Innate and Adaptive Immunity in Experimental and Human Gonoccocal Infection

描述(由申请人提供)：这一性传播感染合作研究中心(STI-CRC)题为“女性实验性和人类淋球菌感染中的先天和获得性免疫”的主要目标是提供对与淋球菌感染相关的先天和获得性免疫机制的不同和全面的了解。我们将强调淋球菌感染的基本宿主反应和先天免疫机制，近期目标是提高对淋球菌免疫学和发病机制的了解，长期目标是获得洞察力，以指导预防人类，特别是女性淋病感染的努力。我们将使用一个有前途的动物模型来检验这些机制，并分别采用一种普遍的翻译方法，强调免疫生物学和临床流行病学之间的联系，以评估候选疫苗在人类中的潜力，然后在人源化的小鼠感染模型的背景下进行研究。提出了五个研究项目和四个服务核心。在第一个项目(Douglas T Golenbock，MD，PL)中，我们将解决脂多糖(LPS)生物学中的一个基本和基本的问题，以了解LOS衍生的脂质A与其直接配体的结合，该配体是激活和允许Toll样受体(TLR)4信号的中间结构。在项目2(Robin Ingalls，MD，PL)中，我们将确定是否淋球菌LOS自然发生突变或TLR4适配器MAI的多态导致宿主对感染的炎症反应的差异。在项目3(Caroline A Genco，PhD，PL)中，我们将研究TLRs和胞质核苷酸寡聚域(NOD)蛋白受体(NOD样受体[NLRs])作为奈瑟菌配体的受体所起的作用。在项目4(Sanjay Ram，MD，PL)中，我们将研究替代补体途径(ACP)增强分子的新角色。备解素分别通过LOS和LOS阻断TLR4介导的信号转导，放大潜在保护性疫苗诱导的抗体功能。在项目5(Peter A.莱斯，医学博士)中，我们将在淋球菌感染的人源化小鼠模型中检验淋球菌LO的多肽模拟作为潜在的候选疫苗，同时也确定针对候选的天然抗体是否保护暴露的女性免受淋球菌感染。该中心将有五个核心(临床、实验室、动物统计和行政)，为5个项目中的4个或5个项目提供支持。 项目1：GONOCCOCAL病中的TLR4/MD-2信号通路(Golenbock，D) 项目1说明(申请人提供)：淋病奈瑟菌可引起多种疾病综合征，包括盆腔炎[pID]。产后疼痛会导致慢性盆腔疼痛、宫外孕和不孕症。这些由淋病奈瑟菌引起的综合症，通常都有由炎性细胞介导的强烈炎症。这种炎症主要是内毒素(LOS)与内毒素受体复合体TLR4和MD-2相互作用的结果。内毒素受体的成分早在十年前就被发现，但仍不清楚：1)脂类A与MD-2结合如何导致活性受体复合体的形成；2)信号随后如何传递，导致促炎介质的产生，如TNFa和IL-1β。在这项提案中，我们描述了确定MD-2一旦与脂质A结合，如何获得激活TLR4的能力的计划。这种方法建立在我们成功提纯MD-2的基础上，MD-2是一种含有7个半胱氨酸残基的小分子，有形成不活跃的多聚体的臭名昭著的趋势。我们计划在没有和存在激活配体的情况下解析MD-2的结构，以及在TLR4存在的情况下解析MD-2的结构，以确定TLR4/MD-2的构象变化是如何诱导信号传递的。然后我们将把精力集中在TLR4相关的适配分子上，这些分子参与细胞信号传递。我们之前已经分析了被称为MAI的适配器蛋白中的5个单核苷酸多态(SNPs)(TLR2和TLR4都使用)。两个SNP：S180L和D96N是人们非常感兴趣的。作为NIH资助的另一个项目的一部分，我们已经开始培育携带这些病变的小鼠，并正在对患者样本进行D96N的筛查。携带D96N或S180L等价物的MAI基因敲除小鼠和敲入小鼠将由Ingalls博士在GC感染的小鼠模型中进行测试(PL，项目2)。我们将对MyD88中已知的6个SNPs进行类似的分子遗传学研究，MyD88是与MAI相互作用的下游接头，也是至少8个TLR的重要接头。如果其中任何一个SNP显示出表型，我们将产生敲入小鼠并筛选患者样本以确定相关性。最后，我们将试图通过生化手段定义MAI和MyD88的相互作用，最终尝试使MAL/MyD88二聚体共结晶。

DESCRIPTION (provided by applicant): The major objectives of this Sexually Transmitted Infections Co-operative Research Center (STI-CRC) entitled "Innate and Adaptive Immunity in Experimental and Human Gonococcal Infection in Women" are directed to providing a diverse and comprehensive understanding of the innate and adaptive immune mechanisms associated with gonococcal infection. We will emphasize basic host responses and innate immune mechanisms in infection with Neisseria gonorrhoeae with the immediate aim of improving understanding of gonococcal immunology and pathogenesis and a longer term goal to gain insights that will direct efforts to facilitate the prevention of gonococcal infections in humans, particularly women. We will use a promising animal model to examine these mechanisms and separately employ a generalized translational approach that will emphasize the links between immunobiology and clinical epidemiology to assess the potential of vaccine candidates in humans, which will then be investigated in the context of the humanized mouse model of infection. Five research projects and four service cores are proposed. In the first project (Douglas T Golenbock, MD, PL), we will address a basic and fundamental question in lipopolysaccharide (LPS, called lipooligosaccharide [LOS] in the case of N. gonorrhoeae) biology to understand the binding of LOS derived lipid A with its direct ligand that serves as an intermediary structure to activate and permit signaling of toll-like receptor (TLR)4. In Project 2 (Robin Ingalls, MD, PL), we will determine if naturally occurring mutations in gonococcal LOS or polymorphisms in the TLR4 adaptor Mai, account for differences in the host inflammatory response to infection. In Project 3 (Caroline A Genco, PhD, PL), we will examine the role that TLRs and cytosolic nucleotide oligomerization domain (NOD) protein receptors (NOD-like receptors [NLRs]) play as receptors for Neisseria ligands. In Project 4 (Sanjay Ram, MD, PL), we will examine novel roles for the alternative complement pathway (ACP) enhancing molecule. Properdin, in blocking TLR4 mediated signaling by LOS and separately, in amplifying potentially protective vaccine induced antibody function. In Project 5 (Peter A. Rice, MD), we will examine peptide mimics of gonococcal LOS as potential vaccine candidates in a humanized mouse model of gonococcal infection while also determining if natural antibodies against the candidates protect exposed women from gonococcal infection. The Center will have five Cores (Clinical, Laboratory, Animal Statistical, and administrative) providing support to 4 or 5 of the 5 Projects. PROJECT 1: THE TLR4/MD-2 SIGNALING PATHWAY IN GONOCCOCAL DISEASE (Golenbock, D) PROJECT 1 DESCRIPTION (provided by applicant): Neisseria gonorrhoeae causes a variety of disease syndromes including pelvic inflammatory disease [PID]. PID can lead to chronic pelvic pain, ectopic pregnancy and infertility. Those syndromes caused by N. gonorrhoeae have, in common, intense inflammation mediated by inflammatory cells. This inflammation is primarily the result of the interaction of Neisserial LPS (LOS) with the LPS receptor complex: TLR4 and MD- 2. The components of the LPS receptor were identified a decade ago, yet it is still poorly understood, 1) how the binding of lipid A to MD-2 results in the formation of an active receptor complex, and, 2) how a signal is subsequently transmitted resulting in the production of proinflammatory mediators such as TNFa and IL-1¿. In this proposal, we describe plans to determine how MD-2, once bound to lipid A, acquires the ability to activate TLR4. The approach builds upon our success in purifying MD-2, a small molecule with 7 cysteine residues that has a notorious tendency to form inactive multimers. We plan to resolve the structure of MD-2 in the absence and presence of activating ligand, and in the presence of TLR4 to determine what conformational changes in TLR4/MD-2 induce signaling. We shall then focus our energies on TLR4-related adapter molecules involved in cell signaling. We have previously analyzed 5 single nucleotide polymorphisms (SNPs) in the adapter protein known as Mai (used by both TLR2 and TLR4). Two SNPs are of great interest: S180L and D96N. As part of another NIH funded project, we have begun to generate mice carrying these lesions and are screening patient samples for the presence of D96N. Mai knock out mice and knock-in mice carrying the mouse equivalent of D96N or S180L will be tested in the mouse model of GC infection by Dr. Ingalls (PL, project 2). We will perform similar molecular genetic studies of the 6 known SNPs in MyD88, the downstream adapter that interacts with Mai and an important adapter for at least 8 of the TLRs. Should any of the SNPs display a phenotype, we will generate knock-in mice and screen patient samples to determine relevancy. Finally, we will attempt to define the interaction of Mai and MyD88 by biochemical means, culminating in an attempt to co crystallize the Mal/MyD88 dimer.