Virus neutralization assay

Virus neutralization was evaluated by a focus reduction neutralization test. Vero E6 cells were seeded at 2 × 10⁴ cells/well in a 96-well plate 24 h before the assay. 200 focus-forming units of virus were preincubated with serial dilutions of heat-inactivated sera for 1 h at 37°C before infection of cells for 2 h. The virus/antibody mix was then removed and foci were left to develop in presence of 1.5% methylcellulose for 2 d. Cells were fixed with 4% formaldehyde and foci were revealed using a rabbit anti–SARS-CoV-2 N antibody (gift of Nicolas Escriou, Institut Pasteur, University Paris Cité, CNRS UMR 3569, Innovation Laboratory: Vaccines Unit, Paris, France) and anti-rabbit secondary HRP-conjugated secondary antibody. Foci were visualized by diaminobenzidine staining and counted using an Immunospot S6 Analyser (Cellular Technology Limited). Prepandemic serum (March 2012) was used as negative control for sera titration and was obtained from an anonymous donor through the ICAReB platform (BRIF code no. BB-0033-00062) of Institut Pasteur that collects and manages bioresources following International Organization for Standardization 9001 and NF S 96-900 quality standards.

The percentage of virus neutralization was calculated as (100−[(#foci sample/#foci control)*100]). Sera IC₅₀ were calculated over seven fourfold serial dilutions from 1/10 to 1/40,000 using the equation log (inhibitor) vs. normalized response−−variable slope in Prism 9 (GraphPad software LLC).