Replication Potential and Cell-tropism of Residual Plasma HIVs

描述(申请人提供)：尽管使用目前可用的抗逆转录病毒疗法(ART)进行了长期有效的治疗，但患者体内的艾滋病毒复制仍在残留水平上，甚至低于超灵敏临床病毒载量分析的检测下限(每毫升血浆约50个vRNA拷贝)。这种持续性残留病毒血症的临床意义和细胞来源仍不清楚。虽然残留病毒仍然不能在存在ART的情况下确定生产性感染，但尚不清楚它们是否由能够在治疗中断后产生具有感染性和复制能力的病毒颗粒的遗传物质组成。如果是真的，那么重要的是，残留的病毒可能会在低依从性治疗期间潜在地促进患者抗药性的演变，并在停止抗逆转录病毒治疗时引发艾滋病毒的快速传播。为了研究这些可能性，我们准备了这一R03应用程序，目的是获得关于有效治疗患者中存在的残留血浆病毒的复制潜力、辅助受体使用和细胞趋向性的知识。我们将在这个应用中测试两个假设：假设1是大多数RV具有复制能力，并且可以在没有ART的情况下很容易在激活的CD4T细胞中感染和复制，这是我们基于从一名抑制ART患者那里获得的初步数据而制定的。假设-2是关于残留病毒如何潜在地建立一个慢性储存库，并在一种内在稳定和增殖的细胞类型中持续存在，可能是在单核-巨噬细胞系的造血干/祖细胞中。这是根据我们和其他人在该领域发布的数据制定的。为了验证我们的假设和实现我们的总体目标，我们将追求两个具体的目标：在目标1中，我们将使用我们设计的一种新的方法，从残留的血浆vRNA开始分子克隆残留病毒，并测试克隆的病毒在体外感染靶细胞时的感染性和辅助受体的使用。在目标2中，我们将测定克隆的残留病毒在体外对CD4T细胞、巨噬细胞、CD34+干细胞和原代星形胶质细胞的复制能力，并与相应的CD4T细胞原代分离株进行比较，以帮助揭示体内残留病毒的来源。这项拟议研究的基本原理是，一旦我们在体外克隆和表征残留病毒的表型，我们就可以努力了解它们在抗逆转录病毒治疗患者的耐药性、病毒载量波动、持续免疫激活和次优免疫恢复中所起的作用。我们期望拟议的项目将产生有用的试剂和关键的新信息，使我们能够探索抑制ART期间低水平病毒血症的临床意义。

DESCRIPTION (provided by applicant): In spite of prolonged, effective treatment using currently available antiretroviral therapy (ART), HIV replication continues at residual levels in patients, even below the detection limit (~50 vRNA copies per ml of plasma) of ultrasensitive clinical viral load assays. The clinical significance and the cellular source of this persistent residual viremia still remain unclear. Although residual viruses remain incapable of establishing productive infection in presence of ART, it is not clear whether they consist of genetic materials capable of producing infectious and replication-competent virus particles after therapy-interruption. If true, then the significance is that residual viruses could potentially contribute o the evolution of drug-resistance in patients during low-adherence to therapy and also spark rapid spreading of HIV when ART is discontinued. To investigate these possibilities, we have prepared this R03 application with the objectives to obtain knowledge about the replication potential, coreceptor usage and cell-tropism of residual plasma viruses present in patients on effective therapy. We will be testing two hypotheses in this application: the hypothesis 1 is that most RVs are replication-competent, and can infect and replicate readily in activated CD4 T cells in absence of ART, which we have formulated based on our preliminary data obtained from a patient on suppressive ART. The hypothesis-2 is about how residual virus can potentially establish a chronic reservoir and persist in an intrinsically stable and proliferating cell-type, perhaps in haematopoietic stem/progenitor cells of monocyte-macrophage lineage. This is formulated based on our and others published data in the field. To test our hypotheses and accomplish our overall objectives, we will pursue two specific aims: in Aim 1, we will clone residual viruses molecularly starting from residual plasma vRNAs using a novel method that we designed, and test the cloned viruses for their infectivity and coreceptor usage during infection of target cells in vitro. In Aim 2, we will determine replication potential of cloned residual virues in CD4 T cells, macrophages, CD34+ stem cells and primary astrocytes in vitro and compare with that of corresponding primary virus isolates from CD4 T cells, in order to help reveal the residual viruses' source in vivo. The rationale for the proposed research is that once we clone and characterize residual viruses phenotypically in vitro, we can put efforts to understand what role they play in drug resistance, viral load blips, persistent immune activation and sub-optimal immune recovery in patients on ART. It is our expectation that the proposed project will generate useful reagents and critical new information that will allow us to probe the clinical significance of low-level viremia during suppressive ART.