Construction And Immunogenicity Of Recombinant Fowlpox Virus Expressing Respectively Major Immunogenic Genes Of NDV,IBDV And FMDV

Newcastle disease virus (NDV) infection results in Newcastle disease (ND), ahighly epidemic and most costly disease, which greatly can threaten poultry. Vaccineinoculation is a safe and effective way to prevent NDV. Infectious bursal disease(IBD) caused by IBD virus is highly cute and contagious disease of chook. Recently,emerge of very-virulent IBDV (vvIBDV) and variant isolate bring out enormousdifficulties because of breaking through protection of parent antibody. Due to lowimmuogeniciy of inactivated vaccine and disperse and returning virus of live vaccine,current focus is development of promising vaccine.Foot and mouth disease (FMD), as a violently artiodactylous infection, iscaused by Foot and mouth disease virus (FMDV). At present, FMD is prevalent inthe world and the FMD virus exists in the form of seven different serotypes: O, A, C,Asia1, and South African Territors1(SAT1), SAT2and SAT3. Historically, FMDSerotype O and Asia1had been spread in part of China. Now it has serious threatthat still occasionally happen in the many countries around China and Southeast Asiahappen.Poxvirus, which permit large exogenous gene be inserted, displays obviousadvantage and distinct progress show that poxvirus can induce immune response ofchook and mammalian against invaded antigen. The selection of what genes can beused for antigen expressed in virus vectors need to be made a decision. A possibleoptimizing heterogenic expression route is to use natural poxvirus promoter. Inaddition, further knowledge for competition among expressed antigens inserted inpoxvirus will help construct multivalent rFPV to prevent various pathogens. We canselect various formatted vaccine containing DNA, protein and inactivated vaccinefollowing to going to investigate co-immunization.The aim to establish to fowl pox virus shuttle transfer plasmid pMTB18-F-F, pMTB18-F-HN, pMTB18-F-VP0, pMTB18-VP0-VP2and pUTAL-F-VP2, the fourgene, F and HN gene of NDV, VP0and VP2of IBDV, was be inserted downstreamof bi-directed promoter of reconstructive poxvirus transfer vector carrying linedpromoter PE/L and P7.5. Above recombinant cassettes were cotransfected with poxvirus282E4, respectively. After serial passage of positive recombinant virusconstruct, expression and biological activity of fusion protein was identified by PCR,RT-PCR and IFA. Exogenous transcription of these construct virus was constantlydetermined by PCR. Indirect immunofluorescence and immune enzymes detection ofrFPV-F-VP0, rFPV-F-F, rFPV-F-HN and rFPV-F-VP2were carried out by usingserum of anti-NDV as the first antibody. Correspondingly, the serum of anti-IBDVwas used for the same detection of rFPV-F-VP0, rFPV-VP0-VP2and rFPV-F-VP2.Five rFPV can effectively express and product has promising biological activity.In present study, this five recombinant poxvirus vaccine was investigated byhumoral immunity and cellular immunity index detection and assessed on vaccineimmunogenicity. These results show that our constructed poxvirus rFPV inducedSPF chicken producing special ELISA antibody against NDV or IBDV. Antibodylevel increased following to the second week and reach the peak at the five-six weekafter vaccine inoculation. The data of antibody level is higher compared with thecontrol group. In the test of multiplication capacity of T lymphocyte of spleen andTh1-like cytokine (IFN-γ and IL-2) and Th2-like cytokine (IL-4and IL-10), therFPV can induce SPF chicken to produce special immune response. The challengeexperiment showed that rFPV-F-HN could partially prevent SPF chicken againstNDV and SPF chicken could reduce damage degree of IBDV-JL01/2007.In our lab, we successively construct FMDV vUTAL3CP1(O serotypev),UTAL-P1-2A-3C-IL18(Asia-I serotype), vUTA2-P1-2A-3C (Asia-Ⅰserotype) andvUTA2-P1-2A-IL18(O serotype) foxvirus vaccines and FMDVGS115/pPIC9K-VP1-2A-CTE (O serotype). GS115/pPIC9K-VP1-2A-CTE (Asia-Iserotype) recombinant protein expressed in yeast and nucleic acid vaccinepVIRIL18P1(O serotype) and pVAXI-P1-2A-3C (Asia-I serotype). Above vaccinecould induce special humoral and cellular immunity of FMDV in mice and hadefficient immunogenicity. Accordingly, we immunize guinea pig and detected level of humoral and cellular response level by using prime-boost strategy in this study.These results showed that the best effect is to employ the protocol of nucleic acidvaccine pVIRIL18P1as the first immunization and recombinant poxvirus as boostimmunization. The obvious cellular effect can be found in the test of immunizationof recombinant vUTA2-P1-2A-3C-IL18. in our experiment, DNA vaccine extendedits most effective response in the first immunization and recombinant poxvirus canbe used to boost immunization. At the same time, we found that poxvirus couldincrease immune response by self-adjvant activity.We evaluated humoral and cellular response by combined immunization withseveral vaccine O serotype vaccine pVIRIL18P1/vUTAL3CP1and vUTAL3CP1/vUTAL3CP1, Asia-I serotype vaccine pVAXI-P12A3C/vUTA2-P12A3CIL18andvUTA2-P12A3CIL18/vUTA2-P12A3CIL18in animal model of pig. Nucleic acidvaccine(500μg/portion) was coated with chitosan and poxvirus vaccine(107PFU)added adjuvant QS-21made a mixture with freeze-drying protective agent. Both thedifferent vaccine were used for two inoculation of28days interval in35days piglet.Subsequently, we confirm their good immune response by serial detection containingindirect ELISA, LPB-ELISA, ELISPOT, T lymphopoiesis and cytokine detection.The further investigation confirmed the immune protocol is best by DNA vaccine asfirst immunization and recombinant poxvirus boost immunization. In conclusion, weprobed into novel vaccine design and immune strategy aiming to NDV, IBDV andFMDV based on reconstructed vector and co-immunization in present study. Inaddition, recombinant DNA vaccine carrying bi-directed promoter and immune testwere as deep research objects. These studies provide good foundation ofdevelopment of recombinant vaccine and of pre-clinical experiment for poultry andmammalian.