RESUMO
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.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Imunidade Celular , Imunidade Humoral , Proteínas Recombinantes de Fusão/imunologia , Animais , Epitopos/imunologia , Adjuvante de Freund , Hepatite C/prevenção & controle , Humanos , Lipídeos , Camundongos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
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.
Assuntos
Antivirais/administração & dosagem , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/administração & dosagem , Ribavirina/administração & dosagem , Vacinas de DNA/administração & dosagem , Vacinas contra Hepatite Viral/administração & dosagem , Adulto , Antivirais/efeitos adversos , Biomarcadores/sangue , Células Cultivadas , Cuba , Método Duplo-Cego , Esquema de Medicação , Quimioterapia Combinada , Feminino , Hepacivirus/genética , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Esquemas de Imunização , Interferon alfa-2 , Interferon-alfa/efeitos adversos , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Ribavirina/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Vacinas de DNA/efeitos adversos , Vacinas contra Hepatite Viral/efeitos adversosRESUMO
Approximately 170 million people are infected with the hepatitis C virus (HCV) worldwide. Infection with this pathogen is persistent in more than 80% of cases, frequently developing severe forms of liver damage such as cirrhosis and hepatocellular carcinoma. No preventive vaccine is available against HCV, and current treatment based on the combination of pegylated interferon and ribavirin is effective in â¼55% of patients infected with genotype 1, the most prevalent genotype. This review analyzes several factors influencing the achievement of a sustained virological response, namely undetectable HCV RNA at 6 months after conclusion of therapy. Particularly, the relevant issue of age and duration of infection is discussed in detail. Indeed, the final decision for starting treatment should be a case-by-case point. However, the cost-benefit analysis seems to indicate that in patients who are motivated and without contraindications, starting the treatment as early as possible is probably the best choice for success.
Assuntos
Hepacivirus/fisiologia , Hepatite C Crônica/terapia , Interferons/uso terapêutico , RNA Viral/análise , Ribavirina/uso terapêutico , Animais , Análise Custo-Benefício , Hepacivirus/patogenicidade , Hepatite C Crônica/imunologia , Humanos , Imunidade , Interferons/economia , Medicina de Precisão , Ribavirina/economiaRESUMO
CIGB-230, a mixture of a DNA plasmid expressing hepatitis C virus (HCV) structural antigens and a HCV recombinant capsid protein, has demonstrated to elicit strong immune responses in animals. The present study evaluated the plasmid biodistribution after the administration of CIGB-230 in mice, as well as toxicity of this vaccine candidate in rats. In the biodistribution study, mice received single or repeated intramuscular injections of CIGB-230, 50 microg of plasmid DNA mixed with 5 microg of Co.120 protein. Plasmid presence was assessed in ovaries, kidney, liver, pancreas, mesenteric ganglion, blood, and muscle of the injection site by a qualitative polymerase chain reaction. The toxicology evaluation included treatment groups receiving doses 5, 15, or 50 times higher, according to the body weight, than the expected therapeutic clinical dose. During the first hour after repeated inoculation, a promiscuous distribution was observed. However, 3 months later, plasmid could not be detected in any tissue. There was an absence of detectable adverse effects on key toxicology parameters and no damage evidenced in inspected organs and tissues. These results indicate that CIGB-230 is nontoxic at local and systemic levels and no concerns about persistence are observed, which support clinical testing of this vaccine candidate against HCV.
Assuntos
Hepacivirus/imunologia , Hepatite C/prevenção & controle , Vacinas de DNA/farmacocinética , Vacinas de DNA/toxicidade , Vacinas contra Hepatite Viral/farmacocinética , Vacinas contra Hepatite Viral/toxicidade , Animais , Feminino , Hepacivirus/genética , Antígenos de Hepatite/genética , Antígenos de Hepatite/imunologia , Hepatite C/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Testes de Toxicidade , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologiaRESUMO
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.
Assuntos
Proliferação de Células , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Antígenos da Hepatite C/imunologia , Hepatite C Crônica/imunologia , Imunoglobulina A/sangue , Leucócitos Mononucleares/imunologia , Adulto , Formação de Anticorpos , Antivirais/uso terapêutico , Proteínas do Capsídeo/imunologia , Proliferação de Células/efeitos dos fármacos , Feminino , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Humanos , Interferon-alfa/uso terapêutico , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Ribavirina/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/virologia , Regulação para Cima , Proteínas do Envelope Viral/imunologia , Proteínas não Estruturais Virais/imunologiaRESUMO
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.