The Prevention Of Anti-propionibacterium Acnes Antibody To Actinobacillus Pleuropneumoniae Infection And The Screening And Identification Of Common Epitopes Between The Two Bacteria

Actinobacillus pleuropneumoniae (APP) is a Gram-negative bacterium that can cause porcinepleuropneumonia and results in large economic losses to the porcine industry worldwide. APP hasbeen divided into15serovars, with a lack of cross-protection between the main serovars resultingin slow progress in the development of vaccines. Propionibacterium acnes (PA), a Gram-positivecorynebacterium, has immunomodulatory activity, can activate the mononuclear phagocyte system,and is able to induce the generation of inflammatory cytokines, thereby protecting against avariety of pathogenic bacteria infections.Previously, we screened the differential genes of main serotypes of APP, and accidentallyfound that there is a very strong cross immune response between PA and APP. PA can preventagainst of the APP infection largely in a humoral immunity dependented manner. Anti-P.acnes IgGwhich produced by B cells after PA immune can significantly increase the survival rate of APPinfected mice. The higher levels of the antibody produce the better protection rate of mice.However, how anti-P.acnes antibodies work in this process and what exactly epitope that canproduce these antibodies to prevent APP infection is not clear yet.In order to reveal how anti-P.acnes antibodies work in this process and look for the commonepitope between the two bacteria and promote the vaccine research of APP, we first builtneutrophils and macrophages depletion mice model respectively and then challenged with APPafter passive immunization with anti-P.acnes antibodies, assessed the role of neutrophils andmacrophages in the process of anti-P. acnes antibody protection of hosts from infection withheterologous APP by the protection rate and the burden of APP in the lung tissue and lungpathological changes. Then we built a PA genome-wide phage display random library and tookanti-APP serum as solid phase target molecule to screening this library. Predicted B cell epitopesof these screened proteins and compared them with APP1to look for high homological epitopesfor synthesis. The high quality epitopes that selected from the synthetic peptides by screeningthem with anti-P.acnes serum and anti-APP serum were coupled with KLH and then the IgG levels,the serological reactivity to anti-epitope peptides serum of PA and APP1, IL-4, IFN-γ level weremeasured by ELISA. The proportion of CD4+/CD8+T cells in the spleen and the survival rate ofmice challenged with APP after immunization with epitope peptides were determined. Finanlly,the immune effect of epitope peptides and their ability to prevent APP infection were analyzedcomprehensively.Experiments show that anti-P. acnes IgG has an effect similar to anti-APP IgG which significantly promoted the phagocytosis of APP by macrophages. Macrophages are essential foranti-P. acnes antibodies prevent APP infection for anti-P. acnes IgG cannot against APP infectionin macrophages depletion mice model. However, anti-P. acnes IgG can against APP infection nomatter neutrophils depleted or not, indicating neutrophils are not essential for the process bywhich anti-P. acnes antibody prevents APP infection in mice. We built a PA genome-wide phagedisplay libraries database with a105capacity from which we got10common antigenic proteinsbetween PA and APP by screening this libraries. After predicted B cell epitopes and madesequence alignment with APP1, we received six B cell epitopes, A2, Ba1, Ba2, Bb4, Bb5andC1,which have high homologous rate with APP1. Besides A2, the remaining five epitopes all haveserological reactivity to anti-P. acnes antibody and anti-APP antibody. These five peptides caninduce a certain level of antibody after immunization in mice. Ba1, Bb5and C1antiserums but notBa2and Bb4antiserums have specificity reactivity to PA and APP. Ba1and Bb5antiserums canrecognize APP1as determined by immunofluorescence. The IL-4levels increased after Ba1,Bb5and C1immunization in mice, but the IFN-γ levels raised only after Bb5immunization. Theseindicated that Ba1and C1induced Th1/Th2mixed immune response and Bb5tend to Th2immuneresponse. The ratio of CD4+/CD8+T cells which immunization with Ba1and Bb5are higher thanthat of the control group, suggesting CD4+T cells was the dominate cells and humoral immunityplayed a leading role in this process. Ba1or Bb5can protect mice against APP1,the survival rate is80%. When combined Ba1with Bb5, the efficacy of Ba1and Bb5against the APP5is90%, and80%for APP1.This study preliminary illustrated the way of anti-P. acnes antibody worked in prevention ofAPP infection and obtained and identified two effectively protective common B cell epitopesbetween PA and APP. Therefore this study lays a foundation for the research of APP vaccines andalso provides beneficial theoretical basis for the research of heterogeneous vaccine.