We evaluated safety and HIV-specific immune responses in healthy adult volunteers who received a priming immunization with a 4-plasmid or 6-plasmid candidate HIV-1 DNA vaccine and subsequently received a booster immunization with a matching candidate HIV-1 rAd5 vaccine. This was a non-randomized, exploratory Phase I evaluation using a roll-over design and provided the first opportunity to boost subjects with rAd5 who had been primed in prior studies with DNA vaccines. This small group of rollover study participants allowed an early evaluation of the DNA prime-rAd5 boost vaccination concept in humans. This study focused on the detailed evaluation of vaccine-induced immune responses. While interpretations should be tempered by the relatively small study size, exploratory nature of the analysis, and use of historical controls for the rAd5 only comparison, there are a number of significant observations that merit additional investigation. Larger studies are being conducted to provide a more robust statistical assessment of general vaccine immunogenicity, and to determine how the observations made in these studies relate to vaccine efficacy.
Both components of the vaccine had been well-tolerated and were immunogenic as single agents in previous Phase I clinical trials 
. The combination vaccine was also well-tolerated, and the sequence of DNA priming and rAd5 vector boosting was far more immunogenic than either the DNA or rAd5 vaccine alone. HIV-specific T cell responses, detected by both ELISpot and ICS were present in 100% of the subjects and were 5-fold higher than in subjects immunized with rAd5 vector alone. The T cell responses were (i) biased toward CD8+
more than CD4+
; (ii) more polyfunctional than T cell responses induced by DNA alone; (iii) sustained for >6 months; (iv) directed against multiple epitopes biased toward MHC class I restriction; and (v) specific for multiple antigens (Envelope > Gag > Pol/Nef). The relative response to Envelope antigens was also similar to that seen after DNA immunization alone (EnvA
EnvB > EnvC). Antibody responses were also induced in 100% of the subjects and were >100-fold higher than in subjects immunized with a comparable dose of rAd5 vector alone. The augmented antibody response did not have significant neutralizing activity to tier 2 viruses () 
Two observations suggest that the improved response after rAd5 vector boosting was related to immunological priming by DNA. First, the pattern of the response after rAd5 vector boost of relatively dominant Env responses, intermediate Gag responses, and relatively lower Pol responses was similar to that observed after the 4-plasmid DNA immunization 
. Secondly, multivariate correlates analysis showed the response measured after DNA priming was the best predictor of IFN-γ ELISpot and CD8+
T cell ICS magnitude after rAd5 vector boosting. In addition, preclinical data and recently reported human data using a different rAd5 vector suggest that augmented HIV-specific T cell responses are not seen in subjects immunized with a second dose of rAd5 
The mechanisms underlying the benefit of prior DNA immunization are unknown. HIV-specific CD4+
T cell responses measured by ICS were persistent following DNA immunization, and their presence may have aided in the rapid expansion of the CD8+
T cell response during boosting. Interestingly, there was no apparent expansion of HIV-specific CD4+
T cells following rAd5 boosting (). Therefore, it is equally likely that the augmented CD8+
T cell responses after rAd5 vector boosting were simply a reflection of the expansion of DNA-induced CD8+
T cell responses. This conclusion is supported by the finding that CD8+
T cell responses were noted in subjects immediately after DNA priming, and that the Nef-specific responses were still detectable in some individuals more than 2 years later. Since nef
was not included in the rAd5 vector, DNA immunization alone was responsible for these responses. Of note, there was no difference in the post-boost response patterns in subjects primed with 4 mg vs. 8 mg of DNA in VRC 004, which is consistent with the primary responses measured after DNA immunization 
The use of heterologous platforms to initially prime and subsequently boost an immune response has been shown to be effective for eliciting antibody responses 
but is especially attractive for augmenting T cell responses 
. The combination of DNA priming and recombinant viral vector boosting has been shown to induce potent immune responses and protection against several pathogens, including Ebola and Malaria 
, and has also been shown to reduce virus load and delay disease progression in macaques challenged with SHIV or SIV 
. One reason for this success may be that combining two different modalities in a heterologous prime/boost regimen appears to induce responses that differ from those induced by repeated dosing of either component alone. Specifically, a recent report showed that T cell responses to an HIV candidate vaccine expressing Gag, Pol, and Nef in a heterologous DNA/rAd5 regimen were different from those induced by a homologous rAd5/rAd5 regimen in humans 
. The heterologous DNA/rAd5 induced a greater Gag-specific CD4+
T cell response than the homologous rAd5/rAd5 regimen. In addition, both the Gag-specific CD4+
T cell responses induced by DNA/rAd5 had a greater frequency of IL-2 producing cells and more phenotypic diversity in the DNA/rAd5-induced T cells than in those induced by rAd5/rAd5. The homologous rAd5/rAd5 regimen also appeared to induce a greater degree of polyfunctional T cell responses than seen in the current study following a single dose of rAd5.
Another important consequence of using heterologous vector combinations is to focus the immune response on the recombinant gene product instead of the vector. Pre-existing vaccinia immunity is known to significantly diminish the response to recombinant replication-competent and replication-defective vaccinia-derived vectors 
, and pre-existing adenovirus immunity has been reported to diminish the magnitude of immune responses induced by adenovirus vectors 
. This effect potentially limits the ability to use multiple-dose regimens of homologous vectors. DNA immunization is not subject to anti-vector immunity and has the potential to establish an expanded precursor frequency of antigen-specific cellular immune responses in the majority of subjects. Therefore, by priming with DNA or other novel vectors it may be possible to lower the threshold for vector-based boosting of immune responses. The impact on modifying the threshold above which vector-based boosting occurs may be different for antibody responses compared to T cell responses, and this question will be addressed in future studies.
T cell subsets can be defined both by function and surface phenotype; the protective efficacy of a vaccine might be determined by which subsets are elicited. As we show, many of the T cell characteristics desirable for a vaccine against HIV were induced by DNA priming and rAd5 boosting. A high proportion of antigen-specific CD4 and CD8 T cells express CD127, suggesting that they are long-lived memory T cells capable of homeostatic maintenance – substantiated by the long term stability of the response. Further, the proportion of cells that express PD-1 declined over time (and the proportion co-expressing CD57+ was relatively low), consistent with maintenance of cells that can proliferate following antigenic challenge. Finally, we observed a mixture of TCM and TEM/TEF, providing a balance of self-renewing cells and cells capable of a rapid effector response.
The combination of DNA priming and rAd5 boosting increased the polyfunctionality of the vaccine-induced T cell response (). Although the biological significance of polyfunctional T cell responses in humans is unknown, in vivo
animal model studies show that such qualitative immunological differences can impact upon vaccine-elicited protection 
. Polyfunctional cells are thought to be more efficacious for two reasons: first, they bring to bear, on each target cell, a wider range of effector functions simultaneously (as opposed to requiring the simultaneous recruitment of multiple effector cells). Second, each polyfunctional cell expresses as much as an order of magnitude more of certain effector functions (such as IFN-γ or TNF; Figure S1
). Ultimately, the value of vaccine-induced immune responses, and whether they achieve sufficient magnitude, functionality, breadth, and durability in the right location to impact the outcome of HIV infection, can only be determined in the setting of a clinical study evaluating efficacy.
An efficacious T cell vaccine against HIV will need to cover the extensive sequence diversity inherent in the current epidemic. While there are many ways to evaluate the breadth of a T cell response 
, we chose to take a rigorous approach by mapping individual epitopes targeted by the vaccine-induced response, and evaluating the ability of the epitope-specific T cells to recognize circulating clade variants. We found that there was wide variation in the number of epitopes targeted (1–10 epitopes, ), most were in Env and Gag (); and the T cell responses against those epitopes recognized all or most major clade variants (). It is unclear whether it is better for a vaccine to elicit multiple strain-specific T cell responses as opposed to fewer broadly cross-reactive responses to selected epitopes in which mutations incur a fitness cost to the virus. It is possible that the inclusion of three separate Env antigens in the vaccine focused the T cell response to common sequences within the three antigens. This could have decreased the number of epitopes targeted, but increased the likelihood of cross-clade recognition by the response.
Epitope-specific responses may vary in their in vitro
and in vivo
impact upon HIV replication 
; a vaccine would ideally elicit responses to those epitopes known to be associated with better viral control. Most prominent among these is the HLA B57-restricted response to GagTW10 
. It is encouraging that this response was elicited by the vaccine in both volunteers who express HLA B57 (). Indeed the GagTW10 functional avidity and pattern of clade variant recognition in these two volunteers was similar to what has been reported in HLA B57-expressing long-term non-progressors ( and unpublished results). The basis for the relative response hierarchy between antigens (EnvA
EnvB > Gag
EnvC > Pol) is not known.
In the “Step” study (HVTN 502) evaluating the efficacy of repeated homologous boosting with the Merck rAd5 vector expressing Gag, Pol, and Nef, it was found that induction of Gag- and Pol-specific CD8+
T cells was not sufficient to prevent HIV infection or result in lower set-point viral load levels in men who became infected despite vaccination. Surprisingly, in men who were uncircumcised or had pre-existing neutralizing antibodies against Ad5, the repeated homologous rAd5 immunization increased the risk of HIV infection above that of placebo recipients 
. The underlying biological mechanism for increased infection rates is unknown, but the results have called into question the potential role for rAd5 vectors in particular, and the value of vaccine-induced T cell mediated immunity for HIV in general. In contrast, a Phase III study (RV144) evaluating a recombinant canarypox vector expressing Env, Gag, and parts of Pol and Nef (vCP1521) in combination with a subunit glycoprotein (rgp120) in 16,402 volunteers in a general population cohort in Thailand showed a 31.2% reduction in acquisition 
. The failure of the Merck rAd5 vector and partial success of the ALVAC/rgp120 prime-boost approach has highlighted the need for a greater depth of understanding of vector biology, as well as the importance of ongoing discovery efforts to define basic aspects of HIV immunity and pathogenesis 
Limitations and Conclusions
The major limitation of this study was its size. Only 14 subjects were evaluated by this intensive approach. The analysis required a large number of PBMCs and apheresis of subjects at key time points. The significant laboratory and personnel resources required also limited the study size. Another limitation was that boosting of subjects previously primed with DNA was opportunistic. Therefore, the study was not randomized and the prime-boost interval was variable. The interval was longer than that utilized in subsequent randomized trials.
In light of the concerning outcome of the Step study and encouraging results from the RV144 Phase III trial, it is important to expand our understanding of effector T cell function as it relates to HIV immunity. It will be critical to develop more sophisticated assays for future efficacy trials to assure that antibody and T cell functions associated with either favorable or adverse outcomes can be defined. Better assays are needed to assess proliferative capacity and kinetics of response, breadth and depth of response, functionality of response including both cytolytic and noncytolytic virus suppression activity, and localization of immune responses beyond just the blood compartment. These approaches in combination with more detailed assessment of antibody function, host genetics, breakthrough viral sequences, and a systems biology approach to analyze the transcriptional and proteomic profile associated with HIV immunity should be applied to future efficacy evaluations in humans to advance the goal of vaccine development.
The 6-plasmid DNA/rAd5 vector prime-boost combination has recently been evaluated in larger multicenter, international studies. Based on preclinical data showing a survival benefit in SIV-challenged macaques, favorable immunogenicity profile with different specificity and composition than that induced by the Merck vaccine, favorable safety profile in clinical studies to date, design features of the rAd5 vector backbone that differ from the Merck rAd5 vector, and the different antigen content and vaccine schedule, the heterologous prime-boost combination of DNA/rAd5 is being evaluated in a Phase II test-of-concept efficacy trial designed to determine whether the frequency, magnitude, functionality, breadth and durability of the vaccine-induced T cell response is sufficient to impact HIV-1 infection and/or disease progression.