A novel chimpanzee serotype-based adenoviral vector as delivery tool for cancer vaccines

Abstract

The use of adenovirus (Ad) as vaccine vectors is hindered by pre-existing immunity to human Ads in most of the human population. In order to overcome this limitation, uncommon alternative Ad serotypes need to be utilized. In this study, an E1-E3 deleted recombinant Ad based on the chimpanzee serotype 3 (ChAd3) was engineered to express human carcinoembryonic antigen (CEA) protein or rat neu extracellular/transmembrane domains (ECD.TM). ChAd3 vectors were tested in CEA transgenic (CEA.Tg) and BALB/NeuT mice, which show immunologic tolerance to these antigens. ChAd3 is capable of inducing an immune response comparable to that of hAd5 serotype-based vectors, thus breaking tolerance to tumor associated antigens (TAAs) and achieving anti-tumor effects. Of importance is that ChAd3 can overcome hAd5 pre-existing immunity and work in conjunction with DNA electroporation (DNA-EP) and other Ad vaccines based on common human serotypes.

Figures

Fig. 1

Schematic representation of the vectors. CEA and rat neu ECD.TM expression cassettes were inserted by homologous recombination in E1–E3 deleted hAd5 and ChAd3 vectors as indicated.

Fig. 2

ChAd3 and hAd5 show comparable expression of CEA in vitro and in vivo. (A) HeLa cells were infected at the indicated ratio vp/cell. 48 h later secreted CEA was measured in the supernatant by ELISA. Grey diamonds and white square symbols show average CEA expression mediated by hAd5-CEA and ChAd3-CEA, respectively. (B) CEA.Tg mice were injected i.m. with the indicated amount (vp) of either hAd5 or ChAd3. Three days later, mice were bled and circulating CEA levels were measured by ELISA. Grey dots indicate the geometric mean of the group.

Fig. 3

Characterization of the immunogenic properties of ChAd3-CEA. (A) Kinetics of the immune response. Groups of 6–8 CEA.Tg mice were immunized with 1 × 1010 vp of either hAd5-CEA or ChAd3-CEA vector. At the indicated time point, mice were bled and PBMCs were stimulated with a pool of peptides covering the C-term of CEA (pool D) and analyzed by intracellular staining for IFNγ. Group average with standard deviations (error bar) is shown. (B) Vaccine potency of ChAd3-CEA. Groups of 6 CEA.Tg mice were vaccinated with the indicated vp of either hAd5-CEA or ChAd3-CEA vector. Thirty days after the injection, PBMCs were analyzed for CEA-specific response by intracellular staining for IFNγ. Histograms and error bars show group average and standard deviations, respectively. (C) hAd5 and ChAd3 vector mixed modality. Groups of 6 CEA.Tg mice were immunized either with 108 vp of hAd5-CEA or ChAd3-CEA vector or mock injected. Thirty days later, mice were boosted with the indicated vector and 2 weeks later, PBMCs were analyzed for CEA-specific response by intracellular staining for IFNγ. White circles indicate the immune response measured per single animal; gray dots represent the group geometric mean. Mice with responses above 0.1% CD8+/IFNγ+ (dashed line) are considered responders. Student's t-test, p = 0.05 for hAd5/hAd5 vs. hAd5/ChAd3 or ChAd3/ChAd3 groups.

Fig. 4

ChAd3-CEA vaccine overcomes hAd5 immunity. (A) Top scheme. Groups of CEA.Tg mice were pre-injected with 1010 vp of wild type hAd5 and immunized either with hAd5-CEA or ChAd3-CEA (1010 vp). The average (±SD) anti-hAd5 titer of the group is shown. Two weeks later, the immune response against CEA was measured by intracellular staining for IFNγ⋅ (B) Pre-exposure to wt hAd5 does not impair ChAd3 vaccine potency. Groups of 10 CEA.Tg mice received 4 weekly DNA-EP with pV1J-CEA or left untreated (mock). Two weeks after, animals were injected with 109 vp of either of the two vectors and the immune response against CEA was measured 14 days later by intracellular staining for IFNγ. (C) Kinetics of immune response. Treated mice were bled at the indicated time points and PBMCs were analyzed by intracellular staining for IFNγ. Mice with a CEA-specific response >0.1% CD8+/IFNγ+ were considered responders.

Fig. 5

ChAd3-_neu.ECD.TM immunogenic properties and anti-tumor effects. (A) T-cell responses. Groups of 10 BALB/NeuT mice were vaccinated at the 8th and 9th week of age. The immune response against rat neu 15.3 epitope was analyzed at week 11 and 14. Open circles indicate the response of individual mice, filled circle denotes the group mean. A mouse with T-cell reactivity of at least 0.1% was considered a responder. Student's t-test at week 11, p < 0.05 for hAd5wt+hAd5 vs. hAd5 alone or hAd5wt+ChAd3 or ChAd3 alone. (B) Antibody titer. Anti-rat HER-2/neu titers were determined as described in materials and methods. At least 10 mice were included in each group. Open circles indicate the response of individual mice, filled circle denotes the group mean. Student's t-test, p < 0.0003 for hAd5wt+hAd5 vs. hAd5 alone or hAd5wt+ChAd3 or ChAd3 alone. (C) Anti-tumor effect. Mice were immunized as described in the text and tumor development followed from week 15 onwards. At least 10 mice were included in each group. Percentage of tumor-free mice is shown. Group symbols are shown in graph legend. Log-Rank test, hAd5wt+ChAd3-ECD.TM vs. hAd5wt+hAd5-ECD.TM: p = 6.27 × 10−06.