The long-term goal of this project is the development of an adequate database concerning the antigenic, protein, lipopolysaccharide and molecular composition of ehrlichiae of medical importance. Such information would form the foundation for studies of the unusual host immune reactions stimulated by these agents, the function of ehrlichial proteins in intracellular life and the contributions of such proteins toward host cell injury. The increasing numbers of human patients with severe morbidity or fatal outcome when infected with ehrlichiae attests to the need for such studies. With the recent development of an efficient method for propagation of E. canis, the previous obstacles to in-depth modern investigation have been removed. The proposal developed herein allows for development of specific polyclonal and monoclonal antibodies to identify antigens specific for or shared among ehrlichiae, and the determination of protein and lipopolysaccharide composition of these molecules. Such basic studies will establish a framework for investigating host immune responses toward B-cell epitopes on these antigens. The ultrastructural localization of antigens by specific antibodies will identify bacterial surface components in a location most likely to interact with host cell components. While B-cell reactive antigens may be used as markers in various experiments, T-cell epitopes are more likely to be important in immune reactions against intracellular bacteria such as E. canis. The evaluation of canine T-cell responses elicited by acute E. canis infection and by specific surface-exposed major immunodominant protein antigens may provide an approach toward understanding the abnormal cellular immune responses and lack of persistent immunity to ehrlichiae. To understand the molecular biology of these antigens, one or more which are major surfaced-exposed proteins and contain T-cell epitopes will be identified in a genomic library of E. canis, and the recombinant DNA will be sequenced. Fundamental studies such as these will provide the tools and practical basis necessary to dissect immune responses to component antigens, to study the function of proteins in the intracellular life of the ehrlichia, and to investigate the pathogenic mechanisms which involve these proteins and cause host cell injury.
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