Studies On The Immune Effects Of Attenuated Salmonella Expressing HA2-scFvDEC205 Against H9N2 Subtype Avian Influenza Virus

Avian influenza?AI?is a highly harmful avian infectious disease caused by avian influenza virus?AIV?.The avian flu epidemic has caused huge losses to the poultry industry worldwide.In the 19th century,chickens died in large numbers and the disease was later named Avian influenza.With the prevalence of AI,there are many subtypes of AIV,and the H9N2 subtype is one of the low pathogenic subtypes.Over the long term,H9N2 has been mutated into one of the more popular AIV subtypes,so how to better prevent AI has been a research hotspot.Hemagglutinin?HA?is an important protein on AIV,the key to AIV invasion of cells.Studies have shown that HA decomposes HA2 in the body.HA2 is an important conserved antigen,and it has been a research hotspot for researching general-purpose vaccines.Dendritic cells?DCs?play an important role in antigen presentation.CD205/DEC-205 is a C-type lectin transmembrane protein on the surface of DCs,and when DEC-205 acts as an endocytic receptor,it enhances antigen uptake and antigen presentation by DCs.A number of literatures have documented that DEC205 plays such a role in antigen-targeting DCs.Most of the drugs currently on the market are whole virus inactivated vaccines,and the vaccines are mainly obtained by chicken embryo culture or cell culture.However,the chicken embryo culture method is complicated and the ovalbumin is easily left to some extent.People who are allergic to eggs cannot be immunized in this way,and the way of cell culture is easy to contaminate and costly.Therefore,it is an important issue to develop a safe and simple bird flu vaccine.This study focused on the DNA vaccine for the prevention of H9N2 avian influenza by attenuating delayed lytic Salmonella delivery of avian influenza HA2 antigen.The specific research contents of this experiment are as follows:?1?Construction and Evaluation of Recombinant Lytic Salmonella H9N2 Subtype AvianInfluenza Virus HA2 Antigen Targeting DCsThe target fragment scFvDEC205-HA2 and scFvDEC205-HA2-dimer were obtained by PCR amplification using the synthetic plasmid?DEC205-HA as a template.The dimer could bind to the double scFvDEC205 and HA2,and then the two target fragments were ligated with the pYA4545 vector to construct the eukaryotic expression plasmids pYA4545-HA2-scFvDEC205and pYA4545-HA2-scFvDEC205-dimer,respectively named HA2-scFvDEC205 and HA2-scFv DEC205-dimer.The plasmid was transfected into HEK-293T cells by liposome,and total protein was extracted after 48 hours of culture.Western-blot and indirect immunofluorescence confirmed the target protein has been expressed successful and then the recombinant plasmids have been transferred into Salmonella?11218,which were named PYL9 and PYL11,respectively.?2?Evaluation of immune efficacy of strains pYL9 and pYL11Sixty mice were randomly divided into 6 groups,10 mice in each group.The group was divided into the pYL9 group,the pYL11 group,the existing?11218?pYA4545?group?Empty vector group?,?11218?pYA4545-HA2?group?HA2 group?.Meanwhile,the saline group and an inactivated vaccine group were also established.Except for the Vaccine group,the other groups were boosted once every 14 days after the first immunization.There was a total of 4 times of oral immunization,the dose was 1×10⁹CFU/only.The mice in the Vaccine group were immunized by subcutaneous injection in the neck at the first immunization and immunized only once.The spleen,mesenteric lymph nodes and PP nodes were taken after the fourth immunization.The maturation and differentiation of DC and the differentiation of T cells in each group of mice were analyzed by flow cytometry.Mouse sera were taken on the 21st and35th day of immunization,and the antibody titer and cytokine levels in the serum of mice were detected by ELISA.After one week of four immunizations,7 mice were randomly selected for challenge test.The dose of 10⁵EID₅₀ was 50?L/only,and the weight change and mortality of the mice were recorded within 14 days after the challenge.Later,the lungs of the mice were taken to make pathological sections in order to observe the pathological damage of the lungs in each group.The results showed that compared with the Saline group and the Empty vector group,the mice immunized with Salmonella pYL9 and pYL11 were found to have a significant increase by5.1%,5.4%?P<0.01?in the proportion of CD3⁺CD4⁺T cells in the spleen of the mice.Compared with the Empty vector group and the HA2 group,the proportion of IL-4+cells in the pYL11 group was significantly increased by 1.49%and 1.63%?P<0.05?.Compared with the Empty vector group,the IFN-?⁺cell content in the pYL11 group was significantly increased by1.25%?P<0.01?.Compared with the HA2 group,the IFN-?⁺cell content in the pYL11 group was significantly increased 1.61%?P<0.001?.Compared with the Saline group,the proportion of CD3⁺CD8⁺T in the spleen of the pYL11 group also increased significantly by 2.56%?P<0.01?,and compared with the Empty vector group,the IFN-?⁺T cell content of the pYL11 group increased significantly by 0.49%?P<0.05?.Compared with the Empty vector group,the expression of CD83 in the pYL11 group was significantly increased by 0.41%?P<0.05?,and the CD86 expression was significantly increased by 34.8%?P<0.001?.In the mesenteric lymph nodes,the CD80 expression of DCs in the pYL11 group was significantly increased by 1.67%?P<0.05?,which compared with the Empty vector group.Compared with the Empty vector group,the CD80 expression of DCs cells in the pYL11 group was significantly increased by0.42%?P<0.05?.ELISA analysis of serum antibody titers and cytokine levels showed that on the21st and 35th day of immunization,antibody titers in the pYL11 group were significantly increased by 0.6%and 0.28%?P<0.05?which compared with the Empty vector group.Compared with the HA2 group,the serum IFN-?level in the pYL11 group was significantly increased?P<0.01?.After challenge,the body weight of each group began to decline.The weight decreased to the lowest on the seventh day,and the weight loss of the pYL11 group was lighter.Survival curve results showed that the survival rate of pYL11 group was 85.7%,the survival rate of Vaccine group was 85.7%,and that of Empty Vector and Saline group were 57.1%and28.5%,respectively.HE staining results of lung pathological sections of the mice showed that the pathological changes of the pYL11 group and the pYL9 group and the Vaccine group were mild.In the Saline group and the Empty vector group,the lungs of the mice had severe pulmonary interstitial thickening and increased inflammatory cell infiltration.In summary,the proportion of CD3⁺CD4⁺T cells and CD3⁺CD8⁺T cells of mice in pYL9 and pYL11 group were significantly increased after immunization.The levels of IL4⁺T cells and IFN-?⁺T cells increased significantly,and the expression of CD80,CD83 and CD86 in DCs increased significantly,and antibody levels in serum increased.After the challenge,the mice in the Salmonella group had less lung lesions than the control group.These results indicated that Salmonella pYL9 and pYL11 had protective effects against H9N2 infection in mice,and pYL11group had better effect.