The specificity and immune effects of the rAd5 neutralizing antibodies of subjects receiving DNA/rAd vectors have been analyzed in this study. We found that the molecular target of neutralization differs depending on whether Ad5 immunity is generated from natural infection or from vaccination with replication-defective viral vectors. Antibodies generated by natural infection are directed primarily to fiber components, while vector exposure elicits responses primarily to capsid proteins other than fiber. Injection of vector into Ad5 seropositive individuals elicits antibodies to both fiber and capsid in nearly all subjects. The presence of antibodies to fiber in naturally infected individuals reduces vaccine-induced immunity to HIV-1 Gag, whereas the response to Env is not substantially affected, although the vaccines generated detectable immune responses to both Gag and Env in Ad5 seronegatives. This observation may be caused by differential trafficking and processing of an aggregated protein in contrast to a transmembrane glycoprotein that could affect antigen presentation. It is also possible that Ad5 immune effects are underestimated in this study because of the DNA immunization.
As shown in this study and documented elsewhere, Ad5 neutralizing antibodies are prevalent in humans. The seroprevalence seems to vary among populations from different continents, and up to 85% of sera can be seropositive for Ad5 (13
). The only study prior to the one reported here that had assessed the contribution of individual capsid-specific antibodies to viral neutralization in humans from natural infection with Ad did not describe the frequency of samples positive for specific capsid proteins (18
). Instead, a population median titer was presented, which is an assessment of avidity, not frequency. In addition, there was no statistical significance of the apparent titer differences presented, and supporting data were only from immunization of mice with rAd5. Taking the findings together, it is unclear whether the median titers across the populations of human samples could be extrapolated to determine immunodominant targets for neutralization. Thus, based on the frequency assessment in the study presented here, neutralizing antibodies to the fiber protein are more common than antibodies to hexon. However, the frequency of hexon antibodies was still significant and must be taken into account when designing the next generation of rAd vaccine vectors.
Chimeric rAd5-based vectors have been demonstrated to overcome vector neutralization (5
). Our results support the use of chimeric Ads with non-Ad5 fibers in combination with other non-Ad5 capsid proteins. Modification of capsid while retaining an Ad5 fiber alone, as is done with mutant hypervariable region Ad5 vectors, is unlikely to fully overcome preexisting neutralizing antibodies from natural Ad infection. The results also emphasize the contribution of anti-fiber antibodies to neutralization generated by natural viral exposure, consistent with previous studies showing the importance of anti-fiber antibody and its synergy with anti-penton antibody for neutralization (7
). These antibodies are likely targeted to the fiber knob (5
). Since the modification of the hexon protein has been shown to affect the distribution of cellular transduction in vivo (20
), it will be important to assess the anti-Ad and anti-transgene immune responses to capsid-modified rAd vaccine vectors.
Vaccination of naïve volunteers with rAd5 rarely generated neutralizing antibodies targeted solely to fiber. This result may reflect differences in the exposure to fiber and hexon in natural infection compared to vaccination. During natural infection, new fiber molecules are transcribed from viral genes in excess of the amount of fiber incorporated into virions, and they may be released from infected cells that undergo cytolysis. The significant late protein synthesis, particularly of fiber, from infected cells not only increases the fiber antigen content, but also exerts biological effects on intercellular adhesion or signaling through cellular receptors that will result in a very different set of immune and inflammatory responses than in E1,E4-deleted vectors. In contrast, vaccination with the replication-incompetent rAd5 vector would result in exposure to smaller amounts of fiber, as the viral proteins introduced in vivo would be restricted to preformed virus, the incoming replication-incompetent virions of the E1,E4 replication defective vector. An Ad virion is composed of 36 fiber protein monomers and 720 hexon protein monomers (15
), and vaccine recipients would be expected to respond better to the more abundant hexon protein. Viral replication in vivo may also affect the presentation of epitopes to B cells and stimulation of helper and memory T cells. Vaccination with rAd5 vectors in volunteers previously seropositive to Ad5 generated sera that targeted all capsid components, including fiber, showing the mobilization and recall of more diverse B-cell target repertoires to fend off the vaccine vector.
Many studies have used intramuscular injection of rAd5 vectors to generate Ad5 immunity in an effort to mimic natural exposure to infectious virus in animal models. Our study shows that vaccination with rAd vectors generates a profile of neutralizing antibodies different than natural infection, presumably due to the differences of route, amount of viral exposure, and capability of viral replication. Such differences call for caution in interpreting animal model data from studies of vector preexposure with respect to their effect on vaccine immunogenicity. In fact, these studies better reflect the effect of repeated intramuscular administration of vector rather than the effect of prior natural exposure.
While some data are available regarding the effect of preexisting neutralizing antibodies on the immunogenicity of rAd5-based vectors (4
), the specificity of these antibodies and correlation with immune stimulation has not been analyzed. In previous trials, there was a trend toward suppression of immunity in the presence of preexisting Ad5 neutralizing antibodies, especially for HIV-1 Gag-specific enzyme-linked immunospot assay responses. Here, we show that such an effect blunted both CD4+
T-cell Gag responses in seropositive volunteers, but we could not detect a significant effect on Env responses after DNA prime/rAd5 boost vaccination. As the response was not completely abolished in seropositive populations (4
), it is likely that the effective dose of vector was decreased but not completely inactivated. It is tempting to suggest that efficient HIV Gag antigen presentation may need a higher level or longer time of antigen expression than other antigens. In support of this hypothesis, it has been shown that the immunogenicity of HIV-1 Gag generated by DNA vaccination can be more dependent on the formation of viral-like particles than other antigens (1
). The results shown here suggest that rAd5-based vectors, especially Gag-encoding vectors, are less likely to stimulate HIV-1 immunity in an Ad5 seropositive population; such a suppressive effect may also have contributed to the lack of rAd5 vaccine efficacy in the recent STEP trial. These data also document qualitative differences in immunity between Ad5 infection and vaccination that may affect the responses to subsequent rAd5 vector exposure. Possibly, these responses could also differentially affect the proinflammatory responses to vaccination that might affect susceptibility to infection, a possibility raised in the STEP study. These results also document a biological basis for differences between responses to immunization with rAd5 vaccines in Ad5 naïve and seropositive subjects that are relevant to proposed DNA/rAd5 efficacy studies; such studies should be preferably performed in Ad5 seronegative subjects.
In summary, this study highlights differences in the specificities of neutralizing antibodies generated by natural infection compared to vaccination. Furthermore, we have shown that anti-Gag cellular immune responses are more sensitive to the effects of preexisting neutralizing antibody than those directed against Env when using these vaccine formulations. The identification of the Ad5 vector proteins targeted by human neutralizing antibodies also suggests that next generation chimeric Ad vectors be developed without rAd5 hexon and fiber, which could avoid preexisting immunity and increase the likelihood of eliciting cellular immunity to HIV-1 antigens encoded in the vaccine.