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INTRODUCTION | The HIV-1 envelope (Env) trimer is protected by multiple mechanisms of humoral evasion, including conformational masking, glycan shielding, and sequence variation.Stabilization of the Env trimer through the incorporation of a disulfide (SOS) between residues 501 of gp120 and 605 of gp41 and the substitution of an isoleucine to a proline (IP) helped fix the trimer into a prefusion-closed conformation recognized by broadly neutralizing antibodies but not by non-neutralizing antibodies.While vaccine elicitation of tier-2 neutralizing responses has now been reported in multiple vaccine-test species including non-human primates, | PMC10491024 | ||
RESULTS | PMC10491024 | |||
Sorting of VRC 018 B cells from donor N751 reveal that only a small fraction of elicited antibodies recognize the glycan-dense surface of the Env trimer | In the Vaccine Research Center (VRC) 018 clinical trial (To quantify the number of cells binding to the glycan-dense Env-trimer surface relative to those bound the glycan-free base, we performed cell cytometry on immunoglobulin G-positive (IgGFrom two plates of single-sorted B cells, 96 wells each, specific for the glycan-base BG505 trimer, we sequenced immunoglobulin genes and cloned them into an expression vector utilizing a method for rapid assembly, transfection, and production of immunoglobulins (RATP-Ig). | PMC10491024 | ||
Vaccine-elicited human antibodies N751-2C06.01 and N751-2C09.01 neutralize HIV strain BG505 | VIRUS | We further characterized the purified antibodies for neutralization activity and found that the antibodies N751-2C06.01 and N751-2C09.01 (named for donor-lineage.clone, hereafter referred to as 2C06 and 2C09) neutralized both BG505 and a version of BG505 virus with glycan at residue 611 missing (BG505.N611Q), found to be especially sensitive to fusion-peptide-directed antibodies (We used SPR to measure the binding of the antigen-binding fragments (Fabs) of 2C06 and 2C09 to BG505 DS-SOSIP trimer. Both bound tightly, with K | PMC10491024 | |
Cryo-EM structure of N751-2C06.01 in complex with BG505 DS-SOSIP reveals the antibody to bind at the fusion-peptide site of vulnerability | To define recognition details, we determined the cryo-EM structure of 2C06 in complex with BG505 DS-SOSIP. We obtained a 3D-reconstruction map at 2.95 Å from 645,442 particles utilizing C1 symmetry (Antibody 2C06 bound at the fusion-peptide site of vulnerability (The fusion peptide was flanked by CDRs H1 and H2 on one side and CDR H3 on the other (The 2C06 epitope also included S241, which is an Overall, the cryo-EM structure of 2C06 in complex with BG505 DS-SOSIP revealed 2C06 to bind at the fusion-peptide site of vulnerability, with heavy-chain CDRs accounting for most of the interactions with the fusion peptide. | PMC10491024 | ||
Cryo-EM structure of N751-2C09.01 in complex with BG505 DS-SOSIP reveals epitope similarity to N751-2C06.01 | To understand the differences and similarities between antibodies 2C06 and 2C09, we determined the cryo-EM structure of 2C09 in complex with BG505 DS-SOSIP. 2C09 binding to the envelope trimer did not induce noticeable asymmetry, and we obtained a 3D-reconstruction map at 2.81 Å with C3 symmetry from 445,733 particles (The fusion peptide in the 2C09 bound complex also formed a U-shape conformation, except for its two N-terminal residues (512 and 513), which turned back to interact with antibody. As a result, fusion-peptide binding to 2C09 involved residues 512–520 interacting almost exclusively with heavy-chain CDRs (The heavy chain of 2C09 contributed about 70% of the total epitope surface. The CDR H3 of 2C09 was four residues shorter than that of 2C06 and, unlike in the 2C06-Env complex, did not reach the neighboring gp41; this difference accounted for most of the difference in total BSA ( | PMC10491024 | ||
Antibodies 2C06 and 2C09 form a reproducible antibody class | Antibodies 2C06 and 2C09 originated from similar heavy-chain variable genes, IGHV3-64D and IGHV3-64, respectively (The similarity in origin genes and in Env-trimer recognition suggested that 2C06 and 2C09 might be members of a reproducible antibody class. Indeed, most residues involved in trimer binding were identical between 2C06 and 2C09, discounting the different length in CDR H3. Both 2C06 and 2C09 also showed low levels of somatic hypermutation (1% and 3%, respectively, based on nucleotide sequence) (To confirm whether 2C06 and 2C09 were members of the same antibody class, we swapped the heavy and light chains of the two antibodies, as such swapping has in the past confirmed class membership. | PMC10491024 | ||
Antibody 2C06 partially accommodates, but antibody 2C09 clashes with, glycan241 | MUTANT | We next investigated the parameters governing breadth of recognition by antibodies 2C06 and 2C09. As described in the aforementioned structural analysis, both 2C06 and 2C09 epitopes involved S241, a rare glycan hole in BG505 among HIV isolates; specifically, in both 2C06 and 2C09, heavy-chain residues D61 and R64 were within 4–5 Å from S241 side chain, and PISA analysis indicated both 2C06 and 2C09 to bury 14–22 ÅWe therefore created an S241N mutant of glycan-base BG505 (glycan-base BG505-N241), which introduced To understand the roles of glycan241 in the recognition of Env trimers by antibodies, we compared the known structures of fusion-peptide-directed antibodies in complex with Env trimers. Except for PGT151, | PMC10491024 | |
High-entropy epitope residues contribute to the strain specificity of 2C06 and 2C09 | MUTANTS | While recognition of the 241 glycan hole explains much of the strain specificity of 2C06 and 2C09, there were seven strains other than BG505 in our 208-isolate panel missing glycan241, and we next investigated whether HIV sequence diversity of the epitope residues contributed to the strain-specific neutralization of these antibodies.We calculated the BSA-weighted average entropy of the epitope residues for fusion-peptide-directed antibodies as well as other broad HIV-neutralizing antibodies. We observed a negative correlation between epitope entropy and neutralization breadth for non-fusion peptide HIV-neutralizing antibodies (To identify epitope residues in BG505 that contribute to strain specificity, we analyzed Shannon entropy and BSA of epitope residues of 2C06 and 2C09 (To confirm the observed interactions of the epitope residues with substantial entropy, we mutated H85 or G644 in BG505 DS-SOSIP to the more frequent utilized amino acids among HIV-1 isolates and assessed the mutants for binding to 2C06 and 2C09 (Overall, the above analyses suggested that fusion-peptide-directed neutralizing antibodies elicited by BG505 Env immunization can partially accommodate the glycan hole at position 241. Those that do, however, encounter other BG505 strain-specific residues, proximal to fusion peptide, and their inability to accommodate variation at these other positions contributes to their strain specificity. | PMC10491024 | |
DISCUSSION | As noted above, the HIV Env trimer evades antibody-mediated neutralization by conformational masking, glycan shielding, and sequence variation. Env trimers stabilized in the prefusion-closed conformation, however, induce recognition of the neutralization-susceptible shape of Env, and their use in human vaccine trials such as VRC 018We previously observed in the VRC 018 clinical trialIt will be interesting to see how prefusion-stabilized Env-trimer immunization can be altered to overcome glycan shielding and sequence variation. One way to overcome immunogenicity issues related to glycan shielding might be to mask the exposed protein base on the trimer with | PMC10491024 | ||
Limitations of the study | somatic hypermutations | This study describes antibodies isolated from a single donor at a single time point. The quality of isolated antibodies is also limited by the source of the study, the VRC 018 clinical trial, which was designed for the primary goal of testing the safety of BG505 DS-SOSIP adjuvanted by alum as a vaccine candidate. As a result, the antibodies isolated have limited somatic hypermutations and low neutralization potency with strain specificity. | PMC10491024 | |
STAR★METHODS | PMC10491024 | |||
RESOURCE AVAILABILITY | PMC10491024 | |||
Lead contact | Further information and requests for resources and reagents should be directed to and will be fulfilled by Peter D. Kwong ( | PMC10491024 | ||
Materials availability | Plasmids generated in this study are available upon request. | PMC10491024 | ||
Data and code availability | Cryo-EM maps have been deposited to the EMDB with accession codes EMD-29725 and EMD-29731, and fitted coordinates have been deposited to PDB with accession codes 8G4M and 8G4T.This paper does not report original code.Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. | PMC10491024 | ||
EXPERIMENTAL MODEL AND SUBJECT DETAILS | PMC10491024 | |||
Serum samples | INFECTIOUS DISEASES, ALLERGY | The samples were collected under the Vaccine Research Center’s (VRC), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health protocol VRC 018 ( | PMC10491024 | |
Cell lines | HEK293F, Expi293F and FreeStyle 293-F cells were purchased from Thermo Fisher Scientific. The cells were used directly from the commercial sources following manufacturer suggestions as described in detail below. | PMC10491024 | ||
METHOD DETAILS | PMC10491024 | |||
Preparation of Env trimers and antigen-specific probes | BG505 DS-SOSIP and ConC trimers were expressed in stable CHO cell lines and purified by non-affinity chromatography. | PMC10491024 | ||
B cell sorting | Cryopreserved PBMC from VRC 018 clinical trial ( | PMC10491024 | ||
Rapid assembly, transfection, and production of immunoglobulins (RATP-Ig) | The sorted B cells were subjected to RATP-Ig following the procedures as described previously. | PMC10491024 | ||
AlphaLISA screening of RATP-Ig supernatants | RATP-Ig supernatants from glycan-base BG505-positive B cells were diluted into AlphaLISA buffer (PBS +0.05% Tween 20 + 0.5 mg/mL BSA), and 5 μL of each were transferred to an OptiPlate-384 assay plate (white opaque, PerkinElmer, Waltham, MA). To each well was added 10 μL of biotinylated Env trimer probe at 10 nM final concentration and 10 μL of anti-human Fc IgG (PerkinElmer, Waltham, MA) acceptor beads at 10 μg/mL final concentration. The mixtures were incubated at room temperature (RT) for an hour and then added 25 μL of streptavidin donor beads (PerkinElmer, Waltham, MA) at 40 μg/mL final concentration. The plate was incubated at RT for 30 min in the dark, and the AlphaLISA signal was read using a SpectraMax i3x multi-mode microplate reader (Molecular Devices, San Jose, CA). Only nine supernatants out of two plates of RATP-Ig supernatants were identified as double-positive for binding BG505 DS-SOSIP and glycan-base BG505, likely due to both false positives in B cell sorting and low level of Ig expression in RATP-Ig for some antibodies. | PMC10491024 | ||
Antibody preparation | DNA encoding antibody heavy- and light-chain variable regions were synthesized and cloned into the pVRC8400 vectors. | PMC10491024 | ||
Env-pseudovirus neutralization assays | single-round infection | Monoclonal antibody neutralization was assessed based on the single-round infection assay of TZM-bl cells with HIV-1 Env-pseudoviruses as described previously. | PMC10491024 | |
Enzyme-linked immunosorbent assay (ELISA) | AVGIGA | Anti-fusion peptide ELISA was performed by coating 96-well plates (Costar High Binding Half-Area; Corning, Kennebunk, ME) overnight at 4°C with 50 μL/well of 2 μg/mL FP8v1-rTTHC (AVGIGAVF), FP7v1-rTTHC (AVGIGAV), or FP6v1-rTTHC (AVGIGA) in PBS. Between each subsequent step, plates were washed five times with PBS-T (PBS plus 0.05% Tween) unless otherwise stated. Plates were blocked with 100 μL/well of in-house B3T blocking solution (30 mM NaCl, 10 mM Tris-HCl, 0.2 mM EDTA, 0.66% fetal bovine serum [FBS], 0.4% bovine albumin, 0.014% Tween 20, 0.004% thimerosal) for 1 h at 37°C. Next, 50 μL/well of serially diluted 2C06 or 2C09 antibody (7-point, 5-fold, 2 μg/mL starting concentration) were added to the plates and incubated for 1 h at 37Competition ELISA for antibody binding to BG505 DS-SOSIP was performed by coating 96-well plates (Costar High Binding Half-Area; Corning, Kennebunk, ME) overnight at 4°C with 50 μL/well of 2 μg/mL snowdrop lectin from | PMC10491024 | |
IgG binding affinity measured by Carterra | A medium density HC30M sensor chip was preconditioned with 1-min pulses of each of three solutions: 50 mM NaOH, 1 M NaCl, and 10 mM Glycine pH 2.0. After preconditioning, the chip was activated with a 10-min injection of a freshly prepared 1:1:1 (v/v/v) mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 0.1 M | PMC10491024 | ||
Bio-layer interferometry (BLI) | AHC | An Octet RED instrument (FortéBio) instrument was used to measure recognition of isolated antibodies to HIV-1 BG505 DS-SOSIP, glycan-base BG505, ConC and glycan-base BG505-N241 trimers. Isolated antibodies (50 μg/mL) were immobilized for 300 s on AHC biosensor tips and equilibrated for 60 s in Octet Kinetics Buffer (Sartorius Corporation) prior to measuring association with HIV-1 trimer diluted in Octet Kinetics Buffer (100 μg/mL). The association of HIV-1 trimers were recorded for 300 s. All BLI experiments were conducted at 30°C and were performed in duplicate. Parallel correction to subtract systematic baseline drift was carried out by subtracting the measurements recorded for a loaded sensor incubated in Octet Kinetics Buffer. Response levels of sensors were analyzed using Octet and GraphPad Prism 9 software. | PMC10491024 | |
Surface plasmon resonance measurements of Fab binding affinity | Binding affinities and kinetics of 2C06 and 2C09 antibodies were assessed by surface plasmon resonance (SPR) on a Biacore S-200 (GE Healthcare) at 25°C in HBS-P+ buffer (10 mM HEPES, pH 7.4, 150 mM NaCl and 0.05% surfactant P-20). Glycan-reactive 2G12 IgG was immobilized onto a CM5 chip by amine coupling to 8000–10000 response units (RU). BG505 DS-SOSIP at 25 nM were captured to the sample channel at 300 RU on the 2G12 sensor chip. Serial two-fold diluted 2C06 and 2C09 Fabs were passed through the sample and reference channels for 180 s followed by a 300 s dissociation phase at 30 μL/min. The surface was regenerated by flowing 3 M MgCl | PMC10491024 | ||
Cryo-EM sample preparation and data collection | Samples for cryo-EM grid preparation of the 2C06-BG505 DS-SOSIP complex were produced by first mixing 15 μL of purified BG505 SOSIP at 4 mg/mL with 55 μL of 2C06 Fab at 1 mg/mL; n-Dodecyl β-D-maltoside (DDM) was added to have a final concentration of 0.005% (w/v) to prevent preferred orientation and aggregation during vitrification, and the mixture was incubated on ice for 20 min. Cryo-EM grids were prepared by applying 3 μL of sample to a freshly glow discharged carbon-coated copper grid (CF 1.2/1.3 300 mesh). The sample was vitrified in liquid ethane using a Vitrobot Mark IV with a wait time of 30 s, a blot time of 3 s, and a blot force of 0. Cryo-EM data were collected using Leginon softwareFor preparing the 2C09-BG505 DS-SOSIP complexes, Env was mixed with the antibody Fab at 1 to 1.2 M ratio at a final total protein concentration of ~3 mg/mL; DDM (1 mM stock solution) was added to a final concentration of 0.1 mM. 2.7 μL of the mixture was pipetted to a Quantifoil-gold 2/2 holey carbon grids, glow discharged for 30 s in a PELCO easiGlow Glow Discharge Cleaning System prior to cryo-grid preparation, and the grid was blotted for 2.5–4 s and plunge frozen into liquid ethane using a Vitrobot Mark IV (ThermoFisher). Cryo-EM data were collected on a Titan Krios operating at 300 kV, equipped with a K2 Summit detector (Gatan) operating in counting mode. Data were acquired using SerialEM 4.0.The movie frames were aligned and dose-weighted | PMC10491024 | ||
Model building and refinement | For structural determination, a model of the antibody Fab was generated using AlphaFold2 python notebook ( | PMC10491024 | ||
Antibody recombination and sequence signature analyses | EVENT | IgBlast was used to annotate antibody germline gene and to analyze V(D)J recombination event.The precursor frequency was calculated using OLGA software. | PMC10491024 | |
QUANTIFICATION AND STATISTICAL ANALYSIS | Cryo-EM data were processed and analyzed using CryoSparc. Cryo-EM structural statistics were analyzed with Phenix and Molprobity. Statistical details of experiments are described in | PMC10491024 | ||
Supplementary Material | PMC10491024 | |||
ACKNOWLEDGMENTS | AIDS, Rosales-Zavala, Cancer | INFECTIOUS DISEASES, ALLERGY, AIDS, FRANK, PLUMMER, CANCER | We thank Jonathan Stuckey for assistance with figures, members of the Virology Laboratory and Vector Core, Vaccine Research Center, for discussions and comments on the manuscript, and David Ho and members of the Aaron Diamond AIDS Research Center for helpful comments. We thank Preeti Apte, Anita Arthur, Allison Beck, Nina Berkowitz, Pamela Costner, Jennifer Cunningham, Aba Eshun, Ingelise J. Gordon, Cynthia S. Hendel, Renunda Hicks, LaSonji Holman, Katherine Houser, Brenda Larkin, Floreliz Mendoza, Abidemi Ola, Karen Parker, Sarah Plummer, Ro Shauna Rothwell, Jamie Saunders, Olga Trofymenko, Xiaolin Wang, William Whalen, and Alicia T. Widge of the VRC 018 clinical trial team for providing the human PMBC samples. We thank the VRC Production Program for providing ConC and BG505 Env trimers; the VRP Production Program includes Nadia Amharref, Frank J. Arnold, Nathan Barefoot, Christopher Barry, Elizabeth Carey, Ria Caringal, Kevin Carlton, Naga Chalamalsetty, Adam Charlton, Rajoshi Chaudhuri, Mingzhong Chen, Peifeng Chen, Yue Chen, Nicole Cibelli, Jonathan W. Cooper, Hussain Dahodwala, Marianna Fleischman, Julia C. Frederick, Haley C. Fuller, Jason G. Gall, Isaac Godfroy, Deepika Gollapudi, Daniel Gowetski, Krishana Gulla, Joe Horwitz, Althaf Hussain, Vera Ivleva, Tina Khin, Lisa Kueltzo, Gabriella Lagos, Q. Paula Lei, Yile Li, Venkata Mangalampalli, Gabriel Moxey, Sarah O’Connell, Aakash Patel, Erwin Rosales-Zavala, Elizabeth Scheideman, Nicole A. Schneck, Zachary Schneiderman, Andrew Shaddeau, William Shadrick, Shamitha Shetty, Brad Tippett, Alison Vinitsky, Calvin Webber, Sara Witter, Lu Yang, Yanhong Yang, and Yaqiu Zhang. This study used the Office of Cyber Infrastructure and Computational Biology High Performance Computing cluster at the National Institute of Allergy and Infectious Diseases, Bethesda, MD. Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Part of the cryo-EM data collection was performed at the Columbia University Cryo-Electron Microscopy Center, which was funded in part by the Frederick National Laboratory for Cancer Research, NIH, under contract HHSN261200800001.SUPPLEMENTAL INFORMATIONSupplemental information can be found online at DECLARATION OF INTERESTSThe authors declare no competing interests. | PMC10491024 |
REFERENCES | PMC10491024 | |||
Sorting of VRC 018 B cells from donor N751 identifies antibodies 2C06 and 2C09 that neutralize BG505 | Langmuir | (A) VRC 018 clinical regimen and a flowchart for B cell sorting and antibody screening.(B) Single B cell sorting with glycan-base BG505 trimer probes reveals only a small fraction of memory B cells from VRC 018 clinical trial to be directed to the glycan-dense surface of the Env trimer versus its glycan-free base.(C) AlphaLISA screening of RATP-Ig supernatants from sorted B cells for antibodies that bind both BG505 DS-SOSIP and glycan-base BG505. Data were measured in triplicates; error bars represent standard error of the mean (SEM).(D) Apparent affinity measurement of top antibodies from AlphaLISA screening for binding to BG505 DS-SOSIP and glycan-base BG505 trimers. Antibody IgGs were expressed and purified, and their binding to trimers was measured by Carterra. 2G12 and VRC01 were used as positive controls. Motavizumab was used as negative control, and no binding was observed.(E) Antibodies 2C06 and 2C09 neutralize BG505, but no other tested strains. Asterisk denotes tier status for BI369.9A unknown but resistant to antibodies 17b, 48d, F105, 3074, and 447-52D that neutralize only laboratory-adapted strains.(F) Affinity of antibody Fabs binding to BG505 DS-SOSIP trimer, measured by SPR.Data in (D) and (F) were measured once and the curves fitted with a simple 1:1 Langmuir binding model for the reported mean ± SEM. See also | PMC10491024 | |
Cryo-EM structure of N751-2C06.01 in complex with BG505 DS-SOSIP reveals antibody recognition at the fusion-peptide site of vulnerability | (A) Cartoon representation of 2C06 in complex with BG505 DS-SOSIP. 2C06 bound at the fusion-peptide site and interacted with fusion peptide (FP), shown in red. Antibody heavy chain (H) is shown in blue, light chain (L) in pale cyan, gp41 in dark gray, and gp120 in light gray.(B) Density map for fusion peptide N-terminal residues. The structure is shown in cartoon representation, with fusion peptide and antibody side chains interacting with fusion peptide shown in sticks. Color codes are as in (A).(C) 2C06 epitope details. Env trimer is shown as surface with subunits in different shades of gray and the epitope surface in magenta. Antibody CDRs and FRs involved in binding are shown in cartoon representation, with interacting side chains shown in sticks. The glycan hole at S241 is on the edge of the epitope and is labeled.(D) Overall sequence variation of HIV-1 group M Env glycoproteins. Normalized sequence entropy is mapped on the trimer surface, with absolutely conserved residues (entropy of 0) in white and the most diverse residues (entropy of 1) in purple.(E) HIV-1 Env sequence variation around the 2C06 epitope. 2C06 epitope is shown as a blue outline. Part of the 2C06 epitope is highly variable (labeled). The residue at 241 is well conserved with over 96% of isolates having an See also | PMC10491024 | ||
Cryo-EM structure of N751-2C09.01 in complex with BG505 DS-SOSIP reveals epitope similarity to N751-2C06.01 | (A) Cartoon representation of 2C09 Fab in complex with BG505 DS-SOSIP. 2C09 bound at the fusion-peptide site and interacted with fusion peptide extensively. Heavy chain (H) is shown in orange, light chain (L) in yellow-orange, fusion peptide (FP) in red, the rest of gp41 in darker gray, and gp120 in lighter gray.(B) Density map showing well-ordered density for fusion peptide. The structure is shown in cartoon representation, with side chains of fusion-peptide residues 512–520 shown in sticks. Some side chains of the antibody interacting with fusion peptide are also shown in sticks.(C) Epitope details of antibody 2C09 shown for one of the binding sites on the trimer. Env trimer is shown as surface, with subunits in different shades of gray and the epitope surface in magenta. Antibody CDRs and FRs involved in binding are shown in cartoon representation, with interacting side chains shown in sticks. The glycan hole at S241 is on the edge of the epitope and is labeled.(D) Sequence conservation around the 2C09 epitope. Normalized sequence entropy of HIV-1 group M is mapped on the trimer surface, with absolutely conserved residues (entropy of 0) in white and the most diverse residues (entropy of 1) in purple. 2C09 epitope is shown as an orange outline. The outline of 2C06 is shown in blue for comparison.(E) Structure superposition between 2C06 and 2C09 complexes. The two structures were aligned by the gp41 subunit bearing the fusion peptide shown. The fusion peptide in both structures were in similar conformation except the first two residues, which in 2C09 (darker red) folded back down to interact with the antibody and in 2C06 extended up to bind the C-terminal helix of neighboring gp41 (top left).See also | PMC10491024 | ||
Antibodies 2C06 and 2C09 form a reproducible class | (A and B) Analysis of junction regions and sequence alignment of 2C06 and 2C09. Germline gene nucleotide and amino acid residues are shown in black. The somatic hypermutations are colored red. Nucleotides removed by exonuclease trimming are crossed out. Germline amino acid differences between IGHV3-64 and IGHV3-64D are labeled with green highlight. Fusion-peptide contacts (red highlight), glycan contacts (green rectangles), and additional trimer contacts (underlined) are highlighted.(C) Heavy-chain germline gene usage of 138 antibodies isolated from donor N751.(D) Binding analysis of chimeras swapping heavy and light chains between 2C06 and 2C09. Data were measured once.(E) Sequence signatures of the reproducible 2C06 and 2C09 class for fusion-peptide binding. HV, heavy-chain variable; LV, light-chain variable. See also (F) Calculated precursor frequency of 2C06/2C09 antibody class for healthy donors HIP1–HIP3 (see | PMC10491024 | ||
Antibody 2C06 partially accommodates, but antibody 2C09 clashes with, glycan241 | PyMOL | MUTANT | (A) BLI binding analysis of 2C06 and 2C09 with Env trimers with or without glycan241 in comparison with other fusion-peptide-directed antibodies. Both BG505 DS-SOSIP and glycan-base BG505 have serine at 241. Glycan-base BG505-N241 is an S241N mutant of glycan-base BG505 containing (B) Structures of VRC34.01 (PDB: 6nc3) and ACS202 (PDB: 6nc2), both in complex with AMC011 Env, which contains glycan241, reveal that both antibodies interact favorably with glycan88 and glycan241, shown in different shades of green. Neutralization breadths were for half-maximal inhibitory concentration (IC(C) Structural alignment of Env trimers from the PDB with two or more sugar residues built for glycan241. The structures were aligned by the gp120 subunit bearing the glycan241 shown. Antibodies, if present in the structure, are not shown for clarity. Glycan88 and glycan241 are shown in different shades of green, with the same view as in other structural panels of the figure. PDB: 6olp, 6vo3, 7rsn, 7rso, 6ohy, 7n6u, 6vrw, 6myy, 7llk, 6u59, 5vn8, 6p65, 6vy2, 7lua, 6okp, 7l6o, 6nc3, 6nc2.(D) Antibodies 2C06 and 2C09 clash with the glycan241 conformation seen in all known Env structures containing glycan241. The structures were aligned with that of ACS202-AMC011 complex by the gp120 subunit bearing the glycan241 shown. Clashes between antibody 2C06 or 2C09 and glycan241 in the ACS202 complex are shown as red disks in PyMOL ((E) Antibodies derived from mouse immunizations, vFP16.02 (PDB: 6cdi), and from non-human primates, 0PV-c.01 (PDB: 6nf2), A12V163-a.01 (PDB: 6n1v), and A12V163-b.01 (PDB: 6mgp), all of which clash with the glycan241 conformation in ACS202. A different glycan241 conformation, similar to that for 2C06 and 2C09, could be modeled, which avoids any clashes. Binding of 0PV-c.01 allows for more space for multiple conformations of modeled glycan241. Hash mark denotes that A12V163-b.01 neutralizes only at ICSee also | PMC10491024 |
Recognition requirements for high-entropy residues contribute to strain specificity of 2C06 and 2C09 | (A) While neutralization breadth generally correlates with epitope entropy, antibodies directed to the fusion-peptide sites of vulnerability have similar epitope entropy. Neutralization breadth represents the percentage of the 208-strain panel neutralized with IC(B) Analyses of sequence entropy and contact surface areas of residues in strain-specific and broadly neutralizing fusion-peptide-directed antibodies. Epitope residues with normalized entropy above 0.4 are listed, as well as residue S241, which has low entropy but with low frequency as serine among HIV-1 isolates. The contact atoms and areas for VRC34.01 were calculated from PDB: 5i8h (VRC34.01 also binds AMC011 Env, which has valine85). Entropy values were calculated with Shannon Entropy-One ((C) H85, with high Shannon entropy of 0.733 and 2.5% frequency, has tight interactions with antibody heavy- and light-chain residues in both structures of the 2C06 complex (2C09 has nearly identical interactions, see See also KEY RESOURCES TABLE | PMC10491024 | ||
Highlights | HIV-1 neutralizing antibodies 2C06 and 2C09 from VRC 018 clinical trial are isolatedCryo-EM structures of 2C06 and 2C09 in complex with HIV envelope trimer are determined2C06 and 2C09 binding requires a glycan hole at 241 and BG505-specific residuesAntibodies 2C06 and 2C09 form a reproducible class, recognizing fusion peptide | PMC10491024 | ||
Background: | toxicity, nonbacterial breast inflammation | PATHOGENESIS, PLASMA CELL MASTITIS, PCM | These authors contributed equally to this work.Plasma cell mastitis (PCM) is a nonbacterial breast inflammation with severe and intense clinical manifestation, yet treatment methods for PCM are still rather limited. Although the mechanism of PCM remains unclear, mounting evidence suggests that the dysregulation of immune system is closely associated with the pathogenesis of PCM. Drug combinations or combination therapy could exert improved efficacy and reduced toxicity by hitting multiple discrete cellular targets. | PMC10063228 |
Methods: | PCM | We have developed a knowledge graph architecture toward immunotherapy and systematic immunity that consists of herbal drug–target interactions with a novel scoring system to select drug combinations based on target-hitting rates and phenotype relativeness. To this end, we employed this knowledge graph to identify an herbal drug combination for PCM and we subsequently evaluated the efficacy of the herbal drug combination in clinical trial. | PMC10063228 | |
Results: | RECURRENCE, PCM | Our clinical data suggests that the herbal drug combination could significantly reduce the serum level of various inflammatory cytokines, downregulate serum IgA and IgG level, reduce the recurrence rate, and reverse the clinical symptoms of PCM patients with improvements in general health status. | PMC10063228 | |
Conclusions: | RECURRENCE, PCM | In summary, we reported that an herbal drug combination identified by knowledge graph can alleviate the clinical symptoms of PCM patients. We demonstrated that the herbal drug combination holds great promise as an effective remedy for PCM, acting through the regulation of immunoinflammatory pathways and improvement of systematic immune level. In particular, the herbal drug combination could significantly reduce the recurrence rate of PCM, a major obstacle to PCM treatment. Our data suggests that the herbal drug combination is expected to feature prominently in future PCM treatment. | PMC10063228 | |
Funding: | C. Liu’s lab was supported by grants from the Public Health Science and Technology Project of Shenyang (grant: 22-321-32-18); Y. Yang’s laboratory was supported by the National Natural Science Foundation of China (grant: 81874301), the Fundamental Research Funds for Central University (grant: DUT22YG122), and the Key Research project of ‘be Recruited and be in Command’ in Liaoning Province (2021JH1/10400050). | PMC10063228 | ||
Research organism | PMC10063228 | |||
Introduction | TCM | DISEASES, PLASMA CELL MASTITIS, PCM | Plasma cell mastitis (PCM) represents a serious inflammatory condition of breast that occurs in young and middle-aged females at nonpregnant and nonlactating period (Traditional Chinese Medicine (TCM) has a rather long history for the prevention and treatment of complex diseases in eastern Asia (Knowledge graph has emerged as an advanced technology in the field of artificial intelligence that is able to connect entities in a graph based on their existing intricate relationships ( | PMC10063228 |
Methods | PMC10063228 | |||
Construction of knowledge graph toward immunotherapy | We employed data mining techniques to collect and compile 240 targets of immunotherapy and systematic immunity from the PubMed database. Next, we collected and compiled 345 herbal drug entities officially released by the National Health Commission of China and the National Administration of Traditional Chinese Medicine. The intricate relations between the herbal drug entities and the immunotherapy targets were extracted from the PubMed database. These intricate relations were subjected to further manual curation. We used 13 ontology terms to describe the intricate relations (edges) in the knowledge graph. Moreover, 64 attributes of the medicinal properties for the herbal drug entities were collected and compiled from the Pharmacopoeia of China. Finally, we built the knowledge graph using Neo4j and Py2Neo tools that consists of 895 nodes and 2197 edges. | PMC10063228 | ||
Design of the clinical trial | PCM | In brief, 160 female patients diagnosed with PCM at the Shengjing Hospital Affiliated to China Medical University were recruited in the clinical trial between January 2021 and February 2022. The patients were divided 1:1 into experimental group (EG) and control group (CG). It is noteworthy that, in order to demonstrate the therapeutic effects of TCM drug combination, we selected the patients who were treated with Western medicine in the real world during the same period. Therefore, the two groups of patients were divided into TCM treatment group (EG) and Western medicine treatment group (CG). There was no significant difference in baseline data such as age, body mass index, clinical classification, marriage, and child-bearing history between the two groups ( | PMC10063228 | |
Clinical trial protocol | The clinical trial for the herbal drug combination was registered at | PMC10063228 | ||
Measurement of serum inflammatory cytokines by ELSIA assay | STERILE | Venous blood of the CG and EG was collected in a sterile non-anticoagulant test tube before and after treatment. The immune transmission turbidimetry was used according to the procedure of CRP kit, and automatic biochemical analyzer was used to detect the level of CRP. The levels of serum cytokines were measured by ELISA (Elabscience) according to the manufacturer’s instructions. | PMC10063228 | |
Measurement of serum immunoglobulin level | STERILE, PCM | The venous blood of PCM patients in the two groups was collected in a sterile non-anticoagulant tube before and after treatment. The serum IgG and IgA were measured by rate scattering turbidimetry using Array 360 System automatic-specific protein analyzer (Beckman Company, USA). | PMC10063228 | |
Assessment of clinical symptoms of PCM patients | PATHOLOGY, PCM | The clinical symptoms were evaluated by an attending physician with board certification in pathology. The patients were scored before and after treatment according to the standard rating scale for PCM ( | PMC10063228 | |
Statistics | All data were evaluated as mean ± SEM. Statistical analysis of the quantitative multiple group comparisons was performed using ANOVA, followed by Tukeyʼs test, whereas pairwise comparisons were performed using the | PMC10063228 | ||
Results | In a previous study, we collected and compiled 240 targets for immunotherapy and systematic immunity from literature data ( | PMC10063228 | ||
Snapshot of the medical knowledge graph toward immunotherapy. | The knowledge graph was constructed using Neo4j and Python tools (
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CONSORT flowchart diagram of the clinical trial for the herbal drug combination. | Next, we extracted the subgraph for the herbal drug combination mentioned above and created a network diagram for the drug combination using Cytoscape tools ( | PMC10063228 | ||
The score statistics of 10 rounds of random herbal drug combinations (1000 random combination from each round) for the treatment of plasma cell mastitis. | It is noteworthy that the scoring results of the 10 rounds are presented as normal distributions. The herbal drug combination identified for the clinical trial was among the top 20 choices in all the 10 rounds and is marked with red dots. | PMC10063228 | ||
Pathway analysis for the potential cellular targets of the herbal drug combination via Reactome Knowledgebase. | The statistically significant pathways are highlighted and displayed in yellow color (p<0.05).Efficacy was assessed every two cycles, and the results were summarized after 6 mo of treatment. The baseline characteristics are shown in | PMC10063228 | ||
The change of percentage for numerous immunological cytokines, including IL-2, IL-4, IL-6, IL-10, IL-17, CRP, IFN-γ, IL-1β, and TNF-α, from the control group and the treatment group (with Traditional Chinese Medicine [TCM] treatment). | TNF-α | Notably, a few key cytokines such as IL-2, IL-4, IFN-γ, IL-1β, and TNF-α were significantly downregulated in the treatment group compared to the control group.
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Patients’ raw data of serum cytokines from the control group (without Traditional Chinese Medicine [TCM] treatment) and the treatment group (with TCM treatment) in the clinical trial ( | PMC10063228 | |||
The change of percentage for IgA level and IgG level from the control group and the treatment group (with Traditional Chinese Medicine [TCM] treatment). | PMC10063228 | |||
Patients’ raw data of serum IgA and IgG level from the control group (without Traditional Chinese Medicine [TCM] treatment) and the treatment group (with TCM treatment) in the clinical trial ( | PMC10063228 | |||
The change of scores from items, including symptom, pain, and living quality. | pain | The scores of symptoms, pain, and living quality from the treatment group were significantly improved compared to the control group.
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Patients’ raw data of symptom scores from the control group (without Traditional Chinese Medicine [TCM] treatment) and the treatment group (with TCM treatment) in the clinical trial ( | PMC10063228 | |||
Patients’ raw data of pain scores from the control group (without Traditional Chinese Medicine [TCM] treatment) and the treatment group (with TCM treatment) in the clinical trial ( | PMC10063228 | |||
The comparison of score of life quality between different groups. | Control group (CG) baseline, experimental group (EG) baseline, CG treated with methylprednisolone, and EG treated with herbal drug combinations. | PMC10063228 | ||
Comparison of operation rate, recurrence rate, and incidence of adverse reactions between the two groups (EG: experimental group; CG: control group). | PMC10063228 | |||
The comparison of whole breast for six representative plasma cell mastitis (PCM) patients in the experimental group (EG) before treatment and after treatment. | ( | PMC10063228 | ||
Clinical symptom scores between the two groups in the clinical trial (EG: experimental group; CG: control group; The clinical symptom rating scale for plasma cell mastitis (PCM) is displayed in | PMC10063228 | |||
Discussion | pain | RECURRENCE, BREAST CANCER, ADVERSE EVENTS, PCM, PATHOGENESIS | With the increasing amount of biomedical data, the traditional drug discovery campaign has been revolutionized with the aid of artificial intelligence techniques to accelerate the process and reduce the cost (Although the pathogenesis of PCM remains largely unclear, there have been numerous reports implicating that the overactivation of immunoinflammatory pathways plays an important role in the development of PCM (We want to remind the reader that a third arm (a placebo group) added to the clinical study might be useful to fully reveal the therapeutic effects of the herbal drug combination. Unfortunately, we were unable to add a third arm due to some ethical concerns. This is because PCM is a rather acute, severe, and intense clinical manifestation of breast. Without any treatment, the serious inflammatory condition of PCM may quickly advance into breast cancer. Therefore, this is a limitation of our clinical study, and we hope to design more delicate clinical trials in the future to demonstrate the therapeutic effects of herbal drug combination. Nevertheless, the detailed analysis of three indicators, including symptom score, pain score, and score of life quality, between the EG treated with herbal drug combination and the CG treated with methylprednisolone in the clinical study suggests that the herbal drug combination might be more effective in reversing the clinical conditions of PCM compared to the methylprednisolone treatment (see In this study, our results revealed that the herbal drug combination identified by knowledge graph could suppress a few key immunoinflammatory cytokines, enhance the systematic immune levels, and significantly reduce the recurrence rates of PCM patients. Note that recurrence has become one major obstacle after surgical resection for PCM treatment in clinical practice. On the other hand, hormone therapy may increase the risk of side effects for PCM patients. Therefore, our approach of herbal drug combination may provide a new avenue for PCM treatment with less recurrence rate and reduced incidence rate of adverse events. | PMC10063228 |
Conclusion | human disorders | RECURRENCE, PCM | In summary, we report the identification and clinical assessment of an herbal drug combination toward PCM. We demonstrated that the herbal drug combination holds great promise as an effective remedy for PCM, acting through the regulation of multiple cellular targets and immunoinflammatory pathways, which leads to the improvement in systematic immune level. In particular, the herbal drug combination could significantly reduce the recurrence rate of PCM, a major obstacle to PCM treatment. Our data suggests that the herbal drug combination is expected to feature prominently in the future PCM treatment. Moreover, these promising results underscore the potential of knowledge graph to identify drug combinations or other novel therapeutics across various types of human disorders. | PMC10063228 |
Ethical statement | The protocol was approved by the Institutional Review Board (IRB) of the China Medical University (approval number: 2021PS024T). This study was registered with | PMC10063228 | ||
Acknowledgements | Cancer | CANCER | Y Yang’s laboratory was supported by the National Natural Science Foundation of China (grant: 81874301), the Fundamental Research Funds for Central University (grant: DUT22YG122), and the Key Research project of ‘be Recruited and be in Command’ in Liaoning Province (2021JH1/10400050); C Liu’s lab was supported by grants from the National Natural Science Foundation of China (no. 81572609), China Medical University Major Construction Project (no. 2017ZDZX05), and Liaoning Colleges Innovative Talent Support Program (Cancer Stem Cell Origin and Biological Behavior). | PMC10063228 |
Additional information | PMC10063228 | |||
Competing interests | Senior editor, No competing interests declared.No competing interests declared.is an employee of China Resources Sanjiu Medical & Pharmaceutical.is an employee of Shanghai BeautMed Corporation.Reviewing editor, | PMC10063228 | ||
Author contributions | Conceptualization, Resources, Supervision, Funding acquisition, Project administration.Data curation, Supervision, Investigation, Project administration.Validation, Visualization, Project administration.Data curation, Software, Visualization, Methodology.Software, Methodology.Validation, Project administration.Resources, Validation, Visualization.Validation, Investigation, Visualization.Validation, Investigation, Visualization.Data curation, Software.Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing. | PMC10063228 | ||
Ethics | Clinical trial registration ClinicalTrials.gov: NCT05530226.Human subjects: The protocol was approved by the Institutional Review Board (IRB) of the China Medical University (approval number: 2021PS024T). This study was registered with | PMC10063228 | ||
Additional files | PMC10063228 | |||
Baseline charateristics and rating scale for the clinical trial. | ( | PMC10063228 | ||
Clinical protocol for the clinical trial study. | PMC10063228 | |||
Some detailed information for the six patients displayed in | PMC10063228 | |||
Python source code for the scoring system of knowledge graph to assess and select appropriate drug combinations. | PMC10063228 | |||
Data availability | Figure 1–3 are computational and therefore no data have been generated. In addition, Figure 4—source data 1, Figure 5—source data 1, Figure 6—source data 1, Figure 6—source data 2 and Figure 6—source data 3 contain the numerical data used to generate the figures. | PMC10063228 | ||
References | ’, pain, human disorders, Astragalus’, Fructus forsythiae’, breast inflammation, human disease, Danshen’, TCM | PLASMA CELL MASTITIS, BREAST CANCER, DISEASE COURSE, DISEASE, MINOR, SYNDROME, SYNDROME, BREAST INFLAMMATION, PCM, PATHOGENESIS, PLASMA CELL MASTITIS | This study presents a valuable identification of an herbal drug combination for treating plasma cell mastitis (PCM), a breast inflammation with severe and intense clinical symptoms. The data were collected and analyzed using a solid approach in a clinical trial of 160 patients (NCT05530226). They can be used to understand how herbal drug combinations could help manage PCM patients.Our editorial process produces two outputs: (i)
Thank you for submitting your article "A herbal drug combination identified by knowledge graph alleviates the clinical symptoms of plasma cell mastitis patients: a non-randomized controlled trial" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:(1) Please address Reviewer 1's comments about the patient groups and confounding factors as well as expand the rationale for the evaluation of the clinical trial as noted for example, by reviewer 2's comments about the combination drug score.(2) Please address the relatively minor comments of all three reviewers.
Figure 2 looks like it could be moved to supplementary data.Figure 3 text was too small to read.The presented changes in figures 5 and 6, while statistically significant do not demonstrate significant difference. Perhaps a third, placebo, arm is needed whereby the steroids and the herbal groups induce immune changes not seen in the placebo group? That data would be clear and convincing, but as presented it is difficult to understand if this is a medication effect o=r the natural course of disease in these patients.
Below are some specific points for the manuscript,1. It would be necessary to elaborate the advantages of employing knowledge graph to design drug combinations for a specific human disease in the Discussion section of the manuscript.2. In Figure 4, the authors claimed that 'a few pathways related to systematic immunity' were identified. In page 8, line 158, the authors should discuss in length for the specific roles of these pathways in PCM pathogenesis.3. The drug combination in the clinical trial didn't obtain the highest score (Figure 2). The authors need to explain this in the Discussion section.4. Figure 1, Supplement 1, the authors need to explain the color scheme (pink, yellow and green etc) for the entities.5. Page 6, line 133, please explain and specify the term 'syndrome differentiation'. The reader who are not in TCM domain may not understand the term.6. In page 6, the authors claimed that the scoring system is able to identify 'herbal drug combinations that are able to hit most discrete cellular targets'. The authors need to explain and elaborate why this criteria is crucial for the identification of drug combinations for PCM in the Discussion section.7. The authors need to provide more details for their clinical study. For example, more details are required for the patients in Figure 6.
This is a rather interesting study that introduced a new concept in the domain of TCM and herbal drugs. Below are some specific suggestions which I think may help to improve the manuscript,1. The herbal drug combination used in the clinical study didn't obtain the highest score during the scoring process (Figure 2). The authors need to explain and elaborate this in a paragraph.2. In Figure 3, the network diagram analysis from the knowledge graph revealed that NLPR3, IL-17, TLR4, STAT3, IL-6, iNOS and TLR2 are possible cellular targets for the herbal drug combination. The authors need to elaborate or provide some references that NLPR3, IL-17, TLR4, STAT3, IL-6, iNOS and TLR2 are relevant to PCM pathogenesis.3. The authors need to explain and elaborate 'penalty function', in page 14.4. Line 326, the authors mean 'validated via cellular model'?5. Line 329, the author refers to 'systematic immunity'?6. Page 17, line 347, 'The herbal drug combination was prepared as granules in the following formulae', The authors need to explain how this formula was determined.7. In Figure 8, the authors need to provide more details for the clinical pictures in a paragraph. For example, age, treatment period and brief descriptions of the symptoms etc.8. The authors need to explain why the drug combination consisting of eight herbal drugs is selected in their study. Page 7, line 137, the authors made a statement that 'eight drugs are regarded as essence combination'. The authors need to explain and elaborate this point in the manuscript.9. Is it feasible that the authors could include chemical ingredients of the herbal drugs into their knowledge graph for the identification of herbal drug combination? The authors should elaborate more for this point in the manuscript.Reviewer #1 (Recommendations for the authors):Figure 2 looks like it could be moved to supplementary data.This has been fixed. Per the referee’s suggestion, we have move Figure 2 to supplementary data.Figure 3 text was too small to read.This has been fixed. Per the referee’s comment, we have rearranged and enlarged the text in Figure 3 (now Figure 2 in draft-track-change). Please see draft-track-change for more details.The presented changes in figures 5 and 6, while statistically significant do not demonstrate significant difference. Perhaps a third, placebo, arm is needed whereby the steroids and the herbal groups induce immune changes not seen in the placebo group? That data would be clear and convincing, but as presented it is difficult to understand if this is a medication effect o=r the natural course of disease in these patients.We want to thank the referee for this very insightful and expert comment. We fully agree with the referee that a third arm (placebo group) added in the clinical trial may be useful to further reveal the therapeutic effects of herbal drug combinations. Indeed, we met some difficulties to add the placebo group in the clinical trial and unfortunately, we were unable to obtain the ethnical approval from the Research Ethnical Committee of our university should we want to add the placebo group in the trial. This is because plasma cell mastitis (PCM) is a rather acute, severe and intense clinical manifestation of breast. Without any treatment, the serious inflammatory condition of PCM may quickly advance into breast cancer. In addition, the patients may feel tremendous pain without treatment due to the quick development of PCM. Therefore, the Research Ethnical Committee raised concerns about the placebo group and we were unable to add the placebo group in the trial. Nevertheless, we fully agree with the referee that a third arm could be useful and we elaborate this limitation of our clinical study in a paragraph in the Discussion section of our manuscript. Moreover, we conducted additional analysis of the clinical data. The detailed analysis of three indicators including symptom score, pain score and score of life quality between the experiment group treated with herbal drug combination and the control group treated with methylprednisolone in the clinical study suggest that the herbal drug combination might be more effective to reverse the clinical conditions of PCM as compared to the methylprednisolone treatment (see Figure 6 —figure supplement 1-2-3). Please see below or draft-track-change, line 234-246 for more details.“We want to remind the reader that a third arm (a placebo group) added in the clinical study might be useful to fully reveal the therapeutic effects of the herbal drug combination. Unfortunately, we were unable to add a third arm due to some ethnical concerns. This is because plasma cell mastitis (PCM) is a rather acute, severe and intense clinical manifestation of breast. Without any treatment, the serious inflammatory condition of PCM may quickly advance into breast cancer. Therefore, this is a limitation of our clinical study and we hope to design more delicate clinical trial in the future to demonstrate the therapeutic effects of herbal drug combination. Nevertheless, the detailed analysis of three indicators including symptom score, pain score and score of life quality between the experiment group treated with herbal drug combination and the control group treated with methylprednisolone in the clinical study suggest that the herbal drug combination might be more effective to reverse the clinical conditions of PCM as compared to the methylprednisolone treatment (see Figure 6 —figure supplement 1-2-3).”Reviewer #2 (Recommendations for the authors):Below are some specific points for the manuscript,1. It would be necessary to elaborate the advantages of employing knowledge graph to design drug combinations for a specific human disease in the Discussion section of the manuscript.Many thanks for this critical and helpful comment. Per the referee’s suggestion, we have elaborated the advantage of the knowledge graph towards the design of drug combinations in the Discussion section of the manuscript. Please see below or draft-track-change, line 214-222 for more details.“The advantage of employing knowledge graph for drug discovery lies in the capabilities of revealing structured associations between drug entities, cellular targets, biological pathways and phenotypes for human disorders. This is useful for scientists to identify new indications or phenotypes for existing drugs, or so-called drug repurposing. With the aid of scoring function or recommendation system, knowledge graph can also be used to design and identify drug combinations for a specific disease. Herein, for the first time, we introduced and employed the concept of knowledge graph to identify herbal drug combinations for the severe Plasma cell mastitis (PCM) with unmet medical needs.”2. In Figure 4, the authors claimed that 'a few pathways related to systematic immunity' were identified. In page 8, line 158, the authors should discuss in length for the specific roles of these pathways in PCM pathogenesis.We want to thank the referee for this insightful comment. This has been fixed. Per the referee’s suggestion, we have discussed in length for the specific roles of the related pathways in PCM. Please see below or draft-track-change, line 214-222 for more details.“For instance, ‘Toll-like receptors cascades’, ‘Adaptive immune system’, ‘Cytokine signaling in immune system’ and ‘Innate immune system’ are critical cellular pathways for systematic immunity which are directly associated with the pathogenesis of PCM. Moreover, the ‘MAP kinase activation’ pathway is associated with cellular defense and innate immunity which are also crucial for the development and inflammatory conditions of PCM.”3. The drug combination in the clinical trial didn't obtain the highest score (Figure 2). The authors need to explain this in the Discussion section.Thanks for this expert and helpful comment. This has been fixed. The drug combination was chosen for the further clinical study for two reasons. First, this drug combination was among top twenty combinations in each round of calculations. Second, we asked experts in TCM to inspect the top twenty combinations on the basis of ‘syndrome differentiation’ as described in Pharmacopoeia of China and finally the combination consisting of eight herbal drug entities including ‘Fructus forsythiae’, ‘Herba violae’, ‘Uniflower swisscentaury root’, ‘Danshen’, ‘Astragalus’, ‘Taraxacum’, ‘Liquorice’ and ‘Honeysuckle’ was selected for further clinical study. Please see below or draft-track-change, line 144-151 for more details.“The drug combination was chosen for the further clinical study for two reasons. First, this drug combination was among top twenty combinations in each round of our calculations. Second, we asked experts in TCM to inspect the top twenty combinations on the basis of ‘syndrome differentiation’ as described in Pharmacopoeia of China and finally the combination consisting of eight herbal drug entities including ‘Fructus forsythiae’, ‘Herba violae’, ‘Uniflower swisscentaury root’, ‘Danshen’, ‘Astragalus’, ‘Taraxacum’, ‘Liquorice’ and ‘Honeysuckle’ was selected for further clinical study (see Figure 2).”4. Figure 1, Supplement 1, the authors need to explain the color scheme (pink, yellow and green etc) for the entities.Thank you for this critical comment. This has been fixed. We have explained the various color scheme in the figure legend. Please see page 22, in draft-track-change for more details.5. Page 6, line 133, please explain and specify the term 'syndrome differentiation'. The reader who are not in TCM domain may not understand the term.We are grateful for this critical comment. This has been fixed. Per the referee’s suggestion, we have explained the term ‘syndrome differentiation’ in the manuscript. Please see Please see below or line 134-138, in draft-track-change for more details.“Here, syndrome differentiation refers to the very basic principle of identifying and treating disease in TCM. Syndrome (Zheng) is the presentation of the pathological changes during a specific disease course including the location, cause and nature of a disease. Moreover, ‘Jun-Chen-Zuo-Shi’ refers to the rules guided by syndrome differentiation to select multiple herbal drug entities to treat a specific disease in TCM.”6. In page 6, the authors claimed that the scoring system is able to identify 'herbal drug combinations that are able to hit most discrete cellular targets'. The authors need to explain and elaborate why this criteria is crucial for the identification of drug combinations for PCM in the Discussion section.Many thanks for this critical comment. Per the referee’s suggestion, we have explained why the criterion of the scoring system is crucial for the identification of drug combinations in the manuscript. Please see below or line 249-252 in draft-track-change for more details.“This is because PCM is a rather complex disease which pathogenesis may involve multiple targets and immunoinflammatory pathways. Hence, we made the hypothesis that drug combinations that can act on most discrete targets or pathways related to PCM might be more effective.”7. The authors need to provide more details for their clinical study. For example, more details are required for the patients in Figure 6.This has been fixed. We have provided detailed information for the six patients in Figure 6 as supplemental materials (SI_patients_information).Reviewer #3 (Recommendations for the authors):This is a rather interesting study that introduced a new concept in the domain of TCM and herbal drugs. Below are some specific suggestions which I think may help to improve the manuscript,1. The herbal drug combination used in the clinical study didn't obtain the highest score during the scoring process (Figure 2). The authors need to explain and elaborate this in a paragraph.Thanks for this expert and helpful comment. This has been fixed. The drug combination was chosen for the further clinical study for two reasons. First, this drug combination was among top twenty combinations in each round of calculations. Second, we asked experts in TCM to inspect the top twenty combinations on the basis of ‘syndrome differentiation’ as described in Pharmacopoeia of China and finally the combination consisting of eight herbal drug entities including ‘Fructus forsythiae’, ‘Herba violae’, ‘Uniflower swisscentaury root’, ‘Danshen’, ‘Astragalus’, ‘Taraxacum’, ‘Liquorice’ and ‘Honeysuckle’ was selected for further clinical study. Please see below or draft-track-change, line 144-151 for more details.“The drug combination was chosen for the further clinical study for two reasons. First, this drug combination was among top twenty combinations in each round of our calculations. Second, we asked experts in TCM to inspect the top twenty combinations on the basis of ‘syndrome differentiation’ as described in Pharmacopoeia of China and finally the combination consisting of eight herbal drug entities including ‘Fructus forsythiae’, ‘Herba violae’, ‘Uniflower swisscentaury root’, ‘Danshen’, ‘Astragalus’, ‘Taraxacum’, ‘Liquorice’ and ‘Honeysuckle’ was selected for further clinical study (see Figure 2).”2. In Figure 3, the network diagram analysis from the knowledge graph revealed that NLPR3, IL-17, TLR4, STAT3, IL-6, iNOS and TLR2 are possible cellular targets for the herbal drug combination. The authors need to elaborate or provide some references that NLPR3, IL-17, TLR4, STAT3, IL-6, iNOS and TLR2 are relevant to PCM pathogenesis.We are grateful for this critical and helpful comment. This has been fixed. We have provided references for these putative targets. Please see page 8, line 161-162 in draft-track-change for more details.3. The authors need to explain and elaborate 'penalty function', in page 14.This has been fixed. We have explained the ‘penalty function’ in the manuscript. Please see below or page 13, line 339-342 in draft-track-change for more details.“Herein, the penalty function is used to ensure that the herbal drug entities in the combination didn’t violate the contraindication rules in Pharmacopoeia of China according to the medicinal attributes of herbal drugs.”4. Line 326, the authors mean 'validated via cellular model'?This has been fixed. Here we mean ‘validated via cellular model’.5. Line 329, the author refers to 'systematic immunity'?This has been fixed. Herein we refer to 'systematic immunity'.6. Page 17, line 347, 'The herbal drug combination was prepared as granules in the following formulae', The authors need to explain how this formula was determined.This has been explained. Please see below or page 19, line 393-398 in draft-track-change for more details.“The formula was determined by TCM experts on the basis of ‘syndrome differentiation’ as described in Pharmacopoeia of China. Furthermore, the herbal drug combination in the form of granules was provided and prepared by Shengjing Hospital Affiliated to China Medical University according to the standard requirement of clinical study by National Medical Products Administration (NMPA).”7. In Figure 8, the authors need to provide more details for the clinical pictures in a paragraph. For example, age, treatment period and brief descriptions of the symptoms etc.This has been fixed. We have provided detailed information for the six patients in Figure 6 as supplemental materials (SI_patients_information).8. The authors need to explain why the drug combination consisting of eight herbal drugs is selected in their study. Page 7, line 137, the authors made a statement that 'eight drugs are regarded as essence combination'. The authors need to explain and elaborate this point in the manuscript.We are thankful for this critical and helpful comment. This has been fixed. We chose to identify drug combinations with eight entities because ‘formulae’ consisting of eight drugs are regarded as ‘essence combination’ in TCM field. Please see page 7, line 140-142 in draft-track-change for more details.9. Is it feasible that the authors could include chemical ingredients of the herbal drugs into their knowledge graph for the identification of herbal drug combination? The authors should elaborate more for this point in the manuscript.Many thanks for this insightful and helpful comment. We fully agree with the referee that the inclusion of chemical ingredients from the herbal drugs may impact the outcome of our analysis and design. Unfortunately, the inclusion of chemical ingredients in the knowledge graph is rather technically difficult due to the limited and incomplete datasets for the herbal drugs in the field of TCM. Nevertheless, our strategy captures the prominent feature of design for drug combinations towards a complex disease such as PCM. In the future, we plan to include multiple types of omics data such as genomic, transcriptomic, proteomic, metagenomic or metabolomics data into the knowledge graph to reveal novel targets and enable novel drug discovery. Please see page 12, line 252-260 in draft-track-change for more details. We hope this could clarify the concerns. | PMC10063228 |
1. Introduction | sore throat, fever, inflammation, URTIs, infection, scratchy | SORE THROAT, UPPER RESPIRATORY TRACT INFECTIONS, INFLAMMATION, FLU SYMPTOMS, INFECTION, COMMON COLD, COLD, INFLUENZA | Introduction: Upper respiratory tract infections (URTIs) are caused by bacteria or viruses, with the most common causes being the common cold and influenza. The high occurrence of URTI means therapies that are effective with minimal side effects are in constant demand. Palmitoylethanolamide (PEA) is a signaling lipid previously shown to be effective in improving the incidence of URTIs. The aim of this study was to assess the effectiveness of PEA (Levagen+) on URTI incidence, duration, and severity. Methods: Participants (Upper respiratory tract infections (URTIs) are caused by an infection of the mucosal lining of the upper airway. URTI symptoms include coughing, sneezing, stuffy or runny nose, fever, and scratchy or sore throat [Palmitoylethanolamide (PEA) is an endocannabinoid-like bioactive signaling lipid that is part of the To date, most literature stating the efficacy of PEA on cold and flu symptoms is based on theoretical evidence or animal studies, rather than human clinical studies. Previous human clinical studies have indicated that PEA is an effective treatment in reducing cold and flu symptoms [Recent human clinical studies have focused more on the potential effects of PEA on inflammation associated with COVID-19. PEA has been shown to be effective against respiratory symptoms caused by increased inflammation associated with COVID-19 [However, it is important to note that both endogenous levels of PEA and exogenous PEA administration have previously been reported to be insufficient in mitigating a significant clinical response due to poor absorption, resulting in low plasma concentrations [ | PMC10609976 |
2. Methods | cancer, HIV, or the chronic, sore throat, allergic, mood disorders, fever, malignancy, URTIs, cognitive damage, cough, scratchy | SORE THROAT, NEUROLOGICAL DISORDERS, ADVERSE EVENTS, CHRONIC ASTHMA, EVENT, RECRUITMENT, MULTIPLE SCLEROSIS, EVENTS, INFLAMMATORY RESPONSE | This was a double-blind, randomised, placebo-controlled trial conducted over 12 weeks that utilised an active group (Levagen+) and a placebo group (maltodextrin). Potential participants were provided with the participant information sheet, and following initial screening via a telehealth consultation, acceptable participants gave their written consent to participate in the study and completed baseline measures. This trial was registered with ANZTCR: number ACTRN12620000846921.Four hundred and twenty-six participants aged between 18 and 65 years old were recruited from databases and public media outlets. Participants were included in the study if they were able to provide informed consent and agreed not to take other supplements or medications aimed at preventing URTIs for the duration of the trial (e.g., Echinacea, Vitamin C, zinc, Tamiflu, or Relenza). Exclusion criteria included those with cognitive damage; serious mood disorders or neurological disorders, such as multiple sclerosis; or those with an unstable or serious illness (e.g., renal, hepatic, gastrointestinal, cardiovascular, diabetes, thyroid gland function, malignancy, lung conditions, chronic asthma). Participants were also excluded if they had experienced acute sickness in the previous 2 months; were active smokers or abused nicotine or drugs; had chronic alcohol use (>14 alcoholic drinks per week); were allergic to any of the ingredients in the active or placebo formula; were pregnant or lactating women; were medically prescribed medications that could affect the immune and/or inflammatory response; had participated in a related clinical trial in the 1 month prior; or had treatment for cancer, HIV, or the chronic use of steroids in the past year.Once enrolled, participants were randomly allocated to one of two groups: either the active (PEA) or placebo group. Randomisation was performed using random allocation site (Sealedenvelope.com, accessed on 10 July 2020) by an individual who was not involved in the trial to ensure both participants and investigators were blinded to the allocation. Those in the PEA (Levagen+During the study period, participants were asked to complete the SF-8 questionnaire as a measure of health-related quality of life every 4 weeks (baseline, week 4, week 8, and week 12). SF-8 was scored according to If participants experienced the onset of URTI symptoms (e.g., cough, sneezing, stuffy or runny nose, fever, scratchy or sore throat, or nasal breathing), they were required to record their daily symptoms online, including severity, using the WURSS-24 questionnaire for the duration of the event or up to 2 weeks (whichever occurred first). If the participant’s symptoms continued for more than 2 weeks, they were asked to stop recording the event and seek medical advice. Once symptoms of an event subsided, participants were asked to continue taking the trial product for the remaining duration of the trial period and record any subsequent URTI events.The primary outcome measure for this study was a change in URTI incidence between groups. Secondary outcome measures included changes in URTI duration, severity (as measured by WURSS-24), general health (as measured by SF-8 questionnaire), number of days off work, and any product tolerance or adverse events.The sample’s size was calculated using G*power (v3.0.10), accounting for an α probability of 0.05 and powered to 0.95 for a 20% difference in URTI incidence (i.e., 30% vs. 24%); the resulting effect size was 0.74. Group sizes of at least 41 URTI incidents were required; therefore, up to 500 participants were to be recruited with the aim of achieving a minimum of 82 URTI events. Once 82 URTIs were recorded, recruitment into the study was closed, and those enrolled completed the study. Analysis was performed using IBM Statistics (version 25.0 for Windows, IBM, Chicago, IL, USA). Differences between the number of URTIs and symptoms reported per group were assessed using chi-square tests. Changes in URTI symptom severity and duration were analysed using Wilcoxon rank sum (Mann–Whitney U) tests. Statistical significance was set at | PMC10609976 |
3. Results | cough, diarrhoea, hoarseness, scratchy throat | ADVERSE EVENTS | Four hundred and twenty-six participants enrolled in the study, with 398 participants completing full trial requirements. There were 19 withdrawals in the active group and 9 in the placebo group. The Levagen+ group reported four adverse events (diarrhoea, A total of 87 participants experienced at least one URTI during the study, with a total of 103 URTI episodes recorded. The Levagen+ group reported significantly fewer URTI episodes (39 and 64, respectively; The number of sick days per episode ranged from 2 to 14 days in both groups, with no significant difference in the median number of sick days between the two groups. For participants reporting a URTI, the Levagen+ group reported a significantly lower severity score for scratchy throat and cough, with hoarseness and the ability to breathe easily trending towards significance (Comparisons between groups for the number of people reporting a specific symptom from the WURSS-24 showed no significant difference between groups for any outcome measure (There was no significant difference between groups for the number of people who thought they were on the active product (83 vs. 92 in the Levagen+ and placebo groups, respectively). Similarly, no significant difference was observed in the number of people willing to take the study product again (80 vs. 91 in the Levagen+ and placebo groups, respectively). | PMC10609976 |
4. Discussion | headaches, fever, infections, pain, sore throats, infection, scratchy throats | DISEASE, EVENT, INFECTIONS, LONG COVID, INFECTION, COMMON COLD, EVENTS, FLU | The aim of this study was to assess the effectiveness of PEA (Levagen+) on URTI incidence, duration, and symptom severity in otherwise healthy adults over a 12-week period. Both groups were equally matched, with no between-group differences in participant demographics (A study by Masek and colleagues (1974) supplemented adults with 600 mg of Levagen+ three times per day (1800 mg of Levagen+ per day) for 12 days. Following supplementation, those with supplementation had fewer episodes of fever and pain and fewer reported headaches and sore throats compared with a placebo group [Kahlich and colleagues [The results of the studies conducted by Masek and colleagues support our findings of a decrease in scratchy throats, sick days, and the total number of URTI episodes in the PEA group. However, the present study did not find a change in reported pain or fever. Based on the mode of action and its anti-inflammatory properties, it would be expected that changes in pain and fever would be observed in the Levagen+ group. Although no significant difference was observed for reported fevers, fewer people in the Levagen+ group reported experiencing a fever. Only 12.8% (5 out of 39) of people in the Levagen+ group who reported an event reported experiencing a fever compared to 25% (16 out of 64) in the placebo group. Fewer people reporting fever may be due to one of two possibilities: Either Levagen+ prevented people from developing a fever or the etiology of illnesses may have been different between groups. As both groups reported an equivalent number of other symptoms (There are several other possible reasons for the lack of significance of the reported fever in the present study compared to that of Masek. The first is the difference in dose (1800 mg vs. 600 mg per day). Despite Levagen+One factor potentially affecting the severity of illnesses, and a potential limitation of this study, was that it was conducted during the COVID-19 pandemic. During this period, participants likely experienced fewer URTI events than normal due to isolation, social distancing, and additional sanitation measures being taken. The additional health measures during COVID-19 potentially affected both the number of infections and the severity. People may have been able to rest and recover more during COVID-19 due to both increased isolation and the ability to work from home. The ability to stay home more may have allowed people to rest more and therefore recover faster, limiting the severity of the infection and therefore potentially limiting fever development. However, as all participants were exposed to the same COVID-19 conditions, the results can reasonably be expected to represent the effectiveness of Levagen+, and the only difference due to COVID-19 may be a lower percentage of people experiencing a URTI and symptoms being less severe.COVID-19 was not a focus of the current study, but it is possible that some of the participants involved experienced COVID-19 while taking the study’s product. The effect that Levagen+Another limitation of the study was our inability to collect biological samples. Due to the COVID-19 pandemic, we opted to exclude biological sample collection in order to minimize the potential risk participants might have in traveling to a collection center. Had we been able to collect biological samples, we could have tested for the specific infection the participants had while reporting symptoms. The detection of the infectious agent would have helped classify each reported event into different disease states, enabling us to better understand the effect of Levagen+ in different etiologies. It is possible that PEA may work better in some infections than others. The collection of blood samples would also have allowed us to analyse various pathways that PEA is reported to act upon (e.g., inflammatory cytokines NF-κB and mast cells).Severity classification is another potential limitation of this study. Due to the number of participants reporting events, grouping events into different severity classifications (e.g., total impact from the WURSS-24 questionnaire) in order to conduct statistical analysis was not possible. It is plausible that PEA may act better on different severities due to its mode of action. For example, PEA has demonstrated the ability to increase β-enzyme activity, which, in turn, enhances the synthesis of the endocannabinoid 2-Arachidonoylglycerol, suggesting that an increased endocannabinoid tone may modulate mast cell degranulation [Future studies into the efficacy of PEA would benefit from looking at specific etiologies and severity within URTIs. Further research on the effect of PEA on fever would require larger numbers that focus specifically on the temperature development of participants during an illness. Another study looking specifically at people experiencing COVID-19 or people experiencing Long COVID symptoms would help determine whether PEA is able to be as effective relative to COVID-19 as it is with respect to the common cold and flu. | PMC10609976 |
Author Contributions | A.R.: Conceptualization, methodology, software, formal analysis, resources, data curation, writing—review and editing, visualization, supervision, project administration, and funding acquisition. R.S.: Investigation, writing—original draft, and writing—review and editing. D.B.: Conceptualization, methodology, data curation, writing—original draft, writing—review and editing, visualization, supervision, and project administration. All authors have read and agreed to the published version of the manuscript. | PMC10609976 | ||
Institutional Review Board Statement | The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board Bellberry Limited, Committee H on 6 July 2020: approval number 2020-04-340. | PMC10609976 | ||
Informed Consent Statement | Informed consent was obtained from all subjects involved in the study. | PMC10609976 | ||
Data Availability Statement | The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. | PMC10609976 | ||
Conflicts of Interest | The authors declare no conflict of interest. | PMC10609976 | ||
References | EVENT | SF-8 outcome measures for each question (Q1 to Q8). There were no significant differences for any question at any collection time point.SF-8 scoring based on participants’ responses to each question.Summary of participants’ baseline demographics.Values are represented as mean ± SD.Trial event outcome measures.Symptom outcome measures for trial participant data reporting symptoms during a reported event.Values represented as severity; * significantly different from placebo ( | PMC10609976 |
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