Construction And Immunogenicity Study Of Multi-Epitopes DNA Vaccines And Recombinant Fowl Pox Virus Against Influenza A Virus

The recent emergence of multiple strains of highly pathogenic avian influenza virus(HPAI) co-exist in poultry and their subsequent transmission to human in southeast Asia and Middle East have raised concerns about the potential pandemic spread of leathal disease.In this study we selected multiple antigen dominant epitopes based on the bioinformatics, designed and synthesized a multiple epitopes gene which contain multiple epitopes of some influenza virus subtypes named Epi gene.On the basis of our laboratory's materials and engeneering accumulation, we constructed a multiple epitopes chimera against H3,H5,H7 and H9 subtypes of influenza A virus on the framework of fusion gene of H5H7HA.Eukaryotic expression vector pVAX1 and reconstructed fowl pox virus transfer vector pUTA2 was utilized to construct the recombinant DNA vaccine pVAX1-H5-Epi-H7,pVAX1-H5HA-Epi,pVAX1-Epi and pVAX1-H5HA and shuttle vector pUTA2-H5-Epi-H7 of recombinant fowl pox virus.Then the shuttle vector pUTA2-H5-Epi-H7 and the FPV 282E4 strain was co-transfected with CEF cell under the three-time selctions of Brdu. After secreening by single plaque and PCR identifaction recombinat Fowl Pox virus rFPV-H5-Epi-H7 was gained,eventually.The products biologic activity of recombinant vaccines in vitro was analysed by RT-PCR,Western blot and IFA,respectively.BHK and CEF cells was firstly transfected with the recombinant eukaryotic expression vector pVAX1-H5-Epi-H7,pVAX1-H5HA-Epi,pVAX1-Epi, pVAX1-H5HA and recombinat fowl pox virus, respectively. Then the whole RNA of the cell was extracted by RNA extraction Kit, the exogenous gene's mRNA was detected by RT-PCR; Western blot analyse of the supernatant of the lysate of BHK and CEFcells finding the two intresting protein bands of 153kDa and 26kDa, and they are equal to the theoretical size; at the same time ,the reconstructed plasmids transfected with the BHK cell was analysed by indirect immunofluorescence assay(IFA) and found the expressed foreign protein could specially recognize the homologous positive serum ,not only the specially fluorescence of H5 and H7HA were detected in the pVAX1-H5-Epi-H7 and rFPV-H5-Epi-H7 transfected cells,but also H3 and H9HA epitopes were also detected under the fluorescence microscope.The immunogenicity of the reconstructs of DNA and fowl pox virus was preliminary analysed in the BALB/c mice and the immunology index were detected. In the same time the reconstructs of pIRE-H57-Epi,pIRE-Epi and vFA7935L18 was set as parallel control in total 12 groups,three times immunization at 2 weeks interval. Blood samples was collected from Vena caudalis every week and serum was made to detected antibody of ELISA.The mice was killed two weeks after the third immunization,blood samples were collected from ophthalmic artery ,the spleen was harvested by asepsis to make spelenic T lymphocyte suspension to be used in the lymphocyte proliferation assay and subgroups analyse of splenic lymphocytes,as well the IFNγ-ELISPOT analyse.The results show that multiple epitops recombinats vFA7935L18, vFPV-H7- EPI-H5,pVAX1-H5-Epi-H7,pIRE-H57Epi-IL18 could all stimulate specially antibodies anainst H3,H5,H7,H9HA. Reconstructed fowl pox virus was superior to DNA reconstructs at whole. Lymphocyte transformation test show that reconstructs with Epi could stir up stronger cellular immune reconpose than the others groups.Fluorescence activated cell sorter found that CD4+ and CD8+ T lymphocyte percentage increased obviously, are statistical significant with controls. IFN-γELISPOT results show that groups of vFA7935L18, vFPV-H7- EPI-H5, pVAX1-H7-Epi-H5, pVAX1-H5-Epi, pIRE-H57-Epi, pIRE-Epi stimulated more spots(>70) than controls(<40) at a statistical significance(<0.01).while pVAX1-H5 and pVAX1-H7 also stimulated more than 60 spots.But reconstructs of multi-epitopes didn't have statistical significance each other..The results above show that multiple epitopes chimera of reconstructed fowl pox virus and DNA against H3,H5,H7 and H9 subtypes of influenza A virus were not only could stir up humoral immunoresponse against H5,H7 as well H3,H9 subtypes of influenza A virus,but also strong celluar immunoresponse.This study made some benefical exploration in the mutiple subtypes of influenza A virus genetically engineering vaccine based on the epitopes design proposal. We do think it is a feasible programe with regard to the variant instability of influenza virus.