Pseudovirus production and neutralization assays

To produce pseudoviruses, plasmids encoding Env were co-transfected with an Env-deficient genomic backbone plasmid (pSG3∆Env) in a 1:2 ratio with the transfection reagent PEI (1 mg/mL, 1:3 PEI:total DNA, Polysciences) into HEK 293T cells [44, 45]. Pseudoviruses were harvested 72 h post transfection for use in neutralization assays.

Neutralizing activity was assessed using a single round replication pseudovirus assay with TZM-bl target cells, as described previously [44, 45]. Briefly, the antibody or HIVIG (HIV hyperimmune globulin) was serial diluted in a 96 well flat bottom plate and pre-incubated with virus for 1 h at 37 °C. Cells at a concentration of 20,000 cells/well (supplemented with 10 μg/ml DEAE-Dextran for all donor virus variants) were added to the virus/antibody mixture and luminescence was quantified 72 h following infection via lysis and addition of Bright-Glo™ Luciferase substrate (Promega). Dose–response curves were fitted using nonlinear regression (GraphPad Prism) to determine IC₅₀ values.