Immunogenicity of heterologous prime-boost regimens involving recombinant adenovirus serotype 11 (Ad11) and Ad35 vaccine vectors in the presence of anti-ad5 immunity

Abstract

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. A potential solution to this problem is to utilize rAd vaccine vectors derived from rare Ad serotypes such as Ad35 and Ad11. We have previously reported that rAd35 vectors were immunogenic in the presence of anti-Ad5 immunity, but the immunogenicity of heterologous rAd prime-boost regimens and the extent that cross-reactive anti-vector immunity may limit this approach have not been fully explored. Here we assess the immunogenicity of heterologous vaccine regimens involving rAd5, rAd35, and novel rAd11 vectors expressing simian immunodeficiency virus Gag in mice both with and without anti-Ad5 immunity. Heterologous rAd prime-boost regimens proved significantly more immunogenic than homologous regimens, as expected. Importantly, all regimens that included rAd5 were markedly suppressed by anti-Ad5 immunity. In contrast, rAd35-rAd11 and rAd11-rAd35 regimens elicited high-frequency immune responses both in the presence and in the absence of anti-Ad5 immunity, although we also detected clear cross-reactive Ad35/Ad11-specific humoral and cellular immune responses. Nevertheless, these data suggest the potential utility of heterologous rAd prime-boost vaccine regimens using vectors derived from rare human Ad serotypes.

FIG. 1.

Immunogenicity of rAd5, rAd35, and rAd11 vectors expressing SIV Gag. (A) Naïve C57/BL6 mice and (B) mice with anti-Ad5 immunity were immunized with 10⁹ vp rAd5-Gag, rAd35-Gag, or rAd11-Gag or a sham vector control. Gag-specific CD8⁺ T-lymphocyte responses were assessed by D^b/AL11 tetramer binding assays at multiple time points following immunization. The rAd5-Gag responses are depicted in the open circles.

FIG. 2.

Immunogenicity of low doses of rAd5, rAd35, and rAd11. Naïve mice were immunized with (A) 10⁸ vp or (B) 10⁷ vp rAd5-Gag, rAd35-Gag, rAd11-Gag, or a sham vector control. Gag-specific CD8⁺ T-lymphocyte responses were assessed by D^b/AL11 tetramer binding assays. The rAd5-Gag responses are depicted in the open circles.

FIG. 3.

Heterologous rAd prime-boost regimens. (A and B) Naïve mice and (C and D) mice with anti-Ad5 immunity were primed at week 0 with (A and C) 10⁹ vp rAd35-Gag or (B and D) 10⁹ vp rAd11-Gag and then boosted at week 4 with 10⁹ vp rAd5-Gag, rAd35-Gag, rAd11-Gag, or a sham vector control. In mice with anti-Ad5 immunity, the rAd5-Gag prime, rAd5-Gag boost regimen was also included as a negative control. Gag-specific CD8⁺ T lymphocyte responses were assessed by D^b/AL11 tetramer binding assays following the boost immunization. The rAd5-Gag boost responses are depicted in the open circles.

FIG. 4.

Heterologous DNA-rAd prime-boost regimens. (A) Naïve mice and (B) mice with anti-Ad5 immunity were primed at week 0 with 50 μg plasmid DNA vaccine expressing Gag-Pol-Nef and then boosted at week 4 with 10⁹ vp rAd5-Gag, rAd35-Gag, or rAd11-Gag. Gag-specific CD8⁺ T-lymphocyte responses were assessed by D^b/AL11 tetramer binding assays following the boost immunization. The rAd5-Gag boost responses are depicted in the open circles.

FIG. 5.

Cross-reactive Ad35/Ad11-specific immunity. Mice with (A) anti-Ad35 immunity or (B) anti-Ad11 immunity were immunized with 10⁹ vp rAd5-Gag, rAd35-Gag, or rAd11-Gag. Gag-specific CD8⁺ T lymphocyte responses were assessed by D^b/AL11 tetramer binding assays. The rAd5-Gag responses are depicted in the open circles.

FIG. 6.

Cross-reactive Ad35/Ad11-specific NAbs. Mice were immunized either (A) once or (B) twice with 10¹⁰ vp rAd5-Empty, rAd35-Empty, or rAd11-Empty. Serum was assessed for Ad5-, Ad35-, and Ad11-specific NAbs 4 weeks following immunization.

FIG. 7.

Adoptive transfer of Ad35-specific IgG. Donor mice were immunized twice with 10¹⁰ vp rAd35-Empty or saline control. IgG was purified from serum, and 500 μl was adoptively transferred to naïve recipient mice prior to vaccination with (A and B) 2 × 10⁸ vp rAd35-Gag or (C and D) 2 × 10⁸ vp rAd11-Gag. Mice that received no adoptive transfer were used as an additional control (None). Gag-specific cellular immune responses were assessed by (A and C) D^b/AL11 tetramer binding assays and (B and D) IFN-γ ELISPOT assays using pooled Gag peptides and individual AL11 and KV9 epitope peptides.

FIG. 8.

Adoptive transfer of Ad35-specific CD8⁺ T lymphocytes. Donor mice were immunized twice with 10¹⁰ vp rAd35-Empty or saline control. CD8⁺ T lymphocytes were purified from splenocytes, and 5 × 10⁷ cells were adoptively transferred to naïve recipient mice prior to vaccination with (A and B) 2 × 10⁸ vp rAd35-Gag or (C and D) 2 × 10⁸ vp rAd11-Gag. Gag-specific cellular immune responses were assessed by (A and C) D^b/AL11 tetramer binding assays and (B and D) IFN-γ ELISPOT assays using pooled Gag peptides and individual AL11 and KV9 epitope peptides.