SARS-CoV-2 Neutralizing Antibody Assay

The neutralization assay was performed to measure neutralizing antibody titers in the serum of vaccinated mice as previously described for influenza viruses^{42 (link),43 (link)}. To measure neutralizing antibody titers against SARS-CoV-2, monolayers of Vero TMPRSS2 cells were prepared in 6-well plates by seeding 1.425 × 10⁶ cells/well in 3 mL DMEM and incubated overnight at 37 °C in 5% CO₂. Mouse sera were inactivated at 56 °C for 30 min and serially diluted two-fold in 96-well microplates to which 100 PFU of SARS-CoV-2 was added and incubated for 1 h at room temperature. Following washing of Vero TMPRSS2 cells with DMEM_anti, (containing 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 20 μg/mL of gentamicin) the virus-serum mixture was transferred to 6-well plates containing the Vero TMPRSS2 cells and incubated at 37 °C in 5% CO₂, with shaking every 15 min. Finally, warmed L15 overlay medium [consisting of Leibovitz L-15 (Life Technologies Corp., Carlsbad, NY, USA) with glutamine supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.028% (w/v) NaHCO₃ and 0.9% (w/v) agarose] was added to the 6-well plates. The plates were incubated for three days at 37 °C in 5% CO₂ and the neutralizing antibody titer was determined as the reciprocal of the highest dilution that prevented the growth of plaques to 50% of that obtained in the control wells.