The Research Of Immunoenhance With Flagellin As An Adjuvant To Rabies Vaccine

Rabies, also known as hydrophobia, is the only human fatality rate almost up to 100% of the zoonotic and central nervous system diseases caused by rabies virus, which is threatening people’s life seriously. So far, there is no effective treatment to rabies. Emergency vaccination is one of the most effective treatments against rabies virus infection. Currently, the used animal rabies vaccines in our country are mainly attenuated live vaccine. However, due to the presence of potential safety problems, such as virulence reversion, mutation, WHO has explicitly pointed out that attenuated vaccine is not suitable to domestic animals. Therefore, to develop safer and more effective rabies vaccine is very important for prevention and treatment of the rabies. Adenovirus expression system, as its advantage and wide apply range, has been used in the development of genetic engineering vaccine of rabies.Flagellin is a major component of flagella. Flagllein can start the MyD88, IRAK and IKK and activate the NF-kB signal paths eventually by binding with TLR5 expressed on the top and basolateral of epithelial cells, monocytes and immature dendritic cells, which can trigger the production of cytokines(such as TNF-α, IL-8) so that the host acquires innate and passive immune response. Thus, flagellin, used as a TLR5 receptor type of vaccine adjuvants, has a good application prospect.In this study, flagellins FljB, FliC and FljB’-FliC were successfully expressed and purified by Prokaryotic expression system. BALB/c mice were immunised with purified protein and whole-killed rabies vaccine(WKRV). The animal test was organized into five groups(10 mice each) :(1) PBS;(2) WKRV;(3) WKRV with 10 μg FljB;(4) WKRV with 10 μg FliC; and(5) WKRV with 10 μg FljB’-FliC. Each group was immunized twice(days 0 and 14) at two-week intervals. Blood samples were obtained on days 0, 7, 14, 21, 28, and 35. The serum IgG antibodies were measured by Animal RABV IgG Antibody Assay ELISA kits. IL-4 and IFN-γ levels in splenocytes were also measured by using ELISPOT kits. As shown in the results of antibody levels, FljB is a promising adjuvant to WKRV. Humour immune response of mice in group that immunized with WKRV and FljB are higher than in group immunised with WKRV and FliC whose effect is insignificant. In addition, FljB’-FliC do not display an immunoadjuvant property with regard to WKRV. As shown in the results of cytokines, FljB can induce stronger Th1 and Th2 immune responses than FliC can. However, FljB’-Fli C cannot enhance cellular immune responses.Adenovirus expression system was used in this study to construct recombinant replication-defective adenovirus Ad-G, Ad-GB and Ad-GC, which could express the rabies virus G protein, G protein with flagellins FljB and G protein with flagellins FliC, respectively. Recombinant adenoviruses were used to immunize BALB/c mice to evaluate vaccine effects. The inoculate frequency was once after two weeks and three times in total. Blood samples were obtained on days 0, 14, 28, 35, and 42. The serum IgG antibodies in the mice sera were measured by Animal RABV IgG Antibody Assay ELISA kits. IL-4 and IFN-γ levels in splenocytes were also measured by using ELISPOT kits. At the same time, the mouse splenocytes were detected the levels of CD3+CD4+T cells and CD3+CD8+T cells by FCM assay. As shown in the results of antibody levels, FljB and FliC can enhance humour immunity responses. Besides, the function of FljB is better than FliC. As shown in the results of cytokines, FljB and FliC can enhance celluar immunity responses. Besides, the function of FljB is better than FliC. As shown in the results of FCM, FljB and FliC can induce the production of Th cells and Tc cells increasingly.All in all, whether in inactivated rabies vaccines or in the genetic engineering vaccine, both FljB and FliC have immunoenhance function. However, the enhancement function of FljB is stronger than FliC. The results suggested that FljB is a promising adjuvant, which has a reference function for the development of a safer and more effective rabies vaccine.