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Plasmids expressing variants of the hepatitis C virus (HCV) core, E1 and E2 proteins individually or as polyproteins were administered to BALB/c mice. All plasmids induced a detectable and specific antibody response. Antibody titres against core, E1 and E2 proteins, 19 weeks after primary immunization, ranged from 1:50 to 1:4500 depending on the inoculated plasmid and the HCV antigen evaluated. Constructs expressing HCV envelope proteins as polyprotein variants including the core amino acid region induced statistically stronger antibody responses than plasmids encoding individual E1 and E2 proteins. Particularly, the pIDKE2 plasmid, expressing the first 650 amino acids in the viral polyprotein, induced a potent and multispecific antibody and lymphoproliferative response against HCV core, E1 and E2 proteins. Anti-E2 antibodies generated by pIDKE2 immunization were cross-reactive to hypervariable region-1 peptides from different genotypes. Immunization with the pIDKE2 also generated a positive cellular immune response against the core antigen, determined by interferon-gamma enzyme-linked immunospot (ELISPOT) assay, and induced detectable levels of interferon-gamma but not interleukin-4 in vaccinated mice. The detection of both antibody and cytotoxic T-lymphocyte responses, potentially targeted to circulating or cell-infecting virions respectively, in mice vaccinated with the pIDKE2 plasmid is very attractive for the effective eradication of HCV infection.

Production of immunogenic hepatitis C virus (HCV) envelope proteins will assist in the future development of preventive or therapeutics applications. Only properly folded monomeric E2 protein is able to bind a putative cellular co-receptor CD81, but this interaction may modulate cell immune function. Recombinant E2 proteins, similar to the native form, but lacking undesirable immunoregulatory features, might be promising components of vaccine candidates against HCV. To obtain E2 suitable for structural as well as functional studies, a recombinant E2 variant (E2680) was produced in Pichia pastoris cells. E2680, comprising amino acids 384 to 680 of the HCV polyprotein, was secreted into the culture supernatant in the N-glycosilated form and was mainly composed of disulfide-linked multimers. Both monomeric and oligomeric forms of E2680 were recognized by conformational-sensitive MAb H53. In addition, antibodies in sera from 70% of HCVpositive patients were reactive against E2680. By immunizing E2680 in BALB/c mice, both a specific cellular immune response and anti-E2680 IgG antibody titers of 1:200,000 were induced. Our data suggest that recombinant E2680 could be useful to successfully induce strong anti-HCV immunity.

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In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine profile in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-γ producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.

Immunological response against envelope protein E1 is very important in natural hepatitis C virus (HCV) infection, although it is insufficient to clear the viraemia. The HCV genomic region encoding the first 149 amino acids of the envelope E1 protein (E1(340), amino acids 192-340) was expressed in Escherichia coli (to a level of 30% of the whole cellular proteins) and purified to 85%. We measured the immune response in rabbits and mice as well as the reactivity against 37 human sera raised against the whole recombinant protein and E1-encoding peptides. From this, 51.1% of human sera were found to react with E1(340). High-level antibodies against E1(340) were obtained in rabbits and mice when immunized. These antibodies had a similar peptide-recognition pattern to that described previously for human sera. The most reactive region was located at the N-terminus of the E1 protein. Cellular immunity in mice was evaluated by delayed-type hypersensitivity assay. It revealed the induction of a CD4+ T-cell-mediated response by this protein. This E1(340) protein and the animal-derived anti-E1 sera are immunological tools that could aid in the monitoring and development of anti-HCV therapies.

In the present study, we evaluated the capability of the plasmid pIDKE2, encoding the HCV (hepatitis C virus) structural proteins Core, E1 and E2, to induce immune response against HCV antigens after injection into rabbits and Macaca irus (crab-eating macaque). Animals were immunized intramuscularly with different amounts of plasmid on weeks 0, 3 and 8. Monkeys received a booster dose on week 46. All rabbits immunized with pIDKE2 generated a positive antibody response and, particularly in rabbits immunized with 2 mg, antibody titres reached values above 1:1500 and 1:400 against the core and the envelope proteins, respectively, 28 weeks after primary immunization. The antibody response in monkeys developed slowly, but antibody titres greater than 1:3500 against HCV structural antigens were detected at week 52. Moreover, anti-E2 antibodies recognized synthetic peptides covering the HVR-1 (hypervariable region-1) from different isolates corresponding to different genotypes. Additionally, a specific lymphoproliferative response against Core and E2 was detected in two out of three monkeys immunized with pIDKE2. The other monkey had a specific proliferative response to E1. Taking all these data together, immunization with pIDKE2 is able to elicit both humoral and cellular immunity against HCV structural antigens in animal models other than mice.