Background:  The high prevalence of pre-existing anti-Ad5 immunity, particularly in the developing world, may represent a critical limitation of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1. We have recently reported that Ad5-specific neutralizing antibodies are primarily directed against the Ad5 hexon protein, suggesting the potential utility of hexon modification strategies to develop rAd5 vectors that circumvent anti-Ad5 immunity.

Methods:  We constructed novel chimeric rAd5 vectors in which the 7 short hypervariable regions (HVR) on the surface of the Ad5 hexon protein were replaced with the corresponding HVR from the rare adenovirus serotype Ad48 (rAd5HVR48). C57/BL6 mice and rhesus monkeys were pre-immunized twice with rAd5-Empty to induce high levels of anti-Ad5 immunity (serum Ad5-specific neutralizing antibody titers of 8192 to 16,384) and were then vaccinated with 10⁶ to 10¹¹ vp rAd5 or rAd5HVR48 vectors expressing SIV Gag. Gag-specific cellular and humoral immune responses were assessed by tetramer binding assays, ELISpot assays, intracellular cytokine staining assays, and ELISA.

Results:  In naïve mice, rAd5-Gag and rAd5HVR48-Gag vectors proved comparably immunogenic, even when inoculated at dose-limiting levels. Importantly, in mice with high levels of anti-Ad5 immunity, the immunogenicity of rAd5-Gag vectors was dramatically abrogated at all doses tested, whereas the immunogenicity of rAd5HVR48-Gag vectors was not detectably suppressed, demonstrating that rAd5HVR48 vectors effectively evaded anti-Ad5 immunity. Then, 12 Mamu-A*01-negative rhesus monkeys, either with or without anti-Ad5 immunity, were inoculated with a single high dose of 10¹¹ vp of each vector. In monkeys with Ad5-specific neutralizing antibody titers comparable with those found in sub-Saharan Africa, Gag-specific cellular immune responses elicited by rAd5-Gag vectors were substantially suppressed by more than 4-fold, whereas responses elicited by rAd5HVR48-Gag vectors were not compromised.

Conclusions:  These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are highly focused on epitopes within the 7 short hexon HVR in both mice and nonhuman primates. Moreover, these studies show that novel recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. HVR-chimeric rAd5 vectors may prove useful as second-generation rAd vector-based vaccines for HIV-1 and other pathogens.