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

描述（由申请人提供）：实现治愈HIV-1的一个主要障碍是存在持续性病毒感染的储库，其中静息记忆CD 4 + T细胞是最大和最好的研究。在这些细胞中，HIV-1作为抗逆转录病毒治疗（ART）难治的潜伏前病毒存在，并且免疫监视不可见，可以重新唤醒并传播感染。近年来，一直在推动开发针对潜伏感染的CD 4 + T细胞的疗法。“休克和杀死”方法试图用不会引起整体T细胞活化的“抗潜伏”药物冲洗潜伏的前病毒，并在ART存在的情况下防止冲洗的病毒传播感染。然而，最近的证据表明，这种做法可能是不够的。我们的长期目标是开发直接靶向和耗尽潜伏感染的CD 4 + T细胞而不重新激活前病毒的策略。我们的中心假设是，表面标志物差异表达的潜伏感染的CD 4 + T细胞可以用来实现这一目标。在NIH资助的资助下，我们分析了用我们的体外潜伏模型产生的潜伏感染和未感染的CD 4 + T细胞的转录组，并确定了33个差异表达的表面标志物。我们还发现，在微阵列筛选中鉴定的受体之一--CD 2--在潜伏期内以更高的水平表达。 本申请提出进一步进行这些研究，目的是建立一组细胞表面标志物，当组合使用时，其将允许以高特异性潜在地鉴定 体内感染静息记忆CD 4 + T细胞。因此，在特定目标1中，我们建议使用我们的体外潜伏期模型来验证微阵列研究中差异表达的所有表面标志物，并了解为什么潜伏感染的细胞呈现特定的表达谱。具体目标2提出验证在先前具体目标中鉴定的标志物，并产生一组表面受体，以高特异性确定HIV-1患者外周血和淋巴结中潜伏感染的CD 4 + T细胞。这些研究的预期结果是建立一组细胞表面标志物，其组合将在体内以高特异性定义潜伏感染的静息记忆CD 4 + T细胞。这些标记物可用于监测临床环境中潜伏性储库的大小，以在实验室环境中富集潜伏感染的CD 4 + T细胞，并且可能作为治疗靶点以耗尽潜伏性储库而不重新激活前病毒。这项工作是与VGTI-Florida的Nicolas Chomont合作进行的，他开发了超灵敏的技术来测量临床样本中整合的HIV-1前病毒，以及离体再激活后的病毒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.