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.

Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV

“…To analyze the antibodies present in the serum of immunized animals, mice sera was obtained by centrifugation of blood samples at 3600 rpm for 20 min at 4 ℃. ELISA assays were performed as previously described, using total IgG, IgG1, IgG2c and IgG3 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) [ 25 ]. The 96-well plates (NUNC Maxisorp) were coated with 2 μg/mL of a commercial E2 protein from genotype 1a (SinoBiological, Beijing, China), E2 aggregates or E2.C8A monomers diluted in PBS (Invitrogen, Carlsbad, CA, USA) and incubated at 4 ℃ overnight.…”

“…In immunized mice, MVA-HCV elicited strong, broad, and polyfunctional HCV-specific CD8 + T cells [ 23 , 24 ]. Furthermore, an heterologous prime/boost immunization protocol involving a prime with DNA-based vaccines consisting of alphavirus DNA-launched replicons (DREP) followed by a boost with MVA-HCV, elicited potent HCV-specific CD8 + T cell responses, together with broad and polyfunctional HCV-specific CD4 + T cells [ 25 ]. However, these immunization protocols induced low antibody titers to HCV antigens, and more optimized protocols are needed to boost the humoral responses.…”

Hepatitis C Virus Vaccine: Challenges and Prospects

“…Despite the challenges faced in HCV vaccine design, there have been a variety of different vaccine approaches investigated with a small number of these candidates reaching human trials (summarized in Table 1). Currently, DNA and peptide-based vaccine candidates are actively being investigated in murine models [154][155][156][157][158]. One recently reported peptide candidate consisted of overlapping peptides derived from the p7 protein which induced antigen-specific CD4+ T cells and cytotoxic CD8+ T cells capable of targeting p7 expressing hepatocytes in vivo [154].…”

“…This study has been the first to show the immunogenicity of p7 when used as the sole target in a vaccine. Additionally, a DNA-based vaccine has been shown to induce CD4+ T cell and CD8+ T cell responses and elicit T cell memory in mice; however, a non-neutralising Ab response was also observed [156]. These vaccine candidates are in an early stage of development and therefore the following sections will discuss candidates that have been more extensively studied.…”

“…Subunit, virus-like particle (VLP), viral vector, peptide and DNA vaccine approaches are listed with the vaccine target, genotype (Gt) and isolate from which the candidate was derived, tested species and the humoral and cellular immune responses were reported. DREP-HCV/MVA-HCV core, E1, E1, p7, NS2, NS3 Gt1a H77 mice non-neutralising IgG yes yes [156] pVax-N3-NS5b NS3, NS4, NS5b Gt1b, Gt3a mice N/A yes yes [157] * N/A (not applicable) for vaccine candidates that are not designed to elicit HCV-specific B cell responses. † N.D (not determined) in the study.…”