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: 10357547
• 负责人: Nadia R Roan
• 金额: $ 85.54万
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357547
• 关键词: Phenotypic; mechanistic; analysis; vivo; HIV

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 联合抗逆转录病毒疗法（ART）可以抑制HIV复制，降低死亡率 在HIV感染者身上。然而，ART并不能消除潜伏的HIV储存库，因此有效的病毒 抑制需要终身ART施用。因此，开发一种消除或实现无ART的方法， 水库的控制是研究的首要任务。实现这一目标的一个挑战是， 了解潜伏感染细胞的表型和功能特性，这些细胞在人们的生活中持续存在， 感染了艾滋病毒表征体内潜伏感染细胞的一个挑战是不能直接将这些细胞表型化。 细胞，因为缺乏将它们与未感染的细胞区分开来的通用生物标志物。因此，唯一的办法 直接使潜伏细胞表型化的方法是离体刺激大量患者来源的细胞， 以诱导潜伏细胞表达病毒蛋白。虽然这允许识别，因此 通过FACS对再活化的细胞进行表型分型，测量的表型与原始表型不同。 由于离体刺激改变了基因表达，因此潜在感染的细胞的表型。在这里，通过应用 基于伪时间的生物信息学方法，称为PP-SLIDE，对成对的未刺激和刺激的 通过高维单细胞分析方法（CyTOF，单细胞 RNAseq），我们推断潜伏感染细胞在其原始刺激前状态下的表型，并使用该 方法来绘制体内潜在的水库。在目标1中，我们将使用PP-SLIDE对CyTOF表型细胞进行分析， 比较存在于临床匹配的男性和女性的血液和组织中的潜在感染细胞。在 目标2，我们将表征目标1中鉴定的标记物以及从一个或多个细胞中鉴定的标记物的程度。 在通过单细胞RNAseq分析的细胞上实施PP-SLIDE的无偏方法，富集储库细胞 携带HIV和基因完整的有复制能力的HIV的细胞，因为这些细胞可能是最有可能 重要的是控制或消除无ART病毒控制。在目标3中，我们将描述 允许潜伏感染的细胞持续存在，重点关注抗原和稳态驱动的克隆 扩增CD 4 + T细胞。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伏细胞的“图谱”，我们的研究将提供前所未有的定义， 储库细胞的特征持续存在，揭示了是否有任何储库细胞特征与解剖学（血液）相关， vs.组织）或生物性别（男性与女性），并告知驱动水库维护的机制。 这些知识对于设计有针对性的方法以实现普遍的艾滋病毒治愈非常重要。