Immunoproteomic Analysis And Study On Subunit Vaccine Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia, a highly contagious respiratory infection in pigs, and all the 15 serotypes are able to cause disease and result in severe losses in the swine industry worldwide. The predominant serotypes in China were serotype 1,2,3 and 7 and the genome sequence of serotype 3 strain JL03 have been determined by our lab. Based on these findings, Current study focused on screening conserved antigens from Actinobacillus pleuropneumoniae by reverse vaccinology, and developed subunit vaccines for cross protection in combination with O/W adjuvant MF59.The obtained findings were as follows:1. Identification of immunogenic proteins from Actinobacillus pleuropneumoniaeIn this study, the immunoproteomic approach was applied to the analysis of extracellular and outer membrane proteins of A. pleuropneumoniae JL03 serotype 3 with convalescent sera for the identification of novel immunogenic proteins for A. pleuropneumoniae. A total of 33 immunogenic proteins were identified from outer membrane (17 proteins) and extracellular proteins(16 proteins) of JL03 serotype 3, of which three proteins, MomP1, MomP2, EF-Tu, were identified simultaneously from OMPs and ECPs. Of the 30 identified immunogenic proteins,6 were known antigens and 24 were novel immunogenic proteins. In novel immunogenic proteins,16 proteins have already been shown immunogenic in certain pathogenic bacteria, D15/OmpD, LppB, FrdA, MDH, FepA, FrpB, TufB, PotD, GapA, ZnuA, TIG, DegP, TufB, PsaA, FkpA and PTA, and 8proteins, CbiK, IlvG, FepB, AfuC, FatB, GGBP, CysG and Ttg2D, are reported to be immunogenic proteins for the first time in this study whose functions have been biologically demonstrated in some bacteria.2. Subcellular localization of immunogenic proteins and protective efficacy in miceGenes of ompD, lppB, and fepA were cloned and expression in E. coli BL21, proteins were purified and analysed by Western blot, and they were demonstrated to be localized on surface of cell by indirect immunofluorescence. Immunogenecity of OmpD, LppB and FepA were evaluated in mice model, and results showed 70% protective efficacy from OmpD and LppB, but only 40% from FepA.3. Study on subunit vaccinePurified rApxⅠA,rApxⅡA,rApxⅢA were in combination with rOmpD and rLppB for subunit vaccine preparation, and MF59 adjuvant were equally mixed (V/V) with prepared antigens. Two subunit vaccines were prepared, Vaccine I contained 50μg/ml of rApxⅠA,rApxⅡA,rApxⅢA, rOmpD and rLppB respectively, and vaccineⅡ75μg/ml. VaccineⅠ, vaccineⅡand VaccineⅢ(inavtivated vaccine, containing APP 1,2,7 serotype strains) were inoculated mice. Antibody titer of rApxⅠA,rApxⅡA,rApxⅢA, rOmpD and rLppB in sera from vaccineⅠandⅡgroups showed significantly higher than sera from control and vaccineⅢgroup. Mice were challenged with 5LD50 serotype 1,2,3,7 strains, survival mice of vaccineⅠ,Ⅱgroups had not shown obviously pathologic and histological changes in lungs, and slight histological changes in lungs after challenge with APP serotype 1. Mice of vaccineⅢgroup had not shown obviously pathologic and histological changes in lungs after challenge with APP serotype 1,2,7, but had shown obvious pathologic and histological changes in lungs after challenge with APP serotype 3. VaccineⅠandⅡgroups showed 70%-90% protective efficacy against challenge with APP serotype,1,2,3,7 strains. However, vaccineⅢgroup showed 100% protective efficacy against challenge with APP serotype 1,2,7 strains, but 40% protective efficacy against serotype 3. Data in the present study demonstrated that subunit vaccines induced cross-protection in comparision with vaccineⅢ(inactivated vaccine), and MF59, an O/W adjuvant, was degradable in vivo. Subunit vaccines in this study are epcected to be a novel vaccine for APP, and protective efficacy in pigs needs investigation in further study.