SARS-CoV-2 Pseudovirus Neutralization Assay

Pseudovirus assays were performed as previously described^{31 (link),32 (link),37 (link)}. In brief, lentivirus (HIV-1)-based luciferase-expressing reporter viruses pseudotyped with the SARS-CoV-2 S protein and its derivatives, HEK293T cells (1 × 10⁶ cells), were cotransfected with 1 μg of psPAX2-IN/HiBiT^{46 (link)}, 1 μg of pWPI-Luc2^{46 (link)} and 500 ng of plasmids expressing parental S or its derivatives using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) or PEI Max (Polysciences, 24765-1) according to the manufacturer’s protocol. At 2 days after transfection, the culture supernatants were collected and centrifuged. The amount of pseudovirus prepared was quantified using the HiBiT assay as previously described^{31 (link),46 (link)}. The prepared pseudoviruses were stored at −80 °C until use. For the experiment, HOS-ACE2 cells and HOS-ACE2/TMPRSS2 cells (10,000 cells per 50 μl) were seeded in 96-well plates and infected with 100 μl of pseudoviruses prepared at four different doses. At 2 d.p.i., the infected cells were lysed with a One-Glo luciferase assay system (Promega, E6130), and the luminescent signal was measured using a CentroXS3 plate reader (Berthhold Technologies) or GloMax explorer multimode microplate reader 3500 (Promega).