Lentivirus-based pseudovirus neutralization assay

The pseudovirus neutralization assay was performed as described previously^41,50. To produce SARS-CoV-2 pseudovirus, a codon-optimized CMV/R-SARS-CoV-2 spike (Wuhan-1, Genbank #: {"type":"entrez-nucleotide","attrs":{"text":"MN908947.3","term_id":"1798172431","term_text":"MN908947.3"}}MN908947.3) plasmid, was constructed and co-transfected with plasmids encoding luciferase reporter, human transmembrane protease serine 2 (TMPRSS2)⁵¹, and lentivirus backbone into HEK293T/17 cells (ATCC #CRL-11268), as previously described⁵². Heat-inactivated serum was mixed with the pseudovirus, incubated at 37 °C, and then added to ACE-2-expressing 293 T cells. Cells were lysed after 72 h, and luciferase activity was measured. Percent neutralization was calculated with uninfected cells as 100% neutralization and cells infected with only pseudovirus as 0% neutralization. ID₅₀ titers were determined using a log (agonist) vs. normalized response (variable slope) nonlinear function in Prism v8 (GraphPad).