Development Of Optimized H5 Subtype Avian Influenza DNA Vaccine And Poultry Immunopotentiator

Avian Influenza is an infectious disease caused by avian influenza virus that can infect poultry and wild birds.From July 2017 to August 2018,some countries and regions in Asia,Europe and Africa experienced outbreaks of H5 subtype avian influenza,such as China,Italy and South Africa,which seriously affected the poultry industry.DNA vaccine is a new genetically engineered vaccine.Studies have found that the avian influenza DNA vaccine made by the HA gene of avian influenza virus has a good protective effect on poultry.Due to the low immunogenicity of DNA vaccines,it is urgent to develop new immunopotentiators to enhance the protective efficacy of DNA vaccines.In this study,the HA gene of H5 subtype avian influenza virus A/Goose/Guangdong/P45/2016（H5N6）was optimized according to the chicken codon bias,and the optimized HA gene was named opti-HA.The opti-HA was cloned into the vector p CAGGS-K to obtain the recombinant plasmid,which was named p CAGGSK-opti-HA.The positive plasmid was transfected into 293T cels.Western blot analysis showed that the recombinant plasmid p CAGGSK-opti-HA could efficiently express HA protein,and the expressed target protein is about 64 k Da,which indicated that the recombinant plasmid p CAGGSK-opti-HA could be successfully expressed in 293T cells.In order to study whether the recombinant plasmids of chicken STING,chicken IL-7,chicken IL-8,chicken IL-15,duck STING,and duck IL-8 as immunopotentiators can enhance the immune effect of DNA vaccine.Chicken STING,chicken IL-7,chicken IL-8and chicken IL-15 were optimized according to the chicken codon bias,and the optimized genes were named opti-CKSTING,opti-CHIL7,opti-CHIL8,and opti-CHIL15,respectively.Duck STING and duck IL8 were optimized according to the duck codon bias,and the optimized genes were named opti-DKSTING and opti-DKIL8,respectively.The optimized genes were cloned into the vector p CAGGS-K with HIS or HA tag to obtain the recombinant plasmid,which were named p CAGGSK-opti-CKSTING-HIS,p CAGGSK-opti-CHIL7-HA,p CAGGSK-opti-CHIL8-HA,p CAGGSK-opti-CHIL15-HA,p CAGGSK-opti-DKSTING-HIS,p CAGGSK-opti-DKIL8-HA,respectively.The positive plasmids were transfected into293T cells.Western blot analysis showed that each recombinant plasmid could express the target protein in 293T cells.To evaluate the immune effect of the recombinant plasmid p CAGGSK-opti-HA,p CAGGSK-opti-CKSTING-HIS and p CAGGSK-opti-CHIL8-HA,60 2-week-old chickens were divided into 4 groups,namely PBS group,p CAGGSK-opti-HA group,p CAGGSK-opti-CKSTING-HIS and p CAGGSK-opti-HA co-immunization group,p CAGGSK-opti-CHIL8-HA and p CAGGSK-opti-HA co-immunization group,respectively.The above plasmid were all administered by multiple injections of the leg muscles.The second immunization was performed two weeks after the first immunization.The Hemagglueination inhibition test showed that the serum antibody levels of the PBS control group were all zero.The serum antibody levels of the p CAGGSK-opti-HA group at the two weeks after the first immunization and the two weeks after the second immunization were about 0.42log2 and4.21log2,respectively.The serum antibody levels of the p CAGGSK-opti-CKSTING-HIS and p CAGGSK-opti-HA co-immunization group at the two weeks after the first immunization and the two weeks after the second immunization were about 1.29log2,5.08log2,respectively.The serum antibody levels of the p CAGGSK-opti-CHIL8-HA and p CAGGSK-opti-HA co-immunization group at the two weeks after the first immunization and the two weeks after the second immunization were about 1.41log2,4.50log2,respectively.Therefore,the serum antibody levels in the p CAGGSK-opti-HA group,p CAGGSK-opti-CKSTING-HIS and p CAGGSK-opti-HA co-immunization group,p CAGGSK-opti-CHIL8-HA and p CAGGSK-opti-HA co-immunization group showed an upward trend during the immunization period.During the whole immunization period,the serum antibody levels of the p CAGGSK-opti-CKSTING-HIS and p CAGGSK-opti-HA co-immunization group and the p CAGGSK-opti-CHIL8-HA and p CAGGSK-opti-HA co-immunization group were slightly higher than those of p CAGGSK-opti-HA group.Therefore,the p CAGGSK-opti-HA group,p CAGGSK-opti-CKSTING-HIS and p CAGGSK-opti-HA co-immunization group,p CAGGSK-opti-CHIL8-HA and p CAGGSK-opti-HA co-immunization group could induce good antibodies in chickens.The recombinant plasmids p CAGGSK-opti-CKSTING-HIS and p CAGGSK-opti-CHIL8-HA could increase the antibody titer produced by DNA vaccines in chickens.All the experimental chickens were challenged with 100 ul of 10⁶EID₅₀ avian influenza virus p45（clade 2.3.4.4）on the two weeks after the second immunization.All the chickens died within 3 days after the challenge in the PBS control group,and all the chickens survived and without obvious clinical symptoms in the other experimental groups.Two chickens could shed virus in the p CAGGSK-opti-HA group,one chicken could shed virus in the p CAGGSK-opti-CKSTING-HIS and p CAGGSK-opti-HA co-immunization group and two chickens could shed virus in the p CAGGSK-opti-CHIL8-HA and p CAGGSK-opti-HA co-immunization group.Thus,the recombinant plasmid p CAGGSK-opti-HA as a DNA vaccine can produce a good protective efficacy against the H5 subtype avian influenza virus（clade2.3.4.4）,and when the DNA vaccine is co-immunized with the recombinant plasmid p CAGGSK-opti-CKSTING-HIS,it has the best protective efficacy on the chickens,but all experimental groups had some immunized chickens that could shed virus.