Effects of Cell-Intrinsic Immunity on Establishment and Reversal of HIV Latency

细胞内在免疫对 HIV 潜伏期建立和逆转的影响

• 批准号: 9354580
• 负责人: Satish Kumar Pillai
• 金额: $ 12.5万
• 依托单位: VITALANT
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9354580
• 关键词: Aging; Anti-Retroviral Agents; Antiviral Agents; CD4 Positive T Lymphocytes; Cell Line; Cell Separation; Cells; Chronic; Clinical; Codon Nucleotides; DNA; Data; Drug toxicity; Exhibits; Foundations; Gene Expression; Genetic Transcription; Goals; HIV; HIV Infections; Health; Human; Immune; Immune response; Immunity; In Vitro; Individual; Infection; Inflammation; Integration Host Factors; Lymphoid; Measures; Mediating; Messenger RNA; Modeling; Morbidity - disease rate; Patients; Peripheral; Pharmaceutical Preparations; Population; Protein Biosynthesis; Proteins; Proviruses; RNA; Reporter; Role; Sampling; Sorting - Cell Movement; Spleen; Technology; Tonsil; Viral; Viral reservoir; Virus; Virus Latency; antiretroviral therapy; base; experience; in vivo; inhibitor/antagonist; insight; interest; latent infection; lentivirally transduced; mortality; novel; novel strategies; overexpression; reactivation from latency; research study; transcriptome sequencing; transcriptomics

 DESCRIPTION (provided by applicant): While the advent of antiretroviral therapy (ART) has dramatically reduced the morbidity and mortality associated with HIV infection, viral eradication is not achievable due to the persistence of latently-infected cells during treatment. ART must therefore be taken on a lifelong basis. Accumulating data suggest that HIV- infected individuals often experience persistent immune dysregulation, chronic inflammation, and accelerated aging even in the setting of ART-mediated viral suppression. These realities have created a pronounced interest in developing strategies to eradicate HIV in infected individuals. Identifying host determinants governing viral latency and reservoir size in vivo will be critical in developing effective strategies to clear the latent reservoir and cure HIV infection. Several cell-intrinsic immune factors have been discovered that restrict HIV infection in the absence of antiretroviral drugs. We recently demonstrated that the elevated expression of two particular restriction factors, p21 (inhibitor of HIV transcription) and schlafen 11 (codon usage-based inhibitor of HIV protein synthesis), was strongly associated with decreased HIV latent reservoir size in ART-suppressed individuals. We further demonstrated that select cell-intrinsic immune responses were enhanced in subjects who initiated ART during early versus chronic infection, likely contributing to the reduced reservoir size observed in these individuals. In this R01 project, we will extend our in vivo observations by performing single cell-level analyses, as well as ex vivo and in vitro experiments to characterize the effects of select host restriction factors on the establishment and reversal of HIV latency. In Aim 1, we will apply novel PCR-Activated Cell Sorting (PACS) technology to clinical samples from HIV-infected, ART-suppressed individuals to interrogate single CD4+ T cells for the presence of HIV DNA and RNA. We will then measure the expression of p21 and schlafen 11 in single cells harboring transcriptionally active and inactive proviruses to elucidate the role of these factors in regulating viral latency. In Aim 2, w will use lentiviral transduction to overexpress and silence select cell- intrinsic immune factors i primary cells and human lymphoid aggregate cultures (HLAC). A dual-reporter HIV construct will then be used to examine the effects of these cell-intrinsic immune factors on the establishment of latent HIV infection. Lastly, in Aim 3, we will manipulate p21 and schlafen 11 expression in a cell line model of HIV latency and in HIV-infected primary cells. Cells will then be treated with a comprehensive panel of latency reversal agents, to examine the effects of p21 and schlafen 11 manipulation on the efficacy of these agents and on the dynamics of viral reactivation. This study will yield valuable insights into how cell-intrinsic immunity may serve as a foundation of novel curative strategies for HIV infection.

 描述(申请人提供)：虽然抗逆转录病毒疗法(ART)的出现极大地降低了与艾滋病毒感染相关的发病率和死亡率，但由于在治疗期间潜伏感染的细胞持续存在，因此无法根除病毒。因此，艺术必须终身学习。越来越多的数据表明，即使在ART介导的病毒抑制的情况下，艾滋病毒感染者也经常经历持续的免疫失调、慢性炎症和加速衰老。这些现实使人们对制定根除感染者艾滋病毒的战略产生了明显的兴趣。确定控制体内病毒潜伏期和储存库大小的宿主决定因素将是开发 清除潜伏的蓄水池和治愈艾滋病毒感染的有效策略。已发现在缺乏抗逆转录病毒药物的情况下，几种细胞固有免疫因素可以限制艾滋病毒感染。我们最近证明，在ART抑制的个体中，两种特定的限制因子p21(HIV转录抑制物)和Schlafen 11(基于密码子使用的HIV蛋白质合成抑制物)的表达增加与HIV潜伏储存库大小的减少密切相关。我们进一步证明，在早期与慢性感染期间启动ART的受试者中，选择性细胞内源性免疫反应增强，这可能是在这些个体中观察到的水库大小减少的原因之一。在这个R01项目中，我们将通过执行单细胞水平的分析以及体外和体外实验来扩展我们的体内观察，以表征选定的宿主限制因素对HIV潜伏期的建立和逆转的影响。在目标1中，我们将新的聚合酶链式反应激活的细胞分类(PACS)技术应用于HIV感染者和ART抑制患者的临床样本，以询问单个CD4+T细胞是否存在HIV DNA和RNA。然后，我们将测量p21和schlafen 11在含有转录活性和非活性前病毒的单个细胞中的表达，以阐明这些因素在调节病毒潜伏期中的作用。在目标2中，我们将使用慢病毒转导技术在原代细胞和人类淋巴聚集培养(HLAC)中过表达和沉默选定的细胞固有免疫因子。然后，将使用双报告HIV构建来检查这些细胞固有免疫因子对建立潜在的HIV感染的影响。最后，在目标3中，我们将在HIV潜伏期的细胞系模型和HIV感染的原代细胞中操纵p21和schlafen 11的表达。然后，细胞将使用 全面的潜伏期逆转药物小组，以检查p21和Schlafen 11操作对这些药物的疗效和对病毒重新激活的动力学的影响。这项研究将对细胞固有免疫如何作为艾滋病毒感染的新治疗策略的基础产生有价值的见解。