Immunogenicity of recombinant fiber-chimeric adenovirus serotype 35 vector-based vaccines in mice and rhesus monkeys

Abstract

Preexisting immunity to adenovirus serotype 5 (Ad5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. A potential solution to this problem is to utilize rAd vectors derived from rare Ad serotypes, such as Ad35. However, rAd35 vectors have appeared less immunogenic than rAd5 vectors in preclinical studies to date. In this study, we explore the hypothesis that the differences in immunogenicity between rAd5 and rAd35 vectors may be due in part to differences between the fiber proteins of these viruses. We constructed capsid chimeric rAd35 vectors containing the Ad5 fiber knob (rAd35k5) and compared the immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing simian immunodeficiency virus Gag and HIV-1 Env in mice and rhesus monkeys. In vitro studies demonstrated that rAd35k5 vectors utilized the Ad5 receptor CAR rather than the Ad35 receptor CD46. In vivo studies showed that rAd35k5 vectors were more immunogenic than rAd35 vectors in both mice and rhesus monkeys. These data suggest that the Ad5 fiber knob contributes substantially to the immunogenicity of rAd vectors. Moreover, these studies demonstrate that capsid chimeric rAd vectors can be constructed to combine beneficial immunologic and serologic properties of different Ad serotypes.

FIG. 1.

Construction of rAd35k5 vectors. (A) rAd35k5 vectors were constructed by replacing the Ad35 fiber gene with a chimeric fiber gene consisting of the Ad35 tail, Ad35 shaft, and Ad5 knob. Genetic organization of the Ad35 genome is shown (ITR, E2, E3, E4, L1-L3, L4, L5). Receptor usage was determined by assessing the abilities of rAd5, rAd35k5, and rAd35 vectors expressing SIV Gag to infect cells transfected with (B) the Ad5 receptor CAR or (C) the Ad35 receptor CD46. Transgene expression was assessed by intracellular expression of SIV Gag.

FIG. 2.

Immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing SIV Gag in mice. Naïve C57BL/6 mice were immunized with (A) 10⁹ vp or (B) 10⁸ vp rAd5-Gag, rAd35k5-Gag, or rAd35-Gag. To assess the impact of preexisting anti-Ad5 immunity, C57BL/6 mice were preimmunized with (C) one or (D) two injections of 10¹⁰ vp rAd5-Empty prior to immunization with 10⁸ vp rAd5-Gag, rAd35k5-Gag, or rAd35-Gag. In all cases, Gag-specific CD8⁺ T lymphocyte responses were assessed by D^b/AL11 tetramer binding assays at multiple time points following immunization.

FIG. 3.

Antivector immunity elicited by rAd35k5 vectors in mice. (A) Naïve C57BL/6 mice were primed at week 0 with 10⁹ vp rAd35k5-Gag and were boosted at week 4 with 10⁹ vp rAd5-Gag or rAd35-Gag. Arrows indicate immunizations. Gag-specific CD8⁺ T lymphocyte responses were assessed by D^b/AL11 tetramer binding assays. (B) Serum samples from mice injected with 10⁹ vp rAd5-Gag, rAd35k5-Gag, or rAd35-Gag were assessed in Ad5 and Ad35 luciferase-based virus neutralization assays.

FIG. 4.

Immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing HIV-1 Env in mice. Naïve BALB/c mice were immunized with 10⁹ vp rAd5-Env, rAd35k5-Env, or rAd35-Env. (A) Env-specific cellular immune responses were assessed by pooled peptide and MI10 epitope peptide-specific IFN-γ ELISPOT assays. (B) Env-specific humoral immune responses were assessed by ELISA.

FIG. 5.

Immunogenicities of rAd5, rAd35k5, and rAd35 vectors in rhesus monkeys. Adult rhesus monkeys were primed at week 0 with 10¹¹ vp (A and B) rAd5, (C and D) rAd35, or (E and F) rAd35k5 vectors expressing HIV-1 Env and SIV Gag. At week 12, all monkeys received a homologous boost immunization. (A, C, and E) Env- and Gag-specific cellular immune responses were assessed by pooled peptide IFN-γ ELISPOT assays at multiple time points following immunization. (B, D, and F) Vector-specific NAb titers were assessed by Ad5 and Ad35 virus neutralization assays. Arrows indicate immunizations.

FIG. 5.

Immunogenicities of rAd5, rAd35k5, and rAd35 vectors in rhesus monkeys. Adult rhesus monkeys were primed at week 0 with 10¹¹ vp (A and B) rAd5, (C and D) rAd35, or (E and F) rAd35k5 vectors expressing HIV-1 Env and SIV Gag. At week 12, all monkeys received a homologous boost immunization. (A, C, and E) Env- and Gag-specific cellular immune responses were assessed by pooled peptide IFN-γ ELISPOT assays at multiple time points following immunization. (B, D, and F) Vector-specific NAb titers were assessed by Ad5 and Ad35 virus neutralization assays. Arrows indicate immunizations.

FIG. 5.

Immunogenicities of rAd5, rAd35k5, and rAd35 vectors in rhesus monkeys. Adult rhesus monkeys were primed at week 0 with 10¹¹ vp (A and B) rAd5, (C and D) rAd35, or (E and F) rAd35k5 vectors expressing HIV-1 Env and SIV Gag. At week 12, all monkeys received a homologous boost immunization. (A, C, and E) Env- and Gag-specific cellular immune responses were assessed by pooled peptide IFN-γ ELISPOT assays at multiple time points following immunization. (B, D, and F) Vector-specific NAb titers were assessed by Ad5 and Ad35 virus neutralization assays. Arrows indicate immunizations.

FIG. 6.

CD4⁺ and CD8⁺ T lymphocyte responses. To assess fractionated CD4⁺ and CD8⁺ T lymphocyte responses from the study in rhesus monkeys described in the legend for Fig. 5, pooled peptide IFN-γ ELISPOT assays were performed using CD8-depleted (Depl) and CD4-depleted PBMCs from monkeys at week 16. Monkeys received 10¹¹ vp (A) rAd5, (B) rAd35, or (C) rAd35k5 vectors expressing HIV-1 Env and SIV Gag at weeks 0 and 12.