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Project A: <BR> #1: Confirm relevance of antigens already identified through in vivo-induced antigen technology (IVIAT) to the pathogenicity of Y. pestis and B. abortus. <BR>#2: Characterize common gene products of F. tularensis, B. abortus, and Y. pestis identified through "in silico ortholog-targeted" homology searches using our existing IVIAT data. #3: Begin building a database of tularemia genes up-regulated during infection using an enhanced IVIAT methodology, known as change-mediated antigen technology (CMAT). <P>Project B: <BR> The research objectives are to 1) determine the phenotypes of cells that express cellular prion protein in lymph follicles of tonsil and lymph nodes of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni) and assess the relative expression level on follicular dendritic cells (FDCs); 2) examine the role of the glycosylphosphoinositol anchor in attachment of both cellular prion protein and disease associated prion protein to FDCs and possible release mechanisms that may contribute to systemic dissemination of infectious prions during infection; and 3) explore the hypothesis that disease-associated prion protein is trapped and retained by FDCs as a prion-complement complex, by assay of the prion binding ability of complement component C1q, and whether C1 is activated by the binding.
NON-TECHNICAL SUMMARY: <BR>Project A: Plague and brucellosis represent re-emerging zoonotic diseases in numerous animal species, domestic and wild, in the Western U.S. Although the pathology of these infections has been well studied, the molecular mechanisms by which their associated bacterial agents cause disease remain enigmatic. Bacterial genes up-regulated during infection may encode proteins useful as protective components for vaccines or as diagnostic targets for bacterial animal disease agents. Our research to date, based on the application of in vivo-induced antigen technology (IVIAT) on Yersinia pestis, the bacterium which causes plague and Brucella abortus, the agent of brucellosis in domestic livestock and wild animals, has resulted in the identification of over 30 genes expressed during infection by these disease agents. Furthermore, we have found almost all these genes to encode proteins highly conserved through the course of evolution. To support our hypothesis that these gene products represent a common theme associated with pathogenic processes in different diseases, we will further study these proteins in our laboratory (in vitro and in vivo), in addition to characterizing the common gene products of B. abortus, Y. pestis, and Francisella tularensis (the agent of tularemia) identified through gene homology searches using our existing IVIAT data. We also intend to conduct similar studies on F. tularensis to identify and characterize genes up-regulated during infection using our previously employed gene discovery methodology. <P>Project B: Prion replication in scrapie of sheep and goats and chronic wasting disease (CWD) of deer and elk occurs throughout lymphoid tissue before neuroinvasion and the ensuing fatal neurodegenerative disease. To understand the role of follicular dendritic cells (FDC) of tonsils and lymph nodes in early infection of natural hosts such as mule deer and elk with CWD, we will determine phenotypes of lymphoid follicle cells expressing cellular PrP (PrPC) in deer and elk, develop a cervid-specific monoclonal antibody recognizing FDCs, test both PrPC and infectious prion (PrPSc) for attachment to follicle cells by GPI anchor using PIPLC digestion, and explore the feasibility of developing a binding assay for soluble complement protein C1q and PrPSc.
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APPROACH: <BR>Project A: Selected recombinant Y. pestis and B. abortus in vivo-induced (IVI) proteins from E. coli strains produced in our laboratory will be evaluated as sub-unit vaccine candidates in fully virulent plague- and brucella-challenge mouse models. We have also down-selected several of our IVI genes (Y. pestis and B. abortus) to be characterized in vitro. Specifically, cytopathogenicity of marked mutants of the selected genes, constructed in our laboratory, will be compared to wild-type strains on different eukaryotic cell types. Additionally, the products of four orthologous IVI loci identified in F. tularensis, B. abortus, and Y. pestis, will be further evaluated to determine their role in the pathogenesis (12 gene products total) by assessment of protective immunity induced by these proteins in the respective immunization/challenge animal models, to include the evaluation of cross-pathogen protection. Genes up-regulated during tularemia infection will also be identified using an enhanced IVIAT methodology, followed by serologic screens on DNA libraries of F. tularensis clade A.II, strain WY89 (Western U.S. type-A sub-group) with sera from wild animal hosts. Specifically, immune serum will be generated in surrogate laboratory animals (rabbit) immunized with inactivated tularemia-infected tissues from domestic and wild hosts. The procedure, known as CMAT ("change-mediated antigen technology"), should result in the enrichment of antibody against pre-expressed IVI antigens. Our collaborators at Ft. Detrick (USAMRIID) will also conduct parallel IVIAT screens (using immune rabbit serum) on a DNA library generated from F. tularensis Schu S4, a clade A.I strain (Eastern U.S. type-A sub-group). As our tularemia IVI databases grow, we will reciprocally exchange clones with USAMRIID to test for cross-reactivity. <P>Project B: Immunohistochemistry (IHC) for cellular prion protein (PrP) and cell surface markers that are specific to dendritic cells, B and T lymphocytes, macrophages or follicular dendritic cells (FDCs) will be performed on frozen and fixed sections of tonsil and lymph node tissues from uninfected deer and elk. Double immunofluorescent staining and co-localization of cellular PrP and cell phenotype proteins will be carried out by confocal fluorescent microscopy. PI-phospholipase C (PI-PLC) treatment of unfixed tissue samples will be used to assay for release of disease-associated PrP from FDCs of lymphoid follicle germinal centers, using release of cellular PrP from uninfected lymphoid tissue first to establish digestion and detection conditions. The effect on PI-PLC release of disease-associated PrP using Proteinase K digestion, neuraminidase treatment, N-glycosidase F, and collagenase A digestion prior to PI-PLC treatment will be investigated. The establishment of an assay for binding of disease-associated PrP by complement protein C1q will use C1 complement components isolated from deer and/or elk serum and disease-associated prion extracted from chronic wasting disease infected tissues to pepare a reconstituted C1 system modeled after a human complement system to demonstrate beta-amyloid binding and C1 activation.