MONOCLONAL IMMUNOGLOBULIN-A FOR IMMUNOTHERAPY AGAINST CRYPTOSPORIDUM PARVUM

用于针对隐孢子虫免疫治疗的单克隆免疫球蛋白-A

• 批准号: 5205562
• 负责人: MARIAN R NEUTRA
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5205562
• 关键词: SCID mouse; antiprotozoal agents; disease /disorder model; disease /disorder prevention /control; drug design /synthesis /production; electron microscopy; enzyme linked immunosorbent assay; epitope mapping; hybridomas; immunocytochemistry; immunoglobulin A; immunoglobulin G; immunotherapy; infant animal; intestinal mucosa; laboratory mouse; lectin; monoclonal antibody; mucosal immunity; protozoal antigen; recombinant proteins; spores; western blottings

The goal of this continuing collaborative project is to test the abilities of specific IgA antibodies to provide immune protection against Cryptosporidium parvum in the intestine. We have generated a panel of 6 monoclonal, dimeric IgA antibodies from mucosally-derived hybridomas. They are partially characterized, showing distinct antigen specificities directed against surface components of C. parum, and all show passive immune protection in the infant mouse model. The project has 3 major aims; 1) To continue production and characterization of monoclonal IgAs. We will establish antigen and epitope specificities of the available monoclonal IgAs and identify possible cross-reactivity with antigens and surface components previously identified by others (including 15 and 47 kD antigens, GP900, and the putative lectin studies in Project 1). Additional IgA hybridomas will be generated after oral immunizations with recombinant protein fragments of a protective, glycosylated antigen GP900 (in collaboration with Drs. J. Crabb, ImmuCell Corp. and C. Petersen, UCSF). Epitope specificities will be compared with those of available monoclonal IgGs, polyclonal antisera, and bovine colostral antibodies. 2) To define in detail the capacities of specific monoclonal IgA antibodies to prevent or reverse C parvum infection. Protective capacities of individual IgAs to prevent C. parvum infection in vivo will be quantitatively compared in the infant mouse model. IgA antibodies with differing antigen specificities will be tested for synergistic protection. Parallel tests will be done in vitro suing MDBK and Caco-2 cell monolayers. Treatment of chronically-infected SCID mice with IgA hybridoma "backpack" tumors will demonstrate whether continuous secretion of protective IgAs can reverse established infection in intestine and biliary tree. using protective IgA monoclonals identified above we will test the ability of added IgA monoclonals identified above, we will test the ability of added IgA to enhance passive protection by hyperimmune bovine colostrum (from ImmuCell) in vivo and in vitro. 3). To investigate possible sites and mechanisms of IgA protection at epithelial surfaces. The distribution of antigens recognized by protective IgAs on C. parvum oocysts, sporozoites and intracellular forms during infection of epithelial cells will be analyzed by high LM immunofluorescence and EM immunogold labeling of intact organisms, sections of infected mucosa, and Caco-2 cell monolayers (defined in Project 2). Immunolabeling and ultrastructural analysis will also be used to elucidate the interactions among parasites, protective IgA antibodies, and epithelial cell surfaces during challenge with C. parvum in vivo and in vitro. The detailed evaluation of IgA antibodies proposed in this project will serve to establish the role of secretory IgA in normal mucosal protection against cryptosporidiosis, and could lead to more effective passive immune prophylaxis and immunotherapy in immunocompromised hosts.

这个持续协作项目的目标是测试能力 特定的IgA抗体可提供免疫保护 肠道中的隐孢子虫。 我们已经产生了6个面板 来自粘液衍生的杂交瘤的单克隆，二聚体IgA抗体。 他们 部分表征，显示出不同的抗原特异性 针对C. parum的表面成分，所有人都表现出被动 婴儿小鼠模型中的免疫保护。 该项目有3个主要目标； 1）继续生产和表征单克隆IGA。 我们 将建立可用的抗原和表位特异性 单克隆IGA，并鉴定可能与抗原的交叉反应性和 以前由其他人确定的表面成分（包括15和47 kD 抗原，GP900和项目1中的推定凝集素研究）。 额外的 重组口服免疫后，将产生IGA杂交瘤 保护性，糖基化抗原GP900的蛋白质片段（在 与Drs合作。 J. Crabb，Immucell Corp.和C. Petersen，UCSF）。 将表位特异性与可用单克隆的特异性进行比较 IgG，多克隆抗血清和牛乳突抗体。 2）详细定义特定单克隆IgA的能力 预防或逆转C parvum感染的抗体。 保护能力 在体内预防孢子虫感染的单个IGA将是 在婴儿小鼠模型中进行定量比较。 Iga抗体 不同的抗原特异性将进行协同保护。 平行测试将在体外提起MDBK和CACO-2细胞单层中进行。 用IGA杂交瘤“背包”处理长期感染的SCID小鼠 肿瘤将证明保护性IGA的连续分泌是否可以 在肠道和胆道树中反向建立的感染。 使用 保护性IgA单克隆在上面确定的，我们将测试 添加了上面确定的IgA单克隆，我们将测试添加的能力 IgA通过高免疫牛初乳增强被动保护（来自 Immucell）体内和体外。 3）。 调查IgA保护的可能地点和机制 上皮表面。 保护性识别的抗原的分布 在C. parvum卵囊，孢子菌和细胞内形式的IGAS上 高LM将分析上皮细胞的感染 完整生物的免疫荧光和EM免疫金标记，切片 被感染的粘膜和Caco-2细胞单层（在项目2中定义）。 免疫标签和超微结构分析也将用于阐明 寄生虫，保护性IGA抗体和上皮之间的相互作用 在体内和体外挑战孢子虫期间的细胞表面。 该项目提出的IGA抗体的详细评估将 为确定分泌IgA在正常粘膜保护中的作用 反对隐孢子虫病，并可能导致更有效的被动免疫 免疫功能低下宿主中的预防和免疫疗法。