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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

基本信息

批准号：
10579911
负责人：
Una T O'Doherty
金额：
$ 52.7万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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/10579911
关键词：
Acceleration Acute Affect Antigens Antisense RNA 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 Location Measures Methods Monitor Mutate Oncogenes Open Reading Frames Proliferating Proteins Provirus Integration Proviruses RNA Splicing Resistance Rest Role Sample Size Site Testing Time Toxic effect Transcript Untranslated RNA Viral Viremia Virus Work antiretroviral therapy 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

项目摘要

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.

抗病毒治疗（ART）的出现揭示了CD4+ T细胞中的治疗抵抗库