Construction And Evaluation Of Bivalent Avian Influenza DNA Vaccine

Highly pathogenic avian influenza?HPAI?is a kind of severe disease that can cause acute symptoms and highly transimitted among various animals,posing a serious threat to human health.With the high mutation ratio,only the vaccines which matching the antigen efficiently can provide solid and effective prevention and control of influenza virus.DNA vaccine is constructed by inserting the target antigen gene into a eukaryotic expression vector,and then it is inoculated into the host body to exert the immunoprotective effect of the corresponding antigen.DNA vaccine has the advantages of simple experimental operation,cost saving and time saving.It is currently one of the most promising genetic engineering vaccines for preventing and controlling HPAI and rapidly responding to virus mutations.As for the currently prevalent clade 2.3.2 and clade 2.3.4.4 H5 subtype HPAI viruses,two DNA plasmids p CA-S1246 and pCA-GZ4 were constructed.The plasmid pCA-S1246 expressing HA protein of A/duck/Anhui/S1246/2015?H5N1??AH/S1246?was identified after in vitro expression,and immunizing dose and HI antibody duration assays were performed.At the same time,immunizing dose,heterologous virus challenge protection and HI antibody duration assays were also performed with the DNA plasmid pCA-GZ4 which expressing HA protein of A/chicken/Guizhou/4/2013?H5N1??GZ/4?to evaluate its immune efficacy systematically.The experimental results showed that the effective immunization doses of the DNA plasmids pCA-S1246 and pCA-GZ4 were all 15?g.During the monitoring period,the HI antibody levels of two plasmids were maintained at around 4 log₂ after booster immunization.The plasmid p CA-GZ4 could effectively withstand the lethal challenge of 4different heterologous viruses and coubld be used as a reserve vaccine against the clade2.3.4.4 virus.On the above basis,two bivalent recombinant DNA plasmids pCA-FGZ4-S1246 and pCA-CGZ4-S1246,which express the GZ/4 and AH/S1246 HA proteins were constructed using a fusion protein and double expression cassette strategy,respectively.The expression in vitro analysis showed that the fusion expression plasmid p CA-FGZ4-S1246 could express the fusion protein linked by the small peptide,and the tandem double expression plasmid p CA-CGZ4-S1246 could stably express the GZ/4 HA protein and the AH/S1246 HA protein.The immunopotency test showed that the two plasmids were able to effectively prevent the lethal challenge of GZ/4 and AH/S1246.The plasmid pCA-FGZ4-S1246 only provided partial protection against two virus challenge.The plasmid pCA-CGZ4-S1246 provided full protection against challenge of GZ/4 virus and 60%against challenge of AH/S1246 virus.The results suggested that it was necessary to further optimize the construction strategy of the double-expression cassette plasmid.For the new emergent HPAIV H7N9 virus,a recombinant plasmid pCA-SD098 expressing the HA protein of A/chicken/Guangxi/SD098/2017?H7N9??GX/SD098?was constructed.Using cassette back-to-back method,we also constructed a bivalent DNA vaccine plasmid pCA-GZ4-SD098 which co-expressing HA protein of GX/SD098 and GZ/4.In vitro identification showed that the single plasmid pCA-SD098 could efficiently express the GX/SD098 HA protein,and the double-expression cassette recombinant DNA plasmid pCA-GZ4-SD098 can efficiently co-express H7 and H5 HA proteins.The efficacy experiments showed that the plasmid pCA-SD098 coubld be 100%resistant to the lethal challenge of GX/SD098.The chickens vaccinated with bivalent DNA plasmid pCA-GZ4-SD098,or mix-vaccinated with plasmid p CA-SD098 and pCA-GZ4,could against lethal challenge of both H7N9and H5N1 HPAIV.All of the plasmids could effectively reduce the virus load of low pathogenic H7N9virus infection.The results showed that the bivalent plasmid pCA-GZ4-SD098 could be used as a universal DNA vaccine to prevent and control the currently prevalent H7N9 and H5N1 HPAIV.However,the optimal dose of the plasmid p CA-GZ4-SD098 needs to be further tested.In this study,several DNA plasmids expressing or co-expressing different HA proteins of the currently prevalent H5 and H7 subtype HPAIV were constructed and evaluated.The results showed that back-to-back of double-expression cassette recombinant plasmid and the mixture of two plasmids could induce the effective protection of two kinds of exogenous HA proteins.The bivalent plasmid pCA-GZ4-SD098 could simultaneously defend the lethal challenge of H7N9 and H5N1 HPAIV and was expected to become a universal avian influenza vaccine.The study established a solid foundation for the further exploring of trivalent and multivalent DNA vaccines.