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（由申请人提供）：尽管联合抗逆转录病毒疗法显著改善了HIV相关的发病率和死亡率，但仍存在许多挑战，包括能够在感染后重新激活的多种潜伏病毒储库的长期持续性。 作为治愈倡议的一部分，任何根除这些储库的努力都需要了解HIV在长期携带病毒的组织和细胞中的再活化和复制的动力学和控制。拟议的研究分析了泌尿生殖道（GU）作为艾滋病毒的独特隔室和水库。我们先前的工作已经证明，肾上皮细胞是一个独特的病毒区室，在初步数据中，我们表明，来自男性和女性尿液的病毒RNA序列与来自PBMC和血浆的病毒RNA序列形成不同的簇，这与GU道中的病毒区室化一致。尿液中的病毒检测允许对该隔室进行重复采样，这在病毒反弹研究中特别有用。为了确定雄性动物中尿液衍生序列的来源，将从尿液和精液中同时分离无细胞和细胞相关病毒序列并进行遗传表征。为了评估GU道对病毒反弹的贡献，将在入组ACTG 5345试验（特定目的1）的患者中，在ART期间和治疗中断后，对尿液来源的HIV核酸与PBMC和血浆进行同步和纵向系统发育序列分析。为了评估HIV核酸在上GU道的长期体内持久性，将对ART长期抑制的HIV阳性受试者的肾活检进行DNA和RNA扩增、原位杂交和深度测序。深度测序将检测与感染肾上皮细胞克隆扩增相关的优先HIV整合位点（特异性目的2）。具体目标3将定义肾脏的长期潜力 作为艾滋病毒的潜伏和可再激活的储存库。一种双重荧光病毒构建体，其允许鉴定和纯化含有转录活性或沉默的细胞群， HIV将用于定义病毒再激活（从潜伏期）的生理学和药理学策略以及激活后RTE的命运。在离体方法中，将使用类似的活化策略评估从抑制的HIV-1阳性受试者的尿液中分离和培养的RTE细胞，并与同时衍生的PBMC的刺激进行比较。总的来说，这项工作将进一步表征GU道相关病毒，以及定义特征 潜伏期和转录激活的肾上皮细胞，一个独特的病毒室，可能有助于长期艾滋病毒的持久性。