Optimization of high throughput viral outgrowth assays for the detection of HIV-1 reservoirs

项目摘要 潜伏感染的CD 4 + T细胞是HIV-1根除的主要障碍。 许多策略正在开发，以消除这些细胞，但该领域缺乏一个简单的 灵敏的检测方法来筛选这些细胞。基于PCR的检测无法区分 有复制能力的病毒和有缺陷的病毒之间的关系，最近的研究表明， 目前最先进的培养物测定法仅测量了一小部分， 有复制能力的病毒在这个提议中，我们的目标是开发一种灵敏的检测方法， 其能够从潜伏感染的病毒中选择性地扩增具有复制能力的病毒， CD 4 + T细胞。这样的分析可以告知是否停止的决定 进行治疗干预后，患者中的cART。对这种测定的需要是 最近报告的2名“波士顿患者”接受了 异基因干细胞移植治疗恶性肿瘤PCR检测均未检出HIV DNA 在任一患者中，来自多达2亿个PBMC（<0.07拷贝/106个PBMC），但 在停止cART后，两名患者的病毒血症迅速反弹。这 结果是不可取的，因为它否定了具有较小HIV-1的理论优势， 水库开发一种简单但灵敏的检测方法，可以筛选非常大的 CD 4 + T细胞的数量可能有助于防止类似情况的发生， 发生在未来。 我们将使用我们开发的优化检测来筛选患者的CD 4 + T细胞 在原发感染期间接受ART治疗的患者， 在我们的测定中测量的感染细胞到病毒反弹发生所需的时间 当这些受试者停止ART时。 这项工作对于制定消灭HIV-1的战略具有重要意义 因为该测定将有助于决定是否停止 抗逆转录病毒疗法

Project Summary Latently infected CD4+ T cells represent the major barrier to HIV-1 eradication. Many strategies are being developed to eliminate these cells, but the field lacks a simple sensitive assay that can screen for these cells. PCR based assays cannot distinguish between replication-competent versus defective virus and recent studies have shown that the current state of the art culture assay measures only a small fraction of the replication-competent virus present. In this proposal we aim to develop a sensitive assay that is capable of selectively amplifying replication-competent virus from latently infected CD4+ T cells. Such an assay could inform the decision of whether or not to discontinue cART in patients after a therapeutic intervention is made. The need for such an assay is highlighted by the recent report of the 2 “Boston patients” who were treated with allogeneic stem cell transplantation for malignancies. No HIV DNA was detected by PCR in either patient from as many as 200 million PBMCs (<0.07 copies/106 PBMCs), but after cART was discontinued there was a rapid rebound in viremia in both patients. This outcome is undesirable is it negates the theoretical advantages to having smaller HIV-1 reservoirs. The development of a simple but sensitive assay that can screen a very large number of CD4+ T cells may help prevent the occurrence of a similar scenario from occurring in the future. We will use the optimized assay we develop to screen CD4+ T cells from patients who were treated with ART during primary infection and correlate the number of latently infected cells as measured in our assay to the time it took for viral rebound to occur when ART was discontinued in these subjects. This work will be important for the development of strategies for HIV-1 eradication since the assay will help inform the decision of whether or not to discontinue antiretroviral therapy.