Plaque reduction neutralization test (PRNT50)
A live SARS-CoV-2 Wuhan strain (B303) which was previously isolated from
the SARS-CoV-2 RdRp PCR positive nasopharyngeal specimen (The GenBank
accession number MT675956) was used for the plaque assay. TCID50/ml of
the SARS-CoV-2 used for plaque assay was 2x106. Vero E6 cells (ATCC
CRL-1586) were cultured with DMEM High-Glucose (Gibco, 41966-029)
supplemented with 10% Fetal bovine serum (FBS),(Gibco, 10500064), 1%
Penicillin-Streptomycin and Amphotericin B (Sigma, A2942). The serum of
each donor was incubated with SARS-CoV-2 at the multiplicity of
infection (MOI) 0.01 for 1 hour at 37°C, 5% CO2, and then inoculated
onto the VeroE6 cells at 100% confluency. After incubation for 1 hour
at 37°C, 5% CO2, the serum-virus mixture was discarded. The cell
monolayers were coated with 2% methylcellulose and 5% Fetal bovine
serum (FBS)/ DMEM mixture (1:1). Four days after infection,
methylcellulose and DMEM mixture was discarded. Plates were washed and
cells were fixed with 4% PFA then stained with crystal violet. Plaques
were counted with the naked eye and the Celigo Image cytometer
(Nexcelom, Celigo Image Cytometer 200-BFFL-5C). The virus control was
studied in duplicate in each assay. A negative control (unexposed
unvaccinated individuals’ serum samples) and an assay control were
included in each study 9.