HIV Specific T Cell Priming Stage of disease References Mostly in acute &Kaufmann et al J Virology Lower responses 2004 in chronic High responses in untreated &Wilson et al J Virology 2001 &Los Alamos HIV Immunology Database &Addo et al J Virology 2001 &Frahm et al J Virology 2005 &Los Alamos HIV Immunology Database T cell Type CD4 CD4 CD4 CD4 CD4 CD4 CD8 CD8 Table 2. Correlation of primed T cell responses with in vivo data HIV Protein HXB2 location OLP sequence Epitopes (HLA-restriction) Gag p17 9–26 SGGELDRWEKIRLRPGGK Human Gag p17 Gag p17 Gag p24 Pol INT Gag p17 32–46 70–86 133–150 210–277 9–26 Gag p24 164–181 YVDRFYKTLRAEQASQEV KHIVWASRELERFAV TGSEELRSLYNTVATLY WILLGLNKIVRMYSPTSI* Human Human DRB1*1501, DRB1*1302, DRB1*0401, DRB5*0101, DRB1*1101, DRB1*0701, DRB1*0405, DRB1*0101 DRB1*1501, DRB1*1302, DRB1*0401, DRB5*0101, DRB1*1101, DRB1*0701, DRB1*0405, DRB1*0101 TEKLQKQITKIQNFRVYY Human, DR supermotif SGGELDRWEKIRLRPGGK A* 0301, A3, B*4002, B7, B27, B40, B63 Found in all stages of disease Gag p17 70–85 TGSEELRSLYNTVATL A*0101, A1, A2, A*0201, A*0202, A*0205, A*0214, A24, B*4006, B*0801, B8 doi:10.1371/journal.pone.0004256.t002 epitopes contained within peptides ELKKIIGQVRDQAEHLK in pol IN157-173, RPVVSTQLLLNGSLA in env gp120252-266, and AVLSIVNRVRQGYSPLSF in env gp41700-717 (Figure 6A) when we screened subjects directly ex vivo that were HIV-1 infected less than 6 months. We were unable to confirm more of our novel epitopes in more HIV-infected subjects due to the fragile nature of CD4+ T cells at this early time point of infection and difficulty of obtaining enough CD4+ T cells from acute HIV-infected subjects. With one acute HIV-infected subject from whom we received follow- up samples, we detected CD4+ T cell responses to epitopes contained within peptide AVLSIVNRVRQGYSPLSF in env gp41700-717 at the initial time point however, screening of PBMCs from subsequent time points revealed the rise and decline of responses targeting epitopes within this particular peptide (Figure 6B). Nonetheless, we provide evidence that novel epitopes seen in our in vitro priming may represent T cell responses seen in AHI that disappear in the infected individuals so early on that they are gone in most individual at time of their HIV-1 diagnosis. Taken together, our data establishes that MDDCs presenting either infectious or AT-2 HIV-1 derived antigens can prime polyfunctional CD4+ and CD8+ HIV specific T cells that target a broad repertoire of HIV-antigens. Furthermore, we show that our primed HIV-specific T cells correlate to T cells that arise naturally in vivo in HIV-1 infected individuals, more specifically during AHI and is lost early on. Though noninfectious HIV-1 had slightly higher efficiency in priming, there were no significant differences in terms of the type of responses elicited thus demonstrating that defective viruses, can serve as a source of antigen in vivo. Discussion The cellular immune response developed against HIV-1 is multifaceted. DCs in the lymph nodes probably contribute both to the initial strong T cell responses and at the same time to the defects seen in CD4+ T cells during AHI. One mechanism explaining the efficiency of viral spread is the formation of the infectious synapse whereby DC harboring HIV-1 interacts with T cells [28,38]. In this setting, the activated HIV-specific CD4+ T cell, expressing CCR5 and high levels of CD38, are prime targets for infection in vivo during primary HIV-infection[39], which explains the preferential infection of HIV-1 specific CD4+ T cells[35]. The HIV-1 specific CD4+ T cells rapidly decline two and three weeks following the onset of the acute viral illness. This may depend on that 80–90% of these cells lack CD127 during primary HIV-infection, consistent with a predetermined apoptotic fate [39]. In spite of the rapid loss initially, HIV-1 specific CD4+ T cells do persist in individuals at all stages of HIV-infection and comprise from 0.02%–2% of the peripheral blood CD4+ T cells. The majority of the CD4+ T cells that remain in individuals with persistent viremia have defective proliferation ability due to impaired development of IL-2 producing TCM cells [40], PD-1 and CTLA-4 expression [29,41]. While the exact proportions of infectious and non-infectious virus circulating in vivo remain the subject of some controversy. The role of these noninfectious virions as antigen for priming immune responses has not been explored. Therapeutic vaccination of SIV- infected rhesus monkeys [42,43] and chronically HIV-infected subjects[10] with autologous DCs pulsed with AT2-inactivated virus was associated with reduced viral loads and enhancement of HIV- specific immune responses. Therefore, we hypothesized that DCs may also have the capacity to present antigens from fusion competent nonreplicating virus i.e. AT-2 HIV-1[25,44] to prime HIV-specific T cell responses. Here we show for the first time that AT-2 HIV-1 can serve as an efficient source of antigen for priming both CD4+ and CD8+ T cells and therefore function as a relevant source of antigen for priming in vivo. The continuous presence of viral antigens gives rise to new memory T cells from the existing na¨ıve population as long as the immune system remains functionally intact and/or the existing na¨ıve T cells can respond to HIV-antigens [45] thus explaining the highly heterogeneous population of HIV-specific T cells that exist PLoS ONE | www.plosone.org 8 January 2009 | Volume 4 | Issue 1 | e4256