Replication Potential and Cell-tropism of Residual Plasma HIVs

描述（由申请人提供）：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HIV复制仍保持在残留水平，甚至低于超灵敏临床病毒载量测定的检测极限（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。尽管残留病毒在抗逆转录病毒治疗下仍不能建立生产性感染，但尚不清楚它们是否由能够在治疗中断后产生传染性和复制能力强的病毒颗粒的遗传物质组成。如果这是真的，那么重要的是，残留的病毒可能会在低依从性治疗期间导致患者耐药性的演变，并在停止抗逆转录病毒治疗时引发艾滋病毒的迅速传播。为了研究这些可能性，我们准备了这个R03应用程序，目的是获得有关在有效治疗的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。我们将在本申请中测试两个假设：假设1是大多数rv具有复制能力，并且在没有抗逆转录病毒治疗的情况下可以很容易地感染和复制激活的CD4 T细胞，这是我们根据从一位接受抑制性抗逆转录病毒治疗的患者那里获得的初步数据制定的。假设2是关于残留病毒如何潜在地建立一个慢性储存库，并在本质上稳定和增殖的细胞类型中持续存在，可能是在单核-巨噬细胞谱系的造血干细胞/祖细胞中。这是根据我们和其他人在该领域发表的数据制定的。为了验证我们的假设并实现我们的总体目标，我们将追求两个具体目标：在Aim 1中，我们将使用我们设计的新方法从残留的血浆vrna开始分子克隆残留病毒，并在体外测试克隆病毒在靶细胞感染期间的感染性和辅助受体的使用。在Aim 2中，我们将在体外测定克隆残留病毒在CD4 T细胞、巨噬细胞、CD34+干细胞和原代星形胶质细胞中的复制潜力，并与从CD4 T细胞中分离的相应原代病毒进行比较，以帮助揭示体内残留病毒的来源。这项研究的基本原理是，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、病毒载量突变、持续免疫激活和次优免疫恢复中所起的作用。我们期望该项目将产生有用的试剂和重要的新信息，使我们能够探索在抑制性抗逆转录病毒治疗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. PUBLIC HEALTH RELEVANCE: The currently available antiretroviral therapy (ART) for HIV can reduce viral loads in patients to very low-levels (known as 'residual viremia') which remain undetectable even by the ultrasensitive viral load tests used in clinic, but are usually detectable by more sensitive laboratory techniques. The studies proposed in this application are aimed at understanding of whether residual viremia has any clinical significance, by cloning residual viruses from patients using molecular techniques and testing them for various biological characteristics in laboratory tissue cultures. These studies should provide new knowledge that would be useful in designing new therapeutic strategies in future for complete inhibition of low-level HIV replication in patients on therapy.