The long-term objectives of this project are to identify the molecular components of spotted fever group (SFG) rickettsiae that stimulate protective immunity and the rickettsicidal immune mechanisms that effectively clear the rickettsial infection. Rocky Mountain spotted fever, the most severe and widespread rickettsiosis in the US, continues to kill more than 5% of patients because of diagnostic difficulty and lack of a means for protection. Numerous SFG rickettsioses are prevalent in other parts of the world, and the immune mechanisms are very similar for the resurgent epidemic louse-borne typhus and for murine typhus. The health impact of this research is that it will establish the knowledge and principles to enable the production of effective vaccines against rickettsial diseases.
The specific aims of this project will be to: 1) define the dominant CD8 T-lymphocyte epitopes of rOmpA and rOmpB of Rickettsia conorii at the oligopeptide level and identify the epitopes processed intracellularly and presented on the cell membrane by endothelial cells for activation of CD8 T-lymphocytes, and evaluate these dominant CD8 T-lymphocyte epitopes for protection in the appropriate mouse model(s); 2) map the B-lymphocyte epitopes of rOmpA and rOmpB and determine the role of antibodies in immune protection against R. conorii; and 3) determine the role of the T-lymphocyte targeting chemokines (IP-10, Mig, and fractalkine) produced by rickettsia-infected endothelial cells in the chemotaxis, localization, and activation of effector CD8 T-lymphocytes. The peptides that activate purified CD8 T-lymphocytes will be identified using both macrophages and endothelial cells as antigen presenting cells initially in order to validate the approach using endothelial cells transfected with a eukaryotic expression vector expressing rOmpA and rOmpB peptides. Mapping of the CD8 T-cell epitopes will employ a strategy of subcloning overlapping DNA fragments followed by selection of overlapping synthetic peptides. The B-lymphocyte epitopes of rOmpA and rOmpB will be determined using recombinant peptides of R. conorii rompA and rompB and finally overlapping synthetic oligopeptides. Conformational epitopes will be determined using recombinant peptides from a random peptide library expressing minotopes. The epitopes that stimulate CD8 T-lymphocytes and antibody production will be evaluated for their ability to stimulate protective immunity in mice and by passive transfer of CD8 clones and monoclonal antibodies. The remarkable cross protection between the divergent SFG rickettsiae, R. conorii and R. australis, will be exploited in our strategies to identify the protective epitopes. Because the immune control of rickettsial infection occurs mainly inside infected cytokine-activated endothelial cells which are targets of CTL activity and adjacent to which CD8 T-lymphocytes have infiltrated, the role of chemokines in 3 particular endothelial cell-derived lines will be determined in the chemotaxis, localization, and activation of the effector CD8 T-lymphocytes using a model of the retinal vascular bed in vivo and endothelial cell cultures. The overall outcome of this project will be the expansion of our knowledge of the most important, insufficiently understood elements of rickettsial immunity: CD8 T-lymphocytes, antibodies, stimulating epitopes at the level of synthetic oligopeptides, and the mechanisms of chemotaxis and activation of CD8 T-lymphocytes in the rickettsia-infected lesions.
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