docs/v4_validation_protocol.md

🧪 Phase 12: In Vitro Validation Protocol (v4.0)

This protocol serves as the "Gold Standard" validation bridge for the Typhoid HDT Discovery Suite. It is designed for execution in a BSL-3 Biosafety Laboratory.

1. Experimental Objectives

To determine if AI-prioritized host-directed candidates (Top Hits: IL1B, TNF, IL6, NLRP3, MTOR inhibitors) significantly reduce the intracellular replication of Salmonella Typhi in human macrophages.

2. Model Systems

• Cell Line: Human THP-1 monocytes (differentiated into macrophages using 50nM PMA for 48 hours).
• Bacterial Strain: S. Typhi Ty2 (Wild-type) or Quailes strain.
• Reference HDT: Dexamethasone (Positive control for inflammation dampening) or Metformin (Positive control for Autophagy activation).

3. The Gentamicin Protection Assay (Workflow)

1. Cell Seeding: Seed THP-1 macrophages in 24-well plates (5x10^5 cells/well).
2. HDT Pre-Treatment: Treat cells with prioritized compounds (e.g., Anakinra for IL1B, Infliximab for TNF, or Metformin for MTOR) at 3 concentrations (e.g., 1µM, 10µM, 50µM) for 2 hours.
3. Infection: Infect macrophages with S. Typhi at an MOI (Multiplicity of Infection) of 10:1.
4. Internalization: Incubate for 1 hour at 37°C / 5% CO2.
5. Gentamicin Kill: Wash 3x with PBS; add media containing 100µg/mL Gentamicin for 1 hour to kill extracellular bacteria.
6. Maintenance & HDT Incubation: Replace media with low-dose Gentamicin (10µg/mL) and maintain HDT treatment for 2h, 6h, and 24h post-infection.
7. Lysis & CFU Counting:
   • Lyse cells using 0.1% Triton X-100.
   • Perform serial dilutions and plate on LB Agar.
   • Incubate at 37°C for 24 hours.
   • Outcome Metric: Colony Forming Units (CFU) per mL.

4. Secondary Validations

• Inflammatory Profiling: Measure supernatant IL-1β, TNF, and IL-6 via ELISA.
• Cytotoxicity (MTT/LDH): Ensure the drug concentration does not reduce macrophage viability by >10%.
• Autophagy Visuals: Use LC3-II/p62 Western Blotting for MTOR-pathway candidates.

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Scrutiny Note: This protocol addresses the primary mechanism of Typhoid persistence (intracellular replication in macrophages). Success in this assay is a prerequisite for pre-clinical animal models.