SARS-CoV-2 Spike Protein Pseudotyped Virus Neutralization

SARS-CoV-2 spike protein-pseudotyped lentiviral stocks were produced by the cotransfection of 293T cells with pMDL, pLenti.GFP-NLuc, pcCoV2.S-Δ19 (or variants thereof), and pRSV.Rev as previously described (41 (link)). Virus stocks were normalized by real-time PCR reverse transcriptase (RT) activity (51 (link)). Pseudotyped virus infections were done with 1 × 10⁴ cells/well in 96-well tissue culture dishes at a multiplicity of infection (MOI) of 0.2 as previously described (41 (link)). Luciferase activity was measured after 2 days using the Nano-Glo luciferase substrate (Promega), and plates were read in an Envision 2103 microplate luminometer (PerkinElmer). To quantify antibody neutralization, sera were serially diluted 2-fold and incubated for 30 min at room temperature with pseudotyped virus (corresponding to approximately 2.5 × 10⁷ cps luciferase) in a volume of 50 μl. The mixture was added to 1 × 10⁴ ACE2.293T cells (corresponding to an MOI of 0.2) in a volume of 50 μl in a 96-well culture dish. After 2 days, the medium was removed, and the Nano-Glo luciferase substrate (Nanolight) was added to wells. Luminescence was read in an Envision 2103 microplate luminometer (PerkinElmer).