The Role of Scavenger Receptor gp340 in Mucosal HIV-1 Transmission and Inhibition

清道夫受体 gp340 在粘膜 HIV-1 传播和抑制中的作用

• 批准号: 8607113
• 负责人: Min Lu
• 金额: $ 73.25万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8607113
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affinity; Animal Model; Antiviral Agents; Binding; Binding Sites; Biological Assay; CCR5 gene; CD209 gene; CD4 Positive T Lymphocytes; Cell fusion; Cell membrane; Cell surface; Cell-Matrix Junction; Cells; Cervical; Competence; Complement; Complex; Data; Development; Disease; Effectiveness; Epithelial; Epithelium; Female; Genital system; Glycoproteins; Goals; HIV Envelope Protein gp120; HIV-1; Human; Immunoglobulin Variable Region; In Vitro; Infection; Inhibitory Concentration 50; Intervention; Investigation; Kinetics; Lead; Mediating; Membrane; Membrane Fusion; Methods; Modeling; Molecular; Molecular Target; Molecular Virology; Monoclonal Antibodies; Mucous Membrane; Multi-Drug Resistance; Mus; Mutation; Oral; Peptides; Pharmaceutical Preparations; Prevalence; Process; Property; Protein Chemistry; Protein Engineering; Receptor Cell; Research; Research Project Grants; Resolution; Role; SRCR proteins; Saliva; Specificity; Structure; Surface; T-Lymphocyte; Testing; Thermodynamics; Tissues; Toxic effect; Vagina; Variant; Viral; Virus; base; biophysical chemistry; biophysical techniques; combat; design; disulfide bond; env Glycoproteins; in vitro Model; in vivo; inhibitor/antagonist; insight; interdisciplinary approach; macrophage; neutralizing antibody; neutralizing monoclonal antibodies; novel; prevent; programs; receptor binding; reproductive; scavenger receptor; small molecule; structural biology; syndecan; targeted treatment; transcytosis; transmission process; vaginal transmission; virucide

DESCRIPTION (provided by applicant): There is an urgent need to understand the initiation and dissemination of HIV-1 infection in mucosal tissues, and to develop new methods for targeting early infection to prevent mucosal HIV-1 transmission. HIV-1 infection requires fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoprotein (Env) and host cell receptors. The human scavenger receptor gp340 has been identified as a secreted component in human saliva that inhibits oral transmission of HIV-1 through a specific interaction with the surface gp120 subunit of an Env spike. Surprisingly the gp120 interaction with the membrane-bound gp340 in the female reproductive tract promotes HIV-1 transcytosis through barrier epithelia and thus infection of CD4+ cells. Our goal in this program is to define the molecular target and mechanism of action of soluble gp340-based HIV-1 entry inhibitors. Our discovery effort will be based on preliminary data obtained with the gp120-binding SRCR domain of gp340 that potently inhibits in vitro infection by HIV-1 with nanomolar IC50 values. We propose a comprehensive, interdisciplinary approach that combines protein chemistry, high- resolution structural determination, thermodynamic and kinetic binding analyses, protein engineering, molecular virology, and animal model efficacy studies. In this project we seek to discover novel HIV-1-specific intervention strategies to inhibit mucosal epithelium-Env binding and virus-cell fusion. The Specific Aims are: 1. To understand the structural basis of gp120 binding by gp340 to inform the design and optimization of novel SRCR domain entry inhibitors. (a) Elucidate the structural properties of the SRCR domain fragment both free and bound to the V3 region. (b) Characterize the energetics of the gp120-gp340 interaction. (c) Use mutational analysis and biophysical methods to dissect the structural determinants of the respective binding sites. (d) Define a "minimal" gp120-binding sequence or set of such sequences. (e) Optimize binding affinity of variants of the SRCR domain, and evaluate their anti-HIV-1 activity in a single-round infectivity assay. 2. To determine the mechanisms by which the gp120-gp340 interaction influences Env structure, function and antigenicity. (a) Determine how V3 mutations identified in Aim 1c influence the functional competence of Env. (b) Characterize the effects of the SRCR domain on the receptor-binding properties of gp120 and its reactivities to neutralizing and non-neutralizing monoclonal antibodies. (c) Determine the virucidal activity of select SRCR domain entry inhibitors against diverse primary HIV-1 strains. 3. To test the effectiveness of optimized SRCR domain entry inhibitors to protect against mucosal HIV-1 infection. (a) Characterize the specificity and potency of select SRCR entry inhibitors in an in vitro model of HIV-1 infection of human cervical and vaginal tissue. (b) Use the humanized mouse vaginal transmission model to assess the in vivo potency and breadth of activity of optimized SRCR entry inhibitors alone and in combination with the gp41 fusion inhibitor C52L and the small-molecule CCR5 coreceptor inhibitor CMPD167. 1

描述（由申请人提供）：迫切需要了解 HIV-1 感染在粘膜组织中的起始和传播，并开发针对早期感染以预防粘膜 HIV-1 传播的新方法。 HIV-1 感染需要病毒和靶细胞膜融合，这一过程由病毒包膜糖蛋白 (Env) 和宿主细胞受体介导。人类清道夫受体 gp340 已被确定为人类唾液中的分泌成分，通过与 Env 刺突的表面 gp120 亚基的特异性相互作用来抑制 HIV-1 的口腔传播。令人惊讶的是，女性生殖道中 gp120 与膜结合 gp340 的相互作用促进 HIV-1 通过屏障上皮细胞转胞吞作用，从而感染 CD4+ 细胞。我们此项目的目标是确定基于可溶性 gp340 的 HIV-1 进入抑制剂的分子靶点和作用机制。我们的发现工作将基于 gp340 的 gp120 结合 SRCR 结构域获得的初步数据，该结构域可有效抑制 HIV-1 的体外感染，IC50 值为纳摩尔级。我们提出了一种综合的、跨学科的方法，结合了蛋白质化学、高分辨率结构测定、热力学和动力学结合分析、蛋白质工程、分子病毒学和动物模型功效研究。在这个项目中，我们寻求发现新的 HIV-1 特异性干预策略，以抑制粘膜上皮-Env 结合和病毒-细胞融合。具体目标是： 1. 了解 gp340 与 gp120 结合的结构基础，为新型 SRCR 结构域进入抑制剂的设计和优化提供信息。 (a) 阐明游离和与 V3 区域结合的 SRCR 结构域片段的结构特性。 (b) 表征 gp120-gp340 相互作用的能量。 (c) 使用突变分析和生物物理方法来剖析各自结合位点的结构决定因素。 (d)定义“最小”gp120结合序列或此类序列的集合。 (e) 优化 SRCR 结构域变体的结合亲和力，并在单轮感染性测定中评估其抗 HIV-1 活性。 2. 确定gp120-gp340相互作用影响Env结构、功能和抗原性的机制。 (a) 确定 Aim 1c 中识别的 V3 突变如何影响 Env 的功能能力。 (b) 表征 SRCR 结构域对 gp120 受体结合特性及其对中和性和非中和性单克隆抗体的反应性的影响。 (c) 确定选定的 SRCR 结构域进入抑制剂对不同主要 HIV-1 毒株的杀病毒活性。 3. 测试优化的 SRCR 结构域进入抑制剂预防粘膜 HIV-1 感染的有效性。 (a) 在人类宫颈和阴道组织的 HIV-1 感染体外模型中表征选定 SRCR 进入抑制剂的特异性和效力。 (b) 使用人源化小鼠阴道传播模型来评估优化的 SRCR 进入抑制剂单独以及与 gp41 融合抑制剂 C52L 和小分子 CCR5 共受体抑制剂 CMPD167 组合的体内效力和活性广度。 1