Inhibiting Mucosal HIV-1 Transmission by Host Cell RNA Interference

通过宿主细胞 RNA 干扰抑制粘膜 HIV-1 传播

• 批准号: 8391122
• 负责人: ALEXANDRA L HOWELL
• 金额: --
• 依托单位: WHITE RIVER JUNCTION VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391122
• 关键词: Adult; Affect; Animal Model; Antibodies; Antigen Presentation; Antigen-Presenting Cells; Antigens; Antiretroviral resistance; Area; B-Lymphocytes; Binding; Biological Models; Blood Cells; C Type Lectin Receptors; C-Type Lectins; CCR5 gene; CD4 Antigens; CD4 Positive T Lymphocytes; CXCR4 Receptors; CXCR4 gene; Cell Count; Cells; Centers for Disease Control and Prevention (U.S.); Chemokine (C-C Motif) Receptor 5; Child; Coitus; Control Groups; Dendritic Cells; Deposition; Development; Disease; Dose; Endocytosis; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Foundations; Gastrointestinal tract structure; Genetic Transcription; Genital system; Goals; HIV; HIV Receptors; HIV-1; Healthcare; Healthcare Systems; Human; Human Characteristics; Immune; Immune response; Immune system; Immunologic Deficiency Syndromes; Incidence; Individual; Infection; Invaded; Investigation; Kinetics; Lead; Lectin; Leukocytes; Life; Lymphocyte; Lymphoid Cell; Measurement; Measures; Mediating; Military Personnel; Modeling; Monitor; Mucosal Immune Responses; Mucous Membrane; Mus; Myeloid Cells; Nucleic Acids; Pathogenesis; Pathology; Peptides; Peripheral; Persons; Pharmaceutical Preparations; Plasma; Play; Population; Process; Proteins; RNA; RNA Interference; Reagent; Reporting; Retroviridae; Reverse Transcription; Role; Route; Scientist; Site; Stem cells; Surface; Synapses; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Tetanus Toxoid; Therapeutic; Time; Tissues; Toxic effect; Transfection; Vaccines; Veterans; Viral; Viral Load result; Viral Proteins; Virus; base; chemokine receptor; cytokine; design; functional disability; gastrointestinal; human stem cells; immunodeficient mouse model; in vivo; intercellular cell adhesion molecule; lymph nodes; macrophage; microbicide; mucosal site; nanoparticle; novel strategies; novel therapeutic intervention; particle; pathogen; prevent; public health relevance; receptor; receptor binding; receptor expression; reconstitution; rectal; reproductive; response; trafficking; transmission process; uptake

DESCRIPTION (provided by applicant): The mucosal immune system plays a central role in preventing the transmission of HIV-1 by a combination of rapid, non-specific responses mediated by innate immune cells, as well as by the induction of specific responses mediated by acquired immune cells. Macrophages and dendritic cells (DC) in mucosal tissues express receptors that are triggered by viral proteins and nucleic acids, leading to the secretion of anti-viral factors that destroy the invading pathogen. These professional antigen presenting cells (APC) also endocytose HIV-1 and present viral peptides to responding T cells in association with HLA class I and class II proteins. However, APC also express CD4, CCR5, and CXCR4 receptors and can become productively infected with HIV-1. In addition, DC can capture HIV-1 via viral binding to C type lectins including DC-SIGN (DC-specific ICAM-grabbing non-integrin). Cell-to-cell contact between an HIV-infected macrophage or DC and a T cell during antigen presentation can lead to infection of T cells by HIV-1, either by the secretion of newly- synthesized virus across the viral synapse that forms between these cells ("cis" infection), or by the transfer of unmodified HIV-1 from the DC to the T cell ("trans" infection). T cells that are either directly infected with HIV- 1, or become infected after interaction with infected APC, then migrate to adjacent lymph nodes, leading to the transmission of HIV-1 from mucosal sites to the periphery. The overall goal of these studies is to determine the extent to which mucosal HIV-1 transmission can be inhibited by blocking expression of CCR5 and DC-SIGN on mucosal macrophages, DC and T cells. Our hypothesis is that preventing HIV-1 binding to macrophages, DC and T cells would dramatically reduce infection of these cells, and also the subsequent APC- mediated infection of responding T cells. We propose to use RNA interference (RNAi) with short-interfering RNA (siRNA) to silence expression of CCR5 and DC-SIGN on mucosal leukocytes from the female reproductive and gastrointestinal tracts using an immunodeficient mouse model that is reconstituted with human progenitor cells and expresses all major human immune cell populations. Our specific aims will determine the extent to which silencing CCR5 and DC-SIGN within mucosal tissues of humanized NOD/SCID/BLT mice leads to the inhibition of HIV-1 infection after intravaginal or intrarectal challenge with HIV-1 (specific aim 1). These studies will include defining optimal doses and routes of administration of siRNA, and also determining whether siRNA can inhibit both cell-free as well as cell associated HIV-1 infection. In addition, we will determine whether inhibition of HIV-1 infection across mucosal tissues correlates with the degree of receptor silencing, as additional anti-viral mechanisms may occur as a function of siRNA exposure. In subsequent studies, we will define the anti-viral mechanisms that contribute to the inhibition of HIV-1 infection by determining the extent to which silencing expression of CCR5 and DC-SIGN on leukocytes in mucosal tissues of NOD/SCID/BLT mice enhances immune responses to HIV-1 and to a model antigen, tetanus toxoid (specific aim 2). Studies in this aim will quantify acquired immune responses to HIV-1 and to tetanus toxoid in siRNA-treated mice by measuring humoral and cellular immune responses. Our overall goal is to develop novel approaches to inhibit the transmission of HIV-1 from mucosal sites to the periphery. Silencing cellular receptors important for HIV-1 infection could augment other therapeutic approaches designed to reduce mucosal HIV-1 transmission, including vaccines and microbicides.

描述（由申请人提供）： 粘膜免疫系统通过先天免疫细胞介导的快速非特异性反应以及后天免疫细胞介导的特异性反应的诱导，在预防 HIV-1 传播方面发挥着核心作用。粘膜组织中的巨噬细胞和树突状细胞（DC）表达由病毒蛋白和核酸触发的受体，导致分泌抗病毒因子，消灭入侵的病原体。这些专职抗原呈递细胞 (APC) 还内吞 HIV-1，并将病毒肽呈递给与 HLA I 类和 II 类蛋白相关的应答 T 细胞。然而，APC 也表达 CD4、CCR5 和 CXCR4 受体，并且可以有效感染 HIV-1。此外，DC 可以通过病毒与 C 型凝集素（包括 DC-SIGN（DC 特异性 ICAM 捕获非整合素）结合）捕获 HIV-1。在抗原呈递过程中，感染 HIV 的巨噬细胞或 DC 与 T 细胞之间的细胞间接触可导致 T 细胞被 HIV-1 感染，这可能是通过在这些细胞之间形成的病毒突触分泌新合成的病毒来实现的。细胞（“顺式”感染），或通过将未修饰的 HIV-1 从 DC 转移到 T 细胞（“反式”感染）。 T细胞要么直接感染HIV-1，要么与感染的APC相互作用后被感染，然后迁移到邻近的淋巴结，导致HIV-1从粘膜部位传播到周围。这些研究的总体目标是确定通过阻断粘膜巨噬细胞、DC 和 T 细胞上 CCR5 和 DC-SIGN 的表达可以在多大程度上抑制粘膜 HIV-1 传播。我们的假设是，阻止 HIV-1 与巨噬细胞、DC 和 T 细胞结合将显着减少这些细胞的感染，以及随后 APC 介导的响应 T 细胞的感染。我们建议使用 RNA 干扰 (RNAi) 和短干扰 RNA (siRNA) 来沉默女性生殖道和胃肠道粘膜白细胞上 CCR5 和 DC-SIGN 的表达，使用免疫缺陷小鼠模型，该模型由人类祖细胞重建并表达所有主要的人类免疫细胞群。我们的具体目标将确定在阴道内或直肠内用 HIV-1 攻击后，人源化 NOD/SCID/BLT 小鼠粘膜组织内的 CCR5 和 DC-SIGN 沉默可在多大程度上抑制 HIV-1 感染（具体目标 1）。这些研究将包括确定 siRNA 的最佳剂量和给药途径，并确定 siRNA 是否可以抑制无细胞以及细胞相关的 HIV-1 感染。此外，我们将确定粘膜组织中 HIV-1 感染的抑制是否与受体沉默的程度相关，因为 siRNA 暴露可能会产生额外的抗病毒机制。在后续研究中，我们将通过确定NOD/SCID/BLT小鼠粘膜组织中白细胞上CCR5和DC-SIGN表达沉默增强免疫的程度来定义有助于抑制HIV-1感染的抗病毒机制。对 HIV-1 和模型抗原破伤风类毒素的反应（具体目标 2）。这一目标的研究将通过测量体液和细胞免疫反应来量化 siRNA 治疗小鼠对 HIV-1 和破伤风类毒素的获得性免疫反应。我们的总体目标是开发新方法来抑制 HIV-1 从粘膜部位向周围的传播。沉默对 HIV-1 感染很重要的细胞受体可以增强其他旨在减少粘膜 HIV-1 传播的治疗方法，包括疫苗和杀菌剂。