Nanotrap particle-based assay to quantify HIV-1 in latently-infected T cells

基于 Nanotrap 颗粒的测定法可量化潜伏感染 T 细胞中的 HIV-1

• 批准号: 8874895
• 负责人: Fatah Kashanchi
• 金额: $ 22.29万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-18 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8874895
• 关键词: Adverse effects; Agonist; Allogenic; Antigens; Binding; Biological Assay; Biological Markers; Blood; CD28 gene; CD3 Antigens; CD34 gene; CD4 Positive T Lymphocytes; Cardiovascular Diseases; Cell Count; Cell model; Cells; Cervical; Collaborations; Complex; DNA; Data; Detection; Diagnosis; Diagnostic; Enzyme-Linked Immunosorbent Assay; Fluorescence; Genetic Transcription; Genome; Goals; HIV-1; Hematopoietic stem cells; Highly Active Antiretroviral Therapy; Hydrogels; Individual; Infection; Interruption; Ionomycin; Knowledge; Life; Life Expectancy; Liquid substance; Measurement; Memory; Messenger RNA; Methods; Modeling; Neurocognitive; Outcome; Patients; Peptide Nucleic Acids; Persons; Pharmaceutical Preparations; Phase; Plasma; Population; Proteins; Proviruses; RNA; Reporter; Residual state; Resources; Rest; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Serum; Shock; Specimen; T-Lymphocyte; Technology; Testing; Untranslated RNA; Vaginal Douching; Viral; Viral Load result; Virion; Virus; Virus Diseases; Work; base; design; extracellular; improved; innovation; irradiation; killings; latent infection; memory CD4 T lymphocyte; mortality; nanoparticle; novel; particle

DESCRIPTION (provided by applicant): The use of highly active antiretroviral therapy (HAART) against HIV-1 for the last two decades reduced mortality of the patients via extension of non-symptomatic phase of infection. The current drug compositions are not able to completely eliminate virus, since HIV-1 is capable of maintaining latent infection in stable reservoirs such as resting CD4+ T cells, naive T cells and CD34+ multipotent hematopoietic stem cells. Thus, interruption of the therapy leads to the rapid increase of viral population, whereas a long combinational ART treatment has negative side effects including neurocognitive abnormalities and cardiovascular diseases. Eradication of latent HIV-1 reservoirs could eliminate infection and, therefore, stop ART treatment of the patients. Quantitative assessment of the pool of resting CD4+ memory T cells in HAART-treated patients is essential for determination of strategies to eradicate the latent HIV-1 infection. However, a high variability in the sensitivity of existing assays such as viral outgrowth assays (VOA) and quantitative PCR-based analyses points for a pressing need to develop unified method to rapidly and precisely quantify latent HIV-1 reservoir. In this application, we propose to develop a highly-efficient method to quantitatively assess latent HIV-1 reservoirs in non-viremic HIV-1 patients via the combination of (i) RCR-based detection of circulating viral TAR RNA to diagnose the reservoir and (ii) improve VOA followed by the analysis of infectivity of Nanotrap particle-captured HIV-1 to quantify the functionally competent proviral copies. To pursue these aims we propose to use innovative Nanotrap technology originally developed to concentrate and preserve low abundance proteins, peptides, nucleic acids and whole viruses from complex biofluid matrices for diagnostic purposes. The Nanotrap hydrogel particles of about 800 nm in size will be used to capture and concentrate both the exosome-packaged circulating TAR RNA in patients' plasma samples (aim 1) and progeny virus produced by reactivated CD4+ T cells from TAR-positive patients in VOA to subsequently use nanoparticle-bound virus for infection of the reporter cells in order to quantify initial replication competent proviral copies (aim 2). These aims are supported by recent data from our group which indicated that 1) serum samples from aviremic HIV-1 patients contain detectable amounts of non-coding HIV-1 TAR RNA, suggesting the presence of transcriptionally-competent provirus in cells; 2) the extracellular TAR RNA is present in exosomes and can be effectively concentrated from serum using nanoparticles; and 3) the effective concentration of HIV-1 virions from supernatants by nanoparticles allows for enrichment of fully infectious HIV-1 that can be further quantified using functional reporter assay. We expect that results of the proposed study will allow rapid assessment of latent HIV-1 reservoirs to determine strategies for reactivation of the latent HIV-1 reservoir and elimination o reinfection.

描述（由申请人提供）：过去二十年使用针对 HIV-1 的高效抗逆转录病毒疗法（HAART）通过延长感染的无症状阶段降低了患者的死亡率。目前的药物组合物不能完全消除病毒，因为HIV-1能够在稳定的储存库中维持潜伏感染，例如静息CD4+T细胞、初始T细胞和CD34+多能造血干细胞。因此，治疗的中断会导致病毒数量迅速增加，而长期的联合 ART 治疗会产生负面副作用，包括神经认知异常和心血管疾病。根除潜伏的 HIV-1 储存库可以消除感染，从而停止对患者的 ART 治疗。对接受 HAART 治疗的患者的静息 CD4+ 记忆 T 细胞库进行定量评估对于确定根除潜伏 HIV-1 感染的策略至关重要。然而，高变异性 现有检测方法（如病毒生长检测 (VOA) 和基于 PCR 的定量分析）的敏感性表明，迫切需要开发统一的方法来快速、精确地量化潜在的 HIV-1 病毒库。在此应用中，我们建议开发一种高效的方法，通过结合以下方法来定量评估非病毒血症 HIV-1 患者中的潜在 HIV-1 储存库：(i) 基于 RCR 的循环病毒 TAR RNA 检测以诊断储存库和(ii) 改进 VOA，然后分析 Nanotrap 颗粒捕获的 HIV-1 的感染性，以量化功能上合格的原病毒副本。为了实现这些目标，我们建议使用创新的 Nanotrap 技术，该技术最初是为了浓缩和保存复杂生物流体基质中的低丰度蛋白质、肽、核酸和整个病毒而开发的，用于诊断目的。尺寸约为 800 nm 的 Nanotrap 水凝胶颗粒将用于捕获和浓缩患者血浆样本中外泌体包装的循环 TAR RNA（目标 1）以及美国之音 TAR 阳性患者重新激活的 CD4+ T 细胞产生的子代病毒随后使用纳米颗粒结合的病毒感染报告细胞，以量化初始复制能力的原病毒副本（目标 2）。这些目标得到了我们小组最近数据的支持，该数据表明：1）来自无病毒血症 HIV-1 患者的血清样本含有可检测量的非编码 HIV-1 TAR RNA，表明细胞中存在具有转录能力的原病毒； 2) 胞外 TAR RNA 存在于外泌体中，可以使用纳米颗粒从血清中有效浓缩； 3) 纳米粒子对上清液中 HIV-1 病毒粒子的有效浓度可以富集完全感染性的 HIV-1，并可使用功能性报告分析进一步定量。我们预计拟议研究的结果将允许快速评估潜在的 HIV-1 储存库，以确定重新激活潜在的 HIV-1 储存库和消除或再感染的策略。

项目成果

The Role of Exosomal VP40 in Ebola Virus Disease.

外泌体 VP40 在埃博拉病毒疾病中的作用。

• 发表时间: 2017-04
• 影响因子: 3.1
• 作者: Pleet, Michelle L;DeMarino, Catherine;Lepene, Benjamin;Aman, M Javad;Kashanchi, Fatah
• 通讯作者: Kashanchi, Fatah

Enhanced detection of respiratory pathogens with nanotrap particles.

使用纳米颗粒增强呼吸道病原体的检测。

• 发表时间: 2016-10-02
• 影响因子: 5.2
• 作者: Shafagati, Nazly;Fite, Katherine;Patanarut, Alexis;Baer, Alan;Pinkham, Chelsea;An, Soyeon;Foote, Benjamin;Lepene, Benjamin;Kehn
• 通讯作者: Kehn