Complementary Mechanisms of Protection Against Pneumococcal Infection

引起肺炎及相关发病率和死亡率的主要原因是粘膜病原体--链球菌 肺炎。从上呼吸道粘膜开始，感染从定植开始，然后可以 并发肺炎和侵袭性血液感染。其中最突出的抗体 粘膜部位是IgA1亚类。肺炎球菌衣壳的IgA1抗体，它的主要毒力因子， 是对殖民、症状感染和接种疫苗的反应而产生的。免疫球蛋白A1的可变区 结合到生物体，恒定区域结合到吞噬细胞(例如，肺泡巨噬细胞和 中性粒细胞)。细菌表面表达一种肺炎链球菌酶，即IgA1蛋白酶。IgA1结合 在可变区和恒定区之间的桥接铰链上被蛋白酶切割到胶囊上， 从而抑制了IgA1支持吞噬、杀死和清除生物体的能力。残渣 保留在表面的可变区修饰细菌的表面并增强与上皮细胞的结合 受体，可能促进与生物体的定居。这种对保护性宿主反应的颠覆 肺炎链球菌使年长的退伍军人更容易受到严重感染。 防止细菌对宿主反应的失活可以通过一组患有 侵袭性肺炎球菌病，在血清中产生中和蛋白酶裂解能力的免疫球蛋白 IGA。此外，我们已经产生了鼠源性单抗(MMabs)，可以结合并中和一些人的 蛋白酶。我们建议推进我们对该酶的结构-功能关系的理解 并考虑将该蛋白作为主要或辅助候选疫苗的可行性。在这 在上下文中，我们假设： 1)中和抗体识别酶上的保守表位。 2)蛋白水解酶中和抗体与人IgA1结合并阻止上皮细胞结合 通过抑制IgA1裂解在体外和鼻腔攻击体内定植。 3)IgA1蛋白水解酶抗体通过间接保护小鼠免受肺炎链球菌致死性黏膜感染。 抑制与细菌表面结合的人IgA1的切割，并直接通过表面结合和 调节有机体的吞噬作用。 为了解决这些假设，我们建议实现以下具体目标： 目的1.鉴定蛋白酶中和单抗(MAb)靶向的表位及其功能 基因保护。 目的2.鉴定蛋白酶中和单抗(MAb)靶向的表位及其功能 基因保护。 目的3.检测蛋白酶特异性单抗对黏膜后致死性感染的保护作用 在体内的挑战和潜在的保护机制。 这项工作是为了确定蛋白酶中和抗体的靶标，地理和 肺炎球菌蛋白水解酶的分子多样性，以考虑这些研究的概括性，以及 蛋白酶结合和中和对IgA1效应器防止定植和支持功能的能力 肺炎链球菌在小鼠体内和体外的吞噬和杀灭作用。这些研究提供了 重要的基础和临床前数据，用于考虑蛋白酶在疫苗疾病预防中的作用， 有可能是心理治疗。

A leading cause of pneumonia and related morbidity and mortality is the mucosal pathogen, Streptococcus pneumoniae. Beginning at the upper respiratory mucosa, infection begins with colonization which can then be complicated by pneumonia and invasive bloodstream infections. The most prominent antibody in these mucosal sites is the IgA1 subclass. IgA1 antibodies to the pneumococcal capsule, its primary virulence factor, are generated in response to colonization, symptomatic infection and vaccination. The variable region of IgA1 binds to the organism and the constant region binds to phagocytes (e.g., alveolar macrophages and neutrophils). A pneumococcal enzyme, IgA1 protease, is expressed on the surface of the bacteria. IgA1 bound to the capsule is cleaved by the protease at the bridging hinge between the variable and constant region, thereby inhibiting the ability of IgA1 to support phagocytosis, killing and clearance of the organism. Residual variable regions that remain on the surface modify the bacteria's surface and enhance binding to epithelial cell receptors, likely promoting colonization with the organism. This subversion of the protective host response to S. pneumoniae predisposes older veterans to increased risk for serious infection. Preventing the bacteria's inactivation of the host's response can be achieved by a subset of patients with invasive pneumococcal disease who generate IgG in serum that neutralizes the protease's ability to cleave IgA. Moreover, we have generated murine monoclonal antibodies (mMabs) that bind and some neutralize the protease. We propose to advance our understanding of the structure-function relationships of the protease and consider the feasibility of advancing this protein as a primary or adjunctive vaccine candidate. In this context, we Hypothesize that: 1) Neutralizing antibodies to IgA1 protease recognize conserved epitopes on the enzyme. 2) Protease-neutralizing antibodies with human IgA1 and prevent epithelial cell binding in vitro and colonization with intranasal challenge in vivo by inhibition of IgA1 cleavage. 3) Antibodies to IgA1 protease protect mice against fatal mucosal infection with S. pneumoniae indirectly by inhibiting cleavage of human IgA1 bound to the bacterial surface and directly by surface binding and mediating phagocytosis of the organism. To address these Hypotheses, we propose to pursue the following Specific Aims: Aim 1. Characterize the epitopes targeted by protease-neutralizing monoclonal antibodies (Mabs) and their genetic conservation. Aim 2. Characterize the epitopes targeted by protease-neutralizing monoclonal antibodies (Mabs) and their genetic conservation. Aim 3. Determine the ability of protease-specific Mab's to protect against fatal infection after mucosal challenge in vivo and the mechanisms underlying protection. This work is directed to determine the targets of the protease-neutralizing antibodies, the geographic and molecular diversity of pneumococcal proteases to consider the generalizability of these investigations, and the ability of protease binding and neutralization on IgA1 effector functions to prevent colonization and to support phagocytosis and killing of S. pneumoniae both in vitro and in vivo in mouse models. These studies provide important basic and pre-clinical data for considering the role of the protease in vaccine disease prevention and, potentially, therapy.