DESCRIPTION: (provided by the applicant): The goal of this research is the molecular and immunologic characterization of the 160 kDa complement regulatory protein (CRP) of the protozoan parasite, Trypanosoma cruzi, the causative agent of Chagas disease. After infection of the vertebrate host, the bloodstream stage trypomastigote is capable of evading host alternative and classical complement activation pathways, allowing dissemination to targettissues and intracellular replication. This is accomplished through the production of adevelopmentally regulated surface glycoprotein, CRP that restricts complement activation atthe level of C3 convertase formation. Our hypothesis is that immunologic neutralization of theCRP contributes to destruction of the extracellular bloodstream stage, and thus will contribute to protection from disease. We have shown that immunization with plasmid DNA encoding the full length CRP structural gene elicits neutralizing antibodies and protects against a lethal challenge in a murine model of Chagas disease. Three specific aims are designed to optimize the design of vaccine candidates based on the CRP gene and to characterize the immune responses to the protein, in vivo. Specific Aim 1 will provide a molecular analysis of the active domains of CRP and the binding interaction between CRP and its ligand, the complement component C3b. The functional analysis will be carried out using mutant CRP constructs expressed in mammalian cells and assayed for complement restriction activity and C3b binding. In addition, we will use neutralizing anti-CRP monoclonal antibodies and CRP-binding peptides to provide a finer map of activity. Specific Aim 2 will focus on an evaluation of CRP-DNA based vaccines in a murine model of Chagas disease. In these studies we will compare three expression vectors, optimize immunization protocols, and evaluate the range of protection using multiple mouse and parasite strains. In addition, we will examine oral and mucosal vaccine delivery systems and mucosal, sub-lethal challenge regimens. The goal of Specific Aim 3 is to analyze the immune effector mechanisms involved in the pmtective anti-CRP response. The role of immune antibodies will be evaluated in DNA immunized mice to determine if conformationally dependent epitopes are essential to the induction of a protective response. Passive transfer and challenge studies will be conducted and we will test vaccinated mice for the presence of CRP-specific cytotoxic T cells using CRP-expressing target cells. The successful completion of these aims will provide the first functional and immunological evaluation of a critical virulence factor of T. cruzi and provide information and reagents for the use of CRP as a vaccine candidate.
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