A chimeric protein encompassing hepatitis C virus epitopes is able to elicit both humoral and cell-mediated immune responses in mice.

Hepatitis C virus (HCV) infection is a worldwide health problem. Vaccines against this pathogen are not available and advances in this field are limited because of the high genetic variability of the virus, inaccessibility of animal models, and incomplete definition of immunological correlates of protection. In the present work, a chimeric protein, Eq1, encompassing HCV amino acid regions from structural antigens, was generated. Eq1 was expressed in GC-366 bacterial cells. After cell disruption, Eq1 was purified from the insoluble fraction by sequential steps of differential solubilization and metal chelating affinity chromatography. Eq1 was specifically recognized by anti-HCV positive human sera. Moreover, immunization of BALB/c mice with different doses of Eq1 formulated either in Alum or Freund's incomplete adjuvant elicited both humoral- and cellular-specific immune responses. Doses of 20 µg of Eq1 induced the strongest cell-mediated immune responses and only the formulation of this dose in Alum elicited a neutralizing antibody response against heterologous cell culture HCV. All these data together indicate that Eq1 is immunogenic in mice and might be an interesting component of vaccine candidates against HCV infection.

Ratio of HCV structural antigens in protein-based vaccine formulations is critical for functional immune response induction.

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.

Recombinant in vitro assembled hepatitis C virus core particles induce strong specific immunity enhanced by formulation with an oil-based adjuvant.

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 pro file 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-gamma 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.

Induction of IgA and sustained deficiency of cell proliferative response in chronic hepatitis C.

AIM: In the present study, antibody and peripheral blood mononuclear cells (PBMC) proliferative responses against hepatitis C virus (HCV) antigens were evaluated in HCV chronically infected patients. METHODS: Paired serum and PBMC samples were taken six months apart from 34 individuals, either treated or not, and tested by enzyme-linked immunosorbent assay (ELISA) and carboxyfluorescein succinimidyl ester staining. RESULTS: Over 70% of the patients showed specific IgG and IgM against capsid, E1 and NS3, while HVR-1 was recognized by half of the patients. An increase in the levels of the anti-capsid IgM (P = 0.027) and IgG (P = 0.0006) was observed in six-month samples, compared to baseline. Similarly, a significantly higher percent of patients had detectable IgA reactivity to capsid (P = 0.017) and NS3 (P = 0.005) after six months, compared to baseline. Particularly, IgA against structural antigens positively correlated with hepatic damage (P = 0.036). IgG subclasses evaluation against capsid and NS3 revealed a positive recognition mediated by IgG1 in more than 80% of the individuals. On the contrary, less than 30% of the patients showed a positive proliferative response either of CD4+ or CD8+ T cells, being the capsid poorly recognized. CONCLUSION: These results confirm that while the cellular immune response is narrow and weak, a broad and vigorous humoral response occurs in HCV chronic infection. The observed correlation between IgA and hepatic damage may have diagnostic significance, although it warrants further confirmation.

Immunization with a recombinant fowlpox virus expressing a hepatitis C virus core-E1 polyprotein variant, protects mice and African green monkeys (Chlorocebus aethiops sabaeus) against challenge with a surrogate vaccinia virus.

HCV (hepatitis C virus) is a worldwide health problem nowadays. No preventive vaccine is available against this pathogen, and therapeutic treatments currently in use have important drawbacks, including limited efficacy. In the present work a recombinant fowlpox virus, FPCoE1, expressing a truncated HCV core-E1 polyprotein, was generated. FPCoE1 virus generally failed to elicit a humoral immune response against HCV antigens in BALB/c mice. By contrast, mice inoculated with FPCoE1 elicited a positive interferon-gamma secretion response against HCV core in ex-vivo ELISPOT (enzyme-linked immunospot) assays. Remarkably, mice inoculated with FPCoE1 significantly controlled viraemia in a surrogate challenge model with vvRE, a recombinant vaccinia virus expressing HCV structural antigens. In fact, 40% of the mice had no detectable levels of vvRE in their ovaries. Administration of FPCoE1 in vervet monkeys [Chlorocebus (formerly Cercophitecus) aethiops sabaeus] induced lymphoproliferative response against HCV core and E1 proteins in 50% of immunized animals. Monkeys immunized with FPCoE1 had no detectable levels of vvRE in their blood, whereas monkeys inoculated with FP9, the negative control virus, had detectable levels of vvRE in blood up to 7 days after challenge. In conclusion, recombinant fowlpox virus FPCoE1 is able to induce an anti-HCV immune response in mice and monkeys. This ability could be rationally employed to develop effective strategies against HCV infection by using FPCoE1 in combination with other vaccine candidates or antiviral treatments.

Neutralizing antibodies and broad, functional T cell immune response following immunization with hepatitis C virus proteins-based vaccine formulation.

HCV is a worldwide health problem despite the recent advances in the development of more effective therapies. No preventive vaccine is available against this pathogen. However, non-sterilizing immunity has been demonstrated and supports the potential success of HCV vaccines. Induction of cross-neutralizing antibodies and T cell responses targeting several conserved epitopes, have been related to hepatitis C virus (HCV) clearance. Therefore, in this work, the immunogenicity of a preparation (MixprotHC) based on protein variants of HCV Core, E1, E2 and NS3 was evaluated in mice and monkeys. IgG from MixprotHC immunized mice and monkeys neutralized the infectivity of heterologous HCVcc. Moreover, strong CD4+ and CD8+ T cells proliferative and IFN-γ secretion responses were elicited against HCV proteins. Remarkably, immunization with MixprotHC induced control of viremia in a surrogate challenge model in mice. These results suggest that MixprotHC might constitute an effective immunogen against HCV in humans with potential for reducing the likelihood of immune escape and viral persistence.

HCV-specific immune responses induced by CIGB-230 in combination with IFN-α plus ribavirin.

AIM: To analyze hepatitis C virus (HCV)-specific immune responses in chronically infected patients under triple therapy with interferon-α (IFN-α) plus ribavirin and CIGB-230. METHODS: CIGB-230 was administered in different schedules with respect to IFN-α plus ribavirin therapy. Paired serum and peripheral blood mononuclear cells (PBMC) samples from baseline and end of treatment were analyzed. The HCV-specific humoral response was tested by enzyme-linked immunosorbent assay, neutralizing antibodies were evaluated by cell culture HCV neutralization assays, PBMC proliferation was assayed by carboxyfluorescein succinimidyl ester staining and IFN-γ secretion was assessed by enzyme-linked immunospot. Data on virological and histological response and their association with immune variables are also provided. RESULTS: From week 12 to week 48, all groups of patients showed a significant reduction in mean leukocyte counts. Statistically significant reductions in antibody titers were frequent, but only individuals immunized with CIGB-230 as early add-on treatment sustained the core-IgG response, and the neutralizing antibody response was enhanced only in patients receiving CIGB-230. Cell-mediated immune responses also tended to decline, but significant reductions in IFN-γ secretion and total absence of core-specific lymphoproliferation were exclusive of the control group. Only CIGB-230-immunized individuals showed de novo induced lymphoproliferative responses against the structural antigens. Importantly, it was demonstrated that the quality of the CIGB-230-induced immune response depended on the number of doses and timing of administration in relation to the antiviral therapy. Specifically, the administration of 6 doses of CIGB-230 as late add-on to therapy increased the neutralizing antibody activity and the de novo core-specific IFN-γ secretion, both of which were associated with the sustained virological response. CONCLUSION: CIGB-230, combined with IFN-α-based therapy, modifies the immune response in chronic patients. The study provides evidence for the design of more effective therapeutic vaccine interventions against HCV.

Hepatitis C virus (HCV) core protein enhances the immunogenicity of a co-delivered DNA vaccine encoding HCV structural antigens in mice.

In the present study, recombinant HCV (hepatitis C virus) core proteins enhanced the immune response elicited by a co-delivered DNA vaccine encoding HCV core and envelope proteins. A mixture of the plasmid pIDKE2 and Co.173, a protein comprising the first 173 amino acids of HCV core, in particular induces a strong humoral response, including antibodies that recognized peptides representing hypervariable region I from different viral isolates. Moreover, positive lymphoproliferative responses against the HCV structural proteins, encoded by the plasmid, were detected after two doses with this mixture. When the HCV core protein used in the mixture with pIDKE2 was Co.120, a protein comprising the first 120 amino acids of the viral antigen, a strong humoral response and a positive lymphoproliferative response were also detected. The effectiveness of this formulation was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV core protein. A 2 log reduction in vaccinia-virus titre was observed in mice immunized with the mixture of pIDKE2 and Co.120. Humoral and cellular immune responses elicited for the mixture of pIDKE2 with either Co.173 or Co.120 was stronger and more diverse than those generated by individual components. In conclusion, our results indicate that formulations comprising both DNA constructs and protein subunit vaccine candidates are able to elicit strong humoral and cellular immunity against several antigens. Particularly, HCV core protein might be used as a feasible vehicle/adjuvant for DNA vaccines.

Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast.

Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384-605 or aa 384-746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.

In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein.

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.

Recombinant in vitro assembled hepatitis C virus core particles induce strong specific immunity enhanced by formulation with an oil-based adjuvant

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 pro file 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.