Adenovirus-based vaccines: comparison of vectors from three species of adenoviridae

Abstract

In order to better understand the broad applicability of adenovirus (Ad) as a vector for human vaccine studies, we compared four adenovirus (Ad) vectors from families C (Ad human serotype 5 [HAdV-5; here referred to as AdHu5]), D (HAdV-26; here referred to as AdHu26), and E (simian serotypes SAdV-23 and SAdV-24; here referred to as chimpanzee serotypes 6 and 7 [AdC6 and AdC7, respectively]) of the Adenoviridae. Seroprevalence rates and titers of neutralizing antibodies to the two human-origin Ads were found to be higher than those reported previously, especially in countries of sub-Saharan Africa. Conversely, prevalence rates and titers to AdC6 and AdC7 were markedly lower. Healthy human adults from the United States had readily detectable circulating T cells recognizing Ad viruses, the levels of which in some individuals were unexpectedly high in response to AdHu26. The magnitude of T-cell responses to AdHu5 correlated with those to AdHu26, suggesting T-cell recognition of conserved epitopes. In mice, all of the different Ad vectors induced CD8(+) T-cell responses that were comparable in their magnitudes and cytokine production profiles. Prime-boost regimens comparing different combinations of Ad vectors failed to indicate that the sequential use of Ad vectors from distinct families resulted in higher immune responses than the use of serologically distinct Ad vectors from the same family. Moreover, the transgene product-specific antibody responses induced by the AdHu26 and AdC vectors were markedly lower than those induced by the AdHu5 vector. AdHu26 vectors and, to a lesser extent, AdC vectors induced more potent Ad-neutralizing antibody responses. These results suggest that the potential of AdHu26 as a vaccine vector may suffer from limitations similar to those found for vectors based on other prevalent human Ads.

FIG. 1.

Construction of the AdHu26 molecular clone. The graph shows the molecular clone of AdHu26 in which orf6 of E4 was replaced with that of AdHu5. Enzyme sites that were used to construct the clone are shown.

FIG. 2.

Receptor binding specificity of AdHu26. CHO-CAR and CHO-HVEM cells were infected with 10⁴ vps/cell of AdHu5GFP or AdHu26GFP. Cells were incubated for 24 h and evaluated microscopically (A). CHO-CAR or CHO-HVEM cells were incubated for 2 h with the Ad vectors expressing GFP, washed several times, and then incubated for an additional 22 h (2 h). Other cultures were incubated in the presence of AdGFP vectors for 24 h. Ad vectors were used at 10³ or 10⁴ vps per cell. Cells were trypsinized, stained with a live cell dye, and evaluated by flow cytometry. The histograms show the levels of GFP expression over the numbers of events (B). A 0.2% solution of red blood cells from a rhesus macaque was incubated with serial dilutions of AdC1, AdHu26, or saline without virus. The picture shows the original wells (C).

FIG. 3.

Prevalence and titers of neutralizing antibodies to AdHu26 in human sera. Human sera collected in the United States (n = 100), Thailand (n = 200), Ivory Coast (n = 200), Nigeria (n = 193), Cameroon (n = 26), Uganda (n = 59), and South Africa (n = 40) were tested for neutralizing antibodies to AdHu26; the same sets of sera from the United States, Uganda, and South Africa were also tested for neutralizing antibodies to AdHu5, AdC7, and AdC6. (A) Prevalence of positive sera. Black bars, percentage of serum samples that were positive at the lowest serum dilution of 1:10; gray bars, percentage of serum samples that were positive at dilutions at or above 1:40. (B) Average antibody titers of the positive sera.

FIG. 4.

Human T-cell responses to AdHu5 and AdHu26. T-cell responses to AdHu5 and AdHu26 were measured by flow cytometry following whole-vector stimulation. (Left panel) Magnitudes of the CD4⁺ or CD8⁺ T-cell responses producing IL-2, IFN-γ, and/or TNF-α to AdHu5 and AdHu26 in 15 healthy subjects. Lines represent the average ± SEM. (Right panel) Representative flow plots of the T-cell response to no stimulation (top left) and staphylococcus endotoxin B (SEB) (bottom left) controls as well as AdHu5 (top right) and AdHu26 (bottom right).

FIG. 5.

Transgene product-specific CD8⁺ T-cell responses to AdHu26 vector vaccination in mice. Groups of four BALB/c mice were immunized with varied doses of an AdHu26 vector expressing gag of HIV-1. Three weeks later the mice were bled and the frequencies of CD8⁺ cells and tetramer staining-positive CD8⁺ T cells from individual mice were determined. (A) Numbers of tetramer staining-positive (tet⁺) CD8⁺ cells/10⁷ PBMCs. In addition, PBMCs from individual mice were stimulated for 5 h with the immunodominant epitope of Gag and then stained for CD8 and intracellular IFN-γ. (B) Frequencies of gag-specific IFN-γ-positive CD8⁺ cells as a proportion of all circulating CD8⁺ cells. (C) Comparison of CD8⁺ T-cell responses to gag using different Ad vectors for immunization. Mice were immunized with 10¹⁰ vps of vector; the frequencies of tetramer staining-positive CD8⁺ T cells were determined 21 days later. (D) Frequencies of gag-specific CD8⁺ T cells tested 21 days after they were primed with different Ad vectors at 10⁸ vps or 21 days after booster immunization given 2 months after the cells were primed.

FIG. 6.

Preexposure to AdHu26 dampens the transgene product-specific CD8⁺ T-cell response to an AdHu26 vector. Mice were immunized with 10¹¹ vps of an AdHu26 vector expressing the rabies virus glycoprotein. Two weeks later, sera were harvested and tested for neutralizing antibodies to AdHu26. All of the serum samples had titers in excess of 1:80. Mice were then vaccinated with 5 × 10⁹ vps of an AdHu26gag vector (Ad26/Ad26gag), and naïve control mice were vaccinated with the AdHu26gag vector at the same time (−/Ad26rab). Two weeks later, the frequencies of gag-specific CD8⁺ T cells in blood were determined. A group of naïve mice (Naive) was tested in parallel. Preexposed animals developed significantly lower responses than non-pre-exposed animals (P = 0.0007). The graph on the left shows the numbers of tetramer staining-positive CD8⁺ cells/10⁷ PBMCs. The same samples were tested for the production (+) or lack of production (−) of IFN-γ (I), MIP-1α (M), and TNF-α (T) in response to the epitope. The graph on the right shows the frequencies of CD8⁺ T cells with different cytokine profiles. The asterisk indicates a statistically significant difference in the responses of preexposed and non-pre-exposed animals.

FIG. 7.

Transgene product and capsid-specific antibody responses to AdHu26. To obtain neutralizing antibodies, groups of ICR mice were immunized with various doses of the AdHu5rab.gp (n = 8), AdC6rab.gp (n = 8), AdC7rab.gp (n = 8), or AdHu26rab.gp (n = 16) vector. They were bled 4 weeks later. (A) Titers of neutralizing antibodies to rabies virus were compared with those in a reference serum sample allows expression of the data in IU. Closed squares, results from individual mice, ×, averages. (B) Neutralizing antibody titers to the Ad vector used for immunization from individual mice (squares) and averages (×). (C) Protection against rabies virus challenge. The same groups of mice described for panel A were challenged with 10 LD₅₀s of rabies virus at 4 weeks after vaccination. Survival was recorded over time. Animals were checked for a total of 3 weeks. All deaths occurred within 14 days after challenge.