Neutralization assay

Neutralization assays were carried out with SARS-CoV-2 pseudotyped particles (CoV2pp), generated in Benhur Lee’s laboratory [31]. CoV2pp carries vesicular stomatitis virus as viral backbone, bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG-rLuc), and expresses SARS-CoV-2 spike protein on its envelope.

ACE2 and TMPRSS2 expressing 293T cells (293T-ACE2+TMPRSS2 clone F8-2), were used for these assays [31]. Cells were maintained with DMEM high glucose with 10% FBS and were seeded in a 96-well plate the day before infection.

Patient sera were heat inactivated at 56°C for 30 minutes and serially diluted in DMEM high glucose with 10% FBS. Serum neutralizations were performed by first diluting the inactivated sample 8-folds and continuing with a 2-fold serial dilution. A pre-titrated amount of pseudotyped particles (diluted to give approximately 5x10⁵ Relative Luminescent Units) was incubated with a 2-fold serial dilution of patient sera for 30 minutes at room temperature prior to infection. Approximately 20 hours post-infection cells were processed for detection of luciferase activity. Cells were lysed and transferred to white, F-bottom 750 Lumitrac plates (Greiner, 655074). Plates were read via the GloMax Navigator Microplate Luminometer (Promega, GM200) using the Renilla Luciferase Assay System Luciferase Assay System (Promega, E2820).

Absolute inhibitory concentrations (absIC) values were calculated for all patient sera samples by modeling a 4-parameter logistic (4PL) regression with GraphPad Prism 8. The 4PL model describes the sigmoid-shaped response pattern. For clarity, it is assumed that the response can be expressed so that the slope increases as the concentration increase. Absolute inhibitory concentration (absIC) was calculated as the corresponding point between the 0% and 100% assay controls. Fifty % and 80% inhibition were defined by the controls for all the samples on the same plate. For example, the absIC50 or absIC80 would be the point at which the curve matches inhibition equal to exactly 50% or 80% of the 100% assay control relative to the assay minimum.