Pseudovirus Neutralization Assay Protocol

HIV pseudovirus (PSV) was produced in HEK 293 T cells (ATCC, Manassas, VA, CRL-3216) and used in TZM-bl neutralization assays as previously described (Goo et al., 2012 (link)). Briefly, monoclonal antibodies were incubated with PSV at 37°C for 1 hr and TZM-bl cells (obtained through the NIH AIDS Reagent Program, Germantown, MD, catalog #8129 from Dr. John C. Kappes, and Dr. Xiaoyun Wu) were added at 10,000 cells/well and the assay was harvested after 48 hr at 37°C by adding Gal-Screen substrate (Thermo Fisher Scientific, catalog #T1028) as previously described (Simonich et al., 2019 (link)). IC₅₀ values represent the half maximal inhibitory concentration in ug mL⁻¹ at which 50% of the virus was neutralized. All neutralization data are the average of at least two independent experiments, each performed in technical duplicate (see Figure 2—source data 1; Figure 4—source data 1; Figure 5—source data 1; Figure 6—source data 1; Figure 7—source data 1; Figure 8—source data 1). IC₅₀ values that deviated from the technical replicates by >10-fold were excluded as outliers from the average IC₅₀ calculation.