Cellular and viral determinants of the persistent HIV reservoir

持久性艾滋病毒储存库的细胞和病毒决定因素

• 批准号: 10512041
• 负责人: Nadejda S Beliakova-Bethell
• 金额: --
• 依托单位: VA SAN DIEGO HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-10-01 至 2025-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10512041
• 关键词: Address; Adherence; Antibodies; Biological Assay; Biological Markers; Biological Specimen Banks; Blood; Blood specimen; CCR5 gene; CD4 Positive T Lymphocytes; CXCR4 gene; Cell model; Cells; Centers for Disease Control and Prevention (U.S.); Chronic; Clinical; Collaborations; Coupled; DNA; Data; Development; Diagnosis; Enrollment; Experimental Designs; Flow Cytometry; Future; Gene Expression; Genes; Genetic Transcription; Gifts; Goals; HIV; HIV Infections; Healthcare Systems; Heterogeneity; Human body; Immunophenotyping; In Vitro; Individual; Infection; Length; Lymphoid Tissue; Membrane Proteins; Memory; Outcome; Pathway interactions; Patients; Persons; Phenotype; Population; Proteins; Proviruses; RNA; RNA Splicing; Research; Sampling; Stimulus; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Testing; Tissue Sample; Tissues; Transcript; Translating; Treatment-related toxicity; Tropism; United States; Variant; Veterans; Viral; Virus; antiretroviral therapy; biomarker identification; biomarker selection; cell type; cohort; cost; improved; in vivo; innovation; latent HIV reservoir; protein biomarkers; purge; response; single-cell RNA sequencing; transcriptome sequencing; transcriptomics; viral rebound

Eradication of the latent HIV reservoir remains the major stumbling block to achieving cure. To eliminate this reservoir, accurate definition (a biomarker) of latently infected cells of different types and within different tissues is highly needed. Several biomarkers proposed previously were able to very modestly enrich (~10-fold) for latently infected cells, but failed to capture the substantial portion of the latent reservoir. Without the detailed characterization of latently infected cells, identification of a suitable biomarker to capture the majority of the reservoir cells will remain challenging. Our long-term goal is to identify a biomarker of HIV latency that can be translated into strategies to target latently infected cells for elimination. The overall objectives of this application are to identify cellular and viral determinants of the persistent HIV reservoir and to test selected biomarkers for their ability to capture latently infected cells in vitro and ex vivo. Our central hypothesis is that a successful biomarker will be represented by reservoir determinants identified individually for cells of different phenotypes and states. The term “phenotype” refers to the canonical phenotypic subsets defined with widely used surface protein markers (for example, maturation phenotype – central memory; functional phenotype – T helper 17). The term “state” refers to a cell state more broadly defined by the cell's total transcriptomic signature: gene sets and pathways that are actively expressed. The rationale of the proposed research is the expected improvement in the efficiency of the reservoir capture when heterogeneity of the reservoir cells is taken into account. We will test our central hypothesis by pursuing the following specific aims: (1) Identify cellular determinants of different reservoir subsets and test selected biomarkers for cell enrichment in vitro; (2) Identify viral determinants of different reservoir subsets in vitro; (3) Validate the reservoir determinants and selected biomarkers using samples from people with HIV. To identify cellular and viral determinants of the persistent reservoir, latest innovations in RNA sequencing (RNA-Seq) technologies will be used. Single cell RNA-Seq coupled with immunophenotyping will be used to characterize the phenotypes and states of cells that can be infected with either CXCR4- or CCR5-tropic virus. Genes that can discriminate between latently infected and uninfected cells will be identified individually within each cell type. To inform on how the identified cellular determinants of the persistent reservoir relate to the type of provirus that they define, proviral activity in different cell types will be characterized using single cell RNA-Seq data, full length sequencing of HIV transcripts, and the PrimeFlow assay to quantify responsiveness of provirus to reactivation stimuli. Biomarkers will be selected from sets of cellular determinants of the HIV reservoir in different cell subsets. Antibodies against these proteins will be tested for the ability to efficiently capture the latently infected cells. Our ultimate goal is to ensure that identified biomarkers can accurately define latently infected cells in clinical samples, and specifically in different tissue compartments, where as much as 98% of the persistent reservoir resides in vivo. In collaboration with the Last Gift cohort, we will have a unique opportunity to conduct studies to validate the identified determinants and biomarkers using blood and lymphoid tissue samples from persons with HIV. When these studies are complete, we will have identified biomarkers that can be used to capture latently infected cells in vitro and ex vivo, with the efficiency of at least 500-fold greater than is currently achievable. These results will be significant because identified biomarkers can be used to isolate reservoir cells from different tissues to provide better characterization of the latent reservoir across the human body. In the future, these biomarkers can serve as a platform for development of strategies to target latently infected cells for elimination. Such research is important to address the needs of people living with HIV, including the large cohort of HIV- infected patients within the national VA Healthcare System.

根除潜伏的艾滋病毒储存库仍然是实现治愈的主要障碍。消除这种 储库，不同类型和不同内的潜伏感染细胞的准确定义（生物标志物） 组织是非常需要的。先前提出的几种生物标志物能够非常适度地富集（~10倍） 潜伏感染的细胞，但未能捕获潜伏水库的实质性部分。未经 潜伏感染细胞的详细表征，鉴定合适的生物标志物以捕获大多数 储层细胞的生长仍将是一个挑战。我们的长期目标是确定HIV潜伏期的生物标志物， 可以转化为针对潜伏感染细胞的消除策略。这一总体目标 应用程序是确定细胞和病毒的决定因素的持久性艾滋病毒水库和测试选定的 这些生物标志物在体外和离体捕获潜伏感染细胞的能力。我们的中心假设是， 成功的生物标志物将由针对不同细胞单独鉴定的储库决定因子来表示 表型和状态。术语“表型”是指由广泛的遗传修饰定义的典型表型子集。 使用表面蛋白标记物（例如，成熟表型-中央记忆;功能表型- T 助手17）。术语“状态”是指更广泛地由细胞的总转录组学特征定义的细胞状态。 特征：活跃表达的基因组和途径。拟议研究的基本原理是 当储集层单元的非均质性被消除时， 考虑了我们将通过追求以下具体目标来检验我们的中心假设：（1）识别 不同储库亚群的细胞决定因素，并测试用于体外细胞富集的选定生物标志物;（2） 体外鉴定不同储库亚群的病毒决定簇;（3）鉴定储库决定簇， 使用来自HIV感染者的样本选择生物标志物。为了确定细胞和病毒的决定因素， 为了实现持久储存，将使用RNA测序（RNA-Seq）技术的最新创新。单细胞 RNA-Seq结合免疫表型分析将用于表征细胞的表型和状态， 可以感染CXCR 4或CCR 5嗜性病毒。基因可以区分潜在的 将在每种细胞类型中单独鉴定感染和未感染的细胞。告知如何识别 细胞决定因素的持久水库涉及类型的前病毒，他们定义，前病毒的活动， 不同的细胞类型将使用单细胞RNA-Seq数据，HIV全长测序， 转录物，和PrimeFlow测定来定量前病毒对再激活刺激的响应性。生物标记 将从不同细胞亚群中HIV储库的细胞决定簇组中选择。抗体 将测试针对这些蛋白质的抗体有效捕获潜伏感染细胞的能力。我们的最终 目标是确保鉴定的生物标志物可以准确地定义临床样品中的潜伏感染细胞， 特别是在不同的组织区室中，其中多达98%的持久性储库驻留在体内。 与“最后的礼物”队列合作，我们将有一个独特的机会进行研究，以验证 使用艾滋病毒感染者的血液和淋巴组织样本确定了决定因素和生物标志物。当 这些研究完成后，我们将确定可用于捕获潜伏感染的生物标志物， 细胞在体外和离体，与至少500倍以上的效率是目前可实现的。这些 结果将是重要的，因为确定的生物标志物可用于从不同的细胞中分离储库细胞 组织，以更好地表征整个人体的潜在储库。未来这些 生物标志物可以作为开发靶向潜伏感染细胞以消除的策略的平台。 这种研究对于满足艾滋病毒感染者的需求，包括大量艾滋病毒感染者的需求非常重要， 在全国VA医疗保健系统内感染的患者。