A novel adenovirus type 6 (Ad6)-based hepatitis C virus vector that overcomes preexisting anti-ad5 immunity and induces potent and broad cellular immune responses in rhesus macaques

Abstract

Success in resolving hepatitis C virus (HCV) infection has been correlated to vigorous, multispecific, and sustained CD8(+) T-cell response in humans and chimpanzees. The efficacy of inducing T-cell-mediated immunity by recombinant serotype 5 adenovirus vector has been proven in many animal models of infectious diseases, but its immunogenicity can be negatively influenced by preexisting immunity against the vector itself. To evaluate the less prevalent adenovirus serotype 6 (Ad6) as an alternative vector for and HCV vaccine development, we have generated serotype 5 and 6 adenoviral vectors directing expression of the nonstructural region of HCV (MRKAd5-NSmut and MRKAd6-NSmut). Immunogenicity studies in mice showed that the two vectors induced comparable T-cell responses but that only MRKAd6-NSmut was not suppressed in the presence of anti-Ad5 immunity. In contrast, preexisting anti-Ad5 immunity dramatically blunted the immunogenicity of the serotype 5-based HCV vector. Furthermore, MRKAd6-NSmut showed equivalent potency, breadth, and longevity of HCV-specific T-cell responses in rhesus macaques as the corresponding Ad5-based vector over a wide range of doses and was capable of boosting DNA-primed animals even if administered at low doses. These data support the use of the MRKAd6-NSmut for anti-HCV immunotherapy and, more generally, for the Ad6 serotype as a better genetic vaccine vehicle than Ad5.

FIG. 1.

Prevalence and cross-reactivity of anti-Ad5 and anti-Ad6 neutralizing antibodies. (A) Ad5 and Ad6 seroprevalence. Individual NAb titers against Ad5 or Ad6 present in whole sera from 93 European individuals are shown. The dashed line corresponds to NAb titers of 200. (B) Correlation between neutralization titers measured in the same panel of sera from European individuals with MRKAd5 and MRKAd6 in SEAP neutralization assay. Statistical evaluation was done by Spearman correlation coefficient (rho = 0.12; P = 0.91).

FIG. 2.

NS polyprotein expression and processing by infection of HeLa cells with MRKAd5-NSmut and MRKAd6-NSmut. Western blotting of whole-cell extracts from HeLa cells infected at different MOIs with MRKAd5-NSmut or MRKAd6-NSmut. Whole-cell extracts were normalized for total protein content before loading on gel. Mature NS3, NS5A, and NS5B products (indicated by arrows) were detected with specific antibodies. Extract from mock-infected cells was used as a negative control (mock). Molecular weight markers are reported on the left side of each panel.

FIG. 3.

Immunogenicity of MRKAd5-NSmut and MRKAd6-NSmut in naïve and in Ad5 preimmune mice. (A) Immunogenicity of MRKAd5-NSmut and MRKAd6-NSmut in naïve mice. IFN-γ ELISPOT responses to the NS3h peptide pool and the CD8⁺ peptide G-1480 in C57BL/6 mice immunized with MRKAd5-NSmut and MRKAd6-NSmut at doses of 10⁷ and 10⁹ VP (indicated at the bottom). Results are expressed as the number of IFN-γ SFC per 10⁶ splenocytes and are reported on the vertical axis. Each bar represents the average of the response to the NS3h peptide pool (gray bars) and CD8⁺ G-1480 peptide (black bars) measured in groups of 10 immunized mice. Response to DMSO is represented by the white bars. Average number of spots in DMSO control samples was 2 per 10⁶ splenocytes. (B and C) Immunogenicity of MRKAd5-NSmut and MRKAd6-NSmut in mice with preexisting anti-Ad5 immunity. C57BL/6 mice were preimmunized with 10¹⁰ VP of Ad5 expressing an unrelated antigen. Groups (n = 5) of naive mice or mice with anti-Ad5 immunity were immunized with 10⁸ VP of MRKAd5-NSmut or MRKAd6-NSmut, and responses against NS3h peptide pool were assessed by IFN-γ ELISPOT assay. Results are expressed as the number of IFN-γ SFC per 10⁶ splenocytes and are reported on the vertical axis. Each bar represents the individual response of animals immunized with MRKAd5-NSmut (B) or MRKAd6-NSmut (C). Anti-Ad5 neutralization titers were between 200 and 4,000 except in mouse m10 (NAb of 50).

FIG. 4.

Effect of adenovirus vector boosting injection in rhesus macaques. IFN-γ ELISPOT responses are shown for individual monkeys after priming (T = 8 weeks, gray bars), before boosting (T = 24 weeks, white bars), and after boosting (T = 28 weeks, black bars). Each bar represents the total anti-NS response, calculated by adding up positive responses to individual NS peptide pools and correcting for DMSO background. Results are expressed as the number of IFN-γ SFC per 10⁶ PBMC. Groups A, C, and E were immunized with MRKAd5-NSmut at the indicated doses (top left). Groups B, D, and F were immunized with MRKAd6-NSmut at the indicated doses (top left).

FIG. 5.

Strength and longevity of the anti-HCV T-cell responses induced in rhesus macaques by MRKAd5-NSmut and MRKAd6-NSmut. The immune response induced by different doses of the two vectors were measured by IFN-γ ELISPOT assay. The geometric mean of the total anti-NS response for the different immunization groups during 50 weeks of follow-up is shown. The total anti-HCV IFN-γ ELISPOT response was calculated for each time point and each animal by adding up positive responses to the individual NS peptide pools and correcting for DMSO background. Numbers on the vertical axis represent the number of SFC per 10⁶ PBMC, and arrowheads indicate immunization dates. MRKAd5, MRKAd5-NSmut; MRKAd6, MRKAd6-NSmut.

FIG. 6.

Bulk cytotoxicity assay in rhesus monkeys immunized with MRKAd5-NSmut and MRKAd6-NSmut. PBMC collected at T = 35 weeks (7 weeks after boosting) were restimulated in vitro with VacHCV-NS and then tested for their ability to kill autologous B-LCLs pulsed with either DMSO alone or with the individual NS peptide pools (NS3p, NS3h, NS4, NS5A, NS5B-I, and NS5B-II). Results are expressed as the percentage of specific lysis at each effector-to-target (E:T) ratio tested.

FIG. 7.

ELISPOT responses in naked DNA-primed, MRKAd6-NSmut-boosted rhesus monkeys. (A) DNA priming and MRKAd6-NSmut (10⁸ viral particles) boosting in rhesus monkeys. ELISPOT responses are shown for individual monkeys after priming (T = 12 weeks), before boost (T = 24 weeks) and after boost (T = 28 weeks). Each bar represents the total anti-NS response, calculated by adding up the positive responses to individual NS peptide pools and correcting for DMSO background. Results are expressed as the number of IFN-γ SFC per 10⁶ PBMC. (B) Strength and longevity of anti-NS ELISPOT responses induced in DNA primed- and /adenovirus-boosted monkeys over time. The line represents the geometric mean of total anti-NS ELISPOT responses of the six animals during a 50 week follow-up. The total anti-NS ELISPOT response was calculated for each time point and for each animal by adding up the positive responses to the individual NS peptide pools and correcting for DMSO background. Numbers on the y axis represent the number of IFN-γ SFC per 10⁶ PBMC. Arrows indicate immunization dates.