We next studied the fate of vaccine-induced CD4 T-cell responses using the peptides that were recognized after immunization. Specific CD4 cell activities directed to the seven epitopes after rMVA injection (in four hybrid DNA plus rMVA immunized animals from group 1) were not recalled from fresh PBMC during acute or chronic infection (Fig. 1c). These data show that vaccine-induced CD4 and CD8 T-cell responses have distinct fates after SHIV challenge.
As vaccine-induced IFN-γ-secreting CD4 T cells were not recalled after SHIV challenge, we analysed the proliferative capacity of both CD4 and CD8 T cell subsets after infection. We used non-replicative whole SIV particles as an antigen source. The particles were inactivated with aldrithiol-2, which preserves the fusogenic capacities of the viral envelope. Viral peptides can therefore be loaded on both MHC class II molecules (by the classical exogenous pathway) and on MHC class I molecules (by an alternative pathway) [16,17]. The proliferating T cells detected may thus be CD4 or CD8. To monitor the T-cell proliferative response against a SHIV-unrelated recall antigen, we used PPD, a mixture of BCG antigens. The macaques were previously immunized with BCG before priming with control or hybrid DNA.
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.
Next, we asked whether SHIV-specific CD4 cell responses were detectable in the lymph nodes. It is possible that SHIV-specific CD4 T cells home to the lymph nodes instead of circulating in the peripheral blood. We compared the presence of SHIV-specific T cells in PBMC and lymph node cells after in vitro peptide restimulation (Table 3 and Table 4). SHIV-specific CD4 T cells were found in none of the lymph nodes, but were detected in PBMC in one case (Table 4). In contrast, SHIV-specific CD8 cell activity was found in both PBMC and lymph nodes (Table 3). SHIV-specific CD4 T cells thus do not relocalize in lymph nodes after SHIV infection.
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.
In conclusion, SHIV infection does not recall vaccine-induced IFN-γ-secreting SHIV-specific CD4 T cells, but does recall their CD8 counterparts. In most cases, these SHIV-specific vaccine-induced CD4 T cells were detected neither in the peripheral blood nor in lymph nodes after infection. In contrast, CD4 T-cell reactivity against the SHIV-unrelated antigen HBs remained detectable.
Our objective was to investigate the fate of vaccine-induced CD4 and CD8 T cells after a mucosal SHIV challenge. The priming/boosting immunization protocol induced the expansion of both specific CD8 and specific CD4 T cells able to proliferate and to secrete IFN-γ. After intrarectal challenge with SHIV 89.6P, immunized animals demonstrated the early control of viral replication and stable CD4 T-cell counts . However, none of the vaccine-induced SHIV-specific CD4 T cells were recalled during acute infection (zero out of six epitopes). Most of them (five out of six epitopes) became undetectable in peripheral blood and lymph nodes, even after in-vitro peptide stimulation. In contrast, infection efficiently boosted most of the vaccine-induced CD8 T-cell responses (five out of seven epitopes). CD4 T-cell responses specific for control recall antigens were persistently detected in infected animals. These results show that vaccine-induced SHIV-specific CD8 and CD4 T cells have distinct fates after partly controlled SHIV infection.
SHIV infection clearly recalled and boosted SHIV-specific DNA plus rMVA-induced CD8 T-cell responses. This is consistent with previous results showing that 57% of vaccine-induced CD8 T-cell responses were recalled by SIVmac239 infection . Our results support the use of DNA/MVA prime-boost protocols, given that the role of virus-specific cytotoxic T cells in the control of primary SHIV infection is now widely accepted .
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.
In summary, our results indicate that vaccine-induced helper and cytotoxic T cells have distinct fates after SHIV infection. Whereas the CD8 T-cell repertoire is similar after vaccination and infection, virus-specific CD4 T cells specifically undergo either a functional impairment or a depletion in blood and lymph nodes during acute infection. Therefore, in vaccinated animals, SHIV-specific CD4 T-cell reactivity induced by the vaccine before infection do not persist after challenge. Despite this, immunization confers a partial control of viral replication and the complete preservation of CD4 T-cell counts during acute infection and up to one year after challenge. The effects of the loss of vaccine-induced CD4 T-cell response on clinical status at long term and on the persistence and the quality of virus-specific CD8 T-cell responses remain to be determined. These results may have important implications for future investigations in the AIDS vaccine field, especially for the evaluation of new vaccine candidates, both in preventive and therapeutic trials.
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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