ISOLATION AND EXPRESSION OF COCCIDIOIDES T CELL ANTIGENS

球孢子菌病是一种T细胞免疫介导的疾病 被证明在宿主防御中起着关键作用。无论是临床还是 实验数据支持这一结论。这是一种不同寻常的特征 霉菌病是由补体检测到的高滴度抗体 固恋，是一个不良的预后征兆。因此，确定哪些C. 免疫性抗原刺激T细胞反应而不是抗体反应 对于随后分离生物体的大分子是必不可少的 这会引起免疫保护。在拟议的研究中，我们使用了一种 一种新的分子系统鉴定方法 重组T细胞反应蛋白(RTPs)的研究 这些大分子的免疫保护性在小鼠模型中的作用 球孢子菌病。我们早期的研究结果表明， 强大的T细胞反应蛋白在1)转化过程中表达 腐生到寄生阶段，2)球体的各向同性生长，3) 内孢子萌发。在此基础上，我们将构建三个相应的cDNAs 从上述寄生阶段分离出的表达文库。 文库将用现有的抗细胞抗体进行筛选 壁和全细胞制剂已被证明是T细胞 免疫淋巴结增殖反应(ILNP)试验。反应性克隆 并将每一株的虫体cdna连接到pCMV上。 哺乳动物表达载体。对BALB/c小鼠进行皮下免疫 表达免疫球虫蛋白的pCMV plus基因插入片段。老鼠是 用酶联免疫吸附试验监测免疫球虫粗制抗体的产生 抗原，然后牺牲以评估其T细胞的反应性 ILNP检测。反应性克隆的cDNA亚克隆到原核细胞中 表达载体(如pSE40)，免疫亲和纯化RTP 用获得的相应的特异性小鼠抗血清进行层析 作为祭品，如上所述。RTP在小鼠身上的反应性进一步测试 ILNP检测与T细胞系、患者淋巴细胞增殖反应 化验。所选的cDNA用于筛选原始表达 文库，或隐翅虫基因组文库，以分离全长基因。 来自该基因cDNA将用于表达RTP，以便进一步 如上对其反应性进行评价。最终，这种方法将 获得可用于小鼠免疫保护的多个RTP 对抗金黄色葡萄球菌的挑战。免疫保护性重组蛋白 通过这种方法获得的是合格的人类疫苗候选者 治疗球孢子菌病。

Coccidioidomycosis is a disease in which T-cell mediated immunity has been shown to play a critical role in host defense. Both clinical and experimental data support this conclusion. An unusual feature of this mycosis is that high titers of antibody, as detected by complement fixation, are a poor prognostic sign. Therefore, determination of which C. immitis antigens stimulate T-cell responses rather than antibody responses is essential for subsequent isolation of macromolecules of the organism that elicit immunoprotection. In the proposed research, we have used a novel molecular approach to the systematic identification of the recombinant T-cell reactive proteins (RTPs), an evaluation of the immunoprotective properties of these macromolecules in a murine model of coccidioidomycosis. Results of our earlier studies have indicated that potent T-cell reactive proteins are expressed during 1) transition of the saprobic to parasitic phase, 2) isotropic growth of spherules, and 3) endosporulation. On this basis, we will construct three corresponding cDNA expression libraries with mRNA isolated from the above parasitic phases. The libraries will be screened with existing antiserum raised against cell wall and whole cell preparations which have been shown to be T-cell reactive in immune lymph node proliferation (ILNP) assays. Reactive clones are isolated and the C. immitis cDNA of each is ligated into the pCMV mammalian expression vector. BALB/c mice are immunized subcutaneously with the pCMV plus cDNA insert which expresses the C. immitis protein. Mice are monitored by ELISA for production of the antibody against crude C. immitis antigen and then sacrificed for evaluation of their T-cell reactivity in ILNP assays. The cDNA of reactive clones is subcloned into a prokaryotic expression vector (e.g., pSE40), and the RTP is purified by immunoaffinity chromatography using the corresponding specific murine antiserum obtained as sacrifice, as above. The RTP is further tested for reactivity in murine ILNP assays and T-cell lines, an in patient lymphocyte proliferation assays. The selected cDNAs are used to screen the original expression library, or a C. immitis genomic library, to isolate the full-length gene. The cDNA from that gene will be used for expression o the RTP for further evaluation of its reactivity as above. Ultimately, this approach will yield multiple RTPs which can be evaluated for immunoprotection in mice against C. immitis challenge. Immunoprotective recombinant proteins obtained by this approach are qualified candidates for a human vaccine against coccidioidomycosis.