SARS-CoV-2 S Pseudotype Neutralization Assay

Codon-optimized full-length spike (S) protein of the original Wuhan-Hu-1 isolate with D614G mutation (D614G) was cloned into a pCAGGS vector. This codon-optimized D614G vector was used as a template for site-directed mutagenesis to incorporate the S variants, listed in Table 1. To make SARS-CoV-2 full-length S-pseudotyped recombinant vesicular stomatitis virus ΔG (VSVΔG)-firefly luciferase virus, BHK-21/WI-2 cells (Kerafast) were transfected with the S expression plasmid and subsequently infected with VSVΔG-firefly luciferase as previously described (11 (link)). For the neutralization assay, serially diluted serum samples were mixed with pseudovirus and incubated at 37°C for 45 min. The virus-serum mix was subsequently used to infect A549-hACE2-TMPRSS2 cells (12 (link)) for 18 h at 37°C before addition of ONE-Glo reagent (Promega) for measurement of the luciferase signal by relative luminescence units (RLUs). The percentage of neutralization was calculated based on the RLUs of the virus-only control and subsequently analyzed using the four-parameter logistic curve in Prism v.8 (GraphPad Software, Inc.).