We evaluated the clinical benefit afforded by an SIV-specific cellular immune response elicited in rhesus monkeys through a plasmid DNA prime/recombinant E1-deleted, E3-inactivated adenovirus serotype 5 (rAd) boost vaccination regimen (13–16). The monkeys selected for study did not express the MHC class I allele Mamu-A*01 because of its association with particularly efficient control of SIV/SHIV replication (17, 18). Six monkeys in each of five experimental groups received different vaccination regimens (Fig. 1) (16). The monkeys were monitored prospectively using pooled peptide Elispot and intracellular cytokine staining assays as measures of cell-mediated immunity, and for neutralizing antibody responses (13–15). Monkeys were then challenged by intravenous route with SIVmac251 and monitored for viral replication and changes in CD4⁺ T lymphocyte subsets (16, 19).

The vaccination regimens elicited high-frequency SIV-specific cellular immune responses, with greater responses detected in the monkeys that received the DNA prime/rAd boost than those that received only rAd immunizations (Fig. 1). After virus challenge, there were no statistically significant differences in any parameters of clinical outcome between the various groups of experimentally vaccinated monkeys. Therefore, all 24 experimentally vaccinated monkeys were subsequently treated as a single group and compared to the 6 sham-vaccinated control monkeys.

Plasma SIV RNA levels were significantly lower in the experimentally vaccinated monkeys compared with the controls at the peak of viral replication, with this difference persisting through day 112 after virus challenge (Fig. 2, A and B). Thereafter, no significant difference was observed in plasma viral RNA levels between the experimentally vaccinated and control monkeys. Although the prediction would be that comparable set-point viral load measurements in the vaccinated and control monkeys would be associated with comparable survival, we elected to follow the monkeys for another 750 days. In fact, as late as day 850 after virus challenge, the vaccinated monkeys maintained a statistically significant survival advantage over the control monkeys (Fig. 3A) (16). Therefore, the set-point plasma viral RNA levels did not predict the relative duration of survival in these vaccinated monkeys.

We next evaluated whether other measurements of viral replication or immune function might serve as predictors of survival. A quantitative measure of viral replication during the first 126 days after infection was determined for each animal by integrating the plasma SIV viral RNA levels between days 3 and 126. The vaccinated monkeys had significantly lower values than the controls (Fig. 3B), which suggests that differences in total viral replication during this early phase of infection are associated with differences in long-term survival.

There is increasing evidence that the loss of memory CD4⁺ T lymphocytes during acute infection is important in AIDS pathogenesis (20, 21). To examine this phenomenon in the present study, peripheral blood memory CD4⁺ T lymphocytes were assessed at a single time point, and their association with clinical outcome in the cohort of SIV-infected monkeys was evaluated. We first assessed these values on day 126 after challenge (Fig. 4), a time point at which plasma virus RNA levels were comparable in the vaccinated and control monkeys (Fig. 4A). Although the proportions of naïve and effector memory CD4⁺ T lymphocyte populations were comparable in the peripheral blood of the vaccinated and control monkeys, central memory CD4⁺ T lymphocyte populations were larger in the vaccinated group (Fig. 4, B to D, and fig. S1). Similar immunologic data were obtained on day 112 after challenge (fig. S2). Moreover, when animals were divided into terciles based on magnitudes of absolute peripheral blood CD4⁺ T lymphocyte counts on day 126 after challenge, no differences in survival were seen (Fig. 4E). Yet, when a similar analysis was done dividing monkeys into terciles based on central memory CD4⁺ T lymphocyte counts, a clear survival advantage was apparent for the monkeys with the highest counts (Fig. 4F). Similar results were seen in an evaluation of data generated on day 112 after challenge (fig. S3). These data sets also revealed an association between the survival of infected animals and the preservation of central memory CD4⁺ T lymphocytes after infection in a Cox proportional-hazards model (P = 0.05, likelihood ratio test). No association was observed in these monkeys between central memory CD8⁺ T lymphocyte counts and survival (fig. S4). Thus, although set-point plasma viral RNA levels were indistinguishable between the vaccinated and control monkeys, a single determination of the peripheral blood central memory CD4⁺ T lymphocyte count was associated with increased survival in these animals.

We then assessed the ramifications of this preservation of total central memory CD4⁺ T lymphocytes in the vaccinated, challenged monkeys on the SIV-specific immune response. On day 203 after challenge, the total central memory CD4⁺ T lymphocytes remained better preserved in the vaccinated than in the control monkeys, and the SIV Gag-specific CD4⁺ and CD8⁺ T lymphocyte IFN-γ, TNF-α, and IL-2 responses were greater in the vaccinated than in the control monkeys (figs. S5 and S6). Interestingly, however, no difference was observed in the magnitude of the SIV neutralizing antibody responses in the vaccinated and control monkeys as late as 126 days after challenge (fig. S7). Therefore, the preservation of total central memory CD4⁺ T lymphocytes in the vaccinated monkeys was associated with preserved virus-specific T lymphocyte responses.

Finally, we evaluated the contribution of vaccine-elicited immunity to survival in this cohort of monkeys. Dividing the 16 monkeys that received Gag immunogens into halves based on the magnitude of peak vaccine-elicited SIV Gag-specific total T lymphocyte responses measured by IFN-γ Elispot assay (Fig. 4G) and IFN-γ CD4⁺ T lymphocyte responses measured by ICS assay (Fig. 4H), a survival advantage was apparent for the monkeys with the highest frequency vaccine-elicited cellular immune responses. The Elispot data also revealed an association between vaccine-elicited cellular immune responses and survival in a Cox proportional-hazards model (P 0 0.015, likelihood ratio test). Therefore, the magnitude of the immune responses generated by the vaccine was associated with survival after virus infection.

These findings suggest that vaccine protection against high levels of viral replication during only the first weeks following an AIDS virus infection may provide sufficient protection against central memory CD4⁺ T cell loss to confer a survival advantage to infected individuals. Moreover, current models of large human HIV-1 vaccine efficacy trials propose the use of set-point viral load and total CD4⁺ T lymphocyte count as surrogate markers for a beneficial vaccine effect (22, 23). It has been presumed that a lower set-point viral load or a higher set-point CD4⁺ T lymphocyte count after infection will portend a better AIDS-free survival. The results of the present study indicate that set-point viral load and total CD4⁺ T lymphocyte count may not have predictive value in this setting. Rather, the quantitation of central memory CD4⁺ T cells in a vaccine trial several months after infection may be an important immune correlate of long-term protection and predict the efficacy of an HIV-1 vaccine.