HIV Specific T Cell Priming Figure 6. In vitro primed novel HIV epitopes correlates with CD4+ T cell responses seen during primary infection. (A) Summary of responses to the novel epitopes detected in 12 acute and recent HIV-infected subject PBMCs. PBMCs from acute and recent HIV infected individuals were thawed and tested directly ex vivo for reactivity to OLPs 11, 189, 262, 322, 377, and 382 using intracellular staining for IFN-c, TNF-a, and IL-2 after six hour incubation. Reactivity against the peptide was determined when the percentage of cytokine-secreting cells were at least twice the percentage of cytokine-secreting cells in the unstimulated wells. (B) HIV-infected PBMCs from one donor evaluated at 3 different time points after infection (sample one: 2 weeks after onset of retroviral syndrome: acute infection, sample two: 1 year infected: recent infection, and sample three: 2 years infected: chronic infection) were thawed and cocultured with peptide pools OLP 371-381 or OLP 382-392 for 14 days. The peptide activated PBMCs were then assessed for reactivity to each of the individual peptides in the pools used. doi:10.1371/journal.pone.0004256.g006 and were no longer detected at later time points. We have only observed a persisting response to this peptide in one subject that was chronically HIV-infected (data not shown). On the other hand, CD4+ T cell responses to the peptide LELDKWASLWNWFNITNW env gp41601-678 so far remain undetected in acute and chronic HIV-1 infected subjects studied by our group and others (data not shown[19,31,36]). Interestingly, LELDKWASLWNWFNITNW corresponds to a highly conserved membrane proximal region of the gp41 and is only exposed during the fusion of the virus with the host membrane[50]. This epitope lies within a region identified as a CD4-epitope ‘‘hotspot’’ in mice immunized with HIV gp120[51], consistent with the idea that an otherwise immunodominant specificity is selectively eliminated in HIV-infected humans. Of note, this particular peptide also contains epitopes of two broadly neutralizing HIV-1 antibodies (2F5 and 4E10) that have been shown to be polyspecific autoantibodies reactive to phospolipid cardiolipin [52] Thus our inability to confirm responses to this particular peptide in HIV- infected subjects may be in part due to its autoimmune nature. To further understand the skewing of priming to CD4+ T cells, we decided to examine CD4+ T cell responses in acute HIV- infected subjects. Our studies reveal that broad and strong CD4+ T cell responses are readily detected during AHI although some responses vanished within 1–2 months. Of note, CD4+ T cell responses targeting env are strong during this early stage and are detected as early as five days post-infection, whereas env responses are rarely detected in the later stages of infection[53,54]. The mechanistic basis for the apparent preferential disappearance of HIV-specific CD4+ T cell responses of some specificities but not others, despite the ongoing presence of the relevant antigens, is unclear. In the case of HIV-specific CD8+ T cell responses there is also evidence of rapid disappearance of initially expanded cells and high avidity cells during AHI [55,56]. This could be attributed to several mechanisms including mutations in the viral genome, inhibition of the presentation and processing of certain epitopes, and/or the inability of the T cells to adequately respond to survival signals. In the case for HIV-specific CD4+ T cells, an intriguing possibility is that these particular HIV-specific CD4+ T cells may be specifically targeted for deletion by the virus. In fact, we (Figure 5B and 5C) and others [34,35] do provide convincing evidence for this particular theory. Of note, the activation of HIV-specific CD4+ T cell responses coincides with the presence of a widespread activation-induced CD4+ T cell apoptosis so that it is possible that these newly activated HIV- specific CD4+ T cell responses would be eliminated as a consequence of both specific and unspecific mechanisms. Alternatively, loss of HIV-1 specific CD4+ T cells may also be a consequence of chronic immune activation, e.g. due to bacterial translocation [57]. Regardless of the rationale, disappearance of these HIV-specific CD4+ and CD8+ T cell responses during AHI may ultimately contribute to the eventual inability of the immune system to control viral replication. In contrast, the persistence of HIV specific CD4+ T cells in untreated LTNPs highlights the importance of these cells. The helper activities of antigen-specific CD4+ T cells have been shown to mediate the control of many viral infections and are critical in maintaining HIV-1 suppres- sion[1,3,58]. However there is individual heterogeneity in adaptive immune responses among LTNPs and consequently the correlates of immunity remain unknown. Therefore the key to deciphering this mystery may lie with the answer to why these HIV-specific CD4+ and CD8+ T cell responses disappear during acute HIV-1 infection [59]. In summary, our in vitro studies highlight the ability of DCs to efficiently prime na¨ıve T cells and induce a broad repertoire of CD4+ T cells. Our findings provide important evidence showing that in a vaccine setting, AT-2 HIV-1 can not only restimulate memory T cell responses but is also capable of priming broad and polyfunctional de novo T cell responses. And given the recent failure of the Merck STEP Trial [60], the need to explore alternative vaccine platforms such as the therapeutic use of DC pulsed with AT2-HIV becomes critical in attaining the ultimate goal of an HIV vaccine. Materials and Methods Ethics Statement This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the PLoS ONE | www.plosone.org 10 January 2009 | Volume 4 | Issue 1 | e4256