SARS-CoV-2 pseudovirus neutralization assay.

Pseudovirus neutralization experiments were performed following our previous study (20), with minor changes. In brief, SARS-CoV-2 spike variant pseudotyped luciferase reporter viruses equivalent to 8 × 10¹⁰ U reverse transcriptase were prediluted in DMEM (with 2% FBS, heat inactivated) containing titrated amounts of an ACE2-Ig construct or an anti-SARS-CoV-2 antibody. Virus-inhibitor mixtures were incubated at 37°C for 30 min and then added to HeLa-hACE2 cells in 96-well plates and incubated overnight at 37°C. Virus-inhibitor-containing supernatant was then removed, changed to 150 μL of fresh DMEM (with 2% FBS), and incubated at 37°C. Cell culture supernatants were collected for a Gaussia luciferase assay at 48 h postinfection.