Generation And Identification Of Human Monoclonal Antibodies Against The Rabies Virus Glycopreition

Rabies is an acute zoonotic infection disease of central nervous system, mainly for peripheral nerve inflammation and acute encephalitis. This zoonosis is caused by rabies virus(RV) and the mortality of untreated cases is 100%. According to the World Health Organization(WHO), 500,000 people at least are given post-exposure vaccinations and rabies infections result in about 55,000 human deaths worldwide every year. The epidemic situation of rabies in China is up for the last few years and our country has become one of the countries most severely affected by rabies. Therefore, it is of great significance to develop effective drugs to prevent and control rabies.WHO recommends the use of rabies vaccination, and direct wound infiltration with rabies immunoglobulin(RIG) in category III exposures(WHO, 2005). Rabies immunoglobulin including purified human immunoglobulin(HRIG) and equine rabies immunoglobulin(ERIG) are used widely in rabies endemic areas. However, because of high cost of HRIG, anaphylaxis and a potential risk of virus infection of ERIG, and also low yield, ERIG and HRIG are difficult to meet the current needs. To overcome the disadvantages of ERIG and HRIG, new drugs are needed. The fully human or humanized therapeutic monoclonal antibodies provide a new way for the prevention and treatment of rabies and become the consensus of biopharmaceutical experts at home and abroad.In this study,we generated eukaryotic expression vector rBacmid-RVG, and expressed r-RVG in Bac-to-Bac baculovirus expression system successfully. Also, we purified the r-RVG and did some analysis on character and immunogenicity. Furthermore, we used the human IgM transgenic mice to produce anti-r-RVG human monoclonal antibodies and identified the biologic activity of the antibodies. This may be a foundation for further development of vaccine to prevent and control rabies.Methods1. Construction of eukaryotic expression vector rBacmid-RVG: Refer to the sequence of RVG gene of rabies virus CVS-11 strain from GenBank, amplifying the RVG gene with the cDNA. Enzyme digested PCR products recombinantting with plasmid pFastBac-GP67 B and transformed into DH5α competent cells. The positive recombinant pFastBac-GP67B-RVG was isolated and was transfected into DH10 Bac competent cells. And then the positive recombinant rBacmid-RVG was identified by PCR analysis.2. Expression and immunogenicity of r-RVG in Bac-to-Bac baculovirus expression system: The positive recombinant rBacmid-RVG was isolated and transfected into Sf-9 cells. Collect cell culture supernatant and cell pellets after the infected the cell showed siginifican cytopathic effect. DNA extracted from Virallyinfected Sf-9 cells was used to PCR analysis. Proteins were detected by Western blot. The r-RVG were purified by a His TrapHP affinity chromatography column and identified by SDS-PAGE. The r-RVG was used to immunize mice and setted negative control group and blank control group. We identified the IgG titer of anti-r-RVG of sera from mice by using indirect ELISA.3. Preparation of human monoclonal antibodies against r-RVG: The human IgM transgenic mice were immunized with the r-RVG by multiple sites subcutaneous immunization and intraperitoneal immunization. The human antir-RVG hybridoma cell lines were screened and produced by using the classical hybridoma technique.4. Identification of anti-r-RVG human monoclonal antibodies: The specificity and isotype of mAbs was determined by the double antibody sandwich enzymelinked immunosorbent assay(ELISA). Also, analyze the binding properties with the inactivated rabies virus CVS-11.Results1. Construction of recombinant expression vector rBacmid-RVG: Refer to the sequence of RVG gene of rabies virus CVS-11 strain, amplifying the RVG gene with the cDNA. The band is 1,500 bp. The recombinant plasmids pFastBacGP67B-RVG were shown two bands of pFastBac-GP67 B about 4,700 bp and RVG about 1,500 bp by restriction enzyme digestion. PCR identification of recombinant expression plasmid rBacmid-RVG with M13 primers and the band is 3,900 bp.2. Expression, purification and immunogenicity of r-RVG The Sf-9 cells were infected with rBacmid-RVG by transfection reagent(Roch). The western blotting showed that the recombinant protein was presented both in the cell culture supernatant and the cell pellets. PCR identification of DNA from extracted Virally-infected Sf-9 cells with RVG primers and the band is 1,500 bp.The purified protein was analyzed by SDS-PAGE, showed a 58 kDa band. The antibody titer of mouse serum(150 μg /mouse/dose) was 1:25,600, indicating good immunogenicity of the r-RVG. IFA identification indicated that anti-r-RVG antibodies could specifically combine with the rabies virus Flury strain.3. Screening of anti-r-RVG human monoclonal antibodies: We fused SP2/0 and B cells from mouse immunized by r-RVG. Five anti-r-RVG hybridoma cell lines 5D1、9A3、15D6、19E6、6H11 have been established after three times of cloning and they can stably secret human anti-r-RVG mAbs.4. Identification of anti-r-RVG human monoclonal antibodies: The double antibody antibody sandwich enzyme-linked immunosorbent assay(ELISA) demonstrated that the five anti-r-RVG mAbs are humanized immunoglobulin IgM type. The five anti-r-RVG mAbs can recognize the r-RVG specifically in the indirect ELISA and we also proved that three human anti-rabies virus mAbs 6H11, 15D6, 9A3 could specifically combine with the inactivated rabies virus CVS-11 strain.Conclusions1. We constructed the recombinant expression plasmid rBacmid-RVG. Successfully expressed and purified the r-RVG in Bac-to-Bac baculovirus expression system. Provide antigen for screening human antibodies anti rabies virus glycoprotein.2. In this study, five hybridoma cell lines had been established, they can stably produce human anti-rabies virus IgM monoclonal antibodies. These mAbs could specifically recognize the r-RVG and three human anti-r-RVG mAbs could specifically combine with the inactivated rabies virus CVS-11 strain. This may be a foundation for further development of vaccine to prevent and control rabies.