Research on the critical immune mechanisms required for host protection against a number of microbial pathogens has opened up new areas for vaccine development. Parasitic diseases have proven particularly intractable to attempts at vaccine development. One reason has been the capacity of parasites to subvert host defenses. Research focused on understanding the immunologic evasion strategies utilized by pathogens will allow vaccine development directed towards inactivation of these evasion mechanisms. The focus of this research proposal is the elucidation of the molecular mechanisms involved in evasion of complement mediated killing by the protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas' disease. Trypomastigotes prevent complement-mediated lysis by the production of factors which interfere with the activation complement (C) system. One such factor is a complement regulatory protein (CRP) which restricts complement activation at the level of C3 convertase formation. This protein binds the complement components, C3b and C4b, the key subunits of the C3 convertase. C3b affinity chromatography has been developed for the purification of active CRP which has allowed for extensive biochemical characterization of the protein.
The aims of this research are to extend our understanding of the activity of the CRP protein through a molecular characterization of its binding interaction with C3b and C4b. The long range goal is to address the hypothesis that if the CRP provides a critical means of avoidance of complement-mediated clearance, then a directed immune response to this protein which neutralizes its activity will result in significant protection against infection. The functional domains of the CRP will be characterized by partial proteolysis and mapping with monoclonal antibodies which neutralize activity. Fine structure epitope mapping will be accomplished by deletional analysis of the CRP gene, as well as characterization of epitopes recognized by neutralizing monoclonal antibodies with phage- displayed peptide libraries. Based on the information and reagents generated in these studies, protection studies will be conducted to test the protective capacity of CRP and its derivatives in experimental models of Chagas' disease. The molecular characterization of this protein and the determination of the means whereby the parasite is protected from destruction in the bloodstream will provide critical information regarding an early step in the infectious process. Studies such as these, which define the molecular basis of an immune evasion mechanism of the parasites, will identify specific targets for the eventual development of immune intervention strategies.
| Beucher, M; Norris, K A (2008) Sequence diversity of the Trypanosoma cruzi complement regulatory protein family. Infect Immun 76:750-8 |
| Beucher, Margaret; Meira, Wendell S F; Zegarra, Vasthy et al. (2003) Expression and purification of functional, recombinant Trypanosoma cruzi complement regulatory protein. Protein Expr Purif 27:19-26 |
| Sepulveda, P; Hontebeyrie, M; Liegeard, P et al. (2000) DNA-Based immunization with Trypanosoma cruzi complement regulatory protein elicits complement lytic antibodies and confers protection against Trypanosoma cruzi infection. Infect Immun 68:4986-91 |
