Development Of A Bivalent H5N1 And H9N2 Subtype Virus-like Particles Vaccine

Recently,influenza pandemics caused by highly contagious viral pathogens have led to significant fatalities and illnesses throughout the world.Highly pathogenic avian influenza can not only make a great quantity of poultry die,but also infect human being to cause a series of severe respiratory disease.Lowly pathogenic avian flu may cause moderate clinical signs such as respiratory disease,a reduction in egg production,and mortality.Therefore,the outbreak of avian flu will cause significant economic losses and seriously threat to human life and health.With the continuous development of poultry breeding industry and the scale of farm in china,the influenza H9N2 pandemic will affect poultry supply and be responsible for serious economic losses.H5N1 Avian fluenza viruses are highly pathogenic and mortality that highly impact on both human and animal health,H5N1 avian influza has become a matter of great concern to public health.Currently,vaccination remains the most effective counter measure against infection with influenza viruses in china.The main defense against influenza epidemics and pandemics is inactivated virus vaccine.However,there are some potential problems such as the risk of re-pathogenicity and the inactivator of vaccine.The inactivated vaccine is not able to prevent the influenza epidemics and pandemics.Therefore,it is more and more attention to preparate an efficient and safe influenza vaccines.Influenza virus-like particles are evaluated as candidate vaccines,since VLPs are absolutely non-infectious and non-replicating and preservation of native antigenic conformation.Currently,china has not yet appeared VLP vaccine against H5N1,H9N2 subtype avian influenza virus.This study based on the above problems to develop three kinds of a bivalent H5N1 and H9N2 VLP vaccine with different adjuvants.The immunogenicity of bivalent H5N1 and H9N2 VLP vaccines was verified from the aspect of immunology.Research purposes: In this study,two different VLPs were produced in baculovirus expression system.Here we used three adjuvants including liposome,oil and chitosan microspheres to develop bivalent H5N1 and H9N2 VLP vaccines in order to systematically compare the immunogenicity of bivalent VLP vaccines in chicken.Research methods: Proliferation and the titer of H5N1 and H9N2 recombinant baculovirus virus was tested respectively.The HA,M1 proteins in H5N1 and H9N2 VLPs was respectively detected by western blot.Then the HA protein concentrations and titers of two VLPs was determined by quantity ELISA assay and hemagglutination test respectively.To optimize the preparation of liposome was done by both single-factor test and orthogonal test.The entrapment rate,physical stability and safety of liposomes were examined.We developed three bivalent H5N1 and H9N2 VLP vaccines by using liposome,oil and chitosan microspheres and studied relevant quality safety evaluation.Finally,the levels of specific humoral and cellular immune response to bivalent H5N1 and H9N2 VLP vaccines in chicken were detected.Result:1.The HA,M1 proteins in VLP has similar immunogenicity with natural virus of H5N1 and H9N2 by western blot.The HA concentration of H5N1 VLP and H9N2 VLP was 134.42±4.78 g/m L,129.73±4.20 g/m L respectively.It has high immunogenicity and can be used as the material of bivalent VLP vaccine.2.The entrapment rate of liposomes was 83.37%,the remaining water content of the liposomes freeze-dried powder measured by vacuum drying method was 2.5%,its encapsulation rate of 60.78% after cryopreservation for 120 days in 4?.The stability of the oil bivalent VLP vaccine was 95.5%,and the release rate of the microsphere bivalent VLP vaccine in vitro was 83.1%.3.After 28 days of immunization on SPF chicken,the HI titers of anti-H5N1 antibody and anti-H9N2 antibody in liposome group were 9.28Log2 and 9.43Log2 respectively.The HI titer of liposome group was significantly higher than those in oil emulsion and microsphere group(p<0.01).Except for the liposome group,neutralizing antibody titers of oil emulsion and microsphere group were significantly lower than those of the inactivated vaccine group(p<0.01).The neutralizing antibody titers of H5N1 virus and H9N2 virus in liposome group were 1/97,1/103 respectively.The neutralizing antibody titer of liposome group was significantly higher than those in oil emulsion and microsphere group(p<0.01).The results of MTT assay showed that after 28 days of immunization,the SI index of liposome group was not significantly different from that of inactivated vaccine group,but the SI index of liposome group was significantly higher than that of microspheres and oil emulsion group(p<0.01).After 56 days of immunization,the SI index of liposome group was higher than that of microsphere and oil emulsion group(p<0.01).The SI index of T lymphocyte cell stimulated by H5N1 virus and H9N2 virus in liposome group was 4.29,5.65 respectively,a bivalent H5N1 and H9N2 VLP vaccine developed by using liposome could induce effective cellular immune response.Conclusion: We developed the liposomes of bivalent H5N1 and H9N2 VLP vaccine that can induce a strong level of humoral and cellular immune response after vaccination,and provide a reference for the development of VLP vaccine.