Plaque Reduction Neutralization Test (PRNT)

The PRNTs were conducted as described previously [8]. An equal volume of complete media (MEM containing 10% heat inactivated FBS, 2% Pen/Strep, 100X NEAA, 1% HEPES, 0.1% Gentamycin, and 0.2% Fungizone®) containing SARS-CoV-2 was combined with 2-fold serial dilutions of cMEM containing antibody and incubated at 37°C in a 5% CO2 incubator for 1 hour. Afterwards, the combined virus/antibody mixtures were then added to 6-well plates (180 μl/well) containing 3-day old, ATCC Vero-76 confluent cell monolayers and allowed to adsorb for 1 hour in a 37°C, 5% CO2 incubator. Three milliliters of agarose overlay (0.6% SeaKem® ME agarose, 2X EBME with HEPES, 10% heat-inactivated FBS, 100X NEAA, 4% GlutaMAXTM, 2% Pen/Strep, 0.1% Gentamycin and 0.2% Fungi-zone®) per well was then added and allowed to solidify at room temperature. The plates were placed in a 37°C, 5% CO2 incubator for 2–3 days (variant dependent) and then 2 mL per well of agarose overlay stain (0.6% SeaKem® ME agarose, 2X EBME with HEPES, 5% heat-inactivated FBS, 2% Pen/Strep, 0.1% Gentamycin, 0.2% Fungizone®, 5% Neutral Red) was added. Once plaques could be visualized (one to two days post-staining), plates were removed from the incubator and counted on a light box. PRNT50 titers were determined and are defined as the reciprocal of the highest dilution that results in a 50% reduction in the number of plaques relative to the average number of plaques visualized in the cMEM alone (no antibody) wells.