Contruction Of Infectious Bursal Disease Virus-like-particle Displaying Avian Influenza Virus-M2e And Its Immune Efficacy Evaluation

Infectious bursal disease virus (IBDV) is a pathogen of worldwide importance to the poultry industry. IBDV destroys B lymphocytes in the bursa of Fabricius in young chickens, causing both mortality and immunosuppression. IBDV are non-enveloped viruses and the virus icosahedral shell is composed of VP2capisid protein. The VP2protein possesses highly immunogenicity, which can induce specific neutralizing antibody against infection. Expression of VP2alone leads to viral-like particles (VLP) containing20VP2trimers, it is expected to become a good vaccine vector which can be used as antigen delivery carriers to display other antigenic epitopes.Highly pathogenic avian influenza (HPAI) of the H5N1subtype is an epidemic disease and has produced infections that resulted in grand-scale economic losses. The problem was highlighted by the persistent potential heath threat to human. Up to date, five generation of H5subtype avian influenza killed vaccines have been developed to control this disease in China. However, the prophylaxis situation of this disease has yet not been optimized. More attention now is focused on the development of an "universal" vaccine for the protective against variants of one subtype viruses or heterosubtypic viruses challenge, of which the most potential target antigenic site is the highly conserved motif in the extracellular domain of matrix protein2(M2e).In this study, the VP2protein of IBDV was used for the delivery of AIV-M2e epitopes. One copy M2e motif of H5or H9subtype of AIV was inserted into the PBC loops of VP2, or four copies of this M2e motif were fused to C-terminus of VP2, respectively. The recombinant VP2gene were expression via pET-32a(+) Escherichia.Coli (E.Coli) system or pFastBac-1Baculovirus system, respectively. Subsequently, the products of the expression system were purified and prepared as chimeric vaccine to evaluation of its immunogenicity to IBDV and AIV.1. Contruction of recombinant genesAccording to the sequence of IBDV VP2gene published in the GenBank, a series of primers were designed to construction recombinant VP2gene. One copy M2e gene of H5or H9subtype of AIV was inserted into the PBC domain via the way of polymerase chain reaction (PCR), and the recombinant gene named as VP2-BCM2eH5or VP2-BCM2eH9, respectively. Similarly, four copies of these M2e genes were fused to C-terminus of VP2, and an adaptor composed of KK amino acids was also added between the VP2and4M2e genes, these recombinant genes named as VP2-KK-4M2eH5and VP2-KK-4M2eH9. Followingly, the recombinant genes were cloned into pMD19-T vector, and the expected chimeric plasmids were further confirmed by sequencing.2. Expression of recombinant protein in E. Coli system and identificationThe prokaryotic recombinant expression vectors were constructed by four aforementioned recombinant VP2genes cloned into the pET-32a(+) vector, andnamed as pET32a-VP2-BCM2eH5, PET32a-VP2-BCM2eH9, pET32a-VP2-KK-4M2eH5andpET32a-VP2-KK-4M2eH9, respectively. Subsequently, the recombinant expression vectors were further identified by sequencing. The positive recombinant plasmids were transformed into BL21E.coli strain and induced with IPTG to primary expression. The expression conditions were optimized, including induction temperature, IPTG concentration and induction time. The four positive recombinant protein products were identified by refered SDS-PAGE to analysis expression products. And Western-Blotting further characterized the recombinant protein reaction with serum against IBDV.3. Expression protein in baculovirus system and identificationSimilarly, the baculorvirus expression vector were constructed by the T recombinant VP2genes cloned into the pFastBac-1vector and were transformed into competent DH10Bac E.coli cells to obtain. The recombinant bacmids subsequently were transfected into Sf9insect cell to form the recombinant viruses, rBac-VP2-BCM2eH5, rBac-VP2-BCM2eH9, rBac-VP2-4M2eH5and rBac-VP2-4M2eH9. The expected chimeric VP2protein could be detected in the Sf9cell extracts after SDS-PAGE,Western-Blotting and indirect immunofluorescence anlysis with specific serum against IBDV. 4. The immune efficacy evaluation of recombinant proteinThe subunit vaccines were developed with chimeric proteins which expressed by E.Coli or baculovirus system in mineral oil-based adjuvants. Groups of ten2-week-old non-immunized chickens of mixed sex were primed with0.3-ml volume vaccine. These test groups were composed of eight chimeric subunit vaccines groups which developed in this study and six control groups which including IBDV subunit commercial vaccine, H5and H9commercial vaccine, pET32a(+) and pFastBac-1vector control group, PBS placebo control group.The chickens were boosted with same vaccine four-week post-primary immunization and challenged with very virulent IBDV virus four-week post-second immunization. Sera were taken at2,4,6,8-week post-primary immunization to test antibody titer against VP2by virus neutralization in chicken embryo fibroblast and M2e antibody titer by indirect ELISA which coated with synthetic M2e peptide as detection antigen. Antibody titer peak against both VP2and M2e were achieved on four-week post boost stimulus. Chicken vaccined with pBac-VP2-BC-M2eH5. pBac-VP2-4M2eH5and pBac-VP2-4M2eH9were generated better response than other chimeric subunit vaccines. Neutralizing antibody titer against IBDV was1:6400.The IBDV virus challenge showed that the recombinant subunit vaccine provided good protection when compared with the IBDV subunit commercial vaccine. And there were no any clinical sign during observed period of time. This study has laid the foundation for a new generation vaccine of IBDV and AIV.