Cell Surface Marker Combinations to Identify Latently Infected CD4+ Cells In Vivo

描述(由申请人提供)：治愈HIV-1的一个主要障碍是存在持续病毒感染的蓄水池，其中静止记忆的CD4+T细胞是最大的，也是研究得最好的。在这些细胞中，HIV-1是一种对抗逆转录病毒治疗(ART)无效的潜伏前病毒，免疫监视是看不见的，免疫监视可以重新唤醒并传播感染。近年来，针对潜伏感染的CD4+T细胞的治疗方法得到了推动。“休克并杀死”方法寻求用不会导致全球T细胞激活的“反潜伏期”药物冲洗潜伏的前病毒，并在存在ART的情况下防止冲洗的病毒传播感染。然而，最近的证据表明，这种方法可能是不够的。我们的长期目标是制定策略，在不重新激活前病毒的情况下，直接靶向并耗尽潜伏感染的CD4+T细胞。我们的中心假设是，潜伏感染的CD4+T细胞上差异表达的表面标志可以用来实现这一目标。在NIH基金的支持下，我们利用我们的体外潜伏模型生成了潜伏感染和未感染的CD4+T细胞的转录组，并鉴定了33个差异表达的表面标记。我们还发现，在微阵列屏幕上发现的受体之一-CD2-潜伏期表达水平较高 抑制ART下HIV-1感染者静息记忆CD4+T细胞的感染本申请建议进一步进行这些研究，目的是建立一组细胞表面标记，当它们组合使用时，将允许以高特异性潜伏地识别 体内感染静息记忆CD4+T细胞。因此，在特定的目标1中，我们建议使用我们的体外潜伏期模型来验证在微阵列研究中差异表达的所有表面标记，并理解为什么潜伏感染的细胞呈现该特定的表达谱。特异性目标2建议验证前一个特异性目标中确定的标志物，并产生一组表面受体，以高特异性识别HIV-1患者外周血和淋巴中潜伏感染的CD4+T细胞。这些研究的预期结果是建立一组细胞表面标志物，结合起来将定义体内潜伏感染的静止记忆CD4+T细胞。这些标志物可用于在临床环境中监测潜伏库的大小，在实验室环境中丰富潜伏的CD4+T细胞，并可能作为一种治疗靶点，在不重新激活前病毒的情况下耗尽潜伏库。这项工作是与VGTI-佛罗里达州的Nicolas Chomont合作开展的，他开发了超灵敏技术来测量临床样本中整合的HIV-1前病毒和体外重新激活后的病毒RNA。

DESCRIPTION (provided by applicant): A major hurdle in achieving a cure for HIV-1 is the existence of reservoirs of persistent viral infection, with resting memory CD4+ T cells being the largest and best studied. In these cells, HIV-1 rests as a latent provirus refractory to antiretroviral therapy (ART) and invisible to immune surveillance, which can be reawakened and spread the infection. In recent years there has been a push to develop therapies targeting latently infected CD4+ T cells. The "shock and kill" approach seeks to flush latent provirus with "anti-latency" drugs that do not cause global T cell activation, and in the presence of ART to prevent the flushed virus from spreading the infection. However, recent evidence suggests that this approach may be inadequate. Our long-term goal is to develop strategies to directly target and deplete latently infected CD4+ T cells without reactivating the provirus. Our central hypothesis is that surface markers differentially expressed on latently infected CD4+ T cells may be used to achieve that goal. With the support of an NIH-funded grant, we profiled the transcriptome of latently infected and uninfected CD4+ T cells generated with our in vitro latency model, and we identified 33 differentially expressed surface markers. We also found that one of the receptors identified in the microarray screen - CD2 - is expressed at higher levels on latently infected resting memory CD4+ T cells of HIV-1 subjects under suppressive ART. The present application proposes to further pursue these studies with the aim of establishing a panel of cell surface markers that when used in combination will allow to identify with high specificity latently infected resting memory CD4+ T cells in vivo. Thus, in Specific Aim 1 we propose to use our in vitro latency model to validate all the surface markers differentially expressed in the microarray study, and to understand why latently infected cells present that specific expression profile. Specific Aim 2 proposes to validate the markers identified in the previous specific aim, and to generate a panel of surface receptors to define with high specificity latently infected CD4+ T cells in peripheral blood and lymph nodes of HIV-1 patients. The expected outcome of these studies is the establishment of a panel of cell surface markers that in combination will define with high specificity latently infected resting memory CD4+ T cells in vivo. These markers can be used to monitor the size of the latent reservoir in the clinical setting, to enrich latently infcted CD4+ T cells in the laboratory setting, and possibly as a therapeutic target to deplete the latent reservoir without reactivating the provirus. This work is a partnership with Nicolas Chomont at VGTI-Florida, who developed ultra-sensitive techniques to measure integrated HIV-1 provirus in clinical samples, and viral RNA after ex vivo reactivation.