Neutralization of SARS-CoV-2 by Entry-Inhibition

Neutralization of authentic SARS-CoV-2 by entry-inhibition assay was determined using SARS-CoV-2-Nluc, an infectious clone of SARSCoV-2 (based on strain 2019-nCoV/USA_WA1/2020) which encodes nanoluciferase in place of the viral ORF7 and demonstrated comparable growth kinetics to wildtype virus^{58 (link)}. Vero E6 cells were seeded into black-walled, clear-bottom 96-well plates at 2 × 104 cells/well and cultured overnight at 37 °C. The next day, 9-point 4-fold serial dilutions of mAbs were prepared in infection media (DMEM + 10% FBS). SARS-CoV-2-Nluc was diluted in infection media at a final MOI of 0.01 PFU/cell, added to the mAb dilutions and incubated for 30 minutes at 37 °C. Media was removed from the Vero E6 cells, mAb-virus complexes were added and incubated at 37 °C for 24 hours. Media was removed from the cells, Nano-Glo luciferase substrate (Promega) was added according to the manufacturer’s recommendations, incubated for 10 minutes at room temperature and the luciferase signal was quantified on a VICTOR Nivo plate reader (Perkin Elmer).