TOXOPLASMA GONDII--DIAGNOSIS AND PREVENTION IN AIDS

这一竞争性更新的总体具体目标是进一步改善 弓形虫病的免疫治疗 艾滋病 在我们目前的资助期间，我们已经开发了一个实质性的 关于免疫学、分子生物学和 几种重要寄生虫抗原的血清学，特别是P30， 主要膜和排泄/分泌抗原（ESA）的这种义务 胞内病原体 在本提案中，我们计划使用这些 观察，以阐明两个不同的，但互补的具体 目的：寄生虫附着于宿主细胞并被人类内化 吞噬细胞 第一个具体目标是评估P30在以下方面的作用： 寄生附着 我们的初步观察表明，P30是 是T.刚地。 我们计划描述 在分子水平上使用变异的寄生虫配体的性质 P30的分子结构。 我们还计划识别和描述 这种寄生虫分子的宿主细胞受体 在我们的研究 实验室证明某些新糖蛋白能够阻断 寄生虫感染人成纤维细胞。 的机制 这些分子阻断与细胞外基质的附着， 研究了 此外，糖结合分子在 宿主：寄生虫附着将使用经处理的宿主细胞来表征 与各种糖基化抑制剂和突变宿主细胞， 已知的糖基化产物的改变。 第二特定 目的是确定双特异性抗体（bsAb）是否可以靶向T. 弓形虫对人类吞噬细胞的作用，并通过以下方式增强对寄生虫的杀伤力： 这些细胞。 我们已经证明，细胞外T。弓形虫通过 当寄生虫被感染时， 用bsAb调理。 这些双重识别抗体由以下组成 与各种吞噬细胞受体交联的寄生虫特异性臂 包括Fc γ R。 我们计划使用交联P30的bsAb， 各种吞噬细胞受体，以确定哪些受体可以最好地 介导寄生虫内化。 我们还证明， 不能与其他内吞融合的寄生虫液泡 主动渗透后的细胞器可以通过靶向 寄生虫到宿主细胞Fc γ RII。 一旦重要的吞噬细胞 鉴定了速殖子附着和内化的受体， 我们将研究内体和溶酶体与寄生虫的融合， 在这些受体摄取后含有液泡， 这些受体激活杀微生物活性的机制。 这将包括对各种O2依赖性和O2非依赖性 据信参与杀弓形虫活性的机制是 吞噬细胞 所获得的信息将直接关系到 改善弓形虫病患者的免疫治疗 艾滋病

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.