Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines

Abstract

A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01_AE and CRF02_AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.

Detection of HIV-1 P24 Gag in Plasma by a Nanoparticle-Based Bio-Barcode-Amplification Method

“…We determined the breadth of the bio-barcode-amplification assay by measuring the presence of HIV-1 p24 Gag in samples of 60 diverse viruses, including ten viruses from each of the six internationally most prevalent clades A, B, C and D and circulating recombinant forms CRF01_AE and CRF02_AG (obtained from the NIH AIDS Research & Reference Reagent Program [31]). At least one of the three synthetic peptides (12-, 15-and 16-mers) we used to create the mixture of sheep polyclonal antibodies against the HIV-1 p24 Gag protein were preserved perfectly in 56 out of 60 strains.…”

Pilot Studies for Development of an HIV Subtype Panel for Surveillance of Global Diversity

“…6,23 The limited number of virus isolates currently available for assay development, pharmaceutical design, and the evaluation of intervention strategies constrains the ability to correctly target viral infections in different geographic regions and to control the spread of HIV. Virus panels that are currently available for assay development and evaluations were isolated more than a decade ago 24,25 and are no longer fully representative of viruses currently in circulation. Many of these older isolates are incompletely characterized in molecular terms, making it difficult to reliably assess assay comparisons.…”

Monoclonal Antibodies to Phosphatidylinositol Phosphate Neutralize Human Immunodeficiency Virus Type 1: Role of Phosphate-Binding Subsites

“…Appropriate goat anti-human IgG or anti-mouse IgM secondary antibody conjugated to alkaline phosphatase was added, and after the addition of substrate, the plates were read at 405 nm. The methods for isolation, propagation, and titration of HIV-1 isolates and the peripheral blood mononuclear cells (PBMC) (10) and pseudovirus/TZM-bl (21) neutralization assays were used as previously described. Briefly, virus was combined with the neutralization reagent for 30 min and incubated at 37°C.…”

“…The following day, cells were washed three times and incubated at 37°C. After 4 days, extracellular p24 was measured and percent neutralization was calculated as described previously (10). Included in each experiment was a positive neutralization control (USHIVϩ).…”