Research And Development Of Human Monoclonal Antibodies Against Hepatitis C Virus

Background and Aims:Hepatitis C,as one of the most common infectious diseases in the world,seriously threatens human health and safety.Hepatitis C Virus(HCV)is a single-stranded positive-strand RNA virus which transmits mainly through the blood.In clinical treatment of hepatitis C,except classic ribavirin combined with interferon therapy,generic antiviral drugs have been widely used.With the widespread application of monoclonal antibodies in the treatment of tumors and infectious diseases,the researches on monoclonal antibody drugs targeting HCV conserved proteins and key factors have been carried out in domestic institutions and overseas.However,monoclonal antibodies for hepatitis C have not yet appeared on the market due to insufficient specificity or safety of the antibodies.In the present study,we focused on the research and development of human monoclonal antibodies against Hepatitis C virus with the platform of "Human Memory B Cell In Vitro Activation Specific Screening",providing more efficient drugs for treatment of Hepatitis C clinically.Methods and Results:1.Preparation of the antigen protein for HCV: HCV surface E2 glycoprotein was selected as the antigen protein,and the prokaryotic expression plasmid of p GEX-HCV-E2 was constructed.The expression and purification of the antigen protein were curried out using prokaryotic cell system.By exploring the optimal conditions,the antigen proteins(HCV-E2)were sucessefully expressed and purified for subsequent antibody screening.2.Isolation and activation of HCV specific memory B cells: A 50 ml of whole blood of hepatitis C rehabilitation volunteers was collected for monocyte isolation,and the total memory B cells were isolated by cell surface specific markers from the isolated monocytes,and then the memory B cells were activated into plasma cells which secreted antibodies in vitro.Finally,the positive plasma cells secreting specific antibodies were identified by enzyme-linked immunosorbent assay(ELISA)using HCV specific E2 antigen.The results showed that we successfully activated memory B cells in vitro and induced plasma cells to secrete antibodies,and screened positive cell wells by detecting antibodies in the culture supernatant.3.Construction of the antibody gene library: The total RNAs were first extracted from the plasma cells of the activated positive pore and then the c DNA was reversed transcriptionally.The genes of the antibody heavy chain and light chain were cloned into eukaryotic cell expression vector by nested PCR with two rounds of amplification,gel recovery,enzyme digestion and enzyme link,respectively,to complete the construction of the antibody heavy light chain variable region gene library.Through the verification of each key step,we successfully constructed the antibody heavy and light chain gene library.4.Screening of specific monoclonal antibodies: The heavy chain and light chain of the antibody plasmids were firstly co-transfected into HEK 293 T cells,and the antibodies secreted into the medium were screened by ELISA using E2 antigen,and finally the HCV-specific whole human monoclonal antibodies were screened.Our results showed that five specific human monoclonal antibodies against HCV surface E2 proteins were successfully selected from multiple heavy and light chain combination sequences using stepwise screening validation.Conclusions:1.The target antigen protein(HCV-E2)were successfully expressed and purified in the prokaryotic system(E.coli),in which the protein was verified to be sued for further the screening of HCV-specific antibodies.2.Five specific monoclonal antibodies against HCV surface E2 glycoprotein were successfully selected using the unique screening combination mode,provided the basis for the development of monoclonal antibody drugs in the treatment of hepatitis C clinically.