC vaccines, has been extensively studied and systematically addressed over the past two decades, leading to huge gains in this field.The first obstacle was overcome by the finding that monocytes, which are abundantly present in peripheral blood, can be induced to become immature dendritic cells (iDCs) upon culturing in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 [95,96]. Early protocols devised for the generation of DCs from monocytes took up to seven days in culture [96,97], but a desire for more rapid maturation for clinical applications and a concern that DCs may become “exhausted” and unable to produce T-cell polarizing cytokines [98] led to the development of protocols with shorter maturation periods [66,99]. One such protocol developed by our lab for the generation of type-1 polarized DCs required only two days in culture and resulted in mature DC1s capable of T-cell sensitization [66]. Furthermore, this combination of GM-CSF and IL-4, followed by IFNγ, and, then, lipopolysaccharide (LPS) exposure also resulted in a second burst of IL-12 production upon interaction with CD40 ligand, suggesting that the key Th1 polarizing cytokine would be present at the time of DC-T-cell interactions [100]. The remaining requirements can be satisfied by using various combinations of inflammatory mediators during the time of dendritic cell maturation and will be discussed in detail.Dendritic cells are exposed to countless signals in the body, leading to their maturation and influencing their polarization; replicating this ex vivo from monocytes requires precise application of inflammatory mediators, both innate and foreign. Maturing DCs with tumor necrosis factor-α (TNF-α) and cytokines, such as IL-1 and IL-6, leads to efficient upregulation of co-stimulatory molecules (CD83, CD86 and CD58), which are indicative of a mature DC phenotype [101]. Additionally, human peripheral blood monocytes treated with Toll-like receptor (TLR) agonist lipopolysaccharide (LPS) (a TLR4) and peptidoglycan (PGN) (a TLR2 ligand) express markers of DC maturation, CD40, CD83, CD86 and HLA-DR [102], demonstrating that both innate inflammatory markers and inflammatory signals from bacteria can lead to DC maturation.There are numerous in vitro protocols to generate different DC subsets that induce specific T-cell responses, many of which rely on the usage of inflammatory mediators, such as TNFα and type-1 interferons in combination with ligands for pattern recognition receptors, such as the TLR-4 ligand LPS or the TLR-3 ligand poly-I:C [101]. For instance, the induction of a type-1 T-cell response is frequently sought for its antitumor properties, and employing dendritic cells polarized into type-1 DCs (DC1s) to achieve this is well described. A key characteristic of DC1s that is necessary for the induction of type-1 T-cell responses is the production of IL-12p70 [103,104]. This DC1 phenotype can be achieved by cross-linking of CD40 with its ligand, CD154 (CD40L) [105,106,107,108,109,110], or by exposing immature DCs (iDCs) to various inflammatory molecules during their maturation [105,111,112]. High levels of IL-12p70 production by mature DC1s requires at least two signals [113] and is enhanced by IFNγ [114]. In contrast, induction of the related cytokine, IL-23, by DCs in response to microbial stimuli is not enhanced by IFNγ [114] and can be elicited with exposure to only one inflammatory mediator [115]. CD40-CD40L interactions, though, do stimulate the secretion of IL-23 [50,112,116]. The ability to traffic to inflamed tissues to take-up antigen, promptly exit and traffic to secondary lymph organs to prime T-cells is a key characteristic of antigen-presenting cells. For immunotherapy applications, however, DCs would ideally traffic directly to a lymph node to interact with T-cells upon transfer into the patient. Maturation of DCs with inflammatory agents, such as LPS or TNF-α plus CD40 ligand, results in rapid down regulation of CCR1, CCR5 and CXCR1, chemokine receptors important for trafficking to inflamed tissues [117]; minimal expression of these chemokine receptors is desired so that the transferred DCs do not migrate to inflamed tissues. Use of the same maturation agents leads to efficient upregulation of the chemokine receptors, CCR7, CXCR4 and CCR4; two ligands for CCR7, namely CCL19 and CCL21, are known to be expressed throughout the lymphatic system [117,118]. Furthermore, CCR7 and CXCR4 are known to also be expressed on naive T-cells, further supporting the co-localization of mature DCs and naive T-cells to allow for their interaction [117]. Additionally, CCR7 signaling is known to support DC survival, dendrite process formation and antigen uptake, leading to more efficient T-cell responses [119]. Together, these results demonstrate how maturation of DCs with inflammatory cytokines and/or bacterial products promotes chemokine receptor expression that allow appropriate transit towards secondary lymphoid organs.Toll-like receptor agonists, as mentioned previously, are a class of inflammatory mediators that can be used to mature DCs and polarize their cytokine production, ultimately impacting the type of T-cell response elicited. Early work in this field identified the TLR4 ligand, LPS, as a potent inducer of IL-12p70 and IP-10/CXCL10 by dendritic cells, attracting monocytes and Th1 cells and further enhancing the type-1 immune responses [102]. TLR2 agonists, on the other hand, induced IL-23p19 production, which is now known to be associated with type-17 immune responses [102]. Recent work on a subset of DCs, called inflammatory DCs (infDC), demonstrated that the TLR2/TLR1 ligand, Pam3Csk4, led to a DC cytokine profile capable of inducing Th17 cell differentiation from naïve CD4+ T-cells [120]. Interestingly, while lipopolysaccharide (LPS) derived from E. coli typically signals through TLR4 and induces a predominately Th1 response, it has recently been shown that an atypical LPS from P. gingivalis signals through TLR2 and induces slightly more IL-23 production than the typical LPS, demonstrating remarkable specificity [121]. The bacterial cell-wall component, peptidoglycan (PGN), is a TLR2 ligand and stimulates DCs to secrete IL-23 and IL-1β, resulting in Th17 cell induction [29]. The DC17 polarizing effect of PGN was regulated through metabolism into the nucleotide oligomerization domain 2 (NOD2)-ligand muramyl dipeptide (MDP). Stimulation of iDCs with TLR ligands in the presence of MDP resulted in the production of IL-23 and IL-1, promoting IL-17 producing T-cells [29]. Finally, culturing human CD4+ T-cells with anti-CD3 antibodies plus monocytes with the TLR ligand, LPS, has been shown to lead to the induction of IFNγ-secreting cells, IL-17-secreting cells and a third population of dual-secreting cells [42], which could be the most desirable outcome for cancer applications. PGE2 became a component in many DC maturation protocols after it was demonstrated that the expression of CCR7 on in vitro matured monocyte-derived DCs was enhanced by maturation in the presence of PGE2. However, it has since been shown that DC expression of CCR7 can be induced in the absence of PGE2 [122]. The advantage of maturation protocols without PGE2 is that the DCs appear to produce higher levels of the type-1 polarizing factor, IL-12p70 [123,124]. Furthermore, PGE2-matured DCs have been shown to induce Th2 cells [125,126], and PGE2 regulates the production of the immune inhibitory cytokine, IL-10 [125]. Also of importance, PGE2 has been associated with enhanced Treg differentiation, function and attraction via secretion of CCL22 [127,128,129], though some studies also suggest a role for PGE2 in the induction of Th17 cells [71,130,131].Most protocols for the generation of DCs from peripheral blood monocytes rely on the use of GM-CSF and IL-4. However, some recent studies promote the use of IL-15 instead of IL-4 for the generation of T-cell activating DCs [132,133]. While these “IL-15-DCs” resemble Langerhans cells [134], activation of these DCs with TLR agonists results in the generation of both Th17 and Th1 cells [135].These data show that different maturation stimuli can be employed to regulate the level of IL-12p70 production by DCs and induce distinct types of T-cell responses, offering an opportunity to modulate the host immune response. Refer to Figure 1 for an overview of dendritic cell-induced Th1 and Th17 immune responses.Dendritic cell induced Th1 and Th17 immune responses. Toll-like receptor (TLR) ligands are used to mature dendritic cells, and depending on the ligand(s) selected, type-1 dendritic cells (DC1s) or type-17 dendritic cells (DC17s) result. DC1s are characterized by the production of IL-12p70 and induce Th1 immune responses with interferon-γ, granzyme A/B, or perforin secretion. DC17 cells are characterized by the production of a number of cytokines, including IL-23, IL-6, IL-1β and TGF-β, and polarize Th17 immune responses with IL-17A/F, IL-22 and IL-21 production. The cytokines denoted with an asterisk (*) have been reported in the literature to have a role in inducing human Th17 cell differentiation, though there are conflicting reports, and it remains unclear precisely which cytokines are in fact necessary. Finally, there is a third population of T-cells that can be induced by dendritic cells that secrete both IFNγ and IL-17, though the exact mechanism of their differentiation and whether they are directly induced by DCs or are the result of a conversion from Th1 or Th17 cells has yet to be elucidated.Dendritic cell-based immunotherapies hold much promise in manipulating the in vivo immune response to attack and eliminate malignancies. Ultimately, multiple subsets of DC may be needed in successful cancer vaccines. The selection of an appropriate maturation protocol for the DCs is of paramount importance: without the proper ex vivo culture conditions, the DCs may not be mature or capable of activating a lasting T-cell response. Furthermore, the specific agents selected for DC maturation will determine the type of T-cell response elicited and, therefore, must be carefully selected. We feel that producing DCs that can generate both Th1 and Th17 cells, in addition to CD8+ T-cells, will effectively utilize the anti-tumor properties of each of these cells and ultimately may lead to success in the treatment of any number of cancers.Funding support: National Cancer Institute R01 CA096997-02 and Pennies-In-Action.org.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).In this brief review, we discuss immune tolerance as a factor that determines the magnitude and quality of serum antibody responses to HIV-1 infection and vaccination in the context of recent work. We propose that many conserved, neutralizing epitopes of HIV-1 are weakly immunogenic because they mimic host antigens. In consequence, B cells that strongly bind these determinants are removed by the physiological process of immune tolerance. This structural mimicry may represent a significant impediment to designing protective HIV-1 vaccines, but we note that several vaccine strategies may be able to mitigate this evolutionary adaptation of HIV and other microbial pathogens.While HIV-1 is extraordinarily diverse and mutates rapidly, the HIV-1 envelope has conserved regions to which neutralizing antibodies can be made [1]. Inducing antibodies by HIV-1 vaccine candidates is a major goal of HIV-1 vaccine development. However, repeated attempts at HIV-1 envelope immunization of animals and man have induced primarily non-neutralizing or HIV-1 strain-specific antibodies. In this review, we were charged to discuss our recent work in the area of host control of humoral responses to HIV-1; consequently, we have not included much outstanding work by the many other investigators who have contributed to understanding humoral immunity to HIV-1. We shall, nonetheless, point out some of the roadblocks that are currently hindering the successful induction of broadly reactive neutralizing antibodies (bnAbs) to the HIV-1 envelope.Not all HIV-1 envelope epitopes are created equal: some are highly immunogenic and dominate antibody (Ab) responses, whereas others are weakly immunogenic and are sub-dominant [2]. Significantly, the neutralizing epitopes that are shared by multiple HIV-1 clades and elicit bnAb responses are weakly immunogenic and elicit little or no protective immunity in most infected patients [3]. A key question that has occupied the HIV-1 field for the last 5 years is “why are BnAbs so difficult to induce?”Most theories to explain the variability of HIV-1 Env immunogenicity focus on limitations of the antigen-receptor repertoire [4,5,6], epitope shielding by interfering structures [7,8,9], and inflammatory capacity [10,11,12]. Evidence to support each of these hypotheses exist but until recently there has been no concerted effort to focus immune responses on subdominant epitopes to exploit their potential utility as vaccine antigens [2,13,14,15,16,17,18,19]. The properties of immunogenicity and antigenicity are related but distinct: immunogens are capable of inducing humoral or cell-mediated, adaptive immune responses while antigens are structures that specifically bind to the antigen receptors of B- or T cells (BCR or TCR, respectively). Consequently, immunogens are always antigens but antigens are not necessarily immunogenic. The classical example of this dichotomy is the hapten; haptens such as nitrophenyl(acetyl) are bound by BCR and antibodies with great specificity but alone cannot elicit humoral responses.The dissociation of antigenicity and immunogenicity of HIV-1 envelope is the essence of the problem in eliciting bnAb responses. That is, while the HIV-1 envelope gp120 and gp41 components have conserved, neutralizing epitopes that are antigenic, i.e., rare antibodies can indeed bind to Env proteins, they are not immunogenic and do not induce neutralizing antibodies targeted at these sites. Largely, epitopes are identified by elicited antibodies, i.e., epitopes that are both antigenic and immunogenic. For most antigens, this dual definition is not problematic. However, for the conserved HIV-1 antigens that do not elicit protective immune responses, understanding immunogenicity is crucial. For example, the bnAb epitopes in the membrane-proximal external region (MPER) of envelope gp41 can be mimicked by scaffolds and peptide-liposome immunogens that induce antibodies to bind precisely at the bnAb polypeptide epitope [13,20]. However, scaffold-induced Ab does not neutralize HIV-1 and the epitope-specific component of the serum response is only a small minority of the induced Ab. We conclude that at least some HIV-1 epitopes are intrinsically weak immunogens.The role of immune tolerance in the weak immunogenicity of neutralizing MPER epitopes is challenged by the discovery of the 10E8 bnAb [21]. This bnAb recognizes an epitope that overlaps the conserved 4E10 MPER determinant and exhibits great neutralizing breadth [98% (178/181) of HIV-1 isolates tested] in vitro. The 10E8 bnAb was reported to have no affinity for phospholipids, including phosphatidyl choline-cardiolipin, did not label fixed HEp-2 human epithelia cells, nor did it bind several human autoantigens diagnostic for various autoimmune diseases [21]. Nonetheless, in a protein microarray [3], whereas 10E8 was observed not to be polyreactive, it did exhibit strong affinity for a human protein expressed in variety of mature organs as well as in fetal tissues [22]. Whether this autoreactivity is physiologically significant remains to be determined by the generation of 10E8 knockin mice [23,24,25].HIV-1 bnAb also recognize complex epitopes. For example, the 2F5 and 4E10 bnAb bind MPER determinants composed both of membrane lipid and the gp41 polypeptide [26]. In addition, these MPER bnAb epitopes mimic host antigens [3,27] such that immune tolerance mechanisms deplete or anergize reactive B cells [23,24,25,28,29]. Thus, vaccine immunogens that induce HIV-1 bnAbs may necessarily have complex structure (e.g., lipid and polypeptide) and/or be formulated to overcome host immune control mechanisms.HIV-1 bnAbs have unusual but characteristic features, most notably, extraordinary frequencies of V(D)J mutations, that imply unusual developmental histories [2,30]. The frequencies of mutations characteristic of bnAbs include point mutations and insertions and deletions (indels). Point mutations from high levels of somatic hypermutation (SHM) range from 15% to >30%, and demand that we consider why high levels of SHM appear necessary for bnAb production [31]. In addition, a substantial number of bnAbs appear to be poly- and/or autoreactive [2]. For a thorough guide to the origins and characteristics of bnAbs, we refer our readers to a recent review [1] that includes a summary table of 22 bnAbs identified between 1993–2013. In germinal centers (GC), antigen-reactive mature B cells proliferate and express high amounts of activation-induced cytosine deaminase (AID), an enzyme required for immunoglobulin class-switch recombination and V(D)J hypermutation [32]. The clonal evolution of GC B cells is a Darwinian process comprising two mechanisms: SHM and affinity-dependent selection. Selection vets GC B cell populations for increased affinity for the immunogen [33,34]. Indeed, GC B cell survival and proliferation is determined by BCR affinity and the capacity of each B cell to collect and present antigen to local GC T-helper (TFH) cells [35].Clonal selection in GCs depends on relative BCR fitness (affinity and specificity) and changes over the course of the immune response as novel V(D)J mutations exert their effects. A GC represents an “experiment” in clonal evolution with regard to the founding B- and T-cell populations and the order and distribution of the introduced V(D)J mutations. A conundrum of bnAb development is how GC B cells could acquire mutation frequencies of 15%–30% while maintaining their ability to bind antigen and effectively compete for TFH help. In general, as mutant BCR fitness (affinity) increases, it becomes increasingly likely that additional mutations are maladaptive. Reduced BCR fitness in GC leads to rapid clonal elimination [36,37] and there is no reason to believe that the capacity to neutralize multiple HIV-1 clades—or to neutralize at all—provides any selective advantage to GC B cells.At least three hypotheses are currently proposed to explain the high mutation frequencies of HIV-1 bnAb. First, that these mutations are necessary to modify germline Abs so as to meet unusually stringent structural requirements. These structural requirements might include not only high affinity, but also restriction to a core epitope that is poorly recognized by the primary, germline, Ab repertoire [31]. Two alternative hypothesis are influenced by the observations of frequent poly- and/or autoreactivity among bnAbs [2,30]. One alternative hypothesis is that many (most?) conserved HIV-1 neutralizing epitopes have been selected to mimic host antigens; consequently, bnAbs are heavily mutated because the germline Ab/BCR that best recognize these epitopes are lost to immunological tolerance. In this model, affinity maturation for a neutralizing epitope represents de novo mutation and selection acting on weakly cross-reactive, previously mutated B cells [2,3,23,30]. Another, related and non-exclusive possibility is that the structural overlap between HIV-1 neutralization- and host epitopes is close but not complete. In this case, mutated, anergic B-cells with neutralization activity undergo virus-driven, “conflicted” purifying selection that acts on V(D)J residues that remove self-reactivity while maintaining affinity for the neutralization epitope [28]. Autoreactivity has been shown to increase in the human GC B cell compartment as a result of V(D)J mutations that alter antibody specificity [38].Infection by HIV-1 poses a remarkable immunological conundrum: conserved neutralizing epitopes are present on HIV-1 envelope (Env) but rarely elicit protective Ab. The unusual, shared traits of bnAbs suggest an atypical clonal evolution that would normally decrease, not enhance, B-cell fitness [2]; they almost certainly represent the efforts of the immune system to both respond to weakly immunogenic neutralizing epitopes, while avoiding producing antibodies with the polyreactivity, long heavy chain complementarity determining (HCDR3) regions and high levels of SHMs.At least one evolutionary strategy used by pathogens to moderate immunogenicity is host mimickry; immunological tolerance can limit or prevent the production of Ab against microbial epitopes that mimic host structures [30]. For example, Ab elicited by bacterial adhesin FimH of fimbriated pathogens cross-reacts with lysosomal membrane protein-2 (LAMP-2) and causes pauci-immune focal necrotizing glomerulonephritis (FNGN) [39]. Similarly, Campylobacter jejuni lipooligosaccharide (LOS) shares epitopes with mammalian, neuronal gangliosides [40], a mimickry associated with modest Ab responses in a minority of infected patients [41]. Immunization of normal mice with C. jejuni LOS elicits weak, T-dependent Ab responses but these are greatly enhanced in mice unable to generate complex gangliosides [41].More recently, we and our colleagues have demonstrated that HIV-1 Ab responses to two highly conserved, neutralizing epitopes of the gp41 MPER of HIV-1 are suppressed as a consequence of immunological tolerance. The 2F5 and 4E10 epitopes of HIV-1 exhibit significant structural similarity to proteins present in most mammals and the B cells that recognize these shared determinants are lost during their development [3,23,24]. Briefly, Verkoczy et al. has generated knock-in mice that carry the V(D)J rearrangements of the 2F5 or 4E10 bnAbs; these mice support robust early B-cell development, but exhibit a characteristic block at the small pre-B to immature B cell transition that defines the first tolerance check-point [23,24,38]. This developmental blockade is also present in mouse lines carrying the unmutated, i.e., germline 2F5 V(D)J rearrangements, that were expressed by the naïve B cells that gave rise to the high-affinity, mutated 2F5 MPER bnAbs [42].Our studies in knock-in animals defined the mutated and germline 2F5 V(D)J rearrangements as sufficiently autoreactive to trigger physiologic tolerance, but they did not define the self-antigens mimicked by these MPER epitopes [23,24,28]. Using traditional immunoprecipitation and protein microarrays we identified two highly conserved host self-antigens that were avidly recognized by the 2F5 and 4E10 bnAbs: kynureninase (Kynu) and splice factor 3B3 (SF3B3), respectively [3]. Kynu and SF3B3 effectively inhibit the binding of the 2F5 and 4E10 bnAbs to their HIV-1 MPER epitopes; in the case of Kynu, this inhibition is mediated by amino acid identity between the HIV and host protein [3]. A larger survey of HIV-1 bnAbs by these same methods [22] indicates that mimicry of host antigens is common, supporting the hypothesis that viral evolution favors structural similarity with host proteins as a way to mitigate immune responses that diminish transmission [30].Microbial mimicry of host antigens is an effective strategy to mitigate humoral immunity in the infected host. We propose that this evolutionary strategy is more widespread than currently recognized and a principal component of weak neutralizing/protective Ab responses to key microbial epitopes. Indeed, one of the central issues of host-pathogen biology has been whether, or to what extent, self-tolerance limits the B- and T-cell repertoires available for responses to pathogens. Work from the Nussenzweig laboratory has shown that in humans, approximately half of the primary naïve B cell repertoire is lost to the first and second tolerance checkpoints [38,43]. It would be surprising if such substantial losses did not reduce the capacity to react with viruses and other microbes, but the degree to which this happens is not known. If tolerance substantially reduces the antibody repertoire available for protective immunity to pathogens, a new world of (potentially useful) epitopes is hidden by immune tolerance.B cells develop from lineage-specific progenitors that express the V(D)J recombinase [44] and first rearrange the immunoglobulin heavy locus (IGH) gene loci to generate a pre-B cell receptor (pre-BCR). The pre-BCR do not bind antigens but their assembly is necessary for continued cell survival and proliferation. Pre-B cells exit the cell cycle as pre-B II cells, initiate rearrangements in the κ or λ light-chain loci and assemble a mature BCR that binds antigen. The generation of BCRs by combinatiorial association of V (variable), D (diversity) and J (joining) gene segments generates a diverse primary repertoire of BCRs but frequently produces self-reactive B cells [38,43].Indeed, most newly generated, or immature B cells in the bone marrow are autoreactive and must be eliminated or inactivated by immunological tolerance. The remaining B cells mature through the transitional 1 (T1) and T2 stages, which are characterized by changes in membrane IgM and IgD expression and the loss or diminution of markers associated with developmental immaturity. In the periphery, newly formed (T2) B cells are subject to a second round of immune tolerization before entering the mature B-cell pools.Three mechanisms of immunological tolerance are known to deplete B-cell pools of self-reactivity: apoptosis, cellular inactivation by anergy and replacement of autoreactive BCRs by secondary V(D)J rearrangement [45,46]. The majority of lymphocytes committed to the B-cell lineage do not reach maturity as they do not express functional µH polypeptides or because they carry self-reactive BCRs [47,48].Autoreactive B cell numbers decline with increasing B-cell maturity. Tolerance mechanisms, especially apoptotic deletion, operate during the transitional stages of B-cell development, and the number of self-reactive cells decreases substantially after entry into the mature pools. Nonetheless, not all autoreactive B cells are lost; some 20%–25% of mature, naive B cells circulating in human blood express autoreactive BCRs [38,43].The corollary to our proposal that the infrequency of bnAbs production is due—at least in part—to the deletion/inactivation of HIV-1 specific B cells that acquire autoreactivity [3,23,24,25,27] is that HIV-1 infected subjects with autoimmune diseases might be more capable of developing bnAbs [30]. Observations that subjects with systemic lupus erythematosus (SLE) and HIV-1 infection are reported at disproportionately low frequencies support this hypothesis [49,50,51,52] but to date, no direct evidence on this point has been published.Pathways leading to bnAb generation have been traced by following the evolution of B-cell lineages back to their origins, the unmutated, mature IgM+IgD+ B cells that first responded to virus infection [30,53,54]. Can these recently discovered pathways define a method for the generation of protective Ab responses by vaccines?Current HIV-1 vaccines can elicit strain-specific neutralizing Ab but not bnAb; bnAb do arise, however, in approximately 20% of HIV-1-infected individuals, albeit after years of infection [55,56,57,58]. These uncommon bnAb responders provide an opportunity to follow the bnAb response backwards, in effect, reversing B-cell evolution to identify antigen-ligands that might re-create this (or comparable) bnAb pathways in many individuals [2,59,60].Harnessing recent advances in flow cytometry, viral genomics, human B-cell culture, recombinant antibody technology, 454 deep sequencing, and bioinformatics, Liao et al. [54] recently demonstrated the co-evolution of HIV-1 and a bnAb B-cell lineage that produced Ab directed to the virus CD4 binding-site. Sequence analysis of virus and antibody V(D)J genes successively recovered from an infected patient revealed that the unmutated B-cell ancestor of this bnAb lineage strongly reacted with Env of the transmitted/founder virus. This ancestral B cell did not exhibit bnAb activity but the evolution of neutralization breadth increased over time and was associated with viral diversification suggesting immune-mediated selection at the neutralizing epitope [54]. Together, the bnAb and virus sequence data assembled by Liao and his colleagues [54] describe the viral and Ab/B-cell evolution culminating in bnAb production. Importantly, the evolving, mutant BCR of the bnAb lineage could be shown to react strongly with successive HIV-1 Env mutants. This shared pattern of reactivity, immediately provided a starting point for Env vaccine constructs to induce reproducibly bnAb generation [2,53] (Figure 1A).Interestingly, Liao et al. [54] observed that intense virus selection and diversification preceded the development of bnAb activity and that neutralization breadth was associated with the acquisition of poly- and autoreactivity. These findings imply that serial vaccine immunogens optimized for binding to the founders and intermediates of bnAb lineages [2] would likely mimic the natural pathways for bnAb development and be capable of overcoming the limiting effects of immune tolerance on bnAb generation. If so, this novel immunization approach offers a logical strategy for inducing B-cell evolution along rare bnAb pathways that cannot be elicited by conventional, single-immunogen vaccines.Synopsis of proposed vaccine strategies. (A) The B-lineage vaccine approach to eliciting HIV-1 bnAb responses in vaccinees is based on the recapitulation of clonal evolutionary pathways that lead to bnAb in a single HIV infected patient [2,54]. This approach relies on the technical capacity to identify, recover, and characterize bnAb B cells and/or plasmacytes from HIV-1 infected patients. BCR encoding gene rearrangements isolated from single bnAb cells are then used to to follow the bnAb response backwards, in effect, reversing B-cell evolution to identify antigen-ligands that might re-create this (or comparable) bnAb pathways in many individuals [2]. In this way, vaccine ligands can be generated and optimized to select for the desired—bnAb—evolutionary pathway; (B) The ancillary approach is us to identify those cells in the naïve B-cell pools capable of recognizing HIV-1 neutralizing epitopes and/or optimized, lineage design, vaccine immunogens. In this approach, cultured B cells are driven to proliferate by exposure to CD154 and immunogens capable of driving bnAb production can be modified to activate the largest possible pool of specific B cells. In consequence, we can study HIV-1 antigen epitopes without regard to their ability to elicit significant humoral responses: antigenicity minus immunogenicity.As an adjunct to the use of serial, optimized immunogens to elicit known bnAb pathways, we have developed culture systems [60,61] for naïve B cells that permit the dissociation of antigenicity and immunogenicity. In brief, we culture single B cells under conditions that support extensive cell proliferation and efficient plasmacytic differentiation [62]. The specificity of Ab secreted into each clonal culture can be screened rapidly in multiplex assays to determine specificity and avidity; the new recombinant Ab technology makes it a simple matter to generate larger quantities of Ab for neutralization studies. Perhaps most significantly, in addition to mature IgM+IgD+ B cells, we can culture late pre-B cells, immature/transitional (imm/T) B cells, GC and memory B cells, and “anergic” B cells [24,28,61]. We can, therefore, identify and characterize the primary B-cell repertoire before and after the first- and second tolerance checkpoints to determine their role in regulating HIV-1 bnAb production.These cultures allow us to identify and study (in mice and humans) the potential B-cell repertoire—i.e., before the tolerance checkpoints—capable of recognizing HIV-1 neutralizing epitopes and to contrast that with the post-tolerance, expressed repertoire that initiates humoral responses. Cultured B cells are driven to proliferate by exposure to CD154 (CD40L), and although BCR expression is necessary for cell survival, activation by epitope ligands is unnecessary. In consequence, we can study HIV-1 antigen epitopes without regard to their ability to elicit significant humoral responses: antigenicity minus immunogenicity.We are using this novel technology to identify and characterize unmutated BCR/Ab that recognize neutralizing HIV-1 epitopes in the gp41 MPER and gp120 CD4 binding site. The biology and structures of these Env epitopes are exceptionally well characterized and available as highly purified recombinant proteins. We plan on screening vaccine immunogens that initiate BnAb lineages (Figure 1A) for their capacity to react with the broadest possible subset of naïve B cells (Figure 1B). In this way, engineered B-cell lineage immunogens [2] can have the widest possible impact in genetically diverse vaccine populations.The long delayed appearance and infrequency of HIV-1 bnAb production has re-opened one of immunology’s central questions: to what extent does self-tolerance impact immunity to pathogens? It has been widely assumed that self and foreign epitopes are virtually non-overlapping and that whereas tolerance may remove some pathogen-specific B cells, epitope coverage is not significantly affected. We and others have now shown that, at least for some HIV-1 neutralizing epitopes, this is not the case: the overlap between foreign and self-antigens can be significant and has substantial impact on protective immunity. This demonstration opens the way to new vaccine strategies. We note that the host-mimicry by microbial epitopes is not necessarily a barrier to using them in vaccines [2,54,63].The simplest form of a lineage-based vaccine design [2], is offered by the recent work of Liao and colleagues [54]. In this example, a candidate vaccine could be constructed by using serial isolates of mutant HIV-1 Env to drive the evolutionary intermediates of a known bnAb lineage. Whereas this vaccine strategy would be very likely to work in the same infected individual, whether it would be equally effective in genetically dissimilar vaccines remains a crucial question. Nonetheless, the use of a known T/F Env to activate naïve bnAb B-cell ancestors followed by booster immunizations specific, mutated Env variants is an attractive approach to direct the BCR evolution along a pathway that leads to bnAb dominance [2]. Characterization of the primary B-cell repertoire will allow these selected immunogens to be engineered for the broadest possible reactivity that remains consistent with bnAb evolution. This approach will maximize vaccine effectiveness in diverse populations and may even identify naïve B cells that can produce bnAb sooner or with fewer V(D)J mutations.A variation on the theme to the idea of lineage-based vaccines based on established natural histories of bnAb responses [2,54], is the design of HIV-1 immunogens designed to interact with specific antigen-receptors on naïve B cells [17,64]. This rational approach to immunogen design is analogous to that used to develop or modify drugs and depends on the remarkable wealth of structural data available for bnAb and their neutralizing epitopes [17]. It has in common with B cell lineage design the targeting of unmutated ancestor antibodies of bnAb lineages [2]. At present, the high specificity of this approach is both a strength and a weakness: nanoparticles bearing the CD4 binding site epitope recognized by the VRC01 bnAb [64] activate B cell lines expressing the VRC01 Ab and its inferred germline counterpart, but as mice, rabbits, and rhesus macaques lack VH gene segments capable of interacting with the immunogen, the utility of these vaccines has not yet been determined in immunization studies [17,64]. It is now clear that weakly immunogenic, but powerfully neutralizing epitopes exist not just on HIV-1 [65] but also on pathogens as familiar as influenza [66,67] and C. jejuni [41]. We propose that these potentially useful, cryptic epitopes are common on microbial antigens and can offer new and useful targets for vaccine development. The nature of these weak immunogens demand, however, novel vaccine strategies and new ways of thinking about immune activation and high-affinity antibody selection.The authors thank their colleagues in the Departments of Immunology, and Medicine, and the Human Vaccine Institute at Duke University for much appreciated assistance and discussion. This work was supported in part by grants from the NIH and the Bill and Melinda Gates Foundation.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Interrogating immune correlates of infection risk for efficacious and non-efficacious HIV-1 vaccine clinical trials have provided hypotheses regarding the mechanisms of induction of protective immunity to HIV-1. To date, there have been six HIV-1 vaccine efficacy trials (VAX003, Vaxgen, Inc., San Francisco, CA, USA), VAX004 (Vaxgen, Inc.), HIV-1 Vaccine Trials Network (HVTN) 502 (Step), HVTN 503 (Phambili), RV144 (sponsored by the U.S. Military HIV Research Program, MHRP) and HVTN 505). Cellular, humoral, host genetic and virus sieve analyses of these human clinical trials each can provide information that may point to potentially protective mechanisms for vaccine-induced immunity. Critical to staying on the path toward development of an efficacious vaccine is utilizing information from previous human and non-human primate studies in concert with new discoveries of basic HIV-1 host-virus interactions. One way that past discoveries from correlate analyses can lead to novel inventions or new pathways toward vaccine efficacy is to examine the intersections where different components of the correlate analyses overlap (e.g., virus sieve analysis combined with humoral correlates) that can point to mechanistic hypotheses. Additionally, differences in durability among vaccine-induced T- and B-cell responses indicate that time post-vaccination is an important variable. Thus, understanding the nature of protective responses, the degree to which such responses have, or have not, as yet, been induced by previous vaccine trials and the design of strategies to induce durable T- and B-cell responses are critical to the development of a protective HIV-1 vaccine.The path forward to an efficacious HIV-1 vaccine will be expedited via the analysis of immune correlates from past and future HIV-1 vaccine efficacy trials. Approaches that aim either to improve upon current findings from a partially efficacious vaccine strategy or approaches toward the generation of broadly neutralizing antibodies by rational immunogen design strategies are likely to be fast-tracked through careful probing of the intersections of immune correlate analyses within and among human vaccine clinical trials. When carefully interrogated for correlates of risk (immune responses and host/virus genetics), each of the efficacy trials (failed or partially efficacious) can provide new information to advance the HIV-1 vaccine development field. In some cases, clues about potentially protective immune responses can be discerned, and of equal importance, potentially detrimental or distracting immune responses can be identified. Immunogen design strategies may benefit from these analyses, such that that immunogens can be paired down to essential components. Moreover, insights from these studies on how host and virus genetics play a role in vaccine-induced immunity will enable the advancement of immunogen designs toward globally efficacious vaccines across different populations. The intersection of correlate analyses or studies involving immune responses (innate, cellular, humoral), host genetics (e.g., FcR expression, HLA) and HIV-1 virus sieve analyses (virus sequence analysis of all infections (placebo and vaccine groups) to determine if there were specific genetic attributes of infecting viruses that were significantly different between the two groups) are areas of the highest information content for follow-up studies (Figure 1). In this review, the correlate analyses for HIV-1 vaccine efficacy trials are described, with an emphasis on those studies that examine the intersection of different components and analyses.Due to the uniqueness of each efficacy trial and outcome, each study can provide information regarding the minimum bar to overcome to achieve HIV-1 vaccine efficacy. Even when there is no overall vaccine efficacy, the heterogeneity of immune responses among vaccines has enabled follow-up studies to identify associations of immune responses with HIV-1 infection risk in subsets of vaccines. Furthermore, an integral part of understanding immune correlates of protection is to also define patterns of immune responses that associate with non-protective vaccines. One of the major goals for the HIV-1 vaccine field is to define correlates of protection from HIV-1 infection, such that the field has biomarkers that will clearly predict vaccine outcome in HIV-1 vaccine efficacy trials.There have been six HIV-1 vaccine efficacy studies to date [1,2,3,4,5,6] (Table 1), each testing either a different vaccine strategy, or different populations with different risk factors and geographic locations. HIV-1 vaccine efficacy studies have been analyzed for correlates of infection risk; however, a correlate of protection for an HIV-1 vaccine has not yet been identified. Both the non-efficacious vaccines [7,8,9,10,11] and the one partially efficacious HIV-1 vaccine [12,13,14,15] trial have yielded several correlates of infection risk; although more weight is given to those findings from trials where there was overall vaccine efficacy. In total, three of these studies (VAX004, Step, and RV144) found significant correlations with HIV-1 infection risk/incidence and two of these studies (Step and RV144) identified potential sites of immune pressure on the virus (virus sieve).Overlapping regions among distinct analyses are targets for novel insights. The intersection of correlate analyses (shown as overlap of circles) involving immune responses (innate, cellular, humoral), host genetics (e.g., FcR expression, HLA), and HIV-1 virus sieve analyses are areas of the highest information content for follow-up studies. A subset of independent immune response measurements (e.g., cellular and humoral responses top left and right) can also overlap to point to similar potential mechanism(s) of protective immunity. (Ab Form: antibody subclass, isotype, dimeric, etc.) The magnitude and quality of vaccine induced immune responses differ in time (black line) post vaccination and may influence vaccine efficacy.The terminology employed for describing correlates and surrogates for protection from HIV-1 infection have been the focus of ongoing discussion and clarification [16,17,18,19]. It is generally agreed upon that for any one of the correlates of infection risk identified in RV144 to be considered a correlate of protection that reliably predicts protection from HIV-1 infection, it must be tested and proven in another clinical trial either with the same setting or in a different vaccine setting [18]. Ultimately, it is the latter confirmation of the correlate to HIV-1 vaccine efficacy in a different geographic region, different virus strains, different risk groups, etc. that is the most sought after for the development of a global HIV-1 vaccine.HIV-1 vaccine efficacy trials and immune correlates. Vaccine-induced immune responses have been studied to identify immune correlations with infection risk and evidence of virus sieve that can inform the design and evaluation of the next phase of vaccine efficacy trials (shaded boxes indicate reported correlations). Immunizations in two efficacy studies (Phambili/HVTN 503, HVTN 505) were stopped; however, follow-up of participants continues. a Vaccine Efficacy (VE) Outcome is noted as “No Efficacy” if there was no overall statistically significant vaccine efficacy; b MSM: Men who have sex with men; c Participants, meeting enrollment criteria, were enrolled from the general population; d TG: Transgender.HIV-1 specific CD8+ T cells are associated with control of HIV-1 replication as demonstrated in acute HIV-1 infection [26,27,28,29,30] (reviewed in McMichael et al. [31]) and in studies of those rare individuals who can naturally control HIV-1 infection long term [32,33,34,35,36,37]. Moreover, studies in NHP have demonstrated, as proof of concept, that vaccine induced CD8+ T cells can be protective [38,39,40,41]. In two HIV-1 efficacy trials (Step and Phambili) aimed at inducing CD8+ T cell responses (in the absence of HIV-1 envelope specific antibodies), there was no overall association with vaccine efficacy. However, these studies indicated that there were vaccine-elicited CD8+ T cells that could impact virus replication; although not sufficient enough to provide overall decreased risk of infection in the trial (either due to low magnitude/breadth/function of T cell responses and/or pre-existing vector immunity etc.). HVTN 505, also designed to target the induction of CD8+ T cell responses (as well as HIV-1 envelope specific antibodies), was discontinued and unblinded due to lack of efficacy. Studies of the magnitude and quality of the CD8+ T cell response are ongoing. Since there was evidence of increased infections in two of the Ad5 vector based vaccine studies, Step and Phambili [42] but not in HVTN 505 [6], there is a greater emphasis on understanding immune responses to vaccine vectors and impact on subsequent immunity. Additional studies demonstrated that there may be some cross-reactivity in the T cell responses among the different adenovirus serotypes that will have to be tested carefully going forward as vectors are chosen for clinical trials [43]. An alternate interpretation is that perhaps many other types of HIV-1 vaccines induce some level of CD4+ T cell activation; however, the key is to have sufficient overall HIV-1 specific immunity to tip the balance toward protection. Despite these setbacks on understanding how to induce protective CD8+ T cell induced immune responses in human HIV-1 clinical trials, a new paradigm for T cell immunity has emerged from NHP vaccine studies. Picker and colleagues report on a CMV based vaccine regimen that protects NHPs from infection and induces a novel subset of CD8+ T cells that can broadly recognize HIV-1 epitopes through class II restriction [38]. The series of papers by Picker and colleagues provide new hypotheses to test for inducing effective cellular immunity, both in terms of both new immunogen vector design and new subsets of CD8+ T cells to target with vaccine approaches. The Step study [3,44] as well as the Phambili study [4] were test of concept studies (double-blind, randomized, placebo-controlled) for induction of HIV-1 specific T cells responses. These trials tested the concept of a replication defective adenovirus serotype 5 (Ad5) vector with clade B HIV-1 genes (gag, pol nef) (MRKAd5) to decrease virus load in those who became infected. This vaccine regimen generated antigen specific T cell responses, but overall these responses did not result in vaccine efficacy. An increased rate of HIV-1 infection was observed in vaccines compared to placebos that was associated with being uncircumcised and having pre-existing Ad5 antibodies (reviewed in Gray et al. [45]). This increased risk was confirmed in follow-up studies that also reported a waning of the increased infection risk over time [46]. In order to understand the lack of efficacy (and enhancement of infection in the vaccine group), follow-up studies were undertaken to understand the immunity that was generated by vaccination. One of the goals of these follow-up studies was to examine potentially protective responses that may be subdominant in the population but may impact either acquisition or disease progression in some of the vaccines. Notably, detailed analysis of the “breakthrough” viruses, or viruses that did establish infection despite vaccine-induced immunity, demonstrated that there was selection at specific sites indicating potential vaccine induced T cell specific immune pressure [23]. Notably, the antigen specific CD8+ T cells responses induced by Ad5 vaccination were to HIV-1 epitope hotspots that were different than those generated in natural infection and were also directed to highly variable regions that likely can tolerate escape mutations from vaccine induced immune responses [47]. Host genetics also played a role in immunity, since those vaccines with HLA alleles (B*27, B*57, B*58:01), known to be associated with HIV-1 control, had lower viral load [48] and their CD8+ T cells exhibited greater killing in in vitro assays [49]. Recent analysis of those infected within a year of their last vaccination, identified that total T cell breadth and total magnitude of the immune response after the immunization series and prior to infection significantly correlated with lower mean viral load [50]. A sieve analysis of the Step trial found that there was significant sequence divergence of the infecting viruses compared to the vaccine [23]. However, despite an observed anamnestic T cell response (from vaccine-induced immunity) after infection, the specific T cell responses measured as part of the evaluation of the trial did not correspond with the sieve findings [51]. Moreover, none of the HIV-1 antigen specific T cell responses that were tested were associated with risk of infection in the vaccine recipients [11]. Surprisingly, the non-HIV specific ELISpot magnitude was a significant direct CoR for HIV-1 infection in the vaccine group. Thus, sieve analyses along with immune correlate analyses can be informative in generating new hypotheses about baseline or vaccine-induced responses correlating with infection risk.Four of the HIV-1 vaccine efficacy trials induce HIV-1 Env-specific antibody responses by three diverse strategies: recombinant HIV-1 gp120 immunogen alone (VAX003 and VAX004); vector prime with gp120 boost (RV144); and DNA prime, vector boost (HVTN 505). Although VAX003 and VAX004 efficacy trials each tested gp120 protein only as an immunogen strategy to induce humoral responses, these trials were distinct in that they were conducted in different risk populations (injection drug users (IDU) vs. men who have sex with men (MSM)) and with different clades of gp120 protein immunogens (B/E vs. B/B). Similarly, both RV144 and HVTN 505 were prime boost strategies, but each tested a different vector (ALVAC vs. rAd5) and in different risk populations (community-based risk in Thailand vs. MSM in the U.S.). It is unknown whether the same mechanisms of protection against HIV-1 acquisition will be similar or quite distinct among the different risk populations/transmission routes. Thus, there are four major differences across these HIV-1 vaccine efficacy trials that need to be considered when comparing studies: (1) differences in the HIV-1 gp120 sequence content; (2) differences in the approach (prime/boost and vector vs. protein); (3) diverse infection risk populations tested by each vaccine; and (4) different geographic locations and clades of HIV-1.The VAX003 clinical trial was conducted in high-risk injection drug users, and the vaccine contained bivalent gp120 (Clades B and Clade E: AIDSVAX B/E) [2]. As part of the primary analysis of vaccine efficacy for VAX003, pre-specified antibody variables (V2 and V3 binding antibodies to a vaccine strain envelope (A244), gp120 binding antibodies (A244 and MN), CD4 blocking antibodies and neutralizing antibodies to HIV-1 MN) were found not to be correlates of risk. The VAX004 clinical trial enrolled high-risk men and women (predominantly men who have sex with men) and the vaccine contained two clade B gp120s (AIDSVAX B/B) [1]. Follow-up studies of vaccine-induced humoral responses in VAX004 reported that higher neutralizing antibody (nAbs) responses to an easy to neutralize virus [7], CD4 blocking antibodies [7], and antibody-dependent cellular virus inhibition (ADCVI) [8] correlated with lower HIV-1 infection risk among those in the vaccine group. Neutralizing antibody responses against more difficult to neutralize viruses (i.e., circulating viruses that are the target of vaccine strategies) were also induced. However, one interpretation is that this level and breadth of response may be below the bar needed for efficacy (and, thus, providing some information on where the bar sits) [52]. Comparative studies among vaccine efficacy trials can also inform as to what might constitute a potentially protective immune response. Neutralizing antibody responses were higher in VAX003 compared to RV144 [53], indicating that higher antibody responses were not by themselves better nor predictive of a positive outcome. Insights from analyses of host genetics suggest that although there was no overall increased infection in the VAX004 vaccine trial, the Fcγ receptor IIIa genotype (VV genotype, encoded by an allele with a valine (V) a position 158) was associated with an increased rate of HIV-1 infection in low risk vaccines, but not in high risk vaccines [9].RV144 is the only HIV-1 vaccine efficacy trial in which the vaccine showed decreased transmission versus the placebo group, with an estimated vaccine efficacy of 31% [5]. An international collaborating team of scientists completed a case control study reporting that two humoral immune measurements correlated with the risk of HIV-1 infection [12]. IgG antibodies to the V1/V2 region of HIV-1 gp120 correlated with a decreased risk of infection [12,13,14,15], and a plasma HIV-1 envelope-specific IgA score correlated with increased risk of infection in the vaccine arm (decreased vaccine efficacy) [12,54]. Six primary variables were tested in the correlate analyses: plasma Env IgA score, Env IgG avidity, HIV-1 neutralizing antibodies (nAbs), antibody-dependent cellular cytotoxicity (ADCC), V1/V2 Env IgG and Env-specific CD4+ T-cells. Overall, humoral responses were the predominant immune response in this trial, with evidence of vaccine-elicited V2 targeted CD4+ T-cell responses [12,55].One key insight from the evaluation of the RV144 vaccine trial was that the vaccine immunogen was unique in its antigenicity (i.e., exposure of specific epitopes) compared to other envelope proteins [25,56]. Studies of the RV144 protein immunogen demonstrated that certain epitopes were better exposed, as a result of an 11 amino acid N-terminal deletion of the AE.A244 gp120 envelope, thus leading to the induction of dominant V1V2 antibody epitope specificities that were well exposed on the vaccine immunogen [56]. In particular, the A244 gp120 used in RV144 was antigenic for both linear V2 epitopes bound by strain-specific V2 antibodies, as well as for conformational V1V2-glycan epitopes bound by V1V2 broad neutralizing antibodies (BnAbs) [25,56]. Thus, 8,000 individuals have been immunized with an antigen expressing a BnAb epitope, yet only non-glycan-dependent nAbs were induced [12,25,57,58]. Thus, in addition to defining correlates of protection in RV144, a second critical reason to study vaccine responses in RV144 is to understand why BnAbs were not induced by an antigenic immunogen. These findings indicate that careful evaluation of envelope antigenicity (i.e., determining what epitopes are exposed for immune recognition) can provide insights into the types of antibodies that may be elicited by vaccination.Further analyses of the six primary variables in the RV144 correlate analyses using interaction models was one way of examining the role of multiple immune measurements in assessing correlates of HIV-1 infection risk. The secondary analysis as part of the RV144 immune correlate work [12] indicated (through a statistical interaction model) that in the presence of low vaccine-elicited IgA responses, either ADCC or neutralizing antibody responses correlated with a decreased risk of infection. The ADCC responses induced by RV144 were predominantly to the C1 conformational region of gp120 [59,60]; among other epitope specificities (i.e., V2, V3) [25]. Thus, one hypothesis from the RV144 correlate study was that C1 region Env-specific IgA could block C1-specific ADCC by binding to the same epitopes on infected cells, but without the functional engagement of NK cells that is needed to mediate ADCC. Indeed, RV144-induced Env IgA was shown to block C1 region-specific IgG-mediated antibody-dependent cellular cytotoxicity by natural killer cells [54]. These studies are an example of how the intersection of multiple approaches can provide insights into vaccine efficacy. Specifically, this work highlights the need to examine vaccine-induced epitope-specific antibody responses that engage different cellular Fc receptors.Together, the two immune correlates of HIV-1 infection risk in RV144, along with data from the secondary analyses and follow-up studies, indicate that evaluating antibodies that target circulating strains in the population being tested as well as cross-clade epitopes may play a role in understanding vaccine efficacy. Circulating Env IgA responses to the C1 region in gp120 of a circulating strain in Thailand (CRF01_AE) was the strongest statistical correlate of HIV-1 infection risk in RV144. Moreover, IgG responses to the V2 region of CRF01_AE Env significantly correlated with a decreased risk of infection among the different clades of V2 responses tested in the peptide microarray assay [12,15]. Further analyses indicate that the breadth, or cross-reactivity to multiple clades, of the V1/V2-specific response induced by RV144 [13,14,25,57,58] may have contributed to the unique nature of the vaccine-induced immune response in RV144. Further studies in other vaccine trials will be able to test these specific hypotheses to see if vaccine-induced immunity that can broadly target circulating strains will be critical for vaccine-mediated protection from HIV-1 infection.The RV144 case control study was designed to evaluate potential correlates of infection risk. Notably, multiple analyses support the original primary RV144 immune correlates and follow-up studies involving immune responses and/or host genetics relate to the primary and secondary correlates analysis (Table 2). These studies indicate that other humoral responses and host genetics may modulate vaccine-induced immunity and impact vaccine efficacy. Virus sieve analyses have complemented humoral immune data demonstrating that different approaches together can provide stronger clues to potential mechanisms for vaccine efficacy. Although there were only six primary immune measurements tested in the case control analysis, other measurements as part of the RV144 case control study [12] and as follow-up analyses [13,14,15,54] were significantly associated with HIV-1 infection risk (statistical results with p < 0.05) (Table 3). Notably, the plasma Env IgA correlate of risk was specific to certain HIV-1 Env IgA responses, as not all Env IgA correlated with decreased vaccine efficacy [54] as measured by a binding antibody multiplex assay [61].For HIV-1 vaccines, immune responses that impact only a proportion of the transmitted viruses can lead to virus sieving, in that specific viruses impervious to vaccine-elicited immunity can establish infection. These virus sieve analyses in concert with probing vaccine-induced immune response can lead to hypotheses of specific immune responses that may prevent the acquisition of some viruses. Moreover, analyses of viruses escaping from vaccine-induced immunity can provide a roadmap for critical targets on infectious virions that need to be targeted by an HIV-1 vaccine to substantially increase vaccine efficacy. Ongoing analyses of the changing complexity of circulating viruses may be critical for understanding the virus target in specific populations. Additional independent studies examining breakthrough viruses in RV144 vaccines (sieve analyses) and the generation of monoclonal antibodies from the B-cells of vaccine recipients have pointed toward a specific site within the V2 region of the HIV-1 envelope. V2 mAbs derived from RV144 vaccines bound to an epitope centered on K169 of the V2 region [25], and a sieve analysis [24] focused on this region found K169 to be a critical site of immune pressure. Thus, if the infecting strain of HIV-1 matched the RV144 vaccine AE.V2 region at K169, the vaccine efficacy was 48%. Thus, we have proposed that one way to enhance the efficacy of follow-up trials to RV144 would be to include additional Envs or V2 peptides with V2 sequences not covered in the original RV144 envelope sequences. Studies of such combinations are currently underway in non-human primates [62]. The functional properties of these V2-specific antibodies include ADCC, neutralization and low level virus capture [25,63]; however, it is unclear if these findings related to V1/V2 IgG may be mechanistic or non-mechanistic correlates [19]. Thus, the combination of the initial analysis of the V2-directed humoral responses induced by RV144, work identifying that V1V2 (K169) IgG3 correlated with a decreased risk of infection [14], the mAb work that identified the importance of a lysine in position 169 of the V2 loop and virology work that resulted in the demonstration of K169 as a site of immune pressure, together, reinforced the hypothesis that the induction of V2-specific antibodies is important to test as a correlate of protection in further HIV-1 vaccine efficacy trials. RV144 correlate analyses: intersections with virology and host genetics.RV144 secondary and follow-up immune measurements: associations and interactions.Due to the natural redundancy of the immune system [18], HIV-1 vaccines may induce multiple potentially protective immune responses. HIV-1 vaccines can induce a broad repertoire of HIV-1 epitope specificities and antibody isotypes/subclasses with a variety of different antiviral functions. Even in RV144, where V1/V2 Env IgG responses were identified as an immune correlate of risk of HIV-1, there was broad heterogeneity among vaccines in their immune responses with a diverse repertoire of antibody forms, specificities and functions [63]. Some of these diverse immune measurements were not tested in the immune correlates analysis, due to inherent assay limitations, sample limitations and the requirement of minimizing test variables in order to maintain statistical power. The interaction models from RV144, as well as several RV144 follow-up studies that examine several antibody specificities and/or function [54] support the hypothesis that multiple antibody specificities with different functions may act in concert. Evaluating multiple antibody specificities and antiviral functions, in addition to identifying single correlates of infection risk, will be central to identifying mechanistic correlates of protection for HIV-1 toward the goal of identifying an efficacious vaccine strategy. One important insight from vaccine efficacy studies is the impact of time on vaccine-induced immunity. The time point at which vaccine efficacy is determined in a trial can dramatically influence the resulting level of vaccine efficacy. RV144 had a 60.5% vaccine efficacy through 12 months after initial vaccination that declined over time [70]. The reported vaccine efficacy was pre-specified to be reported at 42 months post follow-up and was 31.2% at that time. This higher level of vaccine efficacy at an earlier time point is hypothesized to be due to a higher level or quality of antibody response that decreased over time For example, Env IgG3 responses are induced in RV144, but decline more rapidly than the overall Env IgG response [14]. Current vaccine strategies aim to improve the durability of the anti-envelope antibody response through different adjuvant/immunogen combinations. Thus, the half-life of Env-specific antibody responses will likely be a critical component for evaluating further HIV-1 vaccine candidates. Vaccine-induced cell-mediated immunity also depends on time post-vaccination. Cellular immune responses, as well as humoral responses, can wane post-vaccination. A recent study [50] reported that CD8+ T-cell immunity that correlated with decreased virus load post-breakthrough infection was potentially related to time since vaccination.HIV-1 vaccines that can induce an orchestrated response comprised of all arms of the immune response (innate, cellular and humoral) may prove to be the most efficacious across different populations, genders and routes of transmission. Thus, it is imperative to advance forward with strategies for both effective T- and B-cell responses that likely will be induced through specific engagement of the innate immune system. New technologies and approaches have allowed the field to cast a broader net in evaluating vaccine strategies. The advent of virus sequencing at the single genome level has allowed for a detailed analysis of the specificities of immune responses that can impact virus replication in both human and non-human primates. These virus-specific approaches for that are not sensitive to vaccine-induced immunity compared to those viruses that are sensitive; (2) potential insights into important vulnerable sites (and their structure) on the virion that vaccines should target; and (3) information on whether the described vaccine-induced humoral and cellular immune responses have epitope specificities that match those identified as sieve sites in the virus. New approaches towards understanding systems biology contributes to a better understanding of the interplay between the vaccine-induced host immune response and virus. Taken together, these technological advances have yielded a robust amount of data that has an unprecedented need for innovation in the way the data are analyzed.Analysis of host genetics, viral genetics and immune responses [71,72] have already provided numerous insights into both innate and adaptive responses of virus control and disease progression in HIV-1 infected individuals. Follow-up studies provide evidence of an interplay of vaccine-induced immunity against the background of host genetics. Understanding the role of host genetics and the functional attributes of vaccine-induced immunity can enable a better understanding of which immune correlates will be broadly applicable in a globally efficacious vaccine. Similarly, studies are ongoing to understand if immune responses to one vaccine can be predicted by immunity to another vaccine; thus, also allowing deeper insights into intrinsic population effects that may not be distinct to the vaccine strategy being tested.Currently licensed vaccines for other pathogens are based on empirical studies, and many do not have defined mechanistic correlates of protection. The protective efficacy of some of these vaccines are associated with antibody responses, with the exception of rotavirus and BCG ((Bacillus Calmette-Guerin) tuberculosis vaccine) [18,73]. Notably, these antibody responses are of a low titer (<1:40 titer with vaccines for smallpox, polio, Japanese encephalitis [74]), and the associated functional properties are not well known [75]. Mucosal IgG has been associated with the protection of several vaccines (e.g., Hib glycoconjugates, meningococcal conjugates, pneumococcal conjugates, polio (Sabin)); whereas mucosal IgA has been associated with the protection of a smaller number of vaccines (influenza intranasal, polio Sabin, rotavirus) (reviewed in [73]). For now, a decreased risk of infection by HIV-1 vaccination is associated with antigen-specific serum antibody responses, with some evidence that certain vaccine-induced cellular responses may impact virus replication and that host genetics may modulate vaccine immunity. Analyses of the correlates of HIV-1 infection risk have demonstrated that there are intersections among different analyses (involving immune responses, virus and host genetics), thus providing the areas of highest information content for understanding the potential mechanisms of protective immunity. Identification and rigorous characterization of these areas of overlap are likely to be transformative for the next phase of HIV-1 vaccine design and evaluation.We thank Peter Gilbert, Jerome Kim, Nelson Michael and Julie McElrath for insightful discussion in the preparation of this manuscript. The authors acknowledge funding from the HIV-1 Vaccine Trials Network (HVTN) (5U01 AI46725-05), the Duke University Center for AIDS Research (CFAR) Grant (P30 AI 64518), the Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery (CHAVI-ID), National Institutes of Health/National Institute of Allergy and Infectious Diseases/ Division of AIDS and Bill and Melinda Gates Foundation Grants (1033098, 1032144, 1040758).B.F.H. and G.D.T. have patent applications submitted on portions of the work discussed.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Recent studies indicate that DNA immunization is powerful in eliciting antigen-specific antibody responses in both animal and human studies. However, there is limited information on the mechanism of this effect. In particular, it is not known whether DNA immunization can also enhance the development of antigen-specific B cell development. In this report, a pilot study was conducted using plague LcrV immunogen as a model system to determine whether DNA immunization is able to enhance LcrV-specific B cell development in mice. Plague is an acute and often fatal infectious disease caused by Yersinia pestis (Y. pestis). Humoral immune responses provide critical protective immunity against plague. Previously, we demonstrated that a DNA vaccine expressing LcrV antigen can protect mice from lethal mucosal challenge. In the current study, we further evaluated whether the use of a DNA priming immunization is able to enhance the immunogenicity of a recombinant LcrV protein vaccine, and in particular, the development of LcrV-specific B cells. Our data indicate that DNA immunization was able to elicit high-level LcrV antibody responses when used alone or as part of a prime-boost immunization approach. Most significantly, DNA immunization was also able to increase the levels of LcrV-specific B cell development. The finding that DNA immunization can enhance antigen-specific B cell responses is highly significant and will help guide similar studies in other model antigen systems. DNA immunization was discovered about 20 years ago. While it was initially considered a novel approach to elicit T cell responses, data accumulated in the last decade has further indicated that DNA immunization is also very effective in eliciting antibody responses against both viral and bacterial antigens [1,2,3,4,5,6,7], particularly when included as part of a prime-boost immunization [8]. However, the mechanism by which DNA vaccines elicit high quality antibody responses has not been well studied. One possibility is that DNA immunization can elicit high quality antigen-specific B cell responses. In the current report, a pilot study was conducted to address this possibility using the LcrV (V antigen) from Y. pestis as a model antigen. Y. pestis is a gram-negative bacterium that causes human plague, which may present as one of three forms: bubonic, septicemic, or pneumonic, depending on the route of initial infection. Regardless of the route of infection, the disease results in high mortality (50%–90%) if left untreated [9]. An interest in a prophylactic vaccine against plague extends beyond biodefense applications, as isolated plague outbreaks occur sporadically in both developed and developing countries, and antibiotic-resistant strains have been described [10,11,12]. Currently, there is no widely acceptable vaccine against plague. Live attenuated strains and, more recently, formalin-killed whole cell vaccines have been developed but proved highly reactogenic in humans [13,14]. A killed whole-cell vaccine was licensed in the U.S. but was withdrawn from clinical use because it required multiple doses, was highly reactogenic, and did not protect effectively against pneumonic plague [13,14]. The F1 capsular protein (F1) and the V protein (LcrV, a component of the Y. pestis type-III secretion system) have been established as lead antigens for subunit-based plague vaccines and were shown to induce protection against bubonic and pneumonic plague in several animal models [5,7,14,15,16,17,18,19]. These antigens also elicited antibodies when administered in humans, however, the antibody response levels were moderate [20]. Our previous mouse studies established the feasibility of using DNA immunization to elicit LcrV antibody responses; mice immunized with LcrV DNA vaccines were protected from lethal mucosal challenges [5]. In the current study, the same LcrV DNA vaccines were used. Given mounting evidence from both plague and non-plague vaccines studies showing that protective immunity can be significantly improved when vaccines in different forms are administered in a prime-boost format [21,22,23,24,25,26], both DNA vaccine alone and DNA prime-protein boost approaches were included in the current study. We tested whether the heterologous DNA prime-protein boost approach is more effective than the homologous DNA alone or protein alone immunization approaches in eliciting LcrV antigen-specific B cell immune responses.The codon optimized DNA vaccine (V.opt) expressing the LcrV protein of Y. pestis was constructed, as previously described [27]. A synthetic lcrV gene was cloned into the DNA vaccine vector, pSW3891 [26], at the PstI and BamHI sites downstream of the cytomegalovirus (CMV) immediate early (IE) promoter and its adjacent Intron A [28,29]. The DNA plasmids used in this study were prepared by a Mega purification kit (QIAGEN).The wild type lcrV gene was PCR-amplified from the LcrV DNA vaccine, as previously described [5] and cloned into the E. coli expression vector, pBAD/gIII (Invitrogen), with a His(6)-Tag at the C-terminus fused with V-antigen. The plasmid DNA was transformed into E. coli strain, LMG194, for V antigen expression. LMG194 bacterial culture and protein expression were conducted following instructions from the pBAD/gIII kit from Invitrogen. The LcrV-His(x6) protein was purified from the LcrV expressing LMG194 bacterial lysate using a nickel column. The purified V protein was analyzed by SDS-PAGE and Western blot and used for V protein vaccination and ELISA to detect V-specific antibody responses in mouse sera. Female BALB/c mice of 6–8 weeks old were purchased from Taconic Farms (Germantown, NY, USA) and housed in the animal facility managed by the Department of Animal Medicine at the University of Massachusetts Medical School (UMMS) in accordance with IACUC approved protocol. Mice (5/group) received two immunizations at Weeks 0 and 4 with designated vaccination regimens listed in Figure 1. Each mouse received codon optimized lcrV DNA vaccine (V-opt) (X2), V protein alone (X2), V-protein formulated with Incomplete Freund Adjuvant (IFA) (X2), V-opt DNA prime followed by V protein/IFA boost, or DNA vector alone immunization as the negative control. DNA immunizations were conducted via gene gun using a Helios gene gun (Bio-Rad). V.opt or the pSW3891 vector plasmid was coated onto 1.0-micron gold beads at 2 μg DNA/mg gold. Each shot delivered 1 μg of DNA and a total of six non-overlapping shots were delivered to shaved abdominal skin at each immunization after animals were anesthetized. Protein immunizations were done by intramuscular (i.m.) injection at the quadriceps, one injection site at one leg each with a dose of 1 μg/site (X2 sites). Sera were collected prior to and at two weeks after each immunization and at additional time points as indicated in Figure 1. At Week 16, animals were euthanized and splenocytes and bone marrow cells were isolated for B cell assays.Mouse sera were tested for V-specific IgG antibody responses by ELISA as previously described [5]. Microtiter plates were coated with 100 ng/well of purified recombinant V antigen (1 μg/mL in PBS, pH 7.2) at 4 °C overnight and then washed five times with washing buffer (PBS at pH 7.2 with 0.1% Triton X-100). Blocking was done with 200 µL/well of 4% milk-whey blocking buffer for 1 h at room temperature. After removal of the blocking buffer and another five washes, 100 μL of serially diluted mouse sera was added and incubated for 1 h. The plates were washed five times and incubated with 100 μL of biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) diluted at 1:1,000 for 1 h followed with washes. Horseradish peroxidase-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA), diluted at 1:2,000, was added (100 μL/well) and incubated for 1 h. After the final set of washes, 100 μL of fresh TMB substrate (Sigma Aldrich, St. Louis, MO, USA) was added to each well and incubated for 3.5 min. The reaction was stopped by adding 25 μL of 2 M H2SO4, and the plate was measured at OD 450 nm. The temporal serum antibody response trend was monitored directly by OD value from ELISA and antibody titers at the peak response or selected time points were calculated based on the end titration of serum dilution of immune sera (>2 fold over the control sera).For V-specific IgG isotype analysis, a modified ELISA was conducted as previously described [5]. Serially diluted commercial mouse IgG, IgG1, or IgG2a (Southern Biotech, Birmingham, AL, USA) were coated onto ELISA plates to establish individual standard curves. ELISA, as described above, was performed on the same plates and concentrations of V-specific mouse IgG, IgG1, or IgG2a were calculated according to the standard curves.lcrV-DNA and V-protein immunization groups and vaccine components. Each mouse received 2 immunizations: prime at Week 0 and boost at Week 4 using designated codon optimized V DNA vaccine (V-DNA), V protein (V-Prot), or empty DNA vaccine vector (Vector) as indicated. After isolation of splenocytes and bone marrow cells, they were divided into three portions for different assays: (1) freshly isolated cells were used to measure V-specific ASC; (2) V protein stimulated cells were used to detect memory B cells; and (3) splenocytes or bone marrow cells cultured for 5 days without stimulation were used to detect long lasting ASC. For B cell stimulation, bulk splenocytes or bone marrow cells at 5 million cells/ml were stimulated with V protein (5 µg/mL) + IL-2 (20 units/mL) for 5 days before being used for ELISPOT assays. To conduct V-antigen-specific ELISPOT assays, MultiScreenHTS Filter ELISPOT Plates (Millipore, Billerica, MA, USA) were first coated with V antigen at a concentration of 2 μg/mL in PBS at 4 °C overnight, then blocked as described above. Freshly isolated, stimulated or cultured splenocytes or bone marrow cells (100 μL/well, 500,000 cells/well) in R10 medium with 0.1% β-ME were incubated in duplicate wells for 4 h at 37 °C. The plates were then washed and incubated with 100 μL of biotinylated goat-anti-mouse IgG diluted at 1:1,000 in dilution buffer from the ELISPOT kit above for 1 h. After additional washes, 100 μL of AP-conjugated streptavidin complex diluted at 1:2,000 in dilution buffer was added to each well for 1h at 37 °C. The plates were washed, and spots were developed after a 7 min color reaction using 1-STEP NBT/BCIP. IgG spot-forming cells (SFC) were counted. The results were expressed as the number of SFC per 106 input cells. Student’s t-test was performed to evaluate differences in V-specific antibody and B cell ELISPOT data between any two groups. The current pilot study was conducted, using a mouse model, to determine the effects of DNA immunization on the development of LcrV-specific B cells. Groups of BALB/c mice (5 mice/group) were immunized using one of the following regimens (Figure 1): (1) DNA Alone Group—received two codon optimized LcrV (V-opt) DNA [27] immunizations via gene gun at Weeks 0 and 4; (2) Prime-Boost Group—received LcrV DNA vaccine at Week 0 and the recombinant LcrV protein formulated with adjuvant IFA at Week 4 by i.m. immunization; (3) Protein Alone Group—received LcrV protein with IFA twice at Weeks 0 and 4 without DNA priming immunization; (4) Protein No Adjuvant Group—was similar to the third group except that recombinant LcrV proteins were used alone without any adjuvant; (5) Negative Control Group—received an empty DNA vector without lcrV gene insert. Serum samples were collected prior to the start of immunization and every 2 weeks afterwards until Week 10; samples were then tested for levels of LcrV-specific antibody responses. Animals were terminated at Week 16, three months (12 weeks) after the last immunization and serum samples collected at that time were used to measure the persistence of antibody responses. Spleen and bone marrow were collected for the measurement of LcrV-specific antibody secreting cells (ASC).Gene gun delivery of the LcrV DNA vaccine was highly effective and positive antibody responses in the DNA Alone Group were detected even after a single immunization (Figure 2). The 2nd immunization was able to further increase antibody response levels, which continued to increase for about four weeks. The temporal antibody response pattern in the Prime-Boost Group was similar. In contrast, the Protein Alone Group had delayed and lower level antibody responses after one immunization, but was able to reach the same levels as those in the first two groups after the 2nd immunization. Adjuvant is important for the immunogenicity of LcrV protein vaccines as the LcrV protein alone without IFA (Protein No Adjuvant Group) was not able to elicit the same levels of antibody responses, even after two immunizations. There was no LcrV antibody response in the Negative Control Group. The temporal pattern of antibody responses was measured using a fixed serum dilution; therefore, additional ELISA studies were conducted to measure actual antibody titers (Figure 3). These results further confirm the temporal pattern using fixed serum dilutions. At Week 2, after the first immunization, antibody titers in the DNA primed groups were significantly higher than those in the two groups that received a protein vaccine as the prime (Figure 3A). By Week 6, which was two weeks after the 2nd immunization, LcrV-specific antibody titers in the DNA immunization groups were similar to those in the IFA-formulated recombinant LcrV protein group, but were still much higher than those in the group that received LcrV protein alone without IFA (Figure 3B). The same pattern was observed when the actual amount of LcrV-specific IgG (in μg/mL) was measured (Figure 3C) in addition to traditional end titration titers.V-specific temporal antibody responses in mice immunized with different vaccination regimens: codon optimized V DNA vaccine alone (V-DNA), V DNA vaccine prime followed by V protein boost formulated with IFA (V-DNA+Prot/IFA), V protein formulated with IFA (V-Prot/IFA), V protein alone (V-Prot), or empty DNA vaccine vector alone (Vector). V-specific antibody responses were measured by ELISA at different time points using pooled mouse sera from each group against V protein. Each curve represents mean OD values with standard error of duplicated assays for each mouse group, at 1:500 serum dilution.V-specific IgG responses induced by various V vaccine regimens in mice: codon optimized V DNA vaccine alone (V-DNA), V DNA vaccine prime followed by V protein boost formulated with IFA (V-DNA + Prot/IFA), V protein formulated with IFA (V-Prot/IFA), V protein alone (V-Prot), or empty DNA vaccine vector alone (Vector). Panels A and B: V-specific antibody titers were measured by ELISA against V protein in mouse sera collected at 2 weeks after the prime (1st immunization) (Panel A) or at 2 weeks after the boost (2nd immunization) (Panel B). Panel C: V-specific IgG concentrations at 2 weeks after the boost (2nd) immunization. Each bar represents the mean V-specific IgG titers or concentrations in each group of 5 mice with standard error. Statistically significant differences (p < 0.05) are indicated as “*”, “#” or “^” when comparing V-DNA, V-DNA + Prot/IFA, and V-Prot/IFA groups.Subtypes of IgG antibody responses were also measured (Figure 4). Regardless of the type of LcrV vaccine used, IgG1 levels (Figure 4A) were higher than IgG2a (Figure 4B), indicating a Th-2 type antibody dominated response with approaches used in the current study, which is consistent with the use of gene gun or adjuvant IFA, as reported in the literature [6,30]. However, recombinant LcrV protein with adjuvant IFA elicited a higher ratio of IgG1/IgG2a than that observed for the DNA vaccine groups (Figure 4C). Similar to total IgG responses, both IgG1 and IgG2a antibodies were significantly lower in the Protein No Adjuvant Group (Figure 4C). V-specific IgG1 and IgG2a responses induced by various V vaccine regimens in mice: codon optimized V DNA vaccine alone (V-DNA), V DNA vaccine prime followed by V protein boost formulated with IFA (V-DNA + Prot/IFA), V protein formulated with IFA (V-Prot/IFA), V protein alone (V-Prot), or empty DNA vaccine vector alone (Vector). V-specific IgG1 (Panel A) and IgG2a (Panel B) concentrations were measured by ELISA against V protein in mouse sera collected at 2 weeks after the boost (2nd) immunization. Each bar represents the mean IgG concentrations in each group of 5 mice with standard error. Statistically significant differences (p < 0.05) are indicated as “*”, “#” or “^” when comparing V-DNA, V-DNA + Prot/IFA, and V-Prot/IFA groups. Panel C: IgG1/IgG2a ratios determined at 2 weeks after the boost immunization. Each bar represents the mean IgG1/IgG2a ratios in each group of 5 mice with standard error.However, the longevity of LcrV antibody responses in the DNA vaccine primed groups was better maintained than that in the LcrV protein groups (Figure 5). At Week 16, or 12 weeks after the second immunization, total IgG (Figure 5A) and IgG1 (Figure 5B) levels were significantly lower in the two protein groups than in the two DNA primed groups. IgG2a levels in the two protein groups were also lower but the difference was only significant in the Protein No Adjuvant Group (Figure 5C).V-specific IgG (Panel A), IgG1 (Panel B), or IgG2a (Panel C) concentrations measured at 12 weeks after the boost immunization in mice receiving various V vaccine regimens: codon optimized V DNA vaccine alone (V-DNA), V DNA vaccine prime followed by V protein boost formulated with IFA (V-DNA + Prot/IFA), V protein formulated with IFA (V-Prot/IFA), V protein alone (V-Prot), or empty DNA vaccine vector alone (Vector). Antibody titers were measured by ELISA against V protein. Each bar represents the mean IgG concentration in each group of 5 mice with standard error. Statistically significant differences (p < 0.05) are indicated as “*”, “#” or “^” when comparing V-DNA, V-DNA + Prot/IFA, and V-Prot/IFA groups. B cell ELISPOT analysis was conducted to measure LcrV-specific antibody secreting cells in the bone marrow and spleen of immunized mice. Fresh cells, cells cultured for five days without stimulation, and cells stimulated with LcrV antigen were used in this analysis (Figure 6). Figure 6A shows the representative ELISPOT pictures, which indicate that the overall frequency of LcrV-specific B cells in bone marrow was higher than that in the spleen. The recombinant LcrV protein vaccine alone group without adjuvant had the lowest levels of LcrV B cell responses in both bone marrow and spleen, which is compatible with observed antibody responses. When group average data was analyzed, the DNA prime-protein boost group had the highest LcrV B cell responses in spleen after stimulation, responses that were much higher than observed in the two LcrV protein groups (Figure 6B). In bone marrow, DNA vaccine groups generally had a higher number of LcrV B cells than protein vaccine groups, especially in fresh cells. However, due to the high variation in the B cell ELISPOT analysis in bone marrow cells, there was no statistically significant difference between DNA vaccine groups and protein groups in cultured or stimulated cells (Figure 6C).V-specific antibody secreting cells (ASC) in fresh, 5-day cultured and 5-day stimulated splenocytes and bone marrow cells as measured by ELISPOT. Mice immunized with different V vaccine regimens in mice: codon optimized V DNA vaccine alone (V-DNA),V DNA vaccine prime followed by V protein boost formulated with IFA (V-DNA + Prot/IFA), V protein formulated with IFA (V-Prot/IFA), V protein alone (V-Prot), or empty DNA vaccine vector alone (Vector), as indicated. Panel A: Actual sample wells of V-specific ASC spots splenocytes (upper panel) or bone marrow cells (lower panel). Panel B: Frequency of V-specific ASC per million splenocytes in each group. Panel C: Frequency of V-specific ASC per million bone marrow cells in each group. Data represent the mean spot forming cells (SFCs)/million cells with standard deviation from 5 mice/group. The splenocytes and bone marrow cells were collected 12 weeks after the boost (2nd) immunization. Statistically significant differences (p < 0.05) are indicated as “*”, “#” or “^” when comparing V-DNA, V-DNA + Prot/IFA, and V-Prot/IFA groups.Historically, recombinant V protein immunization was shown effective in eliciting V-specific antibody responses and such antibody responses were responsible for the protective immunity in animal models [15,16,31]. A similar approach has advanced to human studies [20]. At the same time, other novel vaccination approaches have been reported in early preclinical studies. Our previous studies demonstrated that LcrV DNA vaccines could elicit significant levels of both LcrV-specific antibodies and CD8 T cell responses as part of the protective immunity against lethal intranasal Y. pestis challenge in a Balb/C mouse model [5,7,27]. Recent studies have suggested that a heterologous prime-boost vaccination approach, in which the same antigen is delivered sequentially by different types of vaccines, may be more effective in eliciting enhanced immune responses than the homologous prime-boost using the same type of vaccines [21]. We previously reported that DNA vaccine prime-protein or inactivated vaccine boost could significantly improve the overall immunogenicity and functional antibody responses against HIV [3,25] and influenza [22]. In the current study, we evaluated LcrV-specific antibody responses using the DNA prime-protein boost regimen based on our previous LcrV DNA vaccine work [3,5,7,25,27] with a new focus on the quality of antibody responses and, more importantly, the development of LcrV-specific B cells. A comparison of immunizations was conducted in this pilot mouse study with LcrV DNA alone, LcrV protein alone, or a combination of DNA prime-protein boost. We demonstrated that all regimens studied were capable of eliciting LcrV-specific antibody and B cell responses. However, the mice that received the LcrV DNA vaccine prime not only produced overall higher antibody titers but also generated improved B cells responses as measured by ELISPOT for the detection of LcrV-specific antibody secreting cells in fresh or stimulated splenocytes and bone marrow cells. Previous analysis of antibody isotypes suggested that a Th1-type (IgG2a dominant) antibody response may be important in providing better protection [5,27]. In addition to the levels of humoral responses, the mice that received the DNA prime produced more balanced IgG1/IgG2a responses with an increased level of IgG2a titers compared to the mice that received the protein alone immunization. These data highlight the potential of heterologous DNA prime-protein boosts to lead to higher Th1-type responses, which may be beneficial for protective immunity against plague. Besides antibody responses, our previous studies demonstrated that the V DNA vaccine could also induce antigen-specific T cell responses [7] similar to what reported by using other types of plague antigens to elicit T cell immune responses [32]. Another important finding from our study is that the levels of LcrV antibody responses were better maintained in the DNA prime-protein boost group compared to the other groups. This may imply that such a combination immunization approach may be more effective in building antigen-specific memory. To support this finding, the prime-boost approach was more effective in eliciting LcrV-specific B cell responses. It is interesting to observe that the benefit of the DNA prime is shown in the bone marrow with fresh cells, indicating the levels of available LcrV-specific antibody secreting cells, and in spleen with stimulated cells, indicating the levels of LcrV-specific memory B cells. These similar patterns of LcrV-specific B cell responses at different stages of development provided exciting evidence to support the use of DNA immunization to elicit high quality antibody and B cell responses. In summary, results from this study demonstrated that a heterologous DNA prime-protein boost approach elicits an improved antibody response and antigen-specific B cell responses against plague. This finding will have a major impact not only to the development of improved plague vaccines but also to the development of vaccines against other infectious-causing agents that require long term protection and high levels of antigen-specific memory B cell responses. Previously, there was limited information on the ability of DNA immunization to elicit high quality B cell responses. Information presented in this pilot study is highly significant and will set the stage for more in-depth studies with other antigen systems to fully establish this previously unknown mechanism of DNA vaccination.The current report confirms previous findings that DNA immunization is effective in eliciting LcrV-specific antibody responses in a mouse model and further demonstrates that the DNA prime-protein boost approach is also effective in eliciting LcrV-specific B cell development, which is important for the quality and longevity of protective immunity. The study reported here was funded in part by NIH Grant U01AI078073. The authors would like to thank Jill M. Serrano for critical reading and editing of the manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Streptococcus pneumoniae still causes severe morbidity and mortality worldwide, especially in young children and the elderly. Much effort has been dedicated to developing protein-based universal vaccines to conquer the current shortcomings of capsular vaccines and capsular conjugate vaccines, such as serotype replacement, limited coverage and high costs. A recombinant live vector vaccine delivering protective antigens is a promising way to achieve this goal. In this review, we discuss the researches using live recombinant vaccines, mainly live attenuated Salmonella and lactic acid bacteria, to deliver pneumococcal antigens. We also discuss both the limitations and the future of these vaccines. Streptococcus pneumoniae is the most common cause of pneumonia as well as a number of invasive diseases, such as meningitis and sepsis, and non-invasive mucosal diseases, such as otitis media and sinusitis. It causes severe morbidity and mortality worldwide, especially in young children and the elderly [1]. It has been estimated that 14.5 million episodes of serious pneumococcal disease occur each year, resulting in 826,000 deaths in children under 5 years of age, the vast majority of which occur in low-income countries with poor access to health care [1]. The overall rate of invasive pneumococcal disease (IPD) among children and adults is reported to be between 11 and 23.2/100,000 individuals per year [2]. In adults, community-acquired pneumonia, among which 30%–50% are caused by S. pneumoniae [2], is one of the major respiratory health diseases in the USA and Europe [2,3]; it is the most frequent cause of death from infection and poses a heavy burden to healthcare systems worldwide [2,4]. It is estimated that the annual total economic burden of pneumococcal disease among US adults aged over 50 years is about $5.5 billion [5]. The increasing incidence of antibiotic-resistant S. pneumoniae strains worldwide posed another threat to the treatment of infection [2]. The burden of pneumococcal diseases is worsened by increasing numbers of people with chronic disease (sickle-cell disease, chronic renal failure, chronic liver disease, asplenia), HIV or mycobacterial infection, as well as an aging population in many developed countries [2]. Currently, we have two types of vaccines against S. pneumoniae, pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV) [6,7]. Both of them are designed to generate antibodies against capsular polysaccharide (CPS) [8,9]. S. pneumoniae has at least 94 serotypes with different abilities in nasopharyngeal carriage, invasiveness and disease incidence. The PPV is a 23-valent vaccine, which covers 23 commonly encountered serotypes. It is recommended to persons older than 65 years of age and aged ≥2 years at high-risk for pneumococcal diseases. The CPS is a T-cell-independent immunogen. It does not lead to isotype switching and induction of memory B-cell responses, leading to a temporary protection [10]. Capsular vaccines could cause hyporesponsiveness that blunts the immune response to subsequent doses due to the first dose [2]. It is also not very effective in infants and children under 2 years old—a group that is highly susceptible to infection, particularly in developing countries. Several meta-analyses showed that this vaccine is effective in low-risk adults, but not in high-risk groups [11,12,13]. To conquer these problems, the conjugate vaccine, PCV7, was licensed in 2000. Recently, PCV10 and PCV13 were licensed too. These vaccines are composed of pneumococcal polysaccharides conjugated to different protein carriers [6]. A conjugate vaccine can present the peptide or carbohydrate epitopes to carrier-peptide- or carbohydrate-specific T cells, respectively [14], resulting in T cell help for the production of memory B cells [10,15] and robust immune responses [16]. The conjugate vaccine increases enabling it to be used for young children [17]. The introduction of conjugate vaccines has tremendously decreased the rate of IPD and nasopharyngeal carriage by vaccine serotypes in children [15,18,19]. An effective herd immunity was also observed [7,20,21] with total 2/3 IPD reduction [6].At the same time, some non-vaccine serotypes become prevalent in the face of the introduction of conjugate vaccines [22,23,24]. Also, certain high-risk groups have poor immunological responses to some of the polysaccharides in the vaccine formulations [25]. There are also concerns about the conjugate vaccines related to the high cost and complexity of manufacture due to the different prevalent serotypes in different geographical areas and the limited coverage of the current PCV vaccines [26]. Thus, to develop a low-cost, effective vaccine against S. pneumoniae is still urgent. The new vaccine should be able to induce more effective and durable immune responses that could potentially protect against all clinically relevant pneumococcal capsular types and cover some high-risk groups who may not respond well to the current vaccine, while keeping the cost low enough to be used in developing countries. The success of vaccines against other pathogens encourages the scientific community to develop a pneumococcal vaccine based on conserved protein antigens across all capsular types [26]. Different reviews have previously covered topics related to new generations of S. pneumoniae vaccines [6,27,28], animal models [29,30], antigen selection [26,31,32,33] and mechanisms of protection [28,34].In this review, we will focus on developing new-generation pneumococcal vaccines using live bacteria delivering conserved protein antigens. We will discuss two types of live vaccines, live attenuated Salmonella and live lactic acid bacteria (LAB), mainly Lactococcus and Lactobacilli. Recombinant bacterial strains have several advantages. They have intrinsic adjuvant properties and can deliver antigens or DNA vectors with its native form using mucosal routes, which mimic the natural infection process to induce immune responses against the heterologous antigens in both mucosal and systemic sites. The productions of these kinds of vaccines are easier and less expensive than that of protein-based subunit vaccines. Several of the best-characterized candidate S. pneumoniae antigens, including pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal surface protein C (PspC), and pneumolysin (Ply), have been tested in various live vectors including attenuated pathogenic bacteria and nonpathogenic bacteria (Table 1). We will focus on the immune responses induced by these recombinant bacterial vaccines. The detailed properties of different protein antigens tested in live vaccines have been discussed elsewhere [32].Salmonella is a pathogenic bacterium. In order to be used as a live vaccine vector, it should be attenuated by various mutations [35,36]. Furthermore, multiple mutations are introduced to reduce the chance of reverting to display virulence. Salmonella is one of the most widely studied live vectors to deliver protective antigens. Recombinant attenuated Salmonella vaccines (RASVs) can attach to, invade and colonize in deep effector lymphoid tissues after mucosal delivery and therefore remodel the host cells that they target as well as promote immunomodulatory effects to induce immune responses in locations where bacteria persist as well as at systemic sites [37,38,39]. Currently, a phase I clinical trial showed that the three S. typhi vaccine vectors—χ9633, χ9639 and χ9640—delivering pneumococcal antigen PspA were safe and well-tolerated [40]. These achievements were made during the process with a final goal of developing a safe RASV suitable for use in newborns/neonates and infants that induces protective immunity to the diversity of S. pneumoniae strains. Live vectored vaccines for S. pneumoniae. 1. All Salmonella strains are derived from the UK-1 strain, unless otherwise specified; see references for detailed genotypes of strains; 2. The capsular polysaccharide (CPS) type is shown in parenthesis; 3. Mice are 5–8 weeks old, unless otherwise specified; 4. i.g., intragastric; i.n., intranasal; i.p., intraperitoneal; i.t., intratracheal, i.v., intravenous; 5.BAL: bronchoalveolar lavage fluid; IL, intestinal lavage fluid; VL, vaginal lavage fluid; LL, lung lavage fluid; NL, nasal lavage fluid; 6. ELISPOT (Enzyme-linked immunosorbent spot );7. N.A.: not available.Several antigens, PsaA, PspA, PspC and Ply, delivered by different recombinant attenuated S. typhimurium vectors were tested in mice (Table 1). These efforts were dedicated to screening optimal secreting signals [50], antigen coding regions [42], combinations of antigens [62,63], antigen delivery methods [50,62,63], Salmonella genotype for increased immunity and safety [57,58,59,60,80] and concept for testing new technology platforms for Salmonella vaccines [54,56].Ply has activities of cytotoxic/cytolytic and complement activation to facilitate the growth and invasion of S. pneumoniae in lungs during the early stage of infection [81]. It is conserved in both amino acid sequence and antigenicity among clinical isolates [32]. Paton et al. used a S. typhimurium C5 aroA strain to deliver a detoxified Ply-PdB (W433F) by intraperitoneal (i.p.) and oral routes [41]. Both immunization routes resulted in significant IgG responses against Ply. However, only the oral route resulted in IgA responses. Until now, there is no animal protection study with Ply delivered by Salmonella vectors. PsaA is a conserved surface protein present in all 90 S. pneumoniae CPS groups [82,83,84]. PCR-restriction fragment length polymorphism analysis of 80 serotypes demonstrated the conservation of the gene using ten different enzymes throughout the entire length of the gene [84]. Monoclonal antibody studies showed that PsaA is present in 89 serotypes of S. pneumoniae [83]. S. typhimurium vaccine vector delivers PsaA in two ways, one as a prokaryotic synthesized antigen [42], another as a eukaryotic synthesized antigen by delivery of a DNA vaccine (see Section 2.1.3) [64]. Salmonella strain χ9241 encoding full-length PsaA induced significantly high titers of anti-PsaA IgG in serum and IgA in vaginal washes, nasal washes, and lung homogenates [42]. Although the gene was cloned from the CPS type 4 S. pneumoniae Tigr4 strain, Salmonella-PsaA vaccine reduced nasopharyngeal colonization by L82016 (type 6B CPS) and E134 (type 23 CPS) in two strains of mice, BALB/c (haplotype H2d) and C57BL/6 (haplotype H2b), independent of whether mice were immunized by the oral or intranasal (i.n.) route. However, immunization could not reduce lung colonization by pneumococcal strains A66.1 (type 3 CPS) and D39 (type 2 CPS). The Salmonella-PsaA vaccine conferred no protection against i.p. challenge with S. pneumoniae strain WU2 (type 3 CPS). The work also indicated that the full length of PsaA with its native conformation might be important to induce protective immunity, which is different from PspA, in which the α-helical segment is enough to induce protective immune responses [85]. Different from PsaA, PspA is a serologically variable surface protein. It is classified into three families and subdivided into six clades based on the C-terminal 100 amino acids of the α-helical region [86]. PspA family 1 is composed of clades 1 and 2, family 2 is composed of clades 3, 4 and 5. Family 1 and 2 PspA cover over 95% of clinical isolates typed to date [86,87]. Their distribution remains unaltered following the introduction of the PCV7 [88]. Although PspA has variability, different PspAs are cross-protective against different S. pneumoniae strains expressing different CPSes and serologically divergent PspAs [89,90]. PspA, delivered by Salmonella vectors, was tested by different groups using either prokaryotic expression vectors or eukaryotic DNA vectors in mice (Table 1). In our group, Nayak et al. first reported that PspA Rx1 (aa 1–470) with its native secretion signal encoded on a high copy plasmid is delivered by a S. typhimurium SR-11 Δcya Δcrp strain [43]. This construction can induce significant anti-PspA IgG, IgA and IgM antibody titers in sera, vaginal washes and intestinal washes in both Salmonella-susceptible BALB/cJ mice (haplotype H2d) and Salmonella-resistant CBA/N xid mice (haplotype H2k) [43]. Enzyme-linked immunosorbent spot (ELISPOT) analyses showed anti-PspA IgG, IgM and IgA ASCs (antigen-secreting cells) in spleen, peripheral blood and Peyer’s patches. The anti-PspA IgG and IgA can also be detected in rabbit serum and vaginal washes following oral immunization with the RASV strain. Mice immunized with Salmonella-PspA were protected against i.p. challenge with the virulent WU2 strain. The passive protection experiments showed that the immune serum can protect mice against intravenous (i.v.) (CBA/N mice) and i.p. challenge (BALB/c mice) with the WU2 strain. This work set the foundation for the following studies [44,50].Kang et al. replaced the native secretion signal for the PspA Rx1 with the β-lactamase secretion signal [44]. The fusion protein encoded amino acids 3–257 of PspA on a medium copy plasmid with a reduced synthesis level of the Asd selection marker [44]. A new Salmonella SL1344 vaccine strain χ8501 (Δcrp-28 ΔasdA16), delivering this novel plasmid pYA3494, induced serum IgG and vaginal IgA against PspA and conferred protection against i.p. challenge with the WU2 strain. Notably, with only one-time immunization, the IgG response induced by χ8501(pYA3494) against PspA was higher than that against LPS or OMPs, both are indications of the immunogenic potential of the bacteria. This was also observed in a RASV strain with a regulated programmed lysis system vectoring the same bla SS-PspA fusion protein (Table 1) [46]. Further experiments showed that Salmonella vaccine strains delivering secretory proteins induced higher antibody responses than a strain delivering cytoplasmic PspA protein [45]. These data showed that antigen location in RASVs is important to induce antibody responses following oral immunization. Xin et al. further evaluated the effects of different Type II signal sequences (SS), including the N-terminal signal sequence of β-lactamase (bla SS), N- and C-terminal sequence of β-lactamase (bla SS+CT), ompA SS and phoA SS, fused with the α-helical domain of PspA Rx1 (encoding PspA Rx1 amino acids 3–285) on the immune responses [50]. The results showed that the strain carrying plasmid pYA4088 with bla SS–PspA fusion yielded the largest amounts of secreted PspA than other signals. Mice immunized with this construction developed the highest serum IgG and vaginal IgA antibody levels, IL-4 and IFN-γ secretion. Immunized mice were protected against i.p. challenge with a virulent S. pneumoniae strain. Thus, the PspA production level in RASVs is important for the protection against S. pneumoniae challenge [50]. The protection conferred by different Salmonella strains vectoring plasmid pYA4088 has been documented through a series of studies (Table 1). The plasmid was used to test our innovation technologies, regulated delayed in vivo antigen synthesis strategy, and regulated delayed attenuation [54,56,91], as well as in a phase I clinical trial [40]. It was used to further explore the effects of different mutations, such as lipid A and O antigen, on the safety and immunogenicity of Salmonella vaccine vectors [51,53,55,57,58,59,60] (Table 1). These tested mutations either directly contribute towards the construction of S. typhimurium vaccine strain χ9558 and S. typhi vaccine strains χ9633, χ9639 and χ9640 with the same genotype or can be used to further improve S. typhimurium or S. typhi vaccine vectors [80]. Strain χ9558 is the representative of a new generation of RASVs displaying wild-type characteristics at the time of immunization and becoming attenuated after colonization of host tissues. It exhibits an improved safety profile in adult mice, with a reduced ability to cause meningitis when administered orally, i.n., or i.p. [92]. It is totally safe and noninflammatory in newborn mice at doses equal to 107 times the 50% lethal dose of the wild-type parent [91]. In adult mice, the strain χ9558, carrying a pspA expression plasmid, induces significantly higher levels of anti-PspA serum IgG and mucosal IgA antibodies than χ8133, a vaccine strain generated by a traditional way. Splenic lymphocytes from mice immunized with χ9558 produce significantly more IL-4 and IFN-γ secretion cells than that from mice immunized with χ8133, as well as increased levels of both Th1-specific cytokines (IL-2, IL-12, TNF-α) and Th2-specific cytokines (IL-4, IL-5, IL-10). Vaccination with χ9558 confers a greater degree of protection against S. pneumoniae challenge than that with χ8133 (71% vs. 21% survival, p < 0.01) [49]. Strain χ9558(pYA4088) is also immunogenic in infant and neonatal mice born from naïve or immunized mothers when administrated orally or i.n. and induce protective immunity against S. pneumoniae challenge [52].Another plasmid pYA3802 with bla SS-PspA-CT (PspA aa 3–285) was used to probe the protective immune mechanisms of RASVs via the oral route. Park et al. proved that the sIgA is important to RASV-PspA-induced protection against intratracheal (i.t.) challenge using pIgR−/− mice which lack the IgA secretion pathway [48]. Peyer’s patch plays an indispensable role for induction of PspA-specific IgA in both systemic and mucosal compartments. MyD88-mediated innate immunity is not essential for induction of Ag-specific B-cell responses induced by RASV synthesizing T-cell-dependent exogenous Ag, but it is critical for the protection against virulent S. pneumoniae challenge. Influenza infection followed by pneumococcal infection can cause severe pneumonia and this secondary pneumococcal pneumonia is the most common cause of influenza-associated death. Seo et al. tested whether the vaccine against S. pneumoniae could reduce the disease burden caused by seasonal epidemic and pandemic influenza [61]. Mice vaccinated orally with a RASV strain carrying plasmid pYA3802 resulted in attenuated pulmonary inflammation and effective long-term protection against secondary pneumococcal pneumonia after influenza infection [61]. Thus, oral RASV-PspA immunization is not only an efficacious way to protect against respiratory bacterial pathogens, but is also a promising approach against the impact of annual epidemic and pandemic influenza outbreaks. These results highlight the importance of immunizing both the young and elderly populations, which are more susceptible to infection by both S. pneumoniae and influenza, with a RASV against S. pneumoniae.PspC is another candidate surface antigen [93,94]. It plays an important role in the virulence of S. pneumoniae and protects mice against pneumococcal challenge in carriage [95] and sepsis models [94,96]. Xin et al. evaluated PspC from S. pneumoniae strain L82015 fused with different secretion signals as mentioned above [50]. The induced immune responses varied depending on the signal sequence used. Strains carrying the bla SS-PspC-CT fusions yielded the largest amounts of secreted PspC, induced the highest serum IgG and vaginal IgA titers, highest IL-4 and IFN-γ responses, and conferred the greatest protection against virulent S. pneumoniae i.p. challenge than other signal sequences fused to PspC. These results are consistent with the PspA results, which demonstrate that the antigen synthesis levels in live bacterial vectors are critical for induction of protective immune responses against S. pneumoniae. Using LAB as vectors delivering PsaA also confirmed this conclusion [67]. To develop an effective vaccine against S. pneumoniae, multiple antigens are preferred to set blockages during the stages that S. pneumoniae attaches to, invades into and spreads in the host. Salmonella has the capacities to deliver multiple antigens with various approaches: (1) as fusion antigens on one plasmid delivered by one strain [62]; (2) as individual antigens on one vector delivered by one strain; (3) as individual antigens on different vectors delivered by one strain [63]; and (4) as a mixture of multiple strains, each specifying individual antigens [63]. These approaches require optimization of each component. We first tested if a RASV strain delivering one vector encoding PspA fusions could induce protections against multiple S. pneumoniae strains [62]. PspA is grouped into three families due to its diversity [86]. It is necessary to use PspAs from different families to elicit effective cross-protective coverage. Previously, we described the use of PspA from the Rx1 strain, which is from family 1. We chose another PspA from strain EF5668 from family 2. We also included the proline-rich domain of EF5668, which has been shown to encode protective epitopes that cross-protect against a variety of S. pneumoniae strains [94,97]. We evaluated fusion constructions consisting of PspA Rx1 and EF5668 with different orders in one vector to screen the best combination for an anti-pneumococcal vaccine. Both fusions elicited serum IgG and mucosal IgA to both families of PspA and strongly augmented percentage of cells with surface-bounded C3 on strains expressing family 1 and 2 PspAs. One of the fusion constructions, Rx1-EF5668, extended and enhanced protection against multiple strains of S. pneumoniae by i.p., i.v., or i.n. challenge [62]. This fusion construction of antigens from different families represents an important strategy for S. pneumoniae vaccine development. We then evaluated the way to deliver multiple antigen genes in separate vectors in the case that a fusion construct of multiple protective antigens is not the optimal choice when a multivalent vaccine is desired. The major challenge to achieve this goal is that the recombinant vaccine strain should stably maintain two or more expression vectors simultaneously, each carrying a unique selectable marker. To facilitate this strategy, we used a DadB+ vector to deliver the pspC gene, together with an Asd+ plasmid carrying the pspA gene to form a dual-plasmid system, which could deliver multiple antigens in a vaccine strain with Δalr ΔdadB and Δasd mutations [63]. The DadB+ plasmids are compatible with Asd+ vectors in a single vaccine strain without comprising the synthesis of individual antigens. Both plasmids are stable over 50 generations of growth, suggesting that antigen synthesis and delivery in vivo are not compromised in this system [63]. To further reduce the possible recombination between plasmids, a recF mutation was introduced into strains [63]. The Salmonella vaccine strain carrying both PspA and PspC by Asd+ and DadB+ vectors, respectively, induced higher serum and secretory antibody responses than the strain delivering a single antigen or a mixture of two vaccine strains each specifying one protective antigen and offered superior protection against i.p., i.v., or i.n. challenge with different serotypes of S. pneumoniae [63]. The DadB+-Asd+ dual-plasmid system represents another important tool to develop multivalent live recombinant vaccines [63].DNA vaccines encoding psaA and pspA have been shown to be effective in inducing antibody responses and Th1 immunity [98], which are important against pneumococcal infection [98,99,100]. However, preparation and characterization of DNA vaccines need complex procedures [101]. These procedures increase the cost of final products. DNA vaccines also induce poor mucosal responses in the nasopharynx. Zhang et al. used Salmonella to deliver multi-antigen-encoding DNA vaccines encoding psaA and pspA genes [64]. They modified the DNA vector by replacing the selection marker from ampicillin to Asd to better maintain the vector and reduce the safety concern due to the use of antibiotic selection markers. They also eliminated the neomycin-resistance selection marker for the same concern. The modified vector was used to clone psaA and pspA genes. Salmonella delivering DNA vaccines encoding pspA or psaA, either alone or mixed together, significantly reduced S. pneumoniae colonization in nasal washes compared with control. Mice orally immunized with RASV carrying multi-antigen DNA vaccines significantly reduced nasal colonization by S. pneumoniae strain D39 compared to immunization with DNA vaccines administered intramuscularly (i.m.). These findings are related to the high level of sIgA in the nasal washer, as well as systemic IgG antibodies and a shift toward a Th1-mediated immune response [64].One of the main problems with DNA vaccines delivered by live Salmonella vaccines is that the DNA cannot effectively contact with the cytosol and then the nucleus of eukaryotic cells to initiate transcription and translation of encoded antigen genes. Besides those described above, other modifications could be used to increase the efficiency of DNA vectors delivered by Salmonella. Generally, there are two barriers for Salmonella delivering DNA vaccines into the cytosol. The first is that Salmonella resides in Salmonella containing vacuole (SCV) after entering the cell, which isolates Salmonella from other cytosolic components. This problem can be conquered by using a strain with the sifA mutation [102]. SifA is critical to maintain the SCV. Mutating sifA disrupts the vacuoles [103]. The second problem is the cell membrane/wall of Salmonella. This can be conquered by using a regulated delayed lysis in vivo strategy [46]. This strategy enables effective lysis of bacteria to release the bacterial cell components, including DNA vaccines. Combining these two approaches has led to promising results for a influenza vaccine [102]. Due to the lack of an animal model, progress to develop safe S. typhi vaccines for human use is slow. Currently, a clinical trial is still the best measurement of safety and effectiveness of S. typhi vaccines or vaccine vectors. Our intensive work carried out in mice lead to the development of a S. typhimurium strain χ9558 with a balance between safety and immunogenicity in adult, neonatal and infant mice [47,49,52,54,56,91,92]. Based on the results, we constructed three recombinant attenuated S. typhi vaccine vectors, χ9633, χ9639, χ9640, with essentially the same genotype as χ9558 carrying plasmid pYA4088 encoding the α-helical fragment of PspA Rx1 (aa 3–285) [65], but with an additional mutation eliminating the immunosuppressive capsular Vi antigen [65,66]. The vectors were constructed to test the hypothesis that the immunogenicity of live Salmonella vaccines is, at least in part, on its RpoS status. All three S. typhi vaccine strains are similar to the licensed live attenuated typhoid vaccine Ty21a in their abilities to survive in human blood and human monocytes. They are more sensitive to complement and less able to survive and persist in sewage and surface water than their wild-type counterparts [65]. Adult, infant and neonatal mice immunized with these vectors develop immune responses against PspA and Salmonella antigens. The percentages of protection against S. pneumoniae challenge in adult mice immunized with these vectors are between 50 and 81.3% [65,66]. In the pre-clinical setting, they achieved the desired balance between safety and immunogenicity in adult, neonatal and infant mice [65,66]. These strains were tested in a dose-escalation clinical trial from 107 to 1010 CFU to further evaluate the safety and immunogenicity and determine which of the three S. typhi vectors has the optimal safety and immunogenicity profile in human hosts [40]. The results proved that the vaccines are safe and well tolerated. Even in the highest dose group, no subject experienced severe reactions or serious adverse events. The vaccine is also very safe to the environment without any shedding of viable vaccine cells in stools. This is a very important feature because bacteremia and shedding are not acceptable for the development of a vaccine for use in neonates/infants and for use in immunocompromised hosts, especially persons infected with HIV. However, only a limited number of subjects had increased levels of anti-PspA IgA. The inability to stimulate significant immune responses to PspA is not clear. It may relate to the high pre-immunization antibody titers against S. typhi and PspA likely due to previous Salmonella infection and pneumococcal vaccination, possible over-attenuation or limited in vivo slow growth of the attenuated S. typhi strains. In this last regard, the use of the regulated delayed synthesis of PspA in vivo might have been due to too much repression and insufficient cell divisions of the vaccine strains to adequately reduce repressor concentration by cell division. Based on the trial results, the vaccine strains have been further modified to increase protective antigen production and delivery to increase immune responses. An improved version of S. typhi based on the most promising vaccine strain χ9640 carrying vectors encoding multiple protective pneumococcal antigens is being developed and evaluated. Human-restricted S. typhi is the choice for oral human vaccines because it can effectively invade mucosal tissues and enter systemic sites, leading to strong mucosal, humoral and cellular immune responses. S. typhimurium only causes self-limited gastroenteritis in human. It is less capable to invade beyond the gut mucosa in healthy humans and less able to stimulate long-lasting immunity [104]. Thus, it is not actively pursued as an oral human vaccine [105]. To evaluate the attenuation and immunogenicity of S. typhi vaccine strains, infection of mice with S. typhimurium is used as an experimental model because S. typhimurium infection in mice results in typhoid-like diseases in mice, which likens S. typhi infection in human. Although Salmonella shows promise as a vaccine vector and has been extensively tested, there is still no licensed RASV. In addition to the issue of immunogenicity, the key concern associated with RASVs is safety, especially in newborns/neonates, the elderly and those who are immunocompromised or have chronic diseases. Clinical results demonstrated that our RASVs χ9633, χ9639 and χ9640 are safe and non-shedding, but less immunogenic [40]. This is different from what we observed in mice with S. typhimurium as a model. Our S. typhimurium vaccine vector χ9558 carrying plasmid pYA4088 induced significant anti-PspA IgG/IgA antibody titers in mice, S. typhi vaccine strains χ9633, χ9639 and χ9640 with the same genotype carrying the same plasmid did not induce significant anti-PspA IgG/IgA responses in the clinical trial. The next step is to increase their immunogenicity while maintaining safety. Attaining the desired balance between safety and immunogenicity is difficult, especially for S. typhi vaccines, which lack a relevant animal model. To develop a S. typhi vaccine, evaluation of S. typhimurium strains of similar genotype and phenotype in mice is used as a close mimic of S. typhi in humans. Considering the differences between the human and mouse mucosal lymphoid system [106,107], several humanized immune system mouse models displaying classical manifestations of human typhoid fever including meningitis, liver pathology and mortality were developed [108,109,110,111]. However, they still have problems with variations in ability of S. typhi to attach to, invade into, survive intracellularly and distribute into internal effector lymphoid tissues. These disadvantages and the high cost of these humanized mice still limit their current use. Further research and improvement of these humanized mouse models should ultimately aid in developing a safe and effective S. typhi vaccine vectors for humans [108].Currently, the mouse is still the most cost-effective model for testing safety and efficacy of RASVs. Most studies with S. typhi RASVs used adult mice, but a few studies have adopted using newborns and neonates in developing pneumococcal vaccines [52,66,91] (Table 1). We still lack safety and efficiency data in aged or in malnourished mice. Baby mice and aged mice have different T- and B-cell responses that affect induction of optimal immune responses to vaccines [112,113,114,115,116,117,118,119,120,121,122]. In newborn/neonate mice, the presence of maternal antibody enhanced immune responses and protections against S. pneumoniae challenge [52,66]. These responses include increased IgG, IgA and IL-4-secreting levels in mice immunized with S. typhimurium vaccine and enhance levels of mucosal IgA, IFN-γ, and IL-4 in mice immunized with S. typhi vaccines [52,66]. All these factors affect the performance of the vaccines. Considering that the newborn/neonatal and aged people are the main high-risk groups, more efforts should be put into using young and aged mice to evaluate the candidate RASV-S. pneumoniae vaccines. We may need age-specific RASVs for different age groups.LAB are a group of Gram-positive bacteria that produce a common end product—lactic acid—from the fermentation of sugars [123]. These non-sporulating bacteria include species of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus [123]. Due to limited biosynthetic abilities for pre-formed amino acids, B vitamins, purine and pyrimidine, their habitats are restricted to the place, such as intestine, where the required compounds are abundant [123]. They have positive effects on human and animal health [124,125] and were widely used for food without causing any known health problem for thousands of years. This status is considered GRAS (generally recognized as safe). LAB can be used as adjuvants for their immunostimulatory properties [126,127,128,129,130]. This GRAS status of LAB in adults and infants and their abilities to stimulate immune responses make them very attractive candidates for the development of mucosal vaccines [131]. Most LAB induce Th1 type responses, some LAB can induce different arms of the immune response [132], like L. reuteri induces Th2 responses and L. rhamnosus induces Th17 responses [126,129]. Lactobacillus and Lactococcus are the main vaccine vehicles to delivery heterologous proteins or DNA to mucosal tissues (see reviews [123,133,134,135]). Live LAB have been shown to be effective adjuvants to improve the immune responses against respiratory pathogens [75,136]. In vitro, L. rhamnosus can inhibit S. pneumoniae adherence to human epithelial cells [137]. Ingestion of LAB reduces nasal colonization by S. pneumoniae in humans [138]. Oral administration of L. lactis in mice can improve clearance of pathogens from the lungs, reduce lung injuries, and increase survival of mice against S. pneumoniae infection [139,140]. The mechanism is related to an up-regulation of the respiratory innate and specific immune responses, like improved production of TNF-α in bronchoalveolar lavage (BAL) fluid, enhanced recruitment of neutrophils into the alveolar spaces, increased activation of BAL phagocytes, and improved production of BAL IL-4 and IL-10 [141]. These responses stimulate the IgA cycle, increase IgA+ cells in the intestine and bronchus, and increase BAL anti-pneumococcal IgA and IgG levels [141]. Nasal administration of L. fermentum in mice can increase protective responses against S. pneumoniae challenge by stimulation of neutrophil activity or by the increase of the number of activated macrophages and lymphocyte populations in the tracheal lamina propria [142,143]. Nasal administration of L. lactis improves local and systemic immune responses against S. pneumoniae with reduced nasal colonization, increased clearance rate of S. pneumoniae from lungs, reduced dissemination of pneumococci into blood and reduced damage to respiratory tissues, which is also related to the up-regulation of the innate and adaptive immune responses in both local and systemic compartments as well as different cytokine responses [143,144]. These responses increase the pulmonary lymphocyte population, anti-pneumococcal IgA and IgG in bronchoalveolar lavage (BAL) and serum, and phagocyte activation in lungs, blood and bone marrow [143]. Increasing resistance to pneumococcal respiratory infection was shown in both normal [140] and malnourished mice fed with L. casei [139]. However, the ability to induce these responses is varied among LAB species. Thus, different LAB strains are evaluated as vaccines or vaccine vectors delivering pneumococcal antigens against S. pneumoniae [67,76,131,145,146].Several pneumococcal candidate antigens, PsaA, PspA, PspC and PppA delivered by LAB, most in L. lactis NZ9000 or its parent MG1363, have been tested against S. pneumoniae challenge in animal models. The strain NZ9000 is derived from the strain MG1363 with the nisRK genes integrated into the pepN gene, which facilitate the use of the NIsin-Controlled gene Expression system-NICE [147,148]. Two strategies were adopted to develop LAB vaccines against S. pneumoniae: recombinant lactic acid bacterial vectors and non-genetically modified Gram-positive enhancer matrix (GEM) particles. We will focus on the live vaccine strategy. The GEM approach was discussed elsewhere [149,150,151,152].To induce immune responses, especially antibody responses, higher antigen production is preferred. Thus strong promoters are adopted. Although several promoters were used in LAB [133], two promoters were used more in LAB-S. pneumoniae vaccines. One is the lactococcal promoter P1 and another is the nisin-regulated promoter [69,71,153]. The P1 promoter, which was used to express psaA, pspA and pspC [69,71,153], is a constitutive promoter. It is a medium strong promoter from the L. lactis genome [154]. However, continuous high-level production of heterologous proteins could result in intracellular accumulation, aggregation and degradation of proteins in the cytoplasm and lead to deleterious effects to the cells [134]. To solve this problem, two methods were adopted. One way was to use a system that can regulate protein synthesis. The most widely used system is the NICE system. The nisin-regulated promoter system has several advantages. The transcription of the promoter Pnis can be efficiently controlled by the extracellular concentration of the antimicrobial peptide nisin through the two-component regulatory system, sensor NisK and regulator NisR [148,155,156], which provides a simple way to control gene expression. The benefits of this system are: the small size of the promoter, which can be trimmed down to less than 50 bp; hyper-production of protein, which can reach up to 50% of the total protein; tightly controlled gene expression with undetectable protein synthesis without induction; very high dynamic induction range to 1,000-fold dependent on the concentration of nisin and can be used in a variety of LAB [147,157]. For maximum induction, the nisin concentration is 10 ng/mL (3 nM), which is the MIC (minimum inhibitory concentration) value for nisin [148]. As an antimicrobial peptide, nisin can repress the growth of Gram-positive bacteria and is regarded as a food-grade preserver. In strains, like L. lactis F17847, with the NICE system, the induction of antigen synthesis with nisin before immunization is necessary. Other strains, like L. lactis NZ9700, can produce nisin to omit this process. However, it also means that the NICE system is converted into a non-regulated system. Basically, the NICE system is a system that regulates protein synthesis in vitro, not in vivo. Another way to reduce the metabolic burden on the LAB vector is to secret the protein into the periplasm, onto the cell wall or into the supernatant. The secreted protein can directly interact with the environment. Several protein secretion systems in LAB have been discussed [133,134,158]. Currently, the Usp45 signal was most frequently adopted. Usp45 is the most abundant protein secreted by L. lactis. The secretion of Usp45 is through the Sec pathway. Adding negative charge peptides at the N-terminal part of the mature moiety will improve the translocation efficiency across the cytoplasmic membrane [159,160]. Thus, this secretion signal is widely used to deliver heterologous antigens [69,153]. However, some researchers do not use this secretion protein. The reason may lie in the fact that location of the protective antigen protein is not important for induction of the immune response by LAB. Thus, for LAB-delivered antigens, the amount of antigen produced is more important than the location of the antigen, especially when delivered by mucosal routes, i.n., intragastric, and oral routes [161,162,163]. This is in contrast to Salmonella, in which periplasmic secreted antigen induced higher antibody responses than cytoplasmic antigen [44]. Although the surface synthesis protein may increase its presentation to the immune cells, it is also prone to be proteolytic degraded extracellularly or denatured by the acid or bile in the gastrointestinal tract in oral vaccinations [158].Recombinant LAB delivering pneumococcal antigens is mainly by the i.n. route. LAB strains were used to deliver PsaA, PspA and PspC by nasal immunization [67,69,153] and PppA by oral route [73]. In these reports, the plasmid-based antigen gene expression system was used. Until now, there are no reports using chromosomal-based antigen gene expression system in LAB for S. pneumoniae vaccines. Mice intranasally immunized with some species of Lactobacillus synthesizing PsaA developed systemic anti-PsaA IgG and IgA responses and displayed reduced pneumococcal colonization upon nasal challenge [67]. The immune responses depended on the amount of PsaA production, which vary in Lactobacillus from 20 to 250 ng/109 cells [67]. L. plantarum and L. helveticus induce significant IgA responses in nasal and bronchial washes and IgG in serum as well as reduced nasal colonization of S. pneumoniae 6B compared with the saline control group. However, when compared with strains carrying the control vector, only the recombinant L. helveticus led to a significant reduction of pneumococcal nasal colonization. Although L. casei does not generate significant antibody responses, it results in reduced colonization compared with the saline group, but not the vector control. These results reflect that LAB strains have different adjuvant properties. The three LAB strains synthesize similar amounts of PsaA (150–250 ng/109 cells). They persist in the mice nasopharynx after inoculation for three days except L. casei [67]. Short persistence with the low level of antigen production is not enough for the stimulation of antibody responses in nasal and systemic sites. Thus, using LAB for a vaccine needs to consider the protein synthesis levels, persistence and intrinsic adjuvant properties of different LAB [67]. L. casei and L. lactis were used to deliver PspAs. L. casei delivered PspAs from clades 1 and 5 under the control of the constitutive P1 promoter. The PspA synthesized retains in the cytosol [69,70]. Mice intranasally immunized with L. casei-PspA1/5 develop significant anti-PspA IgG, but no IgA in nasal washes, saliva or vaginal washes [69,70]. Previous experiments showed that PspA antigen is not effective for inducing IgA without adjuvant [164,165], thus L. casei seems not to display adequate adjuvant activity [69]. The anti-PspA1 antibody can effectively bind to PspA clade 1 and clade 2 and induce different amounts of complement deposition on the pneumococcal surface depending on the serotypes and PspA clades of S. pneumoniae. Mice immunized with L. casei-PspA1 show increased survival times when compared to mice immunized with saline against lethal i.p. pneumococcal challenge [69]. However, the percentage of protection against i.p. challenge was only 33.3% although the mice were immunized six times [69]. L. casei-PspA1 bacteria can be recovered up to five days after the i.n. inoculation with 109 CFU on two consecutive days. The presence of PspA antigen does not affect the ability of strain colonization in the nasal pharynx [69]. Another construction with L. casei delivering PspA5 conferred protection against i.n. pneumococcal challenges in mice. This was accompanied by the increased secretion of IFN-γ by lung cells against invasive pneumococcal challenge [70]. Intranasal immunization with L. lactis with intracellularly produced PspA using the NICE system induced not only serum anti-PspA IgG, but also lung lavage anti-PspA IgA [68]. This result further strengthens the conclusion that LAB strains have different adjuvant activities. Immunization with L. lactis-PspA significantly protected mice against i.p. challenge with the S. pneumoniae TIGR4 strain than protein administered intranasally or control groups did. The protection induced by L. lactis-PspA is similar to that induced by PspA/Alum administered by the subcutaneous (s.c) route. This was attributed to the Th1-mediated immune responses induced. In an intranasal challenge model, L. lactis-PspA afforded the highest protection among the levels elicited with purified PspA administrated i.n. or s.c. with adjuvant or in control groups [68]. About 20% of control mice survive the challenge suggesting that L. lactis may contribute to non-specific host immunity.Ferreira et al. reported that L. casei-PspC (from CPS type 6B) without an SS cannot confer significant protection against i.n. pneumococcal challenge although it induces IFN-γ secretion in lung cells and IL-17 secretion in both lung and spleen cells [70]. This may relate to the low homology of the PspC amino acid sequence between the vaccine (CPS type 6B) and the challenge strain (CPS type 3). Further, Hernani et al. tested L. casei-PspC with or without an SS using different immunization protocols, intranasal, sublingual and primer-boosting with PspC protein. However, none of these protocols induced significant levels of anti-PspC antibodies in vaginal or nasal washes and serum before challenge [71]. Despite these results, nasal immunization of mice with L. casei-PspC without a SS significantly reduced pneumococcal colonization by strain 0603 with an increase of anti-PspC IgA in the nasopharynx five days after challenge [71]. L. casei carrying cell-wall-associated PspC only marginally reduced pneumococcal colonization after challenge. Thus, the reduced colonization of S. pneumoniae may be attributed to the non-specific adjuvanticity of L. casei. These results show that protection is only achieved by using a PspC with high identity at the N-terminal region to the PspC expressed by the L. casei vaccine strain. Considering the polymorphism of PspC [94], using different PspC molecular types to cover more pneumococcal strains will be necessary [71]. An antigenically conserved antigen, PppA [166], delivered by L. lactis NZ9000 on the cell surface, was tested as live or inactive vaccines using intranasal and oral routes in adult and young mice [72,73,74,75]. Nasal and oral immunizations of L. lactis-PppA induced anti-PppA IgM, IgG and IgA responses in serum and bronchoalveolar lavage fluid in both adult and young mice. The responses are significantly higher in young mice than in adult mice. The challenge results showed that intranasal immunization with L. lactis-PppA could confer protection against homologous S. pneumoniae i.p. challenge by either active immunization or passively by antibody from immunized mice and increased resistance to respiratory infection with different pneumococcal serotypes (3, 6B, 14, 23F) in young and adult mice [72,73,74,75]. Oral immunization with L. lactis-PppA provided cross-protective immunity against four CPS types of pneumococcal strains with reduced lung bacterial counts [73]. Passive protection experiments proved that antibody is critical for protection [72]. There are no antibodies against L. lactis found in the serum or the BAL fluid in adult and young mice immunized with the recombinant strain. Thus, the host immune responses are directed against the protein expressed by L. lactis, not the vector [72]. These researches also show that vaccination at an early age of mice with the L. lactis-PppA strain is more effective [72]. LAB were also used to deliver type 3 or type 14 CPS with the eps natural promoter to express the eps genes or the nisin-induced promoter to express the regulatory genes, respectively [76,77]. The CPS synthesized in LAB either associated with the cells (type 3 CPS) [76] or in the supernatant (type 14 CPS) [77]. The mice immunized with L. lactis expressing 0.5 μg of type 3 CPS or 0.5 μg of purified type 3 CPS from pneumococcus elicited similar titers of T-cell-independent anti-CPS IgM and IgG antibodies in the serum [76]. The LAB did not affect the T-cell-independent nature of the anti-CPS antibody responses [76]. Thus, L. lactis is a potential host for capsular synthesis. However, there is no animal protection study with CPS delivered by LAB vectors. Though LAB are normally ingested orally, most work with LAB delivering S. pneumoniae antigens used i.n. immunization (Table 1). One of the reasons is that LAB are non-invasive bacteria, which implies that antigen delivery to antigen-presenting cells is not as effective as when using invasive attenuated pathogenic bacteria. Intranasal vaccination with recombinant LAB can elicit protective immunity in both mucosal and the systemic compartments [167,168]. To avoid mucosal tolerance, administration of high dose (108) of LAB for consecutive five days is preferred to induce IgA responses [73,141]. Most reports use three or more immunizations (Table 1). This increases the costs of the vaccines. Though LAB have been tested in clinical trials as food supplements/adjuvants for several vaccines, such as rotavirus vaccine, oral polio virus vaccine, influenza vaccine and oral cholera vaccine [169,170,171], it is unknown if the necessary doses to use for humans will be feasible and cost effective [131]. Also, for the nisin induction system, pre-induction and extensive washing to remove nisin are required before immunization. Thus, the complex immunization procedure should be addressed with the development of a new protein synthesis system and better procedures for preparing the LAB vaccines. Another issue related to the LAB vaccine is the safety concerns of i.n. immunization. I.n. immunization of mice with recombinant LAB induces excellent immune responses to the expressed antigens. Since the cribriform plate is a thin, well-hidden bone in the nasal cavity with numerous perforations for allowing passage of the olfactory nerves to the brain, there exists a potential route for bacteria to enter the cranial cavity if administered by i.n. route. Immunization with Salmonella by i.n. route results in brain colonization if the bacteria is not fully attenuated [92]. The GRAS feature of LAB is mainly based on the oral route. Thus, it would be helpful to check whether there is a problem using i.n. immunization with LAB-vectored vaccines. Another disadvantage with the LAB-S. pneumoniae vaccines is the use of antibiotic-resistance markers, which are considered unacceptable in live vaccines due to the potential for antibiotics in the final product and the possible contamination of the environment with recombinant drug-resistant bacterial strains. The regulatory agencies also prohibit the usage of antibiotics in vaccine formula. Two antibiotics are commonly used in the LAB vectors. The first one is erythromycin. Erythromycin can inhibit the protein synthesis by binding the 50s subunit of the bacterial 70s rRNA complex. Most plasmids used in LAB vaccines have an erythromycin-resistance selection marker. This antibiotic is necessary to select the recombinant plasmid. LAB strains with recombinant plasmid are grown with erythromycin prior to immunization to maintain the plasmid. Adding the antibiotics not only increases the costs of the final product, but also raises the concern about the plasmid stability. The most used pTREX vectors have poor segregational stability in the absence of antibiotic selection [172]. LAB could lose the recombinant plasmid in vivo and lead to compromised immune responses. To conquer this problem, the use of the balanced-lethal strategy could be attempted with LAB vaccines. Another is the nisin. Nisin is a polycyclic lantibiotic produced by L. lactis to eliminate other competing Gram-positive bacteria. It is commonly used as a safe food preservative against bacteria, yeast, and molds. Nisin can bind to lipid, dissipate the membrane potential, induce efflux of cytoplasmic components and inhibit bacterial cell growth [173]. It is used as the inducer for LAB strains with the NICE regulatory system. Induction with nisin and extensive washing is thus required for synthesis of the antigens before inoculation into the immunized hosts [72], which adds complexity to the production process and increases the costs. Both erythromycin and nisin are broad-spectrum antibiotics against many bacteria. Although nisin is not a big problem for the LAB vaccines, the use of erythromycin raises concerns that this antibiotic may interfere with the normal flora in the human intestinal tract or nasopharynx. It was reported that the erythromycin-resistance gene can easily transfer from LAB to Listeria spp. at a frequency as high as 10–4 [174]. Nisin can also exert some immunomodulatory effects at high concentration [175]. Therefore, LAB vaccine with biocontainment properties to prevent their spreading of heterologous DNA is necessary. To conquer this problem, auxotrophic bacterial strains complemented by a wild-type gene in a cloning or expression vector was developed, such as the purine, threonine, pyrimidine, thymidine and alanine auxotroph [176,177,178,179,180]. Although these balanced-lethal systems were developed for the food industry, and their application in vaccine research have not been reported, they paved the way for their application in vaccine research. Salmonella need to be attenuated to achieve a balance between safety and immunogenicity for vaccine application. It is not necessary to generate and evaluate mutations in LAB due to their GRAS feature. However, LAB have different abilities to modulate the immune system, the careful selection of LAB should be noted as a key factor that influences the results. Different LAB strains induce distinct cytokine profiles and exert different effects on the immune system [126,181,182,183]. Immunostimulating properties of LAB have been proved to be strain-, dose-, and even growth-phase-dependent [139,140,183,184]. Only L. casei CRI 431, L. lactis NZ9000 and L. rhamnosus CRL1505 have proved to be able to increase the resistance of mice to challenge with respiratory pathogens. A human study showed that L. rhamnosus GG has different immune modulation functions [185]. It stimulates immune functions in healthy persons, but down-regulated an inflammatory response in allergic persons. An oral S. typhi vaccine administrated with L. rhamnosus GG induced higher numbers of IgA-secreting cells, while with L. lactis induced higher numbers of CR3 receptor expression on neutrophils [183]. L. lactis-PspA can induce effective IgA responses, but L. casei-PspA is poor for induction of an IgA response [68]. Although the PsaA is synthesized on the surface of different LAB, including L. lactis, L. casei, L. plantarum and L. helveticus (Table 1), only lactobacilli lead to a decreased pneumococcal recovery from the nasopharynx upon a colonization challenge, but not L. lactis due to its low level of PsaA synthesis, which is not enough for inducing adequate humoral responses [67]. Thus, the selection of LAB should consider the intrinsic properties and the appropriate doses of each LAB. Optimum dose, frequency and duration of treatment for using LAB vaccines should be carefully compared and demonstrated through rigorously designed studies. Bacillus Calmette-Guérin (BCG), a live attenuated strain of Mycobacterium bovis, was used as an effective vaccine for M. tuberculosis. It has been given to 3 billion people worldwide since 1948, with a very low incidence of serious complications, even for young children and infants [186]. Besides the common benefits as a bacterial vector [186], it has the immunostimulatory properties that can augment the immune responses against routine immunizations in infant [187]. This live attenuated vaccine establishes a persistent infection and induces both cellular and humoral immune responses. Currently, BCG is shown effective in preventing the several forms of TB in toddlers, which may be a benefit for delivering pneumococcal antigen for newborns and infants since they are the main target population to prevent S. pneumoniae infections. BCG was used to deliver PspA antigen in the cytoplasm, associated with cell membrane or in a secreted form. Although the peak antibody titers elicited by BCG expression pspA with or without a secretion signal did not differ markedly, protective responses were observed only in mice immunized with BCG expressing pspA with its native signal peptide, which leads to the exportation of PspA, or as a fusion with the Mtb19 lipoprotein signal peptide, which results in it being anchored to the cell membrane. These results were observed in both BALB/c and C3H/HeJ mice using an i.p. challenge model [78]. The antiserum can also passively protect CBA/B (Xid) mice, which are highly sensitive to S. pneumoniae challenge [188], against other S. pneumoniae virulent strains exhibiting heterologous PspAs and CPS types [78]. Thus, the BCG-PspA is another potential live vaccine for inducing humoral immune responses against pneumococcal infections. However, the induction of cellular immune responses were not addressed in this report. Recently, progress in rBCG research may pave the road for further use of BCG as an effective vaccine vector for S. pneumoniae [189]. There are a few reports using viral vectors to deliver pneumococcal antigens. Arévalo et al. used replication-defective recombinant adenoviruses Ad5 (rAds) to deliver PspA, PsaA and PdB, either individual or combined [79]. rAds can direct high levels of viral gene expression in mammalian cells and induce strong immune responses [190]. The rAds used here cannot replicate in the host due to the lack of the packaging elements [191]. The results show that mice intranasally immunized with rAds carrying each of the three antigens develop robust antigen-specific serum IgG responses. Mice immunized with rAds carrying three antigens develop slightly reduced antibody responses against PspA, PdB and PsaA compared with the mice immunized with rAd carrying the individual antigen at 6 and 10 weeks. Two-dose vaccination induced stronger antibody responses, but cannot increase them further by a third boosting. rAd/PdB alone does not reduce the lung colonization carriage. Both rAd/PdB+rAd/PsaA or rAd/PdB+rAd/PspA can lead to reduced lung colonization of S. pneumoniae. rAd/PdB+rAd/PspA+ rAd/PsaA is most effective in reducing the bacterial load in the lung after challenge. Protein-based vaccines are the future for S. pneumoniae vaccine research [26]. However, protein vaccines have problems of high costs related to their complex manufacturing process. Iyer et al. reported that candidate antigens, such as PsaA, Stkp, PcsB, show different requirements for stability when combined with different adjuvants and excipients [192]. PsaA needs sodium phosphate to be stable when it absorbs to Alhydrogel, but StkP does not need. Thus, for a multi-antigen vaccine, separate storage of each protein for long-term storage stability might be necessary, leading to further cost increases for a mixed protein vaccine. Recently, a fast-dissolving tablet formulation of a live attenuated enterotoxigenic E. coli was developed [193]. The tablets rapidly disintegrate in vivo but preserve the bacteria at 2–8 °C for at least 12 months with only 0.4 log10 loss of viability during storage. These results provide a practical option for formulating ETEC vaccines or other live bacterial vaccines for oral immunization and help to facilitate delivery of lifesaving vaccines, particularly in low-resource settings [193]. Our experiments showed that RASV vaccine strains stocked at −80 °C do not change their titers after storage for five years [194]. Whether there is a change in the immunogenicity still needs to be probed. These results along with other attributes discussed above demonstrate the cost benefits of using recombinant live vector technologies. S. pneumoniae causing disease involves multiple steps, including attaching to, invading into and spreading in the host [27]. An effective vaccine could block any one of these steps, but preferably all of them. S. pneumoniae also have over 90 serotypes with different pathogenic attributes. The vaccine should protect against infections by most of the serotypes. The ideal vaccine should include multiple antigens to stop multiple steps of infection and confer protection against multiple serotypes. In the future, work should focus on delivering multiple antigens and test protection with multiple challenge models against multiple serotypes. Currently, the challenge studies in evaluating recombinant live vaccine vectors used serotypes 2, 3, 4, 6B, 14, and 23F (Table 1), which did not include some serotypes, like 1, 5, 6A, 7F, 9V, 18C, 19A, 19F in the 13-valent conjugate vaccine. An effective vaccine needs to protect against all these serotypes. The inclusion of multiple antigens protecting against S. pneumoniae strains in multiple families with regard to the PspA and PspC antigens is therefore important. The inclusion of conserved antigens such as PsaA and Ply and the conserved proline-rich domains of PspA and PspC should further augment the protective efficacy of these vectored vaccines [62,63].Much progress has been made in improving the technologies to design, construct and evaluate live bacterial vectored vaccines against S. pneumoniae. These improvements enhance safety, tolerability and effectiveness in inducing protective immunity. Three S. typhi vaccines based on these technologies have been tested in a phase I clinical trial. As for LAB based vaccines, there is still room for improvement. In the future, combining the benefits of LAB and Salmonella will be possible. With the advancement of knowledge about bacteria—not only Salmonella and LAB, but also other bacteria interacting with hosts—and by using new technologies, we should finally be able to develop safe, efficient, and relatively inexpensive needle-free vaccines against S. pneumoniae, as well as other pathogens. We thank Erika Arch for help in assembling this article and Josephine Clark-Curtiss for editorial and content advice. Research was supported by grants from the Bill and Melinda Gates Foundation, the National Institutes of Health, the United States Department of Agriculture and the Ellison Medical Foundation. The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The advantages of genetic immunization of the new vaccine using plasmid DNAs are multifold. For example, it is easy to generate plasmid DNAs, increase their dose during the manufacturing process, and sterilize them. Furthermore, they can be stored for a long period of time upon stabilization, and their protein encoding sequences can be easily modified by employing various DNA-manipulation techniques. Although DNA vaccinations strongly increase Th1-mediated immune responses in animals, several problems persist. One is about their weak immunogenicity in humans. To overcome this problem, various genetic adjuvants, electroporation, and prime-boost methods have been developed preclinically, which are reviewed here. DNA vaccination represents a novel means of expressing in vitro antigens for the generation of both humoral and cellular immunities against a wide spectrum of infectious agents, including viruses, bacteria, parasites, and tumors [1,2,3,4]. These vaccines have elicited protective immunity in a number of preclinical disease models [5,6,7,8,9]. DNA vaccines contain genes encoding the protein of the pathogen itself. Such plasmids neither replicate in the mammalian host nor integrate themselves within the chromosomal DNA.DNA vaccination was first demonstrated when young mice were inoculated with a plasmid expressing human growth hormone (hGH) [10]. In the initial experiment, the hGH gene was injected in the skin of the ear, with the intention of synthesizing the hGH protein for gene therapy. Several immunized mice showed substantial levels of antibodies, two months after the injection of the plasmid DNA (pDNA) into their cells, where the pDNA was read and the hGH protein was synthesized. Similar to the immune responses observed in a viral infection or an attenuated virus vaccination, the intracellular production of protein or peptide antigen induces high levels of Th1-type responses. However, due to the limited amount of protein synthesis in the body, the Th2-type immune responses are elicited at low levels. Although some experimental trials aimed at facilitating clinical or preclinical studies have evoked an immune response against microbial diseases, the usefulness of this technique has not been conclusively proved in multiple animal models [4]. A DNA vaccine to protect horses from West Nile virus, as well as other DNA Vaccines, has been approved for veterinary use [11,12,13]. Bar-Or and coworkers [14] successfully elicited antigen-specific tolerance, with a DNA vaccine encoding myelin basic protein, in patients with multiple sclerosis (MS). Although MS is known to be a demyelinating neurodegenerative disorder of the central nervous system in humans [14,15,16], the mechanism underlying the activity of this vaccine is yet to be fully resolved. DNA vaccines have many potential benefits despite their weak immune responses in humans. Methods such as in vivo electroporation have improved the efficacy of these vaccines. However, an optimal strategy for safe, reproducible, and pain-free DNA vaccination is yet to be developed.DNA vaccines elicit good levels of immune response when highly expressing vectors are used. These plasmids usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene. Several promoters or enhancers are sometimes included to improve mRNA stability and increase protein expression. Plasmids also include a strong transcriptional termination signal [1,2,3]. Some multicistronic vectors are designed to express several immunogens or to express a number of immunostimulatory proteins.An effective vector design is important for maximal protein expression [16]. Optimizing the codon usage of mRNAs in eukaryotic cells is an ideal way to enhance protein expression. As pathogens often show different AT codon usages, changes in the gene sequence to enhance the more effective codons in the target species improve the gene expression. Another important factor in the construction of DNA vaccines is the choice of the promoter. Although the SV40 promoter has been widely used [2], the expression rate of DNA vaccines has been increased using the cytomegalovirus (CMV) immediate early promoter [1,2]. Furthermore, several studies showed that a chimera CMV promoter with a chicken β-actin intron (CAG promoter) generates a higher expression of CMV protein by several folds [2,4,17]. Inclusion of the cis-acting transcriptional elements (CTE) from monkey virus with rev increased the envelope expression. The rev+CTE constructs have distinct advantages for increasing the expression rates. When an envelope sequence from East-African subtype A virus was inserted into an expression vector, it increased the expression rates of the CMV promoter [1,2,17]. Additional modifications to increase the expression rates include the insertion of enhancer sequences, synthetic introns, and adenovirus tripartite leader sequences, and modifications to polyadenylation and transcriptional termination sequences. The design of the DNA sequences or the optimized codon usages can be targeted to various cellular compartments to improve the Th1 or Th2 responses. The addition of N-terminal ubiquitin signals increases immunogenicity [4,11,18], while a conformational change of the amino acids in the protein sometimes enhances the immune responses. The optimized codon usage also enhances protein expression [1,2,18].DNA vaccines have been used to immunize animals by using a number of methods. The type of T-cells raised is influenced by the method of delivery, type of immunogen expressed, as well as targeting of different lymphoid cells. Each method has distinct advantages for immune activation. However, no single method was successful in enhancing the protective effect and properly regulating the induction of a steady immune response.Generally, needle injections are used to induce Th1 responses. Ulmer et al. [19] demonstrated that pDNA encoding viral nucleoprotein (NP) of influenza virus could induce cytotoxic activity regardless of the viral antigen shifts and consequently protect animals from lethal infection. Numerous studies have revealed that these needle injections of pDNA are very effective against viral infections in animal models [2,4,20,21]. The popular approaches for the injection of pDNA by using a needle involve the use of bupivacaine, hypotonic solution, or saline, or sometimes the use of an electroporation method. Immunization is usually performed by intramuscular injection in the skeletal muscles, or by intradermal injection, with the DNA being delivered to the extracellular spaces or into the cells. The delivery into the cells is assisted in some cases by electroporation [22], or by temporarily damaging muscular fibers with bupivacaine or hypertonic solutions. Hence, it is rather difficult to obtain a constant immune response [2]. In addition, the immune responses to these needle methods are also affected by many factors, including needle type, muscle type, age of the animal, and the speed of injection [2,3]. The rapid implantation of vaccine-loaded polymer films is used for carrying DNA and biodegradable polycations into the epidermis, which is rich in immune cells, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following a brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin for weeks. These “multilayer tattoo” DNA vaccines induced immune responses against a model HIV antigen, which were comparable to electroporation in mice. These vaccines also elicited an increase in the gene expression in non-human primate skin, which was 140-fold higher than the level elicited by an intradermal DNA injection [22,23]. At present, this type of needle injection is widely used in animal studies.The gene gun is often used as it increases the Th2 responses [24,25,26]. The pDNA that has been coated on the surface of the gold, is introduced into the cells by using compressed helium as an accelerant [2,24]. This method is effective as it takes advantage of the molecular weight and the safety of gold. Saline injections require 2–20 μg pDNA per mice, whereas gene gun deliveries require only 1–3 μg pDNA to increase an effective immune response [24,25,26]. Most of these results were observed using mice. For clinical trials in humans, it is important to decrease the dose of pDNA because the quantities vary from species to species. For example, primates require approximately 10 times more pDNA than mice. Moreover, saline injections require more pDNA because the pDNA is delivered to extracellular spaces of the target tissue (normally, muscle cells), whereas gene gun injects pDNA directly into the cells [26]. Due to the weak immunogenicity of pDNA, this immunization method must be performed several times and in many places to elicit a potent immune response. Another delivery method is the i.n. inhalation of pDNA through the nasal and lung mucosa. Tadokoro et al. [27] investigated tissue distribution of DNA plasmids by i.n. administration, using a fluorescence in situ hybridization (FISH) method. Their study revealed that the DNA plasmids localized in the alveoli of the lung, liver, spleen, regional lymph nodes, kidney, fetus, and esophagus in the administrated mice. The HIV plasmids were detected two to four weeks after administration. The messenger RNA of HIV env gene was detected in the lung, liver, and spleen, while type 1 HIV proteins were detected in the lungs [27]. DNA vaccination by i.n. as well as intravaginal administration of constructs with HIV genes induced high levels of Th2 immune responses against HIV antigens. The level of mucosal IgA antibodies detected in the feces and vaginal fluid was significant in i.n. administration. This route of administration also resulted in significant levels of HIV-1-neutralizing antibodies in feces and serum [28]. In addition, i.n. immunization with the hemagglutinin gene of influenza pDNA induced a protective immune response against influenza virus in mice models. In addition, secretary IgA antibody was produced at significant levels by high doses of i.n. DNA vaccination in mice [29]. Cytokine assays revealed that i.n. administration of this DNA vaccine induced mainly Th2 immune responses [2,12,28]. A major advantage of the i.n. administration is the ability to increase the dose in order to enable several applications for a more effective DNA vaccination in mice [29,30]. One important point of the i.n. method is the strong induction of secretory IgA antibody. The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness, and cost-effectiveness. However, the levels of immune responses it elicits are relatively low. Liu et al. [31] administered HIV-1 DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes by using fast-acting adhesives. Their findings revealed that the topical application of HIV-1 DNA vaccine induced an immune response against HIV-1 envelope antigen. Skin biopsy of the immunized mice showed significant activation of dendritic cells (DCs), suggesting that the topical application method is an efficient route of DNA vaccination [31]. Watabe et al. [32] reported the expression of a matrix (M) gene of the influenza virus by applying the DNA vaccine several times on the mouse skin, after removal of the keratinocytic layers. Immunization using this method induced M-specific antibodies and cytotoxic T lymphocyte (CTL) response to acquire resistance against the influenza virus. They further found that simultaneous topical application of GM-CSF expression plasmids or liposomes plus mannan produced a stronger immune response and enhanced the protective effect [28]. The ocular inflammatory disease has been treated by topical administration of pDNA encoding IL-10 or other genetic adjuvants [33]. DNA vaccination against herpes simplex virus-2 (HSV-2) by administration through vaginal mucosa has also been reported [34]. In addition, this study was the report of the DNA vaccination through the vaginal region by using NanopatchTM in mice systems [34]. These studies suggested that these immunizations are useful for mice against HSV-2 infection or might be effective in HIV infection. Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations [3] and biodegradable microspheres [3,16]. We previously reported that in mice, the immune responses were transferred into the offspring when the mothers were intravenously injected with liposome-encapsulated DNA vaccine [35]. Though the uptake of pDNA was observed in the fetuses, the actual transfer occurred only during early pregnancy. During the early days after conception with cationic liposomes, the injected plasmid was detected in the tissues of the fetus, consistent with a transplacental transfer. Offspring mounted stronger antigen-specific immune responses than controls and they were protected against homologous influenza virus after vaccination. Moreover, such immune responses were stronger in the offspring when the mothers were injected with DNA plasmid during the early days after coitus. These results suggest that DNA-vaccinated mothers confer the antigen-specific immunity to their progeny.The delivery of DNA vaccines by the needle-free Biojector® device induces Th2-type immune response as well as IFN-gamma ELISPOT and CD8+ T cell responses, when boosted with recombinant adenovirus or vaccinia virus vector vaccine. The needle-free delivery of DNA using a CO2-powered Biojector® device was found to be a useful method of immunization.Graham et al. [36] reported that the immune response to this CO2-powered Biojector® method is enhanced by boosting with recombinant Ad5 vaccines. Although the side effects are minimal, this method is stronger than needle injection in human phase I trial. In a small number of human trials, no HIV-specific antibody responses were detected, even as low-magnitude HIV-specific T-cell responses were detected in approximately 50% of the vaccinees. This initial product led to the development of a 4-plasmid multiclade HIV DNA vaccine, indicating that more effective techniques are necessary for the use of this needleless method [37].The EP method induces one of the strongest Th1 responses. This method induces an immune response that is 10 times or more stronger than the response induced by other pDNA vaccination methods used to immunize animals [38]. However, this method has a drawback owing to the high voltage of electricity used, which delayed its application in clinical medicine [2,38,39]. Although some modified methods have been reported recently, more effective methods are needed.The immunogenicity of DNA vaccine was increased by EP method in mice [40,41]. Zhou and coworkers [42] reported the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs), by fusing soluble PD1 with simian immunodeficiency virus (SIV) antigen. Intramuscular immunization (i.m.) via EP of the fusion DNA in mice resulted in consistently high frequencies of HIV-specific, multifunctional, long-lived cytotoxic CD8+ T cells and robust anti-SIV antibody titers. Soluble PD1-based vaccination potentiated CD8+ T cell responses by enhancing the antigen binding and uptake in DCs and activation in the draining lymph nodes. Their findings suggest that PD1-based DNA vaccination by EP method could be used against HIV and other pathogens.Donate et al. [43] recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. They evaluated the MEA for its use in DNA vaccination by using hepatitis B virus infection model. The plasmid encoding hepatitis B surface antigen (HBsAg) was delivered intradermally with the MEA to the guinea pig skin. The results indicate an increase in protein expression following plasmid delivery using the MEA compared to injection alone. In another study, pDNA vaccination using skin EP elicited robust humoral and CD8+ T-cell immune responses while limiting the invasiveness of the delivery method. The authors of that study compared the ability of homologous prime/boost DNA vaccinations by skin EP and i.m. injection, to elicit immune responses by ELISPOT assay, and studied the complexity of CD4+ T-cell responses. They found that DNA vaccinations by skin EP and i.m. injection were capable of eliciting both single and multifunctional vaccine-specific CD4+ T cells, which is important for protection from HIV infection. Although the amount of DNA delivered by skin EP was five-fold lower, it elicited a significant increase in the magnitude of multiple-cytokine producers suggesting that the skin EP is a useful method of vaccination against various infectious agents [38,44].Electroporation gene therapy has been used in preclinical and clinical trials of melanoma. Delivery of IL-12 by EP method resulted in significant necrosis of melanoma cells in a majority of treated tumors, and significant lymphocytic infiltration, as observed in the biopsies obtained from patients in several cohorts. In addition, the responses to untreated lesions suggested the induction of a systemic response following therapy [45]. Bordbar et al. [46] described the use of EP-mediated DNA immunization to identify important protective epitopes of the large VAR2CSA protein from Plasmodium falciparum, which has been implicated in the pathology of placental malaria. Immunization of mice and rabbit with DNA plasmids induced a high titer of antisera and also induced the generation of protective antibodies. The EP-mediated HIV DNA vaccine increased the HIV-specific cell-mediated immunity by a magnitude of 70-fold over that of HIV-specific cell-mediated immunity elicited by intramuscular injection, as measured by gamma interferon ELISPOT assay. Intracellular cytokine staining analysis for ELISPOT responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines. Delivery of a pDNA encoding IL-12 or IL-2 by using electroporation was demonstrated to be effective. This may be the first report of phase 1 clinical trial using the EP method [45]. DNA immunization is able to raise a range of TH responses by inducing synthesis of a variety of cytokines. A major advantage of DNA vaccines is the ease with which they can be manipulated to modify the type of T-cell that influences a Th1 or Th2 response by addition of several cytokine plasmids.To develop a more potent DNA vaccine, immunomodulatory effects of the administration of IL-2, GM-CSF, IL-12, IFN-gamma, or various expression plasmids were investigated [47,48]. When the vaccine and expression plasmids were incorporated into cationic liposomes [47,49] and administered to mice, the antigen-specific delayed-type hypersensitivity response [48] and cytotoxic T lymphocyte activity were significantly increased. The expression of the cytokines increased for a week or more, when the vaccines were administered in a plasmid form. An analysis of serum HIV-1-specific IgG subclasses showed a significant drop in the IgG1/IgG2a ratio in the group that received the plasmid cytokine-vaccine combination. These results demonstrate that the IL-2 expression plasmid strongly enhances the HIV-1-specific immune response via activation of T helper type-1 cells [49]. Cytokine assays revealed that the HIV-1 DNA vaccine plus IL-12 plasmid induced mainly Th2 immune responses [29,49,50]. Co-administration of pro-inflammatory agents (various interleukins, TNF, and GM-CSF) and Th2-inducing cytokines enhance antibody responses [28], whereas pro-inflammatory agents and Th1-inducing cytokines decrease humoral responses and increase cytotoxic responses, which is more important in the protection of viral infections. Co-stimulatory molecules such as B7-1, B7-2, and CD40L are also sometimes administered. Stimulated macrophages secrete IL-12, IL-18, TNF-α, IFN-α, IFN-β, and IFN-γ, while stimulated B-cells secrete IL-6 and IL-12 [28,50,51].Co-administration of cationic liposomes greatly enhanced the immune responses, and the antibodies against HIV-1 persisted for a long time. Co-administration of the DNA vaccine with IL-12- and GM-CSF-expressing plasmids induced high levels of HIV-specific CTLs and an increase in delayed-type hypersensitivity when administered even by the i.n. route using mice [28,29].The advantages of using genetic adjuvants are their low cost, simplicity of administration, as well as the avoidance of unstable recombinant cytokines and potentially toxic “conventional” adjuvants (such as alum, calcium phosphate, monophosphoryl lipid A, cholera toxin, cationic and mannan-coated liposomes, QS21 [52], carboxymethylcellulose, and ubenimex). Plasmid encoding B7-1 (a ligand on APCs) has successfully enhanced the immune response in anti-tumor models. A mixture of plasmids encoding GM-CSF and the circumsporozoite protein has enhanced protection against subsequent challenges. GM-CSF was proposed to cause DCs to present antigen more efficiently and enhance IL-2 production and TH cell activation, thus driving the increased immune response [53]. The timing/frequency of administration, the dose, as well as the combination of genetic adjuvants are important factors. In addition, the optical gene expression and the amount of pDNA vaccination should also be considered. Sasaki et al. [52] compared a DNA vaccine encoding env of HIV-1 and evaluated the QS-21 saponin adjuvant. Vaccination via the i.n. and i.m. routes elicited comparable systemic immune responses, and QS-21 consistently enhanced antigen-specific serum immunoglobulin G2a (IgG2a) production, delayed-type hypersensitivity reaction, and cytolytic activity of splenocytes. Secretory IgA production and cytolytic activity of the mesenteric lymph node cells were preferentially elicited by i.n. immunization, and QS-21 augmented these activities. The enhancement of humoral and cellular immune responses, by QS-21, was abrogated by treatment with anti-IL-2 and anti-IFN-gamma monoclonal antibodies.Arai et al. [54] examined the adjuvant effect of 8-bromocyclic AMP (8 Br-cAMP) on an HIV-1 DNA vaccine. Administration of the DNA vaccine and 8 Br-cAMP combination, via i.m. and i.n. routes in mice, enhanced both HIV-1-specific humoral and cellular immunities when compared to immunization with the DNA vaccine alone. Furthermore, when administered via the i.n. route, the combination was found to strongly induce the production of secretory IgA antibody. The adjuvant effect of 8 Br-cAMP on the DNA vaccine probably occurs via enhancement of CMV promoter activity of the vaccine.The efficiency of DNA immunization can be improved by stabilizing the DNA against degradation and by increasing the efficiency of the delivery of DNA into the antigen-presenting cells [3]. This has been demonstrated by coating biodegradable cationic microparticles with DNA. Such DNA-coated microparticles can be as effective at raising CTL as the recombinant vaccinia viruses, especially when mixed with alum. Particles that are 300 nm in diameter appear to be the most efficient for uptake by antigen-presenting cells [3].Unmethylated CpG motifs are prevalent in bacterial but not vertebrate genomic DNAs. Mycobacterium DNA has been reported to increase the adjuvant reactions in cancers. Oligodeoxynucleotides containing CpG motifs activate host defense mechanisms, inducing active innate and acquired immune responses [55,56]. Bacterial stimulatory CpG (CpG-S) motifs frequently increase the immune responses. Additionally, CpG-S motifs are hypomethylated and this enhancement occurs only by using bacterial DNA. In contrast, nucleotide sequences that inhibit the activation of an immune response (termed CpG neutralizing or CpG-N) are frequently observed in eukaryotic genomes [55,56]. The innate system works synergistically with the adaptive immune system induced by DNA vaccination. CpG-S motifs induce polyclonal B-cell activation and enhance cytokine expression and secretion. The Toll-like receptor (TLR) family functions as a mediator of innate immunity. The human TLR9 (TLR9) expression in human immune cells correlates with the responsiveness to bacterial CpG motifs. Immunostimulatory CpG motifs induce the expression of the TLR9 protein in human non-responder cells of CpG motifs [57,58,59]. However, most of the currently reported evidences for the existence of immunostimulatory CpG motifs come from murine studies. Hence, experimental data from other species could provide vital clues, because different species may require different DNA-flanking sequences. Recombinant alphavirus-based vectors have also been used to improve DNA vaccination efficiency. Against the Foot-and-mouth disease the model animal were reported to be effective in guinea pigs [60]. In addition, the alphavirus-based vector vaccine delivered by adenovirus has reported to induce sterile immunity against classical swine fever using rabbits and pigs [61]. The genes encoding the antigen of interest are inserted into the alphavirus replicon, replacing structural genes but leaving non-structural replicase genes intact. The Sindbis virus and Semliki Forest virus have been used to build recombinant alpha virus replicons. Unlike conventional DNA vaccinations, the alpha virus vectors kill transfected cells and are transiently expressed. This may be due to the high levels of protein expressed by this vector, replicon-induced cytokine responses, or replicon-induced apoptosis, leading to enhanced antigen uptake by dendritic cells.Abdulhaqq et al. [62] studied novel therapeutic and prophylactic DNA vaccines. To improve their vaccine constructs, they employed methods of RNA/codon optimization and antigen consensus to enhance the expression and cellular/humoral cross-reactivity, respectively. In addition, they studied the potential of various molecular adjuvants to skew Th1/Th2 responses, enhance cellular/humoral responses, and improve protection in various animal models. Subsequently, they observed enhancement of immune responses by the electroporation method and by the use of genetic adjuvants.GenScript is a venture company that designs and produces optimized genes that can alter both naturally occurring and recombinant gene sequences to achieve the highest possible levels of productivity in any given expression system. The OptimumGene™ algorithm manufactured by this company takes into consideration a variety of critical factors involved in different stages of protein expression, such as codon adaptability, mRNA structure, and various cis elements. Highly pathogenic avian influenza viruses are a continuous threat to chicken and human beings. The recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated to combat this threat. To comply with biosafety issues, the G gene was deleted from the VSV genome. The resulting vaccine vector VSVΔG (HA) was propagated on helper cells, resulting in the generation of trans-VSV G protein, and the chickens were vaccinated with a single intramuscular dose of the infectious replicon particles. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented the shedding of the target virus and transmission to sentinel birds. Subsequently, a self-amplifying RNA vaccine was developed [63,64,65].The non-viral delivery of a 9 kb self-amplifying RNA encapsulated within lipid nanoparticles substantially increased immunogenicity, as compared with the delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses that were comparable to those elicited by a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, their results suggest that a comprehensive evaluation of non-viral technologies to deliver self-amplifying RNA vaccines is warranted [66].Prime-boost strategies have been successful in inducing protection against malaria and simian immunodeficiency virus (SIV), as observed in many studies [67,68,69,70]. For boosting the recombinant protein, recombinant poxviruses, adenovirus type 5 [71], adenovirus type 5/35 [18,72], or other vaccines have been used [73,74]. Prime-boost strategies with recombinant protein have increased both the neutralizing antibody titer and the antibody avidity, and increased the persistence of weak HIV-1 envelope or other protein immunogens [3,75]. Recombinant virus boosts have been shown to be very effective for activating Th-1 responses [76]. Priming several times with DNA vaccine induces a weak but long-lasting immunogen in the host, while boosting with the recombinant virus or protein, induces a high level of immune responses.Priming of mice with pDNA encoding Plasmodium yoelii circumsporozoite surface protein, followed by boosting with a recombinant vaccinia virus expressing the same protein, induced significantly higher levels of antibody and CTL activity and provided higher levels of protection, as compared to the observations in mice immunized and boosted with pDNA alone [68,69,75]. These responses were further enhanced with a mixture of cytokine plasmids and boosting with recombinant vaccinia virus. An effective prime-boost strategy for the macaca malarial parasite P. knowlesi has also been demonstrated. Rhesus monkeys were primed with a multicomponent, multistage malarial DNA vaccine [76]. The DNA vaccine encoded 2 liver-stage antigens: the circumsporozoite surface protein (PkCSP) and sporozoite surface protein 2 (PkSSP2). They were then boosted with a recombinant canarypox virus encoding all four antigens (ALVAC-4). Immunized monkeys developed antibodies against sporozoites and infected erythrocytes. Partial protection against sporozoite challenge was achieved, and the parasitemia was significantly reduced, compared to the observation in control monkeys. Although these models are not ideal for extrapolation to malarial treatment in humans, they are important for pre-clinical trials [77,78].After DNA vaccinations, the boosting vaccines are usually a protein vaccine, a live-attenuated virus or a viral vector to help suppress or clear infections. The genetic optimization of synthetic plasmid constructs and their encoded antigens, in vivo electroporation-mediated vaccine delivery, as well as co-delivery with molecular adjuvants have collectively enhanced both transgene expression and the elicitation of vaccine-induced immunity. In addition, the development of prime-boost regimens has significantly contributed to DNA vaccine immunogenicity, and clinical trials using prime-boost vaccination are now in progress [79].Genetic immunization using pDNA has been studied for over 23 years with significant progress. Efficacy studies against many microbial infections by using different animal models have been reported. As the DNA vaccines are easy to prepare and quick to design, which facilitates their mass production, they are considered to be one of the most ideal vaccines. However, the efficacy and safety of these vaccines have not been comprehensively analyzed. Although the first DNA vaccine has been reported, the finer details regarding the method of vaccination, the adjuvant, and the genetic structure of the vaccine are still inconclusive. At present, one of the best combinations for DNA vaccination would be the use of electroporation with proper genetic adjuvant followed by boosting with attenuated virus vector vaccines. However, more clinical trials are needed to prove that DNA vaccination can induce the satisfactory level of effective immune responses in humans. The immune responses measured thus far were not as robust as anticipated from the preclinical studies. This may be due to the differences in the selection of infectious agents, as the antimicrobial immune responses are different for various infectious agents. For example, HIV-infected patients with high viral counts mounted a modest T-cell response with a DNA vaccine encoding several HIV antigens, resulting in no effect on viral count.At present, DNA vaccines induce strong Th1 immune responses but weak Th2 responses. A combination of booster vaccines could be used to address this concern. The developments of more effective and safety clinical studies of delivery methods are expected. The Table 1 summarizes the advantage and disadvantage of presently reported methods of DNA vaccine. The protective immune responses in malarial infection are different as the protective epitopes are different at different stages of the protozoan parasite, which renders preparation of a proper vaccine difficult. In HIV and influenza infections, the surface antigenic epitopes change frequently, and the use of internal protein antigens might be useful in DNA vaccine development. In these cases, the protein sequences of internal and surface conservative regions should be used for the preparation of DNA vaccines. Hence, the major challenge is to develop DNA vaccines that are potent enough to comprehensively protect against these infections. To overcome these hurdles, more effective adjuvants, administration methods, or other boosting vaccines are needed.Advantages and disadvantages of DNA vaccine administration methods.The DNA vaccination is a new method and has various advantages and brings a new impact into the vaccine field. In this manuscript we mainly discussed the vaccine’s concept, history, method, and the preclinical issues of the DNA vaccine. In addition, many clinical trials are new being carried out and a more conclusive DNA vaccination for humans will hopefully appear soon.Our works reported in this review were supported in part by a grant-in-aid for the Ministry of Education, Science, Sports, Culture of Japan, and the Ministry of Health and Welfare of Japan.The authors have declared that no competing interests exist.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).In late October 2011, the Monroe County Department of Public Health (MCDPH) was notified of a suspected case of meningitis in a 9-year old girl from Monroe County, NY. Laboratory testing at the New York State Department of Health (NYSDOH) Wadsworth Center confirmed the identification of Haemophilus influenzae serotype e (Hie) isolated from the patient’s cerebrospinal fluid (CSF) using real-time polymerase chain reaction (RT-PCR). The universal immunization of infants with conjugate H. influenzae type b (Hib) vaccine has significantly reduced the incidence of invasive Hib disease, including meningitis, one of the most serious complications for infected children. Not surprisingly, as the epidemiology of invasive H. influenzae continues to change, non-Hib serotypes will likely become more common. The findings reported here underscore the importance for clinicians, public health officials, and laboratory staff to consider non-Hib pathogens in pediatric cases of meningitis, especially when initial investigations are inconclusive.During the pre-Haemophilus influenzae serotype b (Hib) conjugate vaccine era, Hib was the cause of more than 95% of invasive H. influenzae disease among younger children [1]. Further, meningitis occurred in about two-thirds of these children with invasive Hib disease, with 15%–30% of survivors having serious neurological sequelae such as hearing impairment, mental retardation, seizure disorder, cognitive and developmental delay, and paralysis. The Center for Disease Control and Prevention Emerging Infection Program’s Active Bacterial Core Surveillance (ABCs) system suggests that Hib currently accounts for a rate of 0.20/100,000 cases among children <5 years based on provisional 2012 data [2]. The universal recommendation for the immunization of infants in the United States beginning at two months of age with conjugate H. influenzae type b (Hib) vaccine began in 1991 [3]. This has significantly reduced the incidence of invasive Hib disease, including meningitis, one of the most serious complications for infected children. As the epidemiology of invasive H. influenzae continues to change, non-Hib serotypes such as Haemophilus influenzae serotype e (Hie) are increasing. We present the case of a patient who was clinically diagnosed with bacterial meningitis but all routine laboratory cultures showed no growth at the local lab. We then discuss the importance of public health involvement when a bacterial meningitis case is identified but etiology of the organism is unknown. On October 24, 2011 (Day #1), a previously healthy 9-year old girl with a 2-week history of upper respiratory tract infection symptoms, including a nonproductive cough and negative strep throat culture, presented to a pediatric office with fever (Tmax of 103 degrees F), headache, stiff neck, irritability, and no rash. She was presumptively diagnosed with bacterial meningitis and given a single dose of intramuscular ceftriaxone (50 mg/kg) before being transferred to a local emergency department. At the hospital, cerebrospinal fluid (CSF) obtained through lumbar puncture revealed a cloudy appearance, with a white blood cell (WBC) count of 2,942/mm3 (polys 88%), glucose of 5 mg/dL, and protein of 196 mg/dL; gram stain initially showed no organisms except, on cytospin, four gram-negative bacilli (not coccobacilli) were noted; cultures were negative. A head computed tomography (CT) scan with contrast suggested acute on chronic pansinusitis; subsequent magnetic resonance imaging (MRI) of the brain with contrast also demonstrated pansinusitis with no evidence of intracranial abscess or tracking from the sinuses. The patient was started on intravenous ceftriaxone, gentamycin, and vancomycin; no steroids were administered, given the unclear gram stain results and low suspicion for Hib meningitis. The patient was transferred to the pediatric intensive care unit (PICU). Since meningococcal disease could not be ruled out, the local health department was contacted to initiate an investigation. The patient significantly improved and was transferred from the PICU to a regular pediatric floor on October 26 (Day #3); at that time, her antibiotic regimen was switched to ceftriaxone alone as the initial negative CSF culture following only a single dose of ceftriaxone suggested that the organism was susceptible to that antibiotic. On October 30 (Day #7), she was discharged to home. On follow up with her pediatrician on November 2 (Day #10), she reported some mild headaches and fatigue but, otherwise, had no neurological deficits and was doing well. She completed a 14-day course of ceftriaxone.On October 26, 2011 (Day #3), Monroe County Department of Public Health (MCDPH) staff interviewed the patient’s mother to determine potential exposure and need for post exposure prophylaxis (PEP) for bacterial meningitis due to invasive H. influenzae and/or meningococcal disease. The patient’s family received prescriptions for PEP: ciprofloxacin for the parents and rifampin for the younger sibling. The New York State Immunization Information System (NYSIIS) confirmed that the patient and the younger sibling were up to date in their immunizations, including the Hib conjugate vaccine (with doses given at 2, 4, 6 and 15 months old). On October 27, 2011 (Day #4), laboratory testing at the New York State Department of Health Wadsworth Center identified Hie from the patient’s CSF using RT-PCR. No further action was taken by MCDPH staff regarding potential PEP.In the post-Hib conjugated vaccine era, Waggoner-Fountain et al. first reported on four cases of non-type b encapsulated H. influenzae meningitis diagnosed in children here in the United States (two due to Hie and two due to H. influenzae type f [Hif]) [4]. Among these cases, only one child was school-aged: a 6-year old boy. Despite his age, he had only received a single dose of Hib conjugated vaccine at 15 months old. The boy’s CSF revealed a WBC count of 3,440/mm3, glucose of 34 mg/dL, and protein of 80 mg/dL. Laboratory testing identified Hie in his blood and CSF; he was treated with ceftriaxone for ten days and fully recovered. For the post-Hib conjugated vaccine era, we are not aware of any other published cases of meningitis due to Hie among previously healthy school-aged children here in the U.S.; however, Ladhani et al. reported Hie meningitis amongst two previously healthy children in England and Wales [5]. The identification of Hie is likely influenced by the serotyping method used, as Satola et al. have shown that PCR is the gold standard for capsule typing [6]. Indeed, slide agglutination serotyping (SAST) may be unreliable and, in particular, may overestimate the frequency of H. influenzae type a (Hia). This finding may account for the relatively high percentage of Hia noted among children with invasive disease in one study [7]. As surveillance for H. influenzae has improved during the post-Hib conjugate vaccine era, including referral of isolates for serotyping and wider use of PCR, research has shown that serotype e is increasing [5,8]. This case report underscores the importance of using real-time PCR as a rapid and accurate test to diagnose acute bacterial meningitis when cultures are negative. The changing epidemiology of invasive H. influenzae disease underscores the success of the Hib conjugate vaccine; it also suggests there might be a need to develop vaccines that cover other serotypes in the future.For invasive H. influenzae, current guidelines specify chemoprophylaxis with a four-day course of rifampin for household contacts of cases involving Hib but no other serotypes [9]. At this time, it is not clear what the efficacy is for chemoprophylaxis in non-b typeable disease, such as Hie [10].Indeed, knowing the risk of secondary transmission from invasive non-Hib index cases will require more data from continued surveillance efforts. We would like to recognize Christina Hidalgo, MPH, Glenda L. Smith, BS, Jillian Karr, MPH and Kari Burzlaff, MPH for epidemiology support from the New York State Department of Health Western Regional Office.B.S.K., A.C.W., B.B., J.L.N., D.S., J.R. and D.D.M. participated in the drafting and critical revision of the manuscript for important intellectual content. D.H. provided material support. All authors approved the final version. The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Seven-valent pneumococcal conjugate vaccine (PCV7) introduction and routine pediatric use has substantially reduced the burden of Streptococcus pneumoniae disease worldwide. However, a significant amount of disease burden, due to serotypes not contained in PCV7, still exists globally. A newly recognized serotype, 6C, was until recently, identified and reported as serotype 6A. This review summarizes the serotype epidemiology of pneumococcal disease pre- and post-introduction of PCV7, available post-marketing surveillance data following the introduction of higher valency pneumococcal vaccines (PCV10, PCV13) and future prospects for the development of new pneumococcal vaccines.Streptococcus pneumoniae is a leading cause of disease in children worldwide. Common manifestations of pneumococcal disease include meningitis, bacteremia, pneumonia and otitis media.Development of glycoconjugate vaccines has overcome challenges to elicit protective immune responses in infants and young children. A seven-valent pneumococcal conjugate vaccine (Wyeth Pharmaceuticals Inc., Pearl River, NY, USA, Prevnar) (PCV7) is licensed in over 90 countries and is prequalified by the World Health Organization (WHO) [1]. Introduction and routine infant use of PCV7 resulted in significant reductions of invasive pneumococcal disease (IPD) among vaccinated children. A benefit of universal PCV7 use was an indirect (herd) effect, which is thought to be attributed to reduced vaccine type colonization in the nasopharynx following vaccination and, in turn, decreased transmission of these pneumococci from vaccinated to susceptible (unvaccinated) individuals. The benefits of herd immunity were in part offset by increases in nasopharyngeal colonization of serotypes not contained in PCV7 [2,3]. Replacement by non-vaccine serotypes is a concern for both the individual and the population as a whole, due to the probability of transmission and potential to cause disease.Although routine PCV7 use has led to significant decreases in the incidence of pneumococcal disease, due to the seven vaccine types, a substantial proportion of pneumococcal disease burden, due to serotypes not included in PCV7, still exists. The selection of serotypes included in higher valency pneumococcal conjugate vaccines (PCVs) was consequently based on the need for a broader coverage of serotypes that have become more frequent causes of pneumococcal disease following PCV7 immunization, while continuing to maintain protection against disease, due to PCV7 serotypes. Currently, a 10-valent (GlaxoSmithKline Biologicals, Synflorix) (PCV10) and a 13-valent (Wyeth Pharmaceuticals Inc., Prevenar 13) (PCV13) pneumococcal conjugate vaccine is licensed and available in many countries. Synflorix contains the seven serotypes in PCV7 in addition to serotypes 1, 5 and 7F. Prevenar 13 contains the PCV10 serotypes plus serotypes 3, 6A and 19A. This review summarizes: (a) changes in the serotype epidemiology of invasive pneumococcal disease (IPD), including complicated pneumonia, prior to and following the introduction of PCV7 and higher valency PCVs; (b) available post-marketing IPD surveillance data that reflect the direct impact on the vaccinated/vaccine-eligible age group of children following the introduction of higher valency PCVs; and (c) future prospects for the development of new pneumococcal vaccines.The seven serotypes (14, 6B, 19F, 23F, 18C, 4, 9V) contained in PCV7 were among the most common global causes of invasive pneumococcal disease (IPD) among children <5 years of age. In many countries where PCV7 was implemented in the pediatric immunization schedule and national uptake was high, significant decreases in vaccine serotype IPD and relative increases in reported non-PCV7 serotype IPD were observed and led to a net decrease in the incidence of IPD overall [1,4,5,6]. In countries where overall PCV7 vaccine uptake was low, serotype 14 was the most frequently reported cause of IPD among the seven serotypes [1]. IPD attributable to serotypes contained in PCV10 and PCV13, respectively, globally represent approximately 75%–85% and 80%–90% of IPD among children <5 years of age [1,7]. The data presented below are mainly from studies in Europe and the Americas.Following PCV7 introduction, serotype 19A emerged in many countries as a leading cause of non-PCV7 IPD. In 2000, PCV7 was licensed in the U.S. and recommended for all children <2 years of age and for children two years to five years of age at risk for pneumococcal disease, due to an underlying medical condition. In 1998, serotype 19A accounted for 2.5% of all invasive isolates in children <5 years of age, and in 2005, it accounted for 36% of all isolates in this age group [8]. Between 1998–1999 and 2005, the largest increase in age-specific incidence of IPD 19A occurred among children <5 years of age (6.7 cases/per 100,000 population). The proportion of serotype 19A isolates that were non-susceptible to multiple antibiotics also increased, a result partly from clonal expansion of sequence type (ST)199, during the same time period [8,9]. In 2007, 42% (n = 180/427) of non-PCV7 serotype IPD in children aged <5 years in the U.S. was due to serotype 19A. Serotypes 7F and 3 were responsible for the remainder of IPD caused by non-PCV7 serotypes in children aged <5 years [10]. The incidence of PCV7 serotype IPD in 2007 was <1 cases/per 100,000 population among children aged <5 years [6]. Observed increases in the incidence of serotype 19A disease are likely multifactorial, as similar trends have also been reported in countries without national immunization programs [11,12]. Other factors, such as antimicrobial use and the development of multidrug resistant serotypes, secular trends and/or the emergence of specific clones may have also contributed to changes in 19A disease rates [8,11,13].The incidence of serotype 6A IPD in children <5 years of age was generally observed to decline after PCV7 introduction, which in part was attributed to cross-protection by 6B vaccine antigens [14]. However, serotype 6A IPD continued to be common globally in the post-PCV7 era, which may have been in part due to serotype 6C isolates that were previously identified and reported as serotype 6A [5,6,15]. Serotype 6C is a new recognized serotype within serogroup 6. Studies suggest that PCV7 does not cross-protect against serotype 6C [16,17,18]. In Europe, retrospective analyses of archived samples that were obtained from IPD surveillance before and after PCV7 introduction indicated that the prevalence serotype 6C IPD in the post-PCV7 period overall was associated with clonal expansion of the ST224-complex. The increased proportion of 6C isolates that were non-susceptible to antibiotics was concurrent with the emergence of the ST386-complex in the post-PCV7 period [19,20]. Serotype 6C PCR-screening of the archived 6A invasive disease isolates in the U.S. and Europe indicated that serotype 6C invasive disease in children is less common than in adults. However, since the magnitude of the serotype 6C absolute incidence rate increase was very small, definitive conclusions about the trends in 6C invasive disease are limited. PCV13, which contains serotype 6A and 7F vaccine antigens, has the potential to confer cross-protection against non-PCV13 serotypes 6C and 7A [21]. Opsonophagocytic antibody (OPA) titers to serotype 6C, measured from sera from infants who had participated in a PCV13 clinical trial, were ≥1:8 to serotype 6C and were measurable in 96% of PCV13 recipients. Furthermore, all of the PCV13 immune serum samples tested had detectable OPA titers ≥1:8 for both serotypes 7F and 7A [21]. Serotype 7F and 7A share serogroup-specific epitopes.Serotype 6D has also been described within serogroup 6 [22]. Serotype 6D until recently had been identified and reported as serotype 6B [17,18,23]. To date, serotype 6D is a rare cause of pneumococcal infections in children and adults in the U.S. [20,24], but is relatively common in Asia [25].In an international study, serotype distribution was assessed from longitudinal IPD surveillance data at sites located in Australia, New Zealand, Israel, Uruguay, North America and Canada [26]. Twenty-one surveillance databases that contained data about the rate of IPD reported for at least two years before and one year after PCV7 introduction were identified. Site-specific rate ratios (RR) were calculated using the observed IPD rate for each post-PCV7 year divided by the expected IPD rate extrapolated from the pre-PCV7 rate. For databases in which 6C isolates were differentiated from 6A, the distribution of true 6A isolates was weighted by the size of the surveillance site; 6C was included in analyses as a non-PCV7 vaccine serotype (NVT). For databases in which 6C was not reported separately from 6A isolates, the distribution of serotypes 6C and 6A was estimated from surveillance data available in the same geographic region or from global estimates reported in the pre-and post-PCV7 time periods. Among hospitalized children <5 years old, pooled analyses showed that the overall IPD rates decreased by year 1 after PCV7 introduction (RR 0.55, 95% CI 0.46–0.65) and remained relatively stable through year 7 (RR 0.49, 95% CI 0.35–0.68). Point estimates for VT IPD rates (i.e., PCV7 and 6A) decreased annually through year 7 (RR 0.03, 95% CI 0.01–0.10). Point estimates for rates of IPD caused by non-PCV7 serotypes contained in higher valency vaccines (i.e., serotypes 1, 3, 5, 7F, 19A) were apparent by 2–3 years after PCV7 introduction (compared to the expected IPD rates for the same corresponding serotypes) and continued to increase through year 5 (RR 3.65 95% CI 2.50–5.34) [26].In South Africa and The Gambia, PCV7 was included in the national immunization programs (NIPs) in 2008 and 2009, respectively. Serotype data from invasive disease isolates obtained during a clinical trial conducted prior to PCV7 introduction indicated that 65% of IPD cases among Gambian children aged 2–29 months resulted from serotypes contained in PCV13; serotypes 1 and 5 accounted for 18% of cases and 19A accounted for 9% of cases [27]. The most prevalent clone among serotype 1 invasive disease isolates identified from hospital surveillance or from clinical trials conducted in The Gambia between 1996 and 2005 was ST618 [28]. The incidence rate of serotype 1 IPD can vary considerably, due to secular trends and its propensity to cause epidemics. Among South African infants 1–3 months of age, 78% of IPD cases identified during a clinical trial conducted in 1998–2001 were due to serotypes contained in PCV13; serotypes 6A and 19A accounted for 14% and 9% of cases, respectively [29]. To date, longitudinal IPD surveillance data in West and South Africa following PCV7 introduction in the NIP are not available.In Latin America, PCV7 was implemented in several NIPs in the year 2008 (Mexico, Uruguay), the year 2009 (Peru, Costa Rica) and the year 2010 (Panama, El Salvador, Ecuador). Higher valency PCVs (PCV10, PCV13) replaced PCV7 in the NIPs in Latin America mainly in the year 2011. Prior to PCV7 introduction, common serotypes in Latin America were 14, 6B, 1 and 5, as assessed in a multinational hospital-based IPD surveillance study. In Mexico, among children <5 years of age, the proportion of IPD due to serotypes contained in PCV13 decreased from 77% in the year 2007 to 65% in the year 2011; the proportion of 19A IPD reported four years after PCV7 introduction in the NIP was 3.5 fold higher than pre-PCV7 introduction (year 2007) [30]. In Uruguay, the proportion of IPD among children <5 years of age decreased from 58% (in the year 2007) to 21% (in the year 2011) for PCV7 serotypes and increased from 35% to 44% for the six additional non-PCV7 types during the same time period. The relative proportion of serotype 1 IPD among children <5 years of age in Uruguay increased from 5% in the year 2007 to 17% in the year 2011; the relative proportion of serotype 3 IPD in this age group increased from 14% during the same time period [30].Estimating the proportion of community-associated pneumonia (CAP) due to S pneumoniae is challenging. Clinical presentation of lower respiratory tract infection and chest X-ray findings are non-specific, and thus, a diagnosis of pneumococcal pneumonia based on signs and symptoms alone can be difficult to distinguish from pneumonia due to other respiratory pathogens. Culture of blood samples has low sensitivity for detecting cases of pneumococcal pneumonia and might not be routinely collected. However, the majority of CAP cases are not associated with bacteremia. In children, sputum samples are not easily obtained.An increased occurrence of pneumococcal empyema, due to emerging serotypes, and the continued prominence of serotype 1 as a cause of severe pneumonia post-PCV7 introduction support the added potential benefit of higher valency pneumococcal vaccines to prevent a greater proportion of pneumonia cases, including pneumonia associated with complications. Pneumonia complicated by the development of empyema (alone or with parapneumonic effusion) increased among hospitalized children with pneumonia since the late 1990s [31,32,33]. Before the availability of PCV7, data from tertiary hospitals in the U.S. and Europe showed that empyema due to S. pneumoniae among hospitalized children with pneumonia was predominately due to serotype 1, with serotype 14 also identified as a common cause [31,32,33]. Following the introduction of PCV7 in infants and young children, the rates of pediatric IPD due to empyema increased, due to relative increases in cases caused by non-PCV7 serotypes [32,34]. In a study conducted at a tertiary hospital in the U.S., the incidence of empyema was 8.5/100,000 prior to the PCV7 introduction and increased to 12.5/100,000 after PCV7 introduction. At the time the study was conducted, 88% percent of children in Utah had received three doses of PCV7 by 35 months of age. The proportion of empyema caused by non-PCV7 serotypes increased from 62% to 98% during the study period, with serotype 1 reported as a prominent cause of empyema before and after PCV7 introduction (50% and 33%, respectively) and serotypes 3 (27%) and 19A (26%) identified as emerging causes of severe pneumonia post-PCV7 introduction. Molecular analyses indicated that the sequence type representing each of three serotypes (1, 3, 19A) was present before PCV7 introduction, suggesting that the increase in the incidence of empyema was a result of serotype replacement by non-PCV7 serotypes [32].Similar to the U.S., the prominence of serotype 1 and its association with pneumococcal pneumonia complicated by empyema was observed prior to PCV7 introduction in Europe. The emergence of other non-PCV7 types accounting for complicated pneumonia reported in Europe was noted during the time period after PCV7 introduction, as well [35,36]. In a study conducted in Italy, empyema or other pneumonia complications (e.g., parapneumonic effusion) was reported in 162 of 753 (21.5%) children hospitalized with pneumonia. At the time of the study, 41% of the participants had been vaccinated with PCV7 prior to enrollment. Of the 80 identified pneumococcal bacteremic pneumonia cases, >67% of cases were due to non-PCV7 types, with 32.5%, 15% and 12.5% of cases attributed to serotype 1, 19A and 3, respectively. Of children with complicated pneumococcal pneumonia, 50% were associated with serotype 1. All cases of serotype 1 pneumococcal pneumonia occurred in children older than two years of age, while pneumonia due to 19A was common in younger children (median age: 3.1 years (range: 10 months to 3.7 years)). All of the pneumonia cases due to PCV7 serotypes occurred in children who were not vaccinated with PCV7. In a study conducted at a pediatric tertiary hospital in Spain, the most common cause of pneumococcal parapneumonic empyema was serotype 1 (42%) followed by serotypes 7F, 3, 19A and 5. Forty-six percent of children had received at least one dose of PCV7 [36].In Europe and North America, the serotypes included in PCV10 and PCV13 are estimated to cover approximately 80%–85% and 85%–90% of IPD, respectively, in children <5 years of age; serotypes 19A, 7F, 1 and 6A are prominent causes of IPD in regions that previously included PCV7 in the NIP [1,7,37]. In Africa, serotypes 1, 5 and 6A are common causes of IPD, and both PCV10 and PCV13 are estimated to cover >70% of the serotypes [7].In Latin America, PCV7 was not widely used, and IPD caused by PCV7 serotypes currently represent approximately 55%–60% of the disease burden [7]. Use of PCV10 or PCV13 would potentially increase coverage to approximately 75% and 80%, respectively [7]. In Latin American countries that have included PCV7 in their NIP, IPD due to serotypes 1, 5 and 6A are becoming more prominent [1].PCV10 and PCV13 were first approved in 2008 and 2009, respectively. Currently, both PCV10 and PCV13 are approved in >100 countries and prequalified by WHO [7,38]. The most commonly administered regimens include two or three doses in infancy followed by one dose in the second year of life (2 + 1, 3 + 1) or three infant doses with no toddler dose (3 + 0) [1,39].In Germany, PCV10 and PCV13 were introduced in the NIP in April, 2009, and December, 2009, respectively, which was approximately three years after PCV7 introduction [1,38]. PCV10 and PCV13 are administered at two, three, four and 11–14 months of age. At the time that PCV13 was introduced in the NIP, approximately 50% of children had been immunized with PCV10. By 2011, approximately 85 of children were receiving PCV13. Prior to the introduction of PCV10, 37% and 68% of IPD in children <2 years of age were caused by serotypes contained in PCV10 and PCV13, respectively. The most common serotypes pre-PCV10 introduction were 7F, 19A and 1. In 2011, approximately two years after PCV13 introduction, six cases of IPD due to non-PCV7 serotypes (1, 3, 5, 6A, 7F, 19A) were reported in children <2 years of age compared to 28 cases reported in 2009. Reports of serotype 19A IPD decreased from 15 cases in 2009 to one case in 2011 [40].In the United Kingdom (UK), PCV13 replaced PCV7 in the national immunization program (NIP) in April, 2010, which was approximately four years after routine infant immunization with PCV7 began (September 2006). PCV13 was administered at two, four and 12 months of age. Prior to PCV13 introduction in the NIP (September 2009, to April 2010), the proportion of IPD cases due to the six non-PCV7 types was 69% (n = 191/277) compared to 27% (n = 82/303) of non-PCV7 types present in the first year after PCV7 introduction. The most prevalent serotype pre-PCV13 introduction was 19A followed by serotype 7F and 1 [41]. Approximately three years after the introduction of PCV13, the cumulative weekly cases of IPD caused by the six additional serotypes in PCV13 in children <2 years of age decreased to approximately the same as the number of cases prior to the introduction of PCV7 [42]. Decreases in the number of cumulative cases were mainly due to 19A and 7F [43].In the U.S., PCV13 replaced PCV7 in the routine infant schedule in approximately March, 2010. Infants received PCV13 at two, four, six and 12–15 months of age. Furthermore, a supplemental PCV13 dose was administered to children who were fully vaccinated with PCV7. Preliminary IPD surveillance data (April, 2010, to March, 2011) indicated trends towards reduced rates of 19A and 7F IPD in children <2 years old, compared to matched calendar quartiles during a time period (2006–2008) prior to PCV13 introduction. During the first year after PCV13 introduction, there was no change in the rates of serotype 3 IPD and too few cases of serotype 1, 5 and 6A to evaluate any potential effects [44].In Latin America, several countries included PCV10 or PCV13 as the first PCV in their NIPs in the year 2010 (Brazil, Columbia), the year 2011 (Chile, Nicaragua, Honduras) or the year 2012 (Argentina, Paraguay). In Chile, the proportion of PCV10 serotype IPD among children <5 years of age declined from 74% (n = 209/282) in the year 2010 to 52% (n = 88/168) in the year 2012. In five countries, PCV7 was introduced in the NIP during 2009–2010 and then replaced by PCV10 (Peru, Ecuador) or PCV13 (Costa Rica, Panama, El Salvador) in the year 2011. In Mexico, PCV7 was introduced in the NIP in 2008 and replaced by both PCV10 and PCV13 (year 2010 and 2011, respectively). The number of IPD cases due to the six non-PCV7 serotypes that occurred among Mexican children <5 years of age declined from 40% (n = 49/124) in 2008 to 13% (n = 14/105) in 2012 [30].During the interim period following PCV7 introduction, but prior to PCV13 introduction, decreases in PCV7-serotype IPD in countries with established PCV7 immunization programs were also associated with relative increases in IPD, due to serotypes not covered by currently available multivalent PCVs (PCV10, PCV13), such as 22F, 33F, 15B/C and 11A [24,45,46,47].In the U.S., PCV7 was recommended for routine infant immunization in 2000. In 2006–2007, serotypes 22F and 33F and 15B/C were the most common causes of non-PCV13 serotype IPD among children <5 years of age and cumulatively accounted for 14% of IPD in this age group overall. In contrast, prior to PCV7 introduction (1998–1999), serotypes 22F, 33F and 15B/C altogether accounted for 2.5% of IPD among children <5 years of age [6]. Four non-PCV13 serotypes comprised a cumulative total of 32% of the penicillin nonsusceptible (PNS) isolates in 2007: 15A (11%), 23A (8%), 35B (8%) and 6C (5%) [45].In Europe, serotype 22F was one of the most frequently reported non-PCV13 serotypes in 2010 among children <5 years of age [24]. In Norway, PCV13 replaced PCV7 as a routine childhood vaccine starting in 2010. Prior to PCV13 introduction (2007 to 2009), serotypes 22F, 15B/C and 38 were among the increasing causes of non-PCV13 serotype IPD. During the same time period, there was rapid clonal expansion of ST433, ST199, and ST393, respectively. The emergence of serotypes 15 B/C was associated with the expansion of other clones; however, the most common sequence type continues to be ST199 [48]. In the UK, PCV7 was introduced in 2006. During the time period between 2000–2006 and 2008–2010, rates of IPD were assessed from cases identified through national surveillance. The analyses of cases took into account potential biases due to missing data (serotype and age of patient) and changes in case ascertainment, such as case identification via molecular methods and routine screening for serotype 6C isolates, during the surveillance period. In 2008–2010, the average numbers of non-PCV13 serotype IPD cases reported among children <5 years of age were highest for serotypes 22F, 15B/C and 33F (n = 34, 22 and 15 cases, respectively). During the time period from 2000–2006 to 2008–2010, the incidence rate (adjusted for potential biases) of IPD for non-PCV13 serotypes 22F and 15C increased by approximately three-fold among children <5 years of age [15].In Australia, PCV7 was recommended in 2005 for non-Aboriginal children as a three-dose infant schedule (ages two, four and six months) without a toddler dose. Vaccine coverage was 88% among non-Aboriginal children in 2005. In a study conducted in Western Australia, the overall incidence of IPD in children <5 years of age decreased in 2005–2007 compared to 2002–2004, and an increase in the frequencies of non-PCV13 serotype IPD (6% to 17%) was reported during the same time period. The most common non-PCV13 serotypes in 2007 were 15B/C and 11A [49]. In contrast, in a study conducted in Southern Australia, no substantive increases in non-PCV13 serotypes were reported in children <5 years of age during the time period from 2002–2004 to 2007–2009 [50].In Canada, a national surveillance study was conducted during 2007–2009 to assess the baseline epidemiology prior to PCV13 introduction. Of the 800 invasive disease isolates obtained during the surveillance period, serotypes 22F (6.0%) and 11A (4.4%) were the most common non-PCV13 serotypes overall (all age groups). The proportions of serotype-specific IPD varied by age group (<2 years, three to 16 years, 17 to 49 years, ≥50 years). Among children <2 years of age, the most common non-PCV13 serotypes causing IPD included 15B (10.0%), 23A (6.7%), 22F (5.0%) and 35B (5.0%) [47].In a case-based study conducted at eight pediatric hospital centers in the U.S., invasive disease isolates were prospectively identified during a surveillance period beginning from January 1, 2007, through December 31, 2011. In 2010–2011 (post-PCV13 introduction), non-PCV13 serotypes 33F (n = 16) and 22F (n = 12) followed by serotypes 12, 15B, 15C and 23A (n = 7 for each of the serotypes) were the most common causes of invasive pneumococcal infection. In total, the six serotypes described above accounted for 20% of cases per year in 2010 and in 2011, compared to 10% of cases per year in 2007–2008 (post-PCV7 introduction, but prior to PCV13 introduction). Serogroup 11 accounted for six IPD cases in 2011, compared to zero to four serotype 11 IPD cases/per year in 2007–2010 (pre-PCV13 introduction) [51].In light of the present epidemiology of pneumococcal disease, PCV10 or PCV13 immunization in infants has the potential for significantly reducing the global pneumococcal disease burden that exists today. However, invasive disease serotypes not covered by currently available PCVs are already evident [15,45,47,49] and might become prominent causes of reported disease as circulating vaccine invasive serotypes decrease in countries using PCV10 or PCV13. In turn, a 15-valent PCV, which includes serotypes 22F and 33F, is being developed to offset some of the projected replacement serotypes that are anticipated to accompany routine PCV10 or PCV13 use [52]. In the long run, as the geographic distribution of predominant serotypes changes, effective vaccine coverage provided by PCVs may not be optimal worldwide. Furthermore, manufacturing complexity and the high cost of PCVs limit the ability to sustain production in developing countries. Alternative strategies for the development of serotype-independent pneumococcal vaccines that include common proteins are underway. While many potential choices for vaccine antigens are in the preclinical stages of development, there are a growing number of investigational pneumococcal protein-based vaccines that have recently been or are currently being evaluated in clinical trials [53,54,55,56,57]. General categories of protein-based vaccines in development include serotype-independent subunit vaccines comprised of purified proteins, proteins antigens that are expressed by recombinant bacteria, combination vaccines that include pneumococcal protein antigens in addition to conjugate components and a whole-cell vaccine.Protein-based, serotype-independent subunit vaccines could circumvent the issue of serotype replacement by directly targeting proteins that are highly conserved among a diversity of pneumococcal serotypes. Many of the vaccine candidates that have been studied are proteins that are involved in pathogenesis and can be present on the surface of intact pneumococci or a component that contributes to cell lysis. A noncapsular protein-based vaccine that contains several pneumococcal proteins in a single formulation could potentially provide broader protection, for example, if antibodies to each of the proteins antigens confer protective immunity through one or more mechanism or if the proteins are expressed at different stages of pathogenesis.Investigational multicomponent protein-based subunit vaccines that contain combinations of pneumococcal surface protein A (PspA), pneumococcal choline-binding protein A (PcpA) and PhtD (polyhistidine triad protein D) have recently been or are currently being evaluated in Phase 1 clinical trials [53,56]. A phase 1 clinical trial designed to evaluate the safety and immunogenicity of a DNA vaccine containing a PspA gene expressed in an attenuated Salmonella typhi vector has been completed, as well [58]. PspA is among the earliest studied virulence factors. Although PspA is structurally variable, it is expressed on the surface of many clinically relevant pneumococcal strains. Cross-protection against strains expressing heterologous PspAs has been shown in pre-clinical studies [59]. PcpA is thought to have a role in adherence to host cells, particularly respiratory epithelium, which would be a favorable characteristic in light of the increased attention to pneumococcal pneumonia prevention. Polyhistidine triad protein D (PhtD) is expressed on the surface of many pneumococcal serotypes and has an amino acid sequence that is highly conserved. Antibodies elicited to individual Pht proteins (PhtA, PhtB, PhtD and PhtE) are cross-reactive with antibodies to other proteins within the Pht family [60]. Possible functions of Pht proteins in pathogenesis include adhesion to host cells and downregulation of the complement pathway by binding to factor H. In animal models, immunization with PhtD in combination with other protein antigens has been shown to reduce nasopharyngeal colonization and protect against pneumococcal infection, including pneumonia and invasive disease [61]. Another conserved virulence factor, pneumolysin, is a known intracellular toxin that has also been shown in animal sepsis and pneumonia models to have an enhanced protective effect when given in combination with other proteins [62]. Derivatives of pneumolysin toxoid have been or are currently being evaluated in clinical trials (denoted as dPly or PlyD1 hereafter) [55,56].A combined vaccine that includes pneumococcal proteins as vaccine antigens in addition to polysaccharide-conjugated components is another possible approach to broaden the scope of protection. In an animal model, anti-PhtD antibodies in conjunction with anticapsular antibodies had an additive protective effect against sepsis following intranasal challenge, compared to the effect elicited by anti-capsular antibodies alone [63]. An investigational vaccine containing PhtD, dPly antigens and pneumococcal polysaccharide-conjugated components is in development, and a phase 2 trial was conducted to evaluate the safety and immunogenicity of this vaccine in children 12–23 months of age [64]. An additional role of pneumococcal protein(s) might be as a carrier protein(s) for conjugated vaccine components.A pneumococcal whole-cell vaccine (WCV) comprised of killed S. pneumoniae organisms enable the simultaneous presentation of multiple surface protein antigens. A vaccine that could provide direct protection against pneumococcal disease and possible indirect protection by reducing nasopharyngeal colonization is an approach that takes into consideration the capability of a WCV to elicit both humoral and cellular-mediated immune responses. In animal models, a WCV derived from killed unencapsulated bacterium was shown to induce antibodies that could passively protect against lethal challenge and to stimulate IL-17A-mediated responses, leading to reduced density of pneumococcal colonization in the nasopharynx and middle ear [65,66]. A phase 1 clinical trial with the objective of evaluating the safety of an aluminum-adjuvanted WCV was completed in 2013 [57].PCV7 has been highly effective in reducing global pneumococcal disease burden. In countries where PCV7 was implemented and uptake has been high, significant decreases in vaccine serotype IPD and relative increases in reported non-PCV7 serotype IPD have been observed. The net decreases in the overall incidence of IPD strongly support the benefits of PCV7 vaccination.The magnitude of increased rates of pneumococcal non-vaccine serotype invasive disease reported after the implementation of PCV7 varied among geographic regions and by the extent of vaccine uptake. While shifts in epidemiology temporally followed PCV7 introduction, the extent to which the changes were due to vaccination is unclear. Available baseline pre-PCV7 IPD surveillance data are limited, which makes it difficult to discern if the changes in serotype distribution primarily represented true replacement disease or resulted from non-vaccine factors, such as secular trends or antibiotic selection pressure. Changes in surveillance methods or vaccine uptake could also lead to artificial fluctuations in overall IPD incidence. Ultimately, it remains uncertain to what extent an increase in the prevalence of non-PCV7 serotypes would translate to complete replacement of pneumococcal disease by these serotypes, compared to the overall rates of clinical disease caused by PCV7 vaccine types.A significant proportion of pneumococcal IPD that globally exists today can be prevented by the extended serotype coverage provided by PCV10 and PCV13. Importantly, the introduction of PCV10 and PCV13 can potentially have a broader impact on pneumococcal CAP, as serotypes contained in both vaccines represent serotypes that are prominent causes of bacterial pneumonia in children and adults. Preliminary surveillance data post-PCV10 and PCV13 introduction indicate trends towards a decreased incidence of IPD due to 19A and 7F among vaccinated children. Ongoing surveillance is essential to assess the effectiveness of PCV10 and PCV13 to prevent PCV7 and non-PCV7 serotype IPD. Long-term surveillance is likewise necessary to monitor for emerging and increasingly prevalent pneumococcal invasive serotypes, including non-PCV13 serotypes, such as 15B/C, 22F, 33F and 23A.Protein-based pneumococcal vaccines potentially can overcome some of the limitations of polysaccharide conjugate vaccines. In concept, broader vaccine protection could be achieved via inducing antibodies to protein antigens that are conserved among a diverse number of serotypes, as well as proteins that significantly contribute to pathogenesis. Pre-clinical data with candidate vaccines based on subunit protein antigens, combinations of conjugate and pneumococcal protein antigens or whole-cell pneumococci indicate that antibodies elicited by candidate protein antigens can prevent pneumococcal infections in animal models of invasive and mucosal disease. An increasing number of candidate protein-based pneumococcal vaccines have recently been or are currently being evaluated in phase 1 and phase 2 clinical trials. Additional studies are needed to further evaluate how vaccine-induced changes to pneumococcal serotype distribution in the nasopharynx affect disease transmission potential and long-term impact on the incidence of pneumococcal disease.The authors would like to thank Hua Hua Tong for her critical review and helpful advice.The authors declare no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The current study examined and compared the willingness of young Black men who have sex with men (YBMSM) to accept pre-exposure prophylaxis (PrEP), adult male circumcision, and condoms for reducing their risk of HIV acquisition. The majority (67%) reported unprotected receptive anal sex in the last six months. About three-quarters (71%) would accept using PrEP if it was 100% effective. Cost influenced PrEP acceptance with 19% indicating acceptance at $100 per month co-pay. Of those not circumcised, 50% indicated willingness if circumcision was 100% effective. Acceptance of circumcision decreased markedly to 17% with co-pays of $100. About 73% of men were willing to use condoms if they were 100% effective and 50% indicated a willingness at the cost of $10 per month. The findings suggest that condom use promotion strategies should remain at the forefront of public health efforts to control HIV incidence among YBMSM.The southern region of the United States comprises the largest percentage (18%) of Black Americans and accounts for 46% of all new HIV diagnoses and more than 55% of HIV prevalence [1]. Young Black men who have sex with men (YBMSM) represent 73% of HIV incidence among all Black men and 37% of all MSM [2,3]. YBMSM aged 13–29 years are the only subgroup to have experienced a continuous increase in HIV incidence rates during the last three years [4]. Based on these marked racial/ethnic disparities, many questions remain about the acceptance of newly developed as well as established HIV prevention strategies for YBMSM.The use and effectiveness of condoms against HIV and other sexually transmitted infections (STI) are empirically supported, showing a 59% decrease in STI acquisition with accurate and consistent condom utilization [5,6,7,8,9]. Despite this effectiveness, continued disparities have increased the need for additional approaches to HIV prevention [10]. In recent years, biomedical strategies have reemerged as promising efforts in this regard.Pre-exposure prophylaxis (PrEP) is an empirically supported antiretroviral medication consumed prior to HIV exposure to prevent potential acquisition [11]. Results from the PrEP Initiative study showed a 44% reduction in HIV risk transmission among MSM and eventually led to the release of federal guidelines and FDA approval for PrEP as an HIV prevention strategy [12,13,14]. PrEP acceptability has varied between 30% and 80% [15,16,17] and its uptake has been influenced by demographic characteristics (e.g., age and education), sexual risk behavior and perception of risk [18,19].Another biomedical strategy is adult male circumcision [20,21], which has been recommended as part of a comprehensive approach to HIV prevention [22]. However, effectiveness data have varied among MSM [23,24,25] with a paucity of data specific to Black MSM [26,27].The continued increase of HIV in YBMSM has created a need to understand what prevention strategies are most acceptable to this population. Therefore, the purpose of this study was to describe the willingness of YBMSM to accept the use of condoms, PrEP, and circumcision for reducing their risk of HIV acquisition.Participants were recruited between 15 January 2013 and 14 February 2013, through banner advertisements on the Black Gay Chat website. These advertisements were restricted to residents of Mississippi, Louisiana, Alabama and Georgia. Website visitors who clicked on a banner ad were redirected to the internet-survey for completion. The survey was developed through the Qualtrics online system and included no accessibility limitations (i.e., desktop or mobile preferences). Young men were eligible if they had sex with a man in the past six months, were 18–39 years of age, and identified as being African American or Black. Incentives were not provided. The survey was anonymous and assessed questions assessing demographics, sexual risk behavior and determinants in the utilization of HIV prevention methods. The Institutional Review Board at the University of Kentucky approved all study protocols. Data were analyzed using frequency distributions.The sample consisted of young Black men who have sex with men (YBMSM) (N = 95), ages 18–39 years (mean = 26.8, SD = 5.66). In the last six months, 72% reported insertive anal sex and 74% reported receptive anal sex. The majority (71%) reported engaging in at least one act of anal sex that was not condom-protected. During the last six months, 67% reported at least one instance of engaging in unprotected receptive anal sex and 56% reported engaging in one instance of unprotected insertive anal sex (See Table 1).Characteristics of the Study Sample, African American Men, Aged 18–39 (N = 95).Nearly three-quarters (71%) of the men were willing to accept PrEP if it was 100% effective. Willingness to accept this method decreased with a lower level of effectiveness: 75% effectiveness (43%) and 50% effectiveness (21%). Cost had an influence on men’s willingness to accept PrEP: 19% were willing to accept the medication with a personal cost of $100. Table 2 provides greater details.The majority (75%) of the participants were circumcised. Of those young men who were not circumcised (n = 24), 50% indicated a willingness to be circumcised if this procedure was 100% effective in avoiding HIV infection. Acceptance of circumcision as an HIV prevention strategy decreased markedly to 17% with a personal cost of $100. Table 3 provides more information regarding the decline in acceptance based on cost and effectiveness.Acceptance of pre-exposure prophylaxis (PrEP) as a Safe Sex Measure (N = 95).Acceptance of Circumcision as a Safe Sex Measure (N = 24).The majority of the men (73%) were willing to use condoms if they were 100% effective, with 50% indicating this willingness to accept this prevention strategy at a cost of $10 per month. Table 4 provides greater detail about these findings. Acceptance of condoms as a safe sex measure (N = 95).Regardless of the HIV prevention method being offered, small personal costs have a substantial adverse influence on acceptance of PrEP, circumcision or condom use. Generally, the level of acceptance for all three methods was low, unless the method was rated at 100% efficacy and provided at minimal cost to the participant. The findings suggest that even under ideal circumstances (100% effective and free) a large proportion of men may not be willing to use any of these methods. This observation led to a post-hoc analysis that calculated the percent of men who would not accept the method even under both ideal circumstances (100% efficacy and free). This analysis was achieved through the use of a contingency table. These findings showed that 27% would not accept PrEP, 42% of those not circumcised would refuse do so, and 21% would not use condoms. These values are high given that the ideal circumstances are unlikely to exist, with the possible exception of condom use.Findings regarding PrEP are particular intriguing. The current findings are similar to those from other studies that examined barriers to PrEP acceptance [18]. Previous studies have shown that government funding to assist in the accessibility of PrEP could be a facilitator to the acceptance of this HIV prevention method [18]. Cost-effectiveness has been one of the primary considerations in the use of public funds for these prevention strategies. Delivery of PrEP was found to be a cost-effective strategy for high-risk populations [28,29], but acceptance among YBMSM may alter this equation. Resources to assist in subsidizing personal costs to YBMSM may be needed to enhance uptake of these prevention strategies [30]. PrEP effectiveness has been established from clinical trials when combined with condom use, HIV testing and other established prevention methods [15,30,31]. Although the current evidence supports this strategy, further research is needed regarding whether YBMSM most at-risk of HIV will indeed seek out a provider to give them PrEP at a price they can afford.These findings are limited based on the validity of self-reported data. The participants were a sample of men who opted into an online banner-ad survey and therefore the findings are subject to selection bias. Convenience sampling and restrictions to the southern region of the U.S. limits the generalizability of the findings to other populations of MSM. The results are based on a small sample size and therefore further research is warranted. Additionally, the findings provide limited insight to the participants’ knowledge of HIV prevention methods. This information could be a facilitator or barrier to their decision to prefer certain safe sex methods and should be further examined in future research.Biomedical approaches to HIV prevention, such as the use of PrEP and circumcision, will ultimately require patient acceptance. Availability alone may not be an adequate response. Given optimal circumstances (i.e., 100% effective and no personal costs) PrEP and circumcision are less acceptable to YBMSM than condom use. Because these optimal circumstances may never exist, findings suggest that condom use promotion strategies should remain at the forefront of public health efforts to control HIV incidence among YBMSM. Further, the study findings suggest that HIV preventive measures offered to YBMSM may not be widely embraced, including condom use. Apathy about preventing HIV infection may be a barrier working against efforts to innovatively protect this population. Thus, the role of behavioral science in HIV prevention is one that can complement and enhance emergent biomedical strategies.Support for this study was provided by the DDI endowment made to Crosby.All authors contributed equally to this work. R.C., R.D., and L.S. formed the study. All the authors contributed to the development of the study instruments. R.C. and A.G. supervised the data collection. All of the authors reviewed and analyzed the data. All of the authors contributed to the writing of the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).DNA vaccination has been studied in the last 20 years for HIV vaccine research. Significant experience has been accumulated in vector design, antigen optimization, delivery approaches and the use of DNA immunization as part of a prime-boost HIV vaccination strategy. Key historical data and future outlook are presented. With better understanding on the potential of DNA immunization and recent progress in HIV vaccine research, it is anticipated that DNA immunization will play a more significant role in the future of HIV vaccine development. More than 20 years have passed since the introduction of the concept of the DNA vaccine when several groups of scientists independently reported the use of this novel technology to elicit immune responses in small animal models against either a marker protein [1] or various model viral antigens [2,3,4,5,6,7]. The HIV-1 DNA vaccine was not only among this first group of initial reports [2,3], but was also one of the first DNA vaccines tested in non-human primates [8,9,10,11] and the first tested in humans [12,13,14]. One reason for the excitement towards the discovery of DNA vaccination was the potential of DNA vaccines to elicit T cell-mediated immunity. In the last several decades, the role of T cells in protective immunity has been increasingly realized by basic immunologists yet it was also frustrating to see limited progress in eliciting T cell immune responses by vaccination, especially with the use of traditional vaccines. Inactivated, subunit, or recombinant protein vaccines, which represent the majority of licensed human vaccines, are known to be poorly immunogenic towards the elicitation of T cell immune responses, especially CD8+ T cell responses. In theory, live attenuated vaccines are capable of eliciting high quality T cell immune responses but given safety concerns associated with a modified live pathogen, the selection of this form of vaccine has been declining since the middle of the 20th century.The introduction of DNA vaccination in the early 1990s provided a completely new opportunity for novel vaccine efforts in inducing T cell immune responses [15]. Given the challenge of developing an effective HIV vaccine and the understanding that cell-mediated immunity (CMI) is critical in the control of infection in HIV-1 positive patients [16,17], the HIV vaccine field welcomed the DNA vaccine concept and has contributed to the many further advancements of DNA vaccine technology.This review will focus on various optimizations of DNA vaccine technology from the last 20 years including results from key representative studies. More importantly, a vision will be presented on the transition from using DNA immunization, mainly for the induction of T cells, to the promising future of using this technology to elicit high quality antibody responses in the post-RV144 HIV vaccine landscape.The first human DNA vaccine study was done in HIV-1 infected patients as a Phase I clinical trial for safety analysis, followed by studies in healthy, HIV-1 negative volunteers [12,13]. These studies confirmed the overall safety profile for DNA vaccines but the immunogenicity results were disappointing. The magnitude and frequency of T cell immune responses, as the main objectives in these early clinical trials, were low; HIV-1 antigen-specific antibody responses were generally low or below detection.In order to improve the immunogenicity of DNA vaccines in humans, one key strategy, which is still part of many current clinical HIV-1 DNA vaccine formulations, is the use of novel “molecular adjuvants” [18,19]. Unlike traditional adjuvants, which are usually chemical compounds formulated with protein-based vaccines, molecular adjuvants, similar to DNA vaccines, are DNA plasmid-based expression vectors with gene inserts coding for various cytokines that can stimulate the host immune system. One unique advantage of molecular adjuvants is that they can be co-delivered with DNA vaccine plasmids without any extra formulation work. They can transduce cells at the site of DNA vaccine inoculation, and express cytokines to serve as adjuvants for DNA vaccines [19]. Traditional adjuvants elicit a broad spectrum of immune responses while gene-based adjuvants, in general, express only one particular cytokine that focuses on a key immune regulatory pathway. Based on the profile of immune responses to be elicited, molecular adjuvants can be grouped as Th1 or Th2 adjuvants. Th1 adjuvants include DNA plasmids expressing IL-2, IFN-γ, IL-12, and IL-15, which are used mainly to augment cellular immunity. For example, in clinical trial HVTN044, when IL-2 was delivered in the form of a DNA plasmid coding for the IL-2 fusion protein along with HIV vaccines, enhanced T cell responses were observed [20]. Molecular adjuvant IL-12 was able to increase CD8+ T cell responses in mice [21], control viremia, and improve clinical outcome following SHIV challenge in a non-human primate model [22]. IL-15, unlike most Th1 adjuvants, showed a balanced effect in augmenting both cellular and humoral immunities. This makes IL-15 an attractive adjuvant to be included in HIV-1 DNA vaccines. However, there was no significant increase in the response rate with the addition of IL-12 or IL-15 in a recently completed clinical trial HVTN070 [23]. Other molecular adjuvants express Th2 cytokines, such as GM-CSF, IL-1, and IL-4, and are used to enhance humoral immunity [19]. DNA vaccines, when used in combination with GM-CSF, improved antigen-specific antibody responses and enhanced lymphoproliferation in mice [24]. In rhesus macaques, co-delivery of GM-CSF-expressing plasmid and SIV DNA vaccines followed by a MVA boost resulted in enhanced binding antibody and neutralizing antibody responses, and increased protection against SIV challenge [25]. Whether Th2 molecular adjuvants are able to improve the immunogenicity of HIV-1 DNA vaccines in human is under active clinical investigation. Over the past two decades, significant technical improvements to the design of DNA vaccine vectors have contributed to much improved antigen expression and immunogenicity of HIV-1 DNA vaccines in both animal and human studies.First, codon optimization of immunogen genes [26,27,28,29,30] was found highly effective in elevating immunogen protein expression as shown by in vitro experiments, and enhancing T cell and antibody responses in vivo. Because HIV-1 uses a codon preference that is significantly different from that used in mammalian cells, it was demonstrated that modified codon usage matching that used in mammalian cells, without changing the coding amino acid sequences, can better match the tRNAs preferentially used in mammalian cells and, thus, enhance the protein expression of DNA vaccine immunogens [31].Second, replacing the HIV-1 Env leader sequence with signal peptides from other mammalian proteins was able to greatly improve the protein expression of Env-based HIV-1 DNA vaccines; however, more than one mechanism may be involved in this improved production of Env immunogens by DNA vaccines. Because the main purpose of Env in an HIV-1 vaccine design is to elicit protective antibody responses, certain mammalian leaders that can produce a larger quantity of secreted soluble Env proteins, compared with the original Env leader, will lead to higher antibody response levels. It is also likely that the highly unusual and defective nature of the original HIV-1’s Env leader sequence, i.e., multiple charged amino acid residues, may be responsible for the overall low level production of Env proteins and limited secretion of Env out of cells. As shown by early efforts in improving the production of Env recombinant proteins, the leader sequence of the human tissue plasminogen activator (tPA) was able to greatly improve the expression and secretion of HIV-1 by the mammalian cell expression system [32,33]. When the same tPA leader was used for Env DNA vaccines, higher in vitro Env immunogen expression and in vivo Env-specific antibody responses were observed in mice [34].Third, viral promoter efficiency plays a critical role in improving gene transcription of HIV-1 DNA vaccines. In early studies, strong promoters were derived from certain human oncogenic viruses, including LTR from Rous sarcoma virus and the SV40 early promoter [35]. However, in the last 20 years, intermediate-early gene 1 promoter adopted from human cytomegalovirus (HCMV), a non-carcinogenic virus, has been widely used with high efficiency in most DNA vaccine designs including HIV-1 DNA vaccines [4]. Furthermore, adding an intron A sequence from HCMV to an immediate downstream region of the HCMV promoter was able to further enhance the immunogenicity of HIV-1 DNA vaccines [32]. The other important promoter used for HIV-1 DNA vaccines is the CMV enhancer/promoter with the HTLV-1 R region (CMV/R), the regulatory R region from the 5' long terminal repeat (LTR) of human T cell leukemia virus type 1 (HTLV-1). As a transcriptional and posttranscriptional enhancer, this additional R region substantially enhanced transgene expression by 5 to 10 fold and further improved the cellular immune response [36]. Not only are the above three key elements of vector design individually important for a highly immunogenic HIV-1 DNA vaccine, they can also be combined in the same DNA vaccine to achieve a synergistic effect for immunogen expression and immunogenicity of HIV-1 Env DNA vaccines [34,37].There are additional DNA vaccine designs that have shown various levels of enhancing effects on HIV-1 DNA vaccines, such as the use of a C3d sequence at the C-terminus of the HIV-1 Env insert [38]. It was thought that C3d could help elicit antibody undergoing more rapid avidity maturation [39]. However, inclusion of C3d did not demonstrate a synergistic effect in a codon-optimized Env DNA vaccine [40] and later was not included in HIV DNA vaccines.Two failed phase III HIV-1 vaccine trials using recombinant gp120 proteins (AIDSVAX) [41,42,43] led to the conclusion in the HIV vaccine field that a monomeric gp120 immunogen may not be effective in eliciting protective immune responses. Designing more effective HIV-1 Env immunogens is a major challenge for the development of next generation HIV-1 vaccines. The DNA vaccine approach has become a highly useful tool in this line of work.Because gp160, the full length form of the HIV-1 Env glycoprotein, is membrane-anchored and hard to be expressed as a secreted protein, most recombinant protein-based HIV-1 Env vaccines use the gp120 or gp140 forms, which include only the extracellular portion of the Env protein and, therefore, neither form is in its natural trimer status. The DNA vaccination approach offers a unique opportunity to conduct a direct comparison among gp120, gp140, and gp160 forms of Env immunogens.When gp120, gp140, and gp160 DNA vaccines were compared directly in a rabbit study, gp120 was the most immunogenic form in eliciting antibodies against Env protein, gp140 was less effective, and gp160 was the least immunogenic [44]. Presumably, gp160 is not secreted in vivo, possibly reducing its immunogenicity as evidenced by lower levels of binding and functional antibodies compared with sera elicited by gp120 and gp140 DNA vaccines. In this study, gp140 with an intact cleavage site between gp120 and gp41 was used; however, gp140 can also be designed with the natural cleavage site mutated as a non-cleavable gp140. When DNA vaccines expressing either cleavable or non-cleavable gp140 forms were compared for their immunogenicity in rabbits, the non-cleavable gp140 was somewhat more immunogenic in eliciting binding antibodies than the cleavable gp140, but the cleavable gp140 (the natural form of gp140) was more effective in eliciting neutralizing antibodies, indicating the cleavable gp140 may be more effective in preserving conformational and functional epitopes [45], which was supported by results from non-DNA HIV Env design studies [46].While we remain mindful of the non-trimer nature of gp120 immunogens, there is no direct DNA vaccine immunogenicity comparison data that has shown significant benefits to using other forms of Env designs to elicit a better quality of antibodies, such as neutralizing antibody activities against Tier 2 more resistant viral isolates. DNA vaccines have been used to test Env immunogens with modified sequences. One well-studied DNA vaccine with a modified immunogen is a gp145 DNA vaccine developed by the Vaccine Research Center at US NIH [47]. In this design, multiple deletions at the cleavage site, at the fusogenic domain, and in the interspace between heptad repeats 1 and 2 were introduced based on the idea that it can stabilize and expose the functional domain of the protein that is present in an extended helical structure [47]. The original data from this mice study showed that it failed to generate consistently high level antibody responses. In phase 1 clinical studies VRC 009 and VRC 010, gp145 DNA vaccines were used as the priming immunization followed by an Ad5-based viral vector vaccine boost [48]. High ELISA binding titers were identified in human immune sera against the autologous gp145 immunogens but no data on its recognition of natural Env proteins (gp120 or other forms) was provided. Only 28% of vaccinees generated low levels of serum neutralization activity against two most sensitive viruses, SF162 and MW965.26. No neutralizing activities were detected against the other selected primary virus isolates, raising the possibility that modified Env immunogens with multiple deletions may have negatively impacted the integrity of Env immunogens and the resulting immune sera may not recognize the original Env immunogens.DNA vaccines were also used to test other novel Env immunogen designs, such as Env immunogens with consensus sequences [49]. However, it is frequently ignored that Env consensus sequences only cover constant regions; variable regions of Env are not part of the consensus sequence because the variable region sequences are too variable to reach a consensus. Instead, the natural sequences of the variable regions from certain HIV-1 isolates were used to combine with the consensus sequences of constant regions to form final full length Env sequences [50]. However, no broadly neutralizing antibodies were elicited with such consensus Env immunogens.Antigen engineering is also important for DNA vaccines expressing HIV-1 antigens other than Env. For example, the immunogenicity and type of immune response elicited by a wild type Gag DNA vaccine are quite different from another Gag DNA vaccine that includes an additional tPA leader sequence [51]. DNA vaccines encoding full length wild type Gag expressed an intracellular Gag antigen and generated high level Gag-specific T cell responses whereas adding a tPA leader sequence led to a secreted Gag protein along with a greatly enhanced Gag-specific antibody response but decreased Gag-specific T cell responses compared to the wild type Gag DNA vaccine design.One of the unique technical advantages for DNA vaccines is the possibility of delivering more than one DNA vaccine components at the same time. The daunting sequence diversity of HIV-1 is a key challenge to any candidate vaccines to elicit the broad antibody and cellular immune responses. While it is highly desired, there is limited progress in designing an immunogen that can effectively elicit a broad neutralizing antibody response. An alternative strategy, which has been adopted in many licensed human vaccines, is the polyvalent formulation including similar immunogens from a given pathogen with different immunological subtypes [52]. The difficulty of applying this strategy to HIV vaccine development is the fact that there are no clear serotypes or immunotypes based on HIV-1 Env immunogen. However, a pilot study using the DNA prime-protein boost approach in rabbits to compare the immunogenicity between monovalent and polyvalent gp120 formulations expressing primary Env antigens from several major subtypes of circulating HIV-1 viral isolates provided promising data to support the use of polyvalent Env formulations as part of the overall HIV vaccine development effort [53]. At the peak level of post final boost immunization, rabbit immune sera elicited by the polyvalent gp120 formulation neutralized 67.86% of a multiclade panel of tested viruses, while the monovalent vaccine elicited sera neutralized only 38.39% viruses. More strikingly, for non-clade B viruses, the polyvalent sera neutralized 56.25% of viruses from clade A, C, D, and E; while the monovalent group had a frequency of only 14.06% neutralization capability. A similar idea but requiring additional molecular modifications is the use of “designer’s immunogen” genes encoding several “mosaic” domains, assembled from fragments of natural HIV-1 sequences via a computational optimization method [54]. This approach may be more effective to elicit broad T cell immune responses because mosaic immunogens maximize the coverage of potential T-cell epitopes (peptides of 9 aa length) for a viral population. This approach not only greatly increases the coverage of viral diversity compared to natural sequence vaccine candidates, but also elicits enhanced breadth and depth of epitope recognition of variant sequences of CD8+ T lymphocyte epitopes. In mice, a three-set mosaic Env antigens delivered by DNA vaccines elicited increased breadth of CD8+ T cell epitopes compared to a DNA vaccine encoding natural Env immunogens [55]. Another study used polyvalent mosaic immunogens derived by in silico recombination of natural strains of HIV-1. Rhesus monkeys immunized with this type of mosaic DNA prime and recombinant vaccinia virus boost vaccine regimen elicited increased breadth and depth of epitope recognition of CD8+ T cell responses, compared to consensus immunogen [56].DNA vaccines, when first introduced in the early 1990s, were mainly delivered by simple intramuscular needle injection in small animal models. However, DNA immunization using conventional needle delivery appeared less immunogenic in non-human primates and humans. With recent advancements in DNA vaccine delivery technologies, DNA plasmid delivery methods can be grouped into two major categories: chemical delivery approaches and physical delivery approaches. For chemical delivery approaches, DNA plasmids are dissolved in various solutions, with or without polymer carriers (such as lipid, biopolymer, or other chemical compounds), and are delivered by conventional intramuscular or intradermal needle injections, transdermal patches, or through direct mucosal administration. The common feature of such deliveries is that DNA molecules are dissolved in chemical solution and cells at targeted tissues take up DNA plasmids by a low efficiency system.For the physical delivery approaches, the delivery of DNA vaccines utilizes various physical forces, such as shock wave, high pressure gas, and electrical pulse. In general, the physical approaches require special devices that can produce the external force. The most well known examples are gene gun or an electroporation device. Due to the use of such physical forces, DNA molecules are more likely to be delivered directly inside the cells of the target tissue so the efficiency of DNA vaccines is improved.The gene gun, one of the early physical delivery approaches, uses high pressure gas to deliver DNA plasmid-coated gold particles into the epidermal layer of the skin, an area rich with antigen presenting cells. Only a small amount of DNA is needed for this approach. The limited dose of DNA vaccines that can be delivered per shot requires multiple shots at each immunization. The gene gun was the first DNA vaccine delivery approach that showed balanced humoral and T cell immune responses in humans, including positive antibody responses at protection levels against hepatitis B surface antigens [57,58]. Using a similar approach, DNA vaccines expressing the HA antigen of seasonal influenza viruses were able to elicit protective levels of hemagglutinin inhibition (HI) antibody responses [59]. There is limited information on the use of the gene gun to deliver HIV-1 DNA vaccines in humans because the gene gun device has been under the control of various pharmaceutical companies and not available for broad human clinical studies.Biojector, a high pressure, needle-free device, that shoots the DNA plasmid solution into the skin, also provides improved DNA vaccine delivery by directly transfecting DNA into the cells of targeted skin tissue [60]. This device was compared to traditional needle delivery in a phase I clinical trial [61] in the context of DNA prime followed by rAd5 boost immunizations. This study revealed a higher response rate and 3-fold higher magnitude of T cell responses for Biojector delivery compared to the traditonal needle delivery approach.The electroporation (EP) approach involves delivery of short electrical pulses after needle injection of DNA vaccines. These pulses serve to increase DNA uptake by cells, leading to increased antigen expression and immunogenicity. EP delivery of AIDSVAX, a multi-gene HIV-1 DNA vaccine candidate, increased the magnitude of HIV-1-specific cell-mediated immunity by up to 70-fold over intramuscular injection, as measured by T cell γ-IFN response in humans [62]. A more recent study also confirmed the potency of the electroporation delivery method; the HVTN080 trial showed that 3 times PENNVAX-B (3 mg) delivered via EP elicited a 39% higher positive intracellular cytokine staining (ICS) response rate compared to three vaccinations with PENNVAX-B (6 mg) administered by standard intramuscular injection [23]. One weakness for the EP approach is the high dose of DNA plasmids required for such delivery and the need to include both needle injection and electric shock by an EP device. Furthermore, molecular adjuvant was still included in the above DNA vaccine formulation despite the use of EP delivery, making the whole formulation/delivery package very complicated and potentially expensive.The relative immunogenicity of traditional needle injection, gene gun, and electroporation was compared in a mouse model using HA antigen of avian influenza subtype H5N1 [63]. Both gene gun and electroporation methods were found more immunogenic than traditional needle injection. Interestingly, electroporation and needle injection elicited Th-1 biased antibody responses whereas gene gun induced a Th-2 dominated antibody response. These findings provide valuable information for further selection of DNA vaccine delivery methods for human applications.Since effective T cell immune responses have been correlated with the control of acute viremia in infected subjects, it was attractive to develop an HIV-1 vaccine that is able to induce a robust T cell response. Although early study results showed that HIV-1 DNA vaccines were able to effectively elicit both CD4+ and CD8+ T cells responses in small animal and non-human primate models [2,3,64,65,66], the immunogenicity of DNA vaccines was poor when used alone in humans. The progress of viral vector-based vaccines has stimulated the idea of combining DNA and viral vector vaccines in a prime-boost format. The common feature of DNA and viral vector vaccines, both being gene-based vaccines, is their abilities to present endogenous antigens to stimulate T cell immune responses. DNA vaccine has been matched with several well developed viral vector vaccines to maximize HIV-1-specific T cell immune responses.One highly potent viral vector vaccine for generating cellular immunity in humans is the adenovirus platform. The most widely used is the Ad5 viral vector. Two human studies comparing the induction of HIV-1-specific CTL responses generated by DNA plasmid vaccines or recombinant serotype 5 adenoviral vector (Ad5) vaccines showed the plasmid DNA was four times less potent in magnitude and response rate than Ad5 vaccines that contained similar HIV antigen cassettes. Further, the concept of DNA prime-Ad5 boost regimen was tested in the non-human primate (NHP) model, the most effective T cell responses were elicited by either Ad5 vector used alone or as a booster after DNA priming compared to DNA or MVA vector alone [67]. Similar results was replicated in a phase I clinical trial, the comparative analysis showed that Gag-specific T cell responses elicited by the DNA prime-Ad5 boost vaccine measured by ELISpot were similar to Ad5/Ad5, but higher than observed with the DNA/DNA regimen [68].However, the wisdom of using the Ad5 vector is now being challenged following several clinical trials showing disappointing results. The STEP trial tested the replication defective MRKAd5 vector to deliver a gag/pol/nef vaccine that did not provide any protection in a phase IIb trial and instead, may have caused increased viral acquisition in men with pre-existing Ad5 immunity and in uncircumcised men [69]. More recently, the HVTN505 trial, which used a DNA prime-Ad5 boost immunization regimen, also failed to show any protection with this regimen and had higher numbers of HIV-1 infection in vaccine recipients than the placebo recipients [70] although Ad5 seropositive and uncircumcised men were excluded from the HVTN505 trial. While more recent analysis may not show a statistical significance to confirm the risk of using Ad5 vector, it is difficult to anticipate the wide use of the Ad5 vector for HIV-1 vaccine studies in the near future, and future studies may need to use other serotypes of adenoviral vectors that target different cell receptors. Pox viral vectors are developed for novel HIV-1 vaccine applications because of the rich knowledge accumulated from the success of using vaccines to eradicate smallpox globally and the availability of several well characterized recombinant pox vectors. Of note, the RV144 clinical trial, which achieved 31% efficacy against HIV-1 infection, used a canary pox vector as the priming vaccine.One important pox vector platform is the modified vaccine virus Ankara (MVA). Researchers from Oxford University (Oxford, UK) used a DNA vaccine, composed of several T cell epitopes for HIV-1 including a fragment encoding the gag antigen as priming, followed by a MVA vaccine boost expressing the matched antigen. However, only low level T cell responses were generated, which may be due to limitations of epitope vaccines delivered by DNA vaccines [71,72,73,74,75,76]. The HVTN065 trial later tested a DNA vaccine containing several HIV-1 antigens as the priming immunization, followed by boost with a recombinant MVA expressing HIV-1 antigens. This DNA prime-MVA boost vaccine was well tolerated and produced detectable and reproducible HIV-1-specific cellular immunity in humans [77,78]. In this regimen, DNA priming was responsible for the induction of HIV-1-specific T cell immune response following the boost of MVA. The T cell responses were polyfunctional; about 50% of the HIV-specific CD4+ and CD8+ T cells induced in vaccinated subjects produced more than three cytokines. Furthermore strong T cell proliferation, as well as robust production of the T cell growth factor IL-2 by HIV-1 specific CD4+ and CD8+ cells was observed. A phase IIs clinical trial HVTN205 using a similar DNA prime-MVA boost regimen has been completed and the immune responses are currently under analysis. In addition to MVA, extensive research also went into NYVAC, a highly attenuated vaccinia virus vector. Results from the EuroVacc 02 trial using DNA prime-NYVAC boost demonstrated the safety and high immunogenicity of this platform [79]. The DNA and the NYVAC both expressed fused Gag-Pol-Nef and gp120 Env subunit of Clade C isolate, CN54. CD4+ T cell responses were detected in 90% of DNA prime-NYVAC boost vaccinees, which was superior to responses induced by NYVAC alone (33% of responders). However, the T cell responses were predominantly mediated by CD4 T cells, while only a low magnitude of CD8+ T cells specific for HIV-1 antigens Gag, Pol, and Nef were detected.Clinical trials were also conducted using a HIV-1 DNA vaccine prime followed by boost with another pox vector, the recombinant fowlpox viral vector. In these studies, DNA vaccines pHIS-HIV-B or pHIS-AE encoding gag, pol, env, tat, vup and rev were delivered as the priming vaccine followed by a fowlpox vaccine boost (rFPV-HIV-M3) expressing gag and pol. This regimen was not effective in inducing positive HIV-1-specific T cell response in humans, despite the high immunogenicity revealed in pre-clinical study in a non-human primate model [80,81,82].It is not clear why MVA or NYVAC is more effective than a fowlpox vector in the above studies. Furthermore, additional pox vectors based on less attenuated vaccinia viruses have been tested either alone or in combination with a DNA vaccine prime [83,84,85,86,87]. They showed promising immunogenicity results in animal studies including NHP models. Tiantan pox vector, developed by the China CDC, has been tested in humans as part of an HIV-1 DNA vaccine prime-pox vector boost regimen but data are not yet available. There is no comparative study to determine similarities and differences among different pox viral vectors and more importantly, how each vector system can further expand HIV-1-specific immune responses in hosts primed with HIV-1 DNA vaccines.Based on the early observation that HIV-1 DNA vaccines not only elicited T cell responses but also induced antigen-specific antibody responses [2,3], several studies further tested the idea of using DNA prime-protein boost immunization to further improve the level of HIV-1 Env-specific antibody responses in mice, guinea pigs, and even, rhesus monkeys [88,89,90]. While these early studies in the mid-1990s established the feasibility of combining DNA and recombinant protein vaccines in one immunization regimen, the true potential and exact mechanism of DNA priming to induce high quality Env-specific antibody responses were not yet fully appreciated; even after additional extensive research in the last decade, we have only just started to realize how much DNA immunization can contribute to the development of an effective AIDS vaccine in the context of a DNA prime-protein boost strategy.Among a few DNA prime-protein boost studies conducted in healthy human volunteers, the “DP6-001” study provided the most comprehensive and promising immunogenicity data [91]. In this study, participants received three times priming immunizations with a polyvalent HIV-1 DNA vaccine, including six DNA plasmids (five expressing different primary gp120 antigens from clades A, B, C, and E and one expressing a clade C Gag antigen), followed by two times boost with a polyvalent recombinant gp120 protein vaccine (five primary gp120 antigens matching those used in the DNA prime). Although the DNA vaccine components were administered via intramuscular or intradermal needle injection without using an adjuvant or a physical delivery device (such as gene gun or EP), positive gp120-specific CD4+ T cell responses were detected in all volunteers at the end of three DNA immunizations, and were further boosted by the gp120 protein boost. It was also important to note that these effector memory CD4+ T cells were multifunctional, secreting IFN-γ, IL-2, and TNF-α. The subpopulation positive for CD154 maintained proliferative potential and could rapidly develop into mature effector CD4+ T cells [92]. Interestingly, this phenotype was also seen in long term nonprogressors or aviremic HIV-1 infected patients on highly active antiretroviral therapy (HAART). In the higher dose group (1.2 mg for Gag-expressing DNA vaccine component), positive Gag-specific CD4+ T cell responses were also detected at the end of three DNA immunizations, a response that was rare in previous DNA vaccine studies when no adjuvant or EP device was used. Antigen-specific CD8+ T cells were also observed in vaccinated participants [92].However, the most significant finding was that high-titer gp120-specific antibody responses (end titration titers of 1:105, equivalent to Env-specific antibody titers in chronically infected HIV-1 positive patients) were detected in immune sera of 100% of the DP6-001 trial participants following one or two gp120 protein boosts [91]. All of these immune sera had a broad reactivity recognizing gp120 antigens from HIV-1 subtypes A to E. High level neutralizing activities were easily detected against pseudotyped viruses expressing Env from the sensitive viruses (TCLA isolates and SF162), activities that were better than observed in a previously reported DNA prime-Ad5 vector boost vaccine trial [93], in which no neutralizing activities were detected even against these sensitive viruses. There are good levels of ADCC activities in vaccinees’ sera [94]. When DP6-001 trial volunteer immune sera were tested against the more difficult to neutralize pseudotyped viruses from clades A to E and positive NAb was determined as greater than 50% inhibition, sera from approximately 1/3 of the volunteers were able to neutralize 80%–100% of this pseudotyped virus panel; the other 1/3 of the immune sera was able to neutralize 50%–79% tested viruses, and the remaining 1/3 of the immune sera neutralized 25%–49% of the tested viruses; serum from only one volunteer could neutralize only one pseudotyped virus [91]. In addition, a neutralization assay was conducted against the most difficult to neutralize Tier 2 viruses, but the neutralizing activities were very low. In summary, the DP6-001 trial raised high level binding antibody and broad neutralizing antibody responses. Additional assays have been done to show high level anti-V2 antibodies in DP6-001 volunteers [45], similar to RV144 trial results. The DP6-001 trial also revealed a rare event with high level skin reactogenicity among volunteers including possible skin vasculitis, particularly after the protein boost [95]. This is very different from other DNA vaccine studies where the overall safety profile has been excellent. One possible reason is the use of a strong adjuvant, QS-21, in the protein boost; QS-21 is well-known for its potential in eliciting various adverse events. Future studies with other adjuvants with improved safety profiles are needed in DNA prime-protein boost studies.The finding of high titer and high quality antibody responses in the DP6-001 trial was supported by another phase I DNA prime—Env protein boost clinical trial. A two-valent DNA vaccine prime, including one expressing an HIV-1 Gag and one expressing a V2-deleted gp140, were formulated in polylactice-coglycolides and delivered by intramuscular needle injection, followed by boost immunization using the recombinant gp140 protein matching that used in DNA prime. Compared to volunteers who received gp140 protein alone immunizations, DNA priming generated skewed Th1 phenotype polyfunctional Env-specific CD4+ T cells, a higher frequency of Env-specific memory B cells, and a higher titer of neutralizing antibodies and ADCC [96]. However, the breadth of neutralizing activities in this trial was limited, presumably due to limited valency of Env immunogens included in the formulation and/or a potential negative impact of V2 deletion to the quality of antibody responses.Following the antibody results from DP6-001 trial, additional in vitro and in vivo studies further revealed the role of DNA prime immunization in generating high quality antibody responses.In a rabbit study using the same formulation and immunization schedule as DP6-001, it was observed that rabbit immune sera elicited by recombinant gp120 protein alone mainly recognized the V3 eptiope while rabbit immune sera from the DNA-primed group exhibited unique binding against six additional gp120 epitopes, half of which contain residues that either are part of the CD4 binding site (CD4bs) or are involved in the binding with the CD4bs targeting neutralizing monoclonal antibody (mAb), b12 [97]. This finding indicated that DNA delivery of the gp120 immunogen is more effective than recombinant gp120 protein in eliciting conformation-sensitive epitopes. This finding was further validated by a comparative analysis with sera samples from three human HIV-1 vaccine studies: HVTN041 (recombinant gp120 protein alone vaccine formulated with potent AS02A adjuvant), HVTN203 (canarypox vector prime-gp120 protein boost), and DP6-001 study as discussed above (DNA prime-gp120 protein boost) [94]. Of note, HVTN203 is an early phase clinical study that used the same immunization regimen as the RV144 efficacy trial. HVTN041 sera had the highest binding antibody responses against the linear V3 epitope and high neutralizing activities against sensitive viruses but with limited breadth against other pseudotyped viruses. HVTN203 sera had lower V3 titers but otherwise a similar profile as the HVTN041 sera. DP6-001 sera demonstrated a higher frequency of positive neutralizing activities against more resistant viral isolates and a much higher frequency of CD4bs-specific antibody responses compared to HVTN041 and HVTN203 sera [94]. While the exact mechanism of how DNA priming is able to elicit CD4bs type antibody responses in both rabbit and human studies is unclear, it is possible that in vivo expression of HIV-1 envelope glycoprotein immunogens by DNA vaccination may have an advantage of maintaining the conformation of this sensitive protein compared to recombinant Env proteins manufactured by the traditional in vitro production and purification process. However, more recent small animal study results further indicate that DNA priming may also contribute to a more complicated process involving B cell development and innate immune mechanisms. It was demonstrated that DNA priming immunization was responsible for the improvement of the avidity of gp120-specific antibodies in rabbit immune sera [94]. The magnitude and profile of serum cytokines following recombinant gp120 protein immunization are changed with the addition of a priming step with gp120-expressing DNA vaccine [98].The “Combination DNA and protein HIV vaccine” as it was called several years ago [99] may have other alternate regimens. For example: can the process be reversed by using a protein prime-DNA boost regimen to achieve the same enhanced immune responses? Furthermore, the idea of delivering DNA and protein at the same time has been tested in multiple studies. In an early mouse model, it was shown that adding a DNA vaccine component may actually inhibit cytokine responses elicited by protein vaccines [100,101,102,103,104]. However, as shown by three more recent animal study reports, the combination of DNA and protein vaccines, when delivered at the same time, was also more immunogenic than each component used alone [105,106,107]. This finding raised a number of interesting theoretical and practical questions. First, in this regimen, multiple immunizations were given, therefore, the DNA delivered at the early immunizations actually served as priming for the immune system, which was later boosted by the protein vaccine component. Therefore, in some way, this is a staggered DNA prime-protein boost vaccination regimen. However, since protein was also given during the first and later immunizations, one issue that needs to be determined is whether any priming effects by the protein vaccine may interfere with DNA priming. Second, the DNA vaccine may have a dual role as it can serve as an adjuvant through innate immunity pathways also in addition to its role of delivering a subunit antigen. Therefore, it needs to be determined if co-delivery of the DNA and protein vaccines results in an adjuvant effect for the DNA or if it acts as an antigen delivery tool or both. Finally, recent papers did not provide convincing evidence that co-delivery is better than sequential delivery for DNA and protein vaccines. One study design flaw is the use of larger total DNA and protein doses for co-delivery when compared to sequential delivery. Furthermore, co-delivery actually increases the number of immunizations for either DNA or protein components when compared to sequential immunizations, and thus, is not surprising to see improved immunogenicity. More studies in this area with better designs will provide useful guidance to develop the most effective approach for taking advantage of the unique features of both DNA and protein vaccines.After 20 years’ effort, HIV-1 DNA vaccines have been systemically optimized from vector and antigen design to the inclusion of molecular adjuvants. However, their true utility in human studies was confirmed by two key events: (1) the use of DNA vaccine to prime the host immune system, especially for the induction of high titer and high quality Env-specific antibody responses, and (2) the availability and experience of using physical delivery approaches such as an EP device in humans to enhance the delivery and immunogenicity of DNA vaccines, which proved more effective for T cell immune responses.The role of DNA vaccines as a priming immunization will be compared with the use of viral vectors. Results from the RV144 trial may validate the use of a pox vector to prime the host, but also opens the door for priming with DNA vaccines. The setback with Ad5 vectors reminded us of the potential unrealized risk of using viral vectors. Table 1 compares the differences between DNA and viral vector vaccines. The relative immunogenicity of DNA vaccines may be lower than viral vector vaccines but the expression of additional antigens from viral vector vaccines may cause complications from pre-existing immunity against these antigens and also prevent repeated use of viral vectors. Immune responses elicited by DNA vaccines are highly focused and can be boosted further by repeated immunizations. DNA vaccines have been proven safe in repeated human studies and it is easy to manufacture and transport DNA vaccines, important features for global applications. While the DP6-001 trial showed good cross-clade neutralizing antibodies, high level and broadly cross-reacting binding antibodies, including ADCC, were also identified in the volunteers [91,94], as recently shown in RV144 trial volunteers. Future clinical studies will shed light on the relative merit between these two leading prime-boost HIV vaccine strategies.The use of EP revived the enthusiasm of DNA vaccination in humans but more questions need to be answered. The safety, public acceptability, cost, and finally, the magnitude of improved immunogenicity require further study. Furthermore, the incorporation of molecular adjuvants in the EP delivery approach also needs to be addressed.Features of DNA vaccines and viral vector-based vaccines.This study was supported in part by NIH grants U19 AI082676 (S.L.), P01 AI082274 (S.L.), R01 AI065250 (S.L.), and R33 AI0879191-04 (S.W.). The authors would like to thank Dr. Jill M. Serrano for her careful reading and editing of the manuscript.All authors (Shan Lu, Shixia Wang, and Yuxin Chen) contributed to data collection, literature review, and writing of the manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Since the initial proof-of-concept studies examining the ability of antigen-encoded plasmid DNA to serve as an immunogen, DNA vaccines have evolved as a clinically safe and effective platform for priming HIV-specific cellular and humoral responses in heterologous “prime-boost” vaccination regimens. Direct injection of plasmid DNA into the muscle induces T- and B-cell responses against foreign antigens. However, the insufficient magnitude of this response has led to the development of approaches for enhancing the immunogenicity of DNA vaccines. The last two decades have seen significant progress in the DNA-based vaccine platform with optimized plasmid constructs, improved delivery methods, such as electroporation, the use of molecular adjuvants and novel strategies combining DNA with viral vectors and subunit proteins. These innovations are paving the way for the clinical application of DNA-based HIV vaccines. Here, we review preclinical studies on the DNA-prime/modified vaccinia Ankara (MVA)-boost vaccine modality for HIV. There is a great deal of interest in enhancing the immunogenicity of DNA by engineering DNA vaccines to co-express immune modulatory adjuvants. Some of these adjuvants have demonstrated encouraging results in preclinical and clinical studies, and these data will be examined, as well.The RV144 recombinant canary pox vector, ALVAC/gp120 vaccine efficacy trial was the first to demonstrate a reduction in the risk of HIV acquisition by an HIV vaccine [1]. This vaccine-mediated efficacy, although moderate and appearing to wane over time, has reinvigorated the HIV vaccine field and renewed confidence towards the development of an effective HIV vaccine. There is a lot more work that needs to be done to develop an efficacious HIV vaccine, and the recently reported failure of the HIV Vaccine Trials Network (HVTN) 505 DNA/adenovirus 5 vaccine is a sobering reminder of the challenges we face towards realizing this goal [2].Post RV144, at least two strategies of vaccine development can be identified. The first consists of building upon the poxvirus prime, subunit protein boost employed in RV144 with the goal of enhancing immunogenicity and increasing efficacy, and the second involves pursuing diverse vaccine regimens to identify more effective vaccine strategies [3]. Currently, the main types of vaccines being developed in the clinic for HIV use recombinant protein subunit vaccines, such as the glycoprotein (gp) 120-protein fragment of the HIV envelope tested in the RV144 study, recombinant virus-vectored vaccines, such as ALVAC, New York Vaccinia Virus (NYVAC), modified vaccinia Ankara (MVA) and adenovirus serotypes, and DNA vaccines, typically used to prime immune responses in heterologous prime-boost vaccine modalities. Vaccine modalities based on DNA prime comprise a significant fraction of the current scheduled or ongoing Phase I and II HIV vaccine trials across the world (Figure 1). While plasmid DNA has demonstrated limited efficacy as a stand-alone vaccine, DNA in combination with viral vectors/protein shows a striking synergy in immune responses compared to either component alone. Innovations in the DNA platform with improved DNA delivery methods, such as electroporation and adjuvanting DNA with immunomodulatory molecules, have enhanced DNA immunogenicity. Here, we will briefly review the immunogenicity and efficacy studies using DNA as a prime in non-human primates and focus on pre-clinical and clinical studies of DNA/MVA HIV vaccines.Phase I/II clinical trials (ongoing/scheduled) of HIV vaccines. (Left) the table shows Phase I/II HIV vaccine trials by vaccine modality obtained from the International AIDS Vaccine Initiative (IAVI) database of vaccine candidates in clinical trials [4].In this section, we provide a brief timeline of benchmark studies leading to the development of DNA/MVA HIV vaccines beginning with the use of plasmid DNA to induce immunity against influenza more than two decades ago (Figure 2). The first use of naked plasmid DNA as an expression vector was demonstrated in 1990 [5]. In 1992, Tang et al. employed DNA as a simple means to elicit immune responses against non-self antigens [6]. Mice were immunized intradermally with plasmid encoding human growth hormone (HGH) using a gene gun approach. Remarkably, DNA immunization induced serum HGH Ab (antibody) responses, which were augmented by a booster shot. This simple and unique technique of genetic immunization generated considerable excitement for two reasons; first, DNA immunization would overcome the time-consuming need for protein purification necessary for protein immunizations; and second, DNA encoding viral proteins could serve as a vaccine against viral infections.The timeline of benchmark studies resulting in the development of DNA as a prime for the DNA/MVA HIV vaccine modality. The timeline of key studies resulting in the clinical application of DNA as an immunogen for DNA/MVA HIV vaccines. HGH, human growth hormone; NP, nucleoprotein; CTL, cytotoxic T-cell; Ab, antibody; HA, hemagglutinin; Env, envelope; IM, intramuscular; GM-CSF, granulocyte macrophage colony stimulating factor; SIV, Simian immunodeficiency virus; SHIV, Simian/Human immunodeficiency virus.The latter possibility was quickly realized and elegantly demonstrated by Ulmer et al. in 1993 [7]. cDNA encoding the influenza A nucleoprotein (NP) was injected intramuscularly at a dose of 100–400 μg at zero, three and six weeks in mice. Immunization with 100 μg NP DNA resulted in NP-specific antibodies and cytotoxic T-cell (CTL) responses, which resulted in protection against a heterologous influenza challenge. Around the same time, Robinson et al. demonstrated the efficacy of a hemagglutinin (HA)-expressing DNA plasmid in an avian influenza model [8]. Additional studies in mice demonstrated that gene gun delivery of HA-encoding DNA-coated gold particles to the epidermis conferred superior protection compared to intramuscular, intravenous and intranasal routes of DNA inoculation [9]. Together, these data showed for the first time that the injection of naked DNA encoding a conserved viral protein was a simple, yet effective way to induce CTL responses and high-titer antibodies. DNA-induced immunity was durable and protective, resulting in enhanced protection from a heterologous, highly virulent influenza challenge.In the same year, Wang et al. demonstrated the ability of plasmid DNA to induce anti-HIV immune responses [10]. Immunizations in mice comparing DNA versus protein immunogens showed that DNA immunization elicited higher titers of antibody against functionally important, diverse regions of HIV Env, V3 loop, immunodominant and fusogenic regions of gp41 and the conformational CD4 binding site. Subsequent studies by Peet et al. also showed higher V3 antibody titers in response to DNA compared to protein immunization [11]. The qualitative differences in antibody responses to DNA versus protein immunogens could be attributed to the expression of viral proteins in conformations that mimic natural infection in DNA-transfected cells. This could result in the effective presentation of important epitopes to the immune system. Indeed, it is this feature of DNA that makes it highly desirable as an immunogen.In all, the data showed that DNA was immunogenic in mice. However, data in primates was lacking. Previous studies had shown that non-human primate (NHP) muscle had the ability to take up injected plasmid DNA, albeit at a lower level than mice [12]. Comparison of plasmid DNA expression in mice versus monkey muscle demonstrated about 30-fold lower expression in monkeys compared to mice. While differences in body size and/or muscle histology across species could contribute, it raised questions about the potential immunogenicity of DNA in humans. This concept was formally tested in primates: cynomolgus monkeys were inoculated with 100 μg of plasmid DNA expressing HIV gp160 protein [13]. After four DNA immunizations, humoral and cellular responses were induced, indicating that DNA was immunogenic in primates and would likely be immunogenic in humans. The immunogenicity raised by DNA was shown to be protective; Boyer et al. demonstrated DNA vaccination protected chimpanzees from acquisition of a highly attenuated, heterologous HIV-1 challenge [14]. These data together with other seminal studies in the field, described later in this review, paved the way for the development of DNA/MVA HIV vaccines [15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33].The mechanisms by which intramuscular DNA injection primes an immune response to encoded foreign antigens is the subject of intense discussion for two main reasons: first, the muscle has relatively fewer professional antigen presenting cells (APC) [34,35]. Unlike the skin, which contains Langerhans dendritic cells (DC), the muscle has low frequencies of DCs and macrophages. Indeed, intradermal DNA immunization raises higher magnitude immune responses compared to intramuscular DNA vaccination [22]. Second, myocytes are not professional APCs and, in general, express low levels of major histocompatibility (MHC) Class I and co-stimulatory molecules, and this raises questions as to their effectiveness in priming CD8 T-cells. As a result, there has been a great deal of interest in dissecting the mechanisms involved and key cellular players in DNA immunization, and numerous studies have assessed the relative contribution of myocytes versus APCs in priming CTL responses after DNA immunization [35,36,37].The current paradigm is that the immunogenicity of DNA vaccines results from the cooperative action of three possible mechanisms of antigen presentation: (1) transfected myocytes presenting antigen to CD8 T-cells directly; (2) cross-priming by professional APCs, such as DCs, by the transfer of antigen from transfected myocytes; and (3) direct transfection of APCs [35,36]. Scenarios 2 and 3 seem to be dominant mechanisms among the three. Presentation of antigen to T-cells in Scenarios 2 and 3 would require the migration of antigen-loaded APCs from the site of vaccination to the T-cell zone of the draining lymph node for presentation to T-cells. This would be especially critical in the context of a DNA vaccine encoding cell-associated antigen. However, for secreted antigens, e.g., in the form of virus-like particles (VLPs), one can envision the diffusion of VLPs to the secondary lymphoid organs.The relative contribution of somatic cells versus APCs in antigen presentation would depend on the DNA formulation, method of administration, promoter driving antigen expression in DNA plasmid, the form of DNA expressed antigen (secreted, cell-associated) and other attributes of the host [37,38,39]. For instance, co-injection of adjuvants either expressed by plasmid DNA or in the vaccine formulation could improve the magnitude of immune responses by augmenting the recruitment of APCs to the site of vaccination and enhancing co-stimulatory capacity. Delivery of DNA by electroporation also induces significantly stronger immune responses, in part by increasing the transfection efficiency of myocytes (and, potentially, APCs) by about 100-fold [40]. An understanding of the mechanisms by which plasmid DNA primes immune responses is critical to the design of more immunogenic and effective DNA vaccines. As will be discussed in subsequent sections, multiple means have been employed to improve immune responses primed by DNA vaccines, and these studies have contributed to our understanding of key characteristics facilitating effective immune priming.The ability of naked DNA encoding antigen to induce T- and B-cell responses is remarkable in its simplicity and relative safety. DNA, however, is poorly immunogenic, and the magnitude of immune response is typically low [15,40]. Indeed, antigen load realized by DNA immunization limits immune responses. Therefore, there was a great deal of interest in employing DNA delivery methods that would overcome this limitation. Improved DNA delivery methods exist, such as transfection agent bupivacaine administered either in conjunction with or prior to DNA immunization [15,41], gene gun immunization to deliver DNA-coated gold micro-particles in the skin [9,17] and the use of electroporating agents to enhance DNA immunogenicity [40,42,43]. Because immunity conferred by DNA vaccines is durable and can be boosted by many heterologous vaccine vectors, strategies to boost immune responses primed by DNA have been remarkably successful.Letvin et al. evaluated the immunogenicity and efficacy of DNA vaccine with and without a protein boost in primates [16]. Monkeys received three shots of 1–2 mg plasmid DNA encoding HIV gp160 Env gene from the HXBc2 clone of HIV IIIB, followed by boosting with DNA + Env protein from the parental HIV IIIB strain. This vaccination regimen elicited high antibody titers against gp120, which played a role in complete protection from an intravenous challenge with a simian/human immunodeficiency virus (SHIV) HxB2 challenge, suggesting that this immunization regimen induced sterilizing immunity against HIV. While the challenge model was not rigorous due to the fact that SHIV HxB2 replicates at low levels and uses an Env gene derived from a neutralization-sensitive T-cell line culture laboratory strain of HIV, this immunization strategy provided the proof-of-concept that DNA-primed antibody responses could be augmented by a protein boost.Soon thereafter, Robinson et al. demonstrated that boosting with a live viral vector was superior to protein boosting [19]. The data showed a synergy between DNA prime and poxvirus vector boosting, which enhanced both antibody and cellular immune responses. A comparison of intradermal or gene gun delivered DNA prime followed by protein or fowlpox virus boost expressing Gag, Pol, Env, and Nef genes of SHIV-IIIb showed that intradermal DNA priming followed by fowlpox boosting protected 3/4 of animals from two consecutives homologous SHIV–IIIb challenges. Two thirds of protected animals contained a subsequent highly pathogenic SHIV 89.6P challenge. This study demonstrated that boosting DNA primed responses with a live, replication defective viral vector augments humoral immune responses. Indeed, a prior study by Fuller et al. demonstrated that single inoculation of recombinant vaccinia vector expressing gp160 resulted in a striking enhancement in gp120 specific IgG titers primed by DNA, compared to boosting with DNA [17]. These data indicated that recombinant vaccinia vectors could synergize with DNA to enhance immune responses.MVA is a highly attenuated virus derived from the vaccinia virus strain, Ankara [44]. Its ability to infect multiple cell types, including professional APCs, and inability to initiate a second round of replication in mammalian cells makes it a safe and immunogenic expression vector. More so, MVA is safe even in immune compromised individuals. As a vector, MVA has many desirable properties. It has a large capacity for added DNA and is a highly efficient expression system; the latter is due to the fact that in infected cells, the block in virus assembly occurs after DNA replication and protein synthesis [45]. Several studies showed that intramuscular injection of recombinant MVA expressing foreign genes elicits strong cellular and humoral responses [46,47].In 2001, Drs. Robinson and Amara reported that immune memory established by a DNA/MVA vaccine consisting of two DNA primes and a single MVA boost (DNA DNA MVA; DDM) vaccine regimen blunts acute viremia and rapidly controls set point viremia to below the level of detection and provides long-term viral control of a high-dose pathogenic SHIV 89.6P intrarectal infection in rhesus macaques [22]. The vaccine did not protect from mucosal SHIV 89.6P challenge, but reduced peak and set-point viremia, protected from CD4 T-cell loss and prolonged survival [48]. Subsequent studies comparing a Gag-Pol-Env vaccine to a Gag-Pol vaccine demonstrated better viral control, with the former indicating anti-Env binding Ab together with CD8 T-cells was necessary for containment of virus [49]. Follow up studies demonstrated that long-term viral control was associated with the maintenance of low breadth and low frequency IFNγ, IL-2 co-producing anti-viral T-cells [48]. In two animals, mutational escape of the virus in CD8 Gag epitopes led to ineffective immune control and re-emergence of virus, demonstrating the critical role of vaccine-induced CD8 T-cells in controlling virus [50]. Indeed, in a subsequent study, we documented that transient CD8 depletion in animals with <80 copies of virus per milliliter of plasma resulted in a greater than three-log-fold increase in viral titers, which was controlled after the reemergence of anti-viral CD8 T-cells [51]. Thus, memory responses engendered by a Gag-Pol-Env DDM vaccine resulted in the containment of acute viral infection by rapid recall of memory T- and B-cells. Vaccine induced protection was maintained in 22/24 animals for up to 200 weeks post challenge.In an effort to understand the influence of DNA prime on immune responses boosted by MVA and the contribution to protection, we compared immune responses and protection in DNA/MVA (DDM modality) and MVA-only (MMM modality) vaccines [52]. More recently, we also compared the immunogenicity of a DDMM modality with the MMM modality [53]. These studies demonstrated that MVA vaccination following a DNA prime elicits strong CD4 and CD8 T-cell responses. In contrast, priming with MVA (in the absence of DNA) elicits weak CD4 and CD8 T-cell responses, which are not boosted efficiently by repeated MVA immunizations. On the other hand, antibody responses are boosted in both modalities (in the presence or absence of DNA primes), resulting in strong T-cell and antibody responses in the DDMM group and moderate T-cell and strong antibody response in the MMM group.In the DDM study, we found the evidence for DNA vaccine priming qualitatively different immune responses compared to MMM vaccine. Post SHIV 89.6P challenge, the binding titers against HIV gp140 were comparable between the two groups. Interestingly, however, neutralizing Ab titers against homologous, as well as heterologous Envs were slower to emerge post challenge in the MMM regimen compared to the DDM regimen. Plasma viral load was comparable between vaccine groups, but examination of infected CD4 T-cells by intracellular p27 staining showed a higher frequency of infected cells in the MMM group. Furthermore, slower contraction of both cellular and humoral responses in the MMM group indicated the possibility for sequestered/persistent virus within tissues. Despite these differences, both vaccine regimens achieved similar viral control [52]. These promising data along with other seminal studies in the field shaped the concept of a DNA/MVA heterologous prime-boost vaccine regimen for HIV.In this section, we will review some of the molecular adjuvants used to augment immune responses induced by DNA, discuss known mechanisms of action and examine the efficacy of adjuvanted DNA vaccines in preclinical studies.Among the first experimentation of adjuvanted DNA was a mouse study showing the augmentation of T- and B-cell responses to hepatitis virus core protein (HCV) by co-immunizing with DNA plasmids encoding HCV antigen and either interleukin-2 (IL-2), interleukin-4 (IL-4) or granulocyte macrophage colony-stimulating factor (GM-CSF) [54]. All adjuvants enhanced HCV seroconversion; 80% of mice receiving adjuvanted HCV DNA made detectable anti-HCV antibodies compared to 40% of mice immunized with HCV DNA alone. Co-immunization with IL-2 DNA resulted in the strongest induction of CD4 T-cell responses as measured by ex vivo proliferation of splenocytes in response to rHCV nucleocapsid protein. Furthermore, spontaneous CTL activity in splenocytes was also strikingly enhanced by co-immunizing with IL-2. Effector T-cells derived from mice co-immunized with IL-2 secreted the highest levels of IL-2 and IFN-γ, while IL-4 adjuvanted effectors secreted IL-2 and IL-4, but not IFN-γ. These data indicate that adjuvants can alter not only the magnitude, but also the quality of DNA elicited immune responses.IL-2 has also shown promise as a DNA adjuvant in preclinical and clinical studies. Studies in non-human primates showed that IL-2 adjuvanted DNA significantly attenuated disease by decreasing set-point viremia in a SHIV 89.6P challenge model [55]. This effect was largely mediated by the induction of strong and durable cytolytic CD8 T-cell responses. In a clinical study, higher T-cell response rates were observed with IL-2 adjuvanted DNA (IL-2 administered as a fusion protein with immunoglobulin), when IL-2 was given 48 h post-vaccination compared to concurrent administration [56]. This could reflect a greater need for IL-2 during early T-cell responses relative to the requirement by innate cells.There have been a wide range of other molecular adjuvants tested to enhance the immunogenicity of DNA, including IL-12 and IL-15, transcription factors, such as interferon regulatory factors, growth factors and co-stimulatory molecules administered either as soluble forms or via expression vectors [40]. These data demonstrate that: (1) increasing local cytokine production at the site of antigen administration can enhance the immunogenicity of DNA; (2) the type of cytokine adjuvant (TH1 vs. TH2) can influence the quality of the CD4 helper response, which, in turn, can impact humoral responses; (3) chemokines and growth factors that induce the migration of APCs to the immunization site increase DNA immunogenicity; and (4) an ideal adjuvant augments both cellular and humoral immune responses. Thus, adjuvanting DNA vaccines provide a strategy to effectively manipulate the immune response based on the infectious agent and the host. In our laboratory, we have found promising results with two adjuvants, which are discussed in the following sections.The capacity of GM-CSF to recruit, induce expansion and stimulate the differentiation of APCs makes it highly desirable as an adjuvant for DNA immunizations [57,58]. An elegant series of experiments by Haddad et al. showed that the delivery of pGM-CSF at the same site of immunization was critical to enhance immunogenicity [59]. Using Plasmodium yoelii model in mice, they demonstrated that pGM-CSF enhanced immunogenicity and protection by inducing the local influx of APCs, and this effect was not duplicated by injecting pGM-CSF either intravenously or at a distant intramuscular site. Thus, local and paracrine effects of GM-CSF at the site of vaccination appear to be a critical factor in enhancing immunogenicity. This is a clinically important characteristic, as it reduces the chance for off-target effects and resulting toxicity.Plasmid encoded GM-CSF has been demonstrated to be an effective DNA adjuvant in several DNA/MVA immunization studies. First, we examined GM-CSF adjuvanted DNA in a DDM modality [60]. GM-CSF adjuvant was included only during the DNA primes. Immunogens were derived from the chimeric SHIV isolate, SHIV 89.6, and animals were challenged with a high dose SHIV 89.6P, seven months after the MVA boost. Interestingly, GM-CSF did not significantly enhance the titer of anti-Env antibody, but enhanced the quality of the antibody response. GM-CSF adjuvanted animals demonstrated an earlier peak in neutralizing Ab responses after infection, which resulted in four times lower viral titers at Week 2 post infection. Thus, the GM-CSF adjuvanted DDM vaccine regimen resulted in acute viral containment. In a follow up study using the DMMM vaccination modality, we found that adjuvanting DNA with GM-CSF enhanced the avidity of anti-Env binding antibody that was associated with the enhanced control of peak SHIV 89.6P viremia [61].In a third study, we tested the efficacy of GM-CSF adjuvanted DNA prime in a DDMM vaccination modality with SIV239 immunogens and repeat, moderate-dose intrarectal challenges with a relatively neutralization sensitive SIVsmE660 virus [62]. Adjuvanting DNA with GM-CSF resulted in the protection of 70% of the vaccinated animals compared to 25% and 0% in unadjuvanted and control groups. Correspondingly, an enhancement in the B-cell response was observed in the GM-CSF adjuvanted group with a higher avidity of anti-Env binding antibody, increased titers of neutralizing and Antibody-dependent cell mediated cytotoxicity (ADCC) activities and increased binding titers of anti-Env IgA in rectal mucosa. The avidity of anti-Env IgG for challenge Env was identified as a strong correlate of protection.In all three studies, protection conferred by DNA adjuvanted with GM-CSF appeared to be mediated by the effects of GM-CSF in the B-cell compartment, as the magnitude of T-cell responses were not significantly enhanced by GM-CSF. It is possible, however, that the quality of the B-cell helper CD4 responses may have been enhanced by GM-CSF, although this was not directly determined. Another possibility was that GM-CSF mediated the enhancement in DC maturation and function, especially that of myeloid DCs, which express receptors for GM-CSF, which could play a role in augmenting the quantity and quality of anti-Env B-cell response. The schematic in Figure 3 outlines the points of action of GM-CSF in adjuvanting immune responses. The GEO-D03 DNA vaccine that co-expresses HIV-1 clade B proteins, Gag, protease, RT, gp160 Env, Tat, Vpu and Rev, as non-infectious VLPs and human GM-CSF [63], has completed a human Phase I study in the U.S.The co-stimulatory role of CD40L on T-cells and B-cells makes it a highly desirable adjuvant. Pre-clinical studies in our laboratory have shown promising results with CD40L-adjuvanted DNA/MVA vaccines in rhesus macaques (work in progress). CD40L is a type II transmembrane protein of the tumor necrosis factor super family, expressed transiently by activated CD4 T-cells [64]. Its receptor, CD40, is constitutively expressed by numerous cell types, most notably APCs, such as immature DCs and B-cells. Engagement of CD40 on DCs by CD40L induces upregulation MHC-class II and co-stimulatory molecules, secretion of inflammatory cytokines, such as IL-12, and maturation and survival of DCs; factors central to the initiation of a cellular immune response. Ligation of CD40 on DCs is critical for DC licensing in order to prime antigen-specific CD8 T-cells. Several studies have shown that systemic administration of agonistic anti-CD40 antibody results in CD8 T-cell-mediated tumor eradication. These results support a model in which the induction of strong stimulatory signals by CD40L licenses DCs to prime CD8 T-cells [65,66]. Adjuvant activity of GM-CSF in modulating T- and B-cell responses. GM-CSF influences critical steps in antigen presentation, which could enhance vaccine-induced T-and B-cell responses. GM-CSF increases the recruitment of myeloid progenitor cells, induces their differentiation and maturation, resulting in enhanced Class II expression and antigen presentation. Enhanced migration of activated APCs to lymphoid tissue could enhance vaccine-induced T- and B-cell responses.CD40L also plays an important role in inducing antibody responses by promoting B-cell proliferation and immunoglobulin class switching [67]. In naive B-cells, ligation of CD40 together with B cell receptor (BCR) engagement induces clonal expansion and differentiation to short-lived plasmablasts or into rapidly proliferating germinal center (GC) B-cells [68]. Ligation of CD40 on GC B-cells via CD40L on TFH cells is necessary for affinity maturation and class switching to IgG. Continuous CD40 signaling together with input from the cytokines, IL-21 or IL-4, is required for GC B-cell proliferation, and removal of CD40L results in plasma cell differentiation [69].The stimulatory functions of CD40L in inducing cellular and humoral immune responses led to many strategies for using it as a vaccine adjuvant. However, systemic administration of CD40L is associated with hepatotoxicity and other side effects [70]. Therefore, local and transient expression of CD40L by means of vaccine expression vectors is more suitable for use of CD40L as a vaccine adjuvant. In mice, co-immunization of plasmid DNA expressing secreted HIV Gag together with an expression vector expressing soluble multimeric form of CD40L resulted in enhancement in CD8 cytolytic responses [71]. Gomez et al. demonstrated that soluble hexameric CD40L protein (sCD40L) administered during both DNA prime and NYVAC boosting potentiated both cellular and humoral responses in mice [72]. sCD40L increased the frequency of antigen-specific T-cells by two-fold in the DNA/MVA regimen and by two-fold in the DNA/NYVAC regimen. Adjuvanting with CD40L also decreased the dose of antigen required by 10-fold during the DNA prime. CD40L has also been shown to enhance the immunogenicity of ALVAC HIV vaccines in mice [73].In our laboratory, we designed CD40L-adjuvanted DNA/SIV vaccines to co-express macaque CD40L with the native trimeric form of SIV Env on the membrane of the transfected cell/VLP. This expression strategy promotes the multimerization of the ligand, which is critical for its adjuvant activity [74]. Figure 4 conceptualizes the mechanism by which CD40L expressed by soluble VLPs adjuvants T- and B-cell responses in vivo. We observed that CD40L adjuvant enhances protection from the acquisition of both neutralization-sensitive (SIVsmE660), as well as -resistant (SIVmac251) intrarectal repeat dose SIV challenges. In addition, the CD40L adjuvanted animals that became infected following SIVmac251 challenge showed better viral control, did not develop AIDS and showed enhanced survival. These results strongly support clinical testing of CD40L adjuvanted DNA/MVA HIV vaccine.Immune enhancement by CD40L adjuvanted DNA/MVA HIV vaccines. The schematic conceptualizes the mechanisms by which the co-expression of membrane CD40L on virus-like particles (VLPs; produced by DNA transfected cells or MVA infected cells) expressing HIV Env enhances cellular and humoral responses. DCs and B-cells both receive co-stimulatory signals from VLPs by the ligation of trimeric membrane-bound CD40L (on VLP) and CD40 (on DC or B-cells). VLPs bind to DCs via the interaction of gp120 (on VLP) and CD4 (on DC). This interaction is likely to jump-start CD8 T-cell responses by lowering the threshold for DC activation. Second, VLPs can also bind Env-specific B-cells via the interaction between gp120 (on VLP) and the B-cell receptor (surface Ig). Engagement of the BCR together with CD40 ligation results in the activation and differentiation of naive B-cells. In the germinal center, CD40 signaling enhances affinity maturation, class switch recombination and differentiation to memory B-cells. This model predicts that CD40L-delivered co-stimulation signals will enhance T- and B-cell responses to vaccine antigen. Schematic not drawn to scale; VLPs enlarged relative to immune cells for clarity.The immunogenicity and safety of plasmid DNA in animal studies provided the impetus for clinical testing of DNA encoding HIV proteins in humans. Over the years, a number of studies have been performed in HIV uninfected and infected volunteers; these studies have not only contributed to our understanding of immunogenicity and the safety of DNA vaccines in humans, but have also yielded insights into strategies to enhance DNA-induced immune responses. In this section, we review the immunogenicity of selected human studies that have employed DNA either alone or as a prime in a heterologous prime-boost vaccination regimen.Among the first clinical trials of DNA HIV vaccines was a therapeutic vaccine study by Calarota et al. determining the ability of plasmid DNA encoding HIV nef, rev and tat in raising CD8 T-cell responses in symptom-free HIV-infected patients [75]. The vaccine regimen consisted of three DNA immunizations over a six-month period. Interestingly, even a low dose of 100 µg of DNA induced CTL responses in eight out of nine immunized patients. Although responses were transient and no decrease in viral load occurred, the study provided the proof-of-principle that immunization with DNA could induce CD8 T-cell responses in HIV infected patients. The results suggested that a higher dose of DNA could yield more robust T-cell responses. This strategy was tested in a dose-escalation trial by MacGregor et al., where HIV-individuals were immunized with 100, 300 or 1,000 µg of DNA encoding Env and Rev of HIV-1 MN isolate [41]. Fairly robust T-cell responses were induced at a 1-mg DNA dose with an estimate of 0.1% of CD4s responding. While no antibody responses to Env were observed, the potential ability of DNA to prime a TH1 CD4 response indicated that combining DNA vaccine with vectors that express antigens at higher levels could enhance T- and B-cell immune responses. Studies by Mulligan et al. demonstrated that immunization with DNA expressing HIV antigens was safe and well tolerated in HIV negative individuals and DNA vaccination predominantly induced CD4 T-cell responses [24].Combining DNA prime with MVA boost engendered a striking T-cell response. Goonetilleke compared the immunogenicity of two DNA primes followed by a single MVA boost (DDM) to two MVA boosts (MM) [26]. While no T-cell responses were observed after the DNA primes, boosting with MVA significantly augmented T-cell responses primed by DNA, resulting in a striking increase in IFNγ+ antigen-specific T-cells. On the other hand, human volunteers in the MM modality showed no detectable increase in antigen-specific T-cells. Subsequently, a study by Goepfert et al. showed that two DNA primes followed by two MVA boosts induced vigorous, polyfunctional, broad and durable T-cell responses in 90% of vaccinated individuals [32]. Numerous clinical studies have shown that responses primed by DNA are significantly boosted using replication deficient viral vectors [27,76,77,78] and over multiple studies, the following unifying themes emerge.Foremost, DNA vaccination is safe and well tolerated in most individuals, and no clinical trial to-date has reported the integration of plasmid DNA with the host chromosome. Second, immune responses after DNA priming, despite being low to non-detectable, are induced and significantly amplified by subsequent heterologous boosts with viral vectors. A single DNA prime is insufficient, and >2 DNA primes do not increase immune responses over two DNA primes. Third, intramuscular DNA immunization primes TH1 CD4 T-cell responses; replication-deficient viral vectors can significantly enhance T- and B-cell responses primed by DNA. Fourth, combining a DNA prime and a viral vector-boost modality with protein immunization may result in the induction of potent and persistent T- and B-cell responses.Since the inception of DNA as a tool to induce immune responses two decades ago, continued advancements and innovations in this vaccine platform are paving the way for its clinical use. The strength of plasmid DNA as an immunogen lies in its ability to mimic natural infection; DNA transfected cells express viral proteins in “natural” conformations, which could result in the effective presentation of important epitopes to the immune system. DNA is excellent at priming both T- and B-cell responses and is especially good at inducing durable CD4 T-cell responses. While the magnitude of these responses is typically low, the use of adjuvanted DNA, electroporation and/or boosting with heterologous vectors represent attractive strategies to augment DNA-primed immune responses. The way forward lies in the understanding of the types of protective immune responses primed by DNA and identifying strategies to harness them to build better vaccines.The authors acknowledge the contribution of graphic artist Deepali Gupta at deepali.gupta@pentadesk.com for the generation of the figures.S.S.I. and R.R.A. contributed towards writing of the manuscript The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The editors of Vaccines would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2013:
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+              Abel, Kristina
+              De Val, Bernat Pérez
+              Isaguliants, Maria
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+              Acedo, L.
+              Dean, Gregg
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+              Ackerman, Margaret E.
+              Dempsey, Amanda F.
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+              Agadjanyan, Michael
+              Derdeyn, Cynthia A.
+              Knudsen, Maria L.
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+              Agwale, Simon M.
+              DiClemente, Ralph J.
+              Kobinger, Gary
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+              Beckett, Charmagne G.
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+              Biacchesi, Stéphane
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+              Lavail, Katherine Hart
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+              Long, Elisa F.
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+              Chang, Yung-Fu
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+              Chiappori, Alberto A.
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+              Clayton, Christine
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+              Metzger, Dennis W.
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+              Mitchison, Timothy J.
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+              Reisinger, Stephanie J.
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+              Roxanne, Clark
+              Swamy, Geeta
+               
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+              Rudolph, Carsten
+              Tomaras, Georgia D.
+               
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Med-MDPI/vaccines/vaccines-02-01-00181.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The attenuated live M. bovis Bacille-Calmette-Guérin (BCG) is still the sole vaccine used against tuberculosis, but confers only variable efficacy against adult pulmonary tuberculosis (TB). Though no clear explanation for this limited efficacy has been given, different hypotheses have been advanced, such as the waning of memory T-cell responses, a reduced antigenic repertoire and the inability to induce effective CD8+ T-cell responses, which are known to be essential for latent tuberculosis control. In this study, a new BCG-based vaccination protocol was studied, in which BCG was formulated in combination with a plasmid DNA vaccine. As BCG is routinely administered to neonates, we have evaluated a more realistic approach of a simultaneous intradermal coadministration of BCG with pDNA encoding the prototype antigen, PPE44. Strongly increased T- and B-cell responses were observed with this protocol in C57BL/6 mice when compared to the administration of only BCG or in combination with an empty pDNA vector, as measured by Th1-type spleen cell cytokine secretion, specific IgG antibodies, as well as specific IFN-γ producing/cytolytic-CD8+ T-cells. Moreover, we observed a bystander activation induced by the coding plasmid, resulting in increased immune responses against other non-plasmid encoded, but BCG-expressed, antigens. In all, these results provide a proof of concept for a new TB vaccine, based on a BCG-plasmid DNA combination.Live M. bovis Bacille-Calmette-Guérin (BCG) is currently one of the most widely used vaccines (annually, 120 million vaccine doses worldwide, with four billion vaccinated to date), and still, the only available vaccine against tuberculosis (TB). Indeed, BCG has been administered to neonates in the context of the Expanded Program on Immunization (EPI) since 1974, as it confers protection against miliary TB and TB meningitis in young children with a reduced risk of disease development of 50% [1]. Moreover, its extensive safety record in humans, heat stability and low production cost makes it particularly attractive. BCG presents, however, a highly variable and insufficient protection efficacy against pulmonary TB, the most common and contagious form of the disease [2]. In 2012, 8.6 million new TB cases and 1.3 million TB deaths (among 0.3 million HIV-associated TB deaths) were estimated [3]. A clear explanation for the poor protective efficacy of BCG against pulmonary TB is still not available, though a number of studies have addressed different hypotheses, such as the waning of the memory T-cell response [4], the variability of the administered BCG strains [5], the responses to a more limited antigenic repertoire as compared to the one of Mycobacterium tuberculosis (Mtb) [6] and the influence of pre-existing immunity to antigens shared with non-tuberculous mycobacteria [7]. Another possible explanation for this poor efficacy is linked to the limited ability of the BCG vaccine to induce effective CD8+ T-cell responses compared to Mtb, probably because its lack virulent RD-1 antigens CFP-10 and ESAT-6, which are known to facilitate mycobacteria translocation to the cytosol [8]. Even if their precise contribution in host defense against Mtb remains unclear, the role of CD8+ T-cell responses in controlling Mtb growth, especially during latency, is considered essential [9]. CD8+ T-cells exert an antimycobacterial function by producing cytolytic and microbicidal effector molecules and also contribute to the activation of infected macrophages through their production of the Th1-type cytokines, IFN-γ and TNF-α [10,11].In the quest for an efficient vaccine against TB, most strategies rely on the improvement of BCG by replacing it with other recombinant strains of attenuated mycobacteria or on prime-boost immunization protocols. The latter are based on attempts to enhance/boost previously BCG-induced immunity with subunit vaccines based on immunodominant antigens, either as viral-vectors, such as AdAg85A and MVA85A, or as recombinant fusion proteins from Mtb formulated in adjuvants promoting Th1-type responses [12].Plasmid DNA-based vaccines are another class of promising sub-unit vaccines that can be used in the context of novel TB vaccines to generate MHC Class I and II-restricted immune responses [13]. When combined in a classical BCG-prime DNA-boost vaccination strategy, numerous preclinical studies have shown an increase of BCG potency against Mtb [14,15,16,17]. However, in most of these reports, protective efficacy was only measured during a short-term post-infection period. Alternatively, other studies showed increased specific CD4+ and CD8+ T-cell responses by priming with DNA and boosting with BCG [18,19,20]. Moreover, we have previously shown in a murine long-term survival study that priming with an Ag85A-encoding plasmid DNA prior to BCG vaccination could significantly increase BCG-induced protective efficacy, while boosting with the same plasmid did not [21].Because of the wide clinical use of BCG in neonates, prior administration with a different vaccine is considered as an unrealistic goal. To our knowledge, there are no studies that attempted to directly mix a DNA vaccine with the live M. bovis BCG, instead of the classical prime-boost regimens. In the context of other diseases, some studies took advantage of the adjuvant properties of BCG, formulating DNA vaccines with BCG cell wall polysaccharide and/or nucleic acid fractions [22,23]. In these studies, enhanced cellular and humoral responses were induced, with the activation of TLR signaling pathways and Th1-type cytokine secretion. However, for optimal protective responses against Mtb, the viability of BCG is critical [24]. In this context, recent studies showed how live, but not killed, bacteria can induce significant expression of Type I IFNs and IL-1β from macrophages and dendritic cells, probably through bacterial mRNA sensing [25].Here, we have evaluated the vaccine potential of intradermal co-administration of live BCG with a prototype tuberculosis DNA vaccine encoding PPE44 (Rv2770c), a putative virulence factor of Mtb. By differential mRNA display, it was found that this protein is overexpressed in virulent Mtb H37Rv, as compared to the attenuated Mtb H37Ra strain, and weakly expressed by BCG [26]. Furthermore, ppe44 expression shows high quantitative variations in clinical isolates selected to represent the major phylogenetic lineages of the M. tuberculosis complex, and more specifically, strains of the Beijing type demonstrate high ppe44 expression. PPE44-specific immune responses can be detected in mice acutely, chronically and latently infected with Mtb; the antigen contains well-characterized MHC Class II- and Class I-restricted epitopes and confers protection against Mtb H37Rv when administered as a protein or pDNA vaccine [27].Female C57BL/6 mice aged 6–8 weeks were bred and kept at the WIV-ISP experimental animal facilities (Ukkel site, Brussels), complying with the Belgian legislation that transposes European Directive 2009/41/EC, repealing Directive 90/219/EC (EC, 2009).A schematic timeline representing the experimental protocol is depicted in Figure 1. Priming was performed in adult mice by the intradermal route (ID) with a fresh mix of 106 CFU of live M. bovis BCG (strain Pasteur GL2) and 100 µg of non-coding plasmid DNA (namely, pV1J.ns-tPA [28]), or plasmid DNA encoding PPE44 (namely, pV1J.ns-tPA-PPE44) or plasmid DNA encoding OVA (namely, pCI-OVA, a generous gift of Dr. Joseph Thalhamer and Dr. Richard Weiss [29]). Intradermal injections were carried out 1–2 cm distal from the tail base with a maximum total volume of 100 µL. After 3 weeks of resting, two further intradermal boosts at 3-week intervals were administered with the same 100 μg plasmid at the same site. Two additional control groups consisted of mice primed intradermally with only BCG (106 CFU/mouse) and of mice immunized intramuscularly (IM) three times with 100 µg coding plasmid at 3-week intervals. Intramuscular injections were performed in both quadriceps muscles with 2 × 50 μL, after anesthesia with ketamine/xylazine.BCG/pDNA co-vaccination protocol. Priming was performed in adult mice with a mix of 106 CFU live M. bovis BCG and 100 µg coding or non-coding plasmid DNA by the intradermal route in the tail (ID). After 3 weeks of resting, two further boosts at 3-week intervals were administered with 100 μg of plasmid at the same site. Two additional control groups consisted of one prime-immunized intradermally only with BCG (106 CFU/mouse) and another intramuscularly (IM) immunized three times only with 100 µg coding plasmid at 3-week intervals. Animals were sacrificed 3 weeks after the second DNA boost to analyze cellular immune responses in spleen and inguinal lymph nodes and humoral responses in serum.Throughout this study, the immunogenicity of BCG/pDNA co-vaccination was evaluated by ex vivo restimulation of splenocytes from vaccinated mice, sacrificed 3 weeks after the last immunization. Organs were removed aseptically and homogenized. Leucocytes (4 × 106 cells/mL) were cultivated at 37 °C in a humidified CO2 incubator in round-bottomed microwell plates in a volume of 200 μL RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 5 × 10−5 M 2-mercaptoethanol, 1% penicillin-streptomycin and 10% fetal calf serum (FCS). Six mice per group were individually analyzed for spleen cell cytokine productions specific to purified recombinant E. coli-expressed PPE44 (Rv2770c), Ag85A (Rv3804c) and PstS-3 (Rv0928) purified in our unit [30], culture filtrate from M. bovis BCG and Imject® Ovalbumin (Thermo Scientific, Waltham, MA, USA). The final concentration of the recombinant proteins and culture filtrate was 5 µg/mL. Furthermore, synthetic peptides at a final concentration of 10 µg/mL were used, namely a peptide spanning the predicted PPE44 Db restricted epitope (amino acids 257–265) and the PPE44 I-Ab restricted epitope (amino acids 1–20) [27]. Spleen cell culture supernatants were harvested after 24 h (for IL-2) and 72 h (for IFN-γ, TNF-α and IL-17A), when peak values of the respective cytokines can be measured. Supernatants were stored frozen at −20 °C until analysis. IFN-γ was detected using an enzyme-linked immunosorbent assay (ELISA) with purified rat anti-mouse IFN-γ as the capture antibody and biotin-labelled rat anti-mouse IFN-γ as the detection antibody (BD Pharmingen, Franklin Lakes, NJ, USA). Plates were revealed with O-phenylenediamine dihydrochloride substrate (OPD; Sigma-Aldrich, St. Louis, MO, USA); the reaction was stopped with 1 M H2SO4, and the optical density was read at 490 nm. IL-2, TNF-α and IL-17A were detected using commercial ELISA kits (eBioscience, San Diego, CA, USA).One week after the second DNA boost, mice were intravenously injected with carboxyfluorescein diacetate (CFSE)-labelled target cells. Target cells were prepared as described before [31]. Briefly, spleens from naive C57BL/6 mice were removed aseptically and homogenized, washed in RPMI, resuspended at 20 × 106 cells/mL in RPMI-10% FCS and incubated for 1 h at 37 °C/5% CO2, either alone or with 10 μg/mL of the Db-restricted peptide, PPE44257–265. After incubation, cells were washed, resuspended in RPMI at 20 × 106 cells/mL and labelled for 10 min at 37 °C in the dark with succinimidyl ester of carboxyfluorescein diacetate (CFSE) (Sigma-Aldrich) either at 2 μM (unpulsed cells; CFSElow) or 20 μM (peptide-pulsed cells; CFSEhigh). The staining reaction was stopped by the addition of an equal volume of RPMI supplemented with 10% of FCS; cells were then washed and resuspended in PBS at 100 × 106 cells/mL. To a 1:1 mix of CFSElow/CFSEhigh, 20 × 106 cells were adoptively transferred. Adoptively transferred mice were sacrificed 18 h later, spleens removed and homogenized and the erythrocytes depleted by lysis in ammonium chloride solution, and the cells were washed and resuspended in PBS for acquisition on a FACSCalibur cytofluorometer. To evaluate the percentage of specific lysis, the ratio of CFSEhigh/CFSElow in vaccinated mice was compared to the ratio in transferred naive control mice. For each experimental group, six mice were tested.Sera of vaccinated mice were collected at the time of immune analysis. Levels of specific antibodies in individual sera were determined by ELISA. For that purpose, 96-well plates were coated overnight with the specific recombinant protein at 500 ng/well in borate buffer. Adsorption sites were saturated during 1 h with 5% skimmed milk in PBS. Serial two-fold dilutions of sera were added for 2 h. Then, a peroxidase-labelled secondary rat anti-murine IgG (LO-MK-1 for IgG, purchased at Experimental Immunology Unit, Université Catholique de Louvain, Brussels, Belgium) was added for 1.5 h. Plates were developed following the same protocol used for cytokine detection, as previously described.GraphPad Prism software was used to perform statistical analysis. One-way ANOVA and Tukey’s post-test were performed to demonstrate statistical differences.The production of IFN-γ, IL-2, TNF-α and IL-17A was evaluated in the culture supernatants of spleen cells isolated from vaccinated and control groups after restimulation with recombinant PPE44 or the PPE441–20 peptide. We have previously described that PPE441–20 is an immunodominant T CD4+ epitope recognized after BCG immunization, as well as after DNA vaccination [27]. As shown in Figure 2, PPE44-specific IFN-γ and IL-2 levels measured in the spleen of BCG/pDNA-PPE44 (ID) co-vaccinated C57BL/6 mice were greatly increased and significantly higher than the levels measured in mice of the control groups immunized with BCG alone or with a combination of BCG with a non-coding, empty pDNA vector. The levels induced by the BCG/pDNA-PPE44 combination were also significantly higher than the levels induced after three intramuscular pDNA-PPE44 administrations. TNF-α could be detected in cultures stimulated with recombinant PPE44 protein, but not with PPE441–20 peptide. In addition, increased TNF-α levels were observed in BCG/pDNA-PPE44 immunized mice compared to the levels achieved after vaccination with BCG, BCG combined to non-coding pDNA or intramuscular vaccination with pDNA-PPE44. Finally, no significant differences in specific IL-17A levels were observed between the different experimental groups.Specific Th1-type cytokine production induced by BCG/pDNA co-vaccination. IFN-γ, IL-2, TNF-α and IL-17A levels (pg/mL) were measured by ELISA in 24 h (IL-2) or 72 h (IFN-γ, TNF-α and IL-17A) culture supernatants of splenocytes from vaccinated C57BL/6 mice, after restimulation with the PPE44 MHC Class II-restricted synthetic peptide spanning amino acids 1–20 (PPE44[1–20]) or the recombinant PPE44 protein (rPPE44). RPMI, non-stimulated cells. ID, intradermally. IM, intramuscularly. Data are presented as the mean ± standard error of the mean and representative of two independent experiments, including six mice per group. * p < 0.05; ** p < 0.005.Specific IFN-γ was detected in spleen cell culture supernatants from mice vaccinated intramuscularly with pDNA-PPE44 and mice co-vaccinated with BCG/pDNA-PPE44 after stimulation with the synthetic PPE44257–265 peptide (Figure 3A). We have previously reported that PPE44257–265 spans a predicted Db-restricted epitope and on the in vivo cytotoxic T lymphocyte (CTL) activity against PPE44257–265 in C57BL/6 mice vaccinated with pDNA encoding PPE44 [27]. Hence, we verified whether BCG/pDNA-PPE44 co-vaccination could also induce effective CD8+ T-cells by assessing their in vivo cytolytic activity. PPE44257–265 peptide-pulsed, CFSE-labelled spleen cells from naive C57BL/6 mice were adoptively transferred as targets in vaccinated mice. As expected, a specific lysis of about 23% was measured by flow cytometry in mice vaccinated three times intramuscularly with pDNA-PPE44. In addition, a similar level of specific lysis was observed in mice intradermally coimmunized with BCG/pDNA-PPE44 (Figure 3B). In contrast, no PPE44-specific CTL activity could be detected in mice vaccinated with BCG alone or in mice vaccinated with BCG/empty vector, confirming the inability of the attenuated M. bovis BCG vaccine to induce detectable CD8+ T-cell responses.Specific CD8+ T-cell responses induced by BCG/pDNA co-vaccination. (A) IFN-γ levels (pg/mL) were measured by ELISA in 72 h culture supernatant of splenocytes cultured with the PPE44 MHC Class I-restricted short peptide spanning amino acids 257–265 (PPE44[257–265]). RPMI, non-stimulated cells. Data are presented as the mean ± standard error of the mean and representative of two independent experiments, including six mice per group. * p < 0.05. (B) In vivo-specific cytotoxic T lymphocyte (CTL) activity. Unpulsed splenocytes (carboxyfluorescein diacetate (CFSE)low) and peptide-pulsed splenocytes (CFSEhigh) from naive mice were intravenously transferred to the immunized mice 18 h before flow cytometry analysis. Splenocytes were pulsed with the PPE44 MHC Class I-restrictedshort peptide spanning amino acids 257–265. The percentages of specific lysis in the immunized mice were compared to the ratio in transferred naive control mice. Data represent the mean ± standard error of the mean (n = 3–6/group). ** p < 0.005. ID, intradermally. IM, intramuscularly.In order to find out whether the BCG/pDNA-PPE44 combination could affect the responses specific to antigens expressed by BCG other than PPE44, spleen cells were stimulated with two other BCG antigens, recombinant PstS-3 (Rv0928) and recombinant Ag85A (Rv3804c), or BCG culture filtrate. PstS-3 is one of the three putative phosphate transport receptors of Mtb, known to be an immunodominant B- and T-cell antigen of M. bovis BCG in H-2b haplotype mice [32]. PstS-3 is a potential tuberculosis vaccine candidate, and we have previously shown that mice vaccinated with PstS-3 DNA demonstrated significant and sustained reduction in bacterial CFU numbers in spleen and lungs for three months after Mtb challenge [33]. As shown in Figure 4A, the combination of BCG with pDNA-PPE44 increased the IFN-γ response to recombinant PstS-3 antigen about two-fold. IFN-γ responses to another immunodominant BCG antigen with a very promising vaccine potential, the mycolyl-transferase Ag85A, were even more strongly increased by the BCG/pDNA-PPE44 combination, and responses against BCG culture filtrate (in which Ag85A is one of the most abundant proteins) were equally augmented. Mice co-vaccinated with BCG and control DNA showed the same response as mice vaccinated with BCG alone.Mycobacteria-specific IFN-γ responses against non plasmid-encoded, but BCG-expressed, antigens after BCG/pDNA co-vaccination. IFN-γ (pg/mL) was measured by ELISA in 72 h culture supernatant of spleen cells cultured with mycobacterial recombinant proteins PstS-3 and Ag85A, BCG culture filtrate (BCG CF) and with recombinant PPE44 (A) or purified ovalbumin (B). Data are presented as the mean ± standard error of the mean and representative of two independent experiments, including six mice per group. * p < 0.05 in comparison with BCG (ID) and BCG/control pDNA (ID) groups.In order to find out more about the bystander activation observed in the BCG/pDNA-PPE44 group, mice were vaccinated with a combination of BCG and a plasmid DNA encoding an unrelated, non-mycobacterial antigen, i.e., ovalbumin (OVA). As shown in Figure 4B, BCG co-vaccination with pDNA-OVA also induced increased IFN-γ responses specifically to the three BCG-encoded antigens, PstS-3, PPE44 and Ag85A, and to BCG culture filtrate. IFN-γ levels were two-fold higher against recombinant PstS-3, eight-fold higher against PPE44 and about four-fold higher against Ag85A in mice that had been vaccinated with the BCG/pDNA-OVA combo, as compared to mice only vaccinated with BCG or BCG/control pDNA. As expected, IFN-γ responses to purified OVA were only detected in mice vaccinated with pDNA-OVA or with the BCG/pDNA-OVA combination, but not in naive or BCG vaccinated mice. IFN-γ levels in the supernatant of unstimulated spleen cells were highest in mice vaccinated three times with pDNA-OVA (in blue), which may have contributed to some extent to the mycobacteria-specific IFN-γ responses to PstS-3, PPE44 and Ag85A observed in this group. Interestingly, OVA-specific IFN-γ titers in the BCG/pDNA-OVA animals were higher than in the mice vaccinated only with pDNA-OVA, although both groups had received the same plasmid dose three times.Total anti-PPE44 and anti-OVA IgG levels were measured in sera (Figure 5). BCG/coding-pDNA coimmunization increased the specific antibody levels to the corresponding antigen as compared to the levels observed in both BCG control groups and to levels observed after intramuscular vaccination with coding pDNA. Anti-PPE44 end-point titers in BCG/pDNA-PPE44 coimmunized mice were about thirty-fold higher than in BCG or BCG/control pDNA vaccinated mice and about nine-fold higher than in pDNA-PPE44 vaccinated mice. Anti-OVA end-point titers in coimmunized mice were about twenty-fold and nine-fold higher when compared, respectively, to BCG or BCG/control pDNA vaccinated mice and to coding pDNA vaccinated mice. Anti-PPE44 and anti-OVA end-point titers measured in BCG vaccinated mice and BCG/control pDNA co-vaccinated mice were not significantly different from the titers measured in naive mice. As for the IFN-γ responses, antibody titers in the BCG/pDNA coimmunized animals were higher than in the mice vaccinated with coding plasmid.Specific antibody production induced by BCG/pDNA co-vaccination. Anti-PPE44 (A) and anti-OVA (B) IgG-isotype antibodies were measured by ELISA in serum harvested three weeks after the last immunization. Optical density levels of serial two-fold serum dilutions are presented as the mean ± standard error of the mean and representative of two independent experiments, including five to six mice per group.BCG improvement strategies are needed and under analysis for the development of a more efficient tuberculosis vaccine. Th1-type responses are essential in anti-tuberculosis protection, but CD8+ T-cell responses also play a very important role, particularly in the protection against reactivation of a latent tuberculosis infection [9,34,35]. In this study, we have shown that the weak potential of BCG to trigger MHC Class I-restricted immune responses can be overcome by coimmunization with a plasmid DNA vaccine. Induction of strong CD8+ T-cell responses is perhaps the most important hallmark of DNA vaccines, making them particularly attractive as vaccines against viral and other intracellular pathogens [12].We have described a new BCG/pDNA co-vaccination protocol that is effective in inducing significantly higher CD4+ Th1 and IFN-γ producing/cytolytic CD8+ T-cell responses in C57BL/6 mice as compared to animals immunized with BCG alone or in a combination with a non-coding pDNA vector. As a prototype antigen, we used the PPE44 (Rv2770c) antigen, a putative virulence factor of Mtb, against which specific immune responses can be detected in mice acutely, chronically and latently infected [27]. The PPE protein family of Mtb, characterized by Pro-Pro-Glu motifs near the N-terminus of their sequence, includes 69 proteins rich in glycine and together with the PE (Pro-Glu) protein family, represents approximately 10% of the coding genes scattered throughout the Mtb genome. Proteins of this family have been found to be T-cell immunodominant antigens in animal models and humans, eliciting very high cellular responses and making them of potential interest for vaccine development, e.g., PPE18 (Rv1196), found in the fusion polyprotein of the TB subunit vaccine candidate, Mtb72F [36,37], and PPE14 (Rv0915c) [38]. PPE18 and PPE14 share 14 identical amino acids in their NH2-terminal 20-amino acid sequence with PPE44, a region which encodes the I-Ab restricted Th1 epitope, against which strongly increased responses were found in the BCG/pDNA-PPE44 combination. It is possible that cross-reactive responses against PPE14 and PPE18 are induced by the BCG/pDNA-PPE44 immunization (which could increase the protective efficacy), but more work is needed to formally demonstrate this.An antigen dose effect could be partially responsible for the increased PPE44-specific responses, as this protein is relatively weakly expressed in BCG [39]. Antigen load contributes to the development of specific CD8+ T-cell responses, in terms of kinetics and magnitude [40]. Furthermore, the differentiation of long-lived memory CD8+ T-cells in vivo was reported to correlate with high antigen dose and the stability of T-cell-DC contacts [41]. In this context, it is worth mentioning that a two hundred-fold higher dose of M. bovis BCG than of Mtb is needed to induce similar CD8+ T-cell responses [8]. On the other hand, highly increased immune responses can also be detected in mice and pigs immunized with a combination of BCG and pDNA encoding the mycolyl-transferase Ag85A, which is among the most strongly expressed proteins in the culture filtrate of the BCG vaccine [42].A strong bystander activation was observed with the BCG/pDNA-PPE44 protocol, resulting in higher responses also to other BCG-expressed antigens. Furthermore, this bystander activation was observed in mice vaccinated with the BCG/pDNA-OVA combination, but not in mice vaccinated with the BCG/control pDNA, suggesting that antigen-specific cytokine responses were elicited in vivo by the booster vaccinations with coding pDNAs, which created an immunostimulatory Th1 cytokine environment that amplified responses induced by the BCG priming to these antigens. Bystander activation of T-cells has been best described for CD8+ T-cells, and in this compartment, the release of IFN and IFN inducers leads to the production of IL-15, which mediates the proliferation of CD8+ T-cells [43,44]. Activated CD4+ T-cells of unrelated specificity can also undergo bystander activation, probably involving IL-2 and IL-7 as potential cytokine mediators [45]. It is tantalizing to speculate that similar bystander activation could also be stimulated in other combination protocols using plasmid DNA boosters and live, attenuated bacterial or viral vaccines.Besides the bystander activation caused by the plasmid DNA boosters, BCG also exerted adjuvant effects on the plasmid DNA-induced responses. Thus, OVA-specific IFN-γ titers were higher in BCG/pDNA-OVA than in pDNA-OVA vaccinated mice, although both groups had received the same plasmid dose three times. Differences in administration route, i.e., intradermal versus intramuscular, are unlikely to play a role, as we have found comparable immune responses to the pDNA administered by either route [46]. The BCG cell wall is composed of several pathogen associated molecular patterns (PAMPs) that are able to interact with different pathogen recognition receptors (PRRs), such as TLR2, TLR4 and TLR9, resulting in the induction of innate immune responses [47]. Plasmid DNA vaccines also have intrinsic adjuvant properties, because they can activate TLR9 through their bacterial CpG motifs and TBK1-dependent innate immune signaling pathways through their double-stranded structure [48]. Therefore, it is tempting to speculate that there might be synergies on TLR-induced innate immune responses provided by BCG and pDNA, knowing, for example, that TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mtb [49]. Moreover, TLR9 signaling seems to be critical for the induction of effective CD8+ T-cell responses through cross-priming following the initial pDNA immunization [50,51]. On the other hand, CD4+ Th1 responses and T CD8+ responses were not different in animals vaccinated with BCG alone or in combination with non-coding pDNA, indicating that the adjuvant effects of the vector backbone were minimal [28].BCG is routinely administered by the intradermal route, and we have confirmed here that this route can also be used for plasmid DNA vaccines [52]. This intradermal route is particularly attractive, as skin-associated lymphoid tissues contain a wide variety of specialized cells able to enhance immune responses, such as dermal (langerin expressing) dendritic cells and macrophages, keratinocytes and Langerhans cells [53]. In conclusion, this study has given a proof of principle that the routine BCG immunization protocol could be improved by formulating BCG with pDNA vaccines encoding protective Mtb antigens. The prototype antigen encoded by the pDNA vaccine used in this study could be replaced by other antigens or combination of antigens, such as the latency-associated antigens, against which only poor responses are induced by BCG vaccination [6]. Another approach could be to potentiate CD8+ T-cell responses by using a plasmid DNA vaccine encoding a fusion of immunodominant MHC Class I-restricted epitopes. Although Th1 type immune responses are considered to be essential, the precise correlates of protection against TB are still not fully defined, and excepting studies in non-human primates, there is no proper animal model that can reproduce the reactivation of latent tuberculosis. Long-term survival experiments in mice may give some information, and these experiments are actually in progress.The authors wish to thank Joseph Thalhamer and Richard Weiss (Salzburg University, Salzburg, Austria) for their advice and for providing the ovalbumin-encoding plasmid.This work was partially supported by the 7th EU Framework Program (NEWTBVAC Project).Nicolas Bruffaerts performed all the experiments with the technical help of Fabienne Jurion; Olivier Denis helped with the CTL assays; Nicolas Bruffaerts, Marta Romano and Kris Huygen wrote the manuscript.The authors declare no conflict of interest.

Med-MDPI/vaccines/vaccines-02-02-00196.txt

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.DNA vaccination has once again became elevated to the forefront of efforts aimed at developing vaccines against challenging infectious diseases including HIV/AIDS, emerging strains of influenza as well as SARS. As well, it has a reemerging role as a delivery method for tumor immunotherapy [1,2,3,4,5,6,7,8]. While “first-generation” DNA vaccines were poorly immunogenic, recent technological advances have dramatically improved their ability to drive immunity, including cellular based responses, in preclinical immunogenicity and efficacy studies [9,10,11,12,13,14] as well as in clinical trials [15,16,17,18]. The transfection rate of plasmid DNA and subsequent expression of their encoded antigens (Ages) are significantly enhanced when highly-concentrated plasmid vaccine formulations are delivered through in vivo electroporation (EP), a technology using brief square-wave electric pulses at the vaccination site to facilitate entry and expression of DNA plasmids into transiently permeabilized cells resulting in improved immunogenicity and efficacy of the vaccines [19]. In theory, a cocktail of plasmids could be assembled for directing a highly-specialized immune response against any number of variable antigens (Ag), which, in turn, could induce a more robust and efficacious immune response. In addition, “consensus-engineering” of the Ag amino acid sequences has been effectively used to help bias vaccine-induced immunity towards particular divergent, circulating, or virulent strains such as enhancing protection among divergent strains of HIV and influenza virus [20,21]. Due, in part, to these technological developments, immunization regimens including these “enhanced” DNA (E-DNA) vaccines are extremely customizable and highly versatile.Immunity can be further directed by co-delivery of the vaccine with plasmid-based molecular adjuvants encoding species-specific immunomodulatory proteins. These have typically included cytokines, chemokines, and surface expressed co-stimulatory molecules [18,22,23,24,25,26,27,28,29,30,31,32,33,34]. Such a gene adjuvant approach substantially enhanced immune potency in numerous vaccine studies [16,18,29,35,36]. As a candidate for molecular adjuvant development, transcription factors regulate the gene expression of numerous inflammatory factors and promote activation and maturation of the adaptive immune response [37,38,39]. An established pro-inflammatory mediator is the NF-kappa B protein complex which regulates the expression of cytokines (TNF-α, IL-1β, IL-6, IL-2, etc.), induces DC maturation (characterized by upregulation of CD80, CD86 and CD40), and promotes cell survival by the regulation of Bcl-XL and IAPs, and protecting against TNF-α induced cell death [40,41,42]. This transcription factor consists of five members including p65 (RelA), c-Rel, RelB, p50 (NF-κB1) and p52 (NF-κB2) [42]. Importantly, RelA is a vital component in inflammation and cell survival, possesses transcriptional activating capabilities, and can potently activate kappa B-dependent transcription. Upregulation of this subunit may regulate the gene expression of multiple inflammatory and survival factors that may lead to improved adaptive immunity [43,44,45,46]. An additional candidate for development as a molecular adjuvant is T-bet, a T helper 1 (Th1)-specific transcription factor, which would ideally help to promote the induction of Th1-type immunity. This T-box family member regulates lineage commitment in CD4 Th cells by directly activating transcription of the IFN-γ gene. It also exhibits the property of redirecting committed Th 2 populations to a Th1 phenotype [47,48]. The importance of T-bet in Th1 immunity has been most clearly illustrated and reported in cases where CD4+ T cells lacking T-bet are severely impaired in their ability to produce IFN-γ, yet secrete elevated levels of the opposing Th2 subset cytokines, IL-4 and IL-5. Co-expression of this protein along with a vaccine against tuberculosis has been demonstrated to increase IgG2a antibody (Ab) responses as well as the production of IFN-γ and IL-2 [47]. Thus, a T-bet-expressing molecular adjuvant delivered by E-DNA vaccination would ideally enhance the magnitude and type of immunity induced by immunization. Together, RelA and T-bet present attractive candidates, as molecular adjuvants, for the enhancement of immunity following E-DNA vaccination.In this study, we investigated the ability of molecular adjuvants expressing transcription factors RelA or T-bet, as developed herein, to enhance in mice, immunity of an E-DNA vaccine encoding either HIV-1 Env or Gag. Their potential ability to modify immunity was then assessed. Co-administration of either pRelA or pTbet in conjunction with the pEnv or pGag vaccine significantly increased T cell immunity, as measured by INF-γ production by ELISpot and proliferation. As well, B-cell/antibody levels were enhanced as indicated by an increase in B-cell numbers as well as antigen specific antibody titers. Consistent with these findings, the total amount of antigen specific IgG in serum was increased following the co-administration of plasmids expressing the transcription factors. This study builds on recent successes in demonstrating the potency of E-DNA vaccination and suggests that transcription factors may serve as an effective adjuvant to increase vaccine-induced immunity.The pRelA plasmid DNA constructs encode the full-length mouse NF-κB subunit p65/RelA (GenBank #TF65_MOUSE) and Type-1 transactivator T-bet (GenBank #TBX21_MOUSE), respectively. In addition, the Ig heavy chain epsilon-1 signal peptide (GenBank#AAB59424) was fused to the N-terminus of each sequence, replacing the N-terminal methionine, which facilitates expression [11,49]. Each gene was genetically optimized for expression in mice, including codon- and RNA-optimization, among other proprietary modifications for enhancing protein expression (GenScript, Piscataway, NJ, USA). The optimized genes were then sub-cloned into modified pVax1 mammalian expression vectors (Invitrogen, Carlsbad, CA, USA) under the control of the cytomegalovirus immediate-early (CMV) promoter. These reagents were then used as the molecular adjuvants in this study. The pGag [50] and pEnv [51] plasmids, expressing the HIV-1 proteins Gag and Env respectively, have been previously described.Human Embryonic Kidney (HEK) 293T cells were maintained in Dulbecco’s modified Eagle medium (Life Technologies, Grand Island, NY, USA), supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 IU of penicillin per mL, 100 μg of streptomycin per mL and 2 mM l-glutamine [9]. Briefly, cells were transfected using TurboFection 8.0 (OriGene, Rockville, MD, USA) per the manufacturer’s protocol and subsequently incubated for 24–48 h. Cells were harvested with ice cold PBS, centrifuged and washed, and then pelleted for Western immunoblot analysis [52]. Nuclear extracts (107 cells) were made according to the method of Muthumani et al. [52]. The nuclear proteins from the transfected cells were then dissolved in 20 mM Hepes (pH 7.9) containing 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF and a cocktail of protease inhibitors (Promega Corp, Madison, WI, USA). The protein concentration of each extract was measured by the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA), and extracts were stored in aliquots at −70 °C until used. Standard western blotting analysis was performed. Cells were treated with protein lysis buffer (0.01 M Tris-HCl buffer pH 7.4, containing 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Protease Inhibitor Cocktail tablets; Roche, Indianapolis, IN, USA). Proteins in lysates were then separated using 12% SDS-PAGE [53]. Protein-specific detection antibodies for RelA and T-bet (Cell Signaling Technology, Danvers, MA, USA) were incubated with the blots and expression visualized using the enhanced chemiluminescence (ECL) Western blot detection system (GE Healthcare, Piscataway, NJ, USA).A RelA/p65 expressing vector, which co-expresses luciferase (pNF-κB-Luc) was used to confirm the functionality of RelA/p65, which is necessary before it being used the “adjuvanted” vaccine study. The luciferase reporter assay was performed as described previously [52,54,55]. Briefly, 293T cells (105 cells/well) were seeded in a 96-well plate for 24 h. The cells were then transfected with the RelA/p65 Luc expressing plasmid followed by incubation for 6 h. After incubation, the cell culture medium was removed and replaced with fresh medium. Two days post transfection cells were treated with 20 ng/mL of recombinant TNF-α for 6 h followed by measurement of luciferase activity by using Microlumat plus luminometer (LUMAT LB9501, Berthold Technologies, Oak Ridge, TN, USA). For confirmation of pT-bet function, the production of IFN-γ from pT-bet transfected CD4+ T cells was measured. The impetus for measurement of IFN-γ is based on previously published studies that demonstrated a direct correlation between T-bet and IFN-γ production [56]. Briefly in this analysis naïve CD4+ T cells, isolated from the spleens of Balb/C mice, were purified using a CD4+ T cell isolation kit (Miltenyibiotec, San Diego, CA, USA). These cells were maintained in RPMI media supplemented with 10% FBS, 100 U/mL penicillin and 200 µg/mL streptomycin and subsequently transfected with pT-bet or pVax1 as a negative control. Two days post-transfection, cells were stimulated overnight with anti-CD3 plus anti-CD28 Abs (1 µg/mL). IFN-γ levels in the supernatants collected from the cultured CD4+ T cells were subsequently measured by a standard ELISA [36].Adult female BALB/cJ (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All animal experimentation was conducted according to University of Pennsylvania (UPENN) IACUC approved protocols and performed in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of NIH. UPENN complies with NIH policy as stated in the Animal Welfare Act, and all other applicable federal, state and local laws. Mice were immunized intramuscularly (i.m.) by needle injection into the left-thigh quadriceps muscle with 25 µg of plasmid resuspended in 25 µL of PBS. Vaccinations were immediately followed by EP, at the same site, and repeated at a two-week interval. For EP mediate delivery, a three-pronged CELLECTRA® adaptive constant current Minimally Invasive Device (MID) was used, supplied by Inovio Pharmaceuticals, Inc. (Blue Bell, PA, USA). Specifically, square-wave pulses were delivered through a triangular 3-electrode array (inserted 2 mm intradermally) consisting of 26-gauge solid stainless steel electrodes and two constant-current pulses of 0.1 Amps were delivered for 52 msec/pulse separated by a 1 s delay. During the vaccination/molecular adjuvant administration regimen, and through the termination for the study, all mice were monitored every 3 days for the development of potential adverse effects.Spleens were harvested 7–8 days following the third immunization as previously described [12]. Briefly, spleens were placed in RPMI 1640 medium (Mediatech, Manassas, VA, USA) supplemented with 10% FBS, 1X Antibiotic-Antimycotic (Life Technologies, Grand Island, NY, USA), and 1× β-ME (Life Technologies, Grand Island, NY, USA). Splenocytes were isolated by mechanical disruption of the spleen using a Stomacher machine (Seward Laboratory Systems, Bohemia, NY, USA), and the resulting product was filtered using a 40 μm cell strainer (BD Biosciences, San Jose, CA, USA). The cells were then treated for 5 min with ACK lysis buffer (Lonza, Walkersville, MD, USA) for lysis of RBCs, washed in PBS, and then resuspended in RPMI medium for use in the ELISPOT assay. CD4 naïve T cells were purified from the spleens using a naïve CD4+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). These cells were maintained in RPMI medium supplemented with 10% FBS, 100 U/mL penicillin, 200 μg/mL streptomycin, and stimulated with anti-CD3 plus anti-CD28 (1 μg/mL each). Upon stimulation with anti-CD3 plus anti-CD28 antibodies, cytokine production levels in the culture supernatants of cultured cells were examined by enzyme-linked immunosorbent assay (ELISA) as described previously [3,13].The standard IFN-γ ELISPOT assay used in this study has been previously described [9,11,12]. Briefly, 96-well plates (Millipore, Billerica, MA, USA) were coated with anti-mouse IFN-γ capture antibody and incubated for 24 h at 4 °C (R&D Systems, Minneapolis, MN, USA). The following day, plates were washed with PBS and then incubated for 2 h with blocking buffer (1% BSA and 5% sucrose in PBS). CD4+ or CD8+ T cells (5 × 105 cells/well plated in triplicate) were MACS-purified (Miltenyibiotec, San Diego, CA, USA) from splenocytes and subsequently stimulated with HIV-1 Gag (consensus subtype B) or Env (subtype B (MN)) peptides (15-mers overlapping by 11 amino acids, spanning the lengths of their respective protein (NIH AIDS Reagent Program, Bethesda, MD, USA). After 18–24 h of stimulation overnight at 37 °C in 5% CO2, the plates were washed in PBS and subsequently incubated for an additional 24 h at 4 °C with biotinylated anti-mouse IFN-γ monoclonal antibody (mAb) purchased from R&D Systems (Minneapolis, MN, USA). The plates were then washed again in PBS, and streptavidin-alkaline phosphatase (MabTech, Nacka Strand, Sweden) was added to each well and incubated for 2 h at RT. Lastly, the plates were washed again in PBS followed by incubation with BCIP/NBT Plus substrate (MabTech, Cincinnati, OH, USA) for 5–30 min. Upon completion of spot development based on visual inspection, the plate was rinsed with distilled water and then dried overnight at RT. Spots were enumerated using an automated ELISPOT reader (Cellular Technology, Shaker Heights, OH, USA).Proliferative responses were measured in vitro by incubating 105 splenocytes in culture medium per well in 96-well U-bottom plates in the presence of serial dilutions (5, 1, and 0.1 μg/mL) of recombinant HIV-1 IIIB pr55 (Gag) (NIH AIDS Reagent Program, Bethesda, MD) or HIV-1 MN IIIB gp160 (Env) (Protein Sciences, Meriden, CT, USA) and incubated at 37 °C with 5% CO2. Incorporation of tritiated (3H)-thymidine was measured by pulsing with 1 μCi/well of (3H)-thymidine during a 0–24 h time period as described previously [57]. The plate was then harvested and incorporated 3H-thymidine was measured in a Beta plate reader (Wallac, Waltham, MA, USA). The proliferative response is expressed as a stimulation index (SI), calculated by dividing the mean cpm (counts per minute) of Ag-stimulated wells by the mean cpm of non-stimulated wells.Sera from vaccinated mice harvested 7 days following the third vaccination were tested for antibody responses against recombinant HIV-1 Env (NIH AIDS Reagent Program) by ELISA. Briefly, 96-well ELISA plates were coated with recombinant HIV-1 Env protein (Protein Sciences) and incubated at 4 °C and washed subsequently with PBS and 0.1% Tween-20. Plates were then blocked for 2 h with PBS and 0.2% Tween-20. After removal of the blocking solution, 100 μL of the pre-diluted (1:50, 1:100, 1:500, 1:1000) mouse serum was added and incubated for 1 h. Plates were then washed four times and incubated with a peroxidase-coupled anti-mouse IgG mAb (Sigma-Aldrich, St. Louis, MO, USA). Lastly, plates were washed again followed by addition of 200 μL of substrate solution (R&D Systems, Minneapolis, MN, USA) per well. The optical density at (OD405 nm) was subsequently measured after a 15 min incubation. All assays were performed in triplicate.Muscle tissues (i.e., from the site of injection/vaccination) were removed aseptically, rinsed in Hanks’ balanced salt solution (Life Technologies, Grand Island, NY, USA), minced into approximately 1 × 2-mm squares, and digested in 20 mL of collagenase A (1 mg/mL, Life Technologies, Grand Island, NY, USA) at 37 °C for 45 min, with occasional agitation. The cellular digest was filtered through a sterile 31 μm nylon mesh, centrifuged at 400 g for 10 min, and washed twice in 10% FCS-DMEM. The cell pellet was then resuspended in 4 mL of 10% FCS-DMEM.For flow cytometric analysis, 106 cells from the immunized mice cells were washed in suspension with ice-cold buffer A (PBS/0.1% BSA/0.01% NaN3) and incubated for 20 min at 4 °C with 50 μL of a 1:100 diluted fluorescent-labeled specific antibodies using methods described previously [58]. The fluorescently conjugated Abs utilized were FITC-CD11c, PE-CD4, PE-Cy7-CD45R (B220) (eBioscience, San Diego, CA, USA), Alexa Fluor-750-CD8α, and PerCP-Cy5.5-CD11b (BD Biosciences, San Jose, CA, USA). Cells were washed twice and immediately analyzed on a flow cytometer (Becton Dickinson FACS, San Jose, CA, USA). All incubations and washes were performed at 4 °C with ice-cold buffer A. Cells were gated on singlets and live cells. The flow cytometric data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).Group analyses were completed by a matched, two-tailed, unpaired t-test with all values are presented as mean ± SEM. Mann-Whitney analysis was used to determine statistical differences. All data were analyzed using GraphPad Prism5 Software [59]. Statistically significant differences between groups were defined as * p < 0.1, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The pRelA and pTbet plasmids encode the full-length mouse NF-kappa B subunit p65/RelA and Type-1 transactivator T-bet, respectively. Each was genetically optimized, synthesized, and subcloned into modified pVax1 mammalian expression vectors (Figure 1A). To test for expression of these plasmids, HEK 293T cells were transfected with each and protein production was assessed by standard Western immunoblotting. An approximately 65 kDa protein corresponding to RelA was detected, using a specific Ab, in cell lysates harvested both 24 h and 48 h post-transfection (Figure 1B). Likewise, T-bet was detected as an approximately 56 kDa protein using an anti-T-bet Ab. Binding was specific for their respective proteins since neither bound to lysates from cells transfected with empty vector control plasmid pVax1. These data demonstrate that each of the molecular adjuvants expresses their respective encoded proteins upon in vitro transfection of HEK 293T cells. Further, IκB-dependent transcription was accessed in the HeLa cells luciferase expressing cell system (Figure 1C) to confirm the activation of RelA (p65). An increase in RelA expression as measured by relative luciferase activity was observed in a dose dependent manner. That is, increasing the plasmid from 3 μg to 5 μg or 10 μg resulted in an increase in the relative luciferase activity approximately 1.5 or 2.5 fold. T-bet expression correlates with IFN-γ expression in T cell and NK cells [60] and therefore in this assay IFN-γ serves as surrogate for the functional expression of T-bet (Figure 1D). Molecular adjuvant construction and expression. (A) Mouse RelA or T-bet primary sequences were genetically optimized, synthesized, and then subcloned into modified pVax1 expression vectors. Optimization entailed inclusion of a IgE leader peptide (IgE), preceded by a Kozak sequence, fused at the N-terminus. The figure indicates the restrictions enzymes used for subcloning, the translation initiation site (forward arrow), IgE leader peptide (IgE; hatched bar), protein length (aa), and transgenes (black with white lettering); (B) Protein expression from the nuclear extract was analyzed by Western immunoblotting following transfection of HEK 293T cells with pRelA, pTbet, or empty vector control (pVax1). The relative size (kDa) of the proteins are determined by detection analysis using protein-specific Abs as indicated; (C) Over expression of RelA potently induces κB dependent transcription. HeLa cells were transiently transfected with a NF-κB-dependent luciferase reporter gene together with expression vectors encoding RelA/p65. The cotransfected cells were subsequently grown for 48 h, and the luciferase activity was determined as described in the Materials and Methods; (D). Overexpression of T-bet stimulates production of IFN-γ: Naive CD4 T cells were transfected with either pT-bet or pVax1 and stimulated with anti-CD3 plus anti-CD28 followed the measurement of IFN-γ production by enzyme-linked immunosorbent assay (ELISA) as described Materials and Methods. IFN-γ levels are expressed as μg/mL The contribution of pRelA and pTbet, in terms of enhancing vaccine-induced immunity, was then assessed. Balb/C mice (n = 4/group) were vaccinated three times with 25 µg of pEnv or pGag either with or without 25 µg of pRelA or pTbet, 25 µg of pRelA or pTbet alone, or with 25 µg of a control plasmid (pVax1; Figure 2). The vaccines and adjuvants were delivered in 25 µL of PBS by in vivo EP. Animals were sacrificed on day 35, (i.e., seven days after the third vaccination) followed by isolation of splenocytes for immune analysis by IFN-γ ELISpot. In this assay, HIV-1 Env or Gag peptide pools were used for stimulation of MACS-purified CD4+ or CD8+ T cells and the IFN-γ ELISpot results are displayed in Figure 2. Both CD4+ and CD8+ T-cell responses were observed to be significantly increased in mice vaccinated with pEnv and co-administrated pRelA compared with pEnv alone. Likewise, immunization with pEnv with co-administrated pTbet compared to pEnv alone demonstrated significant increases in CD4+ and CD8+ T cell responses (Figure 2B). Transcription factor adjuvants enhance antigen specific DNA vaccine induced T cell immunity. (A) Balb/C mice (n= 4/group) were vaccinated three times at two week intervals with HIV-1 pGag or pEnv alone, pGag or pEnv with co delivery of either pRelA or pTbet. Other control groups were pRelA or pTbet alone, or a pVax1 control. T cell responses (CD8+ and CD4+) were analyzed by IFN-γ ELISPOT one week following the third immunization and results for IFN-γ+ spot forming cells (SFC) per 106 MACS-purified T cells are indicated following re-stimulation with subtype B HIV-1 Env (B) or Gag (C) peptide pools. Samples were performed in triplicate, error bars represent SEM, and statistically significant values are shown; ** p < 0.01, *** p < 0.001 and ****  p < 0.0001, referring to comparison between the indicated vaccination groups provided in the graph. Experiments were performed twice independently with similar results.To confirm the enhancing effects of these two adjuvants on T cell IFN-γ production for a different Ag, we also vaccinated animals with the HIV-1Gag either with or without pRelA or pTbet, similarly as performed above. Analogous to the pEnv group, CD4+ T cell responses were increased in mice immunized with pGag plus co-administrated pRelA, when compared with mice immunized with pGag alone (Figure 2C). There was an even greater enhancement of the CD8+ T cell response in mice vaccinated with pGag and co-administrated pRelA compared to immunization with pGag alone (Figure 2C). Further, immunization with HIV-1 Gag along with concomitant administration of pTbet demonstrated increased CD8+ T-cell responses when compared to immunization with pGag alone (Figure 2C). However, CD4+ T cell responses were not as significantly increased as observed with co-delivery of pRelA. Also, administration of either pRelA or pTbet alone did not markedly activate either CD4+ or CD8+ T cells against Gag or Env as measured by IFN-γ production. Therefore, these data demonstrate that co-administration of the transcription factor adjuvants promoted enhanced T cell responses against two separate antigens with the data suggesting that expanding the breadth of vaccine-elicited cellular immune responses was stimulated by administration of an immune adjuvant.Since the RelA molecular adjuvant was observed to particularly enhance T cell responses, the proliferative potential of cells immunized in the presence or absence of pRelA was evaluated. Splenocytes from vaccinated animals were harvested at seven days following the third immunization and were then stimulated with their cognate Ag, i.e., either HIV-1 Env or Gag (Figure 3). In pEnv-vaccinated mice, there was a trend towards enhanced proliferation at all Ag doses in mice that also received the pRelA adjuvant when compared to unadjuvanted animals (Figure 3A). This trend was also observed in pGag-vaccinated animals where the overall stimulation index was higher when pRelA was co-delivered (Figure 3B). As well, in both Figure 3A,B, in addition to the overall stimulation index, fold increase graphs are included, with the ‘fold” value being a ratio of stimulation index of the pEnv + pRelA or pGag + pRelA groups divided by stimulation indexes of the pEnv or pGag alone groups. Thus, the stimulation index in pEnv and pGag vaccinated animals was increased by the inclusion of a pRelA adjuvant, at all vaccine doses tested. These responses were specific for the HIV Ags since minimal proliferation was observed in splenocytes from animals that received the pRelA adjuvant alone. Taken together, these results demonstrate that the pRelA DNA adjuvant enhances Ag-specific T cell proliferative responses against two individual specific antigens.Increased T-cell proliferative potential following DNA vaccination plus co-administration of pRelA. Proliferative responses were measured seven days following the third vaccination with either pEnv or pGag alone, pEnv or pGag with pRelA molecular adjuvant, or empty vector control pVax1 alone. Splenocytes were incubated with recombinant HIV-1 Env (A) or Gag (B) at various concentrations: 0.5 (white bars), 1.0 (light gray bars), and 5.0 (dark gray bars) and subsequently pulsed with tritiated (3H)-thymidine for 24 h. Incorporated thymidine was expressed as a stimulation index (SI) calculated by dividing the mean cpm (counts per minute) of Ag-stimulated wells by the mean cpm of non-stimulated wells. Fold increase in SI for pRelA-adjuvanted mice are displayed for each concentration of Env (A, right panel) or Gag (B, right panel). Samples were tested in triplicate. Error bars represent the SEM, and statistically significant values are provided for the indicated group comparison shown in the graphs. **** p < 0.0001. Based on the observed adjuvant mediated increase in T cell IFN-γ and proliferative responses, the effects of these molecular adjuvants on B-cell induction was evaluated. HIV-1 Env-specific IgG was measured in the sera of vaccinated animals seven days following the third vaccination. As indicated, mice received pEnv either with or without co-administered pRelA or pTbet, pRelA or pTbet alone, or a pVax1 control plasmid (Figure 4). Measurable IgG responses were induced by pEnv alone at dilutions ranging from 1:50 to 1:500, but were non longer measurable at a dilution of 1:1000. Importantly, these responses were augmented at all dilutions by the inclusion of the pRelA or pTbet adjuvant when compared to the pEnv group alone. Specifically, differences were observed at the 1:50 sera dilution, where administration of pRelA and pTbet significantly enhanced the induction of HIV-1 Env-specific IgG responses (p = 0.0388 and p = 0.0062, respectively). Enhanced IgG responses were specific for Env since minimal antibody responses were observed in the sera from mice that were administered the pRelA or pTbet adjuvant alone. These data suggest that both transcription factor adjuvants elicited an enhanced humoral immune response that was analogous and consistent with the elevated IFN-γ levels and T cell proliferative responses observed following vaccination with pRelA or pTbet.One potential mechanism for the ability of the transcription factors to enhance antibody responses may be thorough increase in the number of activated B-cells. To access whether this was occurring, the pRelA administered muscle at the site of vaccination was biopsied three days after pEnv immunization with co-administrated pRelA followed by quantification of number of B220+ B-cells at the site of injection. The results indicated that pRelA and pEnv alone caused only a slight increase in B-cell trafficking to the site of injection compared to pVax1 administration alone (Figure 5). This is indicated by the MFI (mean fluorescent intensity) values shown in the individual FACS scans, which are directly proportional to the level of B220+ B cells. However, the addition of a pRelA adjuvant in combination with the pEnv vaccine further enhanced the number of B-cells at the site of injection.Improved B cell responses with pEnv vaccination and co-administered transcriptional molecular adjuvant. B cell/antibody responses were assessed in the sera of vaccinated mice (n = 4/group) seven days following the third immunization with pEnv alone, pEnv in combination with either pRelA or pTbet, each of the molecular adjuvants alone, or with empty vector control plasmid (pVax1). Anti-Env p120 antibody-binding titers were determined by ELISA. Data are presented as the mean endpoint titers. Statistically significant values are indicated; *** p < 0.001 (comparison between pEnv alone and pEnv + pRelA or pEnv + pT-bet) and **** p < 0.0001 (comparison between pRelA alone and pEnv + pRelA or pT-bet alone and pEnv + pT-bet).Molecular adjuvants enhance populations of B-cells at the site of immunization. Cells cultures from the muscle were analyzed by flow cytometry for expression of B220. The isolated cells were incubated in culture media for three days and these cells and then stained with DC subsets (CD11c+/CD11b+), B cells (B220+), T cells (CD4+ and CD8+ subsets), to distinguish monocytes/dendritic, B cells, T cells, respectively. Such differential staining allowed the exclusion of dendritic and T cells from subsequent analysis of B220 expression. Histograms show the B220+ expression on B cells exclusively using a specific mAb as well as an isotype-matched, irrelevant mAb as a control. The profile of an isotype-matched irrelevant Ab, used as a control (shaded area) is also indicated in the panels. MFI = mean fluorescent intensity which is proportional to the level of B220 expressing B cells.The study reported here is the first to report the ability of plasmid-expressed RelA and T-bet to function as molecular adjuvants for antigen specific DNA vaccine induced immunity. While the potential utility of RelA and T-bet are evident, the exact mechanism by which they enhance immunogenicity is currently unknown. For the RelA molecular adjuvant, it is likely that the over expression of this molecule directly drives NF-κB activation, much like many current adjuvants do indirectly. For example, many bacterial-derived carbohydrates such as those present in the GSK AS04 adjuvant and many veterinary adjuvants exert their pro-inflammatory effects through activating this pathway. Likewise, the recently approved MPL adjuvantTLR-4, which activates NF-κB and leads to enhanced Th1 and antibody responses. In the present study, we hypothesized that directly expressing RelA/p65 at the site of injection would potentially impact multiple cell types and multiple signaling pathways within a given cell resulting in NF-κB activation. This could lead to the stimulation of numerous pro-inflammatory signals for the induction of vaccine-specific Th1-type responses. NF-κB is expressed in all hematological cells and the pairing of the p65 subunit leads to multiple functional outcomes including activation. In this study, we also reported an approach to enhance the immunogenicity of DNA-based vaccines using T-bet as an adjuvant. Enhancement of antigen specific immune responses was confirmed by the fact that the T cell immune response was elicited in the T-bet co-administered mice compared to the pVax1 control. The enhanced immunity observed with molecular immune adjuvants may also have been the result of direct transfection of local DCs at the site of injection. It is known that the nuclear translocation of p65 is associated with the activation and maturation of DCs [61]. In addition, NF-kappa B activation controls the expression of critical co-stimulatory molecules such as CD80, CD86 and MHC class II along with pro-inflammatory cytokines such as IL-12, IL-6 and TNF-α [62,63]. Over expression of RelA in local DCs could likely increase cytokine production and co-stimulatory molecule expression, both of which could lead to improved T cell induction. Therefore, it is likely that the inclusion of a RelA-expressing molecular adjuvant could result in NF-kappa B activation in multiple cells at the site of injection, including resident DC and potentially could explain the improved T cell phenotype observed in this study.In addition to stimulation of the T cell responses, enhanced antigen specific IgG production was also observed when a transcription adjuvant was used in combination with the vaccine. For the RelA adjuvant, NF-kappa B activation enhances the expression of adhesion molecules required for immune cell accumulation at the site of inflammation [64]. Further, studies in knockout mice suggest p65 is required for B-cell proliferation in response to BCR signaling [41]. Therefore, the inclusion of pRelA may facilitate the accumulation of Ag-specific cells at the site of vaccination. For the T-bet molecular adjuvant, in vitro over expression in B cell lines has been shown to result in Ab class switching, thereby increasing the level of IgG2a [65]. It is possible that IgG2a responses are enhanced herein since T-bet expression regulates the transcription of mature B cell receptors, which are necessary for the survival of memory B cells. In addition, the differential transcriptional stimulation determined by T-bet may regulate B cell potential for cytokine secretion, trafficking, and survival that ultimately permits flexibility in long-term Ag-specific immunoprotection [65]. Thus, data herein indicate that vaccination with co-expressed transcription factors associated with pro-inflammation and Th1 type development may contribute to the enhancement of vaccine-induced B cell responses.Several viral and bacterial infection models have been tested extensively to define the transcription factors that may have a role in the development, differentiation and maturation of immune cells on particularly the CD8+ T-cell effector and memory populations [46]. Our results are in agreement with a model supporting a role for several transcriptional factors in the enhancement of primary antigen specific cellular and humoral responses. Overall, the work presented here suggests that pRelA and T-bet plays a significant role in creating the immune environment that influences the development and function of the strong vaccine induced CD8+ T-cell response and antibody production.While the molecular adjuvanted E-DNA vaccine approaches presented herein have demonstrated the potential for increased Ag-specific immunity, it is important to consider possible safety issues that may be caused by transcription factor expression. Importantly, while increased NF-kappa B expression can lead to multiple immunological disorders and cancers [66,67] we observed no significant adverse safety events in mice vaccinated with the pRelA or pTbet molecular adjuvants. This may be explained by transient expression of these proteins by plasmid DNA that is known to persist for approximately 14–30 days. In addition, local expression of the plasmid DNA expressed transcription factors may help to drive immunity at the vaccination site while minimizing the possibility of systemic or off-target effects. Thus, it would be unlikely that a transcription factor-encoding genetic adjuvant would induce any serious long-term negative side effects. Furthermore, our group and others have studied extensively the use of various immune modulating plasmid-expressed adjuvants without any evidence for the development of adverse effects. This is an important safety consideration and demonstrates that administration of plasmid based transcriptional factors does not lead to global immune deregulation. However, future studies should further investigate the safety of the proposed adjuvants in preclinical studies utilizing nonhuman primates, before advancing to the clinic. In conclusion, this is the first report, to our knowledge, to demonstrate the ability of several transcription factors, when delivered as DNA expression plasmid, to enhance the immunogenicity of antigen specific DNA vaccines. The use of cellular transcription factors RelA and T-bet as molecular adjuvants for enhancing DNA vaccine-induced immunity was investigated in this report. When co-delivered along with a prototypical DNA vaccine by in vivo electroporation (EP), either of these putative adjuvants stimulated enhanced antigen-specific T and B cell responses as indicated by increased T cell numbers and IFN-γ production, as well as by an increase in antibody levels. This study builds on recent achievements demonstrating the potency of the enhanced DNA (E-DNA) vaccination method and establishes that transcription factors may serve as effective molecular adjuvants to boost vaccine-induced immunity.This work was supported by grants funded to DBW through the National Institutes of Health including NIH-CFAR, NIAIDS-HVDDT and NIH-NIAID-HIVRAD. KM was supported by a National Institute of Health-Center For AIDS Research Training Supplemental Grant-5-P30-AI-045008-13. This work was supported in part by the core facilities of the Penn Center for AIDS Research and the Abramson Cancer Center Core facilities.Conceived and designed the experiments: Kar Muthumani, David B. Weiner. Performed the experiments: Devon J. Shedlock, Colleen Tingey, Lavanya Mahadevan, Natalie Hutnick, Emma L. Reuschel, Sagar Kudchodkar, Seleeke Flingai, Jenny Yan and Kar Muthumani. Contributed reagents/materials/analysis tools: Joseph J. Kim, Kenneth E. Ugen and David B. Weiner. Wrote the paper: Kar Muthumani and David B. Weiner. Obtained funding: Kar Muthumani and David B. Weiner. Study supervision: Kar Muthumani and David B. Weiner. Critical revisions of manuscript: Kar Muthumani, Kenneth E. Ugen and David B. Weiner.The laboratory of DBW has grant funding and collaborations as well as service in scientific review, consulting and advising capacities for commercial entities and therefore there exists possible conflicts associated with this work with Pfizer, Inovio, BMS, Virxsys, Ichor, Merck, Althea, VGXI, J&J, Aldevron, and possibly others. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Modulation of the cytokine milieu is one approach for vaccine development. However, therapy with pro-inflammatory cytokines, such as IL-12, is limited in practice due to adverse systemic effects. Spatially-restricted gene expression circumvents this problem by enabling localized amplification. Intracellular co-delivery of gold nanorods (AuNR) and a heat shock protein 70 (HSP70) promoter-driven expression vector enables gene expression in response to near infrared (NIR) light. AuNRs absorb the light, convert it into heat and thereby stimulate photothermal expression of the cytokine. As proof-of-concept, human HeLa and murine B16 cancer cells were transfected with a HSP70-Enhanced Green Fluorescent Protein (EGFP) plasmid and polyethylenimine (PEI)-conjugated AuNRs. Exposure to either 42 °C heat-shock or NIR light induced significant expression of the reporter gene. In vivo NIR driven expression of the reporter gene was confirmed at 6 and 24 h in mice bearing B16 melanoma tumors using in vivo imaging and flow-cytometric analysis. Overall, we demonstrate a novel opportunity for site-directed, heat-inducible expression of a gene based upon the NIR-absorbing properties of AuNRs and a HSP70 promoter-driven expression vector.In addition to malignant cells, tumors consist of non-transformed host cells, such as fibroblasts and immune cells, vascular tissue, cytokines, and the surrounding extracellular matrix. The tumor microenvironment is a complex interrelated system that plays an important role in malignant cell survival, growth, proliferation, and metastasis. Cancer treatments have historically only targeted the malignant cell itself, and as a result, they rarely prevent recurrence of disease or progression of metastasis [1]. The importance of the tumor microenvironment and the interactions between the different cell types and components has become increasingly recognized. The balance of cellular and cytokine interactions and signaling within this milieu has a major impact on whether the tumor mass regresses or grows, and whether the malignant cells remain localized or metastasize to distant sites. Effective eradication of malignant disease requires therapeutic strategies that focus on the whole tumor as well as metastatic tissue.Gold nanoparticles (AuNP) and nanorods (AuNR) have emerged as attractive nanomaterials for biological and biomedical applications because of their physical and chemical properties. The particles absorb and scatter visible and near-infrared (NIR) light upon excitation of their surface plasmon resonance (SPR) oscillation, which can be tuned over a wide spectral range by changing intrinsic particle parameters such as size and shape [2]. Rod-shaped gold particles have shown promise over spherical shaped AuNP due to fact that they display two separate SPR bands corresponding to their width and length, allowing their longitudinal plasmon bands to range from the visible (600 nm) to the near infrared (1100 nm and up) regions of the electromagnetic spectrum [3]. Their ability to absorb light in the near infrared region, and subsequent conversion of the applied energy into heat, has led to the use of AuNPs and AuNRs for hyperthermia-based applications, including cancer therapy. Photothermal ablation of solid tumors has been investigated in various preclinical models and is currently being evaluated in the clinic [4,5,6]. Nanoparticles can be delivered to the tumor either passively, accumulating in the tumor through the enhanced permeability and retention effect (EPR), or actively targeted to receptors on the tumor or tumor-associated vasculature [7,8,9,10]. The AuNR are easily functionalized with peptides, proteins, antibodies, or nucleic acids for targeting or for creating multifunctional platforms for therapeutic and diagnostic purposes [11,12]. Clinical advantages of using gold nanoparticles include easy synthesis, targeting capability, high biocompatibility, cost effectiveness, and easy clearance from the body [13,14]. They have an excellent track record of being well tolerated in humans and are currently in the process of obtaining FDA approval for clinical use [15,16].Several strategies that allow control of both spatial and temporal expression of transgenes have been developed. Spatial resolution is often achieved by the use of tissue- or cell-specific promoters, or exploitation of the heat-shock response, a temperature-sensitive defense mechanism [17,18,19]. The heat-shock response is mediated by a transcription factor known as heat shock factor (HSF). HSF is synthesized constitutively, but remains dormant under normal conditions. In response to heat, HSF trimerizes and binds with high affinity to heat shock promoters containing specific binding elements, leading to the transcription of heat-shock proteins [20]. Temporal resolution may be under the control of external cues, such as NIR laser light. Induction of heat shock promoter (HSP)-mediated gene expression by laser light is a promising approach for achieving temporal and spatial control of gene expression [21,22]. Other approaches beyond NIR light have considerable technical limitations related to their use of UV, short-wavelength visible (vis), and infrared (IR) laser light, due to poor penetration into biological tissue. Conversely, biological tissue is relatively transparent to light inside the diagnostic window of 700–1100 nm [23]. NIR laser light has been shown to penetrate 10 cm through breast tissue or 4 cm through deep muscle [24]. The ability of nanorods to absorb NIR light makes them particularly well-suited to biomedical applications since the absorbance of the surrounding tissue in this region is low, allowing for minimally invasive delivery of energy to tumor cells that have taken up the AuNR, without inducing damage to intervening and surrounding normal tissue.While cytokine and drug therapeutics are effective against cancer cells, they also cause systemic effects and damage to healthy tissue. To accurately regulate the levels of therapeutic gene expression to achieve enhanced efficacy and minimal toxicity, we are proposing to drive the expression of target genes using a heat-inducible promoter. Our proposed vector system consists of heat generating AuNRs and a therapeutic gene expression vector under the control of the human heat shock promoter 70 (HSP70). We hypothesize that exposure of pathological tissue to a near infrared (NIR) laser source will cause the AuNR to absorb the NIR light and convert it to heat, thus inducing spatially confined, photo-thermal expression of the target gene (Figure 1). Controlled gene expression within the tumor microenvironment could include expression of cytokines, such as IL-12 or interferon gamma, leading to tumor infiltration and activation of immune cells, including antigen presenting cells, natural killer (NK) cells, type 1 helper T cells, and cytotoxic lymphocytes (CTL), resulting in immune targeting of the diseased cells. Alternatively, expression of immuno-suppressive molecules or cytokines, such as IL-10, could promote tissue transplantation, or suicide genes could lead to apoptosis of targeted cells.Wei et al. [25] demonstrated HSP70B-driven expression of 1L-12 using adenovirus. Mice with subcutaneous Hep3B tumors were given an intratumoral injection of adenovirus encoding both heat inducible IL-12 and constitutively expressed granulocyte macrophage colony stimulating factor (GM-CSF). Using external heating of the limb with a water bath, they demonstrated elevated IL-12 levels during 3 separate heating events. While effective, this technique requires whole limb heating. We propose a method to achieve selective heating of diseased or immune cells using non-invasive NIR light and delivery of AuNR to cells of interest.Herein we describe an adjuvant method in which NIR induced hyperthermia is mediated by the cellular loading of nanorods and monitored by the expression of a HSP70 driven reporter within the same cell. Preliminary efficacy studies are presented in nude mice bearing orthotopic B16 melanoma tumors. Target tumor cells are transfected ex vivo with the reporter plasmid and AuNRs prior to transplantation and NIR exposure. We have optimized AuNR-loading into tumor cells and induction of gene expression using a NIR dose sufficient to induce GFP reporter expression, yet low enough to maintain cell viability.Schematic showing near infrared (NIR)-driven hyperthermia mediated therapy. (1) AuNRs complexed with therapeutic HSP70B promoter-driven gene vectors will be injected into mice intratumorally or intravenously with targeting moieties; (2) NIR laser treatment of the tumor site will result in localized heating of the AuNR, which in turn will induce expression of the therapeutic gene; (3) The predicted end result is tumor growth inhibition and tumor mass reduction. The Light Sheer ET Lumenis FDA-approved NIR laser light source and machine specifications are shown to the right. Gold nanorods (AuNR) conjugated to polyethyleneimine (PEI) were purchased from Nanopartz™ Inc., Loveland, CO. The 800 nm NIR light source was an FDA approved clinical diode laser device obtained from Lumenis, Inc. (Lightsheer ET, Lumenis, Inc., Santa Clara, CA, USA) with peak power of 1600 W, laser fluence 10–100 J/cm2 (Figure 1). B16F10-luc melanoma cells, stably transfected with the firefly luciferase gene, were purchased from Caliper (Perkin Elmer, Waltham, MA, USA).The 400 bp minimal human HSP70B promoter fused with the EGFP gene, a kind gift from Dr. Chrit Moonen of Université Victor Ségalen, France, [26] was cloned into the pGL3 vector (Promega, Madison, WI, USA). The construct was confirmed by restriction digestion, and the reporter expression was verified through transfection of HeLa cells as described below.HeLa or B16 cells were transfected with HSP70-pGL3 using Lipofectamine LTX reagent (Invitrogen, Grand Island, NY, USA) at a ratio of 1:4 DNA:lipofectamine, and 24 h later the cells were either left at 37 °C or heat-shocked at 42.5 °C for 30 min. Lipofectamine LTX was chosen to avoid activation of the HSP promoter shown to occur with other transfection reagents [27]. The following day, cells were analyzed for EGFP expression using a LSR Fortessa flow cytometer (Becton Dickinson), or by fluorescence microscopy using a Nikon A1 confocal microscope.B16F10-luc cells were plated in 96-well plates and AuNR were added at increasing concentrations. After 24 h, the cells were washed and lysed. The number of AuNR in each well was determined using UV/VIS spectroscopy. A standard curve was generated by adding serial dilutions of AuNR with known concentration to wells containing saline and cell lysate.To investigate the effect of NIR laser radiation on the induction of transgene expression, we loaded polyethyleneimine (PEI)-conjugated gold rods (10 nm transverse diameter with surface plasmon resonance (SPR) peak of 808 nm) with the HSP70-EGFP vector in the presence of Lipofectamine LTX and incubated HeLa cells with the complexes overnight. The following day, the cells were exposed to varying laser fluencies (25–75 J/cm2) at 10 or 20 pulses using the 800 nm Lumenis laser. Twenty four hours after NIR treatment, the cells were analyzed for EGFP expression and viability by flow cytometric analysis.B16F10-luc melanoma cells were transiently transfected with the HSP70-EGFP plasmid using Lipofectamine LTX, followed 6 h later by addition of PEI-AuNRs at concentrations of 1012 overnight. Cells were injected subcutaneously into the flanks of nude mice. NIR irradiation at varying fluencies was applied to the nascent tumors. In vivo fluorescent and bioluminescent imaging (BLI) was performed at various time points to monitor EGFP and luciferase expression. Excised tumors were analyzed by flow cytometry or fluorescent microscopy for GFP expression.To verify that we could successfully observe heat-shock induced GFP expression in vivo, we first established, as a positive control, B16F10-luc cell lines carrying HSP70-EGFP and AuNPs exposed to heat-shock in vitro prior to being injected into the animal. Heat-shocked cells were exposed to 42 °C in a water bath for 30 min. Negative controls received a sham treatment at 37 °C.To verify in vivo NIR induced gene expression, B16 cells were transfected as above. 2 × 107 tumor cells were injected into the flanks of nude mice the following day and treated with NIR laser within the first hour, initially using laser parameters established in vitro. The experiment was repeated a second time with lower cell numbers, fluencies and duty cycles. Expression of GFP and luciferase in the tumors at 6 and 24 h after injection was tested via in vivo imaging and flow-cytometric analysis.As shown in Figure 2, 60% of HSP70-EGFP transfected B16 cells showed an increase in EGFP expression following heat-shock compared to <1% of transfected cells without heat-shock. This negligible activity in the “off” state confirms that gene expression from the HSP70 promoter is tightly regulated. Figure 2C shows high uptake of the PEI-coated AuNPs during incubation, with 20%‑25% of AuNPs in solution ending up in the cells.Transfection of HeLa cells with the HSP70- Enhanced Green Fluorescent Protein (EGFP) expression vector and quantitative measurement of AuNR uptake in vitro. (A) Cartoon of the expression vector; (B,C) HeLa cells were transfected with the vector using lipofectamine LTX reagent, and 24 h later the cells were either left at 37 °C or heat-shocked at 42.5 °C for 30 min. The following day, cells were analyzed for EGFP expression via flow cytometric analysis or fluorescence microscopy (B); B16-luc cells were plated in 96-well plates and AuNR were added at increasing concentrations. After 24 h, the cells were washed and lysed. The number of AuNR in each well was determined using UV/VIS spectroscopy (C); A standard curve was generated by adding known numbers of AuNR to wells, and a graph showing the number of particles present in the cells at 24 h vs. the number added at 0 h is presented.The results of the in vitro NIR optimization are shown in Figure 3. We found that the optimal dose to reach the highest level of EGFP expression with the lowest cell death/toxicity is achieved by using approximately 1011 AuNR and 10 pulses at 50 J/cm2, with a 30 ms pulse length. In general, cells are driven to increase production of EGFP with increased heating up to some threshold, at which point cell damage rapidly reduces expression and eventually viability. Heating efficiency increases as expected with increased loading of AuNPs (Figure 3A) and pulse numbers (Figure 3B). At a laser fluence of 25 J/cm2 relatively little gene expression occurs, with increases in fluence to 50 J/cm2 inducing increased expression and relatively high viability (approximately 80%). While gene expression was highest at 75 J/cm2, cell viability dropped to 44% and 37% at 6 h and 24 h, respectively. Increasing the length of the pulses from 30 ms to 400 ms actually decreases the GFP expression (Figure 3C; right), demonstrating that peak power is more important than total power. In Figure 3C, the peak power is decreasing (the same energy, 50 J, is spread over a longer pulse), even though the total power is the same. In general, some loss of viability appears as a necessary price for high expression.Cell viability and GFP expression after in vitro NIR treatment. (A) B16-Luc cells (1 × 105 per well in 24-well glass bottom plates) were incubated with HSP70-GFP and 1010, 1011, or 1012 AuNR per well. The following day, the cells were treated with NIR laser with 10 pulses at 0, 25, 50, or 75 J/cm2 (B) B16-Luc cells were incubated overnight with or without 1011 AuNR per well and treated with NIR laser with 10 or 20 pulses. Cells were analyzed for GFP expression and viability by flow cytometery the following day; (C) Mean GFP fluorescence intensity (MFI) and percent viable cells (measured by uptake of propidium iodide) as a function of laser fluence (25, 50 and 75 J/cm2; left), time after NIR treatment (6 h or 24 h; left) and pulse duration (30, 100, or 400 ms; right).Figure 4 shows animals inoculated with heat-shocked cells on the right and sham-heated cells on the left. Both sets of B16F10-luc cells show up well under BLI, but only the heated cells are detected under fluorescent imaging. The signal from the positive controls is strong enough to be detected through the skin of the in vivo by both the IVIS200 and Maestro imaging using FITC filters.In vivo validation studies. B16-luc cells were transfected with the HSP-GFP vector using lipofectamine LTX reagent (Invitrogen), and 24 h later the cells were either left at 37 °C or heat-shocked at 42.5 °C for 30 min in a water bath. The following day, cells were analyzed for EGFP expression via flow cytometry or fluorescence microscopy. Transfected B16 cells either exposed to heat-shock or kept at 37 °C were injected into flanks of nude mice and imaged at 6 h and 24 h for luciferase expression (using the IVIS imaging system) and GFP (using the IVIS and Maestro imaging systems). Mice were injected with D-Luciferin before imaging. The right leg of the mouse was injected with heat-shocked cells whereas the cells injected into the left leg were left untreated.As shown in Figure 5, strong EGFP expression could be detected 6 hours after NIR treatment at both 40 J/cm2 and 50 J/cm2 laser at 30 ms pulse length. This was confirmed with FACS on the 40 J/cm2 and was confirmed to last at least 24 h in the 50 J/cm2 animal. The laser treatment at the higher power seemed to result in more physical damage to the skin of the animal. Bioluminescent imaging (BLI) was also used to monitor luciferase expression in the cells with IVIS200. Interestingly, the luciferase signal was intact on the non-treated left flanks in both mice, but suppressed on the NIR treated right flanks. This finding is in agreement with previous studies reporting that luciferase is highly sensitive to temperature, and thus can function as an indicator of a successful thermal treatment [28]. Further experiments indicated that detectable expression may be achieved with half the gold loading, and using 5 instead of 10 pulses, still at 40 J/cm2. Reducing the laser fluence to 30 J/cm2 or below resulted in no observable expression. Treatment with laser of cells without AuNPs did not result in any increase in EGFP expression or loss of bioluminescence.In vivo imaging of luciferase and GFP expression after NIR laser treatment. (A) B16-luciferase cells were transfected with the HSP70-GFP vector 24 h before NIR treatment and AuNR (106/cell) were added 12 h after transfection. Cells were washed before being harvested and 2 × 107 cells were injected in each flank of two nude mice. Immediately following injection, the right flank of each mouse was treated with NIR laser for 10 pulses at 30 ms at either 40 or 50 J/cm2. The left flank was left non-treated. Mice were imaged at 6 h via IVIS and Maestro in vivo imaging systems. At 24 h after treatment, one mouse was imaged as just described, and the other mouse was sacrificed and the tumor analyzed for GFP expression via flow cytometry analysis (B).Although gene therapy has shown great promise both for cancer and infectious diseases, the major challenge lies in the development of safe and effective delivery systems that can lead to controlled expression of therapeutics. Despite the high transduction efficiency and long-term gene expression of viral vectors, the complexity of their manufacturing and in vivo safety issues remains hurdles to be overcome. We demonstrate a novel opportunity for site-directed, heat-inducible gene expression based upon the NIR-absorbing properties of AuNRs. We have optimized AuNR-loading into tumor cells and induction of gene expression using a NIR dose sufficient to induce GFP reporter expression, yet low enough to maintain cell viability. We have also confirmed induction of gene expression by NIR laser in an in vivo tumor model.Future studies will include delivery of therapeutic genes with heat sensitive promoters using viral nanoparticles (NPs) to prolong the presence of the gene in cells, permitting repeat heating cycles and temporal control of gene expression without the need for continuous gene delivery or ex vivo transfection of cells with the HSP70B promoter plasmid. Simultaneous accumulation of viral NPs and AuNRs or carbon-based NPs in the tumor will be achieved using either targeting ligands or by selective heating of the tumor through non-invasive tissue heating, such as radiofrequency (RF) wave induced hyperthermia which supports increased intratumoral blood flow and nanoparticle accumulation [29]. Tumor tissue is highly susceptible to selective RF-induced heating based on its unique biological composition [30], enabling the use of RF energy to drive gene expression or to promote tumor necrosis. Thus RF-driven hyperthermia is currently being explored as a mechanism for achieving both NP accumulation and for tumor ablation. While NIR light is ideal for treating melanoma or near-surface tumors, RF complements this technique by enabling deep tissue penetration.The authors acknowledge use of the flow cytometry and animal imaging cores at Houston Methodist Research Institute. This research was supported by the National Institute of Health Grant #U54CA151668 and U54CA143837. The authors thank King Li for material support and many helpful discussions, and they are grateful to Scott Holmes for illustrations presented in Figure 1.R.E.S. and B.E.O. contributed study concepts, design, manuscript preparation and editing. Z.-Z.S. designed and cloned the plasmid. Y.-S.K. and H.A.A. acquired data and performed data analysis.None of the authors have any conflicts of interest to report.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag) formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions) can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV) as the test Ag, the combined use of two Toll-like receptor (TLR) ligand adjuvants, CpG oligonucleotides (ODNs) and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs) before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing receptor (PRR) ligands, such as CpG/polyI:C, increases both adaptive and innate responses and represents a promising adjuvant strategy for enhancing the protection of future viral vaccines.Viral diseases present a huge problem for the global aquaculture industry, where infectious diseases are estimated to be responsible for a production loss of ten to twenty percent each year [1]. Among these, pancreas disease (PD) caused by salmon pancreas disease virus, now more commonly referred to as salmonid alphavirus (SAV), is responsible for big economic losses throughout the Norwegian, Scottish and Irish aquaculture industry.SAV strains are grouped into six different subtypes (SAV1-6), based on sequencing and phylogenetic analysis [2]. SAV1 isolates are found in Ireland and the U.K., while SAV3 isolates are exclusively found in Norway and affects both Atlantic salmon and rainbow trout [3,4]. PD histopathological signs are characterized by lesions in pancreatic acinar tissue, heart and, later, also in skeletal muscle [5,6]. Several studies have demonstrated protective immune responses against SAV in salmonids, both experimentally and in the field [7,8], and the protection has been shown to be associated with antibody (Ab)-mediated immunity and neutralizing Ab (nAbs) [7,9,10]. Following passive immunization of salmon parr and post smolts with SAV antisera, the fish are reported to be protected upon re-challenge, which indicates that protective immunity is conferred by NAbs [11]. Since then, various vaccination strategies against SAV have been tested both in Atlantic salmon and in rainbow trout, such as an inactivated SAV vaccine based on a Subtype 1 isolate [12], a recombinant live attenuated SAV vaccine (Subtype 2 isolate) [13] and subunit and DNA vaccines based on an SAV Subtype 3 isolate [14]. An inactivated whole-virus vaccine based on an Irish SAV Subtype 1 isolate has been used in Norway, Ireland and the U.K. since 2007 [15].Most of the commercial finfish vaccines against viruses are, like the above-mentioned SAV vaccine, based on inactivated antigens (Ag) or recombinant subunit proteins formulated in oil emulsions. Oil-based adjuvants are based on creating a depot of Ag, which improves Ag delivery to Ag-presenting cells (APC) or by attracting effector cells to the site of injection. However, side effects due to oil adjuvants have been reported and are expressed both physiologically and morphologically [16,17,18]. Administration of these oil-based vaccines is performed by intraperitoneal (i.p.) injection, and relatively high doses are needed to achieve protection [19]. Hence, more potent adjuvants enabling the use of a lower Ag dose and reducing side effects would advantageously be used for fish vaccines. Many adjuvants are derived from pathogens and act via cell-associated germ line-encoded pattern recognition receptors (PRR) on APCs to provide a danger signal. Such adjuvants induce maturation of the APCs and enhance Ag presentation and associated co-stimulation [20]. Among the best studied PRRs is the Toll-like receptor (TLR) family, of which 17 types have been described in different fish species [21]. The piscine TLRs includes TLR9 recognizing bacterial and viral DNA [22,23] and TLR3 and TLR22, which both recognize double-stranded (ds) RNA [24,25]. Studies by us and other groups have demonstrated that ligands for these receptors, such as synthetic CpG oligonucleotides (ODNs; TLR9 ligand) and polyI:C (TLR3/22 ligand) can stimulate the production of pro-inflammatory cytokines/chemokines and Type I IFNs, which increase the host’s ability to eliminate viral pathogens [26,27]. Further, our group has, in accordance with mammalian studies [28], shown that a combined treatment of CpG/polyI:C induces synergistic upregulation of a wide array of immune genes in Atlantic salmon [26] and significantly enhances protection on its own against SAV [29]. When formulated in an SAV whole-virus Ag formulation [30], the combo significantly increased antibody-mediated clearance of SAV from blood, thus preventing the development of SAV-specific heart lesions. This strongly indicates that a humoral Ab response is important for protection against SAV and that CpG/polyI:C boost this protection.The Novirhabdovirus VHSV (viral haemorrhagic septicaemia virus) glycoprotein (G) DNA-vaccine is an intra-muscularly (i.m.) injected vaccine that has been shown to induce early non-specific, as well as long-lasting specific protection against VHSV in trout [31,32] and seems to act as a genetic adjuvant [33]. Several studies have suggested that the effects of DNA-vaccines are regulated by innate responses through PRR-signalling cascades [34], and although the mechanisms that induce early protective responses by the VHSV-G DNA construct are still unclear, innate signalling are most likely involved. The VHSV-G DNA vaccine has been shown to create a local immune-competent environment at the i.m. injection site [35], and an interesting question addressed here was whether i.m. co-injection of this DNA vaccine simultaneously with an i.p. administered TLR-agonist-adjuvanted vaccine based on an inactivated SAV Ag could provide improved protective responses. Combining several PRR agonists has previously shown synergistic effects when intended as adjuvants, resulting in enhanced and more durable responses to Ag, as well as dose sparing effects [36,37]. To our knowledge, this is the first report of immunity associated with inactivated virus Ag vaccines formulated with mixed TLR agonists and with VHSV-G (vhsG) DNA vaccine as a genetic adjuvant. In the current study, the protective effects between an i.p. injection of SAV Ag with CpG/polyI:C were compared to the same treatment combined with a simultaneous i.m. injection of the vhsG DNA vaccine to see whether additive or synergistic effects were induced. To evaluate vhsG adjuvant effects, several controls were included, whose effects have been investigated earlier [29,30]; SAV Ag was i.p. injected alone or with an oil adjuvant (Montanide ISA763A), formulations of which were co-injected i.m. with vhsG to thoroughly examine the possible effects these adjuvant combinations could have on protection and immune responses against an SAV Subtype 3 challenge. DNA plasmid pcDNA3-vhsG [38] was kindly provided by Dr. Niels Lorenzen and diluted in 1× PBS to a concentration of 0.2 mg/mL. The synthetic dsRNA (poly I:C; Merck, Nottingham, UK) and the phosphorothioate-modified class B CpG oligonucleotide (2006T: TCGTCGTTTTGTCGTTGTCGTT, Thermo Scientific, Ulm, Germany) were dissolved in TE buffer (10 mM Tris, 1 nM EDTA, pH 8) at 5 mg/mL and further diluted 10-fold in the final vaccine formulations (50 µg/dose). SAV Ag was prepared by propagating the SPDV/SAV strain F93-125 in cell culture, which then was formalin inactivated. To demonstrate the potential additive or synergistic effects of the adjuvants tested, the same suboptimal dose of inactivated SAV Ag was used for all formulations. Montanide ISA 763 (Seppic, France) was used to prepare the water-in-oil formulations, by dispersing the water phase (containing the formalin inactivated SAV Ag) into the vegetable oil phase (containing emulsifiers and stabilizers) and emulsified using a homogenizer with an emulsification rotor. All SAV Ag formulations were provided by MSD Animal Health (Bergen, Norway). The experimental challenge study was performed at ILAB’s challenge lab facility at the University of Bergen (Høyteknologisenteret, Bergen, Norway), which fulfil the confinement conditions required for working with GMOs and DNA vaccines. Atlantic salmon, pre-smolt (Fister) with a mean weight of approximately 29 g at time of vaccination were kept in tanks supplied with running fresh water at 11–12 °C and fed with commercial dry feed (Skretting, Stavanger, Norway) based on appetite. The fish were starved for a minimum of 48 h and anaesthetized with metacainum (0.1 mg/mL bath treatment) prior to all handling.Fish were divided into 7 treatment groups (n = 65, 69 or 75, depending on the required sampling size) and a saline injected control group (n = 79). As described in detail in Table 1, three treatment groups were i.p. injected with 100 μL of the SAV1 whole-inactivated virus Ag formulation, formulated with or without oil and/or 50 µg CpG/polyI:C. Moreover, three treatment groups were, in parallel to the i.p. injections, injected i.m. with 10 µg of the PcDNA3-vhsG plasmid diluted in 50 µL PBS (1×). One group received PcDNA3-vhsG plasmid alone, and the control group was injected with 100 μL of PBS. Fish were marked by fin and/or maxilla clipping, and there were no mortalities observed after injection. A total of 570 fish were used and divided into 3 tanks; 1 tank for the SAV cohabitation challenge (422 fish excluding shedders), and 2 tanks for harvesting organs to monitor early immune gene expression (2 × 74 fish). At 6 weeks post vaccination (wpv), 86 Atlantic salmon were injected i.p. with 0.2 mL SAV Subtype 3, each receiving a viral dose of about 1 × 103 TCID50 and added to the SAV challenge tank to serve as shedders (n = 86 corresponds to 20% of the final amount of fish in the tank). The shedders were marked with a red VIE label under the anal fin one week prior to challenge.Treatments, dose regime, number of fish and schedule for the sampling of organs and blood. The treatment that is underlined, PcDNA3-vhsG, was intra-muscularly (i.m.) injected parallel to intraperitoneal (i.p.) injection of SAV Ag treatments. Five extra fish were sampled at 6 wpv and 3 wpc for analyses not included here. hpv; hours post vaccination; wpc; weeks post challenge. 1 The same fish sampled for nsP1 RT-qPCR as for the nAb assay; 2 the same fish were sampled for heart histopathology and for the nAb assay. SAV, salmonid alphavirus; Ag, antigen; nAb, neutralizing antibody.Spleen and head kidney (HK) were harvested from 8 fish per group at 12 and 48 h post vaccination (hpv) and were stored according to the manufacturer’s guidelines on RNAlater (Ambion, Applied Biosystems, Foster City, CA, USA). RNA isolation, cDNA synthesis and RT-qPCR were executed as described previously [26] with minor changes. Four hundred nanograms of total RNA were reverse transcribed (TaqMan Reverse Transcription Reagents kit; Applied Biosystems) into cDNA using random hexamer primers in 30-μL reaction volumes following the manufacturer’s guidelines. Primer and probe sequences and the efficiencies of the assays used in this study are presented in Table 2. cDNA samples (2.5 µL) were analysed in duplicates (target genes) or triplicates (endogenous control) in 20-µL reactions on a 7500 Fast Real-Time PCR system. The Cq-threshold was automatically set to 0.2 for analysis of both endogenous and target genes. Relative expression and statistics were calculated using Relative Expression Software Tool (REST) 2009 [39], which is based on Pfaffl’s mathematical model [40], where individual Cq-values were compared between saline-injected fish (control) and vaccine injected fish (test) and correlated to the endogenous control gene, EF1αβ, and PCR-efficiency. Serum samples from 8 to 15 individuals per group and time point (see Table 1 for details) were collected at 6 wpv and 3 and 6 wpc (weeks post challenge) and examined for SAV-neutralizing activity. To do so, virus initially incubated with diluted sera was left to adhere to Chinook salmon embryo-214 (CHSE) cells, and after 8 days, the presence of cell-associated virus was detected by an ELISA-method, as reported earlier [30]. Individual sera from each group were pooled, and half of the pooled sera were heat inactivated (HI; 56 °C for 30 min). Assays for both HI and not heat inactivated (NHI) sera were repeated 3 times for all samples. Two-fold dilutions of either HI or NHI serum were added in duplicate to a 96-well microtiter plate with maintenance media (MM; Minimum essential medium eagle (MEM) supplemented with 2% Foetal bovine serum (FBS)), giving a final dilution range from 1:20 to 1:640 (1:160 to 1:5,120 for the CpG/polyI:C-treated groups) when 100 µL of virus supernatant SPDV (SAV Subtype 1 isolate, 6,000 TCID50/mL) were added to the wells containing salmon serum dilutions. Neutralizing effects in serum were expressed as the highest reciprocal titres showing a >50% reduction of the positive control OD value using the following formula:
+		
+		
+		
+        To measure SAV levels during the viraemic phase, a quantitative real-time RT-PCR (RT-qPCR) was performed on viral RNA extracted from sera 3 wpc from 10 individuals per group, as described previously [26]. Non-structural protein 1 (nsP1) primers and the probe used for this assay are described in Table 1. Individual Cq-values were transformed to relative numbers by the following formula, where y represents the lowest Cq-value detected (i.e., the highest number of nsP1 transcripts) and where x is any of the other Cq-values detected:A sample was considered infected when it had a relative value between 1.0 × 100 and the cut off value of 3.0 × 10−7 (x = 37.5).Heart samples for detecting SAV-induced lesions were collected from 15 fish/group at 5 and 6 weeks post-challenge and immediately fixed in 3.5% formaldehyde in buffered saline at pH 7.0 (4.0 g NaH2PO4·2H2O, 6.5 g Na2HPO4·2H2O, 100 mL 35% formaldehyde and 900 mL dH2O). To evaluate the severity of SAV-induced heart lesions, a previously defined scoring system was used (no lesion: 0; minimal: 1; mild: 2; moderate: 3; severe: 4), where scores of 2 and more are defined to be specifically induced by an SAV infection [41]. The lesion scoring was done by Marian McLoughlin (Aquatic Veterinary Services, Belfast, Ireland) using “blinded” heart samples.All analyses were done in GraphPad Prism 5.0 if not mentioned otherwise. Differences in protection (SAV nsP1 RT-qPCR and histology) were statistically evaluated by the Kruskal-Wallis rank sum test with p < 0.05 as the significance limit, followed by Dunn’s post hoc test at a 5% level of significance. The histology test parameter used for statistical analysis was the severity of heart lesions, scored on the ordinal scale (0–4). Statistical analysis of the SAV nsP1 RT-qPCR used the individual Cq-values of each group as the test parameters. A modified expression of the relative percent protection score (RPPsc.) [29] was used to evaluate the level of protection against SAV induced by the tested treatments, based on the results obtained with the experimental methods (SAV-induced heart lesions histology or SAV-specific RT-qPCR assay). The advantage with this modified RPPsc. method is that the actual differences in degrees of severity of disease between the affected animals for the treated and control groups are taken into consideration. RT-qPCR data were statistically analysed by the Relative Expression Software Tool (REST 2009 v.2.0.13) [39].Primers and probe sequences for quantitative reverse-transcriptase PCR and PCR efficiency.Fw; forward. Rev; reverse. * The same primers were used for SYBR Green as for TaqMan.Transcription levels of selected antiviral innate genes, as well as selected B- and T-cell markers were analysed at 12 and 48 hpv in HK (Figure 1) and spleen (Figure 2) for the vaccine formulations in relation to the control group. It is worth noting the often high standard deviations underlining the highly individual immunological response, which is common in fish. Tables with average Cq-values are included as supplementary material.Relative expression of (A) antiviral, (B) B- and (C) T-cell markers in head kidney at 12 and 48 hpv for all treatments compared to saline-treated fish (expression is normalized to reference gene EF1aB). Relative expression is presented as histograms (the colour codes for each gene analysed are indicated below the histograms) calculated from fold induction by Pfaffl’s method (see Materials and Methods), and significant up- or down-regulation is based on data from REST 2009. Significant differences for all treatments compared to SAV Ag are highlighted with an *, against SAV Ag vhsG as Δ and against SAV Ag CpG/polyI:C as °, + or −, respectively, indicates the presence or absence of either SAV Ag, oil, CpG/polyI:C or vhsG.Relative expression of (A) antiviral, (B) B- and (C) T-cell markers in spleen at 12 and 48 hpv for all treatments compared to saline-treated fish (expression is normalized to reference gene EF1aB). Relative expression is presented as histograms (the colour codes for each gene analysed are indicated below the histograms) calculated from fold induction by Pfaffl’s method (see Materials and Methods), and significant up- or down-regulation is based on data from REST 2009. Significant differences for all treatments compared to SAV Ag are highlighted with an *, against SAV Ag vhsG as Δ and against SAV Ag CpG/polyI:C as °, + or –, respectively, indicates the presence or absence of either SAV Ag, oil, CpG/polyI:C or vhsG.Results showed that IFNa1, IFNγ and two antiviral proteins induced by IFNs (Mx and Vig-1) were strongly induced by the CpG/polyI:C-adjuvanted treatments, which confirms earlier findings [30]. In detail, for the CpG/polyI:C-adjuvanted treatments, IFNγ were highly upregulated at both time points in HK (Figure 1A; 60–150-fold) and spleen (Figure 2A; 50–100-fold), which was not seen for any of the other treatments. IFNγ is a cytokine secreted by T- and NK-cells and important for both innate and adaptive immune responses against viral infections. Upregulation of IFNa1 in HK and spleen for CpG/polyI:C-treated groups was significantly higher at 12 hpv than at 48 hpv, when a 10-fold reduction was seen. This is consistent with the fact that Type I IFN is an early induced innate antiviral actor, and in accordance, the antiviral genes, Vig-1 and Mx (induced by IFN Type I), accompanied the IFNa1-induction and were still highly expressed at 48 hpv in the CpG/polyI:C-treated groups with approximately a 40-fold induction in spleen and 100-fold for HK. Generally, the antiviral immune gene expression pattern seen for SAV Ag alone and for the formulations without CpG/polyI:C was moderate in both organs (zero- to two-fold in general). In HK, SAV Ag alone induced a low, but significant, upregulation of IFNγ, Vig-1 and Mx at 48 hpv compared to control fish. Antiviral gene responses in HK for vhsG immunized fish were in general moderate and not significantly higher than those in the other groups, except for the CpG/polyI:C-treated groups. Compared to SAV Ag CpG/polyI:C, the immune gene expression patterns for SAV Ag CpG/polyI:C vhsG were similar in both organs, except a slight, but significant, upregulation of IFNa1 and Vig-1 in HK at 48 hpv and downregulation of IFNγ in spleen at 48 hpv. In spleen at 48 hpv, IFNa1 was significantly upregulated in all vhsG-treated groups compared to SAV Ag alone (0.8- to 2.3-fold). Besides that, the expression pattern levels in spleen were similar to those in HK.PAX5, soluble (s) and membrane-bound (m) IgM were chosen as B-cell markers. PAX5 belongs to the family of paired box transcription factors and is present during early B-cell development, but must be repressed for plasma cell differentiation to take place [42]. Expression of mIgM often occurs in parallel to PAX5, and the ratio of mIgM vs. sIgM is important in relation to B-cell development. Early and developing B-cells have higher levels of mIgM and no or low sIgM levels, whereas a shift in their ratio indicates the presence of antibody producing B-cell populations [43]. In general, induction of all three B-cell markers was low at both time points, and the expression pattern varied for the two immune organs. At 12 hpv, a slightly higher expression of PAX5 and mIgM was measured in HK (Figure 1B) for vhsG-treated groups with fold inductions from two to four compared to the other treatments (approximately 1.5 to 2.5). PAX5 was the only B-cell marker induced at 12 hpv in HK of fish treated with SAV Ag, SAV Ag oil or SAV Ag CpG/polyI:C. At 48 hpv, a slight increase of mIgM from 0.8- to 1.5-fold was present for these latter groups, which could indicate a later onset of early B-cell development in groups not receiving vhsG. In spleen (Figure 2B), no clear expression pattern could be seen, and the B-cell markers were hardly detectable until 48 hpv, except for the SAV Ag CpG/polyI:C-treated group, where both PAX5 (2.4-fold) and mIgM (2.3-fold) were induced at 12 hpv. At 48 hpv, the expression of PAX5 was reduced, and both sIgM and mIgM were induced for fish treated with SAV Ag CpG/polyI:C and SAV Ag CpG/polyI:C vhsG.Co-receptors for T-helper cells (CD4) and cytotoxic T-cells (CD8) were used as T-cell markers to further study early adaptive responses. Atlantic salmon contain two shorter CD4 domain molecules (CD4-2a and -2b) in addition to the four classical (CD4-1) domains [44]. To determine which T-cell subsets were activated, both CD4-1 and CD4-2a, in addition to CD8α transcript levels were measured in HK (Figure 1C) and spleen (Figure 2C) at 12 and 48 hpv. As for B-cell markers, the general induction of T-cell markers in both organs was low and, with a few exceptions, their mRNA levels differed little between treatments and sampling time points. As the supplementary data show, the basal levels of CD4-2a (average Cq-values of 29.7 and 27.5 at both time points for HK and spleen, respectively) were higher in both tissues and at both time points compared to CD4-1 (average Cq-values of 33.6 and 32.4 at both time points for HK and spleen, respectively), although CD4-2a levels were not affected in the two organs at either time point. CD4-1 and CD8α were the most highly upregulated in HK at 12 hpv in groups co-injected with vhsG, ~2.3- to four-fold for both markers, which declined to an average of 1.3-fold for both CD4-1 and CD8α over the next 36 h. At 48 hpv, CD4-1 and CD8α levels in HK were significantly higher for the SAV Ag alone treatment (2.6 and 2.2, respectively) compared to the other treatments (0.8–1.4 for CD4-1 and approximately 1.25 for CD8α). In spleen, the overall expression levels of T-cell markers were very low at both 12 and 48 hpv, except at 48 hpv for SAV Ag CpG/polyI:C vhsG and vhsG alone, where both CD4 markers were upregulated. The presence of anti-SAV neutralizing responses in sera was measured at 6 wpv and at three and 6 wpc by a viral neutralization assay. It is well known that heat sensitive factors in serum may augment the neutralizing activity of Ab [45], and therefore, neutralizing activity was measured both with HI and NHI sera. For the NHI sera (Figure 3A), detectable neutralizing antibody titres (nAbTs) were present from 6 wpv for all groups, except groups treated with SAV Ag oil vhsG, vhsG alone or saline. The highest nAbTs were found in the SAV Ag CpG/polyI:C-treated group with titres of 640 before challenge that rose to 1280 and further to 2560 at three and 6 wpc, respectively. Fish treated with SAV Ag CpG/polyI:C that also received the vhsG i.m. injection showed the second highest nAbTs of 640 and 320 at 6 wpv and 3 wpc, respectively and 640 at 6 wpc. While all groups receiving a SAV Ag formulation, except the SAV Ag oil vhsG group, mounted a detectable neutralizing response before challenge, groups receiving either vhsG alone or saline had detectable nAbTs first after challenge. For vhsG alone and saline, the nAbTs are most likely induced upon exposure to the challenge virus, with titres of 80 (3 wpc) and 160 (6 wpc) for vhsG and 160 for saline at both three and 6 wpc. For HI sera, nAbTs were only present in three groups pre-challenge (Figure 3B). CpG/polyI:C-adjuvanted treatments provided the highest responses, with nAbTs ranging from 640 at 6 wpv to 1,280 at 6 wpc for SAV Ag CpG/polyI:C, and for SAV Ag CpG/polyI:C vhsG, the generation of nAbs was consistent with a titre of 160 at all sampling points. No positive sera were found among the fish injected with SAV Ag, SAV Ag oil or SAV Ag oil vhsG, while SAV Ag vhsG had detectable nAb responses both pre- and post-challenge. Fish treated with vhsG and saline, where >80% of the fish in both groups had positive SAV-specific heart lesions at 6 wpc (Figure 4B), showed detectable nAbTs at 6 wpc (80 for both treatments).Six weeks after vaccination, all fish were challenged by cohabitation with an SAV Subtype 3 isolate, and vaccine-induced protection was measured at three, five and 6 wpc (Figure 4). No mortality was observed after challenge. At 3 wpc, during the viraemic phase [46], viral RNA from sera were isolated, and SAV nsP1 transcript levels were detected by RT-qPCR. At 3 wpc, 70% (seven out of 10 fish) of the saline-treated fish had SAV positive sera, thus indicating a successful challenge (Figure 4A). Four of the six water-formulated SAV Ag treatments, namely SAV Ag, SAV Ag CpG/polyI:C, SAV Ag vhsG and SAV Ag CpG/polyI:C vhsG, provided full protection (RPPsc. = 100%) against SAV at 3 wpc, with non-detectable Cq-values. For the two oil-formulated groups, SAV Ag oil and SAV Ag oil vhsG, 20% and 10% of the fish had nsP1 positive sera, leading to RPPsc. of 71.4% and 85.7%, respectively. Furthermore, based on the prevalence of viremia determined by nsP1 RT-qPCR, there was a significant difference between all water- and oil-formulated SAV Ag treatments compared to saline-treated fish, except for SAV Ag oil (RPP.sc. = 71%). The prevalence of nsP1 positive fish in the group treated with vhsG alone (nine out of 10 serum positive fish) was significantly higher than the prevalence in the other treatment groups, except the saline group. Protection for the vhsG alone treatment was less than for the saline injected fish with an RPPsc. of −0.28%.Vaccine-induced anti-SAV neutralizing titres from not heat inactivated (A) and heat inactivated (B) sera, collected at 6 wpv, 3 wpc and 6 wpc (the colour codes for each time point are next to the y-axis). Titres representing a 50% reduction, calculated as described in the Materials and Methods, are shown above the histogram corresponding to each treatment. + or −, respectively, indicates the presence or absence of either SAV Ag, oil, CpG/polyI:C or vhsG.Heart tissue was sampled at five and 6 wpc to evaluate the severity of SAV-induced heart lesions by histological scoring. Protection based on the reduction of the severity of SAV-induced heart lesions was comparable to the protection shown through the reduction of the prevalence of viremia at 3 wpc for all treatments. Here, only histology data for 6 wpc are presented (Figure 4B), given that the prevalence of fish with SAV-specific heart lesions in the control group was highest at 6 wpc (80% vs. 60% at 5 wpc), where 10 out of 12 individuals had severe heart lesions. Further, at 6 wpc, lesions in the group vaccinated with SAV Ag oil vhsG were reduced, and only one out of 15 fish showed moderate lesions (RPPsc. 83.3%). The group treated with SAV Ag oil had an RPPsc. of 58.3%, and five fish showed mild to severe lesions, compared to an RPPsc. of 100% at 5 wpc (not shown), with only two fish showing minimal heart lesions. However, there was no significant difference in protection for that treatment between both time points.Protection against pancreas disease (PD) in vaccinated and control groups based on (A) the reduction of the prevalence of viremia and (B) the reduction in severity of SAV-specific heart lesions. (A) Relative SAV nsP1 expression at 3 wpc measured by SAV nsP1 RT-qPCR for each treatment group. Individual Cq-values were transformed to RelCq numbers, as described in the Materials and Methods. One (1.0 × 100) indicates the highest presence of nsP1 transcripts. Sera below the dotted line (cut off; 3.0 × 10−7) were considered negative, and sera below the solid line had undetected Cq-values. Relative SAV nsP1 values are presented as black (<3.0 × 10−7) and blue (≥3.0 × 10−7) dots. (B) The distribution of individual heart lesion scores assessed by histology at 6 wpc for each treatment group. A score of ≥2 was set as the cut off (indicated by the dotted line). Individual heart lesion scores are presented as black (<2) and blue (≥2) dots. Relative percent protection score (RPPsc.) values corresponding to each group are shown above Graphs A and B. + or −, respectively, indicate the presence or absence of either the SAV Ag, oil, CpG/poly I:C or vhsG DNA construct.How innate and adaptive immunity interact upon vaccination affects the outcome of protection. Especially, TLRs, included in the PRR-family, have emerged as key components of the innate immune system, activating signals critically involved in the initiation and maintenance of adaptive responses. Thus, TLR stimuli can be exploited as powerful adjuvants to elicit both primary and anamnestic immune responses. In light of this, our group used a combination of a selected CpG ODN [47] and a synthetic dsRNA (polyI:C), known to activate PRR-family members specialized in viral and nucleic acid detection (TLR-3, -9, -22 and RIG I), as a model to study the mechanisms of adjuvant action in bony fish. We showed that this TLR-ligand adjuvant combination induced a strong modulation of core response genes and increased levels of nAbs [30].Here, to follow up and to extend this model, the CpG/poly I:C combo was i.p. injected alone or combined with an i.m. injection of the VHSV G DNA genetic adjuvant. Montanide ISA763A was included as a control adjuvant, i.p. injected in combination with the genetic adjuvant. This was to determine if any enhanced protective responses were induced, since oil-adjuvanted formulations previously had provided a lower protection, most likely due to depot effects [29,30]. An inactivated SAV whole-virus formulation was used as the test Ag and injected i.p. with the same suboptimal dose for all formulations, hypothesized to give about 70% protection based on previous unpublished results. However, in the present SAV challenge, fish injected with SAV Ag alone were fully protected (RPPsc. of 100%), thus preventing the detection of differences in protection between non-oil-based treatments. SAV Ag formulated with Montanide ISA763A oil had a lower efficacy (RPPsc. of 58.3%–85.7%) and produced lower nAb titres (25%–70% reduction) compared to the formulations receiving the equivalent water-based formulation, a difference that could be explained by the slower release of the Ag from the vaccine depot for the oil-adjuvanted formulations.Correlating to earlier reports, the present CpG/polyI:C formulation induced a distinctively higher upregulation of early innate antiviral transcripts (IFNa1, Vig-1 and Mx) and also higher titres of nAbs compared to all other treatments [26,29,30]. nAbs are thought to be the primary correlate of protection against SAV [7,9,10], and the results suggest that an efficient clearance of virus mediated by Ab is possible. Interestingly, IFNγ expression levels were highly elevated in the CpG/poly I:C-adjuvanted groups. IFNγ, the hallmark of Th1 responses in mammals, is produced mainly by activated T-cells, NK cells and NKT cells; however, other cells, including macrophages/DCs, as well as B-cells are known to express IFNγ upon CpG-stimulation [48]. In support of this, a recent paper showed that salmon MHCII-positive mononuclear phagocytes, as well as B-cells and putative T-cells showed highly upregulated IFNγ transcript levels upon CpG-stimulation [49]. Since IFNγ has been shown to upregulate TLR9 expression in salmon leukocytes [22], the increased levels of this cytokine may represent a positive feed-back loop, where the secreted IFNγ upregulates the TLR9 receptor, and thereby, the cell’s responsiveness to its own agonist are increased.In higher vertebrates, B-cells activated via PRRs and/or by Ag cross-linking of the B-cell receptor rapidly respond, proliferate and differentiate into IgM secreting short-lived plasma cells (SLPC). T-cell help is needed to induce long-term B-cell memory, represented by memory B-cells or long-lived plasma-cells (LLPC) [50,51]. Bony fish Ab secreting cells (ASCs) are known to possess comparable B-cell subpopulations [52]; however, the understanding of the mechanisms by which these subpopulations are produced and distributed is scarce. Since germinal centres and antibody isotype shifting are not found in bony fish, the classical T-helper function in fish can be questioned. It is thus possible that CpG ODNs (through PRRs) can activate salmon B-cells directly, so that once activated, they start to proliferate and mature into ASCs. This would allow polyclonal activation of the entire B-cell pool, which has been demonstrated for mammalian B-cells [53,54]. Salmon B-cells express TLR9 and are responsive to their own agonist, CpG DNA [55]. Here, the CpG/poly I:C-adjuvanted vaccines were injected into the peritoneum, and recent studies have revealed that IgM-positive cells dominate the peritoneal cavity of unstimulated rainbow trout [56]. Upon inflammatory stimulation, these IgM positive cells were found at high levels 72 h post-injection. Since bony fish B-cells are phagocytic [57], it is possible that the vaccine can be engulfed by B-cells in the peritoneum and directly activated by CpG ODNs to differentiate into ASCs or, alternatively, migrate into secondary immunological organs, such as HK or spleen, and at those sites become ASC. This interesting possibility should be further elucidated in future studies.Moreover, Desvignes et al. [9] have previously suggested that the complement in general might aid in the clearance of SAV. Other studies have since then shown that salmonid Abs are dependent on complement activity to neutralize VSHV and IHNV [58,59], which both are enveloped viruses. SAV is also an enveloped virus; hence, it is possible that complement factors are involved in SAV neutralization, as well. A previous study indeed showed induced levels of complement component C4 (classical pathway) in salmon treated either with saline or with CpG/polyI:C after SAV challenge [29]. In this study, a major fraction of the nAb titres were heat sensitive, underlining the involvement of the complement system in the clearance of SAV. The addition of naive salmon serum as the complement source was evaluated in the virus neutralization assay using HI sera samples. Interestingly, for the sera with low titres, i.e., SAV Ag CpG/poly I:C vhsG, the fresh complement increased nAbs, while for the high titre group (SAV Ag CpG/poly I:C), there was no increase in nAbs (results not shown). A possible explanation for the variation in the results for the different groups could be that in the groups with very high nAbT, the neutralizing activity by the Abs per se is very efficient, and therefore, the complement does not provide any additional effect. In the groups with lower nAbT, the results indicate that combining the complement and Abs increases neutralization, supporting a role of complement-mediated neutralization, for example, by the classical complement pathway. Further studies aimed at elucidating the significance of the complement for the neutralizing responses need to be addressed.B- and T-cell markers were, in general, not, or very modestly, induced for all formulations tested, which could be due to the early sampling time points. The results show that fish treated with SAV Ag CpG/polyI:C had a slight, but notable, increase of PAX5 transcripts levels at 12 hpv in both HK and spleen. In spleen, PAX5 induction occurred in parallel to that of mIgM at 12 hpv, while at 48 hpv, PAX5 expression was reduced and mIgM remained stable. This indicates that an early B-cell development takes place [43]. In rainbow trout, activated B-cells differentiate into plasmablasts and plasma cells (PC), both in HK and spleen, and are distributed through the blood system to peripheral tissues [43,52], where hydroxyurea-resistant PC can migrate back to the anterior kidney and may persist there as LLPCs. In mammals, LLPCs are generated by migration to a supportive niche in the bone marrow [50]. The ability of LLPCs to produce Ab for months to years without the stimulating Ag relies on specialized cues. One suggested cue is Type I IFN, which, when injected as an adjuvant in mice, has been shown to induce both short- (10 dpv) and long-lived (26 wpv) Ab production [60]. It has been suggested that the signals induced by Type I IFN affect either migration to survival niches or differentiation of PC [61]. It may well be possible that CpG/polyI:C through its strong induction of Type I IFN could enhance the generation of a, if present, similar long-lived Ab production in salmon, and our intent is to further investigate this aspect.The immunological mechanisms behind the full protection provided by SAV Ag alone have been reasoned to depend on a T-cell independent (TI) nAb response, as that formulation did not induced an antiviral immune gene response at 5 dpv [30]. Here, a moderate induction of IFNa1, Mx, Vig-1 and IFNγ was evident in HK of fish treated with SAV Ag alone at 12 hpv. Inactivated viral Ags based on other enveloped viruses have been shown to trigger IFN responses [62,63]. Here, the actual mechanism responsible for the IFN responses observed in vivo with SAV Ag alone is unknown; nonetheless, by analogy with other alphaviruses, it can be hypothesized that the inactivated SAV particles present in the Ag preparation might induce the Type I IFN response seen, possibly through binding to the mannose receptor of N-glycans present on SAV E2, as observed for other alphaviruses [64] and other enveloped viruses [63]. The effect that the moderate induction of innate responses has on cellular immunity later in the course of the challenge is complex to interpret, owing to the limited knowledge on cellular immunity against viral infections in fish. Interestingly, NHI sera from fish treated with SAV Ag alone displayed nAb activity, while there was no detectable nAbT in HI sera for the group receiving SAV Ag alone. This variation between NHI and HI sera emphasizes the importance of the complement in the clearance of SAV and also the suggested ability of CpG to activate B-cells polyclonal through TLR 9, based on the high generation of heat-stable nAbs evident here for SAV Ag CpG/polyI:C compared to SAV Ag alone.To our knowledge, very few studies have been performed on teleost to investigate the immunostimulatory and protective effects of an i.p. vaccine injected simultaneously with a DNA-based adjuvant injected i.m. The efficacy of the DNA vaccines based on the glycoprotein G from VHSV and IHNV are well-documented; these vaccines have been shown to induce a long-lasting protection against these viruses [65,66], and a commercial IHNV-G DNA vaccine is approved for use in Canadian aquaculture [67]. An early induced cross-protection after VHSV G DNA vaccination has been seen following infection with nodavirus in turbot (Scophthalmus maximus) [68] and with the heterologous Novirhabdovirus IHNV in rainbow trout. This study also showed that the DNA expressed G protein does not confer protection against bacterial diseases [69], hence emphasizing that the early non-specific protection provided is purely antiviral. In one study on rainbow trout [70], two or four CpG motifs were incorporated into the plasmid backbone along with VHSV G open reading frame, and this modified DNA vaccine gave significantly higher immune responses (Mx and IFNγ) and a significantly higher production of anti VHSV nAb compared to when plasmid without CpG motifs were administered. Based on these findings, it was hypothesized that the G protein might be able to contribute with a non-specific antiviral protection also against an SAV infection. As evident here, VHSV G glycoprotein DNA vaccination showed no additive effects on early protective responses against SAV. Furthermore, co-injecting vhsG with CpG/poly I:C or oil-adjuvanted SAV Ag neither caused additive nor synergistic effects on the immune gene expression. Muscle samples (n = 8) harvested at 48 hpv from vhsG co-injected groups were analysed for vhsG-transcripts, and the levels varied between individual fish, where some individuals had undetectable vhsG mRNA levels (results not shown). The low transcription of vhsG in muscle and the weak effect on innate immune gene expression could be explained by the relative early sampling time points. Indeed, studies on rainbow trout have shown that it can take as long as 14 dpv (earlier samplings at two and 7 dpv did not show any expression) before ISGs are significantly upregulated in spleen [70], and similar results in the liver of rainbow trout [71] and kidney of Japanese flounder (Paralichthys olivaceus) [72] have also been described. Protective immune responses observed after challenge in G DNA-vaccinated rainbow trout have been related to increased Mx expression and other ISGs [31,35,73], preceded by the upregulation of IFN Type I and II [35,74]. This early non-specific protection has been followed by a more specific long-lasting anti-viral response, based on both humoral (nAb) and cellular protective mechanisms (MHCII, T-cells) [73,75,76]. Interestingly, both B- and T-cell markers analysed for all vhsG formulations in this study had the highest expression compared to all other groups and were most distinct at 12 hpv in HK. This could indicate that the glycoprotein G has been expressed, and the high standard deviations indicate a high variation between individuals related to either immune gene expression profile and/or of vhsG expression.In regards to humoral immunity, diverse responses were seen in the different groups i.m. injected with vhsG. While the SAV Ag oil vhsG treatment showed reduced nAbs titres compared to SAV Ag oil, the water-based SAV Ag formulation co-injected with vhsG had higher titres than SAV Ag alone at both 6 wpv and 6 wpc. Finally, a reduction in neutralizing responses was present when vhsG was co-injected with the SAV Ag CpG/polyI:C formulation, and the NHI nAbTs were reduced by half or more at all three time points compared to SAV Ag CpG/polyI:C. These data show that vhsG induced a slightly higher production of nAbs for the SAV Ag-treated fish co-injected with vhsG, and it also suggests the presence of factor(s) that cause a reduction in humoral responses when CpG/polyI:C-adjuvanted SAV Ag is co-injected with vhsG. For both mammals and teleosts, it has been shown that polyvalent vaccination often negatively affects the generation of specific Ab [77,78,79]. Skinner et al. [78] have shown that concurrent i.p. vaccination of a polyvalent oil vaccine with i.m. injection of a rhabdovirus-specific DNA vaccine delayed seroconversion of IHNV-specific nAb compared to DNA vaccination alone. The negative effects on nAbT when SAV Ag CpG/poly I:C was co-injected with the vhsG vaccine can thus have several explanations, such as antigenic competition or Ag immunodominance. However, without the possibility of analysing the presence of vhsG-specific Ab (due to the limited amount of sera remaining), neither explanation can be claimed for certain.To summarize, the vaccine based on a suboptimal dose of inactivated SAV whole virus Ag formulated with CpG/polyI:C induced the highest nAb responses, followed by the combined treatment of SAV Ag vhsG and, finally, the oil-adjuvanted SAV Ag formulation. The expression of several innate antiviral immune genes showed consistently elevated levels in the groups injected with CpG/polyI:C compared to the other adjuvants tested. B- and T-cell markers were, in general, not, or very modestly, induced for all formulations tested. For groups receiving the vhsG DNA vaccine, no antiviral immune gene expression was detected at these early time points, but as indicated by Martinez-Alonso et al. [70], a later induction could be possible. Pre-challenge humoral responses for SAV Ag co-injected with vhsG had slightly higher levels of both heat-sensitive and heat-stable neutralizing factors compared to SAV Ag alone, suggesting a very moderate adjuvant effect of vhsG. No enhancement of nAbs responses was evident when co-injecting vhsG with the TLR-ligand-adjuvanted SAV Ag formulation. Instead, a negative influence was observed, which may result from Ag competition.Overall, these data show that CpG/polyI:C is a potent TLR-ligand combo, which could be used in future salmonid vaccination strategies against SAV. Further work should be aimed at investigating the duration of the efficacy of these TLR-ligands as adjuvants, while at the same time facilitating their administration. One potential strategy may be to protect the TLR-ligand/Ag formulation from degradation by encapsulating them in protective vehicles, such as nanoparticles, as recently described in [80]. Considering the potency of the TLR-ligand combo tested here, one can anticipate that CpG/polyI:C used as an adjuvant in an SAV Ag formulation could provide an Ag dose-sparing effect.The authors would like to thank Niels Lorenzen at the National Veterinary Laboratory (Danish Technical University, Copenhagen, Denmark) for providing the VHSV-G DNA plasmid construct and Barbara Spasowska and Glenn Sundnes at MSD Animal Health Innovation AS for their excellent work with the organization of all fish samples.HLT carried out all of the experiments for this study except the nsP1qPCR, participated in the data-analyses and drafted the manuscript. SV participated in the design of this study, nsP1qPCR, data-analyses and revision of the manuscript. MMcL carried out the histopathology analyses, KEC contributed to the virus neutralization assay. SG provided some of the qPCR assays. PF participated in the design and revision of the manuscript. JBJ participated in the design, interpretation of the data and revision of the manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Adjuvants are crucial components of vaccines. They significantly improve vaccine efficacy by modulating, enhancing, or extending the immune response and at the same time reducing the amount of antigen needed. In contrast to previously licensed adjuvants, current successful adjuvant formulations often consist of several molecules, that when combined, act synergistically by activating a variety of immune mechanisms. These “combination adjuvants” are already registered with several vaccines, both in humans and animals, and novel combination adjuvants are in the pipeline. With improved knowledge of the type of immune responses needed to successfully induce disease protection by vaccination, combination adjuvants are particularly suited to not only enhance, but also direct the immune responses desired to be either Th1-, Th2- or Th17-biased. Indeed, in view of the variety of disease and population targets for vaccine development, a panel of adjuvants will be needed to address different disease targets and populations. Here, we will review well-known and new combination adjuvants already licensed or currently in development—including ISCOMs, liposomes, Adjuvant Systems Montanides, and triple adjuvant combinations—and summarize their performance in preclinical and clinical trials. Several of these combination adjuvants are promising having promoted improved and balanced immune responses.Adjuvants are crucial components of vaccines, both for human and animal applications. Adjuvants were initially developed empirically by co-formulating vaccine antigens with a variety of molecules including oils, salts, and carbons. Our growing understanding of the immune system, however, and in particular the innate immune system, has enabled us to develop adjuvants according to a more rational and focused approach rather than through “trial and error.” Indeed, adjuvant research has become an integral part of vaccine development. It combines a variety of disciplines, including chemistry, biochemistry, molecular biology, and immunology. Many novel adjuvant technologies have been developed or are in the pipeline for future vaccine candidates. Such novel technologies include combination adjuvants, which consist of more than one adjuvant component and which often act synergistically by stimulating and activating a variety of cells and immune mechanisms. Here, we will review several of the best-known combination adjuvants, including liposomes, ISCOMs, montanides, nanoemulsions and Adjuvant Systems, and summarize their performance in preclinical and clinical trials.Cationic liposomes have been studied for many years as delivery vehicles/adjuvants. Liposomes protect antigens from degradation, deliver them to antigen presenting cells (APCs), and can be used for mucosal delivery. Many liposomes mediate retention of the vaccine at the site of delivery, and some have immunostimulatory properties, although frequently other compounds such as toll-like receptor (TLR) ligands need to be incorporated for optimal adjuvanticity. Cationic liposomes may contain the following lipids: dimethyldioctadecylammonium (DDA), 3-(N-[N',N'-dimethylaminoethane]-carbamoyl) cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-(1-[2,3-dioleyloxy] propyl)-N,N,N-trimethylammonium (DOTMA), octadecenoyloxy (ethyl-2-heptadecenyl-3-hydroxyethyl) imidazolinium (DOTIM), N-palmitoyl-d-erythrospingosyl-1-O-carbamoyl spermine (CCS), 1,2-dioleyl-sn-glycero-3-ethylphosphocoline (DOEPC) and 3-tetradecylamino-tert-butyl-N-tetradecylpropion-amidine (diC14-amidine) (reviewed by [1]). The mechanism of action, while not completely understood yet, is dependent on the nature of the lipids in the formulation, and thus varies widely between different liposomes. One common characteristic related to the cationic charge is the enhanced uptake of DNA or protein by target cells [2]. One of the major drawbacks of liposomes as vaccine delivery vehicles has been their instability. While numerous animal trials have been reported, only recently have liposome-based vaccine formulations moved into human clinical trials. This has become possible by the incorporation of helper lipids stabilizing the liposome formulations [2]. DDA has been used as adjuvant in animal trials since 1966 [3], either alone or in combination, and was tested in humans in 1970 and 1973 in combination with tetanus toxoid and alum [4,5]. However, although the vaccine was safe and induced antibody responses, these were not considered robust enough. Consequently, DDA has been combined with other compounds including Emulsigen®, monophosphoryl lipid A (MPL), trehalose dibehenate (TDB), monomycoloyl glycerol (MMG) and polyI:C. We combined DDA with Emulsigen and found robust immune responses in cattle [6]. One of the most promising combinations is DDA with TBD (cationic adjuvant formulation [CAF01]). TDB not only stabilized the liposomes, but also significantly enhanced T-cell responses to Mycobacterum bovis Ag85B-ESAT-6 in mice, in particular T cells producing IFN-γ and IL-17; this correlated with an increase in IgG2 levels, while IgG1 levels remained the same [7]. The immune responses induced by Ag85B-ESAT-6 formulated with CAF01were long-lived and protective [8]. In addition to mediating depot formation of the vaccine formulation, CAF01 appears to promote influx/activation of DCs into the injection site [9]. Interestingly, in a recent report, the antibody and CD8+ IFN-γ responses induced by small unilamellar DDA/TDB liposomes were higher than those elicited by multilamellar DDA/TDB liposomes; however, addition of TLR3 or TLR9 ligand enhanced the immune responses, in particular CD4+ and CD8+ T cells, induced by the multilamellar ones, though this was not found for smaller liposomes [10]. CAF01 has been or is being tested in several phase I clinical trials, one against tuberculosis in combination with Ag85B-ESAT-6 (ClinicalTrails.gov identifier NCT00922363) and two with the HIV peptide cocktail AFO-18 (ClinicalTrials.gov identifier NCT01141205) (Table 1). MPL (a TLR4 ligand) and MMG, two of the other compounds combined with DDA, are both found in bacterial cell walls and promote Th1-biased immune responses. MPL is derived from LPS, a TLR4 ligand, and when included in an Ag85B-ESAT-6—DDA liposome formulation, it enhanced protection against tuberculosis both in mice and in cynomolgus monkeys [11,12]. Prominent inflammatory responses to DDA/MPL were observed in subcutaneously immunized mice, including high local levels of pro-inflammatory cytokines, chemokines and a pronounced influx of neutrophils, monocytes/macrophages and activated natural killer cells [13]. The antigen-specific T-cell responses induced by CAF01 (DDA/TDB), DDA/MPL and DDA/MMG in mice were all comparable. However, whereas all three compounds are immunostimulatory, TDB and MMG have the advantage over MPL of stabilizing the liposomes and are thus more promising. DOTIM was originally used for in vitro and in vivo gene delivery into cells [14]. However, in view of its ability to mediate uptake of DNA into endosomes [15], it was more recently combined with DNA, TLR3 or TLR9 ligand. When co-administered with antigen, the combination of DOTIM and CpG ODN promoted significantly enhanced antigen-specific T-cell responses when compared to delivery of protein with CpG ODN alone [16]. The DOTIM-based liposome in combination with cholesterol and plasmid DNA, designated JVRS-100 adjuvant, promotes pro-inflammatory responses followed by the development of Th1-type responses. Formulations with JVRS-100 have been shown to be efficacious in rodent models against several viruses including hepatitis B virus [17], influenza virus [18], herpes simplex virus-2 (HSV-2) [19], and Rift Valley fever virus [20]. This liposome formulation is also suitable for mucosal delivery as demonstrated in mice where protection from pneumonic tularemia [21] and plague [22] was induced. Currently, JVRS-100 adjuvant is being tested in phase I and phase II clinical trials with an influenza split vaccine (ClinicalTrials.gov identifier NCT00936468; ClinicalTrials.gov identifier NCT00662272) (Table 1). Liposomes and TLR agonists: Clinical studies.Immune stimulating complexes (ISCOM) are ring-like structures containing cholesterol, phosphatidylcholine and saponins, mostly QuilA. ISCOMs have been tested in a variety of species, including small and large animals. The actual ISCOM matrix can directly associate with antigens or be formed first and then added to the formulation at a later time. An important advantage of ISCOMs is their excellent stability for over one year at 4 °C. While the exact mechanism of action is still unclear, ISCOMs have proven to be highly effective when delivered via mucosal surfaces. ISCOMs interact with dendritic cells (DCs) and can enhance cross-presentation of the incorporated antigen [24], followed by an efficient induction of both CD4+ and CD8+ antigen-specific T-cell responses [25]. ISCOM vaccines are known to induce long-lasting antibody responses, a balanced Th1/Th2 response, and induction of cytotoxic T lymphocytes [26]. ISCOMs have been used in combination with a parenterally administered H5N1 influenza vaccine in healthy adults (PANFLUVAC). In this study, 50 μg of a 3rd Generation ISCOMs was combined with various concentrations of recombinant hemagglutinin (HA). The trial is currently underway (ClinicalTrials.gov identifier NCT00868218) (Table 2). In another clinical trial, ISCOMs have been used with the melanoma vaccine NY-ESO-1 ISCOMATRIX in patients with measurable stage III or IV melanoma. Four doses of the vaccine containing 120 μg ISCOMATRIX combined with 100 μg of NY-ESO-1 protein were given to patients with advanced melanoma. The trial is currently ongoing. To improve their efficacy, ISCOMs are often used in combination with immune stimulators such as Cholera Toxin A (CTA) subunit or TLR ligands. For example, fusion proteins have been tested including the CTA1-DD, which combines the enzymatically active CTA1-subunit with a B-cell targeting moiety (DD), derived from Staphylococcus aureus protein A [27,28]. Intranasal immunization of mice with hemagglutinin and neuraminidase from influenza strain PR8 resulted in enhanced IgA and CD4+ T cells [29]. Mucosal IgA and CD4+ T-cell response were enhanced after intranasal administration and consequently provided 100% protection against challenge infection. The mechanism was CTA1 enzyme-dependent and designed to target B cells. Another example includes co-formulation of ISCOM and a TLR9 ligand (CpG ODN). In particular, when used in cancer vaccines, this combination resulted in regression of tumors in a pancreatic cancer mice model using tumor induction by injection of PANC02 tumor cell line transfected by OVA gene [30]. The vaccine, ISCOM+TLR9+OVA antigen, was able to induce strong immunity against the tumor and induce effective memory responses, including a high percentage of CD8+IFNγ+ T cells after re-induction of the tumor. Noteworthy, antigen and ISCOM alone or CpG ODN alone were not efficient to induce anti-tumoral immune response and protection in mice. Another example to improve ISCOMS includes tubular immunomodulatory complexes [31,32] or the association with another adjuvant such as Montanide [33]. For example, when used in combination with a recombinant protein of food and mouth disease virus (FMD) in guinea pigs, higher immune responses were found when ISCOMs and Montanide ISA 206 were used in combination. ISCOMs and IC31®: Preclinical and clinical studies.Montanide-based adjuvants have been used in both veterinary and human vaccines. These oil-based formulations have been successfully commercialized and are now available for animals in a vaccine against FMD. For human application, Montanide-based therapeutic vaccines have been developed for cancer [41]. The mechanism of action for this oil-based adjuvant includes the formation of a depot at the injection site, which enables the slow release of the antigen. Formulated antigens are concentrated and protected against degradation while phagocytosis is stimulated. Montanides can be improved by combining them with other adjuvants or immune modulators. An example of a Montanide-based adjuvant is the Incomplete Seppic Adjuvant (ISA™, SEPPIC Inc. Fairfield, NJ, USA), which acts as oil-emulsion adjuvant, and which has been tested in combination with a number of experimental vaccines including a malaria vaccine [42,43]. Also, Montanides and ISCOMs were tested with a recombinant protein vaccine for FMD virus using guinea pigs [33]. Interestingly, this combination showed the greatest promise in inducing early and long-term protection. Since activation of the innate immune system is one of the best ways to stimulate a systemic immune response, Montanides have been tested in combination with TLR ligands including LPS for TLR4, Poly (I:C) for TLR3, Imiquimod for TLR7 and CpG ODN for TLR9. Montanides have been evaluated in several clinical trials. For example, the Montanide ISA 51 was tested in combination with cancer antigens and cytokines including an evaluation of the effects of local Granulocyte-Macrophage Colony Stimulating Factors (GM-CSF) on skin DCs in melanoma patients [44,45]. Interestingly, low doses of GM-CSF did not enhance its immunogenicity [46]. Montanides and Poly (I:C) have been used for a phase I trial on 28 advanced ovarian cancer patients [47]. Using overlapping long peptides (OLP) from the human cancer-testis antigen NY-ESO-1, patients were treated with OLP alone, OLP+Montanide or OLP+Montanide+polyIC LC. No NY-ESO-1-specific antibodies or CD8+ T cells were detected after vaccination with OLP alone, though they were found in 46% (Ab) and 62% (TCD8+) patients respectively after vaccination with OLP+Montanide, and in 91% (both) patients respectively after vaccination with OLP+Montanide+Poly-ICLC. These results strongly indicate the importance of the adjuvants, especially when combined in a single formulation. In another study in melanoma patients, imiquimod was added to a combination of viral nanoparticles coated with CpG ODN and Montanide. The combination resulted in greater memory and effector CD8+ T cells responses [48]. Finally, CpG ODN was assessed to be efficient in cancer vaccine therapy with the same antigen NY-ESO-1 [49]. Treated patients were able to produce specific CD8+ T cells. Interestingly, for three different leukemia-associated antigens, no positive results were found using the CpG–Montanide combination [50]. Although only tested in very few subjects, it underlines the importance of the choice of adjuvants and antigen. Furthermore, Montanides have been tested in combination with very small size proteoliposomes (VSSP) in the treatment of prostate cancer [51]. VSSP is already included in a combination of adjuvants. However, the authors showed that association of a Gonadotropin Releasing Hormone (GnRH)-peptide with VSSP and Montanide ISA-51 was able to reduce size and weight of both testicles and prostate in a male rat model.Polycationic peptides enhance cellular uptake of proteins or bacterial DNA by cells [52]. IC30, which consists of poly-l-arginine, has been shown to efficiently transport tumor antigens to APCs [53]. IC30 adjuvant activity involves recruitment of MHC class II (+) cells at the injection site and migrating antigen-specific cells to the draining lymph nodes. Subcutaneous injection of IC30 with tumor antigen-derived peptide antigens led to antigen-specific T-cell responses, which were detectable for more than four months after injection suggesting a depot effect [54].Based on the ability of cationic compounds to enhance uptake, IC30 was combined with CpG ODN, a TLR9 agonist. The combination of IC30 and CpG ODN induced stronger antigen-specific T cells responses, which were detectable even one year after injection [55]. Apart from IC30, various other cationic antimicrobial peptides have been developed to treat infections. The potential of an antibacterial cationic peptide KLKL5KLK (KLK) to induce humoral and Th2-type immune responses against co-administered antigens after prime-boost immunizations have been reported [56]. This led to development of the novel adjuvant IC31®, where the potential of KLK to induce Th2 responses and that of TLR9 agonist to induce Th1 responses was exploited. The novel bi-component vaccine adjuvant, IC31® (Intercell AG, Vienna, Austria) consists of a vehicle based on KLK and the TLR9 agonist ODN1a. ODN1a, a single-stranded DNA-phosphodiesther, consists of repeats of the dinucleotides deoxyinosine and deoxycytosine [oligo-d(IC)13] [57]. CpG ODN was replaced by ODN1a due to potential side effects induced by CpG ODN such as production of systemic pro-inflammatory cytokines [58]. The preclinical studies with IC31® in various disease models in animals have shown potent immunogenicity and protection efficacy including antigen-specific humoral and cellular responses. These encouraging results pave the way for the novel combination adjuvant IC31® into clinical trials against various viral and bacterial infections (Table 2). The two components of IC31®, KLK and ODN1a, act via different mechanisms. Subcutaneous injection of fluorescent-labeled OVA, KLK, and ODN1a in mice led to depot formation at the site of injection, which was detectable up to 58 days after injection. However, co-administration of fluorescent-labeled OVA and ODN1a resulted in rapid clearance from the injection site, indicating the potential of KLK to induce a depot at the injection site [59]. IC31® formed a stable complex that protected both adjuvants and antigens from degradation leading to continuous stimulation of the immune system and induction of specific immune responses. In addition, co-administration of OVA with IC31® in TLR9 and MyD88 double knock-out mice completely abolished antigen-specific immune responses suggesting that the immune-stimulatory effects of IC31® depends on TLR9/MyD88 signaling pathways [59]. Since cationic peptides have the potential to enhance uptake of bacterial DNA, KLK may serve as a delivery vehicle to transport ODN1a inside the murine and human DCs to interact with intracellular TLR9 receptors [60]. Further engagement of TLR9 leads to NF-κB activation via the adaptor molecule MyD88, thereby triggering the immune responses. Being the only professional APCs, DCs are specialized in processing and presenting antigens to naïve T cells and induce activation of antigen-specific immune responses [61]. Presentation of antigens on MHC molecules, expression of co-stimulatory molecules, and secretion of cytokines are essential for optimal activation of naïve T cells. IC31® has been shown to upregulate the surface expression of MHC class I and co-stimulatory molecules CD80, CD86, CD40 and CD54 on murine bone-marrow dendritic cells (BMDCs). Further, in in vitro studies using OVA-pulsed BMDC, IC31® induced antigen-specific CD4+ Th cells proliferation and differentiation of OVA-specific CD4+ T cells into antigen-specific Th1 cell (IFN-γ producing) and Th2 cell (IL-4 producing) types [59]. In vivo studies suggests involvement of plasmacytoid DCs (pDCs) in the adjuvant activity of IC31®, as highly activated pDCs with enhanced expression of co-stimulatory molecules were found in the draining lymph nodes of mice injected with IC31®. In addition, IC31® induced potent cytotoxic effector T lymphocytes that killed target cells in an antigen-specific manner [59]. Various combination adjuvants consisting of a variety of molecules have been developed in recent years and are currently being tested in preclinical trials. This includes both dual and triple combinations and includes polymers such as poly(lactic-co-glycolic) acid, chitosan and polyphosphazenes [62,63]. A novel combination adjuvant was developed consisting of innate defense regulator peptide (IDR), polyphosphazene (PP) and TLR ligand, either CpG ODN or PolyI:C. Innate defense regulator peptides are short peptides that stimulate both innate and adaptive immunity and by activating antigen-presenting cells link innate and adaptive immunity. Innate defense regulator peptides are involved in a variety of immune functions including innate immune activation, wound healing, cell recruitment and to some extent, direct antimicrobial control [64,65,66]. Polyphosphazenes are synthetic polymers that are used as carriers for adjuvants and vaccine antigens and that on their own have modest adjuvant activity [67,68]. However, when combined with immune stimulators such as CpG ODN or PolyIC these molecules can form complexes that become highly immunogenic. The combination adjuvant was developed through screening of hundreds of molecules and testing in human, murine and porcine cells. The most promising candidates were assessed in vivo in a variety of animal species including cattle, pigs, sheep, mice and cotton rats in combination with a variety of vaccine antigens. Interestingly, when co-formulated, the adjuvants induced strong expression of cytokines and chemokines—such as Gro1a, TNF-α, IL-6—in stimulated cells and displayed a strong synergistic effect when used in combination [69]. Furthermore, the adjuvant platform can be formulated into microspheres that range in size from 100 nm to 2 μm and that enhanced the mucosal immune response following intranasal immunization. Interestingly, the proper selection of innate defense regulator peptide was found to be critical as some of these peptides could actually decrease the immune response [70]. These microparticles could be lyophilized and stored at room temperature for more than a year without any loss of specific activity [71]. A synergistic effect of the TLR agonist, IDR and PP on the magnitude of the humoral, and specifically cell-mediated, immune responses was demonstrated in murine and bovine models [72,73,74]. The adjuvant platform was highly effective against a variety of infectious diseases. For example, when combined with the Bordetella pertussis antigens pertussis toxoid, filamentous hemagglutinin, and/or pertactin, the vaccine induced protective immune responses in both mice and pigs against lethal infection with B. pertussis [75]. The vaccine induced an earlier onset and longer duration than existing commercial vaccines and was effective already after a single immunization even in the presence of maternal antibodies. Furthermore, the vaccine shifted the immune response to a more balanced or Th1-type response [76,77], Similarly, when the same adjuvant combination was used with a recombinant fusion protein of respiratory syncytial virus, strong immune responses were found that provided protection against infection in mice, cotton rats and lambs [78,79], When used in combination with chlamydia antigens or influenza virus antigens, strong immune responses were detected in vaccinated animals [69,80]. Adjuvant systems™ (GlaxoSmithKline (GSK)) are various combinations of classical adjuvants and immunostimulators specifically designed to tailor the adaptive immune responses against specific pathogens in a target population including children, elderly and immunocompromised individuals. Various adjuvant systems have been developed and a few are in clinical trials. AS03 and AS04 have already been licensed for use in humans. AS03 was approved for H5N1 prepandemic and H1N1 pandemic influenza vaccines and AS04 for human papilloma virus (HPV) (Cervarix™) and hepatitis B virus (HBV) (Fendrix®). AS04 was also licensed for herpes simplex virus (HSV) but was recently terminated due to low efficacy in clinical trials [81].The TLR4 agonist lipopolysaccharide (LPS) is a potent adjuvant but it is very toxic and causes septic shock. MPL is a “detoxified” derivative of LPS isolated from the Gram-negative bacterium Salmonella Minnesota R595 strain. While less toxic, MPL retains the immunostimulatory properties of LPS. AS04 (Adjuvant System 04), is a combination adjuvant containing MPL and aluminum hydroxide. AS04 is licensed for use in HPV and HBV vaccines. Another promising HSV vaccine, gD2/AS04, showed better immune protection and significantly reduced the viral load and viral shedding when compared to the gD2 vaccine adjuvanted with aluminum salts alone in guinea pigs [82]. In clinical trials, gD2/AS04 tested in HSV-1 and HSV-2 seronegative women showed 73% and 74% efficacy respectively against genital herpes infections [83]. However, in a randomized, double-blind phase III clinical trial conducted in 8323, 18–30 year-old, women seronegative for HSV-1 and HSV-2, the HSV-2/AS04 vaccine efficacy was only 20% and it failed to protect the women from genital disease [84].When combined with alum, MPL still retains its ability to interact with TLR4 and activate innate immune responses. This leads to activation of NF-κB and transient production of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ) and chemokines, such as CCL2 and CCL3 at the injection site [85]. CCL2 and CCL3 can promote recruitment of other immune cells, especially monocytes and macrophages, and activation and maturation of APCs, especially DCs at the site of injection. Increasing numbers of mature DCs migrate to the draining lymph nodes and interact with T cells to trigger the induction of antigen-specific adaptive immune responses [86]. MPL induces Th1-type immune responses by promoting IFN-γ production by antigen-specific CD4+ T cells, hence overcoming the shortcoming of alum, which induces only Th2-type immune responses. When combined with alum, MPL-induced stimulation of pro-inflammatory cytokines was neither improved nor inhibited by alum. However, alum did prolong the local cytokine responses induced by MPL at the injection site [85]. Since water-in-oil emulsions were too reactogenic for use in humans, GSK developed AS03, an oil-in-water emulsion. AS03 consists of dl-α-tocopherol, squalene, and polysorbate 80. Oil-in-water emulsions are less reactogenic and have better safety profiles. One such oil-in-water emulsion is MF59, which has been approved for human use in European countries. MF59 consist of squalene, polysorbate 80 and Span 85 and the only difference between MF59 and AS03 is the presence of α-tocopherol, a biodegradable and immunostimulatory oil. α-tocopherol is a form of vitamin E that is easily absorbed in human body [87]. Squalene, another component of AS03, is normally synthesized in the liver and found in various organs with highest secretion by human skin. Squalene is an essential intermediate molecule in the biosynthesis pathways of steroid hormone, cholesterol and vitamin D [88]. The last component of AS03, polysorbate 80 (polyoxy-ethylene sorbitan-20 monooleate) is added to stabilize the emulsion. It is a semi-synthetic molecule derived from naturally occurring fatty acid that is largely used as aqueous formulation stabilizer in the food and pharmaceutical industries [89]. Injecting antigen and AS03 72 h apart or in different limbs resulted in reduced influenza-specific antibody titers suggesting that AS03 adjuvant activity depended on the spatial and temporal co-localization with the antigen [90]. AS03 induced production of NF-ĸB, and subsequently cytokines and chemokines, including CCL2, CCL3, IL-6, CSF3 and CXCL1 in muscle and the draining lymph nodes of mice after injection [90]. Similar to alum and MF59, DCs were not directly activated by AS03. Antigen-loaded DCs, monocytes and granulocytes were found to migrate to the draining lymph nodes after injection of AS03. H5N1 split-virion A/Vietnam/1194/2004 influenza vaccine with AS03(A) resulted in increased production of polyfunctional H5N1-specific CD4+ T cells in volunteers aged 18–60 years [91]. An increase in antigen-specific CD4+ T cells might be responsible for persistence of increased antibody responses and induction of memory B cell responses by AS03. AS02 is a combination of oil-in-water emulsion with MPL and QS21 (MPLA+QS21+o/w emulsion) and was initially developed for malaria vaccines. QS21 has been shown to induce antigen-specific antibody, cell-mediated and CTL responses, including development of immunological memory in nonhuman primates [92,93,94]. AS02 was used for development of the RTS,S candidate malaria vaccine, which includes the circumsporozoite protein (CSP) as a vaccine antigen. RTS,S consists of a hybrid protein that includes a portion of the CSP fused to the hepatitis B virus surface antigen (HBsAg), which is expressed together with unfused HBsAg in genetically engineered yeast cells [95]. In phase I/IIa clinical trials, AS02-based malarial vaccines provided better protection (protecting six out of seven individuals) compared to AS03 and AS04-based formulations in malaria-naïve individuals challenged with P. falciparum through bites from mosquitoes [96]. Recombinant AS02-based malaria vaccine has been tested for safety, immunogenicity and efficacy in various phase I clinical trials. Apart from malaria vaccines, AS02 has been tested with tuberculosis, hepatitis B, HIV vaccines and cancer immunotherapy (Table 3). Adjuvant system (AS): Clinical studies.Malaria vaccine adjuvanted with AS02 has shown great promise. However, to further increase the efficacy and to eliminate the infected hepatocytes, the AS01B adjuvant system has been evaluated by GSK. The AS01 adjuvant system is a liposomal formulation comprised of immune-stimulants MPL and QS21 (MPLA+QS21+a liposomal suspension). When tested in rhesus monkeys, RTS,S/AS01 elicited higher antigen-specific antibody and IFN-γ producing Th1-type responses compared to any other adjuvant formulations [116]. Even when tested with MSP-1 antigen, AS01B adjuvanted vaccine elicited the highest anti-MSP1 antibodies and strong Th1 responses characterized by high numbers of IFN-γ secreting cells that were sustained for 24 weeks after final vaccination [121]. A RTS,S/AS01 malaria vaccine was more efficacious than a RTS,S/AS02A vaccine, inducing increased IgG titers and polyfunctional CD4+ T cells expressing IL-2, IFN-γ, TNF-α, or CD40L [117] (Table 3). In a more recent clinical trial in children, RTS,S/AS01E also induced higher CD4+ T-cell responses as compared to RTS,S/AS02D [119]. In view of the strong induction of cell-mediated immunity, AS01B was also evaluated in a human clinical trial with a TB vaccine in comparison to AS02. In this study, M72 antigen was formulated with either AS02 or AS01. While both M72/AS01 and M72/AS02 had an acceptable safety profile and elicited robust polyfunctional M72-specific CD4+ T-cell and antibody responses, the highest CD4+ T-cell responses were induced by M72/AS01 [118]. Thus, the M72/AS01 was chosen for further vaccine development. Nanoemulsions have a mean droplet diameter of 50–1,000 nm and are particularly suited to mucosal delivery. Although not as extensively studied as liposomes, one nanoemulsion is licensed and several have progressed to human clinical trials. One of the best characterized nanoemulsions is MF59, an oil-in-water emulsion of ~250 nm consisting of the metabolizable oil squalene, Tween-80 and Span 85 (reviewed in [122]). MF59 has an acceptable safety profile and was licensed in 1997 in Europe for an influenza vaccine, thereby becoming the first adjuvant licensed for human applications since alum. MF59 creates a depot effect and directly activates immune cells [123]. Two other oil-in-water nanoemulsions, when formulated with smallpox, HIV-1, influenza, or hepatitis B antigens and delivered intranasally, mediated the production of not only robust virus neutralizing antibodies, but also IFN-γ and TNF-α, as well as elevated IgG2a and mucosal IgA levels. These oil-in-water emulsions, designated W205EC and W805EC, consist of 1% cetyl pyridum chloride (CPC), 5% Tween 20 or Tween 80, 8% ethanol in 64% soybean oil and 22% water (NanoBio Corporation, Ann Arbor, MI, USA). When formulated with smallpox, HIV-1, influenza, or hepatitis B antigens and delivered intranasally, these adjuvants mediated the production of not only robust virus neutralizing antibodies, but also IFN-γ and TNF-α, as well as elevated IgG2a and mucosal IgA levels. W805EC was evaluated in a human clinical trial with Fluzone®, which demonstrated that this adjuvant is well tolerated. Volunteers received a single intranasal dose with increasing antigen and adjuvant amounts. The highest dose (10 µg) of antigen in combination with 20% W805EC, induced increased serum hemagglutination inhibition antibodies reactive with three influenza strains, as well as antigen-specific IgA in the nasal wash [124].MF59 has been combined with either a CpG ODN or a TLR4 agonist, E6020, in an influenza soluble trivalent egg-derived antigen formulation. While addition of CpG ODN or E6020 to MF59 did not increase influenza-specific antibody titers, a shift towards a more Th1-biased response was observed which suggests that co-formulation of MF59 with a TLR ligand may tailor the immune response to be more Th1 biased [125]. In contrast, co-delivery of E6020 within MF59 enhanced the magnitude of the antibody levels, as well as increased the breadth of the response, when formulated with Men B antigens or Men ACWY-CRM conjugate vaccine [126]; furthermore, addition of either E6020 or CpG ODN to the Men B vaccine in MF59 increased the percentage of antigen-specific cells secreting IL-2, TNF-α and IFN-γ, again demonstrating a shift towards a Th1 bias. Recently, CpG ODN was shown to both enhance the hemagglutination and virus neutralization titers, and specifically increase the IgG2a levels, when added to a trivalent influenza vaccine (FCC) formulated in MF59. In addition, a much more pronounced Th1 profile, characterized by a potent IFN-γ response, was induced when CpG ODN was added [127]. This effect of addition of the TLR agonists is of particular significance not only for these two vaccines, but also in general when a particular Th bias is needed for protection.Many of the vaccines that have been recently developed, or will be in the future, consist of highly pure pathogen-derived antigens. This approach is necessary for vaccine development against live-threatening infectious diseases and cancers. For these antigens to induce robust immune responses, formulation with adjuvants is critical. Adjuvants can enhance the speed and magnitude of the development of immune responses, reduce the amount of antigen and/or vaccinations needed, increase cross-protection and reduce non-responsiveness, in particular of specific populations such as the very young or elderly. In view of these multiple roles, the current trend in vaccine formulation against both cancer and infectious diseases is to include at least one and often multiple components to engage PRRs, and thus activate the innate immune response, as well as at least one compound that stabilizes or targets antigens to the appropriate immune cells. Engagement of PRRs by their ligands is critical for induction of the innate immune response which then directs the adaptive immune response to be either Th1-, Th2- or Th17-biased. This ability to tailor the immune response is particularly important for vaccine development against difficult targets such as chronic bacterial or viral infections and cancer. Several classes of PRRs have been identified, including TLRs, NOD-like receptors (NLR), RIG-I-like receptors (RLRs) and C-type lectin receptors (RLRs). As described in this review, TLR agonists have been most extensively studied as adjuvants ands shown to be very promising. The TLR agonists often act synergistically with other classes of adjuvants used in the formulation or even with other TLR or NLR agonists. In fact, it is now known that one of the most effective vaccines, the live-attenuated yellow fever vaccine, activates multiple DC subsets through TLR2, TLR7/8 and TLR9, which leads to a robust balanced immune response. Aluminum salts and muramyl-dipeptides are two examples of NLR agonists used in licensed vaccines and clinical trials, respectively, and as more agonists of these additional classes of PRRs are identified, we can expect more to be tested as adjuvants.Currently there are four licensed vaccines that contain multiple TLR agonists, BCG that contains TLR2 and TLR4 agonists, Cervarix and Fendrix that contain alum and MPL, and Cadi-05 (Immuvac) (Mycobacterium indicus pranii) against leprosy. This demonstrates that it is possible to overcome any real or perceived disadvantages of multi-adjuvanting. One problem is the increased expense and complexity of manufacturing and formulating a vaccine antigen with multiple adjuvants. Another major issue is the potential for more adverse effects in the recipients; however, the individual components of a combination adjuvant often can be used at a lower concentration than when administered as a stand-alone adjuvant. This may actually reduce side-effects and production costs. A third hurdle is the concern of regulatory agents with novel adjuvants and combinations in particular, which hopefully will become more easily alleviated once more information about the mechanisms of action of adjuvants—and vaccines in general—is gained. For instance, the mechanisms of action have been elucidated for several adjuvants, including MF59, alum, MPL (reviewed in [128]); however, efforts in this area, specifically for combination adjuvants, need to be expanded. Systems biology can now be applied to elucidate molecular signatures to new vaccine candidates in comparison to very effective current vaccines such as the yellow fever vaccine and thus predict efficacy and safety [129]; this is another new area of research that will assist in licensing new vaccine formulations.In this review we summarized recent and relevant information about many known and new adjuvant combinations including ISCOMs, liposomes, Adjuvant Systems, Montanides and triple combination adjuvants is clear that combinations of adjuvants have multiple advantages, and are critical to achieve the desired degree of vaccine efficacy against many new, challenging disease targets. Furthermore, there is strong evidence supporting inclusion of at least one PRR agonist. As these pathogens target different organs and tissues and cause different types of pathogenesis, there likely will be a need for a panel of combination adjuvants for specific disease targets, routes of delivery and/or populations. As recently described [128], depending on whether mainly humoral or cell-mediated immunity is required to induce protection against a pathogen, a TLR4 or TLR3 agonist might be selected to tailor the response. In addition, the choice of delivery vehicle should depend on the route of immunization, mucosal or parenteral. When targeting specific populations such as neonates or the elderly, adjuvant selection should take the expression levels of PRRs in these populations into consideration. As more information becomes available on the efficacy, safety and mechanism of action, we can expect an increase in the number of licensed vaccines containing various combination adjuvants. Research in the authors’ laboratories is funded from a variety of funding agencies including the Canadian Institutes of Health Research (CIHR), the National Science and Engineering Research Council (NSERC), the Saskatchewan Health Research Foundation (SHRF), the Alberta Livestock and Meat Agency (ALMA), the Saskatchewan Agriculture Development Fund (ADF), the Pan-Provincial Vaccine Enterprise and the Krembil Foundation. The manuscript was published with permission of the Director of VIDO as manuscript #675.B. Levast wrote the ISCOM and IC31 sections, S. Awate wrote the adjuvant systems section, L. Babiuk critically read the review, G. Mutwiri wrote the Montanide section, V. Gerdts wrote the introduction, IC31 and triple adjuvant sections and S. van Drunen Littel-van den Hurk wrote the liposomes, nanoemulsions and conclusions sections and was responsible for the final submission.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Vaccine efficacy is optimized by addition of immune adjuvants. However, although adjuvants have been used for over a century, to date, only few adjuvants are approved for human use, mostly aimed at improving vaccine efficacy and antigen-specific protective antibody production. The mechanism of action of immune adjuvants is diverse, depending on their chemical and molecular nature, ranging from non-specific effects (i.e., antigen depot at the immunization site) to specific activation of immune cells leading to improved host innate and adaptive responses. Although the detailed molecular mechanism of action of many adjuvants is still elusive, the discovery of Toll-like receptors (TLRs) has provided new critical information on immunostimulatory effect of numerous bacterial components that engage TLRs. These ligands have been shown to improve both the quality and the quantity of host adaptive immune responses when used in vaccine formulations targeted to infectious diseases and cancer that require both humoral and cell-mediated immunity. The potential of such TLR adjuvants in improving the design and the outcomes of several vaccines is continuously evolving, as new agonists are discovered and tested in experimental and clinical models of vaccination. In this review, a summary of the recent progress in development of TLR adjuvants is presented.The main goal of vaccination is to induce immunologic protection from infectious diseases of bacterial, viral and parasitic origin. Host immune responses to a given vaccine antigen can be greatly enhanced by simultaneous administration of an immune adjuvant. Adjuvants are exogenous substances that have a wide variety of nature and origin, ranging from mineral salts, oil and water-based emulsions, polymers, microparticles, liposomes, saponins, microbial products and even cytokines [1,2,3].Despite the importance of their influence on the immune response, the mechanisms of action by which most adjuvants potentiate innate and adaptive immunity have only recently begun to be understood. Adjuvants are generally categorized into delivery systems and immunostimulatory adjuvants. Delivery systems, particulate adjuvants and emulsions including alum [4], water-in-oil and oil-in-water emulsions (i.e., Complete Freund’s Adjuvant (CFA) [5] or MF59 [6]) are thought to generate an antigen depot at the site of injection, which is then slowly released over time (although other factors have been described to contribute to the effect of alum and MF59, for example [7,8]). This process leads to enhanced antigen uptake and presentation by antigen presenting cells (APCs) and induction of high antigen-specific antibody titers. The second category of vaccine adjuvants, immunostimulatory substances, enhances immune responses via a direct effect on immune cell activation and function. These adjuvants induce: (1) upregulation of surface expression levels of the major histocompatibility complexes I and II (MHC I and MHC II) on APCs and enhanced antigen presentation to the T-cell receptor (TCR) (Signal 1); (2) APC maturation/activation and increased surface expression of co-stimulatory molecules (CD40, CD80, CD86) needed for proper activation of naïve T cells (Signal 2); (3) direct and indirect immunomodulation and differentiation of T lymphocytes; 4) recruitment of immune cells at the site of injection and migration to the draining lymph nodes [9]. In addition, both categories of adjuvants induce immune cell responses mediated by inflammatory mediators (i.e., cytokines and chemokines (signal 3)) [9] and surface receptors/adhesion molecules. The convergence of the events elicited by immune adjuvants leads to enhanced adaptive immune responses and subsequent immune protection, particularly through the activation of dendritic cells (DC) and T cells [10]. It has also been established that activation of APCs occurs via specific recognition of microbial products, a step that has been defined as Signal 0 [9], and is required for innate immune responses that guide T helper cells towards Th1-, Th2- and Th17-type differentiation. Th1-type responses are defined by the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α, by high levels of IgG2a/b (or IgG2c), IgG3 and IgA in mice, and IgG1, IgG3 and IgA in humans, cell-mediated immunity (CMI) via both CD4+ T cells and CD8+ cytotoxic T cells (CTLs) (although the latter also require antigen presentation via MHC class I). Th2-type responses are defined by IL-4, IL-5, IL-6, IL-10 and IL-13 and CD4+ T cell-dependent B cell-mediated humoral immunity via induction of IgG1 and IgE/ IgA in mice or IgG4 and IgE in humans [11]. Dysregulation of Th1-type responses to self-antigens or the commensal flora leads to tissue destruction and chronic inflammation, while dysregulation of Th2-type responses is implicated in allergy and asthma. Recently, Th17-type responses, characterized by IL-17 and IL-23 [12] have been described to modulate neutrophil recruitment [13], and B and T cell functions, including those of regulatory T cells (Treg) [14], thus playing a role in vaccine development [15]. Therefore, inclusion of adjuvants in vaccine formulations is important for both stimulation of innate immunity and induction of improved antigen-specific adaptive responses. Various adjuvants have been shown to mediate different types of adaptive immune responses. For example, alum (the first USDA-licensed adjuvant approved for use in humans in the US and present in over 80% of the licensed human vaccines) stimulates Th2-type responses and strong antigen-specific IgG1 and IgE antibody production, but it does not induce CD8+ T-cell immunity and may even inhibit Th1-type immune responses [16]. By contrast, adjuvants such as QS-21 (a saponin from the Soap bark tree Quillaja saponaria in an oil-in-water emulsion), MF59 or Freund’s complete adjuvant (CFA) induce preferentially Th1-skewed responses, or a mixed Th1/Th17-type and Th1/Th2-type immunity [3].In the early 1990s, the potential for a number of bacterial and viral components to act as immune adjuvants has been elucidated by their ability to interact with specific host cell receptors that recognize microbial molecular patterns, the Toll-like receptor family (TLRs) [17]. The role of TLRs in regulation of host innate and adaptive immune responses has been explained by their ability to induce activation of immune cell signaling. In B cells, TLR signaling induces up-regulation of surface markers involved in antigen up-take (MHC I and MHC II) and in cross-talk with T cells (CD40, CD80, CD86), ultimately enhancing antigen-specific antibody production when TLR ligands are used combined with antigens in the context of vaccination. In addition, TLR signaling also plays a role in induction of B- and T-cell memory. In APCs, including B cell, DCs and macrophages, TLR signaling also results in enhanced secretion of both pro- and anti-inflammatory mediators that drive development of T helper cell subsets into Th1-, Th2- or Th17-type, depending on the type of APC involved [18]. Generally, signaling via TLR3, TLR4, TLR7, TLR8 and TLR9 promotes Th1-type immune responses while signaling via TLR2 (along with TLR1 or TLR6) and TLR5 favors Th2-type immune responses [19,20]. TLR ligands also influence Treg development [21]. A direct influence of TLR signaling on Treg development has been shown, due to expression of functional TLRs on these cells, as well as an indirect effect, due to Treg interaction with TLR-activated APCs [22]. TLR signaling can lead to either Tregs functional activation or suppression, depending on the TLR ligand type and effect on antagonistic induction of Th17 cells [21]. This aspect is particularly relevant for cancer, autoimmunity and chronic inflammation, due to the effects of Th17-type cytokines (IL-17A, IL-17F and IL-22) [21,23]. This review discusses the mechanisms of action of TLR agonists with vaccine adjuvant properties and highlights their potential use to improve vaccination against infectious diseases and cancer.Toll-like receptors (TLRs) comprise members of a family of related trans-membrane proteins that recognize microbial and viral products. TLRs have been categorized as pattern recognition receptors (PRRs) that recognize ligands from pathogenic microorganisms (the “pathogen-associated molecular patterns” (PAMPs) [24]), from commensal organisms (the “commensal-associated molecular patterns” (CAMPs) [25]) and endogenous ligands deriving from damaged cells (the “danger-associated molecular patterns” (DAMPs)) [26].The structure of TLRs is that of horse-shoe shaped proteins composed of three domains: an extracellular or cytoplasmic leucine-rich repeat (LRR) domain which mediates ligand recognition, a single trans-membrane domain, and an intra-cytoplasmic domain, the TIR domain, homologous to the corresponding intracellular domain of the IL-1 receptor (IL-1R) Toll/IL-1R [17]. In humans, 10 TLRs have been identified so far. TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 are surface-expressed and recognize extracellular ligands and microorganisms, while TLR3, TLR7, TLR8 and TLR9 are situated on endosomal membranes within the cell and are engaged by intracellular ligands and microrganisms [17]. Ligand binding and TLR homo- or heterodimerization brings the TIR domains of adjacent TLRs together, providing a conformational change necessary to trigger signaling. Binding of additional adaptor proteins is also essential for intracellular cascades. Adaptor proteins include the myeloid differentiation factor 88 (MyD88) [27], the MyD88 adaptor-like protein (Mal/TIRAP), the TIR domain-containing adaptor protein inducing interferon-β (TRIF/TICAM) and the TRIF-related adaptor molecule (TRAM) [28,29] (Figure 1). Negative regulators of TLR function have also been identified and include the Toll-interacting protein (Tollip), IRAK-M, the α- and HEAT-Armadillo-motif-containing protein (SARM) and the B cell adaptor for PI3K (BCAP) [30].Schematic cartoon of Toll-like receptor (TLR) signaling [17,24,27,28,29,30]. Extracellular TLR homodimers (TLR4 and TLR5) are represented in black; heterodimers of TLR2 and TLR1, TLR6 or TLR10 are indicated in black/green. Intracellular homodimers (TLR3, TLR7, TLR8 and TLR9) are indicated in gray.All TLRs except TLR3 require MyD88 recruitment to the TIR domain for signaling activity [27]. TLR2 and TLR4 also require the cooperation of the adaptor protein Mal/TIRAP. In the MyD88-dependent signaling pathway, activation of IRAK4 and IRAK1 (members of the IL-1R-associated protein kinases (IRAKs) [31]) is followed by that of TRAF6 (tumor necrosis factor receptor-associated factor 6 [32]) and RIP (receptor interacting protein [33]), with subsequent signal transfer to a complex made of TAK1 (TGF-β-activated kinase 1), TAB1, TAB2 and TAB3 (TAK1-binding proteins 1, 2 or 3) and, ultimately, activation of NF-κB and, through members of the mitogen-activated protein kinase (MAPK) family (ERK, JNK, p38), activation of AP-1 [34]. The MyD88-dependent TLR signaling pathway leads to host cell responses involved in cell survival/proliferation and immune pathways culminating with immune cell activation, induction of inflammatory mediators and antimicrobial products. Signaling through TLR7, TRL8 and TLR9 also activates a parallel MyD88-dependent cascade through IRF7 (interferon regulatory factor 7 [35]), followed by TRAF6, IRAK4 and TRAF3 activation and leading to type I interferons (IFN) production (Figure 1). TLR3 signaling activates a MyD88-independent pathway via TRIF and TRAF3 [36], leading to activation of IRF3 and resulting in secretion of IFN-β and IL-10 (Figure 1). The TLR3-TRIF signaling also drives activation of MyD88-dependent pathway downstream components, TRAF6 and RIP1, converging on activation of NF-κB and AP-1. Similar to TLR3 signaling, TLR4 can also induce MyD88-independent signaling, although TRIF recruitment is not direct but requires prior activation of TRAM. Downstream cell activation via both TRAF6/RIP1, as well as TRAF3-IRF3, provides an amplification of the cytokine repertoire (Figure 1). TLR3 (CD283) is expressed in endosomal compartments in myeloid dendritic cells (mDCs) and, weakly, in monocyte-derived macrophages [37] and it recognizes viral double-stranded RNA (dsRNA) that is produced during viral replication in infected cells, with the potential contribution of CD14 [38]. A synthetic analog of dsRNA, Poly I:C (polyriboinosinic:polyribocytidylic acid) [39], has similar immunostimulatory properties, inducing TLR3 activation via the TRIF/TRAM pathway and secretion of inflammatory cytokines and IFN-β (Figure 1). However, Poly I:C also interacts with other receptors, such as the retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5) and double stranded RNA-dependent protein kinase [40], which may possibly influence its adjuvant activity. Nevertheless, since TLR3 ligands favor strong cellular Th1-type immune responses, they have been tested as adjuvants in vaccines against viral infections. Activation of dendritic cells by TLR3 agonists not only contributes to induction of innate and adaptive immune responses against microbial pathogens, but also favors NK cell activation and killing of tumor cells by stimulating anti-tumor CD8+ T cells [41]. Poly I:C has been used as an adjuvant in various experimental vaccine models. However, the major draw-backs of Poly I:C are its low stability and toxic side-effects. Pre-clinical studies carried out in primates have shown that Poly I:C is easily degraded by serum nucleases, with a consequent reduction of IFN secretion and anti-tumor activity [42]. Unfortunately, increasing the dosage of Poly I:C has not proven a successful strategy, as this TLR3 ligand is not well-tolerated. Thus, several derivatives of Poly I:C have been synthetized and tested for safety and adjuvanticity, such as Poly ICLC and Poly I:C12U (Table 1).Poly I:C favors antigen cross-presentation to primed CD8+ T cells, due to TLR3-dependent increased MHC class I expression and type I IFN secretion, as well as development of antigen-specific cytotoxic T cell clones [43]. Poly I:C inclusion in an HIV vaccine based on purified recombinant gp120 antigen has shown development of MHC class I-restricted CD8+ cells in vivo [44]; in another HIV vaccine strategy, addition of Poly I:C (and CpG DNA) to DNA encoding for a Gag antigen/anti-DEC205 antibody fusion protein has shown improved mucosal antigen presentation on MHC class I molecules and enhanced CD4+ T cell-mediated immunity [45]. Several studies in experimental animal models support the efficacy of vaccine formulations containing TLR3-based adjuvants [46]. In the quest for vaccines against cancer, the use of Poly I:C has shown enhancement of tumor specific T cell responses [47]. For example, in an ovarian cancer vaccine, Poly I:C enhances DC maturation and IL-12 secretion [48].Poly I:CLC (Hiltonol®) is a synthetic double-stranded polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine carboxymethyl cellulose and is less sensitive to serum-degradation [49]. Poly ICLC induces high IFN-γ secretion and enhances CTL responses and antigen-specific antibody titers, although mild to severe side effects have been reported when it is used at high doses [50,51]. Poly ICLC is currently being tested in clinical trials for both tumors and infectious diseases.Examples of TLR adjuvants, disease models tested in experimental and clinical trials, and human vaccines. Disease models and corresponding vaccines are shown in bold.Poly I:C12U (Ampligen®), a synthetic Poly I:C containing mismatched bases (uracil and guanine), is also immunostimulatory while less toxic than Poly I:C and Poly I:CLC. Intranasal immunization with Poly I:C12U as an adjuvant in a hemagglutinin (HA)-based H5N1 influenza vaccine induces higher levels of protective, specific mucosal IgA and systemic IgG responses than the corresponding adjuvant-free vaccine [53]. In phase II and III clinical trials for HIV vaccines, and in phase I and II cancer vaccine studies, Poly I:C12U has been deemed safe to use and induces maturation of mDCs, secretion of IL-12 and inhibition of IL-10, enhances antigen-specific CTL responses and Th1-type CD4+ T cell responses [54,139]. Poly I:C has also been combined with a cationic adjuvant formulation, CAF01, a liposome-based adjuvant composed of dimethyldioctadeclammonium and trehalose-6,6-dibehenate (DDA/TDB) [140]. The combined Poly I:C-CAF01 adjuvant is called CAF05. Through the effect of Poly I:C, CAF05 enhances CD8+ T cell responses and, through the effect of CAF01, induces a long lasting antigen depot. CAF05 favors Th1-type and Th17-type immunity and antibody responses in animal models of bacterial, viral and parasitic infections, and also has an effect on reducing tumor growth rates [52,141]. TLR4 (CD284) is expressed by the majority of circulating immune cells but its mature form has been characterized in macrophages and mDCs [37,142]. TLR4 signals via both the MyD88-dependent and the (MyD88-independent) TRIF-dependent pathway, leading to a robust IL-12 production, secretion of type I IFNs and a strong Th1-type cellular and humoral immune response (Figure 1). A number of TLR4 ligands has been described, with lipopolysaccharide (LPS) being the first bacterial product shown to interact with this receptor [143]. Other TLR4 agonists include a variety of components from fungi, viruses and parasites and endogenous ligands [144,145,146,147]. The TLR4/LPS molecular interaction has been elucidated in detail [148]. LPS has a hydrophilic polysaccharide component and a hydrophobic lipid A, composed of polyacylated diglucosamine lipids. The lipid A interacts with the TLR4 accessory molecule, lipid A binding protein (LBP) [149], followed by formation of a complex with CD14 (soluble or cell wall-anchored via glycosyl-phosphatidylinositol (GPI)), which is then presented to TLR4 and the myeloid differentiation protein 2 (MD-2) [150]. LPS, along with its molecular derivatives, has been tested in numerous vaccine clinical trials (Table 1). However, despite its strong immunostimulatory effect, an intrinsic toxicity severely limits its use in humans. A detoxified form of LPS, the monophosphoryl lipid A (MPLA) from Salmonella minnesota R595, was developed by Ribi [151]. MPLA retains a potent immunostimulatory activity in vitro and in vivo while lacking toxicity, and is used in a number of complex adjuvants broadly referred to as Ribi adjuvant systems (RAS). For example, synthetic MPL RC-529 (Ribi.529) is used in the human hepatitis B virus (HBV) recombinant antigen vaccine, SupervaxTM [55]. MPLA triggers both the MyD88-dependent and TRAM/TRIF-dependent pathway, although an apparent preferential bias towards signaling via the TRIF-dependent pathway has been reported [152]. Induction of a strong protective Th1-biased immunity and secretion of pro-inflammatory mediators (i.e., TNF-α) by MPLA has been shown for Leishmania and TB vaccine formulations, as well as induction of anti-inflammatory mediators (i.e., IL-10) [61,70]. MPLA has been combined with a variety of other adjuvants, such as QS21 and liposomes (AS01, GlaxoSmithKline (GSK) Vaccines), QS21 and an oil-in-water emulsion (AS02 (GSK)), and alum (AS04 (GSK)) [153] (Table 1). Due to the effect of MPLA, AS01, AS02 and AS04 all induce TLR4-dependent NF-κB activity and cytokine secretion, maturation and trafficking of DCs and monocytes to the draining lymph nodes and antigen-specific T cell activation (although AS04 does not directly activate B or CD4+ T lymphocytes) [154].In experimental and clinical trials, AS01 has been shown to induce Th1-type immunity, improve CD8+ T-cell responses and high antibody titers, for example to TB, varicella zoster virus (VZV), HIV antigens and to the malaria antigen RTS,S (a P. falciparum surface protein fused to the HBV surface antigen (HBsAg)) [63,64,68]. AS02, which has also been tested with HBV, HIV, TB and malaria antigens, elicits a more balanced Th1/Th2 immunity, with lower lymphoproliferative responses and a shorter-lived protection than AS01 [62,65,68]. By contrast, AS02 induces higher CD8+ cytolytic T cell responses than AS04, the MPLA/alum adjuvant. In the AS04 adjuvant, the MPLA/antigen complex is stabilized by the presence of alum, which also favors formation of antigen depot. AS04-containing vaccines have been tested against viral pathogens, including HBV [57], HPV [70,71], herpes simplex virus (HSV) [74] and Epstein-Barr virus (EBV) [75] showing improved protective responses than the corresponding alum-alone containing vaccines. AS04 is part of the HBV vaccine, FENDrix® [58], which has been safely and successfully used in healthy adults and in specific high-risk patients [55,59]. The AS04-containing vaccine against HPV, Cervarix®, is prophylactically used against cervical cancer and is also well tolerated [72,73]. In addition, MPLA-containing adjuvants have been used in cancer vaccine formulations, for example with the MUC1 antigen against prostate cancer or non-small cell lung cancer (NSCLC) [78] (Stimuvax®), or in combination with the adjuvant DETOX® (an oil-droplet complex that contains purified Mycobacterium phlei cell wall skeleton products (CWS) [76]) in a melanoma vaccine (Melacine®). DETOX is also used with the MUC1/ KLH antigen complex (Theratope) in breast and ovarian cancer treatment [79]. AS02 has also been used with the recombinant melanoma-associated antigen 3 (MAGE-A3) in cancer vaccine approaches, with some success [77]. The AS01, AS02 and AS04-adjuvanted vaccines are considered safe [66,67,69]. Based on the success of AS04, and on the different chemical composition of MPLA species, the adjuvant effect of other synthetic lipid A mimetics with different length and degree/type of fatty acid acylation has been examined (Table 1). For example, aminoalkyl glucosaminide 4-phosphates (AGPs), tested against L. monocytogenes, influenza and RSV [81], the E6020 synthetic molecule [155], and the RC-529 (Ribi.529) molecule, structurally similar to the hexa-acyl component of MPL® [65]. These synthetic lipid A mimetics are considered safe. Another synthetic lipid A derivative, glycopyranosyl lipid adjuvant (GLA) has been used in combination with squalene (SE, an oil-in-water emulsion), and shown to induce strong Th1-type responses, enhance antigen-specific responses and have a good safety profile [82]. Lastly, the lipid A derivative, OM-174 from E. coli, has also been tested for its adjuvant effect [80].TLR7 and TLR8 (CD288) are expressed in neutrophils, monocytes, macrophages, eosinophils and B cells (TLR7), plasmacytoid DCs (pDCs) (TLR7), NK cells and T cells (TLR8) and Langerhans cells [156]. Similar to TLR3, TLR7 and TLR8 have an intracellular localization within endosomal compartments in the cells that express these receptors. Engagement of TLR7 and TLR8 leads to signaling through MyD88 ⁄Mal, NF-κB and IRF7 activation and secretion of proinflammatory cytokines, chemokines and other mediators (Figure 1). In DCs, TLR7/TLR8 activation leads to cell maturation/activation, expression of co-stimulatory molecules (CD80, CD86 and CD40), enhanced antigen presentation and secretion of Th1-type pro-inflammatory cytokines (IFN-α, TNF-α and IL-12). pDCs respond to TLR7 activation by secreting IFN-α while mDCs respond to TLR8 activation by producing IL-12 [157]. Both TLR7 and TLR8 induce Langerhans cell differentiation and migration from the skin to the lymph nodes. Signaling via TLR7 induces secretion of Ig, IL-6 and TNF-α by B cells [158] and IFN-γ by NK cells [159]. TLR8 signaling induces T cell proliferation, IFN-γ, IL-2 and IL-10 production, memory T cell activation and also reduces CD4+ Treg-mediated immunosuppression [160]. The ligands for TLR7 and TLR8 include single stranded (ss) RNA enriched for poly-U or poly-GU sequences [161], synthetic imidazoquinolinamines, such as imiquimod (R-837) and resiquimod (R-848) [162] and guanosine analogues, such as loxoribine. While TLR7 or TLR8 agonists are not approved as vaccine adjuvant components, imiquimod and resiquimod have undergone extensive clinical testing in a 5% cream formulation (AldaraTM) for topical treatment of HPV-induced warts, actinic keratoses, basal cell and squamous cell carcinoma, lentigo maligna and molluscum contagiosum [83,84] (Table 1). Both compounds induce strong local secretion of IFN-α, TNF-α, IL-6 and IL-12, as well as cytotoxic T-cell responses. Topical application of imiquimod-containing formulations has also been tested in prostate cancer vaccines, favoring development of specific CTL responses and antibodies [85]. In a melanoma trial, systemic co-administration of imiquimod, a melanoma peptide vaccine and Flt-3 ligand (a DC activator) resulted in enhanced peptide immunogenicity and recruitment of both mDCs and pDCs in the treated areas. Imiquimod topical application also favors development of a T cell-dependent response to intradermal injection of the melanoma antigen, NY-ESO-1 [87]. In various tumor animal models, the combination of DNA-based vaccines and imiquimod treatment has been successful in reducing tumor onset, increasing CTL responses and IgG2a antibody production [86]. In pre-clinical studies on HSV and HIV, antigen-specific T cell responses and antibody secretion are enhanced by imiquimod [88], and in Leishmania infections, macrophage-dependent bacterial killing and resolution of cutaneous lesions have been reported following use of imiquimod [89]. Unfortunately, systemic administration of imiquimod is highly toxic and studies on TLR7/TLR8 adjuvant safety and efficacy are limited by the unresponsiveness of mice to TLR8 agonists for human use [161].In humans, TLR9 (CD289) is expressed by immune cells in intracellular endosomal compartments and its role is particularly relevant in B cells and pDCs [163]. TLR9 signals through the MyD88 pathway via IRAK and TRAF-6 without the contribution of Mal (Figure 1), leading to production of Th1-type pro-inflammatory cytokines (IL-1, IL-6, IL-12, IL-18, TNF-α and IFN-γ), up-regulation of CD80, CD86, CD40 and MHC molecules expression, increased antigen processing/presentation and CD8+ T cell responses [164,165]. In particular IL-12 and type I IFNs induced in pDCs via TLR9 drive a strong Th1-type immunity and CD8+ CTL cytotoxicity, while TLR9-dependent B cell activation leads to increased antigen-specific humoral responses and IgG class switching [166,167]. The ligands for TLR9 are bacterial and viral DNA that contains unmethylated CpG motifs and synthetic oligodeoxynucleotides (ODN) expressing CpG motifs [168]. The synthetic TLR9 ligands retain the immunostimulatory activity of bacterial DNA and are divided in three major classes, based on their structure, biological properties and ability to activate immune cells in vitro [169,170]. Multiple CpG motifs on a phosphorothioate backbone are classified as “K” type ODN (also called “B” type), which are strong inducers of B cell activation, pDCs and monocyte maturation. “D” type ODN (also called “A” type), have a mixed phosphodiester/phosphothioate backbone containing a single CpG motif flanked by palindromic sequences and 3'- and 5'-end poly-G tails that allow formation of concatamers. These CpG ODN activate NK cells. The third category, “C” type ODN, is structurally and functionally similar to both “K” type and “D” type ODN, with both phosphorothioate nucleotides and palindromic CpG motifs, and induce activation of both B cells and pDCs and production of IFN-α. Numerous pre-clinical and clinical studies have been carried out with TLR9 adjuvants (Table 1) [171,172]. The adjuvant activity of “K” type ODN has been explored in vaccine models against malaria [90,91], HBV [94,95], influenza [93] and anthrax [98]. CpG ODN induces a strong specific antibody response to the malarial Apical Membrane Antigen 1 (AMA1) and to the merozoite surface protein 142 (MSP142) (both poorly immunogenic vaccine candidates) [92]. In the case of HBV, the B type CpG ODN, CPG 7909, enhances specific, long-term antibody responses to the Engerix-B® vaccine (recombinant HBsAg vaccine absorbed on alum (Alhydrogel)), as compared to Engerix-B alone [96]. Another CpG ODN, the 1018 immunostimulatory sequence (ISS), has shown to improve the efficacy of the HBV vaccine Heplisav®, with only minor local side effects [97]. By contrast, inclusion of CpG 7909 in the influenza vaccines Fluarix® is considered less substantial, although it enhances IFN-γ secretion and is well tolerated, which is advantageous for reducing the vaccine dosage [93]. CpG-ODN is also used in anti-cancer vaccines and immunotherapy, due to its ability to induce high numbers of tumor-specific cytotoxic CD8+ T cells when co-administered with HPV and melanoma tumor antigens [99,100]. In vaccine trials with the synthetic tumor peptide MART1 (melanoma-associated antigen recognized by T cells 1) (Melan-A) and with the NY-ESO-1 peptide antigen, addition of CpG ODN enhances antigen-specific CD8+ T cell responses [101,104]. CpG 7909 has only shown partial success when used in a MAGE-A3 protein-based vaccine, which has been improved by addition of MPL and QS21 in a liposomal formulation to CpG 7909, the AS15 adjuvant. The AS15-adjuvanted vaccine induces an increased MAGE-A3 delivery to APCs and enhances T-cell immunogenicity [105] (Table 1). However, despite a good safety profile of CpG 7909, intra-tumoral injection of this TLR9 adjuvant has shown scarce results on tumor growth in melanoma and basal cell carcinoma models [102]. Similarly, evaluation of CpG 7909 administration during chemotherapy for NSCLC treatment, or combined with GM-CSF and the tumor antigen, hTERT (human telomerase reverse transcriptase), has not shown a great success rate [103].TLR2 (CD282) expression is relatively ubiquitous in immune cells and is found on the surface of neutrophils, macrophages, monocytes, basophils, T cells, B cells, NK cells and immature DCs [37]. TLR2 dimerizes with either TLR1 or TLR6 and also utilizes other accessory molecules, such as CD36, CD14 and LBP [173,174,175,176]. TLR2-dependent signaling proceeds through the Mal/TIRAP and MyD88-dependent pathway, inducing activation of NF-κB and MAPKs pathways leading to immune cell activation, survival/proliferation, secretion of inflammatory mediators and expression of co-stimulatory molecules (CD80, CD86 and CD40) (Figure 1). TLR2 interacts with structurally diverse ligands. Natural and synthetic lipopeptides and lipoproteins that signal via TLR2 include M. fermentans macrophage-activating lipopeptide (MALP-2), a TLR2/TLR6 ligand [173], the syntetic triacylated lipoprotein, Pam3CSK4, a TLR2/TLR1 ligand [174] and the diacylated lipoprotein, Pam2CSK4, a TLR2/TLR6 ligand [177]. TLR2 also binds peptidoglycans (PG) [178], glycosylphosphatidyl-inositol-anchored structures from gram positive bacteria (lipoteichoic acid, LTA), lipo-arabinomannan from Mycobacteria and lipomannas from M. tuberculosis [179]) and other cell wall components (i.e., β-glucans [180] and zymosan [181]), as well as viral products [182] and some bacterial LPS types (reviewed in the TLR4 section). Endogenous ligands and DAMPs [183] and several lipid-free bacterial proteins have also been described as TLR2 ligands, including porins, toxins, fimbriae [184] and the PPE18 protein from M. tuberculosis [185]. The molecular and structural details of TLR2 interaction with some of its ligands have been elucidated, while other TLR2/agonist complexes are currently being explored [186,187,188]. The adjuvanticity of TLR2 agonists has been characterized as predominantly Th2-biased. The most extensively studied TLR2 adjuvants include MALP-2, Pam3CSK4, Neisseria PorB and E. coli LT-IIa-B(5) and LT-IIb-B(5) (Table 1). A large majority of studies conducted with these adjuvants have been carried out in experimental animal models, and most are not yet approved for routine administration in humans. In experimental and pre-clinical studies, TLR2 adjuvants support DC and B cell responses and T cell activation, including that of antigen-specific CD8+ T cell (CTL), although at relatively modest levels as compared to other TLR adjuvants [189]. Treg functions can also be influenced by TLR2 activation; for example, TLR2/TLR1 signaling may mediate protective mucosal Th17-type responses to pathogens and Treg cells expansion, while TLR2/TLR6 signaling may promote tolerogenic dendritic cells and Treg responses [190]. MALP-2 has been used as an adjuvant in a number of experimental immunization studies with prototype antigens, such as ovalbumin (OVA), but also in disease models. Intranasal administration of MALP-2 in a vaccine model against enterohemorragic E. coli enhances secretion of antigen-specific serum IgG and mucosal IgA, IFN-γ, IL-2 and IL-4 [106]. The synthetic derivative of MALP-2, BPP (S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl polyethylene glycol), also enhances secretion of antigen-specific antibodies and cytokines (TNF-α, IL-10 and MIP-1β), and favors antigen cross-presentation by DCs to CD8+ T cells and cytotoxic T-cell response [191]. Lipoproteins have been also used in experimental and clinical vaccine studies. In a vaccine against Lyme disease, immunization with the B. burgdorferi outer surface lipoprotein A (OspA) with alum has shown enhanced secretion of protective antibodies against a C-terminus epitope of OspA [108]. This Lyme disease vaccine, LYMErix®, was licensed in 1998 after showing a good safety profile in clinical trials. However, potential concerns regarding skewing of Treg responses toward a Th17 phenotype and induction of autoimmune disease have stopped its commercialization [109]. Pam3CSK4 has been used in an anti-malarial vaccine containing several P. falciparum circumsporozoite protein (CSP) B cell epitopes and a universal T cell epitope, demonstrating induction of relatively high titers of peptide-specific IgG and IgG1, IgG3 and IgG4 antibody subclasses in immunized volunteers [110]. Lipid-containing TLR2 adjuvants can be easily conjugated to vaccine antigens, and even antigens themselves can be modified by addition of a lipid-core peptide for TLR2 interaction and direct activation of immune cells [192]. Such a strategy has been employed in a vaccine containing an HBV core antigen CTL peptide and a helper T lymphocyte (HTL) peptide conjugated with a palmitic acid at the N-terminus (Theradigm-HBV) [112]. In a phase I trial, this vaccine has shown higher immunogenicity than the un-palmitoylated vaccine and induction of long-term, dose-dependent, HBV-specific CTL responses in healthy subjects [113]. Phase I and II trials of an HIV-1 lipopeptide-based vaccine have shown similar long-lived, antigen-specific IgGs and specific CTL responses [114]. Porins from Neisseriae, F. nucleatum, Chlamydia, Shigella, Haemophilus and Salmonella have been examined in numerous experimental immunization and pathogen challenge models. Generally, bacterial porins have a rather conserved, trimeric structure consisting of monomers with a high content of β-barrel structure. In the bacterial membrane, porins mediate passage of ions and solutes for organism survival [193]. Purified porins can be formed into stable native preparations, called proteosomes, and are recognized by TLR2 on the surface of immune cells. Porins from Neisseriae, F. nucleatum and Chlamydia have a TLR2/TLR1-dependent adjuvant activity [194,195,196], while the adjuvanticity of Shigella porin and Salmonella OmpS2 is mediated by TLR2/TLR6 signaling [117,197]. Remarkably, Neisseria, F. nucleatum, Chlamydia and Salmonella porins induce Th2-type skewed immune responses [116,173,195] while Shigella porin appears to favor Th1-type responses [198]. The TLR2-dependent porin effects include APC activation/proliferation, increased surface expression of CD80 (Shigella, Salmonella), CD86 (Neisseria, F. nucleatum, Chlamydia), CD40 and MHC II molecules and induction of antigen-specific IgM, IgG and IgA antibodies [118,119,196,199,200]. Neisserial porin proteosomes have been tested as adjuvants in mucosal and systemic vaccinations against different pathogens in both experimental and clinical models without side effects or toxicity [111,120,121,122,123,201]. Toxins from Enterobacteriacee, divided in type I (the cholera toxin (CT) and the E. coli heat-labile enterotoxin I (LT-I)) and type II (the E. coli LT-IIa, LT-IIb and LT-IIc) [202,203,204], are also potent mucosal immune adjuvants, although their clinical development is severely compromised by their high toxicity in humans [205]. Enterotoxins are oligomeric proteins composed of an A subunit, responsible for the enzymatic activity of the toxin, and a pentameric B subunit (B5), which mediates binding to ganglioside receptors on host cells (i.e., GM1, GD1b and GD1a, GQ1 and GT1). Genetically detoxified type I toxin A and B subunits, including the LTK63, LTR192G, LTR72 and LTH44A molecules, have been tested in experimental vaccine models against bacterial, viral and parasitic infections, in cancer vaccines and in clinical trials [107,115,206,207], but their safety remains under scrutiny [208]. Besides binding to ganglioside receptors, the B subunit of type II LT (LT-IIa-B(5) and LT-IIb-B(5)) also binds to TLR2 [209,210], via regions that are normally masked by the A subunit in the whole holotoxin [209]. Interaction of LT-IIa-B(5) and LT-IIb-B(5) with the TLR2/TLR1 heterodimer is facilitated by binding to the GD1a ganglioside and leads to APC activation, secretion of high levels of antigen-specific systemic and salivary IgG and IgA antibodies, memory B cell development, secretion of cytokines (with high IL-4 and low IL-12), repression of Treg development/function and increased Th1, Th17 and especially Th2-type responses [204,210,211,212]. Additionally, LT-IIa induces CD8+ T cell apoptosis, thereby reducing IFN-γ secretion and further influencing Th-type immunity [213]. The TLR2-dependent functions are retained by non-toxic mutants of the LT B(5) subunit, such as LT-IIb(T13I), which fail to bind their ganglioside receptors [214,215]. TLR5 is expressed on the surface of neutrophils, monocytes, mDCs, Langerhans cells, T cells and NK cells [163,216,217]. Signaling through TLR5 via the MyD88 ⁄Mal pathway leads to a strong induction of NF-κB activation and a preferential Th2-type immunity (although a Th1 component can be also present) [218,219] (Figure 1). The ligand for TLR5 is bacterial flagellin and the TLR5-binding region is located in a conserved region of flagellin, the D1 portion [220,221,222,223,224]. A number of experimental models have demonstrated that flagellin, in both soluble monomeric and polymeric forms, has an immune adjuvant effect and induces DC maturation/activation with subsequent up-regulation of CD80, CD83, CD86 and MHC class II, secretion of IL-10 and TNF-α by monocytes and IFN-γ and α-defensins by NK cells, T cell proliferation/activation and antigen-specific CTL responses [224,225]. Although likely not through a direct effect on B cells, but more to a general TLR5-dependent enhancement of APC functions, immunization with flagellin-containing vaccines also leads to enhanced secretion of antigen-specific IgG and local IgA responses [222]. For example, addition of flagellin in an intranasal influenza vaccine in mice has shown enhancement of immune response as compared to the vaccine without flagellin [133,134]. Similar effects have been shown in experimental models of vaccination against Y. pestis [124], West Nile virus [125] and L. monocytogenes [216] (Table 1).A major advantage of this TLR5-dependent adjuvant is its use in fusion proteins with recombinant antigens, which has shown induction of superior immune responses as compared to simultaneous co-administration of flagellin and antigens [19,127,226]. This approach has been used in experimental and clinical trials of vaccines against influenza, using a flagellin/hemagglutinin-based vaccine (VAX125, VAX128) [134,135,136] or a flagellin/matrix protein 2 ectodomain (M2e) vaccine (VAX102) [137,138], and in vaccines against malaria [127,128], vaccinia virus [227], P. aeruginosa [228] and even against dental caries [129]. Although the safety of flagellin-based vaccines is still being evaluated in clinical trials, no major local or systemic side effects have been reported so far. In addition to its use as adjuvant for vaccines against infectious diseases, flagellin has also been used in cancer treatment, where NF-kB and transcriptional regulation of mediators of apoptosis are induced by flagellin via TLR5 signaling. It is thought that reduction of apoptotic cell death may be beneficial for the consequences of radiation treatment in normal tissues. In experimental irradiation studies in rodents and primates, improved survival rates have been observed following vaccination with flagellin-derived polypeptide (CBLB502) [130]. In other studies on potential anti-cancer strategies, flagellin has also shown an enhanced generation of tumor-specific CD8+ T cell immune responses [131].The ultimate goal of vaccination is to generate protection against diseases. Such protective immunity requires induction of different host responses that are elicited by using vaccine formulations containing appropriate antigens and adjuvants. Adjuvants are important components of vaccines and can influence the outcomes of vaccination, particularly by directing host immune responses towards different T helper cell immunity and enhancing both the quality and the quantity of immune response against the antigens. However, major concerns in vaccine adjuvants development include their safety and efficiency. Even though vaccine design is still rather empirical, recent advances in immunology research have expanded our understanding of the mechanisms of action of various adjuvants and greatly improved the chances for successful development of safe and effective interventions to prevent and treat a number of human diseases through modulation of host immune responses. The discovery of TLRs and their role in modulation of innate and adaptive immunity has led to exploitation of their ligands as immune modulators, due to their ability to induce specific immune cell activation and influence host adaptive immunity. The advantage of TLR adjuvants is not only in their ability to preferentially induce Th1 or Th2 responses and development of CD4+ or CD8+ T-cells, but also to modulate B cell activation and enhance antibody secretion to otherwise poorly immunogenic antigens, improving both quality and quantity of specific antibody production. Furthermore, TLR adjuvants appear suitable for enhancing mucosal immunity, an area that is gravely underdeveloped in the current human vaccine strategies. As discussed here, experimental models and clinical trials evaluating TLR agonists as immune adjuvants have identified valuable molecules for use in vaccines against infectious diseases, allergies and cancer immunotherapy (Table 1). In particular, TLR3, TLR4 and TLR9 agonists have been shown to improve a number of vaccines, for example against HBV, influenza, malaria and anthrax, as well as some types of cancer. TLR7/TLR8 agonists are less developed as adjuvants but are already used with success in topical cancer immunotherapy. The efficacy of vaccine formulations containing traditional adjuvants has also been reported to be synergistically improved by the addition of TLR agonists. It is likely that the known TLR ligands described here and potentially other novel TLR ligands with adjuvant effect, could be introduced in human vaccine formulations worldwide in the near future as both stand-alone adjuvant systems or in combination with existing non-TLR adjuvants in the design of next-generation vaccines [19,126,229]. The authors thank Lee M. Wetzler and Munir Mosaheb (Boston University) for critically reading the manuscript, and NIH/NIAID grant R01 AI40944. Deana N. Toussi and Paola Massari contributed towards writing of the manuscript.The authors declare no conflict of interest.