Research And Development Of Human Monoclonal Antibody Drugs Against Rabies Virus

Background and aims:Rabies is a zoonotic B-type infectious disease mainly caused by rabies virus(RABV)and the mortality rate is close to 100%.The rabies mortality rate in China is the second in Asia,and more than 10 billion RMB have been spent for the prevention of rabies annually in China,which has a huge economic burden on society.At present,the prevention of rabies level 3 exposure mainly relays on injections of human or equine anti-rabies virus immunoglobulin,but the former is expensive and the latter has lots side effects,and both of them are difficult to produce in large quantities.Human monoclonal antibodies specific for rabies virus have the advantages of strong targeting,high sensitivity,low toxicity and side effects.At present,there’re rarely monoclonal antibodies on market for rabies.RABV glycoprotein(GP)is the only protective antigen protein on the surface of the virus.Therefore,this project aims to screen human monoclonal antibodies with rabies virus GP fragment using the established human memory B cell activation platform in our lab,providing the efficient drugs for prevention and treatment of rabies clinically.Methods:1.Preparation of antigen protein of rabies virus: Based on the literatures,patents and some other materials,the glycoprotein(GP)of rabies virus CTN-1 was chosen as the antigen protein and the gene sequence was obtained from Genbank(SEQ NO.:FJ959397.1).The sequence of the entire extracellular domain(1-439 amino acid)that includes all antigenic sites was constructed into a GST expression vector and the expression and purification of the protein were carried out with a prokaryotic expression system.The recombinant protein was confirmed by Western bolt and Elisa.2.Isolation and activation of RABV-specific memory B cells: The individuals with higher RABV Ig G content were screened from multiple volunteers who had been vaccinated with rabies vaccine.A 50 ml of whole blood(anticoagulant)was taken from each individual.Memory B cells were first isolated from the blood using magnetic bead sorting kit,then the memory B cells were activated into plasma cells in vitro.Finally,positive plasma cells secreted specific antibodies were identified by Elisa assay using RABV specific antigen.3.Construction of the gene bank of the antibodies against rabies virus: the RNAs were first extracted from the activated plasma cells with RABV-specific antibodies in the positive wells,and the sequences of heavy and light chain variable regions were reversely transcribed into c DNA.The antibody heavy chain and light chain genes were cloned into eukaryotic cell expression vectors by nested PCR with two rounds of amplification,glue recovery and enzyme digestion.4.Screening of the specific human monoclonal antibodies: The plasmid mixture of the whole library was first obtained by bacterial transformation,then the large mixed library with heavy chains or light chains was divided into several parts,then a small heavy chain group and a small light chain group were assembled into complete antibody.Finally,the positive group was screened by Elisa,then the positive result group was divided into smaller components until the single antibody clone was screened,and the single specific human monoclonal antibody was selected step by step through this mode.Results:1.We successfully obtained the antigen protein from E.coli system,and detect the specificity and sensitivity of antigen-antibody binding and detection by Western blot and Elisa,the results indicated that the antigen protein could be used for screening of the antibodies.2.We successfully isolated the RABV-specific memory B cells from the fresh blood of RABV Ig G positive volunteers with the antigen of RABV,and the cells were activated into the plasma cells,in which the antibodies could be secreted,then we select the positive plasma cells.3.We successfully constructed the gene bank of antibodies with heavy and light chain variable region.Five colonies sequences of the specific human monoclonal antibodies against RABV were selected using GP protein.Conclusions:1.We have successfully constructed a prokaryotic expression vector of rabies virus-specific antigen protein,which has been successfully used for antibody screening by using the e.coli prokaryotic system.2.We successfully screened five colonies of the specific human monoclonal antibodies against rabies virus using the GP protein in our lab,which laid the foundation for the development of the antibody drugs for rabies treatment clinically.