Neutralizing antibodies and CD8+ T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors

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 vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. Ad5-specific neutralizing antibodies (NAbs) are thought to contribute substantially to anti-Ad5 immunity, but the potential importance of Ad5-specific T lymphocytes in this setting has not been fully characterized. Here we assess the relative contributions of Ad5-specific humoral and cellular immune responses in blunting the immunogenicity of a rAd5-Env vaccine in mice. Adoptive transfer of Ad5-specific NAbs resulted in a dramatic abrogation of Env-specific immune responses following immunization with rAd5-Env. Interestingly, adoptive transfer of Ad5-specific CD8(+) T lymphocytes also resulted in a significant and durable suppression of rAd5-Env immunogenicity. These data demonstrate that NAbs and CD8(+) T lymphocytes both contribute to immunity to Ad5. Novel adenovirus vectors that are currently being developed to circumvent the problem of preexisting anti-Ad5 immunity should therefore be designed to evade both humoral and cellular Ad5-specific immune responses.

FIG. 1.

Anti-Ad5 immunity suppresses immune responses elicited by rAd5-Env. Groups of mice (four mice per group) in either the absence (A) or presence (B) of anti-Ad5 immunity were immunized with 10⁹ or 10⁷ vp of rAd5-Env vaccine. Vaccine-elicited cellular immune responses were assessed by D^d/P18 tetramer binding to CD8⁺ T lymphocytes. Anti-Ad5 immunity was induced by preimmunizing mice with 10¹⁰ vp of rAd5-Empty 4 weeks prior to rAd5-Env immunization.

FIG. 2.

Ad5-specific humoral and cellular immune responses. Naive mice or mice preimmunized with one or two injections of 10¹⁰ vp of rAd5-Empty were assessed for Ad5-specific NAb titers (A) and Ad5-specific IFN-γ ELISPOT responses (B). ELISPOT assays were performed using splenocytes incubated with Ad5-infected or uninfected syngeneic 3T3 stimulator cells.

FIG. 3.

Control adoptive transfer of splenocytes or serum from naive mice. A total of 5 × 10⁷ splenocytes or 500 μl of serum from naive donor mice was adoptively transferred to groups of recipient mice (four mice per group) prior to immunization of the recipient mice with 10⁸ vp of rAd5-Env. Vaccine-elicited immune responses were assessed by D^d/P18 tetramer binding to CD8⁺ T lymphocytes (A), Env peptide pool and P18 epitope peptide-specific IFN-γ ELISPOT assays (B), and Env-specific ELISAs (C).

FIG. 4.

Adoptive transfer of splenocytes or serum from mice with low levels of anti-Ad5 immunity. A total of 5 × 10⁷ splenocytes or 500 μl of serum from donor mice preimmunized with one injection of 10¹⁰ vp of rAd5-Empty was adoptively transferred to groups of recipient mice (four mice per group) prior to immunization of the recipient mice with 10⁸ vp of rAd5-Env. A group of mice with active anti-Ad5 immunity was included as a control. Vaccine-elicited immune responses were assessed by D^d/P18 tetramer binding to CD8⁺ T lymphocytes (A), Env peptide pool and P18 epitope peptide-specific IFN-γ ELISPOT assays (B), and Env-specific ELISAs (C).

FIG. 5.

Adoptive transfer of splenocytes or serum from mice with high levels of anti-Ad5 immunity. A total of 5 × 10⁷ splenocytes or 500 μl of serum from donor mice preimmunized with two injections of 10¹⁰ vp of rAd5-Empty was adoptively transferred to groups of recipient mice (four mice per group) prior to immunization of the recipient mice with 10⁸ vp of rAd5-Env. A group of mice with active anti-Ad5 immunity was included as a control. Vaccine-elicited immune responses were assessed by D^d/P18 tetramer binding to CD8⁺ T lymphocytes (A), Env peptide pool and P18 epitope peptide-specific IFN-γ ELISPOT assays (B), and Env-specific ELISAs (C). (D) Ad5-specific NAb titers of these mice were assessed before and after the adoptive transfer.

FIG. 6.

Adoptive transfer of purified IgG from mice with high levels of anti-Ad5 immunity. Serum or purified IgG from naive donor mice or donor mice preimmunized with two injections of 10¹⁰ vp of rAd5-Empty were adoptively transferred to groups of recipient mice (four mice per group) prior to immunization of the recipient mice with 10⁸ vp of rAd5-Env. (A) Vaccine-elicited immune responses were assessed by D^d/P18 tetramer binding to CD8⁺ T lymphocytes. (B) Ad5-specific NAb titers of these mice were assessed before and after the adoptive transfer.

FIG. 7.

Adoptive transfer of purified CD8⁺ T lymphocytes from mice with high levels of anti-Ad5 immunity. Purified CD8⁺ T lymphocytes [CD8 (+)] or splenocytes depleted of CD8⁺ T lymphocytes [CD8 (−)] from donor mice preimmunized with two injections of 10¹⁰ vp of rAd5-Empty were adoptively transferred to groups of recipient mice (four mice per group) prior to immunization of the recipient mice with 10⁸ vp of rAd5-Env. A group of mice with active anti-Ad5 immunity was included as a control. Vaccine-elicited immune responses were assessed by D^d/P18 tetramer binding to CD8⁺ T lymphocytes (A), Env peptide pool and P18 epitope peptide-specific IFN-γ ELISPOT assays (B), and Env-specific ELISAs (C). (D) Ad5-specific NAb titers of these mice were assessed before and after the adoptive transfer. This study was repeated three times.