Gal screen

Pseudovirus production and neutralization assays were performed as previously described^{58 (link)} with an alternative cell lysis and B-galactosidase detection system (Gal-Screen; ThermoFisher). Specifically, 85 μl of the 150 μl total volume was removed from each well (50 μl remaining) and 50 μl of Gal-Screen substrate (diluted 1:25 in “Buffer A”) was added to each well. Luminescence was measured after 40 min incubation at room temperature. The mAbs were diluted twofold from 20 to 0.6 μg ml⁻¹. IC₅₀ values represent the concentration (μg ml⁻¹) at which 50% of the virus was neutralized and are the average of two or three independent experiments performed in duplicate.

293T effector cells grown to 75% confluency in 12-well plates were cotransfected with expression plasmids for the respective S protein or empty vector (1.5 µg/well) and the beta-galactosidase alpha fragment (0.5 µg/well) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Subsequently, effector cells were washed, resuspended in 500 µl and added to 293T target cells (96-well format, 100 µl/well, four technical replicates) that were transfected with plasmids encoding ACE2 (0.1 µg/well) and the beta-galactosidase omega fragment (0.1 µg/well), or A549-ACE2 target cells (96-well format, 100 µl/well, four technical replicates) that were transfected with plasmid encoding the beta-galactosidase omega fragment (0.1 µg/well). Beta-galactosidase substrate (Gal-Screen, Thermo Fisher Scientific) was added (100 µl/well) after an additional 24 h of incubation, and samples were incubated for 90 min in the dark at room temperature before they were transferred into white 96-well plates and luminescence was measured using a Hidex Sense plate luminometer (Hidex).

Cell–cell fusion assay was based on the α-complementation of E. coli β-galactosidase^{8 (link)}. Briefly, 293T cells were cotransfected with either HIV-1 Env and the α fragment of β-galactosidase or CD4, CCR5 and the ω fragment of β-galactosidase. Env-expressing cells (2.0 × 10⁶ cells/mL) were mixed with CD4− and CCR5-expressing cells (2.0 × 10⁶ cells/mL). Cell–cell fusion was allowed to proceed at 37 °C for 2 h. Cell–cell fusion activity was quantified using a chemiluminescent assay system, Gal-Screen (Applied Biosystems, Foster City, CA).