Effector Functions of Mucosal IgA

描述（由申请方提供）：本提案的长期目标是更好地了解人类粘膜免疫系统和伊加受体在粘膜免疫应答中的作用。口服疫苗递送提供了优于常规肠胃外途径的显著优势。最值得注意的是，它刺激粘膜免疫反应，在入侵部位对抗病原体。然而，阻碍口服疫苗发展的一个主要限制是缺乏对如何有效地将疫苗抗原呈递给粘膜免疫系统的理解。目前正在研究的克服这种限制的一种有希望的方法是使用分泌型伊加（SIgA）作为疫苗递送载体。SIgA在恶劣的胃肠道环境中极其稳定，并且结合并被肠微折叠（M）细胞摄取，所述肠微折叠（M）细胞专门用于跨派尔集合淋巴结的滤泡相关上皮运输管腔抗原。最近的一项研究表明，当口服给予小鼠时，携带线性抗原表位插入分泌组分的重组SIgA诱导针对抗原的免疫应答。研究还表明，SIgA与小鼠M细胞的结合是由一种新的伊加受体介导的。小鼠M细胞的分子身份目前尚不清楚。也不知道人类M细胞是否表达可比较的伊加受体。因此，本项目的一个重要目标是鉴定通过将肠上皮Caco-2细胞与Raji B细胞共培养产生的人M细胞上表达的伊加受体。由于小鼠和人类之间在伊加受体的表达和功能方面存在重大差异，我们将首先确定人类M细胞是否也表达新的伊加受体，或者已知的伊加受体是否负责IgA与人类M细胞的结合。如果建议存在新的伊加受体，我们将使用两种不同的实验方法鉴定它，即，cDNA表达克隆和免疫沉淀。一旦我们确定了人M细胞伊加受体（新的或已知的），我们将分析其在人肠道活检样品中的表达及其在体外SIgA转胞吞作用中的功能。该项目的另一个主要目标是了解人类M细胞如何调节SIgA转胞吞作用。人伊加是一种高度复杂的糖蛋白，我们将生产定义明确的重组人伊加分子，每个分子在聚糖或蛋白质部分中含有特异性修饰。使用这些抗体，我们将确定人SIgA的基序和表位，控制其结合的效率和运输的人M细胞在体外产生的。此外，我们还将确定由SIgA结合M细胞激活的细胞内信号通路，并确定它们的活性是否调节SIgA转胞吞的效率。在小鼠中开发的M细胞靶向策略通常不适用于人类，因为这两种生物体之间伊加生物学和M细胞表面特征的差异。使用人类M细胞的拟议研究将为开发实用的基于SIgA的人类口服疫苗提供必要的基础知识。 公共卫生相关性：口服疫苗不需要无菌注射设备或高度熟练的医务人员，因此大大减少了免疫接种所需的费用和工作量。拟议的研究将增加我们对口服疫苗如何被人体肠道吸收的理解。它们还将为开发安全有效的无针疫苗提供新的战略。

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.