Immunogenicity Study Of H7N9 Avian Influenza Ha With Transmembrane Modification Subunit Vaccine

H7N9 Avian Influenza（AI）is an acute and highly contagious respiratory disease caused by H7N9 Avian Influenza Virus（AIV）.H7N9 AI seriously harms the development of the poultry farming industry and may infect humans,which poses a great threat to public health safety.Hemagglutinin（HA）protein is the most important antigen protein on the surface of H7N9 AIV.It plays an important role in receptor recognition,fusion with host cell membranes,and pathogenicity.It is the preferred antigen protein for avian influenza subunit vaccines.Studies have confirmed that the transmembrane region of HA protein（Transmembrane,TM）will affect the thermal stability of HA protein,which in turn affects the immunogenicity of HA protein.Therefore,this study used the baculovirus insect cell expression system to express the HA protein of H7N9 subtype AIV and the modified HA（TM）protein of the transmembrane region,and compared the two subunit vaccines before and after the modification of the HA protein transmembrane region by animal experiments Difference in immunogenicity.In this study,the TM of H7N9 subtype AIV HA gene was modified,and then its nucleotide codon was optimized.Baculovirus insect cell expression system was used to express H7N9 HA protein and TM modified protein H7N9 HA（TM）protein.Western-Blot test results show that both proteins have been expressed,the size is about 63 k Da,which is consistent with the expected size.The results of the hemagglutination test showed that the hemagglutination titer of the H7N9 HA protein was 15 log2,and the hemagglutination titer of the H7N9 HA（TM）protein was 14 log2.The H7N9 HA protein and HA（TM）protein were uniformly diluted to a hemagglutination titer of 12 log2 after HA titer determination.After 30 minutes of treatment at different temperatures,the heat resistance of HA（TM）protein was improved,especially at 48℃treatment for 30 min,the H7N9 HA protein had no hemagglutination,and the H7N9 HA（TM）protein still had 33.3%hemagglutination.The H7N9 HA protein and H7N9 HA（TM）protein were diluted to a hemagglutination potency of 12 log2 with SEPPIC ISA 201VG adjuvant to prepare a subunit vaccine.The SPF chickens were randomly divided into 3 groups,12 in each group,which were H7N9HA group,H7N9 HA（TM）group and PBS control group,the vaccine group SPF chickens were immunized with the corresponding HA protein subunit vaccine,and the PBS control group SPF chickens were immunized with PBS,immunized once,the immunization dose is0.3 m L per SPF chicken,and three weeks after immunization 200μL 10⁶ EID₅₀ H7N9 virus was used for the challenge test.The serum collected from the second and third weeks after immunization was used to determine the HI antibody titer.The results showed that:in two week after immunization,the HI antibody titers of the two vaccine groups were above 4 log2,and the H7N9 HA（TM）group was slightly higher In the H7N9 HA group,but the difference was not significant（P>0.05）;in three weeks after immunization the HI antibody titers of the two vaccine groups were above 5 log2,the H7N9 HA（TM）group was slightly higher than the H7N9 HA group,the difference between the two groups was not significant（P>0.05）.After challenge,all SPF chickens in the PBS control group died within 3 days.The H7N9 HA group and H7N9HA（TM）group had no symptoms until the end of the experiment,and the survival rate was100%.Pharyngeal swabs and cloaca swabs were collected for detoxification testing 3,5,7,9 and 11 days after challenge.The detoxification situation is as follows:H7N9 HA（TM）group detected 1 chicken detoxification after the challenge,the detoxification rate of the vaccine group was 8.33%;H7N9 HA group detected 2 chickens detoxification,the detoxification rate of the vaccine group 16.67%.The virus titer of the swabs with positive virus isolation was further measured,and the results showed that the virus titer of the H7N9HA（TM）group was lower than that of the H7N9 HA group.In summary,this study used the baculovirus insect cell expression system to express the H7N9 HA protein and H7N9 HA protein with its TM modified.Studies have shown that the thermal stability of the H7N9 HA（TM）protein is better than the H7N9 HA protein,the subunit vaccine prepared by H7N9 HA（TM）is also superior to the subunit vaccine prepared by H7N9 HA in immunogenicity.The results of this study provide more candidate vaccines for the prevention and control of H7N9 avian influenza.