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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：虽然抗逆转录病毒疗法（ART）的冒险大大降低了与HIV感染相关的发病率和死亡率，但由于治疗过程中潜在受感染的细胞的持续存在，无法实现病毒辐射。因此，艺术必须终生进行。累积数据表明，即使在艺术介导的病毒抑制的情况下，感染HIV的个体也经常会出现持续的免疫失调，慢性感染和加速衰老。这些现实引起了人们对在受感染个体中开发放射性艾滋病毒的策略的明显兴趣。识别主机确定管理病毒潜伏期和体内储层大小对于开发至关重要清除潜在储层和治愈艾滋病毒感染的有效策略。已经发现几种细胞中性免疫因子在没有抗逆转录病毒药物的情况下限制了HIV感染。我们最近证明，两个特定限制因子的表达升高，P21（HIV转录的抑制剂）和Schlafen 11（基于密码子使用的HIV蛋白合成抑制剂）与改善ART抑制个体中的HIV潜伏储层大小密切相关。我们进一步证明，在早期和慢性感染期间开始ART的受试者中，某些细胞中性免疫反应得到了增强，这可能导致这些个体中观察到的参与者大小的降低。在此R01项目中，我们将通过执行单细胞级分析以及离体和体外实验来扩展体内观察结果，以表征某些宿主限制因子对HIV潜伏期的建立和逆转的影响。在AIM 1中，我们将将新型的PCR激活细胞分选（PACS）技术应用于来自HIV感染的，抑制Art抑制的个体的临床样本，以询问单个CD4+ T细胞，以在HIV DNA和RNA中存在。然后，我们将测量具有转录活性和无活性病毒的单个细胞中P21和Schlafen 11的表达，以阐明这些因素在控制病毒潜伏期中的作用。在AIM 2中，W将使用慢病毒转移来表达过表达和沉默选择细胞中性免疫因子I主细胞和人淋巴骨料培养物（HLAC）。然后将使用双重重复蛋白HIV构建体检查这些细胞内部免疫因素对建立潜在HIV感染的影响。最后，在AIM 3中，我们将在HIV潜伏期和HIV感染的原代细胞中操纵P21和Schlafen 11的表达。然后将用A 综合延迟逆转剂的综合面板，以检查p21和Schlafen 11操纵对这些药物和病毒重新激活动力学的有效性的影响。这项研究将对细胞中性免疫如何成为艾滋病毒感染的新型治愈策略的基础产生宝贵的见解。