Tarcha Eric J, Basrur Venkatesha, Hung Chiung-Yu, Gardner Malcolm J, Cole Garry T
Department of Biology University of Texas at San Antonio, Margaret Batts Tobin Building, Rm. 1.308E, 6900 North Loop 1604 West, San Antonio, TX 78249, USA.
Infect Immun. 2006 Jan;74(1):516-27. doi: 10.1128/IAI.74.1.516-527.2006.
Coccidioidomycosis is a respiratory disease of humans caused by the desert soil-borne fungal pathogens Coccidioides spp. Recurrent epidemics of this mycosis in the southwestern United States have contributed significantly to escalated health care costs. Clinical and experimental studies indicate that prior symptomatic coccidioidomycosis induces immunity against subsequent infection, and activation of T cells is essential for containment of the pathogen and its clearance from host tissue. Development of a human vaccine against coccidioidomycosis has focused on recombinant T-cell-reactive antigens which elicit a durable protective immune response against pulmonary infection in mice. In this study we fractionated a protective multicomponent parasitic cell wall extract in an attempt to identify T-cell antigens. Immunoblots of electrophoretic separations of this extract identified patient seroreactive proteins which were subsequently excised from two-dimensional polyacrylamide gel electrophoresis gels, trypsin digested, and sequenced by tandem mass spectrometry. The full-length gene which encodes a dominant protein in the immunoblot was identified using established methods of bioinformatics. The gene was cloned and expressed, and the recombinant protein was shown to stimulate immune T cells in vitro. The deduced protein was predicted to contain epitopes that bind to human major histocompatibility complex class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes induced gamma interferon production by immune T lymphocytes. The T-cell-reactive antigen, which is homologous to secreted fungal aspartyl proteases, protected mice against pulmonary infection with Coccidioides posadasii. We argue that this immunoproteomic/bioinformatic approach to the identification of candidate vaccines against coccidioidomycosis is both efficient and productive.
球孢子菌病是一种由土壤传播的真菌病原体球孢子菌属引起的人类呼吸道疾病。这种真菌病在美国西南部的反复流行显著增加了医疗保健成本。临床和实验研究表明,先前有症状的球孢子菌病可诱导对后续感染的免疫力,T细胞的激活对于控制病原体及其从宿主组织中清除至关重要。针对球孢子菌病的人类疫苗的开发主要集中在重组T细胞反应性抗原上,这些抗原可引发针对小鼠肺部感染的持久保护性免疫反应。在本研究中,我们对一种具有保护作用的多组分寄生细胞壁提取物进行了分级分离,试图鉴定T细胞抗原。该提取物经电泳分离后的免疫印迹鉴定出患者血清反应性蛋白,随后从二维聚丙烯酰胺凝胶电泳凝胶中切下这些蛋白,用胰蛋白酶消化,并通过串联质谱法测序。使用已建立的生物信息学方法鉴定了在免疫印迹中编码一种主要蛋白的全长基因。该基因被克隆并表达,重组蛋白在体外显示出可刺激免疫T细胞。使用基于TEPITOPE的算法预测推导的蛋白含有与人主要组织相容性复合体II类分子结合的表位。与预测的T细胞表位相对应的合成肽可诱导免疫T淋巴细胞产生γ干扰素。这种与分泌型真菌天冬氨酸蛋白酶同源的T细胞反应性抗原可保护小鼠免受波萨达斯球孢子菌的肺部感染。我们认为这种用于鉴定抗球孢子菌病候选疫苗的免疫蛋白质组学/生物信息学方法既高效又有成效。