SARS-CoV-2 Spike Pseudovirus Neutralization Assay

nAbs were quantitated against replication-deficient SARS-CoV-2 ancestral (Wuhan) Spike pseudotyped lentivirus particles as previously described⁶⁰ . Briefly, pseudovirus particles were generated by co-transfecting a Spike expression construct pCG1-SARS-2-S Δ18^{61 (link),62 (link)} and GFP-luciferase vector with lentivirus packaging and helper plasmids into HEK293T cells, using Fugene HD (Promega)^{63 (link)}. Pseudovirus particles were incubated with serially diluted serum or BALF samples at 37 °C, 5% CO₂ for 1 h prior to spinoculation (800 x g, 35 °C) of ACE2 over-expressing HEK293T cells. Seventy-two h post-transduction, cells were fixed and stained with Hoechst 33342 (NucBlue™ Live ReadyProbes™ Reagent, Invitrogen) as per the manufacturer’s instructions, and imaged using the Opera Phenix high content screening system (Perkin Elmer). The percentage of GFP positive cells was enumerated (Harmony® high-content analysis software, Perkin Elmer) and neutralising endpoint titre determined as the dilution required for ≥50% inhibition of infection (EC₅₀), estimated by sigmoidal curve and interpolation in GraphPad Prism software.