The overall specific aim of this competing renewal is to further improve the immunotherapy of Toxoplasmosis in those individuals afflicted with AIDS. During our current funding period we have developed a substantial body of information regarding the immunology, molecular biology and serology of several important parasite antigens, in particular P30, the major membrane and excreted/secreted antigen (ESA) of this obligated intracellular pathogen. In this proposal we plan to use these observations to elucidate two different, but complementary specific aims: parasite attachment to host cells and internalization by human phagocytes. The first specific aim is to evaluate the role of P30 in parasite attachment. Our preliminary observations suggest that P30 is an important attachment factor for T. gondii. We plan to characterize the nature of this parasite ligand at the molecular level using variant molecular constructs of P30. We also plan to identify and characterize the host cell receptor for this parasite molecule. Studies in our laboratory demonstrate that certain neoglycoproteins are able to block parasite infection of human fibroblast cells. The mechanism by which these molecules block attachment to extracellular matrix will be investigated. Moreover, the role of sugar binding molecules in host:parasite attachment will be characterized using host cells treated with various inhibitors of glycosylation and mutant host cells with known alterations in their glycosylation products. The second specific aim will be to determine if bispecific antibodies (bsAb) can target T. gondii to human phagocytes and enhance the killing of the parasite by these cells. We have shown that killing of extracellular T. gondii by human phagocytes can be significantly increased when parasites are opsonized with bsAb. These dual recognition antibodies are comprised of a parasite specific arm cross linked with various phagocytic receptors including FcgammaR. We plan to use bsAb that cross link P30 with various phagocyte receptors to determine which receptors can best mediate parasite internalization. We have also demonstrated that the parasitophorous vacuole inability to fuse with other endocytic organelles following active penetration can be overcome by targeting the parasite to a host cell FcgammaRII. Once the important phagocyte receptors for tachyzoite attachment and internalization are identified, we will investigate both endosome and lysosome fusion with the parasite- containing vacuole following uptake by these receptors, and the ability of these receptors to activate the mechanisms for microbicidal activity. This will include analysis of various O2-dependent and O2-independent mechanisms believed to be involved in toxoplasmacidal activity by phagocytes. The information obtained will be directly related toward improving the immunotherapy of toxoplasmosis in those individuals with AIDS.
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