RESUMEN
BACKGROUND: Hepatitis C virus (HCV) infection is often persistent and gradually advances from chronic hepatitis to liver cirrhosis and hepatocellular carcinoma (HCC). Worldwide, hepatocellular carcinoma is the fifth most common neoplasm. METHOD OF STUDY: the Interferon lambda (IFNL) polymorphisms genotypes (rs8099917, rs12979860 and rs12980275) and the presence of mutations in HCV core protein were analyzed in 59 patients with HCC, and also in 50 cirrhotic patients (without HCC). RESULTS: the rs12980275-AG genotype was associated with HCC on age-adjusted analysis (OR 2.42, 95% CI 1.03-5.69, P=0.043). Core substitutions R70Q and L91M were mainly found in genotype 1b isolates. Furthermore, a borderline level of statistical significance association was found among the presence of amino acid Glutamine (Q) in the position 70 and IFNL3 genotype AG (P=0.054). CONCLUSIONS: the screening of these polymorphisms and functional studies would be useful in clinical practice for identifying groups at high risk of HCC development.
Asunto(s)
Carcinoma Hepatocelular/virología , Hepacivirus/genética , Antígenos de la Hepatitis C/genética , Interleucinas/genética , Neoplasias Hepáticas/virología , Proteínas del Núcleo Viral/genética , Anciano , Carcinoma Hepatocelular/genética , Femenino , Fibrosis/genética , Fibrosis/virología , Humanos , Interferones , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Polimorfismo GenéticoRESUMEN
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.
Asunto(s)
Clonación Molecular , Hepacivirus/genética , Pichia/genética , Procesamiento Proteico-Postraduccional , Proteínas Estructurales Virales/genética , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Hepacivirus/metabolismo , Antígenos de la Hepatitis C/biosíntesis , Antígenos de la Hepatitis C/genética , Antígenos de la Hepatitis C/metabolismo , Proteínas del Núcleo Viral/biosíntesis , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Estructurales Virales/biosíntesis , Proteínas Estructurales Virales/metabolismoRESUMEN
The expression and processing of the Hepatitis C virus core protein (HCcAg) were analyzed in the methylotrophic yeast Pichia pastoris. Two proteins with 21 (p21) and 23 kDa (p23) were detected in immunoblot with a serum from a chronic carrier patient, as the major products for HCcAg. Both proteins, p21 and p23, produced by proteolytic processing in P. partoris, share the same N-terminal region and reacted with a monoclonal antibody towards the first 35 amino acids of HCcAg. The proteolytic processing of the recombinant polypeptide, having the HCcAg and the first 148 aa of E1 protein, was also confirmed by immunoblot analysis using mAbs with HCcAg and E1 specificities, respectively. The 32 kDa glycosilated E1 protein was then immuno-identified, as well as the processed HCcAg. These data demonstrated the usefulness of P. pastoris, as expression system, to study the processing of HCV structural proteins.
Asunto(s)
Pichia/genética , Precursores de Proteínas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Antígenos de la Hepatitis C/genética , Antígenos de la Hepatitis C/inmunología , Antígenos de la Hepatitis C/metabolismo , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Transformación Genética , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunologíaRESUMEN
The immunogenicity of a truncated HCV core protein (Co.120) was studied in BALB/c and C57BL/6 mice, given three intramuscular injections of antigen, adjuvanted with either aluminum hydroxide or Freund's adjuvant. A rapid antibody response was noted after the first dose, with both strains of mice eventually exhibiting comparable levels of anti-core IgG (titers >1:100000), with a mixed IgG1/IgG2a subclass response. Spleen cells from Co.120-immunized mice gave a significant specific proliferative response. IFN-gamma gene expression was also detected after an ex-vivo specific stimulation of spleen cells in all immunized mice. This response was independent of dose, H-2 genetic background or type of adjuvant. The results indicated that immunization with the Co.120 protein elicits a potent anti-HCV humoral and cellular immune response.
Asunto(s)
Hepacivirus/inmunología , Antígenos de la Hepatitis C/inmunología , Proteínas del Núcleo Viral/inmunología , Animales , Especificidad de Anticuerpos , Femenino , Expresión Génica , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/biosíntesis , Antígenos de la Hepatitis C/química , Antígenos de la Hepatitis C/genética , Inmunidad Celular , Inmunoglobulina G/biosíntesis , Técnicas In Vitro , Interferón gamma/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Células TH1/inmunología , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genéticaRESUMEN
DNA immunization is a promising approach in generating immune responses to infectious pathogens in many different animal models. In an effort to augment the anti-[hepatitis C virus (HCV) core] immune response, generated after DNA immunization, the importance of vaccination regimen regarding dose and boosting was investigated in the present study. Balb/c mice were intramuscularly injected with an expression plasmid encoding a truncated variant comprising amino acids 1-176 of the HCV core protein. The highest anti-core antibody titres (1:3700) were detected in mice inoculated with 50-100 microg of core-encoding plasmid. Additionally, we demonstrated that antibody levels induced by a single injection of DNA could be further increased by boosting with a second injection of DNA three weeks after primary immunization. However, administration of additional doses or lengthening of the resting period between inoculations resulted in similar or even weaker anti-core antibody responses. A similar anti-(HCV core) lymphoproliferative response was also detected in animals that had the highest level of anti-core antibodies. These results indicate that, in clinical trials, vaccination regimen might be a critical factor in generating optimal anti-(HCV core) immune responses after genetic immunization.
Asunto(s)
Hepacivirus/inmunología , Antígenos de la Hepatitis C/genética , Plásmidos , Animales , División Celular/inmunología , Femenino , Anticuerpos contra la Hepatitis C/biosíntesis , Antígenos de la Hepatitis C/inmunología , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Linfocitos T/citología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
Antibody reactivities to hepatitis C virus (HCV) antigens and to synthetic peptides derived from different parts of the HCV genome (core, NS4, and NS5) were evaluated in HCV-infected hemodialysis patients. In the RIBA 3 assay, NS5 was significantly less recognizable by sera of hemodialysis patients compared to other HCV-infected subjects. Among hemodialysis patients, those coinfected with hepatitis B virus (HBV) (positive for hepatitis B surface antigen [HBsAg+]) showed a reduction in reactivity to C33 and C100. Sera of only 23% of the hemodialysis patients (37 of 161) reacted with more than three of eight peptides tested, significantly fewer than the 60% (12 of 20) of the sera of other HCV-infected patients tested (P = 0.001). This immunosuppression was also manifested by a reduced frequency of recognition of additional peptides on follow-up. An even more reduced reactivity was observed among the HBV-coinfected patients (HBsAg+). The low-responder hemodialysis patients were not infected with any particular genotype of HCV, and the same HCV genotypes observed in the whole group of hemodialysis patients (1a, 1b, 2a, and 3a) were found circulating in the low-responder group. Even in this low-responder population, the good performance of two peptides (peptide 716, corresponding to a portion of the core, and peptide 59, corresponding to a portion of NS4) corroborates the immunodominance of the conserved epitopes within these peptides.