For pseudovirus production, full-length rev-env cassettes were cloned into one of two mammalian expression vectors, pcDNA 3.1 Directional/V5-His-TOPO (Invitrogen, Carlsbad, CA) or pTarget (Promega, Madison, WI). The amplicon sequence selected for cloning was the one closest to a participant’s consensus that was generated from at least five sequences. The resulting clones were sequenced to ensure an exact match to the original amplicon sequence and where cloned inserts differed from the parental sequence, mutagenesis was performed to ensure a match with the parental sequence. Env-pseudotyped viruses were generated by co-transfecting envelope clones with a clade B backbone pSG3ΔEnv (NIH AIDS Research and Reference Reagent Program) in HEK293T cells as previously described [27 (link),28 (link)]. Pseudovirus functionality was determined by measuring luciferase expression after infecting TZM-bl cells (NIH AIDS Research and Reference Reagent Program, ARRRP). Relative luminescence units (RLUs) of ≥100,000 were considered ideal and 30,000 RLUs were accepted in cases where readings were 2.5 x the background; <2.5 times the background were considered negative.
Other organizations :
National Health Laboratory Service, University of Cape Town, New Mexico Consortium, Los Alamos National Laboratory, Beth Israel Deaconess Medical Center, Duke Medical Center, Botswana Harvard AIDS Institute Partnership, University of the Witwatersrand, Centre for the AIDS Programme of Research in South Africa, University of KwaZulu-Natal, South African Medical Research Council, Perinatal HIV Research Unit, Fred Hutch Cancer Center, South African National Blood Service, Desmond Tutu HIV Foundation, German Center for Infection Research, Walter Reed Army Institute of Research, International Vaccine Institute, University of Pennsylvania, University of North Carolina at Chapel Hill
Pseudovirus functionality measured by luciferase expression in TZM-bl cells
control variables
Clade B backbone pSG3ΔEnv used for co-transfection
HEK293T cells used for pseudovirus generation
positive controls
Pseudoviruses with ≥100,000 relative luminescence units (RLUs)
negative controls
Pseudoviruses with <2.5 times the background RLUs
Annotations
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