Combined adenovirus vector and hepatitis C virus envelope protein prime-boost regimen elicits T cell and neutralizing antibody immune responses

Abstract

Despite the recent progress in the development of new antiviral agents, hepatitis C virus (HCV) infection remains a major global health problem, and there is a need for a preventive vaccine. We previously reported that adenoviral vectors expressing HCV nonstructural proteins elicit protective T cell responses in chimpanzees and were immunogenic in healthy volunteers. Furthermore, recombinant HCV E1E2 protein formulated with adjuvant MF59 induced protective antibody responses in chimpanzees and was immunogenic in humans. To develop an HCV vaccine capable of inducing both T cell and antibody responses, we constructed adenoviral vectors expressing full-length and truncated E1E2 envelope glycoproteins from HCV genotype 1b. Heterologous prime-boost immunization regimens with adenovirus and recombinant E1E2 glycoprotein (genotype 1a) plus MF59 were evaluated in mice and guinea pigs. Adenovirus prime and protein boost induced broad HCV-specific CD8+ and CD4+ T cell responses and functional Th1-type IgG responses. Immune sera neutralized luciferase reporter pseudoparticles expressing HCV envelope glycoproteins (HCVpp) and a diverse panel of recombinant cell culture-derived HCV (HCVcc) strains and limited cell-to-cell HCV transmission. This study demonstrated that combining adenovirus vector with protein antigen can induce strong antibody and T cell responses that surpass immune responses achieved by either vaccine alone.

Importance: HCV infection is a major health problem. Despite the availability of new directly acting antiviral agents for treating chronic infection, an affordable preventive vaccine provides the best long-term goal for controlling the global epidemic. This report describes a new anti-HCV vaccine targeting the envelope viral proteins based on adenovirus vector and protein in adjuvant. Rodents primed with the adenovirus vaccine and boosted with the adjuvanted protein developed cross-neutralizing antibodies and potent T cell responses that surpassed immune responses achieved with either vaccine component alone. If combined with the adenovirus vaccine targeting the HCV NS antigens now under clinical testing, this new vaccine might lead to a stronger and broader immune response and to a more effective vaccine to prevent HCV infection. Importantly, the described approach represents a valuable strategy for other infectious diseases in which both T and B cell responses are essential for protection.

FIG 1

Construction and characterization of recombinant adenovirus vectors. (A) Schematic diagram of expression cassettes inserted into the Ad6 backbone. The N-terminal methionine residue is indicated as Met; tetracycline operator binding sites (TetO₂) within the CMV promoter (CMV) are indicated as T. (B) Western blot detection of E2 protein expression in HeLa cells 48 h after infection with Ad6E1E2p7 or Ad6E2₆₆₂ at different MOI. A lysate of noninfected cells represents the negative control (cells). β-Actin immunodetection is shown for normalization of lysate protein content. (C) Detection of E2 protein by immunohistochemical staining of HeLa cells infected with Ad6E1E2p7 (1) or Ad6E2₆₆₂ (2) or left uninfected (3).

FIG 2

Humoral and cellular immune responses in C57 and BALB/c mouse strains receiving Ad6 or Ad6-protein vaccinations. Sera and splenocytes were collected 2 weeks after the last vaccination (regimens are detailed in Table 1 and schematically reproduced at graph bottom). (A and D) Antibody endpoint titers measured by ELISA using as the antigen E1E2p7 or E2₆₆₂ prepared from extracts of HEK293 cells transfected with plasmids expressing the same sequence as encoded by adenovirus and captured on an ELISA plate with GNA lectin. Titer was defined as the highest serum dilution that resulted in an absorbance value (OD) 5 times the value of preimmune sera. (D) Antibody titers in vaccine-responding animals (i.e., groups PPP on T212 and AA on HCV-1 excluded) measured on homogeneous ELISA antigens were compared by unpaired t test; only statistically significant differences are shown. (B, E, and F) The number of antigen-specific IFN-γ-secreting cells was determined by ELISpot assay on splenocytes. ELISpot data corrected for background are expressed as IFN-γ spot-forming cells (SFC) per million splenocytes. Peptide pools of T212 (B, C, and E) or H77 (F) origin were used. (E and F) T cell responses on each peptide pool from responding animals only (i.e., PPP group excluded) were compared by unpaired t test; only statistically significant differences are shown. Ab titer and ELISpot data are shown as box (median and interquartile range) and whisker (minimum to maximum value) plots. A, Ad6E1E2p7; P, E1E2 protein vaccinations (D to F). (C) IFN-γ intracellular staining and FACS analysis. Data are expressed as percentage of IFN-γ-secreting CD4 or CD8 cells in response to T212 peptides after subtraction of DMSO background, presented as group means ± standard deviations.

FIG 3

Quantification and characterization of humoral responses induced by Ad6 and protein vaccines in guinea pigs. Vaccination regimens are described in Table 1 and schematically indicated at the bottom of each graph (P, protein; A, Ad6E1E2p7; A_t, Ad6E2₆₆₂ truncation). Sera were collected 2 weeks after the second Ad6 or final protein immunization. Data are shown as box (median and interquartile range) and whisker (minimum to maximum value) plots. (A) Antibody endpoint titers measured by ELISA using antigen E1E2p7 or E2₆₆₂ (T212 strain) prepared from extracts of HEK293 cells transfected with plasmids expressing the same sequence encoded by adenovirus and captured on an ELISA plate with GNA lectin, or E1E2 recombinant protein (strain HCV-1) used to directly coat the plate. Titer was defined as the highest serum dilution that resulted in an absorbance value (OD) 5 times the value of preimmune sera. Antibody titers in vaccine-responding animals (i.e., groups AA and A_tA_t, HCV-1 E2 protein excluded) measured on homogeneous ELISA antigens are compared either within groups after Ad and protein immunization strains (paired t test [solid line]) or among groups at the end of vaccination (unpaired t test [dashed line]), with only statistically significant differences shown. (B) Ratios of IgG2a to IgG1 titers, calculated to characterize the Th profile (ratios of >1 indicate Th1 response, and ratios of <1 indicate Th2 response).

FIG 4

Quantification and characterization of neutralizing antibody responses induced by Ad6 and protein vaccines in guinea pigs. Sera were collected 2 weeks after final immunization with the vaccination regimens detailed in Table 1 and schematically depicted at the bottom of each graph (P, protein; A, Ad6E1E2p7; A_t, Ad6E2₆₆₂ truncation. (A) Neutralization endpoint titer (ID50) for HCVpp-H77, with median value and interquartile range presented. (B) Percent neutralization of a panel of chimeric HCV JFH strains expressing structural proteins from diverse genotypes (Gt). Sera were tested at a final 1/500 dilution, and the mean neutralization values are presented, where the dotted line represents the assay cutoff. (C) Schematic diagram of single-cycle HCV transmission assay (18). (D) HCV H77/JFH cell-free or cell-to-cell de novo infection events in untreated (control) anti-HCV Ig (100 μg/ml), guinea pig preimmune sera (black bars) or postimmune sera (gray and white bars) at a final 1/100 dilution. Each bar represents the mean + standard deviation of newly infected targets cells per 10% producer cells. The amount of extracellular virus at the end of the 24-h incubation period was determined by endpoint titration, and the conditions under which >99% of cell-free virus was eliminated are indicated by the horizontal line. Inhibition of cell-cell transmission was defined as reduced infection of naive cells in the absence of cell-free virus, and significant inhibition is indicated with an asterisk. Statistical analyses were performed using a nonparametric one-way ANOVA (Kruskal-Wallis test) or Student's t test in Prism 4.0, where necessary corrections for multiple comparisons were made, and Bonferroni's multiple-comparison test was used to assess the degree of difference from the wild-type positive control.