Construction And Efficacy Evaluation Of H5+H7 Subtype Avian Influenza Virus-Like Particles Vaccine

The H5N1 avian influenza virus(AIV)firstly emerged in Hong Kong in 1997 and caused human infections.Since then,it spread to multiple countries,mainly Asia,Africa,and Europe.As of April 2021,it has infected 862 people and caused 455 deaths(https://www.who.int/influenza/human_animal_interface/H5N1_cumulative_ta ble archives/en/).In addition to the highly pathogenic H5N1 subtype AIV,the highly pathogenic H7 subtype AIV also has a significant impact on poultry and human health.In April 2013,three residents in Shanghai and Anhui Province,China,showed rapidly developing lower respiratory infections and were found to be infected with a new reassortant of AIV known as H7N9 subtype.As of April 2021,the H7N9 influenza virus has infected 1,568 people and caused 616 deaths,with a high case fatality rate(http://www.fao.org/ag/againfo/programmes/en/empres/H7N9/situation_update.ht ml).The highly pathogenic H5N1 and H7N9 AIV seriously threaten the poultry industry and human health.Therefore,a highly safe and effective vaccine is needed to prevent a potential pandemic of H5N1 or H7N9 influenza viruses.Traditional vaccines are mainly inactivated vaccines,which rely on chicken embryos for production.Inactivated vaccines have many defects,such as the insufficient supply of chicken embryos when an influenza outbreak occurs,or causing local or systemic allergic reactions,and short duration of the immune response.Therefore,a safe and effective vaccine is needed to replace the traditional inactivated vaccines.Virus-like particles(VLP)are self-assembled viral structural proteins without viral genetic materials,and their structure is similar to natural viruses.Virus-like particles contain a high density of viral surface proteins,making them enter cells as effectively as natural viruses,and VLP vaccines are highly specific and immunogenic.VLP don’t contain viral genetic components but it can penetrate cells and tissues,making VLP safer than other conventional inactivated vaccines.In addition,the production of VLP doesn’t rely on chicken embryos,which ensures an adequate supply of vaccines in case of an avian influenza pandemic,and VLP also has the advantages of a short production cycle and low production cost.In this study,the H5 VLP vaccine and H5+H7 VLP bivalent vaccine were sucessefully prepared using a recombinant baculovirus insect cell expression system.The virus challenge results showed that both vaccines provided 100%protection for SPF chickens against the HPAIV and were comparable to commercial vaccines.Thus,VLP vaccines provide a potential method for the preparation of H5N1 and H7N9 bivalent vaccines.1.Preparation of H5N1 subtype avian influenza virus-like particles vaccine and evaluation of immunization effectIn this study,the H5 VLP vaccine was generated by co-infection of Sf9 cells with recombinant baculovirus rBac-TT3-HA expressing the HA gene of TT3 strain which belongs to H5N1 subtype HPAIV and rBac-GD15-NA and rBac-GD15-M1 expressing the NA and M1 genes of GD15 strain which belongs to H7N9 subtype HPAIV at the ratio of MOI of 1.Inactivated vaccines prepared from recombinant baculovirus rBac-TT3-HA expressing the HA protein of TT3 virus and the commercial inactivated vaccines were used as controls.Single immunization of four-week-old SPF chickens in a dose of 15μg stimulated effective hemagglutination inhibition(HI)antibodies,virus-neutralizing(VN)antibodies,and IgG antibodies in all vaccine groups.In a challenge protection test with TT3 strain,no clinical signs and deaths were observed in all vaccine groups within 14 days’ observation,while all chickens in PBS control group died within two days,indicating that the H5 VLP vaccine can provide 100%protection against H5N1 HPAIV.Throat and cloacal swabs were collected at 1,3,5,7,and 9 days after the challenge for virus titration.The results showed that no virus shedding was detected in vaccine groups,while all chickens in the PBS control group had virus shedding.Results of viral load in organs showed significant inhibition of virus replication in all vaccine groups.Histopathological change of the mouse lung revealed that all vaccine groups effectively inhibited lung injury in immunized chickens infected with the virus,with H5 VLP vaccine showed the most obvious effect.In conclusion,these results suggest that the H5 VLP vaccine provides complete clinical protection against the highly pathogenic H5N1 subtype AIV and significantly inhibit virus shedding and virus replication in vivo,and obviously reduce the extent of acute lung injury.Therefore,the H5 VLP constructed in this study provides an alternative strategy for the development of a novel H5N1 vaccine.2.Construction of H5+H7 virus-like particles vaccine and evaluation of immunization effectIn this study,the H5 VLP and H7N9 VLP were generated using an insect cell baculovirus expression system,respectively.These two VLPs were mixed at equal concentrations and volumes,and then emulsified with adjuvant at a ratio of 1:2 to prepare a bivalent H5+H7 VLP vaccine.In addition,a commercial inactivated vaccine and mock PBS were used as controls.A single immunization of H5+H7 VLP vaccine at a dose of 15 μg and commercial vaccine induced effective HI antibodies,VN antibodies,and IgG antibodies.Challenge protection test with highly pathogenic H5N1 and H7N9 AIVs was conducted after three weeks’ immunization.No clinical signs or deaths were observed in the vaccine group during the 14 days of observation.All chickens in the H5N1 and H7N9 unimmunized group died within two or five days after the challenge,respectively,indicating that the H5+H7 VLP vaccine provides complete protection against H5N1 and H7N9 highly pathogenic AIVs.Throat and cloacal swabs were collected on days 2,4,and 6 after the challenge for virus titration,and no virus shedding was detected in any of the vaccine groups.Results of viral load in organs revealed significant inhibition of virus replication in all vaccine groups.Moreover,the H5+H7 VLP vaccine was more effective in suppressing the extent of acute lung injury caused by virus infection in immunized chickens compared with the commercial vaccine.In conclusion,these results suggest that the H5+H7 VLP vaccine provides 100%protection against H5N1 and H7N9 highly pathogenic AIVs and significantly inhibits viral shedding as well as virus replication in immunized chickens.In addition,H5+H7 VLP was superior to commercial vaccines in reducing lung injury caused by viral infection.Therefore,the VLP vaccine prepared by co-emulsification of two VLPs in this study provides a reference for the preparation of other bivalent vaccines.