Construction And Immunogenicity Investigation Of Recombinant Rabies Virus Expressing Flt3L

Rabies is a zoonosis caused by the infection of rabies virus(RABV) in the central nervous system. Once the disease occurred, the mortality is almost up to 100%. More than55,000 people died of rabies each year all over the world, and China is one of the countries with high rabies incidence. Currently, there is no effective treatment or medication for rabies, and vaccination can effectively prevent the occurrence of rabies.In this study, mouse Fms-like tyrosine kinase 3 ligand(Flt3L) gene was cloned into the genome of rabies virus strain LBNSE, the recombinant rabies virus(rRABV)expressing mouse Flt3 L was rescued by reverse genetic technology and designated as LBNSE-Flt3 L. The production of Flt3 L in BSR cells infected with LBNSE-Flt3 L was determined by ELISA kits, and the results showed that Flt3 L was expressed in a dose dependant manner. To characterize the rRABV in vitro, virus growth kinetics was examined in BSR cells and no significant difference was found between LBNSE-Flt3 L and LBNSE, indicating that the insertion of exogenous gene Flt3 L did not affect the replication and spread of RABV. To investigate the effect of expressing Flt3 L on virus pathogenicity, mice were injected with LBNSE-Flt3 L through i.c. route. No clinical symptom was observed in infected mice and the weight of infected mice reduced at first and recovered to normal later. Compared with LBNSE, LBNSE-Flt3 L was slightly attenuated, but there is no significant difference.To further investigate the immunogenicity of LBNSE-Flt3 L, mice were injected with106 FFU of LBNSE-Flt3 L, LBNSE, or LBNSE-GM-CSF(constructed in our laboratory previously). The blood from each groups were collected every week from 1 to 10 weeks post immunization to detect the level of virus neutralizing antibody(VNA). The VNA test results showed that the VNA titer of both LBNSE-Flt3 L and LBNSE-GM-CSF reached the peak at 4 weeks post immunization, and all these two groups could induce faster and higher VNA titers compared with LBNSE group. Furthermore, the challenge was carried out at two weeks post immunization with 50 LD50 of CVS-24 through intracranial route.The challenge result showed that both LBNSE-Flt3 L and LBNSE-GM-CSF immunization groups could provide 80% protection, while the LBNSE immunization group only provided 45% protection.In order to study the mechanism by which the Flt3 L enhances the immunogenicity of RABV, the mouse bone marrow-derived dendritic cells were prepared and infected with LBNSE-Flt3 L, and Flow cytometry(FACS) was carried out to determine whetherLBNSE-Flt3 L could activate the DC in vitro. Meanwhile, mice were infected with LBNSE-Flt3 L, LBNSE, or LBNSE-GM-CSF, and the activation of DC, as well as GC B,after immunization were analyzed with FACS. The results showed that LBNSE-Flt3 L could activate significantly more DC compared with the mock or parental virus immunization group both in vivo and vitro, which was similar with that of LBNSE-GM-CSF. In addition, GC B were also found to be promoted in the LBNSE-Flt3 L immunized mice compared with LBNSE immunized group by FACS.Taken together, the rRABV expressing mouse Flt3 L could enhance the immunogenicity by activating DC and B cells in GC, therefore, induce higher VNA titer and provide higher protection. Our study will provide a new reference for the research of rabies vaccine.