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.

消除潜在的艾滋病毒水库仍然是实现治愈的主要绊脚石。消除这一点 水库，不同类型的潜在感染细胞的准确定义（生物标志物）， 组织非常需要。以前提出的几种生物标志物能够非常富裕（〜10倍） 对于潜在感染的细胞，但未能捕获潜在储层的大部分。没有 潜在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 储层细胞的含量将保持挑战。我们的长期目标是确定艾滋病毒潜伏期的生物标志物 可以将其转化为靶向潜在感染细胞以消除的策略。总体目标 应用是识别持续性HIV库的细胞和病毒决定剂，并测试所选的 生物标志物具有在体外和体内捕获潜在感染细胞的能力。我们的中心假设是 成功的生物标志物将由对不同细胞单独确定的储层代表 表型和状态。 “表型”一词是指广泛定义的规范表型子集 使用的表面蛋白标记物（例如，成熟表型 - 中央记忆；功能表型 - T 助手17）。术语“状态”是指细胞总转录组更广泛地定义的单元格状态 签名：积极表达的基因集和途径。拟议研究的理由是 当储层细胞的异质性是 考虑到。我们将通过追求以下特定目的来检验中心假设：（1）确定 不同储层子集的细胞决定剂，并测试所选的生物标志物在体外富集细胞； （2） 在体外鉴定不同储层子集的病毒决定剂； （3）验证储层决定者和 使用来自艾滋病毒患者的样品选择生物标志物。鉴定细胞和病毒决定剂 将使用持久的储层，RNA测序（RNA-Seq）技术的最新创新。单细胞 RNA-seq加上免疫表型，将用于表征细胞的表型和状态 可以用CXCR4或CCR5循环病毒感染。可以区分潜在的基因 在每种细胞类型中将单独识别感染和未感染的细胞。告知如何确定 持续储层的细胞决定剂与它们定义的病毒病毒类型有关 使用单细胞RNA-seq数据，HIV的全长测序将表征不同的细胞类型 转录本和主要流量测定法，以量化病毒对重新激活刺激的反应性。生物标志物 将从不同细胞子集中的HIV储层的一组细胞确定剂中选择。抗体 针对这些蛋白质，将测试有效捕获潜在感染细胞的能力。我们的最终 目的是确保确定的生物标志物可以准确地定义临床样本中的潜在感染细胞，并确定。 特别是在不同的组织室中，其中多达98％的持续储层位于体内。 通过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 使用来自HIV患者的血液和淋巴组织样品鉴定出确定的确定剂和生物标志物。什么时候 这些研究已经完成，我们将确定可用于捕获潜在感染的生物标志物 体外和离体细胞的效率至少比目前可实现的效率高500倍。 结果将很重要，因为鉴定的生物标志物可用于将储层细胞与不同的不同 组织以更好地表征人体的潜在储层。将来，这些 生物标志物可以作为制定靶向潜在感染细胞进化的策略的平台。 这样的研究对于满足艾滋病毒感染者的需求很重要，包括大量的艾滋病毒队列 在国家VA医疗保健系统中感染患者。