This study evaluated differences between homologous priming and boosting with an rAd5 vaccine and heterologous DNA prime/rAd5 boost vaccination. Homologous boosting greatly enhanced Env-specific antibody responses, but did not increase T-cell responses. Despite limited post-prime immune responses, DNA priming significantly enhanced the magnitude of post-boost Env-specific antibody and CD4+ T-cell responses and influenced the cytokine and memory marker profiles of the boosted T-cell responses.
In addition to identifying vaccine-induced T cells based on antigen-specific ex vivo stimulation and cytokine production, we also directly identified activated T cells ex vivo. Recombinant Ad5, but not DNA, induced large percentages of activated CD4+ and CD8+ T cells shortly after immunization, and, consistent with a secondary immune response, the peak response was attained more quickly following the boost (one week) compared with the rAd5 prime (two to three weeks). Although few DNA-Ad5 individuals had detectable post-prime increases in activated cells, most had accelerated post-boost kinetics similar to the Ad5-Ad5 group for both CD4+ and CD8+ T cells, with responses peaking by one week, suggesting a secondary immune response. Thus DNA is likely inducing undetected HIV-specific T-cell responses. In our trial this was especially notable for CD8+ T cells, since no participants had a detectable ICS response at the primary immunogenicity timepoint following DNA vaccination. Evaluation of immunogenicity and potential benefits of DNA vaccination should therefore not be based solely on ELISpot or ICS assays measured directly after DNA vaccination, but should include these or alternate measures following boost.
Homologous priming and boosting is a common strategy for many licensed vaccines, which likely are effective due to induction of humoral immunity that benefits from repeated immunization with the same immunogen. However, the potential benefits of homologous immunization for T-cell responses are not well defined. In our study, the magnitude of antibody responses to gp140 and gp41 were increased significantly after rAd5 boost; conversely, a second vaccination with rAd5 did not enhance T-cell responses, potentially due to anti-vector immunity induced by the first vaccination. However, the divergent effects on cellular and humoral immune responses indicate a more complex mechanism than simply impairing expression of the HIV proteins from the rAd5 boost. Indeed, antigen presentation may be affected by Ad5-specific neutralizing antibodies forming immune complexes with the vector, resulting in an effect on dendritic cell maturation(27
In contrast to homologous rAd5 vaccination, rAd5 boosting of DNA resulted in an increased response rate and magnitude compared with the response detected after the DNA prime, most notably for Env-specific CD8+ T cells. At six months post-boost, the proportion of Env-specific CD8+ T cells expressing CD57 and granzyme B also tended to be higher in the DNA-Ad5 group. CD57 expression is associated with terminally-differentiated T cells with high cytotoxic effector potential, which are armed for a quick response to pathogens(24
). Induction of these cells by a vaccine is likely beneficial to control viral replication at its earliest stage, but the cells must be maintained over time. Although CD57 is proposed to be a marker of replicative senescence(24
), these cells are capable of rapid expansion(29
). We also detect these cells at six months post-boost, indicating their potential for long-term maintenance.
The CD8+ T cells from both treatment groups had similar CD45RA expression, with nearly all post-boost Env-specific cells expressing CD45RA. Non-naïve CD45RA+ T cells have been classified as effector, rather than memory, T cells(23
), but our data and other studies indicate they can still contribute to long-term memory. The majority of CD8+ T cells induced by vaccinia and yellow fever vaccines, known to produce long-lived protection, highly express CD45RA and persist and maintain proliferative ability for many years(22
). In our trial, vaccine-induced CD8+ T cells were detected six months post-boost and thus may similarly contribute to long-term memory.
The magnitude of the CD4+ T-cell response at four weeks and six months post-boost was similar for both treatment groups, but the cytokine profile of Env-specific CD4+ T cells differed at the six-month timepoint. A subset of DNA-Ad5 individuals had high percentages (>50%) of Env-specific T cells that co-produced IFN-γ and IL-2. This might be beneficial since polyfunctionality is linked to mounting a more effective host response(19
). Thus, DNA priming can favorably influence the long-term vaccine-induced cytokine profile, at least in some individuals.
Except for Env-specific CD8+ T-cell responses, a single dose of rAd5 resulted in the highest response rates not only when compared with a second rAd5 dose, but also when compared with rAd5 administered after DNA prime. The dampening of rAd5-induced responses to some HIV proteins following DNA vaccination is possibly due to a DNA-induced suppressive effect. This effect may not be due to DNA as the method of vaccination, but rather due to the design of the DNA-encoded immunogens. The three Env proteins (clades A, B and C) are encoded on different plasmids, each including a secretory sequence. Gag, Pol and Nef are encoded on one plasmid. The NIH Vaccine Research Center (VRC) has recently developed a new DNA vaccine that encodes Gag, Pol and Nef on separate plasmids(31
); trials with this DNA vaccine also include a third DNA vaccination. Those trials do not demonstrate a lower post-boost Gag-specific CD4+ T-cell response rate like we observed(32
), suggesting that these changes may have alleviated any potential suppressive effect for Gag, although not for Pol. Finally, our trial was restricted to Ad5-seronegative participants. In contrast to potential suppression, DNA priming of rAd5 is reported to mitigate a potential decreased response to rAd5 vaccination in Ad5-seropositive individuals(32
The Step Study, a prior efficacy trial of an rAd5-vectored vaccine, failed to reduce HIV infection or reduce HIV viral load(3
). Instead, early analyses revealed an increased incidence of HIV infection in Ad5-seropositive vaccinees. Concern was raised as to whether the vaccine increased the number of activated CCR5+ T cells, thus increasing the availability of target cells susceptible to infection. In our study (restricted to Ad5-seronegative participants), we found an increase in activated T cells, and many activated CD4+ T cells expressed CCR5 (data not shown). However, this increase was short-lived, at least as measured in blood, suggesting only a brief period of increased risk, unless vaccine-induced activated T cells persist at mucosal sites. Although kinetics indicate that the majority of activated cells are likely to be HIV-specific, some may be vector-specific. If so, differences in the design of the rAd5 vectors used in the two trials may affect vector persistence and thus persistence of activated cells. In particular, our VRC rAd5 vaccine has additional Ad5 gene deletions that may decrease vector persistence. Supporting this, a recent study demonstrated that the VRC rAd5 vaccine did not induce expansion of vector-specific CD4+ T cells or increase the activation state of Ad5-specific CD4+ T cells(33
Our data demonstrate no T-cell benefit for repetitive boosting with the same viral vector vaccine. Instead, a single dose of rAd5 induced the highest response rates and magnitudes for many HIV proteins, regardless of DNA priming. However, DNA priming altered the character of the post-boost T-cell response, even without inducing a detectable T-cell response itself. This was most notable for Env, which as noted above has better expression than the other proteins in the DNA vaccine used in this trial(5
). The newer DNA vaccine may extend the benefits of DNA priming to the other proteins. It should also be noted that there are other methods for improving the immunogenicity of DNA vaccines. One is through co-administration of DNA that encodes for cytokines that can potentially serve as adjuvants (34
). Another is through administration by electroporation. Both of these methods may further enhance the priming potential for DNA vaccination (36
Unlike the T-cell response, homologous boosting with the same viral vector dramatically increased Env-specific antibody responses. These antibodies bind to Env but do not neutralize HIV. The recent RV144 trial suggests potential benefits to inducing non-neutralizing antibodies, as nearly all vaccinees developed Env-specific binding antibodies with minimal neutralizing activity(4
). This trial also highlights the potential utility of adding recombinant protein as one component of a vaccine regimen. The protective effect in RV144 was likely antibody-mediated since vaccination was associated with decreased HIV acquisition but not viral control in those who became infected. Our study, considered in the context of these other trials, suggests that vaccine regimens may need to include both heterologous vaccine modalities to optimize T-cell responses and homologous boosting to increase antibody responses.