Effector Functions of Mucosal IgA

描述（由申请人提供）：本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。与传统的肠胃外途径相比，口服疫苗递送具有显着的优势。最值得注意的是，它刺激粘膜免疫反应，对抗入侵部位的病原体。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了解。目前正在研究克服这一限制的一种有前景的方法是使用分泌型 IgA (SIgA) 作为疫苗递送载体。 SIgA 在恶劣的胃肠道环境中极其稳定，可结合肠道微褶皱 (M) 细胞并被其吸收，这些细胞专门负责将管腔抗原转运穿过派尔氏集结的滤泡相关上皮。最近的一项研究表明，当给小鼠口服给药时，携带插入分泌成分的线性抗原表位的重组 SIgA 会诱导针对抗原的免疫反应。研究还表明，SIgA 与小鼠 M 细胞的结合是由一种新型 IgA 受体介导的。小鼠 M 细胞的分子身份目前未知。目前尚不清楚人类 M 细胞是否表达类似的 IgA 受体。因此，该项目的一个重要目标是鉴定肠上皮Caco-2细胞与Raji B细胞共培养产生的人M细胞上表达的IgA受体。由于小鼠和人类之间 IgA 受体的表达和功能存在重大差异，因此我们将首先确定人类 M 细胞是否也表达新的 IgA 受体，或者已知的 IgA 受体是否负责 IgA 与人类 M 细胞的结合。如果表明存在新的 IgA 受体，我们将使用两种不同的实验方法来鉴定它，即 cDNA 表达克隆和免疫沉淀。一旦我们鉴定出人类 M 细胞 IgA 受体（新的或已知的），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 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。人类 IgA 是一种高度复杂的糖蛋白，我们将生产明确的重组人类 IgA 分子，每个分子都包含聚糖或蛋白质部分的特定修饰。使用这些抗体，我们将鉴定人 SIgA 的基序和表位，这些基序和表位控制体外产生的人 M 细胞的结合和运输效率。此外，我们还将鉴定 SIgA 与 M 细胞结合激活的细胞内信号通路，并确定它们的活性是否调节 SIgA 转胞吞作用的效率。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 IgA 生物学和 M 细胞表面特征的差异。拟议的使用人类 M 细胞的研究将为开发基于 SIgA 的实用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技能的医务人员，从而大大减少了免疫所需的成本和工作量。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了解。他们还将为开发安全高效的无针疫苗提供新策略。

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.