Complementary Mechanisms of Protection Against Pneumococcal Infection

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，感染开始于定植，然后可以是 并发肺炎和侵入性血流感染其中最突出的抗体 粘膜部位是IgA 1亚类。肺炎球菌荚膜（其主要毒力因子）的IgA 1抗体， 是由于定植、有症状的感染和接种疫苗而产生的。IgA1可变区 与生物体结合并且恒定区与吞噬细胞结合（例如，肺泡巨噬细胞和 中性粒细胞）。肺炎球菌酶，IgA1蛋白酶，在细菌表面表达。IgA 1结合 被蛋白酶在可变区和恒定区之间的桥接铰链处切割， 从而抑制IgA 1支持吞噬作用、杀伤和清除生物体的能力。残余 保留在表面上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殖民化。这种颠覆性的保护主机响应， S.肺炎使老年退伍军人更易患严重感染。 防止细菌灭活宿主的反应可以通过以下患者的子集来实现： 侵袭性肺炎球菌疾病，在血清中产生IgG，中和蛋白酶的切割能力 IgA。此外，我们已经产生了鼠单克隆抗体（mMab），其结合并且一些中和所述抗体。 蛋白酶我们建议推进我们对蛋白酶的结构-功能关系的理解 并考虑将这种蛋白作为主要或预防性疫苗候选物的可行性。在这 在上下文中，我们假设： 1)IgA1蛋白酶的中和抗体识别酶上的保守表位。 2)蛋白酶中和抗体与人IgA1并阻止上皮细胞结合， 体外和体内通过抑制IgA1裂解用鼻内攻击定殖。 3)IgA1蛋白酶抗体保护小鼠免受沙门氏菌致死性粘膜感染。肺炎间接通过 抑制与细菌表面结合并直接通过表面结合的人IgA 1的切割， 介导生物体的吞噬作用。 为了解决这些假设，我们建议追求以下具体目标： 目标1.表征蛋白酶中和单克隆抗体（Mab）靶向的表位及其 遗传保护 目标2.表征蛋白酶中和单克隆抗体（Mab）靶向的表位及其 遗传保护 目标3.确定蛋白酶特异性Mab在粘膜感染后保护免受致命感染的能力。 体内挑战和保护机制。 这项工作旨在确定蛋白酶中和抗体的靶点，地理和 肺炎球菌蛋白酶的分子多样性，以考虑这些调查的普遍性， 蛋白酶结合和中和IgA1效应子功能的能力，以防止定植和支持 吞噬和杀伤S.在小鼠模型中的体外和体内的肺炎。这些研究提供 为考虑蛋白酶在疫苗疾病预防中的作用提供了重要的基础和临床前数据， 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.