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

描述（由申请方提供）：过去二十年来，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期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能够在稳定的储库如静息CD 4 + T细胞、幼稚T细胞和CD 34+多能造血干细胞中维持潜伏感染。因此，治疗的中断导致病毒群体的快速增加，而长期的联合ART治疗具有负面副作用，包括神经认知异常和心血管疾病。根除潜伏的HIV-1宿主可以消除感染，因此可以停止对患者的ART治疗。定量评估HAART治疗患者的静息CD 4+记忆T细胞库对于确定消除潜伏性HIV-1感染的策略至关重要。然而， 现有检测方法如病毒生长检测（VOA）和基于定量PCR的分析的灵敏度表明迫切需要开发统一的方法来快速和精确地定量潜伏的HIV-1储库。在本申请中，我们建议开发一种高效的方法，通过以下两种方法的组合来定量评估非病毒血症HIV-1患者中的潜伏HIV-1储库：（i）基于RCR的循环病毒TAR RNA检测，以诊断储库;（ii）改善VOA，然后分析Nanotrap颗粒捕获的HIV-1的感染性，以定量功能活性前病毒拷贝。为了实现这些目标，我们建议使用创新的Nanotrap技术，最初开发用于从复杂的生物流体基质中浓缩和保存低丰度蛋白质，肽，核酸和完整病毒，用于诊断目的。尺寸为约800 nm的Nanotrap水凝胶颗粒将用于捕获和浓缩患者血浆样品中的外泌体包装的循环TAR RNA（目的1）和由来自VOA中的TAR阳性患者的再活化的CD 4 + T细胞产生的子代病毒，以随后使用纳米颗粒结合的病毒感染报道细胞，从而定量初始复制能力的前病毒拷贝（目的2）。这些目的得到了我们小组最近数据的支持，这些数据表明：1）来自病毒血症HIV-1患者的血清样品含有可检测量的非编码HIV-1 TAR RNA，表明细胞中存在有转录能力的前病毒; 2）细胞外TAR RNA存在于外来体中，并且可以使用纳米颗粒从血清中有效地浓缩;和3）通过纳米颗粒从上清液中有效浓缩HIV-1病毒粒子允许富集完全感染性的HIV-1，其可以使用功能性报道分析进一步定量。我们希望，拟议的研究结果将允许快速评估潜伏的HIV-1水库，以确定策略，重新激活的潜伏的HIV-1水库和消除再感染。

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.