Tissue culture plates

To measure the potential cytotoxicity of the different CD4mcs on TZM-bl or primary CD4+ T cells, a cell viability assay using CellTiter-Glo^® One Solution Assay (Promega, Madison, WI, USA) was performed. Briefly, TZM-bl or primary CD4+ T cells were seeded at a density of 1 × 10⁴ cells/well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer, Woodbridge, ON, Canada). After 24 h, indicated concentrations of CD4mcs up to concentrations of 100 μM were added to the cells followed by incubation for 48 h at 37 °C; the same volume of its vehicle, DMSO, was added as control. Then, a volume of CellTiter-Glo^® One Solution equal to the volume of cell culture medium present in each well was added, followed by 2 min mixing on shaker and 10 min incubation at room temperature. An LB941 TriStar luminometer (Berthold Technologies, Bad Wildbad, Germany) was used to measure the luciferase activity of each well.

293T cells were transfected with the lentiviral vector pNL4.3 R-E- Luc (NIH AIDS Reagent Program) and a plasmid encoding for the indicated Spike glycoprotein at a ratio of 10:1. Two days post-transfection, cell supernatants were harvested and stored at −80 °C until use. 293T-ACE2 target cells were seeded at a density of 1 × 10⁴ cells/well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer, Waltham, MA, USA) 24h before infection. Pseudoviral particles were incubated with the indicated plasma dilutions (1/50; 1/250; 1/1250; 1/6250; 1/31250) for 1 h at 37 °C and were then added to the target cells followed by incubation for 48 h at 37 °C. Then, cells were lysed by the addition of 30 µL of passive lysis buffer (Promega, Madison, WI, USA) followed by one freeze-thaw cycle. A LB942 TriStar luminometer (Berthold Technologies, Bad Wildbad, Germany) was used to measure the luciferase activity of each well after the addition of 100 µL of luciferin buffer (15 mM MgSO₄, 15 mM KPO₄ [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 µL of 1 mM d-luciferin potassium salt (Prolume, Randolph, VT, USA). The neutralization half-maximal inhibitory dilution (ID₅₀) represents the plasma dilution to inhibit 50% of the infection of 293T-ACE2 cells by SARS-CoV-2 pseudoviruses.