: This proposal is focused upon the intracellular amastigote stage of Leishmania mexicana complex parasites. Vaccination with the amastigote surface P-8 proteoglycolipid complex, expressed in the amastigote stage, confers significant resistance to infection in the murine model. CD8+ and CD4+ T cells as well as INFy, CDld and perforin-mediate mechanisms are critical for protection. In addition, the P-8 glycolipids directly affect macrophage function (cytokine production and parasite uptake). This proposal is focused on elucidating the immune protective mechanisms induced by P-8 vaccination and on the biological roles (structure-function) of the P-8 glycolipids. Specifically the aims are: 1. Determine the mechanisms by which CDld presentation/NKT activation contributes to the protection against Leishmania infection induced by P-8 vaccination. The P-8 glycolipids may act as an inflammatory agent (adjuvant), as well as an """"""""antigen"""""""" -presented via CDld. Using the routine model (blocking antibodies, immune depletion, and immune deficient mice), the role of CDld presentation and NKT cells in the induction and effector phases of the protective immune response will be determined. Further, CDld-P-8 antigen presentation by antigen-pulsed and infected macrophages and dendritic cells will be examined (role of processing, kinetics, CDld-P-8 glycolipid(s) interaction (KB)). These studies should indicate whether the P-8 glycolipids (and possibly other amastigote glycolipids) might represent a new class of leishmanial antigens and determine the importance of NKT cell activation in vaccine-induced protection against leishmaniasis. 2. Investigate the biological function and biochemical characterization of the P-8 amastigote antigen. The P- 8 glycolipids appear to be distinct from previously described leishmanial glycolipids and have biological effects on the parasite's host cell, the macrophage (morphology, parasite uptake; cytokine/chemokine production). Consequently, the biochemical structure of the P-8 glycolipids will be determined using mass spectroscopy and the receptor (TLR; non-TLR) involved in acrophage activation will be determined. Further, the immunologic effects on key antigen presenting cells (dendritic; M(bs) will be determined. These studies should reveal the function of the P-8 glycolipids in vivo and further our understanding of the amastigote-host interaction. ? ?
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