SARS-CoV-2 Neutralization Assay Protocol

Neutralization assay was conducted as described previously⁹ . Briefly, sera from vaccinated individuals were heat treated for 30 min at 56 °C. Sixfold serially diluted plasma, from 1:10 to 1:2430 were incubated with SARS-CoV-2 lineage A(USA-WA1/2020) for 1 h at 37 °C. The mixture was subsequently incubated with TMPRSS2-VeroE6 in a 12-well plate for 1 h, for adsorption. Then, cells were overlayed with MEM-supplemented NaHCO₃, 4% FBS 0.6% Avicel mixture. Plaques were resolved at 40 h post-infection by fixing in 10% formaldehyde for 1 h followed by staining in 0.5% crystal violet. All experiments were performed in parallel with baseline control sera, in an established viral concentration to generate 60–120 plaques/well.