From our comprehensive immunologic analysis of the Step trial, several key findings emerge that bear relevance to the trial outcome and future HIV vaccine design. We demonstrate that the MRKAd5 HIV-1 gag/pol/nef vaccine elicited a higher CD8+ T cell response rate and magnitude than that reported for any of the candidate immunization regimens tested over the past 15 years (1
), although immunologic assays have changed considerably over this period of time. Furthermore, pre-existing Ad5 immunity substantially influenced responses to the vaccine antigens. The potency of HIV-specific CD8+ T cells and the antiviral cytokines they elaborated in vaccinated cases were similar to their matched non-cases. These findings suggest two possible explanations for the disappointing trial results: 1) the characteristics of T cell immunity that might afford HIV-1 protection must be more broadly reactive or qualitatively different than those elicited by this vaccine, or 2) immune responses mounted by T cell-based vaccines alone will not be sufficient to protect against HIV infection or disease. We view that, before concluding the latter, we must exclude the possibility of the former as feasible both in the Step study and future related preclinical and clinical trials.
Since 77% of vaccinated cases generated HIV-specific T cells prior to infection, obviously their mere presence was not sufficient for protection. The first consideration is that the magnitude of response was too low, particularly if a substantial proportion of effectors must migrate to mucosal sites. In , approximately 0.5–1% (in some up to 12%) of circulating CD8+ T cells were HIV-specific at a peak time point prior to infection in cases. Conceivably, a threshold level may be necessary following immunization to provide a recall response that can efficiently control early bursts of replication following HIV exposure. The median percentage of CD8+ T cells induced by the MrkAd5/HIV-1 vaccine is 43% lower than that observed in our investigations of CD8+ T cells in Seattle long-term non-progressors evaluated with the same assay using peptide reagents covering the three gene products (manuscript in preparation). Whether this quantitative difference is a major contributor to the vaccine’s lack of efficacy is uncertain, and may be particularly relevant in persons with pre-existing Ad5 immunity whose magnitudes and response frequencies were lower than those without previous immunity. However, the extrapolation of immune factors associated with control of chronic HIV-1 infection may be misleading as a model for predicting risk of infection, and the magnitude of IFN-γ-secreting T cells has not been shown to correlate with contemporaneous viral load in acute infection (19
). Hence, to inform future T cell-based HIV vaccine design, non-human primate vaccine studies could provide additional insight into whether a threshold quantity of CD8+ T cells exists, regardless of specificities, in order to substantially lower viral load during acute infection.
One leading hypothesis for the lack of efficacy of the MrkAd5/HIV-1 vaccine is that HIV-specific CD8+ T cells generated in cases lacked sufficient breadth to recognize epitopes within the transmitting viral strains. Thus, even if the quantity of HIV-specific CD8+ T cells was adequate, their specificities may have been too narrowly focused on a few epitopes that are distinct from the corresponding sequences within the transmitting strains. (21
). Thus, vaccines that induce CD8+ T cells that recognize multiple diverse epitopes, as demonstrated in some SIV vaccine models (23
), may hold more promise in containing the spread of the heterologous transmitting HIV-1 strain. Our comprehensive evaluation of vaccine-induced T cell determinants and transmitting viral sequences in the cases will shed light on this possibility.
An alternative explanation for the lack of vaccine efficacy is that the antiviral function of the T cell effectors was incapable of controlling viremia, particularly at mucosal sites. We demonstrated that most vaccine-induced CD8+ T cells produced IFN-γ alone or in combination with TNF-α; both cytokines have antiviral activities that can mediate clearance of some infections (25
). Only a small percentage of HIV-specific CD8+ T cells expressed IL-2. Of note, in natural HIV-1 infection, “protective” CD8+ T cells have been associated with antigen-specific proliferation (27
), commonly secrete IL-2 (28
) and have been linked to perforin expression (30
). The proliferative capacity and expression of cytolytic granules will be evaluated in the vaccine-induced CD8+ T cells to determine the possibility of functional impairment. Further, the possibility that a skewed functional profile may have resulted from repeated immunizations is also a concern, one that cannot be addressed in the Step study but can in future clinical trials by varying vaccine dose.
One longstanding concern has been that immunization with MrkAd5 HIV-1 gag/pol/nef vaccine alone may not optimally prime CD4+ helper cells, which are important in maintaining long-term antiviral CD8+ T cell memory. Here we found that the vaccine elicited HIV-specific CD4+ T cells with a Th1-type cytokine profile, similar to those memory CD4+ T cells associated with vaccine-mediated protection in animal models and in successful control of HIV-1 and other chronic infections ((31
) and reviewed in (32
)). In the Step study, approximately 85% of CD4+ T cells secreted IL-2; of these, approximately two-thirds also produced TNF-α and/or IFN-γ. However, the vaccine induced HIV-specific CD4+ T cell responses in just 41%, and only 31% mounted both CD4+ and CD8+ HIV-specific T cells after the full immunization series. Importantly, both cases and non-cases mounted similar response rates and magnitudes. These findings suggest that suboptimal CD4+ helper responses are unlikely to explain the study outcome in all cases. Moreover, such an effect may more likely impact the durability of efficacy, which may be partially addressed in long-term follow up of the study. Of note, other candidate regimens under evaluation, including priming with HIV DNA followed by boosting with recombinant Ad5 or pox vectors containing HIV-1 inserts, typically induce higher CD4+ T helper response rates, which has been a leading argument for advancing these regimens to larger scale trials.
Our exploratory studies to understand why the vaccine may have increased infection risk in the Step study did not provide major insights. Certainly, HIV-specific CD4+ T cells were elicited in 46% of cases prior to infection, and while this has been considered a desired effect, these may preferentially serve as susceptible target cells for HIV-1 infection, as has been reported in HIV-infected individuals (33
). This possibility is supported by a previous study demonstrating enhanced SIV replication and disease progression in non-human primates receiving a varicella-zoster virus vaccine expressing SIV envelope (34
). However, enhanced infection resulting from vaccine-induced SIV- or HIV-specific CD4+ T cells has not been observed in the numerous published nonhuman primate SIV vaccine studies including those involving adenovirus-based candidates or in other clinical HIV vaccine efficacy trials, but whether this possibility was considered or adequately addressed is unclear. Two findings perhaps require further evaluation. One, the Ad5-specific T cell response rates were lower in the cases than the non-cases, suggesting that these cells may have trafficked to mucosal sites, a process known to occur in natural infection, and thus increased the number of susceptible CD4+ T cell targets for HIV. Additionally, circulating CCR5-expressing activated CD4+ T cells were more abundant in persons with high Ad5 titers when assessing cryopreserved PBMC, which may have increased target cell susceptibility to HIV-1 following exposure at mucosal sites. While detectable levels of these cells in blood were not increased in the vaccine arm in comparison to the placebo arm, it remains unclear what may occur in tissue compartments at the site of HIV-1 infection. To address this possibility, studies are planned to examine lower GI tissue and foreskin after immunization for enhanced T cell activation.
The Step study outcome highlights the enormous challenges that lie ahead in developing an efficacious HIV vaccine. One notable issue is defining the immunologic responses that can better predict vaccine efficacy. Although some leads have emerged from examining correlates of immune protection with other efficacious vaccines and with successful control of chronic HIV-1 infection, the immune profile that will provide the most valuable protective response against HIV infection remains an enigma. While the validated assays employed here are a substantial improvement over traditional assays, we recognize that these may be insufficient in identifying the immunologic properties that most closely associate with an immune correlate for protection against mucosal HIV infection. Some key analyses are pending, and at a minimum, measuring the ability of CD8+ T cells to proliferate and to suppress HIV-1 replication is an important next step. In addition, a more thorough understanding of the total effector responses through transcriptional microarray and proteomics also takes priority for available stored specimens. Clues may come from comprehensive analysis of the protective immune correlates in the live attenuated SIV vaccine model in rhesus macaques, but in future clinical vaccine trials we must adopt multiple exploratory studies to gather leads on the response patterns that will be useful to measure. Further analyses of immune responses as predictors of the HIV-1 infection risk are being pursued, including survival analyses those that use the time to HIV-1 infection, and it will be important to verify findings reported here as more cases accrue in the study.
Obviously, an efficacious HIV vaccine must afford protection against heterologous virus, and the enormous variability of HIV-1 creates a major hurdle in designing a vaccine that can induce a sufficiently broad response to permit recognition (35
). This barrier may be one that ultimately compromises the effectiveness of T cell-based vaccines. Future analyses of the Step study samples will reveal the extent of coverage the MrkAd5/HIV-1 vaccine provided, which will guide improved vaccine designs. Strategies hold promise that improve T cell breadth of relevant epitopes using HIV-1 inserts that provide enhanced coverage of circulating strains, such as more centralized or even mosaic HIV inserts (38
). Further, optimizing the functional antiviral responses of the T cells elicited, based on findings from more sophisticated genomics and proteomics approaches (40
), may improve the chances for success in achieving long-term antiviral CD8+ T cell memory against HIV-1 infection. Faced with an epidemic that will be best halted by an effective vaccine, there is no better time to channel knowledge from data, not hindsight or opinion, into careful planning for the next steps in the search for a preventive HIV vaccine.