Selection Of Protective Monoclonal Antibodies Against Aemophilus Parasuis And Identification Of Immunogenic Rotein Of H. Parasuis

Haemophilus parasuis (HPS) is a Gram-negative, non-hemolytic, NAD-dependent bacteriumbelonging to the Pasteurellaceae family. The organism is an important upper respiratory-tract pathogenin swine and the etiological agent of Gl sser’s disease, which is characterized by fibrinous polyserositis,polyarthritis, meningitis and arthritis syndrome or acute pneumonia without polyserositis and acutesepticemia. In recent years, with changes in production such as early weaning and use of three-siteproduction systems, HPS infection has been increasingly implicated as a major cause of nurserymortality in commercial swine herds, especially in specific pathogen free herds.Although Gl sser’s disease can be successfully treated with antimicrobials, resistance toantimicrobials has been reported. Vaccination is an efficient way to control this disease provided thechallenge is a homologous serotype, but major variability has been reported in cross-protection tests.Therefore, development of novel therapeutic methods is necessary. Some studies reported that theantibodies detected in the sera of vaccinated swine were directed only against HPS outer membraneproteins (OMPs), suggesting that OMPs are more immunogenic than other components of HPS. Inrecent years, the development of protein-based vaccines has been given much more attention, andseveral immunogenic OMPs of Gram-negative bacteria have been identified by immunoproteomicsanalys is and protection assays. The identification of novel and more efficient immunoprotectiveantigens is crucial for the development of a monovalent or multivalent subunit vaccine that can protectswine from HPS infection.In the present study, monoclonal antibodies (MAbs) against HPS were generated by fusing spleencells from BALB/c mice with SP2/0murine myeloma cells.1D8which reacted with all15serotypes ofHPS was screened by enzyme-linked immunosorbent assay (ELISA) and Western blot. The bactericidalactivity of1D8was determined by the complement-mediated killing assay. For the protectionexperiment,1D8could protect mice from challenges with lethal dose HPS and prolong survival timesignificantly. For the bacterial elimination assay,1D8could protect mice against homologous andheterologous challenges with nonlethal dose4and5strains which are the most widespread strains ofHPS in China and eliminate bacteria efficiently in vivo. Our results demonstrate1D8hascross-protection ability and protect mice from challenges with different serotype HPS.To find out causes about cross-protection ability of1D8, we identified the immunoreactive proteinfor1D8through2-DE and mass spectrometry analysis. The data suggest that OmpA is the target proteinof1D8. OmpA, a major outer membrane protein of Gram-negative bacteria, is very highly conservedand participates in biofilm formation, bacterial conjugation, bacteriophage binding, cell growth andinvasion of mammalian cells. Several OmpA-like proteins have been identified in other Gram-negativebacteria, including Riemerella anatipestifer, Pasteurella multocida and Leptospirosis. However,information regarding HPS OmpA-induced immune response is limited. On the basis of our presentknowledge of outer membrane proteins regarding adhesion to and invasion of host cells, we hypothesizethat1D8impairs the biological functions of OmpA by disrupting the interaction between HPS and the host cell. Moreover,1D8eliminate the bacteria by antibody-dependent cell-mediated cytotoxicity(ADCC) and complement dependent cytotoxicity (CDC).In our study, monoclonal antibodies against OmpA were generated and identified, and theneutralizing activities of1D8were evaluated using in vitro and in vivo experiments. The resultsdemonstrate the protective roles of1D8raised against OmpA and indicate that OmpA is a promisingcandidate for a subunit vaccine against HPS.