Cellular and viral determinants of the persistent HIV reservoir

根除潜在的艾滋病毒蓄积者仍然是实现治愈的主要绊脚石。要消除这一点 不同类型和不同体内的潜伏感染细胞的准确定义(生物标记物) 纸巾是非常需要的。之前提出的几个生物标志物能够非常适度地富集(~10倍) 对于潜伏感染的细胞，但未能捕获潜伏水库的实质性部分。如果没有 潜伏感染细胞的详细特征，确定合适的生物标记物来捕获大多数 对储集层细胞的研究仍将具有挑战性。我们的长期目标是确定艾滋病毒潜伏期的生物标记物 可以转化为针对潜伏感染细胞的消灭策略。这个项目的总体目标是 应用是识别持久性艾滋病毒储存库的细胞和病毒决定因素，并测试选定的 它们在体外和体外捕获潜伏感染细胞的能力的生物标志物。我们的中心假设是 成功的生物标志物将由针对不同细胞的单独识别的储藏决定因素来代表 表型和状态。术语“表型”指的是广泛定义的规范表型子集 使用的表面蛋白标记(例如，成熟表型-中央记忆；功能表型-T 帮手17)。状态一词是指由细胞的总转录水平更广义地定义的一种细胞状态 特征：活跃表达的基因集和途径。这项拟议研究的基本原理是 当油藏单元的非均质性为 考虑到这一点。我们将通过追求以下具体目标来验证我们的中心假设：(1)确定 不同储集层亚群的细胞决定因素，并测试选定的细胞体外浓缩生物标志物；(2) 在体外确定不同储存库亚群的病毒决定因素；(3)验证储存库决定因素和 使用艾滋病毒携带者的样本选择生物标记物。以确定该病毒的细胞和病毒决定因素 永久性储存库，将使用RNA测序(RNA-Seq)技术的最新创新。单细胞 RNA-Seq结合免疫表型将被用来表征细胞的表型和状态 可感染CXCR4或CCR5嗜性病毒。基因可以潜伏地区分 感染和未感染的细胞将在每种细胞类型中单独识别。告知已确定的 持久储存库的细胞决定因素与它们定义的前病毒类型有关，前病毒活动在其中 不同的细胞类型将使用单细胞RNA-Seq数据进行表征，对HIV进行全长测序 转录本，和PrimeFlow试验来量化前病毒对重新激活刺激的反应性。生物标志物 将从不同细胞亚群中的HIV储存库的细胞决定因素集合中选择。抗体 针对这些蛋白质的能力将接受测试，以有效捕获潜伏感染的细胞。我们的终极 目标是确保已识别的生物标记物可以准确地确定临床样本中潜伏感染的细胞，以及 特别是在不同的组织隔间中，高达98%的持久储存库驻留在体内。 在与最后的礼物队列的合作下，我们将有一个独特的机会进行研究，以验证 使用艾滋病毒携带者的血液和淋巴组织样本确定决定因素和生物标志物。什么时候 这些研究完成后，我们将识别出可以用来捕捉潜伏感染的生物标志物 在体外和体外培养细胞，其效率至少是目前可以实现的500倍。这些 结果将是重要的，因为已识别的生物标记物可以用于从不同的 组织，以便更好地描述人体内的潜伏储存库。在未来，这些 生物标记物可以作为制定策略的平台，以消除潜伏感染的细胞。 这类研究对于解决艾滋病毒携带者的需求非常重要，包括大量艾滋病毒感染者-- 国家退伍军人医疗系统内的感染患者。

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.