Replication Potential and Cell-tropism of Residual Plasma HIVs

残留血浆 HIV 的复制潜力和细胞向性

• 批准号: 8410803
• 负责人: Gautam K. Sahu
• 金额: $ 8.55万
• 依托单位: ROGER WILLIAMS HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8410803
• 关键词: Adherence; Anti-Retroviral Agents; Antiretroviral resistance; Astrocytes; Biologic Characteristic; Biological Assay; CD34 gene; CD4 Positive T Lymphocytes; Cells; Chronic; Clinic; Clinical; Cloning; Data; Detection; Drug resistance; Evolution; Funding; Future; Genetic Materials; Genome; Goals; HIV; Hematopoietic; Immune; In Vitro; Infection; Interruption; Knowledge; Laboratories; Length; Long-Term Effects; Methods; Minor; Molecular; Molecular Cloning; Mutation; Nature; Patients; Pharmaceutical Preparations; Plasma; Play; Production; Proliferating; Publishing; Reagent; Recovery; Relative (related person); Research; Research Project Grants; Residual state; Role; Satellite Viruses; Source; Stem cells; T-Lymphocyte; Techniques; Testing; Tropism; Umbilical Cord Blood; Variant; Viral; Viral Genome; Viral Load result; Viremia; Virion; Virus; Virus Replication; Work; antiretroviral therapy; base; cell type; clinically significant; design; effective therapy; expectation; experience; immune activation; in vivo; interest; macrophage; monocyte; novel; novel therapeutics; particle; stem; tissue culture; viral DNA

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.

描述（由申请人提供）：尽管使用当前可用的抗逆转录病毒疗法（ART）进行了长时间，有效的治疗，但患者的艾滋病毒复制仍在残留水平下继续，甚至低于检测限度（每毫升血浆的〜50 VRNA拷贝）超敏感的临床病毒载量。该持续性残留病毒血症的临床意义和细胞来源仍不清楚。尽管残留病毒仍然无法在ART存在下建立生产性感染，但尚不清楚它们是否包括能够在治疗中断后能够产生感染和复制能力的病毒颗粒的遗传材料。如果是真的，那么重要的是，残留病毒可能有可能导致患者在低遵守治疗期间的药物抗性进化，并在停止ART时也会在ART停止时迅速传播HIV。为了调查这些可能性，我们已经准备了此R03应用，并具有目标，以获取有关患者在有效治疗中存在的残留等离子体病毒的复制潜力，共纤维受体使用和细胞热量的知识。我们将在此应用中检验两个假设：假设1是大多数RV是复制能力的，并且可以在没有ART的情况下在活化的CD4 T细胞中轻易感染和复制，我们已经根据从抑制性ART的患者中获得的初步数据进行了制定。该假设2是关于残留病毒如何可能建立慢性储层并持续存在于本质上稳定且增殖的细胞类型中，也许是在单核细胞巨噬细胞谱系的造血茎/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。为了检验我们的假设并实现总体目标，我们将追求两个具体的目的：在AIM 1中，我们将使用我们设计的新方法从残留的血浆VRNA开始克隆残留病毒，并测试克隆的病毒，并在靶细胞感染的情况下为其感染率和corectoptor使用。在AIM 2中，我们将确定在CD4 T细胞，巨噬细胞，CD34+干细胞和原代星形胶质细胞中克隆的残留病毒的复制潜力，并与来自CD4 T细胞的相应原发病毒分离株进行比较，以帮助揭示残基在VIVO中的残留病毒。拟议的研究的理由是，一旦我们克隆并在体外表现出残留病毒，我们就可以努力了解它们在耐药性，病毒载荷，持续性免疫激活和在ART中患者中的免疫恢复的作用。我们期望拟议的项目将生成有用的试剂和关键的新信息，这将使我们能够在抑制性艺术期间探究低级病毒血症的临床意义。 公共卫生相关性：目前可用于HIV的抗逆转录病毒疗法（ART）可以将患者的病毒负荷降低至非常低的水平（称为“残留病毒血症”），即使在临床中使用的超敏感病毒负荷测试也无法检测到，但通常是可检测的，但通常是 通过更敏感的实验室技术。该应用中提出的研究旨在了解残留病毒血症是否具有任何临床意义，方法是使用分子技术从患者中克隆残留病毒，并对它们在实验室组织培养物中的各种生物学特征进行测试。这些研究应提供新的知识，这些知识将在未来设计新的治疗策略中有用，以完全抑制治疗患者的低水平HIV复制。