Preliminary Study On An Inactivated Vaccine Against H9 Subtype Avian Influenza Virus Based On Mosaic HA Sequence

H9N2 Avian influenza virus(AIV)is widely prevalent in China,which seriously threatens the health of poultry.At the end of the 20 th century,China began to use the inactivated vaccine for proventing H9N2 AIV infection.However,the existing vaccine does not match the antigenicity of the new strain when the H9N2 AIV antigen is mutated or recombined so that a new vaccine strain needs to be screened.This process is time consuming and laborious which needs to be updated almost every 2-5 years.Therefore,the development of a universal vaccine with cross-protection effect has become a hot spot in influenza vaccine research,such as designing a vaccine by a conserved region of influenza virus,a mosaic vaccine,and so on.The Mosaic sequence is a new sequence in which the epitopes contained in the existing target protein sequences are mosaic-incorporated without changing the length and characteristics of the target protein,so that it contains more potential T cell antigens.Mosaic sequences are designed to be assembled using fragments of existing proteins by using genetic algorithms(computation optimization methods),resulting in sequences similar to existing sequences.The HIV vaccine and the H5 subtype AIV vaccine obtained good general protection effects which is designed according to the Mosaic sequence.This paper preliminary studied the application effect of Mosaic sequence in H9 subtype AIV inactivated vaccine.1.Design and analysis of mosaic HA sequence(HAm/H9)of H9 AIVA H9N2 AIV HA protein mosaic sequence(HAm/H9)was designed and synthesized.The epitope coverage of HAm/H9 was analyzed.The HA protein sequence of H9N2 AIV isolates with different evolutionary branches was selected,and the HAm/H9 sequence was used for genetic evolution and homology analysis to predict the B cells and T cell epitopes of HAm/H9.The results showed that the HAm/H9 protein sequence has a good potential epitope coverage;it belongs to the Y280 branch,and its homology range from 86% to 100% with other strains;T cells and B cell epitopes of HAm/H9 are more than other strains'.2.Rescue of recombinant H9N1 AIV expressing HAm/H9 proteinUsing the H1N1 influenza virus PR8 strain as the backbone,the HA sequence of the PR8 strain was replaced by the HAm/H9 sequence by reverse genetic manipulation technique,and the expression plasmid PHW2000-HAm/H9 of HAm/H9 was constructed.Using the "7+1" reverse genetic manipulation,PHW2000-HAm/H9 and the expression plasmids expressing the other 7 genes of PR8(except HA)were co-transfected into 293 T and MDCK polyculture cells,and the recombinant H9N1 AIV strain rPR8-HAm/H9 was rescued and obtained.The growth curves of the recombinant virus and the parental PR8 strain in BHK-21 cells were determined.The results showed that the proliferation titers of the two strains were similar,and the replacement of the HA gene did not change the proliferation characteristics of the virus.3.the immune protection effect of recombinant virus on H9N2 AIVThe chicken embryo allantoic fluid infected with the recombinant H9N1 strain rPR8-HAm/H9 was inactivated with 0.1% formaldehyde to prepare an inactivated vaccine.After the vaccine was tested for stability and safety,it was carried out on the chicken body for immuning protection effect on H9N2 AIV local isolate JM0305 strain.At the same time,the JM0305 inactivated vaccine group was set as a positive control group.The results showed that the HI antibody level reached 6 log2 and the serum neutralizing antibody titer was 1:22 after 2 weeks of booster immunization in the rPR8-HAm/H9 immunized group;the serum antibody also had a neutralizing effect on JM0305;the JM0305 inactivated vaccine was After 2 weeks of booster immunization,the HI antibody reached 6 log2 and the serum neutralizing antibody titer was 1:16.Cross-neutralization test results showed that the serum of rPR8-HAm/H9 immunized group had a certain neutralizing effect on JM0305.The challenge test of JM0305 strain showed that the challenge protection of JM0305 strain was 80% in rPR8-HAm/H9 immunization group and 90% in JM0305 inactivated vaccine group.The results of virus isolation showed that the rPR8-HAm/H9 immunized group could better inhibit the virus isolation of JM0305 strain,and the virus could not be detected on the fifth day after challenge.The result was better than the JM0305 inactivated vaccine control group.In summary,the HA mosaic sequence of H9N2 AIV was designed and synthesized.The H9N1 recombinant strain expressing HAm/H9 protein was obtained by HA gene replacement using H1N1 influenza virus as the backbone.The inactivated vaccine prepared by recombinant virus was found to have good immunoprotective effect against H9N2 AIV JM0305 strain.The next step is to use other branches of H9 isolates for crossprotection determination.It provides new ideas and methods for the follow-up of H9 subtype AIV vaccine,and provides a reference for the study of broad-spectrum vaccine.