HCV And HBV Specific T Cell Immune Response And Molecular Mechanism Research

Hepatitis C is a blood-borne infectious disease caused by the hepatitis C virus(HCV)and it has a worldwide prevalence.According to the World Health Organization report the prevalence of hepatitis C averages 3%around the world.An estimated 170 million people are infected with the hepatitis C virus.Each year,3 million to 4 million new infections are added and 250,000 deaths are caused by HCV infection.Hepatitis C has become an important issue that threats global public health.At present,due to the diversity of HCV genes and the lack of ideal animal and cell infection models,HCV vaccine research still faces many difficulties.T cell responses play a key role in the spontaneous clearance of viruses in acute HCV infection.Studies have show-n that strong HCV-specific CD8+T cell responses occur in self-limited HCV infected patients or chimpanzees,but weaker CD8+T cell responses are observed in chronically infected hosts.To explore the characteristics of T cells in the natural course of HCV infection,and the correlation between T cell immunity and disease progression.We recruited a special cohort from Hebei Province which infected HCV by blood donation during 1970 to 1990.They did not receive any treatment.We used NS3 protein over-lapping peptide library to stimulate PBMCs in vitro.Flow cytometry analysis of T cell cytokine secretion,memory cell type and inhibitory molecule expression,etc.,we explored the characteristics and differences of T cell immunity in self-resolved group and chronic infection group.The results showed that the cytokine secretion levels of CD8+T cells in the self-resolved group were significantly higher than those in the chronic infection group and the healthy control group.HCV-specific CD8+T cells of the two groups were mainly effector memory T cells(TEM)and effector T cells(TEMRA),while the HCV-specific CD4+T cells were mainly effector memory T cells(TEM)and center memory T cells(TCM).Meanwhile,the expression of inhibiting receptors on virus-specific CD8+ T cells of chronic patients was characterized with PD-1high,TIM-3high expression,which was associated with liver injury characterized by abnormal AST or ALT levels and morphopathologic changes.Interestingly,the expression of LAG-3 on CD8+T cells in chronic patients was lower which was negatively correlated with ALT or AST.Our data indicated the characteristics of virus-specific T-cell immunity in long-term infected HCV patients and helped elucidate the role of immune responses in the associated clinical process.At the same time,we also conducted molecular level study of people with HCV chronic infection.We carried out genotyping of the infected HCV virus through molecular biology experiments and obtained 26 whole genome sequences using one generation sequencing and next generation sequencing.According to genotyping results,these individuals were infected with two genotypes,HCVlb(17)and HCV2a(9).For the whole genome sequence,we have found that the sequences of HCVIb or HCV2a had high homology based on the evolution distance analysis of MEGA7.0.Through phylogenetic tree analysis with domestic and foreign HCV sequences,the sequences of HCV2a were relatively clustered and clustered on an independent branch,and the phylogenetic relationship with NDM228 strain which measured abroad was relatively close.The 17 sequences of HCVlb were relatively dispersed.The phylogenetic tree of each protein coding region was also analyzed.The results were basically consistent with the analysis of the entire gene sequence.Through the second-generation sequencing,we obtained the information of the original reads,the splicing sequence,the sequencing depth of each site of each sequence,entropy,mut rate and so on.The second-generation sequencing of 26 sequences has been completed.Data analysis is still under way.We analyze the quasi-species complexity,immune escape mutations of neutralizing antibody epitopes and important T-cell epitopes.We combine clinical data to explore its relevance with liver injury.In addition,the molecular structure of MHC plays an important role in the study of T cell immunity.MHC molecules can limit TCR recognition.Moreover,the conformational coordination between polypeptide and MHC is crucial for the identification of TCR.If the key armio acid is mutated followed with a change in conformation,the binding of the pMHC complex to the TCR is weakened.The same MHC-polypeptide can be recognized by different TCRs,and the flexibility of the MHC-polypeptide complex is also one of the reasons that the TCR can be cross-recognized.It can be seen that the binding ability of MHC-polypeptides and their conformational changes affect TCR recognition.Based on the analysis of the molecular structure of H-2Kd-HBV peptide complex and the structural analysis and analysis of HLA-B*4001,we found that both complexes have a positively charged amino acid R and a negatively charged amino acid E.The formed salt bridge participates in the binding of MHC to polypeptides.We performed single and double mutations on the two amino acids respectively.We did protein refolding,circular dichroism which tested the thermostability of protein,and the recognition of HBV-specific CD8+T cells by H-2Kdtetramer.The results all proved that the amino acid mutation in the salt bridge affected peptide binding to MHC,stability of complexes,and recognition of TCR by peptide-MHC complexes.In summary,this study explored the characteristics of T-cell immunity in the nature course of HCV infection and the correlation between T-cell immunity and the extent of liver damage,and analyzed the full genome sequence of HCV in infected individuals.Moreover,the importance of salt bridges in MHC-peptide binding and TCR recognition was found.This study has certain indicative and guiding significance for the prevetion and treatment of HCV and HBV.