SARS-CoV-2 Pseudovirus Neutralization Assay

A SARS-CoV-2 pseudovirus, incorporating a luciferase reporter gene, was generated similar to previously described approaches^{1 (link),2 (link),21} . To generate pseudoviruses, HEK293T cells were co-transfected with packaging construct psPAX2 (AIDS Resource and Reagent Program), a luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and a plasmid expressing S protein, pcDNA3.1-SARS-CoV-2 SΔCT with lipofectamine 2000. 48 h after transfection, supernatants containing pseudoviruses were collected and filtered through a 0.45-µm filter. To quantify neutralizing activity of human plasma samples or purified IgG stocks, HEK293T target cells, transfected to express human ACE2 (HEK293T-hACE2), were seeded in 96-well tissue culture plates at a density of 1.75 × 10⁴ cells per well and cultured overnight. Three-fold serial dilutions of heat-inactivated plasma or purified IgG samples were prepared, mixed with 50 µl of pseudovirus, incubated for 1 h at 37 °C, and then added to HEK293T-hACE2 seeded wells. After 48 h of incubation, cells were lysed in Steady-Glo Luciferase Assay Reagent (Promega) according to the manufacturer’s instructions. SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction in relative light units was observed relative to the average of the control wells treated with virus only.