We used an assay based on the incorporation of a fluorescent dye, PKH26, into the plasma membrane. This assay allows a phenotypic analysis of proliferating cells by flow cytometry. We could distinguish CD8 T cells (CD3+ CD8+) and CD4 T cells (CD3+ CD8−) within the whole PBMC population. Although both subsets proliferated in response to PPD stimulation (Fig. 2a), only CD8 T cells proliferated with AT2-SIV during chronic SHIV infection (Fig. 2b). We detected no SIV-specific proliferation in the CD4 T cell subset among PBMC from eight SHIV-infected monkeys.
Therefore, consistent with the results obtained using IFN-γ ELISPOT, no SIV-specific proliferation could be detected in the CD4 T-cell subset after SHIV infection, despite the maintenance of CD4 cell immunity specific for recall antigens such as PPD.
We then tried to restimulate the T cells with their cognate peptides to determine whether SHIV-specific CD4 T cells could be expanded in vitro. Only one of the six epitopes recognized by CD4 T cells after immunization induced IFN-γ secretion after in-vitro PBMC restimulation during acute infection (Table 2). Post-infection CD4 T cell counts were remarkably stable in the blood of all the immunized animals in which the SHIV-specific CD4 T-cell responses were not detected (Fig. 1a and ). Therefore, the absence of CD4 T-cell responses cannot be attributed to a global CD4 T-cell depletion.
Finally, to evaluate whether the loss of the CD4 cell response was restricted to SHIV-specific CD4 T cells, we checked if CD4 T cells directed against a recall antigen distinct from SHIV antigens persisted. Immunization with DNA coding for hybrid SHIV/HBsAg particles induced HBs-specific T cells in four out of five animals. These cells were detected in ELISPOT assays after in-vitro restimulation with HBs-derived peptides (Fig. 3a). When positive ELISPOT results were obtained, we performed cell depletion experiments to confirm that IFN-γ secretion was caused by CD4 T cells (not shown). One year after SHIV infection, and 2 years after the last hybrid DNA injection, these HBs-specific CD4 T cell responses were still detectable in the blood of infected animals (Fig. 3a), unlike SHIV-specific CD4 T cells (Fig. 3b). Therefore, only SHIV-specific CD4 T-cell reactivities were lost.
There is increasing evidence that T helper cells also play a role in the control of immunodeficiency virus infection . However, Vogel et al. found that only 14% of vaccine-induced CD4 T cells were recalled by SIV infection . In our study, SHIV infection did not recall vaccine-induced SHIV-specific CD4 T cells. Our results also indicate that SHIV infection can totally eliminate SHIV-specific vaccine-induced CD4 T-cell reactivity from the blood and lymph nodes, because these responses were not detected in most animals even after in vitro culture with peptide. The mechanisms involved in this loss of reactivity remain to be elucidated. A possible hypothesis dealing with viral escape involves point mutations in epitopes recognized by CD4 T cells. Therefore, we sequenced plasmatic viral RNA corresponding to immunizing domains from the gag and nef genes. Sequences were determined both early and late in infection at the population level. During the course of infection, no mutation was observed in the antigenic domains targeted by the T-cell responses (not shown). In addition, the sequence of the peptides used to stimulate PBMC exactly matches to the sequence of the virus that replicates in the monkeys (not shown). Alternatively, the 2 weeks culture of PBMC stimulated by peptides in vitro may result in the preferential killing of virus-specific CD4 T cells by the virus present in culture. However, cell culture performed in the presence of azidothymidine-inhibited viral replication in vitro but did not rescue CD4 T-cell reactivity measured by ELISPOT (not shown). Therefore, SHIV-specific CD4 T cells were not impaired as a result of viral reactivation and infection in vitro. Finally, recent work has underlined the importance of virus-specific CD4+ T cells that secrete IL-2 , a key cytokine that we did not study here. Virus-specific IL2-secreting CD4 T cells represent long-term central memory CD4 T cells . However, a clear correlation between IL-2 secretion and proliferation was also reported [20,21]. The study of two cellular functions, i.e. IFN-γ secretion and proliferation, thus appears relevant to assess CD4 T-cell responses.
A remaining hypothesis supported by our data to explain the loss of reactivity of vaccine-induced CD4 T cells after challenge is that vaccine-induced SHIV-specific CD4 T cells encounter their specific antigen during acute infection, leading to cell activation. Activated CD4 T cells provide the ideal conditions for virus replication , and thus virus-specific CD4 T cells could be preferentially infected, as described in HIV infection . Infected SHIV-specific CD4 T cells could be anergized, show functional impairment in IFN-γ secretion and in proliferation , be lysed by virus-specific CD8 cytotoxic T cells, or be deleted after virus-induced apoptosis . This could explain why SHIV-specific CD4 T cells were undetectable in ELISPOT, intracellular staining and proliferation tests, even after in vitro restimulation. Recent data show that SHIV 89.6P, in contrast to SIV or HIV, preferentially targets naive CD4 T-cells during early infection  by using mainly the CXCR4 co-receptor . This is in agreement with our results as we found that naive CD4 T cells were preferentially eliminated in our unvaccinated control animals, whereas they were preserved in vaccinated animals (not shown). In addition, the results shown in the present study suggest that SHIV 89.6P is able to target pre-existing memory CD4 T cells specific to its own antigens. These results are not mutually exclusive because it was shown that nearly half of memory CD4 T cells bear the CXCR4 receptor . It thus remains possible that these cells become infected by SHIV 89.6P upon activation. The blockade of T-cell co-stimulation during SIVmac239 acute infection resulted in lower levels of proliferating CD4 T cells and lower levels of peak viraemia. These data provide the clear evidence of the contribution of cellular activation to SIV-induced disease enhancement . On the basis of these findings, the induction of a virus-specific T helper response may be both beneficial and harmful. In this context, the data presented here support the idea that inducing virus-specific CD4 T cells in combination with CD8 T cells before infection is not deleterious up to one year after challenge, even if specific T helper cells are preferentially targeted by the virus during acute infection. In humans, it is possible to restore or induce T-helper responses specific for HIV in infected patients. This was observed after therapeutic immunizations [29,30] and after viral rebounds during standardized treatment interruptions . However, these T helper reactivities were only transiently mobilized and became rapidly undetectable. This contrasts with T helper responses specific for HIV-unrelated antigens, which were persistent in the patients . These data suggest that HIV preferentially targets CD4 T cells specific to its own antigens that are activated in vivo, resulting in a functional impairment of these cells. Our observations, in a monkey model in the context of preventive immunization, are consistent with these results.
The authors would like to thank Lucie Da Silva, Geneviève Janvier, Patricia Brochart and Diane Couraud for technical assistance. They also thank Dr Jeffrey Lifson and the AIDS Vaccine Programme for providing AT2-SIV, Dr Michel Morre from Biotech Inflection Point for providing recombinant IL-7, and the NIH AIDS Research and Reagent Program for providing Env peptides. We thank Rémi Cheynier for critical reading of the manuscript.
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