A New Insight into HIV-1 Latency Through a Novel in Vitro System

描述(申请人提供)：艾滋病领域面临的一个主要挑战是根除人类免疫缺陷病毒1型(HIV-1)。这一努力将需要开发新的治疗方法，专门针对驻留在感染细胞内的处于非生产性感染状态的前病毒。反过来，实现这一目标的进展将需要我们对HIV-1潜伏期的生物学知识和理解有一个巨大的飞跃。然而，目前所有研究HIV-1潜伏期的系统仍然严重不足以完成这项任务。这项应用寻求使用一种新的体外模型来促进我们对潜伏感染细胞的独特特征的了解。病毒潜伏期是临床实践中的一个重要问题，也是根除病毒的主要障碍。潜伏感染细胞池是在初次感染期间建立的，并持续整个疾病过程。作为一种休眠前病毒，HIV-1对细胞和体液免疫反应是看不见的，对抗逆转录病毒药物也是耐药的。潜伏的储存库保存了耐药病毒，这些病毒可能在治疗不充分或依从性差的情况下重新出现，并是在结构化治疗中断后观察到的反弹病毒血症的原因。病毒潜伏期也可能通过在病毒传播和辅助受体开关中发挥作用，影响HIV-1感染和疾病进展的自然历史。虽然嗜CCR5的HIV-1毒株在初次感染期间负责病毒传播，并在整个疾病过程中持续存在，但在大约一半的病例中出现的嗜CXCR4毒株与更快的疾病进展有关。假设这两个变种可能表现出不同的建立和重新出现潜伏期的倾向，这可能有助于解释在感染HIV-1期间观察到的辅助受体使用的变化。到目前为止，缺乏有效和可靠的细胞系统来研究HIV-1潜伏期，阻碍了对其生物学的了解和有针对性的治疗策略的发展。我们最近描述了一种新的体外培养系统来研究HIV-1潜伏期的诱导、维持和重新激活。我们的细胞系统概括了一次和二次抗原驱动的免疫反应事件，其中CD4+T细胞被树突状细胞和抗原激活，在体外感染HIV-1，然后在白介素7存在的情况下通过休止期恢复静止。我们已经证明，这些体外产生的潜伏感染细胞缺乏激活标记的表达；不进行细胞增殖，也不维持病毒复制。最后，我们已经证明，在次级抗原刺激后，所有这些活动都会迅速恢复。我们的系统能够回答关键问题，并为潜伏感染细胞的性质提供新的见解。因此，本申请建议确定通过荧光激活细胞分选分离的体外产生的潜伏感染细胞的基因表达谱。此外，我们建议检验CCR5和CXCR4嗜好的HIV-1毒株表现出不同的建立和重新出现潜伏期的倾向的假设。公共卫生相关性：人类免疫缺陷病毒1型(HIV-1)确立了一种称为潜伏期的非生产性感染状态。作为一种休眠的前病毒，HIV-1对免疫反应是看不见的，并且对抗逆转录病毒药物具有耐药性。因此，潜伏期是临床实践中的一个重要问题。根除HIV-1将需要开发针对潜伏感染细胞的新治疗策略。到目前为止，由于对HIV-1潜伏期的了解有限，在这一方向上的进展受到了阻碍。在这一应用中，我们建议利用我们最近开发的体外细胞系统来获得对潜伏感染细胞的独特特征的新见解，以及调节HIV-1潜伏期的诱导、维持和重新激活的机制。我们希望从这些研究中获得的新知识可能会导致针对潜伏感染细胞池的新疗法的设计，并最终根除病毒。

DESCRIPTION (provided by applicant): A major challenge facing the AIDS field is the eradication of the human immunodeficiency virus type-1 (HIV-1). This effort will require the development of new therapies that specifically target the provirus residing within infected cells in a status of non-productive infection. In turn, advances toward that goal will require a quantum leap in our knowledge and understanding of the biology of HIV-1 latency. However, all the current systems to study HIV-1 latency remain severely inadequate for that task. This application seeks to employ a novel in vitro model to advance our knowledge about the distinctive features of latently infected cells. Viral latency is an important problem in clinical practice, and represents a major obstacle to virus eradication. The pool of latently infected cells is established during primary infection and persists throughout the course of the disease. As a dormant provirus, HIV-1 is invisible to cellular and humoral immune responses, and is refractory to anti-retroviral drugs. The latent reservoir archives drug-resistant viruses that can re-emerge in the context of inadequate therapy or poor compliance, and is responsible for the rebound viremia observed following structured therapy interruption. Viral latency may also affect the natural history of HIV-1 infection and disease progression by playing a role in virus transmission and coreceptor switch. Whereas CCR5-tropic HIV-1 strains are responsible for virus transmission during primary infection and persist throughout disease, emergence of CXCR4-tropic strains that occurs in about half of all cases is associated with faster disease progression. The hypothesis that the two variants might display different propensities to establish and re- emerge from latency could help explain changes in coreceptor usage observed during HIV-1 infection. So far, the lack of a valid and reliable cellular system to study HIV-1 latency has hindered progress toward the understanding of its biology and the development of targeted therapeutic strategies. We have recently described a new in vitro culture system to investigate the induction, maintenance and reactivation of HIV-1 latency. Our cellular system recapitulates the events of primary and secondary antigen-driven immune response in which CD4+ T cells are activated with dendritic cells and antigen, infected in vitro with HIV-1, and then brought back to quiescence through a resting phase in the presence of interleukin-7. We have demonstrated that these in vitro-generated latently infected cells lack expression of activation markers; do not undergo cellular proliferation and do not sustain viral replication. Finally, we have shown that all these activities resume promptly following secondary antigen stimulation. Our system is amenable to answer key questions and to provide new insights into the nature of latently infected cells. Therefore, the present application proposes to determine the gene expression profile of in vitro- generated latently infected cells isolated by fluorescence activated cell sorting. Moreover, we propose to test the hypothesis that CCR5- and CXCR4-tropic HIV-1 strains show different propensities to establish and re- emerge from latency. PUBLIC HEALTH RELEVANCE: The human immunodeficiency virus type-1 (HIV-1) establishes a status of non-productive infection known as latency. As a dormant provirus, HIV-1 is invisible to immune responses, and resistant to anti-retroviral drugs. Thus, latency is an important problem in clinical practice. Eradication of HIV-1 will require the development of new therapeutic strategies that target specifically latently infected cells. So far, progress in that direction has been hindered by limited knowledge of HIV-1 latency. In this application we propose to make use of an in vitro cellular system we have developed recently to gain new insight into the distinctive features of latently infected cells, and the mechanisms that regulate induction, maintenance and reactivation of HIV-1 latency. The new knowledge we expect to draw from these studies could lead to the design of new therapies aimed at the pool of latently infected cells, and to virus eradication.