Phenotypic and mechanistic analysis of the in vivo HIV latent reservoir by single-cell technologies

通过单细胞技术对体内 HIV 潜伏病毒库进行表型和机制分析

• 批准号: 10448398
• 负责人: Nadia R Roan
• 金额: $ 84.35万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10448398
• 关键词: Anatomy; Antigens; Atlases; Automobile Driving; Back; Bioinformatics; Biological Markers; Blood; CD4 Positive T Lymphocytes; Cell surface; Cells; Clinical; Clonal Expansion; Cytomegalovirus; DNA sequencing; Data Analyses; Data Set; Development; Exhibits; Face; Flow Cytometry; Fluorescence; Frequencies; Gene Expression; Gene Expression Profile; Genetic Transcription; Genome; Goals; HIV; HIV Genome; HIV Infections; Homeostasis; Immunology; Individual; Infection; Intervention; Knowledge; Lead; Length; Leukapheresis; Link; Location; Maintenance; Maps; Measures; Methods; Molecular; Patients; Persons; Phenotype; Population; Property; Proteins; Regression Analysis; Research; Research Personnel; Research Priority; Role; Sampling; Sorting - Cell Movement; Specificity; Surface; System; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Time; Tissues; Translations; Viral; Viral Proteins; Virus; Woman; antiretroviral therapy; base; biological sex; co-infection; cohort; defined contribution; design; experimental study; high dimensionality; in vivo; latent HIV reservoir; lymph nodes; men; mortality; multidimensional data; novel; patient population; receptor; sex; single cell analysis; single cell technology; single-cell RNA sequencing; tool; trait; viral DNA

PROJECT SUMMARY Combination antiretroviral therapy (ART) can suppress HIV replication and lead to decreased mortality in HIV-infected individuals. However, ART does not eliminate the latent HIV reservoir, so effective viral suppression requires lifelong ART administration. Therefore, developing a way to eliminate or achieve ART-free control of the reservoir is a top research priority. One challenge to accomplishing this is that we still have limited understanding of the phenotypic and functional properties of the latently-infected cells that persist in people living with HIV. One challenge to characterizing in vivo latently-infected cells is the inability to directly phenotype these cells, due to the lack of a universal biomarker distinguishing them from uninfected cells. As a result, the only way to directly phenotype latent cells has been to stimulate a bulk population of patient-derived cells ex vivo in order to induce expression of viral proteins by the latent cells. Although this allows identification and therefore phenotyping of the reactivated cells by FACS, the measured phenotypes are different from the original phenotypes of the latently-infected cells since ex vivo stimulation alters gene expression. Here, by applying a pseudotime-based bioinformatics approach called PP-SLIDE on paired sets of unstimulated and stimulated patient cells deep-phenotyped by high-dimensional single-cell analytical approaches (CyTOF, single-cell RNAseq), we infer the phenotypes of latently-infected cells in their original pre-stimulation state, and use this approach to chart the in vivo latent reservoir. In Aim 1, we will use PP-SLIDE on CyTOF-phenotyped cells to compare the latently-infected cells present in the blood and tissues of clinically-matched men and women. In Aim 2, we will characterize the extent to which markers identified in Aim 1, as well as markers identified from an unbiased approach implementing PP-SLIDE on cells analyzed by single-cell RNAseq, enrich for reservoir cells harboring HIV with genetically-intact replication-competent HIV, for these are the cells that are likely the most important to control or eliminate for ART-free viral control. In Aim 3, we will characterize the mechanisms that allow the latently-infected cells to persist, focusing on the role of antigen- and homeostasis-driven clonal expansion of CD4+ T cells. By combining cutting-edge single-cell analysis tools with high-dimensional data analysis methods to map the “atlas” of in vivo latent cells, our studies will provide an unprecedented definition of the features of reservoir cells that persist, reveal whether any reservoir cell traits associate with anatomy (blood vs. tissues) or biological sex (men vs. women), and inform on the mechanisms driving reservoir maintenance. This knowledge that will be important for designing targeted methods to achieve a universal HIV cure.

项目摘要 抗逆转录病毒疗法（ART）可以抑制HIV复制并导致死亡率降低 在艾滋病毒感染者中。但是，艺术不会消除潜在的艾滋病毒水库，因此有效的病毒 抑制需要终身艺术管理。因此，开发一种消除或无艺术的方法 控制储层是最重要的研究重点。实现这一目标的一个挑战是我们仍然有限 了解持续存在于生活的人的潜在感染细胞的表型和功能特性 与艾滋病毒。表征体内受感染的细胞表征的一个挑战是无法直接表型 由于缺乏通用生物标志物，细胞将它们与未感染的细胞区分开。结果，唯一的方法 直接表型潜在细胞一直是刺激大量的患者衍生细胞的人群，但 诱导潜在细胞表达病毒蛋白。尽管这允许身份证明，因此 FACS重新激活细胞的表型，测得的表型与原始的表型不同 由于离体刺激以改变基因表达，因此受到潜在感染细胞的表型改变了基因表达。在这里，应用一个 基于伪频率的生物信息学方法在未刺激和刺激的配对组上称为pp-slide 患者细胞通过高维单细胞分析方法进行深度型（Cytof，单细胞） rnaseq），我们在其原始刺激状态下推断潜在感染的细胞的表型，并使用 绘制体内潜在储层的方法。在AIM 1中，我们将在细胞型细胞上使用PP-Slide到 比较临床匹配的男性和女性血液和组织中存在的潜在感染细胞。 AIM 2，我们将描述AIM 1中标记的标记以及从一个 无偏见的方法在通过单细胞RNASEQ分析的细胞上实现PP-SLIDE，富含储层细胞 具有一般性复制能力的艾滋病毒的艾滋病毒，因为这些可能是最可能的细胞 对于控制或消除无艺术病毒控制的重要性很重要。在AIM 3中，我们将表征 允许潜在感染的细胞持续存在，重点关注抗原和稳态驱动的克隆的作用 CD4+ T细胞的扩展。通过将尖端的单细胞分析工具与高维数据相结合 分析方法绘制体内潜在细胞的“地图集”，我们的研究将为空前的定义提供 持续存在的储层细胞的特征，揭示了是否有任何储层细胞性状与解剖学相关（血液） 与组织）或生物学性别（男性与女性），并告知驱动储层维护的机制。 对于设计有针对性的方法以实现普遍艾滋病毒治疗非常重要的知识。