SARS-CoV-2 Spike Pseudovirus Neutralization Assay

Pseudoviruses (virus-like particles pseudotyped with the SARS-CoV-2 spike protein) were prepared by co-transfection of HEK293 cells with 1 µg pNL4-3.luc.R-E- plasmid (luciferase expressing HIV-1 with defective envelope protein) (NIH AIDS Reagent Program, ARP2128) and 0.06 mg CMV promoter-driven plasmid encoding the spike protein using Lipofectamine 2000 transfection reagent (ThermoFisher, 11668027), exactly as described [23 (link)]. The infection assay was similarly performed as described [23 (link)], in brief, by pre-incubating pseudovirus with serial dilutions of nBio from either cell culture supernatant or mouse plasma in media at RT for 30 min, prior to addition to HEK293T cells stably expressing full-length human ACE2 protein. The cells and nBio/pseudovirus mixture were incubated at 37 °C with 5% CO₂ for six hours, after which the media was replaced with fresh DMEM (10% FBS and 1% penicillin–streptomycin). After 72 h, DMEM was removed and DPBS (ThermoFisher) was added to cells before mixing with an equal volume of ONE-Glo EX Luciferase Assay System (Promega E8130), shaking for 5 min at room temperature, then reading the luciferase signal using a BioTek Synergy Neo plate reader (BioTek Instruments Inc.). The data were analyzed by GraphPad Prism Version 8.4.3 (GraphPad Software, LLC) to obtain IC₅₀ and IC₉₀ values.