IGG SUBCLASS & FUNCTION OF ANTIPOLYSACCHARIDE ANTIBODY

IGG亚类

• 批准号: 2067506
• 负责人: JOHN R SCHREIBER
• 金额: $ 22.22万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067506
• 关键词: Pseudomonas aeruginosa; antibacterial antibody; bacterial disease; bacterial polysaccharides; bactericidal immunity; chimeric proteins; complement pathway; disease /disorder model; fusion gene; gene mutation; gene rearrangement; human genetic material tag; human tissue; humoral immunity; immunodeficiency; immunoglobulin G; immunoglobulin genes; laboratory mouse; laboratory rat; molecular cloning; monoclonal antibody; opsonin; tissue /cell culture; transfection

Polysaccharide (PS)-encapsulated bacteria are a major cause of infections in humans. Anti-PS IgG antibodies (Ab) are critical to host defense against infection with these bacteria. The function of anti-PS Ab includes complement fixation and opsonization of bacteria for uptake and killing by phagocytes. The mammalian IgG anti-PS Ab response, however is restricted to only one or two subclasses (IgG3 in mice, IgG1 and IgG2 in man). Defining the relative function of anti-PS Ab of different IgG subclasses is crucial to better understanding of immunity to PS-coated bacteria as well as the pathogenesis of several human disease states such as IgG subclass deficiency, and chronic pneumonia in Cystic Fibrosis where inappropriate IgG subclass of anti-P. aeruginosa (PA) Ab may contribute to inability to clear PA from the lung. The relative function of anti-PS Ab of different IgG subclass remains unclear due to previous studies primarily using polyclonal affinity purified Ab exposed to chaotropic agents that alter Fc function, or to the use of monoclonal Ab with different antigenic specificities. In this proposal, we will precisely define the role of IgG subclass in anti-PS Ab effector function. First, we will make murine monoclonal Ab (MAb) directed to O- PS-side chain of PA LPS and to mucoid exopolysaccharide of PA. Next, using sequential sublining, we will make switch variants of these MAb that have identical variable regions but are of different IgG subclasses. The variable region genes will be cloned and ligated to the gamma constant region human genes to make human/murine chimeric transfectomas producing Ab of all four human IgG subclasses that have identical variable regions. The functional characteristics of the murine and chimeric anti-PS Ab will be compared in terms of: 1) ability to fix complement, 2) ability to opsonize PA for uptake by phagocytes, and 3) protective efficacy in animal models of PA infection. Finally, to determine the in vivo importance of the predominant anti-PS IgG subclass, IgG3 deficient mice will be mae by targeting the murine gamma 3 gene for mutation and homologous recombination in embryonic stem cells. These mice will be evaluated for their ability to immunologically respond to PS, PS-protein conjugates, and bacterial infection. These studies will determine which anti-PS IgG subclass functions most efficiently against PS-coated bacteria, better define the mechanism of functional differences between IgG subclasses and explore the immunologic relevance of these differences in vivo. This information will allow more rational strategies of active and passive immunization against PS-coated bacteria and improved treatment of IgG subclass deficiencies.

多糖（PS）包裹的细菌是感染的主要原因 在人类身上。 抗PS IgG抗体（Ab）对宿主防御至关重要 抵抗这些细菌的感染 抗PS抗体的功能 包括补体固定和细菌的调理作用以供吸收， 被吞噬细胞杀死。 然而，哺乳动物IgG抗PS Ab反应 仅限于一个或两个亚类（小鼠中的IgG 3，IgG 1和IgG 2 在人类中）。 不同IgG抗PS抗体的相关功能测定 亚类是至关重要的，以更好地了解免疫PS包被 细菌以及几种人类疾病状态的发病机制， IgG亚类缺乏症和囊性纤维化中的慢性肺炎 当抗铜绿假单胞菌（PA）Ab的不适当IgG亚类可能 导致不能从肺中清除PA。 相对函数 不同IgG亚类的抗PS抗体的数量仍不清楚， 研究主要使用多克隆亲和纯化的Ab暴露于 改变Fc功能的离液剂，或使用单克隆抗体 具有不同的抗原特异性。 在本提案中，我们将 精确定义IgG亚类在抗PS Ab效应子中的作用 功能 首先，我们将制备针对O- PA LPS的PS-侧链和PA的粘液样胞外多糖。 接下来， 使用连续的亚线，我们将使这些单克隆抗体的开关变体 具有相同的可变区但属于不同的IgG亚类。 可变区基因将被克隆并连接到γ 恒定区人基因以制备人/鼠嵌合转染瘤 产生所有四种人IgG亚类的Ab，其具有相同的 可变区 小鼠的功能特征和 嵌合抗PS Ab将在以下方面进行比较：1）固定 补体，2）调理PA以供吞噬细胞摄取的能力，以及3） 在PA感染动物模型中的保护功效。 最后为 确定主要抗PS IgG亚类的体内重要性， IgG 3缺陷型小鼠将通过靶向鼠γ 3基因来制备， 胚胎干细胞中的突变和同源重组。 这些 将评价小鼠免疫应答的能力， PS、PS-蛋白结合物和细菌感染。 这些研究将 确定哪种抗PS IgG亚类对 PS包被细菌，更好地定义功能差异的机制 IgG亚类之间，并探讨这些免疫相关性 体内差异 这些信息将使我们更理性 PS包被细菌的主动和被动免疫策略 和改善IgG亚类缺陷的治疗。