Expression,Purification And Immune-protective Efficacy Of The Duck H7 And H4 Avian Influenza HA Protein

Influenza virus is an important zoonotic pathogen,posing a severe threat to both human and animal health.Poultry are the natural reservoir of influenza viruses.Based on the antigenic differences of the viral HA and NA proteins,16 different HA subtypes and 9 different NA subtypes of influenza viruses have currently been detected in avian species.Among them,H4 subtype avian influenza virus(AIV)is now widely circulating worldwide.Previous studies have demonstrated that the current circulating H4 AIVs can infect and replicate in mammalian host,and more importantly some H4 AIVs can partially transmit between mammals via respiratory droplets,thereby posing a potential threat to human health.H7N9 influenza viruses have severely threatened human health since the first report to cause human infections in 2013.With the deepening of epidemiological surveillance and genetic evolution studies,it was shown that the H7N9 viruses are undergoing complex evolution.A new duck-origin H7N9 virus with different evolutionary lineage of the 2013 H7N9 viruses emerged in 2014,posing a new potential threat to human health.Vaccine immunization is the most effective measure for the prevention and control of influenza epidemics and outbreaks.To deal with the potential threat to human health of H4 and the newly emerged duck-origin H7N9 AIVs,we conducted a study to express,purify and evaluate the immune protective efficacy of the HA protein of an H4 and a duck-origin H7N9 virus.The HA genes of the AIV strains,A/Duck/Jiangxi/S21055/2012(H4N2)and A/Duck/Fujian/S4170/2014(H7N9),were cloned into the baculovirus expression vector pFastBac1,resulting in the construction of the recombinant pFBacH4 HA and pFBacH7 HA plasmids.After transforming DH10 Bac competent cells,the recombinant Bacmid-HA was purified,identified by PCR,and transfected into SF9 insect cells where it was expressed and assembled into recombinant baculovirus.The recombinant baculovirus was used to infect High Five insect cells to express the HA protein in large scale.The expressed HA protein with a purity of over 90% was validated by SDS-PAGE,western-blot and indirect immunofluorescence assay.The hemagglutination assay showed that the purified HA protein possessed good biological activity.In order to evaluate the immune protective efficacy of the purified HA protein of H4 and H7N9 viruses,we carried out the immunization and challenge study in SPF chickens.When boosted at 2 weeks after the initial immunization,the results showed that the serum HI antibody titers were 2-3 log2 at 4 weeks after single immunization with H4 HA or H7 HA protein.The H4 HI antibody titers in the serum of SPF chickens did not increase significantly at two weeks after the second immunization,whereas the H7 HI antibody titers reached 5-6 log2.The SPF chickens were challenged with the homologous viruses at two weeks after the second immunization.It was shown that although immunization with H4 HA protein could not prevent virus shedding,it could shorten the virus excretion period.In contrast,immunization with H7 HA protein could completely prevent virus shedding in the whold observation period,resulting in 100% immune protection efficacy.These results indicated that the immunogenicity of the purified HA protein of H4 and duck-origin H7N9 viruses in this study was different,among which the immunogenicity of the H7 HA protein was favorable,enabling it to provide complete protection to the immunized SPF chickens.This study provides a good technical reserve for the development of subunit vaccine to prevent and control the H7N9 viruses,and also provides necessary reference data for the development of subunit vaccine against H4 AIVs.