Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D

Abstract

Recombinant adenovirus serotype 5 (rAd5) vector-based vaccines are currently being developed for both human immunodeficiency virus type 1 and other pathogens. The potential limitations associated with rAd5 vectors, however, have led to the construction of novel rAd vectors derived from rare Ad serotypes. Several rare serotype rAd vectors have already been described, but a detailed comparison of multiple rAd vectors from subgroups B and D has not previously been reported. Such a comparison is critical for selecting optimal rAd vectors for advancement into clinical trials. Here we describe the construction of three novel rAd vector systems from Ad26, Ad48, and Ad50. We report comparative seroprevalence and immunogenicity studies involving rAd11, rAd35, and rAd50 vectors from subgroup B; rAd26, rAd48, and rAd49 vectors from subgroup D; and rAd5 vectors from subgroup C. All six rAd vectors from subgroups B and D exhibited low seroprevalence in a cohort of 200 individuals from sub-Saharan Africa, and they elicited Gag-specific cellular immune responses in mice both with and without preexisting anti-Ad5 immunity. The rAd vectors from subgroup D were also evaluated using rhesus monkeys and were shown to be immunogenic after a single injection. The rAd26 vectors proved the most immunogenic among the rare serotype rAd vectors studied, although all rare serotype rAd vectors were still less potent than rAd5 vectors in the absence of anti-Ad5 immunity. These studies substantially expand the portfolio of rare serotype rAd vectors that may prove useful as vaccine vectors for the developing world.

FIG. 1.

Novel rAd vector systems. (A) Genomic organization of replication-competent, wild-type Ad26. (B) Genomic organization of replication-incompetent rAd26 vector system with E1/E3 deleted, consisting of an adaptor plasmid (pAdApt26) and a cosmid (pWE.Ad26.ΔE3.5orf6). The E1 region was replaced by a TG cassette in the adaptor plasmid. The E3 region was deleted and the E4orf6 region was replaced by the corresponding region from Ad5 (5orf6) in the cosmid. The numbers indicate Ad26 nucleotide positions, and the region of homology that facilitates recombination between the adaptor plasmid and the cosmid is depicted. The genomic organizations and vector systems for rAd48 and rAd50 are analogous. ψ, packaging signal; E1 to E4, early genes; L1 to L5, late genes.

FIG. 2.

Receptor binding studies. Receptor usage was determined by assessing the abilities of rAd5, rAd11, rAd35, rAd50, rAd26, rAd48, and rAd49 vectors expressing eGFP to transduce (A) CHO cells stably transfected with CAR or (B) B16F10 cells stably transfected with CD46. Untransfected cells were utilized as negative controls. TG expression was assessed by flow cytometry. *, P < 0.005.

FIG. 3.

Seroprevalence studies. Serum samples from 200 adults from sub-Saharan Africa were evaluated for Ad5-, Ad11-, Ad35-, Ad50-, Ad26-, Ad48-, and Ad49-specific NAbs. NAb titers were arbitrarily divided into the following categories: <16 (negative), 16 to 200, 200 to 1,000, and >1,000.

FIG. 4.

Immunogenicity studies using mice. (A, B) Naïve C57BL/6 mice (n = 4/group) were immunized i.m. with 10⁹, 10⁸, or 10⁷ vp rAd5, rAd11, rAd35, rAd50, rAd26, rAd48, or rAd49 expressing SIV Gag. (C, D) C57BL/6 mice with anti-Ad5 immunity were similarly immunized with 10⁹ vp of each vector. Gag-specific cellular immune responses were assessed by D^b/AL11 tetramer binding assays at multiple time points following immunization (A, C) and by IFN-γ ELISPOT assays in response to a Gag peptide pool as well as the CD8⁺ T-lymphocyte epitopes AL11 and KV9 and the CD4⁺ T-lymphocyte epitope DD13 on day 35 (B, D). Mean responses with standard errors are shown.

FIG. 5.

Heterologous prime-boost studies with mice. Naïve mice (n = 4/group) were primed on day 0 with (A) 10⁹ vp rAd35-Gag, (B) 10⁹ vp rAd26-Gag, or (C) 50 μg DNA-Gag and then boosted on day 28 with 10⁹ vp of the rAd-Gag vectors shown. Arrows indicate immunizations. Gag-specific cellular immune responses were assessed by D^b/AL11 tetramer binding assays at multiple time points following immunization. (D, E) Serum Ad5-, Ad11-, Ad35-, Ad50-, and Ad26-specific NAb titers were determined using the mice in the study whose results are depicted in panel (A). (F, G) Serum Ad5-, Ad35-, Ad26-, Ad48-, and Ad49-specific NAb titers were determined using the mice in the study whose results are depicted in panel (B). Mean responses with standard errors are shown.

FIG. 6.

Immunogenicity studies using rhesus monkeys. Adult rhesus monkeys (n = 3/group) were immunized with a single injection of 10¹¹ vp rAd5-Gag, rAd26-Gag, rAd48-Gag, or rAd49-Gag. (A) IFN-γ ELISPOT assays in response to a Gag peptide pool and (B) Gag-specific ELISAs were performed at multiple time points following immunization. Mean responses with standard errors are shown.