Interestingly, the CD4+ T cell epitopes defined in the V2 region contained or fell in between the V2 K169Q and I181L sieve mutations in RV144 vaccine recipients who subsequently became HIV infected [12] (Fig. of the CD4+ responses in the ALVAC-HIV-AIDSVAX B/E prime-boost regimen in the Thai Phase III trial (RV144). Non-transformed Env-specific T cell lines established from RV144 vaccinees were used to determine the fine epitope mapping of the V2 and C1 responses and the HLA class II restriction. Data showed that there are two CD4+ epitopes contained within the V2 loop: one encompassing the 47 integrin binding site (AA179-181) and the other nested between two previously described genetic sieve signatures (AA169, AA181). There was no correlation between the frequencies of CD4+ fine epitope responses and binding antibody. Introduction The modest efficacy achieved by the RV144 Thai HIV vaccine trial [1] and SP600125 the subsequent discovery of correlates of protection [2] has renewed interest in HIV antibody binding, specificity and their functionality in vaccine regimens. HIV vaccines tested previously Rabbit Polyclonal to DGKB in Phase II/III trials while SP600125 eliciting strong CD8+ responses to HIV proteins failed to prevent contamination [3]. Canarypox-based ALVAC-HIV vaccine primary with HIV Env gp120 boosts elicited weak CD8+ responses but induced both strong binding antibodies (bAb) and proliferative capacity by peripheral blood mononuclear cells (PBMC) to HIV Env [4,5,6]. The RV144 correlates of protection study showed that IgG antibody binding to gp70V1V2 inversely correlated with contamination, while IgA antibody binding to Env protein directly correlated with contamination [2]. Although no CD4+ T cell correlates of protection were found in that analysis, there was a pattern for reduced risk of HIV contamination with increasing magnitude of cytokines produced by Env-specific mononuclear cells [2]. In addition, our group has shown that this RV144 vaccine regimen induces CD4+ T cells that are specific to the V2 region of the HIV Env. These cells are also polyfunctional as measured by intracellular cytokine staining (ICS) and have cytolytic capability [7]. It was intriguing to find that ALVAC-HIV-AIDSVAX B/E prime-boost regimen focused the CD4+ T cell response in the same region that gives rise to the IgG bAb that showed a correlation with protection [2]. Due to the paucity of cells collected during RV144 it is impossible to test further other cellular immune parameters in the case control cohort. Despite this limitation, we attempted to further characterize the V2 specific CD4 T cell responses using the non-transformed Env-specific CD4+ T cell lines to determine the fine epitope mapping of the responses to the V2 and C1 region and their class II restriction. Materials and Methods PBMC samples PBMC samples were obtained from the RV144 trial [1] and were selected as described in our previous publication [7]. Briefly, PBMC from 50 RV144 trial participants (40 vaccinees and 10 placebo recipients) collected at 6 months post completion of immunization (V9) were used to establish gp120 specific T cell lines. Thirty T cell lines were generated but only 14 demonstrated a specific response to the gp120A244 SP600125 and were further expanded to increase cell number. Expanded T cell lines were frozen in aliquots of 5 million cells prior to use in the study. PBMC from placebo recipients did not yield any viable T cell lines. In addition, the corresponding plasma collected at 6 months post final vaccination were tested for bAb to gp120 C1 and V2 regions. The RV144 trial is usually registered at www.ClinicalTrials.gov, NCT00223080 and was reviewed and approved by many ethical committees as described in the main clinical paper RV144 [1]. Antigens and Peptides The HIV CRF01_AE derived A244 gp120 used for the establishment of the T cell lines was kindly provided by Marc Gurwith (GSID) and was identical to that contained in the AIDSVAX B/E vaccine. The 138 peptide set of 15C18 aa overlapping by 10C12 aa spanning CRF01_AE isolate CM235.