Generation And Identification Of Human Monoclonal Antibodies Against The Rabies Virus

Rabies is a zoonotic infections disease of central nervous system caused by rabies virus that infects human being as well as animals all over the world. It is so far the only human case fatality rate of up to100%of acute B infectious diseases. China is one of the most serious national rabies harm in the second place. In recent years, an epidemiological research demonstrated that the patients were bitten by dogs and died of rabies significantly increased in China,it has become a serious public-health and social problem.According to the categorization of exposure defined by the World Health Organization (WHO), the most severe cases, category III of patient require rabies vaccination and direct wound infiltration with rabies immunoglobulin (RIG). Now the widely used RIG is HRIG and ERIG, but these immunoglobulins from blood are difficult to meet the current needs because of the high cost and low yield. In addition, the quality of bloo-borne products is difficult to control and there is a potential risk of virus infection and anaphylaxis when using blood-borne immunoglobulin.The fully human or humanized therapeutic monoclonal antibody drugs provide a new solution for the prevention and treatment of post-exposure to the rabies virus, the human monoclonal antibody drugs replace the extensive use of blood-borne immunoglobulin has become the consensus of the clinicians and biopharmaceutical experts at home and abroad.It is the first time that our study group used the human IgM transgenic mice imported from the UK Medical Research Council and Cambridge, combined with the hybridoma technique and enzyme-linked immunosorbent assay (ELISA) high-throughput cross-screening technique (HTS), prepared the anti-rabies virus human monoclonal antibodies and further to identify their biologic activity. The first-phase preparations of mAbs would be the bases for the screening and application studys in terms of immunoprophylaxis neutralized monoclonal antibody and immunotherapy on the rabies.Methods:1. The human IgM transgenic mice were immunized with the inactivated rabies virus (CTN strain) by multiple sites subcutaneous immunization and intraperitoneal immunization.2. The human anti-rabies virus hybridoma cell lines were screened and produced by using the classical hybridoma technique in a combinatory enzyme linked immunosorbent assay high throughput screening (HTS) strategy.3. Ammonium sulfate precipitation method combined with the Protein L affinity chromatography were uesed to purify the human anti-rabies virus mAbs, the purity of the mAbs and the molecular size of heavy chain and light chain were identified by SDS-PAGE. The human mAbs were identified via the double antibody sandwich enzyme-linked immunosorbent assay (ELISA).4. The specificity of mAbs was determined by the indirect ELISA and the Dot Blot assay. In addition, the affinity of mAbs was detected by BiaCore X-100system.5. Expression and purification of rabies virus glycoprotein, and analyze the binding properties of human anti-rabies virus mAbs with the glycoprotein by Western Blot.Results1. Five anti-rabies virus hybridoma cell lines6B2、8G2、9E3、9F2、16B1have been established, they can stably secret human anti-rabies virus mAbs.2. Five human anti-rabies virus mAbs were purified, and the SDS-PAGA results showed that the purified mAbs with high purity, the molecular weight of heavy chain and light chain are75kDa and25kDa, respectively. The double antibody sandwich enzyme-linked immunosorbent assay (ELISA) demonstrated that the five anti-rabies virus mAbs can produce human IgM mAbs.3. The five anti-rabies virus mAbs can recognize the CTN strain of rabies virus specifically in the indirect ELISA and the Dot Blot assays. The equilibrium dissociation constants (KD) of8G2、9E3、9F2are2.32×10-10M、2.62×10-10M and4.06×10-11M, respectively.4. Expression and purification of rabies virus glycoprotein. Western Blot results proved that three human anti-rabies virus mAbs8G2,9E3,9F2could specifically combine with the rabies virus glycoprotein.ConclusionsIn this study, five hybridoma cell lines have been established, they can stably produce human anti-rabies virus IgM monoclonal antibodies. These mAbs could specifically recognize the inactivated rabies virus CTN strain, three of them showed high affinity and could specifically combine with the rabies virus glycoprotein.