Construction And Immunogenicity Of RDNA Against Multi-subtype Influenza A Virus

Influenza virus is the pathogen of influenza, which is a kind of highly contagious disease with the first incidence rate among all of the infectious diseases. In 20th century,4 pandemic ever swept the global, just the 1918 Spanish influenza pandemic led to the death of at least 20 million people worldwide. Severity of influenza epidemics is a formidable public health challenge as well as significant socio-economic burden. The indulge in willful persecution of the avian influenza from 1997 has been waking people as alarm bell. Till now people could not control the disease due to the high variability of influenza virus in spite of the rapid progress of the modern scientific technology. Vaccines are still the main means to prevent influenza. The recent emergence of multiple strains of highly pathogenic avian influenza virus(HPAI) coexist 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. According to the independence of epitopes of influenza virus and the regulation of Kozak, two gene cassette (Epi I and Epi II), which included epitopes of H1,H7 and H9 influenza virus HA gene and epitopes of other influenza virus antigen (HA, NP, NA, M) and some enzyme cut sites, was designed and synthesized. On the basis of our laboratory's materials and gene cassette Epi II, we constructed a multiple epitopes chimera against H1,H3,H5,H7 and H9 subtypes of influenza A virus on the framework of fusion gene of H5HA and H3HA1.Based on the HA gene sequence of JL9/04, HA gene of H5 avian influenza virus was cloned. According to the HA gene of A/Wisconsin/67/2005(H3N2), the HA1 gene of H3 influenza virus was synthesized. Eukaryotic expression vector pVAX1 was utilized to construct the recombinant DNA vaccine pVAX1-NP-Epi I,pVAX1-H5HA-Epi II-H3HA1-M2e,pVAX1-NP,pVAX1-H5HA,pVAX1-H3HA1 and pVAX1-H5HA-H3HA1. In order to testify the expression in mammalian cells, two recombinant plasmids were purified and transfected into Hela cells by lipofection. The products biologic activity of recombinant vaccines in vitro was analysed by IFA.And the transcription capability of recombinant vaccines in vitro was analysed by RT-PCR.Hela cells were firstly transfected with the recombinant eukaryotic expression vector pVAX1-NP-Epi I,pVAX1-H5HA-Epi II-H3HA1-M2e, respectively. Then the whole RNA of the cell was extracted by RNA extraction Kit, the exogenous gene's mRNA was detected by RT-PCR; at the same time ,the reconstructed plasmids transfected with the Hale 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 H5HA/H3HA/NP were detected but also H1HA/H7HA/H9HA were also detected under the fluorescence microscope. These results identified that it's good antigenicity.The immunogenicity of the reconstructs of DNA was preliminary analysed in the BALB/c mice and the immunology index were detected. In the same time the reconstructs of pVAX1 and PBS was set as blank control in total 7 groups, three times immunization at 2-3 weeks interval. Blood samples was collected from vena caudalis every week and serum was made to detected antibody of ELISA. The mice was killed 10days 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 epitopes recombinats pVAX1-NP and pVAX1-H5HA-Epi II-H3HA1-M2e combined immunization could stimulate specific antibodies anainst H1,H3,H5,H7,H9 HA. Lymphocyte transformation test show that reconstructs with Epi II 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. Ratios of all groups were stabile between 1.4-2.0, which indicate there is no abnormalities during the immune response. IFN-γELISPOT results show that groups of pVAX1-NP -Epi I and pVAX1-H5HA-Epi II-H3HA1-M2e combined immunization stimulated more spots(>70) than controls(<30) at a statistical significance(<0.01); groups of pVAX1-H5HA-H3HA1 and pVAX1-NP combined immunization,pVAX1-H5HA -H3HA1 immunity class stimulated more spots(>60) than controls(<30) at a statistical sense(<0.01); while pVAX1-H5HA and pVAX1-H3HA1 also stimulated more than 55 spots. A number of epitope chimeric gene′added can enhanced spleen T lymphocyte secrete IFN-γand increased levels of cellular immunity in mice.The results above show that multiple epitopes chimera of DNA against H1,H3,H5,H7 and H9 subtypes of influenza A virus were not only could stir up humoral immunoresponse against H5,H3 as well H1,H7,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.