Phase 1: Plasma Cohort Selection and Characterization -- Days 1-14
Obtain IRB-approved pooled plasma from two hybrid immunity cohorts: Delta hybrid immunity (vaccination + Delta infection, n=10 donors) and Omicron BA.1/2 hybrid immunity (vaccination + BA.1 or BA.2 infection, n=10 donors). Collect samples 1-4 months post-Omicron infection. Heat-inactivate at 56°C for 30 minutes. Characterize by measuring total IgG, variant-specific binding antibodies using ELISA with BA.5, BA.1/2, and Delta spike proteins. Perform comprehensive neutralization profiling against multiple variants (Delta, BA.1, BA.2, BA.4, BA.5) using pseudovirus assays on ACE2-expressing cells.
...Phase 1: Plasma Cohort Selection and Characterization -- Days 1-14
Obtain IRB-approved pooled plasma from two hybrid immunity cohorts: Delta hybrid immunity (vaccination + Delta infection, n=10 donors) and Omicron BA.1/2 hybrid immunity (vaccination + BA.1 or BA.2 infection, n=10 donors). Collect samples 1-4 months post-Omicron infection. Heat-inactivate at 56°C for 30 minutes. Characterize by measuring total IgG, variant-specific binding antibodies using ELISA with BA.5, BA.1/2, and Delta spike proteins. Perform comprehensive neutralization profiling against multiple variants (Delta, BA.1, BA.2, BA.4, BA.5) using pseudovirus assays on ACE2-expressing cells.
Phase 2: Cross-Variant Neutralization Analysis -- Days 15-24
Conduct detailed neutralization mapping using authentic virus strains in BSL-3 facility. Test plasma dilutions from 1:10 to 1:20,480 against SARS-CoV-2 variants including ancestral WA1, Delta, BA.1, BA.2, and BA.5. Use plaque reduction neutralization test (PRNT) with Vero-E6 cells. Calculate geometric mean titers (GMT) and fold-reductions relative to homologous strain. Perform antigenic cartography analysis to map neutralization landscapes. Validate that both plasma pools show similar BA.5-binding titers but potentially different neutralization breadth.
Phase 3: Antibody Functional Characterization -- Days 25-31
Analyze Fc effector functions using flow cytometry-based assays. Measure antibody-dependent cellular cytotoxicity (ADCC) using NK92-CD16 effector cells and spike-expressing target cells. Assess antibody-dependent cellular phagocytosis (ADCP) using THP-1 monocytes and spike-coated beads. Determine complement deposition (CDC) using spike-expressing cells and complement-sufficient serum. Measure antibody avidity using chaotropic elution with increasing urea concentrations (0-8M). Compare functional profiles between the two plasma pools.
Phase 4: Passive Transfer Optimization -- Days 32-35
Validate plasma transfer protocol using pilot study with TKI mice. Administer 200 μL plasma intravenously and track circulating human IgG levels by ELISA. Confirm sustained antibody levels (>50 μg/mL) at 24-48 hours post-transfer. Test plasma compatibility with no adverse reactions. Prepare SARS-CoV-2 Omicron BA.5 viral stock (titer 10^6 PFU/mL) and verify identity by whole genome sequencing focusing on spike mutations characteristic of BA.5 (L452R, F486V).
Phase 5: Protection Efficacy Challenge -- Days 36-42
Randomize 8-10 week old TKI mice into groups (n=12 per group): vehicle control, Delta hybrid plasma, and Omicron BA.1/2 hybrid plasma. Administer plasma 24 hours before challenge. Challenge intranasally with 10^4 PFU SARS-CoV-2 BA.5 under anesthesia. Monitor daily for clinical signs and body weight changes. Include biomarker analysis by collecting small blood samples at 24 and 72 hours post-challenge for inflammatory markers. Sacrifice at 4 days post-infection for viral load assessment.
Phase 6: Comprehensive Outcome Analysis -- Days 43-52
Quantify lung viral loads using RT-qPCR (N gene) and plaque assay for infectious virus. Perform immunohistochemistry on lung sections using anti-nucleoprotein antibody to visualize infected cells and quantify infection extent. Analyze lung inflammation by H&E staining and inflammatory cytokine multiplex assay (IL-6, TNF-α, IFN-γ, IL-1β). Correlate protection with plasma characteristics including variant-specific neutralization, binding avidity, and Fc effector functions. Perform statistical analysis using mixed-effects models to account for repeated measures and multiple comparisons correction.