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性免疫细胞介导的快速非特异性应答以及诱导获得性免疫细胞介导的特异性应答的组合，在预防HIV-1传播中发挥核心作用。粘膜组织中的巨噬细胞和树突状细胞（DC）表达由病毒蛋白质和核酸触发的受体，导致分泌破坏入侵病原体的抗病毒因子。这些专职抗原呈递细胞（APC）也内吞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CD 4、CCR 5和CXCR 4受体，并且可以被HIV-1感染。此外，DC可以通过与C型凝集素包括DC-SIGN（DC特异性ICAM-抓取非整联蛋白）的病毒结合来捕获HIV-1。在抗原呈递期间，HIV感染的巨噬细胞或DC与T细胞之间的细胞-细胞接触可导致T细胞被HIV-1感染，这是通过新合成的病毒分泌穿过在这些细胞之间形成的病毒突触（“顺式”感染），或通过未修饰的HIV-1从DC转移到T细胞（“反式”感染）。直接感染HIV- 1或在与感染的APC相互作用后被感染的T细胞，然后迁移到邻近的淋巴结，导致HIV-1从粘膜部位传播到外周。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CCR 5和DC-SIGN的表达可以抑制粘膜HIV-1传播的程度。我们的假设是，阻止HIV-1与巨噬细胞、DC和T细胞结合将显著减少这些细胞的感染，以及随后APC介导的应答T细胞的感染。我们建议使用RNA干扰（RNAi）与短干扰RNA（siRNA）沉默表达的CCR 5和DC-SIGN的粘膜白细胞从女性生殖和胃肠道使用免疫缺陷小鼠模型，重建与人类祖细胞和表达所有主要的人类免疫细胞群体。我们的具体目标是确定在人源化NOD/SCID/BLT小鼠的粘膜组织内沉默CCR 5和DC-SIGN导致在用HIV-1进行阴道内或直肠内攻击后抑制HIV-1感染的程度（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，我们将确定是否抑制跨粘膜组织的HIV-1感染与受体沉默的程度相关，因为额外的抗病毒机制可能作为siRNA暴露的函数而发生。在随后的研究中，我们将通过确定NOD/SCID/BLT小鼠粘膜组织中白细胞上CCR 5和DC-SIGN的沉默表达在多大程度上增强对HIV-1和模型抗原破伤风类毒素的免疫应答来确定有助于抑制HIV-1感染的抗病毒机制（具体目标2）。这一目标的研究将通过测量体液和细胞免疫应答来量化siRNA处理小鼠对HIV-1和破伤风类毒素的获得性免疫应答。我们的总体目标是开发新的方法来抑制HIV-1从粘膜部位向外周的传播。沉默对HIV-1感染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，包括疫苗和杀微生物剂。