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

弓形虫作为研究隐孢子虫糖蛋白抗原的模型

• 批准号: 7893819
• 负责人: ROBERTA M O'CONNOR
• 金额: $ 19.92万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2012-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7893819
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affect; Antibodies; Antigens; Apical; Attenuated; Binding; Biological Assay; Biological Models; Biology; Carbohydrates; Cells; Childhood; Complex; Cryptosporidiosis; Cryptosporidium; Data; Developing Countries; Developmental Delay Disorders; Diarrhea; Disease; Disease Outbreaks; Enzymes; Epithelial Cells; Epitopes; Escherichia coli; Future; Gastrointestinal Diseases; Glycoproteins; Goals; Growth; Human; Immune; Immune response; Immunocompromised Host; In Vitro; Infection; Intestines; Invaded; Investigation; Label; Lectin; Life; Measures; Methods; Modeling; Molecular; Monosaccharides; Mucins; Mus; Oligosaccharides; Opportunistic Infections; Organelles; Parasites; Patients; Pattern; Play; Polysaccharides; Population; Post-Translational Protein Processing; Process; Proteins; Proteolytic Processing; Proteomics; Receptor Cell; Recombinants; Relative (related person); Role; Serum; Sporozoites; Staging; Structure; Surface; System; T-Lymphocyte Epitopes; Techniques; Toxoplasma gondii; Vaccines; Virulence Factors; Water; cell mediated immune response; effective therapy; glycosylation; immune function; immunogenic; immunogenicity; pathogen; pressure; public health relevance; research study; therapeutic vaccine; vaccine development; vector vaccine; waterborne

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.

描述（由申请人提供）：隐孢子虫（Cp）是一种普遍存在的水传播病原体，在全世界范围内引起腹泻病。这种病原体会导致免疫功能低下的宿主，特别是艾滋病患者出现危及生命的腹泻病。迄今为止，尚无针对 Cp 的有效治疗方法或疫苗。研究表明，Cp 依赖粘蛋白样糖蛋白附着并侵入肠上皮细胞。这些粘蛋白抗原也是保护性细胞免疫反应的目标。然而，研究这些抗原在宿主细胞侵袭和免疫识别中的作用受到研究这种寄生虫的困难的阻碍，因为这种寄生虫不能在体外进行基因操作或繁殖。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，我们在相关的顶端复合体弓形虫 (Tg) 中表达 Cp 糖蛋白。我们之前的研究表明，Tg 表达的 Cp 粘蛋白与天然糖蛋白类似，经过糖基化、蛋白水解加工和定位。然而，天然抗原和Tg表达抗原之间的大小差异表明糖基化和/或蛋白水解加工可能存在细微差异。在此应用中，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测定中是否与天然抗原具有相似的功能。在这些研究中，我们将使用 Cp 粘蛋白抗原 CpMuc4。我们假设 Tg 表达系统可用于生产重组 Cp 糖蛋白抗原，该抗原将有效模拟天然抗原的功能和免疫原性。具体目的是： 1.探索和比较native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 的蛋白水解加工和聚糖结构。 2.评价Tg-rCpMuc4模拟天然抗原的功能和免疫原性的能力。这些研究将比较天然 rCpMuc4 和 Tg rCpMuc4 与肠上皮细胞结合并抑制体外感染的能力。我们将使用天然和 Tg-rCpMuc4 作为抗原来评估 Cp 感染小鼠和人类的体液和细胞介导的免疫反应。该目标的一个探索性目标是确定表达 CpMuc4 的减毒 Tg 是否可以用作疫苗载体递送系统。这些研究将对 Tg 表达系统作为生产和研究 Cp 粘蛋白抗原的载体进行全面分析。这些研究的结果将为未来的 R01 研究提供初步数据，这些研究涉及子孢子附着和侵袭中涉及的特定表位和宿主细胞受体的鉴定，以及粘蛋白糖表位在宿主细胞识别和免疫反应中的作用。公共卫生相关性：隐孢子虫 (Cp) 是普遍存在的水生寄生虫，可导致全球胃肠道疾病的爆发。目前还没有针对这种寄生虫的疫苗或治疗方法。本研究调查了使用弓形虫来研究 Cp 抗原，这些抗原是隐孢子虫病疫苗中的候选抗原。