Table 1. Primed CD4 and CD8 T cell responses BS874 LHIV #17 MN LHIV Polyclonal T cell Lines BS874 AT2 #19 BS874 AT2 #21 BS874 AT2 #23 BS874 AT2 #24 BS874 AT2 #25 BS874 LHIV #14 BS874 LHIV #35 MP980 LHIV #9 MP980 LHIV #16 MP980 AT2 #7 MP980 AT2 #9 MP980 AT2 #36 MP980 AT2 #48 DR682 AT2 #1 DR682 AT2 #5 DR682 AT2 #7 JP651 LHIV #13 EW836 LHIV #5 EW836 AT2 #4 JN521 AT2 #1 Virus MN AT2 MN AT2 MN AT2 MN AT2 MN AT2 MN LHIV MN LHIV MNLHIV MN LHIV MN AT2 MN AT2 MN AT2 MN AT2 MN AT2 MN AT2 MN AT2 MN LHIV MN LHIV MN AT2 MN AT2 DR682 AT2 #3 MN AT2 Protein gag/p24 gag/p17 gag/p24 gag/p24 gag/p24 gag/p24 pol/RT gag/p24 pol/RT gag/p15 gag/p17 pol/INT env/gp41 gag/p17 gag/p24 env/gp41 env/gp120 gag/p17 pol/INT gag/p24 gag/p17 pol/INT gag/p24 env/gp41 gag/p24 gag/p15 pol/RT gag/p17 gag/p15 gag/p17 OLP Sequence WIILGLNKIVRMYSPTSI TGSEELRSLYNTVATL WIILGLNKIVRMYSPTSI WIILGLNKIVRMYSPTSI YVDRFYKTLRAEQASQEV WIILGLNKIVRMYSPTSI WIILGLNKIVRMYSPTSI MTKILEPFRRKQNPDIVIY WIILGLNKIVRMYSPTSI MTKILEPFRRKQNPDIVIY RNQRKIVKCFNCGKEGHT TGSEELRSLYNTVATL ELKKIIGQVRDQAEHLK LELDKWASLWNFDITN KHIVWASRELERFAV WIILGLNKIVRMYSPTSI AVLSIVNRVRQGYSPLSE RPVVSTQLLLNGLSLA SGGELDRWEKIRLRPGGK TKELQKQITKIQNFRVYY IVQNLQGQMVHQAISPR IVQNLQGQMVHQAISPR AYFLLKLAGRWPVKTIH LKTAVQMAVFIHNFKRK WIILGLNKIVRMYSPTSI LELDKWASLWNFDITN IVQNLQGQMVHQAISPR HIAKNCRAPRKKGCWK YELHPDKWTVQPIVLPEK LEKIEEEQNKSKKKAQQA GKIWPSHKGRPGNFLQSR MGARASVLSGGELDRWEK doi:10.1371/journal.pone.0004256.t001 In our in vitro priming, five out of ten CD4+ T cell responses matched the responses previously documented ex vivo with cells from HIV-1 infected subjects at different stages of disease and a number of these HIV-1 specific CD4+ T cell responses are frequently detected (Table 2: Figure 4B)[19]. The primed p24 epitopes WIILGLNKIVRMYSPTSI and YVDRFYKTLRAE- QASQEV are frequently observed in HIV-infected sub- jects[19,30] and p24 contains several CD4+ T cell epitopes[19]. For instance, the WIILGLNKIVRMYSPTSI p24133-150 response was frequently seen in the majority of the donors we tested and several T cell clones responding to epitope/s located within p24 have been established (data not shown). p24133-150 is contained within the immunodominant region of gag and also contains several known CD8-epitopes including GLNKIVRMY and IILGLNKIVR. Moreover it contains multiple CD4+ epitopes that are promiscuous and can be loaded on to several different HLA DR haplotypes (e.g. ILGLNKIVRMY recognized in DRB1*0101, DRB1*1302, DRB1*1501) a factor that may HIV Specific T Cell Priming contribute to its frequent recognition. In addition, the peptide YVDRFYKTLRAEQASQEV has been reported to be the peptide most recognized by CD4+ T cells in all categories of subjects investigated[19]. Studies on the antigenic specificity of CD8+ T cell responses in HIV-1 infected subjects, especially in chronic infection, are more comprehensive [31,32]. Responses to three in vivo HIV-1 specific CD8+ T cell epitopes described in HIV-1 infected patients were primed in our in vitro system and all were gag specific. They are located within gag p15 and p17, corresponding to TGSEELR- SLYNTVATL p1770-85 and SGGELDRWEKIRLRPGGK p179- 26 and RNQRKIVKCFNCGKEGHT p1521-38 (Table 2). These peptides contain at least five epitopes that have been identified ex vivo in HIV-1 infected subjects at different stages of disease [19,31,36] (Los Alamos NL). CD8+ T cells specific for the TGSEELRSLYNTVATL and SGGELDRWEKIRLRPGGK p17 epitopes have also previously been primed for in vitro using DCs pulsed with liposome complexed HIV-1 protein[11] and DC pulsed with peptide[12]. The CD8+ T cell responses primed with peptide pulsed DCs recognized the epitope KIRLRPGGK within the p17 SGGELDRWEKIRLRPGGK sequence and this is a HLA*A3-restricted epitope[12]. Our response was seen in a HLA* A3-positive subject and thus could be directed to the same CD8+ T cell epitope. Alternatively, it could be a novel epitope response contained within the same region. Thus comparison of our data with ex vivo data obtained from HIV-infected subjects demon- strates that the epitopes seen in our in vitro priming correlates with T cell responses that develop in vivo. Strong and broad HIV-specific CD4 T cell responses can be identified at time of acute HIV-infection but quickly decline irrespective of treatment status The predominantly CD4+ T cell responses and the observation of in vitro primed specificities that had not been previously reported from studies of infected patients prompted us to re- examine in vivo responses more closely, in particular in acute HIV-infected subjects. In order to assess the repertoire of HIV- specific CD4+ T cell responses found in acute HIV infection (AHI), we used fresh CD8-depleted PBMCs from 12 subjects with documented AHI and screened them for HIV-specific CD4+ T cell responses. Peptides were divided in peptide pools correspond- ing to the different HIV proteins before being used in the Elispot assays and responses were analyzed per protein and as total response obtained by summing up the responses to the individual HIV proteins. Furthermore, we monitored changes in CD4+ T cell responses over time. At the first available time point for immunological studies and before initiation of HAART (baseline, BL), all investigated subjects showed a positive, and in most cases robust, HIV-specific CD4+ T cell response (median total response 2,780 SFC/106 CD8-depleted PBMCs; range 315-8646) (Figure 5A). At one month after BL, 8 out of 9 evaluated subjects showed a decrease in magnitude of the HIV-specific CD4+ T cell responses compared to baseline (p = 0.02; Wilcoxon matched pairs test)Further decline was observed 3 months after BL compared to the one month time point in 6 of 7 individuals studied longitudinally (p = 0.04; Wilcoxon matched pairs test). The HIV-specific CD4+ T cell responses waned in both subjects treated with HAART (grey circles) and in individuals who remained without therapy (white circles) and occurred while most subjects were still viremic, showing that the reduction in HIV-specific CD4+ T cell responses occurred in spite of a continuous exposure to the antigen. In order to investigate the responses to the different proteins, we next analyzed the evolution of the CD4+ T cell responses to the Gag, Pol, Env and Nef proteins (Figure 5B). The results demonstrate PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4256