Neutralization assay with pseudotyped virus

Lentiviral supernatants were produced from transfected HEK‐293T cells as described previously (Martínez‐Martín et al, 2009). Briefly, lentiviruses were obtained by co‐transfecting plasmids psPAX2 (gag/pol), pHRSIN‐GFP, and either a truncated S envelope (pCR3.1‐St) (Robbiani et al, 2020) or VSV envelope (pMD2.G) using the JetPEI transfection reagent (Polyplus Transfection). Viral supernatants were obtained after 48 h of transfection. Polybrene (8 µg/ml) was added to the viral supernatants prior to transduction of ACE2⁺HEK293T cells. A total of 15 × 10³ ACE2⁺ HEK293T cells per well in a 96‐well plate were seeded the day before transduction. Serially diluted plasma was incubated with viral supernatant for 1 h at 37°C prior addition to the cells. Cells were centrifuged for 70 min at 1,600 g and left in culture for 48 h and then were resuspended in PBS with 2% FBS and 5 mM EDTA and fixed with 2% paraformaldehyde. GFP⁺ cells were then analyzed on a FACSCanto II flow cytometer (Becton‐Dickinson), and the data were analyzed with FlowJo software (BD).