The Genitourinary Tract as a compartment and reservoir for HIV

泌尿生殖道作为艾滋病毒的隔室和储存库

• 批准号: 9325517
• 负责人: Mary E. Klotman
• 金额: $ 48.18万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-20 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9325517
• 关键词: AIDS clinical trial group; Acute Kidney Tubular Necrosis; Aftercare; Alpha Cell; Anti-Retroviral Agents; Base Sequence; Biopsy; Blood; Body Fluids; Cell Culture System; Cell Death; Cells; Characteristics; Clinical; Clonal Expansion; Consequences of HIV; DNA amplification; Data; Enrollment; Epithelial Cells; Evolution; Female; Genetic; Genetic Transcription; Genitourinary system; Genome; HIV; HIV Infections; HIV Seropositivity; HIV therapy; HIV-1; Human; In Situ Hybridization; In Vitro; Infection; Interruption; Kidney; Kinetics; Modeling; Morbidity - disease rate; Nucleic Acids; Nucleic acid sequencing; Patients; Peripheral Blood Mononuclear Cell; Pharmacology; Phylogenetic Analysis; Physiological; Plasma; Population; Proteins; RNA Sequences; RNA amplification; Regimen; Relapse; Renal tubule structure; Sampling; Satellite Viruses; Seminal fluid; Sequence Analysis; Site; Source; Specimen; T-Lymphocyte; Therapeutic; Time; Tissues; Transcriptional Activation; Urine; Viral; Viral Load result; Viral reservoir; Virus; Virus Latency; Virus Replication; Work; antiretroviral therapy; deep sequencing; in vivo; integration site; kidney cell; male; mortality; peripheral blood; promoter; public health relevance; reactivation from latency; renal epithelium; success; viral DNA; viral RNA; viral detection; viral rebound

 DESCRIPTION (provided by applicant): Despite the dramatic improvement in HIV-associated morbidity and mortality from combination antiretroviral therapy, a number of challenges remains including the long-term persistence of multiple, latent viral reservoirs capable of reactivation in the absence of ART. Any effort to eradicate these reservoirs as part of a cure initiative requires understanding of the dynamics and control of HIV reactivation and replication in tissues and cells harboring the virus long-term. The proposed studies analyze the genitourinary (GU) tract as a unique compartment and reservoir for HIV. Our prior work has demonstrated that renal epithelial cells are a unique viral compartment and in preliminary data we show that viral RNA sequences derived from the urine of males and females form separate cluster(s) from those derived from PBMC and plasma, consistent with viral compartmentalization in the GU tract. Viral detection in the urine allows for repeated sampling of this compartment, which can be particularly useful in viral rebound studies. To determine the source of urine- derived sequences in males, cell free and cell-associated viral sequences will be isolated and genetically characterized simultaneously from urine and semen. To assess the contribution of the GU tract to virus rebound, simultaneous and longitudinal phylogenetic sequence analysis will be performed on urine-derived HIV nucleic acids in comparison with PBMC and plasma, during ART and after treatment interruption in patients enrolled in the ACTG 5345 trial (Specific Aim 1). To assess the long-term in vivo persistence of HIV nucleic acids in the upper GU tract, DNA and RNA amplification, in situ hybridization and deep sequencing will be performed on kidney biopsies from HIV positive subjects that have prolonged suppression on ART. Deep sequencing will detect preferential HIV integration sites associated with clonal expansion of infected renal epithelial cells (Specific Aim 2). Specific Aim 3 will define the long-term potential of the kidney as a latent and reactivatable reservoir for HIV. A dual fluorescent viral construct, that allows identification and purification of cell populations containing transcriptionally active or silenced HIV, will be used to define physiologic and pharmacologic strategies for virus reactivation (from latency) as well as the fate of RTE upon activation. In an ex vivo approach, RTE cells isolated and cultured from the urine of suppressed HIV-1 positive subjects will be assessed using similar activation strategies and compared to stimulation of simultaneously derived PBMC. Collectively, this work will further characterize the GU tract associated virus as well as define characteristics of latency and transcriptional activation in renal epithelium, a unique viral compartment that may contribute to long-term HIV persistence.

 描述（通过应用提供）：尽管抗逆转录病毒疗法的HIV相关发病率和死亡率有了显着改善 缺乏艺术。作为治疗计划的一部分，任何放射性储层的努力都需要了解长期内拥有该病毒的组织和细胞中HIV重新激活和复制的动力学和控制。拟议的研究将泌尿生殖器（GU）分析为艾滋病毒的独特隔室和水库。我们先前的工作表明，肾上皮细胞是一个独特的病毒室，在初步数据中，我们表明，源自雄性和女性尿液的病毒RNA序列与源自PBMC和血浆的尿液形成单独的簇（S），与Gu -Tract中的病毒隔离一致。尿液中的病毒检测允许重复对该室的采样，这在病毒反弹研究中可能特别有用。为了确定男性尿液衍生序列的来源，将分离出无细胞和相关的病毒序列，并简单地从尿液和精液中表征。评估GU道对病毒反弹的贡献，与PBMC和血浆相比，将对尿液来源的HIV核酸进行简单和纵向的系统发育序列分析，在ART和ACTG 5345试验的患者中进行ART和治疗中断后，将对尿液来源的HIV核酸进行贡献（特定目标1）。为了评估上gu核酸，DNA和RNA扩增中HIV核酸的长期体内持续性，将对来自HIV阳性受试者的肾脏活检进行原位杂交和深层测序，这些受试者长时间抑制了ART。深度测序将检测与感染肾上皮细胞克隆扩张相关的优先HIV整合位点（特定的目标2）。特定目标3将定义肾脏的长期潜力 作为艾滋病毒的潜在储备。双荧光病毒构建体，允许识别和纯化包含转录活性或沉默的细胞群体 HIV将用于定义病毒重新激活的生理和药物策略（来自潜伏期）以及激活后RTE的命运。在离体方法中，将使用类似的激活策略评估从抑制HIV-1阳性受试者尿液中分离和培养的RTE细胞，并将其与易于衍生的PBMC刺激进行比较。总的来说，这项工作将进一步描述与Gu道相关的病毒以及定义特征 肾上皮中的潜伏期和转录激活，这是一种独特的病毒室，可能导致长期HIV持久性。