Development Of Combined Vaccine Against H5 Subtype Avian Influenza And Infectious Bursal Disease

H5N1 HPAIV is one major zoonosis pathogen that can not only infect poultry and wild birds,but also infect mammals including humans.H5N1 subtype avian influenza has covered more than 60 countries and regions around the world,resulting in the death of millions of poultry and birds,causing seriously harm to the poultry industry.Infectious bursal disease?IBD?mainly affects immature chickens,and induces immune suppression,leading to the exposure to wide range of viral,bacterial and other pathogen,and also leading to vaccine immune failure.AIV and IBD are important risks of China's poultry industry,vaccination is one of the most effective means to prevent and control the dieases.However,due to the continuous variation of the virus and the increasing demand for large-scale breeding,a number of new vaccines have been developed to overcome the shortcomings of traditional vaccines.Duck enteritis virus?DEV?has a large genome capacity with large number of non-essential regions that allow foreign gene being inserted into.In this study,we intended to develop vaccine against avian influenza or infectious bursal disease,and two combined vaccine against both diseases.HA gene from A/Chicken/Guizhou/4/2013?H5N1??GZ/4?and VP2 gene from vvIBDV was connected by internal ribosome entry site?Internal ribosome entry site,IRES?sequence in a different order,in which HA-IRES-VP2 and VP2-IRES-HA series.The HA and VP2 gene fragments and series gene fragments were then cloned into pENTR-sv40.The insertion of sv40-HA,sv40-VP2,sv40-HA/VP2 or sv40-VP2/HA cassette into the FosT-us78 from the system was done by using LR Clonase II enzyme.Our lab used to establish a system to generate the DEV vaccine strain by using the transfection of overlapping fosmid DNAs.Using this system,we successfully inserted the target gene between the US7 and US8genes of the DEV genome,and constructed the four recombinant viruses,rDEV-H5-8,rDEV VP2,rDEV-HA/VP2 and rDEV-VP2/HA.The in vitro experiments shown that,the inserted gene could be stably inherited and expressed in the recombinant virus without affecting its replication in cells.In order to evaluate the protective efficacy induced by recombinant virus in SPF chicken,3-week-old SPFchickens were immunized with 10³?10⁴?10⁵ TCID₅₀ recombinant virus,and challenged with 100LD₅₀ GZ/4 or 100 LD₅₀ HLJ-0504.The results shown that immunized with 10³ TCID₅₀ rDEV H5-8 or 10⁴ TCID₅₀ rDEV VP2 was able to induce100%protection,while the rDEV-HA/VP2 and rDEV-VP2/HA groups could not induce effective immune protection in both three doses.The antibody test results showed that HI antibody of three groups induced by rDEV H5-8 all turn positive at 2 weeks p.v.,with electroporation reached 4 Log₂ or above;only part of HI antibody turned positive in groups immuned with rDEV-HA/VP2;no HI antibody can be tested in groups that immuned with rDEV-VP2/HA;IBDV antibody of three groups induced by rDEV VP2,r DEV-HA/VP2 or rDEV-VP2/HA were not detected.In order to futher evaluate the protective efficacy,3-week-old SPF chickens were immunized with 10⁵ TCID₅₀ rDEV-H5-8 or rDEV VP2 or 10⁵ TCID₅₀ rDEV H5-8 mixed with10⁵ TCID₅₀ rDEV VP2,and challenged at 1,2,5 weeks p.v.with 100 LD₅₀ GZ/4 or100 LD₅₀ HLJ-0504,and commercial IBDV vaccine VAXXITEK and Gt strain were immunized as control.The results shown that,SPF chicken immune with rDEV 5-8 can induce complete immune protection against AIV at 1week p.v.,without any morbidity,mortality or virus shedding;HI antibody all turned positve 2 weeks p.v..rDEV VP2immunization could not completely avoid the pathological damage of bursa,but could protect the chicken from death 2week p.v.challenging with vv IBDV;no IBDV antibody was detected during the observation of five weeks.Gt strain could induce 100%protection against vv IBDV at 1 week p.v.only with the bursa mildly atrophy;the positive rate of antibody rased to 40%at 5 weeks p.v..VAXXITEK could induce 100%protection against vvIBDV at 1 week p.v.,without any bursa pathological injury,and all antibody turned positive at 2 weeks p.v..Protective efficiency induced by co-immunization of rDEV H5-8and rDEV VP2 against AIV and vvIBDV reached 100%at 5 weeks p.v.;but still could not completely avoid the pathological damage of bursa;the antibody titers were slightly inferior than those induced by rDEV H5-8 or rDEV VP2 alone.rDEV-HA/VP2 and rDEV-VP2/HA still could not induce effective antibody level after co-immunization.In conclusion,the strategy of using IRES to connect HA gene and VP2 gene in series to construct the combined vaccine against AI and IBD is not feasible.However,rDEV H5-8can induce superior immune protection and high level of HI antibody,which can be used as an alternative vaccine for the prevention of AI;rDEV VP2 has a certain immune potency,may have the potential for further development.Moreover,co-immunization of rDEV H5-8 and rDEV VP2 can induce a good immune protection effect against H5AIVand vvIBDV,which can be used as a strategy to develop combined vaccine.