Novel method for evaluating HIV latency and persistence in vivo

评估 HIV 体内潜伏期和持久性的新方法

• 批准号: 9221961
• 负责人: Matthew David Marsden
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-10 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9221961
• 关键词: Address; Animal Model; Animals; Biological Models; Biology; Bone Marrow; Cells; DNA; Disease Progression; Future; Generations; Genetic; Genetic Recombination; Genetic Variation; HIV; In Vitro; Individual; Infection; Latent Virus; Liver; Measurable; Measures; Methods; Modeling; Monitor; Mus; Pathogenesis; Pathogenicity; Phenotype; Plasma; Population; Procedures; RNA; Reporter; Research; Sampling; System; Systems Analysis; Testing; Thymus Gland; Tissue Sample; Tissues; Variant; Viral; Viral reservoir; Virion; Virus; Virus Latency; antiretroviral therapy; comparative efficacy; deep sequencing; effectiveness measure; experimental study; humanized mouse; improved; in vivo; in vivo Model; mouse model; novel; novel strategies; prevent; public health relevance; purge; tool; viral RNA; viral rebound

 DESCRIPTION (provided by applicant): HIV latency represents a key barrier preventing the cure of infected individuals through the use of antiretroviral therapy (ART) alone. Historically, efforts to deplete latently-infected cells have been hampered by a lack of relevant small animal models that can be used to study HIV latency and evaluate methods for eliminating latent virus. To address this issue we recently developed a new in vivo model for HIV latency using the humanized BLT (bone marrow-liver-thymus) mouse. We demonstrated that HIV forms latency within infected BLT mice, and that the latent virus is integrated, activation-inducible, replicatio-competent, and responds to known HIV latency reversing agents (LRAs). This model represents a versatile tool for studying latent HIV. However, challenges still exist within the HIV latency field, particularly in accurately quantifying latently-infected cells and in evaluating activation-elimination approaches that cause significant but incomplete depletion of persistent/latent reservoirs of HIV. Here, we propose to develop a new system for analyzing HIV persistence and latency using genetically barcoded virus. We have created an HIV swarm composed of >200,000 different viral variants that are genetically identical except for the presence of a short non-expressed, phenotypically neutral "barcode" sequence. This barcoded virus replicates efficiently in vitro, is pathogenic in vivo in BLT mice, and has multiple potential applications in HIV cure research. We intend to further develop and test the barcoded virus in the context of the humanized BLT mouse latency model. We will use deep sequencing approaches to evaluate viral diversity following ex vivo stimulations of latently-infected cells from bulk tissue samples, and thereby compare the relative efficacy of different individual or combination LRAs. We will also test our hypothesis that barcoded virus will allow us to monitor the diversity of HIV plasma virus that emerges from reservoirs upon cessation of ART. This would set the stage for in vivo testing of LRAs, with reductions in barcode diversity serving as a new additional measure of the effectiveness of LRAs in a whole animal system. Together, these experiments will provide important new information on HIV latency and will form the basis of future in vivo modelling of HIV cure approaches.

 描述（由申请人提供）：HIV潜伏期是阻止单独使用抗逆转录病毒疗法（ART）治愈感染个体的关键障碍。从历史上看，由于缺乏可用于研究HIV潜伏期和评估消除潜伏病毒方法的相关小动物模型，消除潜伏感染细胞的努力受到阻碍。为了解决这个问题，我们最近开发了一种新的体内HIV潜伏期模型，使用人源化BLT（骨髓-肝脏-胸腺）小鼠。我们证明了HIV在感染的BLT小鼠中形成潜伏期，并且潜伏病毒是整合的、活化诱导的、复制能力的，并且对已知的HIV潜伏期逆转剂（LRA）有反应。该模型是研究潜伏HIV的通用工具。然而，在HIV潜伏期领域内仍然存在挑战，特别是在准确定量潜伏感染细胞和评估激活-消除方法方面，这些方法导致HIV的持久性/潜伏性储库的显著但不完全耗尽。在这里，我们建议开发一个新的系统，用于分析艾滋病毒的持久性和潜伏期使用基因条形码病毒。我们已经创建了一个由> 200，000种不同病毒变体组成的HIV群，这些病毒变体除了存在短的非表达的表型中性“条形码”序列外，在遗传上是相同的。这种条形码化病毒在体外有效复制，在BLT小鼠体内是致病性的，并且具有多种潜在的应用， 艾滋病治疗研究。我们打算在人源化BLT小鼠潜伏期模型的背景下进一步开发和测试条形码化病毒。我们将使用深度测序方法来评估对来自大量组织样本的潜伏感染细胞进行离体刺激后的病毒多样性， 从而比较不同的单独或组合LRA的相对功效。我们还将测试我们的假设，即条形码病毒将使我们能够监测艾滋病毒血浆病毒的多样性，出现从水库停止抗逆转录病毒治疗后。这将设置阶段，在体内测试LRA，减少条形码多样性作为一个新的额外措施的有效性LRA在整个动物系统。总之，这些实验将提供有关HIV潜伏期的重要新信息，并将成为未来HIV治疗方法体内建模的基础。