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Loss of reactivity of vaccine-induced CD4 T cells in immunized monkeys after SIV/HIV challenge

Puaux, Anne-Laurea; Delache, Benoitd; Marconi, Séverinea; Huerre, Michelc; Grand, Roger Led; Rivière, Yvesb; Michel, Marie-Louisea

Basic Science

Background: Immunization protocols involving priming with DNA and boosting with recombinant live virus vectors such as recombinant modified Vaccinia Ankara (rMVA) are considered as vaccine candidates against HIV. Such protocols improve the outcome of simian/human immunodeficiency virus (SHIV) pathogenic challenge in Rhesus monkeys.

Objectives: To investigate the fate of vaccine-induced T cells after a mucosal SHIV challenge.

Methods: We immunized Rhesus monkeys (Macaca mulatta) by DNA priming followed by rMVA boost. After intrarectal challenge with SHIV 89.6P, immunized animals demonstrated early control of viral replication and stable CD4 T-cell counts. We monitored T-cell responses by measuring IFN-γ secretion and proliferation.

Results: Immunization induced strong and sustained SHIV-specific CD4 and CD8 T-cell responses. CD8 T-cell responses were recalled during acute infection, whereas none of the vaccine-induced SHIV-specific CD4 T-cell responses were recalled. Moreover, most of the CD4 T-cell responses became undetectable in peripheral blood or lymph nodes even after in-vitro peptide stimulation. In contrast, we persistently detected CD4 T-cell responses specific for control recall antigens in infected animals.

Conclusion: SHIV 89.6P challenge results in a lack of reactivity of vaccine-induced SHIV-specific CD4 T cells. These results may have important implications in the AIDS vaccine field, especially for the evaluation of new vaccine candidates, both in preventive and therapeutic trials.

From the aINSERM U 370 Carcinogenèse Hépatique et Virologie Moléculaire

bLaboratoire d’Immunopathologie Virale, URA CNRS 1930, Département de Médecine Moléculaire

cUnité de Recherche et d’Expertise Histotechnologie et Pathologie, Institut Pasteur, Paris, France

dLaboratoire d’Immunopathologie Expérimentale, Service de Neurovirologie, DSV/DRM-CRSSA, Commissariat à l’Energie Atomique, Fontenay aux Roses, France.

Correspondence to Marie-Louise Michel, INSERM U 370 Carcinogenèse Hépatique et Virologie Moléculaire, Département de Médecine Moléculaire, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel: +33 1 45 68 88 49; fax: +33 1 40 61 38 41; e-mail:

Abbreviations AT2: Aldrithiol-2; AZT: Azidothymidine; HBsAg: hepatitis B surface antigen; hybrid DNA: hybrid SHIV/HBsAg particles-expressing DNA; Rh: Rhesus macaque; rMVA: recombinant modified Vaccinia Ankara; SHIV: simian/human immunodeficiency virus; Th: T helper.

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The evaluation of candidate AIDS vaccines in simian models has proved that vaccine-induced T-cell responses can modulate viral infection [1–4]. Both CD8 and CD4 vaccine-induced T cells appear to be involved in the partial control of infection in primates [5]. Many studies have shown that cytotoxic T lymphocytes (CTL) are involved in the control of HIV and SIV infections. The CTL response is triggered by HIV-specific T helper lymphocytes and by the generation of cytokines, which are produced by activated CD4 T cells. There is evidence that both CD4 [6,7] and CD8 T-cell responses [8,9] play a role in the control of HIV replication. It is now generally accepted that CD8 T-cell responses play a direct role in the control of viral replication. However, the role of virus-specific T helper cells is less clear. It has been proposed that they play a direct role [10], possibly mediated by cytokine and chemokine secretion and cytotoxicity [7]. Alternatively, T helper cells may increase the induction and persistence of virus-specific CTL and neutralizing antibodies [11].

We recently showed that viral replication can be controlled after a mucosal challenge with a pathogenic chimera of simian and human immunodeficiency virus (SHIV) 89.6P by priming with DNA expressing hybrid SHIV/hepatitis B surface antigen (HBsAg) particles (hybrid DNA) and boosting with a Gag–Pol–Env–Nef–Tat-expressing recombinant modified Vaccinia Ankara (rMVA). The mucosal challenge was only partly controlled as infection occurred in all animals. However, this priming/boosting protocol decreased the viral load by 100-fold and prevented CD4 T-cell depletion during primary infection [12]. In addition, immunizations activated virus-specific T-cell proliferation and IFN-γ secretion. These cells were long lasting and could be detected using in-vitro assays before and at the time of pathogenic challenge. To determine the fate of vaccine-induced T-cell responses after challenge, we studied the dynamics of T-cell responses after immunization and subsequent infection by monitoring the immune response to the DNA-encoded antigens at the epitope level.

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Materials and methods

Immunization and viral challenge

Rhesus monkeys (Macaca mulatta) were immunized and challenged as described previously [12]. The animals were vaccinated with Bacille Calmette–Guérin (BCG) strain 1173P2 (Institut Pasteur, Paris, France) one month before the first DNA immunization. Briefly, five animals were injected with SHIV/HBsAg-encoding hybrid DNA at months 0, 2 and 6 (group 1). Four animals (group 2) were injected with the HBsAg-encoding control DNA at the same timepoints. Ten months after the last DNA injection, both groups received booster injections, consisting of 2 × 108 pfu of rMVA expressing full-length SHIV proteins [12]. Three months later, all immunized animals and control animals [12] were challenged intrarectally with 10 50% intrarectal monkey infectious doses of pathogenic uncloned SHIV-89.6P [13,14].

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Synthetic peptides

Gag and Nef peptides were synthesized by Neosystem (Strasbourg, France). SHIV 89.6P Env peptides were provided by the National Institutes of Health AIDS Research and Reagent Programme. These peptides and their use in in-vitro assays are described elsewhere [12] and span the DNA-encoded domains from Gag, Nef and Env antigens. Two other sets of 15-mers, each overlapping by five amino acids and derived from the hepatitis B virus envelope, were synthesized by Neosystem: pool HBs#2 (nine peptides spanning residues 251–332) and pool HBs#3 (nine peptides spanning residues 328–390).

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In vitro stimulation of peripheral blood mononuclear cells

We generated peptide-specific CD8 and CD4 T-cell lines by stimulating freshly prepared peripheral blood mononuclear cells (PBMC) in vitro with the above-mentioned peptide pools in the presence of cytokines. The culture conditions are described elsewhere [12].

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IFN-γ enzyme-linked immunospot assay

The enzyme-linked immunospot (ELISPOT) assay was used to count antigen-specific IFN-γ-secreting cells among rhesus macaque PBMC [12]. We used synthetic peptides as antigens to assess specific responses. When PBMC were scored positive against a pool of peptides, each peptide from the pool was tested separately in order to map T-cell responses to a single peptide. Ex-vivo assays were performed using fresh PBMC. In-vitro assays were perfomed using PBMC cultured for 14 days with peptides and IL-2. In both cases the T cells were stimulated by an overnight incubation with specific peptides.

ELISPOT assays were performed using total PBMC, CD4-depleted or CD8-depleted PBMC as effector cells. For depletion experiments, PBMC were depleted of CD4 or CD8 cells using dynabeads (Dynal Biotech France, Compiègne, France) following the manufacturer's instructions. The efficiency of the magnetic depletion was evaluated by flow cytometry (FACScalibur, BD Biosciences, Sunnyvale, CA, USA) using FITC-conjugated anti-monkey CD3 (CD3-FITC, clone FN18; BioSource, Clinisciences, Montrouge, France), phycoerythrine-conjugated anti-human CD4 and peridin chlorophyll protein (PerCP)-conjugated anti-human CD8 (CD8α-PerCP, clone SK1; CD4-phycoerythrine, clone M-T477; BD Biosciences) monoclonal antibodies.

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Intracellular cytokine staining

Restimulated PBMC were incubated either with phorbol myristate acetate/ionomycin as a positive control or with pools of peptides in a total volume of 200 μl RPMI 5% fetal calf serum. Brefeldin A was added at a final concentration of 10 μg/ml, and the cells were then incubated 6 h at 37°C. Cells were washed twice with fluorescence-activated cell sorter (FACS) buffer (phosphate-buffered saline bovine serum albumin 1%, sodium azide 0.1 %) and then stained with 2 μl of CD8α-PerCP (clone SK1; BD Biosciences) and 0.5 μl of CD4-phycoerythrine (clone M-T477; BD Biosciences) in 50 μl of FACS buffer for 30 min at 4°C. After two washes with FACS buffer, the cells were fixed with 2% formaldehyde-FACS buffer for 15 min at 4°C. The cells were then washed twice in FACS buffer and once with permeabilization buffer (0.1% saponin in FACS buffer). Cells were then stained with 0.5 μl anti-human IFN-γ-FITC monoclonal antibody (clone MD-1; Biosource) in 50 μl permeabilization buffer for 30 min at 4°C. The cells were washed twice with permeabilization buffer, once with FACS buffer, and then fixed with 1% formaldehyde-FACS buffer. Samples were stored in the dark at 4°C. We acquired 50 000–200 000 lymphocyte-gated events using a FACSCalibur flow cytometer (BD Biosciences) and analysed results with the Cellquest software (BD Biosciences). The background level of IFN-γ staining in PBMC (induced by a control hepatitis C virus peptide) varied from animal to animal but was typically below 0.05% in the CD8 lymphocytes and below 0.02% in the CD4 lymphocytes. Samples were only considered positive if the IFN-γ staining was at least twice as strong as the background or if there was a distinct population of bright IFN-γ-positive cells. Background staining was assessed using a control isotype-matching monoclonal antibody and subtracted from all values.

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Proliferation assay

We measured lymphoproliferative responses by culturing PBMC either with aldrithiol-inactivated SIVMne/HuT78 (300 ng p28CA equivalents per ml, provided by Dr Lifson [15]) or with purified protein derivative (PPD), a mixture of BCG antigens, 10 μg/ml, Statens Seruminstitut, Copenhagen, Denmark.

Freshly isolated PBMC were stained with PKH26 (Sigma-Aldrich, Saint-Quentin Fallavier, France) according to the manufacturer's instructions. Labeled cells were then seeded at a density of 1 × 106 per ml in duplicate in U-bottomed wells containing 200 μl RPMI 1640 with 10% human AB serum and cultured for 7 days. Cells were then washed twice with FACS buffer and stained with 2 μl CD8α-PerCP (clone SK1; BD Biosciences) and 2 μl CD3-FITC (clone FN18; Biosource) in 50 μl FACS buffer for 30 min at 4°C. After two washes with FACS buffer, the cells were fixed with 2% formaldehyde-FACS buffer for 15 min at 4°C. Samples were stored in the dark at 4°C. We acquired 20 000 lymphocyte-gated events as above. The percentage of CD3+ CD8+ (T CD8) or CD3+ CD8− (T CD4) cells that lost PKH26 labeling was determined in each condition. The results are expressed for each cell subset as SI. SI values greater than 2 were considered positive. This cutoff point is twice the median value obtained with PBMC from naive monkeys.

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Induction of IFN-γ-secreting CD8 and CD4 T cells specific to SIV/HIV antigens after immunization

Rhesus monkeys were immunized by priming with DNA coding for HBs/SIV hybrid antigens (hybrid DNA) and followed by rMVA boost (group 1) or by DNA coding for HBsAg (control DNA) and rMVA boost (group 2). We monitored T-cell responses in the PBMC of immunized animals. After 2 weeks of in-vitro stimulation with peptide pools (nine to 13 peptides per pool), we used ELISPOT assays to assess IFN-γ-secreting precursor T cells. Overlapping peptides (mostly 15-mers) that can stimulate both CD4 and CD8 T cells in vitro were used. These peptides are derived from the Gag, Nef and Env domains expressed during immunization with hybrid DNA. A single peptide assay was carried out to determine which peptides were specifically recognized by individual animals. In addition, we used cell depletion assays coupled with the ELISPOT assay or intracellular staining of IFN-γ coupled with cell surface phenotyping and flow cytometry analysis to analyse the phenotype (CD4 or CD8) of specific IFN-γ-secreting T cells. During the month before the challenge, immunized animals exhibited SHIV-specific T-cell responses that were associated with both CD4 and CD8 T cells (Tables 1 and 2). SHIV-specific CD8 T-cell responses were detected in five out of eight immunized animals (Table 1, see column ‘pre-infection’). Recognized peptides mainly derived from Gag antigen, although 1 peptide from Nef was also recognized. SHIV-specific CD4 T-cell responses were detected in three out of eight immunized animals. Interestingly, only animals from immunization group 1 exhibited these CD4 T-cell reactivities (Table 2, see column ‘pre-infection’). Recognized peptides derived from Gag, Env and Nef antigens. In summary, IFN-γ-secreting CD8 and CD4 T cells were activated by DNA prime and rMVA boost immunization and were easily detected in the PBMC of animals before challenge.

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Effect of SIV/HIV challenge on vaccine-induced CD8 and CD4 T-cell responses

Immunized animals were challenged with SHIV 89.6P 3 months after rMVA boost. The outcome of challenge was modulated by immunization, as no decrease in CD4 T-cell counts was observed and as peak viraemia was 100-fold lower in immunized animals than in control animals ([12] and Fig. 1a). We studied the fate of CD8 and CD4 T-cell responses after pathogenic SHIV challenge in all immunized animals, during both the acute and chronic phase of infection. For this purpose, we carried out ex-vivo ELISPOT assays using fresh PBMC from each animal with the peptides listed in Table 1 and Table 2. Fig. 1bFigure 1b and c shows the median and range of vaccine-induced SHIV-specific CD8 and CD4 T-cell frequencies measured ex vivo. Most CD8 T cells appeared to be strongly recalled during SHIV 89.6P acute infection (Fig. 1b). Peptide-specific IFN-γ-secreting T cells were detected during acute infection at high frequencies, i.e. up to 6000 spots per million PBMC. Five of the seven class I epitopes recognized after immunization (distributed among five immunized animals) were recalled during acute infection. These five class I epitopes were recognized throughout the acute and chronic phases of infection, both ex vivo (Fig. 1b) and after in-vitro stimulation (Table 1, column ‘post-infection’). CD8 T cells specific for the remaining two class I epitopes were not recalled during acute infection and were undetectable after in-vitro stimulation (Table 1). One of these CD8 T-cell reactivities was never detected again in blood during SHIV infection, but was found in the lymph nodes late in infection (Rh 970361/Gag peptide 255–269, see Table 3). The other became detectable again in PBMC during the chronic phase of infection (Rh 960837/Gag peptide 195–209, not shown).

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.

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Analysis of specific lymphoproliferation

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.

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Analysis of vaccine-induced CD4 T-cell responses after in-vitro stimulation

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 [12]). 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.

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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 [12]. 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 [18]. 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 [19].

There is increasing evidence that T helper cells also play a role in the control of immunodeficiency virus infection [5]. However, Vogel et al. found that only 14% of vaccine-induced CD4 T cells were recalled by SIV infection [18]. 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 [20], a key cytokine that we did not study here. Virus-specific IL2-secreting CD4 T cells represent long-term central memory CD4 T cells [21]. 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 [22], and thus virus-specific CD4 T cells could be preferentially infected, as described in HIV infection [23]. Infected SHIV-specific CD4 T cells could be anergized, show functional impairment in IFN-γ secretion and in proliferation [24], be lysed by virus-specific CD8 cytotoxic T cells, or be deleted after virus-induced apoptosis [25]. 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 [26] by using mainly the CXCR4 co-receptor [27]. 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 [26]. 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 [28]. 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 [31]. 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 [31]. 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.

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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.

Sponsorship: This work was supported by grants from the Agence Nationale de Recherches sur le SIDA. Fellowships were from Agence Nationale de Recherches sur le SIDA and Ensemble contre le Sida, sidaction.

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CD4 T-cell response; cellular immunity; primate; SHIV; vaccine

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