IMMUNOREACTIVE MACROMOLECULES OF COCCIDIOIDES CELL TYPES

球孢子细胞类型的免疫反应性大分子

基本信息

批准号：
2003254
负责人：
GARRY Thomas COLE
金额：
$ 36.36万
依托单位：
UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-01 至 2002-08-31
项目状态：
已结题

项目摘要

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.

球孢子菌病是西南地区常见的一种全身性真菌感染美国。大多数感染是自限性的，但有些患者发展为播散性球孢子菌病或持续性疾病，其中之一其中可能会危及生命。我们拟议研究的重点是 C. immitis 免疫反应性大分子的表征。这包括刺激 T 细胞和抗体反应的抗原。这种综合方法的逻辑基于我们目前的知识宿主对球孢子菌感染的免疫反应。球孢子菌病是一种 T 细胞介导的免疫被破坏的疾病已被证明在宿主防御中发挥着关键作用，但人们对其知之甚少 T 细胞反应性大分子的精确性质。及早发现原发性球孢子菌病由沉淀素抗体反应指示 C.imitis 抗原。然而，再一次，血清学反应性大分子未知。为了解决这些问题我们使用了识别 C 候选分子的方法。免疫炎通过组合刺激 T 细胞或抗体反应生物化学和重组 DNA 技术。我们将首先识别 C. Immitis 大分子通过使用刺激细胞介导的免疫抗原特异性鼠 T 细胞系。候选分子通过以下方式获得先前证明复杂混合物的分馏在细胞免疫测定中具有免疫反应性。在第二种方法中，强调在拟议的研究中，来自 mRNA 的 cDNA 表达文库 C. immitis 的腐生期和寄生期并在 lambda ZAP 中构建 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的 cDNA 克隆的鉴定蛋白质最终可以导致可以引入的基因的分离转化为适当的表达载体，用于体外或体内生产 T 细胞反应蛋白。我们对早期血清学诊断的研究球孢子菌感染集中在一种 120-Kda 糖蛋白上，该糖蛋白由 3-O-甲基化杂甘露聚糖。后者结合特异性抗C。 immitis 患者 IgM，显然是该药物的碳水化合物部分所特有的真菌属于全身性真菌病原体。我们在这部分的具体目标该项目的目的是分离、纯化和表征免疫反应性细胞外 120-Kda 糖蛋白的寡糖亚组分用于原发性球孢子菌病的血清诊断。我们的总体目标是为了表征 C. immitis 的特异性抗原，其引起体液和细胞免疫反应，希望改善诊断试剂，并最终开发出针对球孢子菌病的疫苗。