T. gondii as a model for investigation of Cryptosporidium glycoprotein antigens

描述(申请人提供)：隐孢子虫(CP)是一种普遍存在的水传播病原体，可引起世界各地的腹泻疾病。这种病原体会在免疫受损的宿主中引起危及生命的腹泻疾病，特别是艾滋病患者。到目前为止，还没有有效的CP治疗方法或疫苗。研究表明，CP依赖于粘蛋白样糖蛋白附着和侵袭肠上皮细胞。这些粘蛋白抗原也是保护性细胞免疫反应的目标。然而，由于研究这种寄生虫的困难，这些抗原在宿主细胞入侵和免疫识别中的作用受到阻碍，因为这种寄生虫不能在体外进行遗传操作或繁殖。为了满足对模拟天然CP粘蛋白翻译后修饰的表达系统的需求，我们在相关的弓形虫顶端复合体(Tg)中表达了CP糖蛋白。我们以前的研究已经证明，TG表达的CP粘蛋白是糖基化的，经过蛋白水解性加工和定位，类似于天然的糖蛋白。然而，天然抗原和TG表达的抗原之间的大小差异表明，在糖基化和/或蛋白分解过程中可能存在细微的差异。在这一应用中，我们建议比较天然和TG表达的CP粘蛋白抗原的翻译后修饰，并确定TG表达的抗原在抗原功能和免疫识别方面是否具有与天然抗原相似的功能。在这些研究中，我们将使用CP粘蛋白抗原CpMuc4。我们推测，TG表达系统可用于生产能有效模拟天然抗原功能和免疫原性的重组CP糖蛋白抗原。具体目的是：1.探索和比较原生和TG-rCpMuc4的翻译后修饰。在这些研究中，我们将研究天然CpMuc4和TG-rCpMuc4的蛋白质降解过程和多糖结构。2.评价TG-rCpMuc4模拟天然抗原功能和免疫原性的能力。这些研究将比较天然和甘油三酯rCpMuc4与肠上皮细胞结合的能力，并在体外抑制感染。我们将使用天然和TG-rCpMuc4作为抗原，评估感染CP的小鼠和人类的体液和细胞免疫反应。这一目标的一个探索性目标将是确定表达CpMuc4的减毒TG是否可以用作疫苗载体递送系统。这些研究将为TG表达系统作为生产和研究CP粘蛋白抗原的载体提供彻底的分析。这些研究结果将为未来R01鉴定与子孢子附着和入侵相关的特定表位和宿主细胞受体，以及粘蛋白糖蛋白在宿主细胞识别和免疫反应中的作用提供初步数据。与公共卫生相关：隐孢子虫(CP)是一种普遍存在的水媒寄生虫，可导致全球范围内的胃肠道疾病暴发。目前还没有针对这种寄生虫的疫苗或治疗方法。这项研究调查了弓形虫用来研究隐孢子虫病疫苗候选的CP抗原。

DESCRIPTION (provided by applicant): Cryptosporidium (Cp) is a ubiquitous waterborne pathogen that causes diarrheal disease worldwide. This pathogen causes life-threatening diarrheal disease in immunocompromised hosts, particularly AIDS patients. To date there is no effective treatment or vaccine for Cp. Studies suggest that Cp relies on mucin- like glycoproteins to attach to and invade intestinal epithelial cells. These mucin antigens are also targets of protective cellular immune responses. However, investigating the role of these antigens in host cell invasion and immune recognition is hampered by the difficulty of studying this parasite which cannot be genetically manipulated or propagated in vitro. To address the need for an expression system that mimics post-translational modifications of the native Cp mucins, we expressed Cp glycoproteins in the related apicomplexan Toxoplasma gondii (Tg). Our previous studies have demonstrated that Tg-expressed Cp mucins are glycosylated, proteolytically processed and localized similarly to the native glycoprotein. However, size discrepancies between the native and Tg- expressed antigens suggests that there may be subtle differences in glycosylation and/or proteolytic processing. In this application we propose to compare the post-translational modifications of a native and Tg- expressed Cp mucin antigen, and determine if the Tg-expressed antigen functions similarly to the native antigen in assays of antigen function and immune recognition. For these studies we will use the Cp mucin antigen, CpMuc4. We hypothesize that the Tg expression system can be employed to produce recombinant Cp glycoprotein antigens that will effectively mimic the functions and immunogenicity of the native antigen. The specific aims are: 1. To explore and compare the post-translational modifications of the native and Tg-rCpMuc4. In these studies, we will investigate proteolytic processing and glycan structure of native CpMuc4 and Tg-rCpMuc4. 2. To evaluate the ability of Tg-rCpMuc4 to mimic the function and immunogenicity of the native antigen. These studies will compare the ability of native and Tg rCpMuc4 to bind to intestinal epithelial cells, and inhibit in vitro infection. We will evaluate humoral and cell mediated immune responses in Cp-infected mice and humans using native and Tg-rCpMuc4 as antigens. An exploratory goal of this aim will be to determine if attenuated Tg expressing CpMuc4 can be employed as a vaccine vector delivery system. These studies will provide a thorough analysis of the Tg expression system as a vehicle for producing and studying Cp mucin antigens. Results from these studies will provide preliminary data for future R01 studies on identification of specific epitopes and host cell receptors involved in sporozoite attachment and invasion and the role of mucin glycotopes in host cell recognition and immune responses. PUBLIC HEALTH RELEVANCE: Cryptosporidium (Cp) are ubiquitous water-borne parasites that cause outbreaks of gastrointestinal disease worldwide. Currently there are no vaccines or treatments for this parasite. This study investigates the use of Toxoplasma gondii to study Cp antigens that are candidates for inclusion in a vaccine for cryptosporidiosis.