Determinants of reservoir contraction and expansion in vivo, ex vivo, and in vitro

体内、离体和体外储库收缩和扩张的决定因素

• 批准号: 10022993
• 负责人: Una T O'Doherty
• 金额: $ 55.65万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-16 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10022993
• 关键词: Acute; Affect; Antigens; Antisense RNA; Antiviral Agents; Antiviral Therapy; Bar Codes; CD4 Positive T Lymphocytes; CD8B1 gene; Categories; Cell Cycle Arrest; Cell Death; Cell division; Cells; Cessation of life; Chronic; Clonal Expansion; Data; Exons; Gene Silencing; Genes; Genetic Transcription; Goals; HIV; HIV Infections; Immune; Immune Targeting; Immune response; Immune system; In Vitro; Individual; Infection; Integration Host Factors; Introns; Lead; Location; Measures; Methods; Monitor; Mutate; Oncogenes; Open Reading Frames; Play; Proteins; Proviruses; RNA Splicing; Resistance; Rest; Role; Sample Size; Site; Testing; Time; Toxic effect; Transcript; Untranslated RNA; Viral; Viremia; Virus; Work; antiretroviral therapy; base; cohort; cytotoxicity; deep sequencing; design; driving force; experimental study; genetic makeup; immune clearance; in vitro Model; in vivo; integration site; latent HIV reservoir; longitudinal analysis; mutant; pressure; promoter; protein expression; transcriptome sequencing

The advent of antiviral therapy (ART) revealed a treatment-resistant reservoir in CD4+ T cells capable of refueling HIV viremia when ART is stopped. This reservoir is a major barrier to achieving a cure for HIV infection. Our recent work suggests the inherent reservoir decay is more rapid than previously recognized. This decay is obscured due to an opposing force that results in proviral clonal expansion. The driving forces behind proviral clonal expansion remain mysterious, but integration into introns of oncogenes may play a role. Overall objective: In this proposal, we dissect the drivers of reservoir contraction (Aim 1) and expansion (Aim 2). Our approach will be to perturb both forces and to study the resulting effects. We will use proviral, integration site, and RNA sequencing to understand how perturbing these forces affects the genetic make-up of proviruses, their propensity to expand, and their expression. Our approach is to perform massive deep sequencing in a few individuals rather than large sample size. We believe our intriguing results validate our deliberate decision to limit sample size to obtain deeper sequence information within each individual. With this approach, we recently provided unprecedented depth and elucidated previously unknown selection pressures. Design and Methods: In Aim 1, we isolate the role of immune clearance by measuring reservoir contraction in vivo and in vitro. We also dissect the cytotoxicity induced by HIV proteins by mutating individuals Open Reading Frames before infecting CD4 T cells with a barcoded virus. In Aim 2, we dissect the drivers of clonal expansion, including HIV-driven cell division, by using a barcoded virus. We also use longitudinal integration site analysis to compare the rate of clonal expansion as well as the “character” of the expanded proviral clones in elite controllers, acutely and chronically infected individuals on ART. The premise of our proposal is largely based on our work showing that there are two counterbalancing forces that cause (1) proviral contraction through viral cytotoxicity and immune clearance and (2) proviral expansion through clonal proliferation. The significance of our proposal includes that it may contribute to growing evidence that the reservoir is more visible than previously realized. We envision this work could lead to approaches that enhance immune clearance or target splicing to reduce reservoir size.

抗病毒疗法的冒险（ART）揭示了CD4+ T细胞中对治疗的储层 停止艺术时能够加毒艾滋病毒血症。该水库是 实现治愈艾滋病毒感染的方法。我们最近的工作表明，继承的储层衰减是 比以前认识的要快。由于对立的力量，这种衰变被遮挡了 导致前毒性克隆扩张。前病毒膨胀背后的驱动力仍然存在 神秘的，但整合到癌基因的内含子中可能起作用。总体目标：在此中 提案，我们剖析了储层收缩的驱动因素（AIM 1）和扩展（AIM 2）。我们的 方法将是扰动两种力并研究产生的效果。我们将使用前病毒， 集成位点和RNA测序以了解这些力如何影响这些力 病毒的基因组成，他们的诺言及其表达。我们的方法 是在几个个体而不是大型样本量中进行大量的深层测序。我们 相信我们有趣的结果可以验证我们故意限制样本量以获得的决定 每个人内的更深序列信息。通过这种方法，我们最近提供了 前所未有的深度和先前未知的选择压力。设计和 方法：在AIM 1中，我们通过测量储层收缩来构成免疫化的作用 体内和体外。我们还通过突变剖析了HIV蛋白诱导的细胞毒性 在AIM 2中，个体在用条形码病毒感染CD4 T细胞之前打开阅读框架。 我们通过使用A剖析克隆扩张的驱动因素，包括HIV驱动的细胞分裂 条形码病毒。我们还使用纵向整合位点分析来比较克隆的速率 扩展以及Elite Controller中扩展的提供商克隆的“角色”，敏​​锐地 以及长期感染艺术的人。 我们的提案的前提在很大程度上基于我们的工作表明有两个 通过病毒细胞毒性和免疫的临时收缩引起的平衡力（1） 清除率和（2）通过克隆增殖的病毒扩张。我们的意义 提案包括，它可能有助于越来越多的证据表明水库更可见 比以前意识到的。我们设想这项工作可能会导致增强免疫力 清除或目标剪接以减小储层尺寸。