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

项目摘要 潜伏感染的CD4+T细胞是根除HIV-1的主要障碍。 人们正在开发许多策略来消灭这些细胞，但该领域缺乏简单的 一种可以筛选这些细胞的灵敏检测方法。基于聚合酶链式反应的检测不能区分 最近的研究表明，复制能力强的病毒与有缺陷的病毒之间的关系 目前最先进的培养方法只测量了一小部分 存在复制能力强的病毒。在这项建议中，我们的目标是开发一种灵敏的分析方法 能够选择性地从潜伏感染的病毒中扩增具有复制能力的病毒 CD4+T细胞。这样的化验可以为决定是否停止生产提供信息 患者在进行治疗干预后进行CART。这种化验方法的必要性是 最近关于两名波士顿患者接受治疗的报告突出了这一点 异基因干细胞移植治疗恶性肿瘤。聚合酶链式反应未检测到HIV DNA 在任何一位患者中，都有多达2亿个PBMC(0.07个拷贝/106个PBMC)，但 停用CART后，两名患者的病毒血症迅速反弹。这 结果是不受欢迎的，因为它否定了拥有更小的HIV-1的理论优势 水库。开发一种简单但敏感的检测方法，可以筛查非常大的 CD4+T细胞的数量可能有助于防止类似情况的发生 发生在未来的。 我们将使用我们开发的优化方法来筛选患者的CD4+T细胞 在初次感染期间接受抗逆转录病毒治疗的患者与潜伏期 在我们的检测中测量的感染细胞到病毒反弹发生所需的时间 当艺术在这些科目中停止的时候。 这项工作对于制定根除艾滋病毒-1战略具有重要意义。 因为化验将帮助做出是否停止的决定 抗逆转录病毒治疗。

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.