Study On VSV Vector Based Bivalent Vaccine For Marburg Virus And Ebola Virus

Marburg Virus and Ebola Virus belong to the genus Filovirus and Filoviridae,and are non-segment single-strand negative RNA viruses with a genome size of about 19 kb.They can cause extremely severe Marburg virus hemorrhagic fever or Ebola hemorrhage Fever in human and non-human primates.The main infection symptoms include organ failure,natural hole bleeding,vomiting,etc.,the mortality rate is 50%-90%.Due to the overlap of outbreak spot of Marburg and Ebola viruses as well as the similar symptoms,we developed bivalent vaccine for the prevention and control simultaneously of the two viral infections.The surface glycoprotein of Marburg virus and Ebola virus is the most important antigen for vaccine development,while vesicular stomatitis virus(VSV)has a low dose of immunity,Safe and efficient,no pre-existing immune and other advantages as a vaccine carrier.Therefore,we chose Angola Marburg virus GP(MGP)and Zaire Ebola GP(ZGP)and their pre-screened highly immunogenic fragments GP2A and L to construct a bivalent recombinant VSV vector based vaccine rVSVΔG-MGP-ZGP.Then Balb/c mice were immunized with rVSVΔG-MGP-ZGP and mouse serum antibodies and lymphocyte factors were measured for evaluating the efficacy of the VSV vector based bivalent recombinant vaccine.(1)Six recombinant VSV vectors and three VSV recombinant viruses were successfully constructedIn this study,different combinations of MGP,ZGP,GP2Δ,and L were cloned into the VSV vector to replace the VSV GP gene and six plasmids were constructed,such as pVSVΔG-MGP-ZGP,pVSVAG-MGP-L,pVSVΔG-GP2Δ-L,pVSVAG-ZGP-2A-L,pVSVAG-MGP R-2A-L,pVSVAG-MGP M-2A-L.2A sequence is a self-cleavage fragment of foot-and-mouth disease virus and MGP,MGP M and MGP R are different strains of Marburg virus.After BHK21 WI2 cells were infected with the recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase,the mixture of transfected plasmids pVSVAG-MGP-ZGP,pVSVAG-MGP-L,pVSVAG-GP2Δ-L and helper plasmids were transfected into the above BHK21 WI2 cells with proper proportion,respectively.RT-PCR,Western blot and indirect immunofluorescence were then used to confirm the expressions of the insert fragments.The results demonstrated that rVSVΔG-MGP-ZGP,rVSVAG-MGP-L and rVSVAG-GP2Δ-L were successfully rescued.(2)The preliminary evaluation of the efficacy of the VSV based biavalent vaccine for Marburg virus and Ebola virus.Balb/C mice were immunized with rVSVΔG-MGP-ZGP once or twice,the level of antibodies and cytokines were measured at 35 days after immunization.The results showed that rVSVΔG-MGP-ZGP induced high antibody titers against MGP and ZGP(1:51200)after the second immunization.The value of IgG2a/IgG1 demonstrated that the first immunization group tended to stimulate Thl cell responses,whereas the second immunization group tended to elicit Th2 cellular responses.These results indicate that the boosted immunity could shift from the Thl-type response to Th2-type response.The proliferation assay for spleen lymphocyte showed that the immune group could induce a certain extent of cellular immune response.When the protein GP2Δ(truncated MGP)and GPΔTM(truncated ZGP)were used to stimulate spleen cells,the level of IL-2 and IFN-y was higher than that of the saline group.Particularly,the level of IFN-y in the GPΔTM-stimulated twice immunization group was significantly increased,but no significant increase was observed in the GP2Δ-stimulated group.These data demonstrated that,this bivalent vaccine not only produced highly specific antibodies against MARV and EBOV GP protein,but also induces the specific cellular immune responses.