Inhibiting Mucosal HIV-1 Transmission by Host Cell RNA Interference

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、非特异性免疫反应和获得性免疫细胞介导的特异性免疫反应的组合，在预防HIV-1的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，导致分泌抗病毒因子来摧毁入侵的病原体。这些专职的抗原提呈细胞(APC)还内吞HIV-1，并将病毒多肽与人类白细胞抗原I类和II类蛋白相关联地递送给反应性T细胞。然而，APC也表达CD4、CCR5和CXCR4受体，并能高效地感染HIV-1。此外，DC可以通过病毒结合包括DC-SIGN(DC特异性ICAM-抓取非整合素)在内的C型凝集素来捕获HIV-1。在抗原呈递过程中，HIV感染的巨噬细胞或DC与T细胞之间的细胞对细胞接触可导致T细胞感染HIV-1，途径是通过这些细胞之间形成的病毒突触分泌新合成的病毒(顺式感染)，或者通过未经修饰的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CCR5和DC-SIGN的表达，可以在多大程度上抑制HIV-1的粘膜传播。我们的假设是，阻止HIV-1与巨噬细胞、DC和T细胞结合将极大地减少这些细胞的感染，以及随后APC介导的反应性T细胞的感染。我们建议使用RNA干扰(RNAi)和短干扰RNA(SiRNA)来沉默来自雌性生殖道和胃肠道的粘膜白细胞上CCR5和DC-SIGN的表达，利用由人类祖细胞重组的免疫缺陷小鼠模型，表达人类所有主要免疫细胞群。我们的特定目标将确定在阴道或直肠内用HIV-1攻击后，在人源化NOD/SCID/BLT小鼠的粘膜组织中沉默CCR5和DC-SIGN在多大程度上抑制HIV-1感染(特定目标1)。这些研究将包括确定siRNA的最佳剂量和给药途径，以及确定siRNA是否可以抑制无细胞和细胞相关的HIV-1感染。此外，我们将确定在黏膜组织中抑制HIV-1感染是否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，我们将通过测定NOD/SCID/BLT小鼠黏膜组织中白细胞上CCR5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。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处理的小鼠对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的方法来抑制HIV-1从粘膜部位传播到外围。沉默对HIV-1感染至关重要的细胞受体可以加强其他旨在减少HIV-1粘膜传播的治疗方法，包括疫苗和杀微生物剂。 公共卫生相关性： 美国疾病控制中心估计，去年美国新增HIV-1感染者超过5.6万人。科学家们还没有生产出预防HIV-1的疫苗，而且现有的抗病毒药物具有显著的毒性，必须终身服用，而且不能治愈这种疾病。开发新的有效方法来预防艾滋病毒-1感染是V.A.医疗保健系统的优先事项。大多数新的艾滋病毒-1感染发生在与感染者发生性行为后的生殖器或身体的胃肠道区域。当HIV-1进入人体后，它通过与细胞表面的某些蛋白质结合来感染细胞。在我们的研究中，我们将确定阻断这些蛋白质是否可以保护一个人免受HIV-1感染。我们将使用一个动物模型，在这个模型中，这些研究可以更容易地进行测试。由于美国退伍军人和现役军人中感染HIV-1的人数很多，而且这种疾病在美国的发病率不断上升，因此拟议的研究与退伍军人的医疗保健问题高度相关。

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. PUBLIC HEALTH RELEVANCE: The Centers for Disease Control estimates that over 56,000 new HIV-1 infections occurred in the U.S. last year. Scientists have not produced a vaccine to protect against HIV-1, and the available anti-viral drugs have significant toxicity, must be taken for life, and do not cure this disease. The development of new and effective ways to prevent HIV-1 infection is a priority for the V.A healthcare system. Most new HIV-1 infections occur within the genital or the gastrointestinal areas of the body following sexual intercourse with an infected person. When HIV-1 enters the body, it infects cells by binding to certain proteins on their surface. In our studies, we will determine whether blocking these proteins can protect a person from becoming infected with HIV-1. We will use an animal model in which these studies can be more easily tested. The proposed studies are highly relevant to Veterans health care issues because of the number of HIV-1-infected individuals in the U.S. Veteran and active military population, and because of the increasing incidence of this disease in the U.S.