The Thai HIV phase III prime-boost trial (RV144) using ALVAC-HIV® (vCP1521) and AIDSVAX B/E® was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT were performed on PBMC from HIV-1 uninfected vaccine (N=61) and placebo (N=10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4+ T cell mediated. Responses were targeted within the HIV Env region, with 15/25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α4β7 integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19/30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4+ T cells, with the majority of responders producing both IL-2 and IFN-γ (12/19; 63%). HIV-Env Ab titers were higher in subjects with IL-2 compared to those without IL-2 secreting HIV-Env specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4+ with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality and functional cytolytic capacity. While the RV144 T cell responses were modest in frequency compared to humoral immune responses, the CD4+ T cell response was directed to HIV-1 Env and more particularly the V2 region.

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Background Clinical trials testing candidate human immunodeficiency virus type 1 (HIV‐1) vaccines have required the use of HIV neutralization assays to detect responses to specific geographic subtypes of HIV‐1. The variability in results seen with current p24 neutralization assay endpoints prompted us to assess the utility of flow cytometry for monitoring the neutralization of HIV‐1 primary isolates. Methods A modified neutralization assay was performed using CD8‐depleted peripheral blood mononuclear cells (PBMC). The cells were fixed, permeabilized, stained with a directly conjugated HIV‐1 p24 monoclonal antibody, and analyzed by flow cytometry. HIV‐1 subtype B′ and E primary isolates were tested using pooled sera or plasma from subtype B′ or E infected patients. Results Primary isolate cultures (without neutralizing antibody) showed from 18% to 42% p24+ cells, depending on the virus. Less than 0.2% p24+ cells were detected in uninfected cultures. Subtype‐specific neutralization of viruses was observed using plasma or serum pools; neutralization ranged from 0% to 99% reduction of infected cells. Conclusions Flow cytometric detection of intracellular HIV‐1 p24 can be used as an endpoint assay to assess neutralization of HIV‐1 subtypes B′ and E primary isolates. This enumerative method has the advantage of identifying intracellular p24 in specific subsets at an early culture timepoint. It also provides an alternative quantitative endpoint for HIV neutralization assays. Cytometry 40:141–150, 2000. © 2000 Wiley‐Liss, Inc.