Innate and Adaptive Immunity in Experimental and Human Gonoccocal Infection

描述（由申请人提供）：这个性传播感染合作研究中心（STI-CRC）的主要目标是“女性实验和人类淋球菌感染中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理解。我们将强调淋病奈瑟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理解，并获得有助于预防人类，特别是妇女淋球菌感染的长期目标。我们将使用一个有前景的动物模型来检查这些机制，并单独采用一种通用的转化方法，该方法将强调免疫生物学和临床流行病学之间的联系，以评估人类候选疫苗的潜力，然后在人源化小鼠感染模型的背景下进行研究。提出了5个研究项目和4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，在淋病球菌中称为低脂寡糖[LOS]）生物学中的一个基本和基本问题，以了解LOS衍生的脂质a与其直接配体的结合，后者作为激活和允许toll样受体（TLR）4信号传导的中间结构。在项目2中（Robin Ingalls， MD， PL），我们将确定淋球菌LOS中自然发生的突变或TLR4接头Mai中的多态性是否解释了宿主对感染的炎症反应差异。在Project 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[NLRs]）作为奈瑟菌配体受体的作用。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。Properdin在阻断TLR4介导的LOS信号传导中，以及在扩增潜在保护性疫苗诱导的抗体功能中发挥作用。在项目5 （Peter a . Rice， MD）中，我们将在人源化的小鼠模型中检验淋球菌LOS的肽模拟物作为潜在的候选疫苗，同时确定针对候选疫苗的天然抗体是否能保护暴露的妇女免受淋球菌感染。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个提供支持。

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.