Potent Anti-hepatitis C Virus (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-Launched RNA Replicons and Modified Vaccinia Virus Ankara-HCV

Abstract

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV or DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune responses through apoptosis, and here we demonstrate that they efficiently trigger apoptosis-related proteins in transfected cells. Immunization of mice with the DREP vaccines as the priming immunization followed by a heterologous boost with a recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV (MVA-HCV) induced potent and long-lasting HCV-specific CD4⁺ and CD8⁺ T cell immune responses that were significantly stronger than those of a homologous MVA-HCV prime/boost immunization, with the DREP-e-HCV/MVA-HCV combination the most immunogenic regimen. HCV-specific CD4⁺ and CD8⁺ T cell responses were highly polyfunctional, had an effector memory phenotype, and were mainly directed against E1-E2 and NS2-NS3, respectively. Additionally, DREP/MVA-HCV immunization regimens induced higher antibody levels against HCV E2 protein than homologous MVA-HCV immunization. Collectively, these results provided an immunization protocol against HCV by inducing high levels of HCV-specific T cell responses as well as humoral responses. These findings reinforce the combined use of DREP-based vectors and MVA-HCV as promising prophylactic and therapeutic vaccines against HCV.IMPORTANCE HCV represents a global health problem as more than 71 million people are chronically infected worldwide. Direct-acting antiviral agents can cure HCV infection in most patients, but due to the high cost of these agents and the emergence of resistant mutants, they do not represent a feasible and affordable strategy to eradicate the virus. Therefore, a vaccine is an urgent goal that requires efforts to understand the correlates of protection for HCV clearance. Here, we describe for the first time the generation of novel vaccines against HCV based on alphavirus DNA replicons expressing HCV antigens. We demonstrate that potent T cell immune responses, as well as humoral immune responses, against HCV can be achieved in mice by using a combined heterologous prime/boost immunization protocol consisting of the administration of alphavirus replicon DNA vectors as the priming immunization followed by a boost with a recombinant modified vaccinia virus Ankara vector expressing HCV antigens.

Keywords: HCV; MVA; alphavirus replicon; immune response; mice; poxvirus; vaccine.

FIG 1

Generation and analysis of HCV protein expression of the different DREP-based HCV vaccine candidates. (A) Scheme of the four novel DREP-based HCV vaccine candidates generated in this study, expressing either core-E1-E2 or p7-NS2-NS3 HCV genes. DREP-C-E1-E2 and DREP-p7-NS2-NS3 were grouped to form the DREP-HCV vaccine candidate, while DREP-e-C-E1-E2 and DREP-e-p7-NS2-NS3 were grouped to form the DREP-e-HCV vaccine candidate. The DREP replicons contain the alphavirus replicase placed under the control of the cytomegalovirus (CMV) promoter, and the HCV genes (either core-E1-E2 or p7-NS2-NS3) were placed under the control of the alphavirus subgenomic promoter (SP). In DREP-e-C-E1-E2 and DREP-e-p7-NS2-NS3, the translational enhancer (e) is placed in frame and upstream of the HCV genes, as indicated. (B) PCR analysis. Primers hybridizing in the DREP-vector regions flanking the place where the HCV genes were inserted were used to confirm their correct insertion. (C) Expression of HCV proteins in human HEK293T cells mock transfected or transfected with DREP-HCV (mixture of DREP-C-E1-E2 and DREP-p7-NS2-NS3), DREP-e-HCV (mixture of DREP-e-C-E1-E2  and DREP-e-p7-NS2-NS3), or empty DREP-Ø at 48 h posttransfection.

FIG 2

In vitro induction of apoptosis-related proteins by the DREP replicase. HEK293T cells were mock transfected or transfected with DREP-Ø, DREP-p7-NS2-NS3, or DREP-e-p7-NS2-NS3 vector. Plasmid nmCas, lacking the alphavirus replicase gene, was used as a negative control. At 4, 24, and 34 h posttransfection, cells were harvested, lysed with 1× Laemmli buffer–β-mercaptoethanol, and fractionated in 10% SDS-PAGE gels. The presence of total and phosphorylated (P-) PKR (A) and eIF2α (B) proteins was detected by Western blotting. Rabbit anti-β-actin and rabbit anti-histone H3 antibodies were used as loading controls. (C) A quantitative ratio of phosphorylated eIF2α/total eIF2α is shown and was determined by quantifying the Western blot bands represented in panel B using the Image Lab software. The dashed line indicates the threshold level for the controls. A.U., arbitrary units.

FIG 3

Immunization schedule. A prime/boost immunization protocol was performed in C57BL/6JOlaHsd mice to study the immunogenicity of DREP-HCV and DREP-e-HCV vaccine candidates, as described in Materials and Methods. Immunization groups are indicated, together with the time points at which animals were immunized (dose and route of administration are shown) and sacrificed to analyze the adaptive and memory HCV-specific T cell and humoral immune responses.

FIG 4

HCV-specific CD4⁺ and CD8⁺ T cell adaptive immune responses elicited in immunized mice. Five mice per group were sacrificed at 10 days postboost, and the splenic HCV-specific CD4⁺ and CD8⁺ T cell immune responses were analyzed by ICS, as described in Materials and Methods. P values indicate significantly differences between results for DREP-HCV/MVA-HCV, DREP-e-HCV/MVA-HCV, and MVA-HCV/MVA-HCV, as indicated (*, P < 0.05; ***, P < 0.001). (A) Magnitude of total HCV-specific CD4⁺ and CD8⁺ T cell adaptive immune responses directed against all HCV antigens. Percentages of CD4⁺ or CD8⁺ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against a mixture of core, E1, E2, p7, NS2, NS3, NS4, and NS5 genotype 1a HCV peptide pools are represented. (B) Breadth of HCV-specific CD4⁺ and CD8⁺ T cell adaptive immune responses. Percentages of core-, E1-, E2-, p7-, NS2-, NS3-, NS4-, or NS5-specific CD4⁺ and CD8⁺ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against each specific HCV peptide pool are represented. (C) Polyfunctionality of total HCV-specific CD4⁺ and CD8⁺ T cell adaptive immune responses directed against all HCV antigens. Responses are divided according to function in combined production of CD107a, IFN-γ, TNF-α, and/or IL-2 and grouped according to the color-coded pie charts, taking into consideration the number of functions (one, two, three, or four).

FIG 5

HCV-specific CD4⁺ and CD8⁺ T cell memory immune responses elicited in immunized mice. Five mice per group were sacrificed at 53 days postboost, and the splenic HCV-specific CD4⁺ and CD8⁺ T cell immune responses were analyzed by ICS, as described in Materials and Methods. P values indicate significantly differences between results for DREP-HCV/MVA-HCV, DREP-e-HCV/MVA-HCV, and MVA-HCV/MVA-HCV, as indicated (***, P < 0.001). (A) Magnitude of total HCV-specific CD4⁺ and CD8⁺ T cell memory immune responses directed against all HCV antigens. Percentages of CD4⁺ or CD8⁺ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against a mixture of core, E1, E2, p7, NS2, NS3, NS4, and NS5 genotype 1a HCV peptide pools are represented. (B) Breadth of HCV-specific CD4⁺ and CD8⁺ T cell memory immune responses. Percentages of core-, E1-, E2-, p7-, NS2-, NS3-, NS4-, or NS5-specific CD4⁺ and CD8⁺ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against each specific HCV peptide pool are represented. (C) Polyfunctionality of total HCV-specific CD4⁺ and CD8⁺ T cell memory immune responses directed against all HCV antigens. Responses are divided according to function in combined production of CD107a, IFN-γ, TNF-α, and/or IL-2 and grouped according to the color-coded pie charts, taking into consideration the number of functions (one, two, three, or four).

FIG 6

Phenotypic profile of adaptive and memory HCV-specific CD4⁺ and CD8⁺ T cells elicited in immunized mice. Five mice per group were sacrificed at 10 (A) and 53 (B) days postboost, and the memory phenotypic profile of splenic HCV-specific CD4⁺ and CD8⁺ T cells was analyzed by ICS, as described in Materials and Methods. (A) Graphs indicate the percentages of T central memory (TCM; CD127⁺ CD62L⁺), T effector memory (TEM; CD127⁺ CD62L⁻), and T effector (TE; CD127⁻ CD62L⁻) adaptive HCV-specific CD4⁺ or CD8⁺ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against all genotype 1a HCV peptide pools. Below the graphs, representative fluorescence-activated cell sorting plots of the memory phenotypic profile of adaptive NS2- and NS3-specific CD8⁺ T cells are represented. CD8⁺ T cells expressing CD127 and/or CD62L are depicted in black as density plots, while the NS2- or NS3-specific CD8⁺ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 are depicted in blue, with their percentages being indicated. (B) Percentages of memory HCV-specific CD4⁺ and CD8⁺ T cells with a T central memory (TCM), TEM, or TE phenotype and expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against all HCV peptide pools. **, P < 0.005; ***, P < 0.001.

FIG 7

HCV-specific humoral immune responses elicited in immunized mice. (A) Levels of HCV E2-specific total IgG binding antibodies were measured by ELISA in serial 2-fold dilutions of pooled serum samples (n = 10 per group) obtained from immunized mice at 10 days postboost. Absorbance values were measured at 450 nm. The mean and standard deviations are indicated. Blue asterisks indicate significant differences between results with DREP-HCV/MVA-HCV and MVA-HCV/MVA-HCV, while red asterisks indicate significant differences between results with DREP-e-HCV/MVA-HCV and MVA-HCV/MVA-HCV (*, P < 0.05). (B) Levels of HCV E2-specific IgG1, IgG2c, and IgG3 isotype antibodies in diluted 1/50 individual serum samples (n = 10 per group) obtained from immunized mice at 10 days postboost. Absorbance (optical density [OD]) values were measured at 450 nm. The mean and standard deviations are indicated.