RESUMEN
Hypervariable region 1 (HVR1) is one of the potential neutralization domains in the E2 glycoprotein of hepatitis C virus (HCV). Point mutations of the HVR1 can lead to humoral immune escape in HCV-infected patients. In this study, we segregated the chronically infected viraemic sera from HCV-infected patients into populations of antibody-free virus and antibody-associated virus (AAV) and mapped potential epitopes within the E1E2 gene junction of AAV sequences (residues 364-430). Furthermore, we generated HCV pseudoparticles (HCVpp) derived from AAV sequences to assess their infectivity. We studied the neutralization potential of virus-free Fab obtained from antibody-virus complexes, in the HCVpp system. We observed selective targeting of clonotypic HCV variants from the quasispecies pool. Moreover, we identified potential neutralizing epitopes within the HVR1 and an additional epitope that overlapped with a broadly neutralizing AP33 epitope (amino acid 412-423 in E2). We observed a marked difference in the infectivity of HCVpp generated using E1E2 sequences isolated from AAV. We document reduction in the infectivity of HCVpp-H77 and HCVpp derived from AAV sequences when challenged with virus-free Fab. Our results provide novel insights into the complexities of engagement between HCV and the humoral immune system.
Asunto(s)
Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis C Crónica/inmunología , Inmunidad Humoral , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Endopeptidasa K/química , Mapeo Epitopo , Hepacivirus/genética , Hepatitis C Crónica/virología , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Pruebas de Neutralización , Suero/química , Suero/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genética , Viremia/inmunologíaRESUMEN
Longitudinal analysis of chronic hepatitis C virus (HCV) infection has shown that the virus has several adaptive strategies that maintain persistence and infectivity over time. We examined four serum samples from the same chronically infected HCV genotype 4a patient for the presence of IgG antibody-associated virus. RNA was isolated from antibody-associated and antibody-free virions. Subsequent to sequence analysis, 27 aa hypervariable region 1 (HVR1) peptides were used to test the humoral immune escape. We demonstrated that differential peptide binding of Fab was associated with a single amino acid change. We provide direct evidence of natural humoral immune escape by HCV within HVR1.
Asunto(s)
Hepacivirus/inmunología , Evasión Inmune , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Mutación Missense , Proteínas Virales/genética , Proteínas Virales/inmunología , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/sangre , Humanos , Estudios Longitudinales , ARN Viral/genética , Análisis de Secuencia de ADNRESUMEN
The humoral immune system responds to chronic hepatitis C virus (HCV) infection by producing neutralising antibodies (nAb). In this study we generated three HCV pseudoparticles in which E1E2 glycoprotein sequence was targeted by the host humoral immune system. We used patient derived virus free Fabs (VF-Fabs) obtained from HCV genotype 1a (n = 3), genotype 1b (n = 7) and genotype 3a (n = 1) for neutralisation of HCVpp produced in this study both individually and in combination. Based on the available anti-HCV monoclonal nAb mapping information we selected amino acid region 384-619 for conformational epitope mapping. Amongst our notable findings, we observed significant reduction in HCVpp infectivity (p<0.05) when challenged with a combination of inter genotype and subtype VF-Fabs. We also identified five binding motifs targeted by patient derived VF-Fab upon peptide mapping, of which two shared the residues with previously reported epitopes. One epitope lies within an immunodominant HVR1 and two were novel. In summary, we used a reverse epitope mapping strategy to identify preferred epitopes by the host humoral immune system. Additionally, we have combined different VF-Fabs to further reduce the HCVpp infectivity. Our data indicates that combining the antigen specificity of antibodies may be a useful strategy to reduce (in-vitro) infectivity.