Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species

Abstract

Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8+ T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.

Figures

Fig. 1

Dose-response immunogenicity of human adenovirus in mice and macaques. Immunological potency of human Ad vectors encoding for HIV-1 gag in BALB/c mice (A). Five animals per group were immunized intramuscularly with escalating doses of each Ad vector. IFN-γ ELISpot was performed on splenocytes collected three weeks later using as antigen a 9mer peptide encoding the HIV gag major H-2 Kd CD8 epitope (AMQMLKETI). Each bar represents the relative potency defined as the minimal Ad vectors dose capable of inducing a HIV-1 gag-specific T-cell response in at least 2 out of 5 animals. Data are shown as the reciprocal of minimal dose. The adenovirus serogroups are shown with different bar colours (white=group B, black=group C, dark grey=group D). Immunological potency of human Ad vectors in macaques (B). Three animals per group were vaccinated intramuscularly with 1010 and 108 vp of each Ad vector encoding for HIV-1 gag. Four weeks after vaccination, T cell responses to a 15mer peptide pool covering gag were measured by IFN-γ ELISpot on PBMC. Data are expressed as IFNγ Spot Forming Cells (SFC) per million PBMC. The mean responses + SEM are shown for each immunization group.

Fig. 2

Phylogenetic analysis of chimpanzee adenovirus. The phylogenetic tree showing the different human adenovirus species (A-F) was obtained by aligning the adenovirus hexon sequences. Human adenovirus (hAd) representative of each species and chimpanzee adenoviruses (ChAd) were included in the analysis. The phylogenetic tree was calculated using the neighbor-joining method as implemented in ClustalX and displayed using drawtree from PHYLIP version 3.69. Alignment positions containing gaps were excluded from the analysis. The alignment of hexon proteins was manually optimized taking into account structural restraints from the Ad5 hexon Xray structure. Bootstrap confidence values are reported at branch points (1000 bootstrap cycles).

Fig. 3

Dose-response immunogenicity of chimpanzee adenovirus in mice and macaques. Immunological potency of chimpanzee Ad vectors encoding for HIV-1 gag in BALB/c mice (A) was measured as described in Fig. 1. Data are shown as the reciprocal of minimal dose. The adenovirus serogroups are shown with different bar colours (white=group B, black=group C, light gray=group E). Immunological potency of human Ad vectors in macaques (B). Three animals per group were vaccinated intramuscularly with 1010 and 108 vp of each Ad vector encoding for HIV-1 gag. Data are expressed as IFNγ SFC per million PBMC. The mean responses + SEM are shown for each immunization group.

Fig. 4

Frequency of IFNγ-secreting CD8 and CD4 T cells in mice and macaques. Frequency of CD8 and CD4 T cells secreting IFNγ in response to stimulation with a gag peptide pool was assessed using Intracellular Staining (ICS) and FACS analysis in mice (A and B, CD8 and CD4, respectively) and macaques (C and D, CD8 and CD4, respectively). Each symbol corresponds to IFNγ+ CD8 or CD4 frequency in individual animals, expressed as the percentage of total CD8 or CD4. Horizontal line represents geometric mean.

Fig. 5

Neutralizing antibody response and lack of cross neutralization among different chimp adenovirus strains. Neutralizing antibody titers to a panel of human and chimpanzee adenoviruses measured in sera collected from a large cohort of Caucasian human healthy volunteers (A). The graph reports the percentage of individuals that show neutralizing titers above (black bar segment) or below (grey bar segment) 200 against each individual adenovirus tested. Lack of cross-neutralization among different chimp adenovirus strains (B). Chimp Ad belonging to different Ad serological groups were selected according to the classification reported in Fig. 2. Mice were pre-immunized twice with 1010 vp of EGFP-expressing vectors (identified by different bar color and pattern) and then vaccinated with 109 vp of HIV-1 gag expressing vectors as reported in the x axis. Three weeks later mice were tested for T-cell response against gag by IFNγ ELISpot. Bars represent the mean+SEM in each immunization group, expressed as IFNγSFC per million splenocytes.

Fig. 6

Longevity of memory T and B cell response in macaques. Three macaques were vaccinated in early 2003 with 1010 vp of ChAd3-gag and boosted 6 years later with the same dosage of PanAd3-gag. IFNγ ELISpot response is shown at peak post prime (4 weeks after vaccination), at the time of long term boost (6 years) and 2 weeks post boost (A). Data are shown as the group mean+SEM response at each time point. Quality of the IFNγ secreting T cell subset, assessed ICS and FACS analysis 2 weeks after PanAd3 long term boost, is shown in (B). Each symbol corresponds to IFNγ + CD4 or CD8 frequency in individual animals, expressed as the percentage of total CD4 or CD8. Horizontal line represents geometric mean. Antibody titers to p24 gag protein, assessed by ELISA and expressed as endpoint titers, are shown in (C) for the three individual animals at the time of PanAd3-gag boost and two weeks later. Horizontal line represents geometric mean

Fig. 7

Immunogenicity of human and chimpanzee adenovirus in humans. IFNγ ELISpot data from human healthy volunteers vaccinated once with the indicated dosage of HCV adenovirus vaccines Ad6-NSmut (n=9, (30)) and ChAd3-NSmut (n=10, (30)) or malaria adenovirus vaccine ChAd63-METRAP (n=39, (31, 32)) are shown as mean+SEM of individual responses to the vaccine inserts (A). Individual responses are obtained by summing each volunteer’s reactivity to peptide pools (six pools for the HCV NS region and six pools for TRAP plus one pool for ME regions of the Malaria vaccine insert). Peak response was measured two or four weeks after vaccination. Dot plots showing IFNγ secretion by CD4 and CD8 T cells from two representative volunteers vaccinated with Ad6 or ChAd3 HCV vaccine vectors are shown in (B). PBMC were stimulated with either a mixture of the three HCV NS pools covering NS3 and NS4 region of the HCV vaccine insert (NS3-4), or with DMSO, the peptide pool diluent, as negative control. Numbers in each plot correspond to frequency of IFNγ + cells over total CD4 and CD8.