VSV–SARS-CoV-2 pseudotype neutralization assay

The pseudotype viral system was based on the recombinant VSV*ΔG-Luc vector in which the glycoprotein gene (G) had been deleted and replaced with genes encoding green fluorescent protein and luciferase⁵⁹. Pseudoviruses were generated as reported previously^60,61. For the neutralization assay, an initial dilution of the compounds was followed by three-fold dilutions in quadruplicates in DMEM-2% (vol/vol) FCS supplemented with 20 μM HSA (CSL Behring). The mixture was mixed with an equal volume of DMEM-2% FCS containing 250 infectious units per well of SARS-CoV-2 pseudoviruses and incubated for 90 minutes at 37 °C. The mix was inoculated onto Vero E6 cells (supplied by the American Type Culture Collection (ATCC), CRL-1586) in a clear-bottom, white-walled 96-well plate during 90 minutes at 37 °C. The inoculum was removed and fresh medium added, and cells were further incubated at 37 °C for 16 hours. Cell were lysed according to the ONE-Glo luciferase assay system (Promega), and light emission was recorded using a Berthold TriStar LB941 luminometer. The raw data (relative luminescence unit (RLU) values) were exported to GraphPad Prism version 8.4.3, and the % neutralization values were normalized to the untreated PsV signal. IC₅₀ values with 95% confidence intervals were estimated by the model of non-linear regression fit with settings for log (inhibitor) versus normalized response curves. Data points are plotted by the mean ± s.e.m. of quadruplicate data.