Construction Of Attenuated Salmonella Expressing H9N2 Subtype Avian Influenza Mosaic HA Protein And Evaluation Of Sequential Immunization

The H9N2 subtype of avian influenza is a low pathogenic type of avian influenza that has a significant impact on egg production in laying hens.Vaccination is now the most effective way to prevent and control avian influenza.Currently,inactivated vaccines are widely used in the market to respond to the H9N2 subtype of influenza,with high safety and long immune duration.However,inactivated vaccines mainly induce humoral immunity,resulting in weaker mucosal immunity,making it difficult to block the virus’s infection of the upper respiratory tract of chickens.In recent years,ferritin nanoparticles have been successfully used as novel vaccine carriers in human influenza,and have great prospects.In this study,avian influenza H9N2 Mosaic HA protein and self-assembled ferritin were fused and expressed to form nanoparticles with ferritin as the core and Mosaic HA antigen on the surface.By orally immunizing chickens with attenuated Salmonella delayed-lysate and combining sequential immunization,purified nanoparticles were immunized intranasally to induce an immune response in the body.The main contents and results are as follows:（1）Construction and verification of fusion expression of Mosaic HA and ferritin plasmidIn this experiment,the Mosaic HA gene sequence of p YL212 plasmid was fused with the ferritin（Ferritin）sequence of p YL264 plasmid to form the target fragment of Mosaic HA-Ferritin,and the plasmid p YL269 was successfully constructed.The target fragments were ligated to attenuated Salmonella vector and E.coli expression vector respectively and transformed into strains to obtain Salmonellaχ11802（p YL272）and BL21（p YL288）.The target protein in BL21（p YL288）was purified by His tag.The successful expression of the target protein was detected by Western blot experiment,and the results of transmission electron microscope confirmed the successful assembly of nanoparticles.（2）Evaluation of the efficacy of sequential immunization with oral-administration oral immunization plus and purified mosaic HA protein against H9N2 avian influenza virusOne-day-old female laying hens were selected as experimental animals and immunized after the level of autoantibody decreased for 21 days.The first exemption day was set to 0d,14d and 28d for second and third exemption.The animals were divided into five groups:p YL272oral immunization group,p YL272 sequential immunization group,BSG control group,p YA4545 empty vector control group and commercial vaccine group.p YL272 oral immunization group,BSG group and p YA4545 group were all orally immunized with1×10⁹CFU/100μL,Vaccine group was intramuscularly injected with 200μL commercial vaccine on 0 d,p YL272 sequential immunization group was given 1×10⁹CFU salmonella/100μL on 0 d and 14 d,and 20μg purified protein was injected intranasally on 28 d.One week after the third immunization,serum and bronchoalveolar lavage fluid were collected to detect the level of antibody in vivo.The levels of cytokines m RNA were detected by fluorescence quantitative PCR（q RT-PCR）after stimulation of HA and Con A proteins in spleen T lymphocytes.Splenic lymphocyte proliferation was detected by CCK8 and flow cytometry.42days after intranasal challenge with 1×10⁶TCID₅₀strain of H9N2 AIV A3 virus,the body weight of the animals was monitored every other day.The serum and bronchoalveolar lavage fluid（BALF）were collected on the 5th day to detect the antibody level.The lung tissue was collected to detect the virus load by q RT-PCR.The swabs of oropharynx and cloaca were collected on the 3rd,5th and 7th day to detect the level of virus excretion in vitro.The results showed that the levels of Ig Y antibody in serum and SIg A antibody in mucous membrane of p YL272 oral immunization group and p YL272 sequential immunization group were increased one week after three immunization.The results of flow cytometry showed that the number of CD8⁺T lymphocytes in p YL272 sequential immunization group was significantly higher than that in BSG group and p YA4545 group.CCK8 lymphocyte proliferation test showed that when stimulated by HA protein,the T lymphocyte proliferation ability of spleen in p YL272sequential immunization group was significantly higher than that in BSG group,and when Con A protein was stimulated in vitro,the level of spleen T lymphocyte proliferation in p YL272sequential immunization group was significantly higher than that in BSG group,and that in p YL272 oral immunization group was higher than that in p YA4545 empty vector group.The results of m RNA detection of cytokines IL-4 and IFN-γin spleen cells showed that the m RNA level of cytokine IFN-γin p YL272 oral immunization group was significantly higher than that in BSG group（P<0.001）.The m RNA levels of IL-4 and IFN-γin p YL272 sequential immunization group were significantly higher than those in BSG group and p YA4545 group（P<0.001）.The results of ELISA detection in bronchoalveolar lavage fluid collected 5 days after challenge showed that the level of mucosal SIg A antibody in p YL272 oral immunization group was significantly higher than that in BSG group and p YA4545 group.After challenge,the titer of pulmonary virus showed that the viral load in p YL272 oral immunization group and p YL272sequential immunization group was lower than that in BSG control group.The results of pathological sections of lung tissue showed that the number of inflammatory cells decreased and pulmonary hemorrhage was alleviated in p YL272 oral immunization group and p YL272sequential immunization group.Fluorescent quantitative PCR detection of oropharyngeal and cloacal swabs collected at 3,5 and 7 days after challenge showed that the detoxification level of BSG group was the highest,while that of p YL272 sequential immunization group was lower.The results showed that p YL272 sequential immunization group could enhance the mucosal immunity and reduce the symptoms of chicken virus infection to a certain extent.To sum up,the recombinant plasmid Mosaic HA-Ferritin was successfully constructed and expressed by delayed lytic Salmonella and sequentially immunized chicks with purified protein intranasally.The results showed that both oral immunization and sequential immunization could significantly enhance the proliferation of chicken splenocytes and the level of cytokines stimulated by antigen protein,and the effect of sequential immunization was better.After challenge,the level of mucosal antibody was higher and the titer and detoxification level of pulmonary virus were lower in sequential immunization group than in oral immunization group,which laid a foundation for the follow-up development of novel H9N2 subtype avian influenza virus nanoparticle vaccine.