Effector Functions of Mucosal IgA

粘膜 IgA 的效应器功能

• 批准号: 8197795
• 负责人: Sherie L Morrison
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197795
• 关键词: Address; Amino Acid Sequence; Antibodies; Antigen Targeting; Antigens; Apical; B-Lymphocytes; Binding; Biology; Biopsy Specimen; Caco-2 Cells; Carbohydrates; Cells; Characteristics; Coculture Techniques; Complex; Confocal Microscopy; Dendritic Cells; Development; Environment; Epithelial; Epithelium; Epitopes; Equipment; Exhibits; Genes; Glycoproteins; Goals; Health Personnel; Human; IgA receptor; Immune response; Immune system; Immunization; Immunoglobulin A; Immunoprecipitation; In Vitro; Injection of therapeutic agent; Intestines; Knowledge; Laboratory Animals; Large Intestine Carcinoma; M cell; Mammalian Cell; Mass Spectrum Analysis; Mediating; Modification; Molecular; Molecular Profiling; Monomeric GTP-Binding Proteins; Mucosal Immune Responses; Mus; Mutate; Nature; Needles; Oral; Organism; Peptide Sequence Determination; Phosphotransferases; Play; Polysaccharides; Property; Proteins; RNA Interference; Raji Cell; Receptor Down-Regulation; Recombinants; Regulation; Role; Route; Sampling; Secretory Component; Secretory Immunoglobulin A; Signal Pathway; Site; Sterility; Structure; Structure of aggregated lymphoid follicle of small intestine; Surface; TC7; Techniques; Vaccine Antigen; Vaccines; Validation; Workload; base; cDNA Expression; combat; cost; expression cloning; gastrointestinal; in vitro Model; inhibitor/antagonist; insight; monolayer; mucosal vaccine; novel; oral vaccine; pathogen; public health relevance; receptor; transcytosis; vaccine delivery

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to gain a better understanding of the human mucosal immune system and the role of IgA receptors in the mucosal immune response. Oral vaccine delivery offers significant advantages over conventional parenteral routes. Most notably, it stimulates mucosal immune responses that combat pathogens at the sites of invasion. However, a major limitation that has hindered the development of oral vaccines has been a lack of understanding of how to effectively present the vaccine antigen to the mucosal immune system. One promising approach currently being investigated to overcome this limitation is the use of secretory IgA (SIgA) as a vaccine delivery vehicle. SIgA is extremely stable in the harsh gastrointestinal environment, and binds and is taken up by intestinal microfold (M) cells that are specialized for transporting luminal antigens across the follicle-associated epithelium of Peyer's patches. A recent study indicated that when administered orally to mice, recombinant SIgA carrying a linear antigenic epitope inserted into the secretory component induced immune responses against the antigen. Studies also showed that the binding of SIgA to mouse M cells is mediated by a novel IgA receptor. The molecular identity of the mouse M cell is currently unknown. It is also not known if human M cells express a comparable IgA receptor. Therefore, an important goal of this project is to identify the IgA receptor expressed on human M-cells generated by coculturing intestinal epithelial Caco-2 cells with Raji B cells. Since there are major differences between mice and humans in the expression and function of IgA receptors, we will first determine if human M cells also express a novel IgA receptor or if known IgA receptors are responsible for IgA-binding to human M cells. If the presence of a novel IgA receptor is suggested, we will identify it using two different experimental approaches, i.e., cDNA expression cloning and immunoprecipitation. Once we identify the human M-cell IgA receptor (novel or known), we will analyze its expression in human intestinal biopsy samples and its function in SIgA transcytosis in vitro. Another major goal of this project is to understand how human M cells regulate SIgA transcytosis. Human IgA is a highly complex glycoprotein and we will produce well-defined recombinant human IgA molecules each of which contains a specific modification in the glycan or protein moiety. Using these antibodies, we will identify the motifs and epitopes of human SIgA that control the efficiency of its binding and transport by human M cells generated in vitro. Furthermore, we will also identify intracellular signaling pathways activated by SIgA-binding to M cells and determine if their activities regulate the efficiency of SIgA transcytosis. M-cell targeting strategies developed in mice are often not applicable to humans because of the differences in IgA biology and the surface characteristics of M cells between these two organisms. The proposed studies using human M cells will provide the fundamental knowledge essential for the development of practical SIgA-based oral vaccines for humans. PUBLIC HEALTH RELEVANCE: Oral vaccines do not require sterile injection equipment or highly skilled medical personnel, thus significantly reducing the costs and workload necessary for immunization. The proposed studies will increase our understanding of how oral vaccines are taken up by the human intestine. They will also provide new strategies for the development of safe and efficient needle-free vaccines.

描述（由申请人提供）：该提案的长期目标是更好地了解人类粘膜免疫系统以及IgA受体在粘膜免疫反应中的作用。与常规肠道途径相比，口服疫苗的交付具有显着优势。最值得注意的是，它刺激粘膜免疫反应在入侵部位打击病原体。但是，阻碍口服疫苗发展的主要局限性缺乏了解如何有效地将疫苗抗原呈现给粘膜免疫系统的。目前正在研究以克服此限制的一种有希望的方法是将分泌IgA（SIGA）用作疫苗输送工具。 SIGA在刺激性的胃肠道环境中非常稳定，并且结合并被肠道微量（M）细胞所吸收，这些细胞专门用于在Peyer斑块的卵泡相关上皮上运输腔植物。最近的一项研究表明，当口服小鼠口服时，重组SIGA携带线性抗原表位，该抗原表位插入分泌成分中会诱导针对抗原的免疫反应。研究还表明，SIGA与小鼠M细胞的结合是由新型IgA受体介导的。小鼠M细胞的分子身份目前尚不清楚。人类M细胞是否表达可比的IgA受体也不知道。因此，该项目的一个重要目标是鉴定通过与拉吉B细胞共培养肠上皮Caco-2细胞产生的人类M细胞表达的IgA受体。由于小鼠与人之间在IgA受体的表达和功能中存在主要差异，因此我们将首先确定人类M细胞是否也表达了新型的IgA受体，或者是否已知的IgA受体负责IgA结合对人M细胞的影响。如果提出了新型IgA受体的存在，我们将使用两种不同的实验方法（即cDNA表达克隆和免疫沉淀）识别它。一旦我们鉴定了人类M细胞IgA受体（新颖或已知），我们将分析其在人类肠道活检样本中的表达及其在体外Siga transctstosis中的功能。该项目的另一个主要目标是了解人类M细胞如何调节SIGA转胞细胞增多症。人IgA是一种高度复杂的糖蛋白，我们将产生明确的重组人IgA分子，每个分子都包含在聚糖或蛋白质部分中的特定修饰。使用这些抗体，我们将确定人类SIGA的基序和表位，这些基序和表位控制其在体外产生的人类细胞结合和转运的效率。此外，我们还将确定通过SIGA结合到M细胞激活的细胞内信号传导途径，并确定其活性是否调节Siga transcytsytosis的效率。在这两种生物之间，小鼠开发的M细胞靶向策略通常不适用于人类。使用人类M细胞的拟议研究将为开发基于SIGA的人类的口服疫苗提供基本知识。 公共卫生相关性：口服疫苗不需要无菌注射设备或高技能的医务人员，因此大大降低了免疫所需的成本和工作量。拟议的研究将增加我们对人类肠道如何采用口服疫苗的理解。他们还将为开发安全有效的无针疫苗提供新的策略。