Generation and titration of HIV-1 pseudoviruses

Immune sera neutralization breadth and potency and antibody avidity indices were evaluated using a highly sensitive, single-round pseudotype virus infectivity assay system. A total of 32 HIV-1 Env-pseudotyped virions from tier 1, 2, and 3 of various isolates (Table ​(Table2)2) were generated using the Fugene 6 transfection method (Promega, Madison, WI, USA). Pseudoviruses were produced by co-transfection of HEK293T cells with an Env-expressing plasmid of different clades and Env-deficient HIV-1 genomic backbone plasmid, pSG3ΔEnv.

HIV-1 Env-pseudotyped virions from various clades.

Pseudoviruses, generated for comparing the data of neutralizing antibody assay were produced by co-transfection of HEK293T cells with an Env-expressing plasmid of different clades and Env-deficient HIV-1 genomic backbone plasmid, pSG3ΔEnv using TMZ-bl neutralization assay.

At 72 h post-transfection, viral supernatants were harvested. Virus stocks were made cell free by low-speed centrifugation and filtration (0.45 μm) and later stored at −80 °C in growth medium containing 20% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). Pseudoviruses were further titrated for luciferase expression in the TZM-bl cells as described previously^56,57 using Bright-Glo luciferase reagent (Promega Madison, WI, USA) and luminescence was measured using a Glomax Explorer luminometer (Promega Madison, WI, USA). Infection was measured by luciferase expression, and 200 × 50% tissue culture infective doses (TCID₅₀) was calculated using Reed-Muench method⁵⁸. These pseudoviruses exhibit a neutralization phenotype that is typical of most primary HIV-1 isolates^59,60.