Develpment And Application Of A Recombinant Avian Influenza Virus (H5/H7) Bivalent Inactivated Vaccine

H5 and H7 subtypes highly pathogenic avian influenza virus(HPAIV)usually cause all of infected poultry deaths and result in disease outbreaks among poultry.The outbreaks of H5 subtype AI has occurred in more than 60 countries and regions in the world since 2003.Meanwhile,both H5 and H7 subtype AIV have caused severe human infections and deaths,posing a great threat to public health.Vaccine immunization is one of the most effective AI prevention and control measures suitable for China's national situation.Since 2014,the H5 subtype AIV isolated from domestic poultry mainly belongs to clade 2.3.4.4.The National Avian Influenza Reference Laboratory has developed a corresponding series of H5N1 subtype Re-8 strain inactivated vaccines which have been widely used in poultry and achieved good results.At the beginning of 2017,H7N9 subtype HPAI outbreaks occurred in chickens in China.Therefore,the AI vaccine against both H5 and H7 subtype AI was urgently needed.This study first systematically identified the biological characteristics of the H7N9 H7-Re1 vaccine strain,including viral titers,the virulence,immunogenicity and so on.The results showed that this vaccine seed had high biosafety,high specificity,good immunogenicity,and could efficiently and stably proliferate in chicken embryos,which suggests it is an ideal vaccine seed for developing the inactivated vaccine against H7 subtype HPAI infection in poultry.Secondly,based on the H5N1 Re-8 strain and H7N9 H7-Re1 strain,H5/H7 bivalent inactivated vaccine was prepared and a series of safety and efficacy evaluations were conducted.The chicken embryo allantoic fluid of H5N1 Re-8 strain and H7N9 H7-Re1 strain were concentrated 2-fold volume,inactivated by formaldehyde and then emulsified according to the antigen volume ratio of 1:1.5 confirmed in this study to prepare the H5/H7 bivalent inactivated vaccine.The safety test results showed that high-dose,single-dose and single-dose repeated inoculation of SPF chickens did not have any adverse reactions within 14 days after inoculation.The minimum immunization dose of the vaccine is 0.1mL,and the H5 and H7 subtype HI antibody mean titers were both above 6log2 3weeks post immunization.There was no significant difference in immune efficacy between intramuscular injection and hypodermic injection in the SPF chickens.Both of them can produce high antibody levels.Antibody levels were up to 9log2 at the peak time(4 weeks post the first immunization),complete protection period was from 14 to 210 days post immunization in chickens injected with one dose(0.3mL)of this vaccine.The results of HI antibody test and challenge results showed that the anti-death protection could be obtained when the HI antibody titers of H5 and H7 reached 3log2 3weeks post immunization,and the complete protection could be obtained when the HI antibody titers reached 4log2 or above.The H5/H7 bivalent inactivated vaccine was tested in chickens,white feather broilers,yellow feather broilers,geese,and meat ducks in the field.The results showed that this vaccine was of good safety in all the field poultry.The vaccine could be well absorbed by different kinds of poultry without local and systemic adverse reactions.The HI antibody titers of layers can be as high as about 9log2 150 days post immunization,and the immunization protection duration can last for 150 days.Immunizing according to the procedure with three doses of vaccine before laying eggs,the HI antibody were higher and the duration is longer in layers.Immunizing once for the commercial white feather broiler and twice for the commercial yellow feather broiler and commercial meat duck can make them obtain immune protection before slaughter.Immunization with the vaccine according to the procedure could induce good HI antibody in layer ducks and breeding geese and protect them against the challenge of H5 and H7 subtype AIVs.In this study,the H5/H7 bivalent inactivated vaccine(H5N1 Re-8 strain + H7N9 H7-Re1 strain)has been developed and evaluated in different poultry.This vaccine exhibited good safety and effectiveness in chickens,ducks and geese.Since September in 2017,the vaccine has been used nationwidely and played an important role against the infection of the H5 and H7 AIVs in poultry and successfully blocking human infection with H7N9 virus.