IMMUNOREACTIVE MACROMOLECULES OF COCCIDIOIDES CELL TYPES

球孢子菌病是一种常见于西南部的系统性真菌感染。 美国。大多数感染都是自限性的，但有些患者 发展为播散性球孢子菌病或持续性疾病， 可能会危及生命。我们在拟议研究中的重点是 中华绒毛虫免疫活性大分子的特性。这 包括刺激T细胞和抗体反应的抗原。 这种集成方法的逻辑是基于我们目前对 球虫宿主对球虫感染的免疫反应。 球孢子菌病是一种T细胞免疫介导的疾病 显示在宿主防御中发挥关键作用，但对其知之甚少 T细胞反应性大分子的精确性质。早期发现 沉淀素抗体反应提示原发性球孢子菌病 对免疫球虫抗原。然而，再一次，准确的性质是 血清反应性大分子(S)未知。要解决这些问题 问题，我们已经使用的方法确定候选分子C。 免疫刺激T细胞或抗体反应的一种组合 生化和重组DNA技术。我们将首先确定C. 免疫大分子刺激细胞免疫，通过使用 抗原特异的小鼠T细胞系。候选分子通过以下方式获得 以前证明过的复杂混合物的分馏 细胞免疫分析中的免疫反应。在第二种方法中，强调 在拟议的研究中，CDNA表达文库来自于 金龟子的腐生期和寄生期及其在lambda ZAP中的构建 II用针对上述免疫反应产生的抗体进行筛选 混合物。编码免疫反应性融合基因克隆的鉴定 蛋白质最终可以导致分离出可以被引入的基因 转化为合适的表达载体，用于体外或体内生产 T细胞反应蛋白。我们早期血清学诊断的研究 球虫感染集中在一种120 kDa的糖蛋白上，该糖蛋白包括 3-O-甲基化杂甘露聚糖。后者与特异的抗C。 免疫球蛋白M，显然是这种碳水化合物部分所独有的 真菌是系统性真菌病原菌中的一种。我们在这一部分的具体目标 该项目的主要内容是分离、纯化和鉴定免疫活性 胞外120kDa糖蛋白的寡糖亚组分(S) 用于原发球孢子菌病的血清诊断。我们的总体目标 是为了表征能诱导体液的免疫球虫的特定抗原 和细胞免疫反应，希望改善诊断 试剂，并最终开发出一种球孢子菌病疫苗。

Coccidioidomycosis is a systemic fungal infection common to southwestern United States. Most infections are self-limiting, but some patients develop disseminated coccidioidomycosis or persistent disease, either one of which can be life threatening. Our focus in the proposed research is the characterization of immunoreactive macromolecules of C. immitis. This includes antigens which stimulate T-cells as well as antibody response. The logic for this integrated approach is based on our current knowledge of the immunological response of the host to coccidioidal infections. Coccidioidomycosis is a disease in which T-cell mediated immunity has been shown to play a critical role in host defense, but little is known of the precise nature of T-cell reactive macromolecules. Early detection of primary coccidioidomycosis is indicated by a precipitin antibody response to C. immitis antigen. Once again, however, the precise nature of the serologically-reactive macromolecule(s) is unknown. To address these problems we have used the approach of identifying candidate molecules of C. immitis which stimulate T-cell or antibody response by a combination of biochemical and recombinant DNA techniques. We will initially identify C. immitis macromolecules which stimulate cell-mediated immunity by using antigen-specific murine T-cell lines. Candidate molecules are obtained by fractionation of complex mixtures previously demonstrated to be immunoreactive in cellular immunoassays. In a second approach, emphasized in the proposed research, CDNA expression libraries derived from MRNA of saprobic and parasitic phases of C. immitis and constructed in lambda ZAP II are screened with antibody raised against the above immunoreactive mixtures. Identification of CDNA clones which encode immunoreactive fusion proteins can ultimately lead to isolation of genes that can be introduced into appropriate expression vectors for in vitro or in vivo production of T-cell reactive proteins. Our research on serodiagnosis of early coccidioidal infection has focused on a 120-Kda glycoprotein that consists of a 3-O-methylated heteromannan. The latter, which binds specific anti-C. immitis patient IgM, is apparently unique to carbohydrate fractions of this fungus among the systemic fungal pathogens. Our specific aims in this part of the project are to isolate, purify, and characterize the immunoreactive oligosaccharide subfractions(s) of the extracellular, 120-Kda glycoprotein for use in serodiagnosis of primary coccidioidomycosis. Our overall goals are to characterize specific antigens of C. immitis which elicit humoral and cellular immune responses in the hopes of improving diagnostic reagents, and eventually developing a vaccine against coccidioidomycosis.