Innate and Adaptive Immunity in Experimental and Human Gonoccocal Infection

描述（由申请人提供）：性传播感染合作研究中心（STI-CRC）的主要目标是“女性实验性和人类淋球菌感染的先天和适应性免疫”，旨在提供与淋球菌感染相关的先天和适应性免疫机制的多样化和全面的理解。我们将强调淋球菌感染的基本宿主反应和先天免疫机制，其直接目的是提高对淋球菌免疫学和发病机制的认识，长期目标是获得有助于预防人类（特别是女性）淋球菌感染的见解。我们将使用一个有前途的动物模型来研究这些机制，并单独采用一种广义的翻译方法，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，然后将在人源化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（道格拉斯T Golenbock，MD，PL）中，我们将解决脂多糖（LPS，在N.淋病）生物学以理解LOS衍生的脂质A与其直接配体的结合，所述直接配体充当中间结构以激活并允许Toll样受体（TLR）4的信号传导。在项目2（Robin Ingalls，MD，PL）中，我们将确定淋球菌LOS中自然发生的突变或TLR 4衔接子Mai中的多态性是否导致宿主对感染的炎症反应差异。在项目3（Caroline A Genco，PhD，PL）中，我们将研究TLR和胞质核苷酸寡聚化结构域（NOD）蛋白受体（NOD样受体[NLR]）作为奈瑟氏菌配体受体的作用。在项目4（Sanjay Ram，MD，PL）中，我们将研究替代补体途径（ACP）增强分子的新作用。备解素在阻断LOS介导的TLR 4信号传导中的作用，以及在增强疫苗诱导的潜在保护性抗体功能中的作用。在项目5（彼得A. Rice，MD），我们将在淋球菌感染的人源化小鼠模型中检查淋球菌LOS的肽模拟物作为潜在的疫苗候选物，同时还确定针对候选物的天然抗体是否保护暴露的妇女免受淋球菌感染。中心将有5个核心（临床、实验室、动物统计和管理），为5个项目中的4个或5个提供支持。 项目1：TLR 4/MD-2信号通路在淋病（Golenbock，D） 项目1描述（由申请人提供）：淋病奈瑟菌引起多种疾病综合征，包括盆腔炎[PID]。PID可导致慢性盆腔疼痛、异位妊娠和不孕。N.淋病通常具有由炎性细胞介导的强烈炎症。这种炎症主要是奈瑟球菌LPS（LOS）与LPS受体复合物：TLR 4和MD- 2相互作用的结果。LPS受体的组分在十年前就被鉴定出来了，但仍然知之甚少，1）脂质A与MD-2的结合如何导致活性受体复合物的形成，以及2）信号如何随后传递导致促炎介质如TNF α和IL-1的产生。在这个建议中，我们描述了计划，以确定如何MD-2，一旦绑定到脂质A，获得激活TLR 4的能力。该方法建立在我们成功纯化MD-2的基础上，MD-2是一种具有7个半胱氨酸残基的小分子，具有形成无活性多聚体的臭名昭著的倾向。我们计划在活化配体存在和不存在的情况下解析MD-2的结构，以及在TLR 4存在的情况下，以确定TLR 4/MD-2中的构象变化诱导信号传导。然后，我们将把精力集中在TLR 4相关的适配器分子参与细胞信号转导。我们以前分析了5个单核苷酸多态性（SNP）的衔接蛋白称为Mai（TLR 2和TLR 4使用）。两个SNPs是非常感兴趣的：S180 L和D96 N。作为NIH资助的另一个项目的一部分，我们已经开始产生携带这些病变的小鼠，并正在筛选患者样本中是否存在D96 N。Ingalls博士将在GC感染的小鼠模型中检测携带小鼠等效物D96 N或S180 L的Mai敲除小鼠和敲入小鼠（PL，项目2）。我们将对MyD 88中的6个已知SNP进行类似的分子遗传学研究，MyD 88是与Mai相互作用的下游衔接子，也是至少8个TLR的重要衔接子。如果任何SNP显示表型，我们将产生敲入小鼠并筛选患者样本以确定相关性。最后，我们将尝试通过生物化学手段来定义Mai和MyD 88的相互作用，最终尝试共结晶Mal/MyD 88二聚体。

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.