Pseudovirus neutralization assay

Pseudoviruses were generated by transfection of 293T cells (ATCC) with an HIV-1 Env expressing plasmid and an Env-deficient genomic backbone plasmid (pSG3ΔEnv), as described previously [57]. Pseudoviruses were harvested 72 h post-transfection for use in neutralization assays. Neutralizing activity was assessed using a single round of replication in a pseudovirus assay with TZM-bl target cells. Briefly, TZM-bl cells were seeded in a 96-well flat bottom plate. To this plate was added pseudovirus, which was preincubated with serial dilutions of antibody for 1 h at 37°C. Luciferase reporter gene expression was quantified 72 h after infection upon lysis and addition of Bright-Glo Luciferase substrate (Promega). To determine IC₅₀ values, dose response curves were fitted using nonlinear regression.