HIV Persistence During Suppressive Antiretroviral Therapy

抑制性抗逆转录病毒治疗期间艾滋病毒的持续存在

• 批准号: 9343942
• 负责人: Frank Maldarelli
• 金额: $ 71.03万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9343942
• 关键词: Age; Anatomy; Anti-Retroviral Agents; Biological Assay; Biopsy; Blood; Bronchoalveolar Lavage Fluid; C-reactive protein; CD4 Positive T Lymphocytes; CD8B1 gene; CXCL10 gene; Cells; Characteristics; Clinical; Clinical Protocols; Clonal Expansion; Collaborations; Colon; Colonoscopy; DNA; Data; Distal part of ileum; Evaluation; Fibrin fragment D; Frequencies; Future; Gastrointestinal tract structure; Genetic Variation; Goals; Gut associated lymphoid tissue; HIV; HIV Drug Resistance Program; HIV Infections; HIV-1; Helper-Inducer T-Lymphocyte; IL2RA gene; Immune; Immune response; Immune system; Immunologics; Immunosuppressive Agents; Individual; Infection; Interleukin-17; Interleukin-6; Intestines; Investigation; Life; Link; Measurement; Memory; Methodology; Patients; Penetration; Peripheral Blood Mononuclear Cell; Persons; Pharmaceutical Preparations; Plasma; Population; Production; Protocols documentation; RNA; Race; Rectum; Reporting; Research Personnel; Sampling; Series; Site; Source; Structure; Surveys; Techniques; Technology; Therapeutic; Time; Tissues; Tonsil; Translational Research; United States National Institutes of Health; Universities; Variant; Viral; Viremia; Virus; antiretroviral therapy; cohort; genome sequencing; ileum; immune activation; in vivo; inflammatory marker; insight; integration site; lymph nodes; mortality; novel; novel strategies; outcome prediction; reconstitution; response; sex; site-specific integration; tool; viral RNA; virus host interaction

Combination antiretroviral therapy (ART) results in marked suppression of viremia in persons with HIV-1 infection. Therapy is not curative, however, & detectable viremia & replication-competent HIV-1 persist despite ART-induced suppression. The origin of persistent viremia on therapy is uncertain; potential sources include ongoing complete cycles of HIV-1 replication, long-lived reservoirs of chronically infected cells, sanctuary sites into which antiretrovirals have poor penetration, or a combination of these possibilities. Understanding the source & mechanisms of viral persistence on ART has critical implications for future therapeutic approaches & strategies for virus eradication. We began our investigation of the source of persistent HIV by developing an assay for viremia (HIV RNA) with single-copy sensitivity & by developing clinical protocols to determine the effects of ART intensification. These studies & others revealed no decrease in persistent viremia after drug intensification, suggesting that persistent viremia may be the product of long-lived reservoirs of chronically infected cells. Others have reported the utility of 2-LTR circles as an indicator of continued HIV replication during ART & increases in such circular forms during intensification with raltegravir. In addition, a survey of anatomic reservoirs revealed a higher HIV RNA to DNA ratio in cells from gut-associated lymphoid tissue (GALT) in the terminal ileum compared to the colon & rectum & decreases in HIV RNA during drug intensification. Such detailed analyses demonstrate the complexities of host-virus interactions & highlight the limitations in our understanding of mechanisms of persistence during suppressive ART._The proposed project represents a focused approach to overcoming our limitations & understanding persistence by quantifying the host & viral contributions to HIV persistence. We are building on prior studies that used sensitive methodologies to quantify & genetically characterize virus in plasma to investigate HIV reservoirs in cellular compartments & are now expanding the range of our analyses by applying new single-cell methodologies & isolating specific cell subsets in blood & tissue from infected individuals._To characterize the host-virus relationship during suppressive therapy, we are quantifying cellular & soluble immune correlates of persistent viremia. We initiated these studies by investigating the level of cellular immune activation before & after initiation of ART. The relative proportion of cellular immune activation markers (e.g., CD8+CD38+DR+cells) were high prior to therapy but declined sharply after ART was initiated; ultimately, HIV RNA levels & levels of immune activation stabilized to a persistent steady state with approximately the same time frame. Previous investigators detected persistent cellular immune activation during ART, but analyses to date have been restricted to 2-3 years on ART. To determine whether immune activation was still elevated after achieving steady-state persistent viremia, we quantified levels of cellular immune activation markers in patients with viremia suppressed for 7 years on ART & age-, sex- & race-matched uninfected controls. A modest but significant level of cellular immune activation (CD8+CD38+DR+ cells) was detectable even after 7 years on ART._We investigated potential causes of persistent immune activation first by characterizing PBMC more fully, quantitating memory & naive, CD38, & DR subsets in CD4 & CD8 lineages. In parallel, we quantitated the levels of persistent viremia. Initial evaluation revealed a strong association between levels of persistent HIV RNA & CD8 memory subsets (r=0.51, p=0.0004) & CD8+CD38+DR+ immune activation (r=0.44, p=0.003). These data indicate that either generalized cellular activation itself drives production of HIV or activation is present in response to persistent viremia._Determining the difference between these two possibilities will offer new insights for therapeutic strategies to eliminate such cellular reservoirs. If generalized activation is the source of persistent viremia, then treatment with agents that stimulate the immune system & increase cellular activation will result in increased HIV production from latent reservoirs, followed by overall decay in viremia. In contrast, if increased immune activation is directly controlling the level of persistent viremia, then further immune activation could result in its decay. With the HVIB Translational Research Unit, we will take a dual approach to define further characteristics of CD4+CD38+ & CD8+CD38+ cell subsets by quantifying additional markers in the long-term suppressed patients. Our prediction is that CD8 markers of activation &proliferation will correlate with viremia, but CD4 markers will not. If so, we will have identified a key distinction between cellular immune activation & viremia before & after introduction of ART._We have now completed measurements of a series of soluble immune activation parameters (D-dimer, sCD14, hsCRP, IL-6, IP-10, sCD163, & are conducting an analysis fo the relative levels of these markers, cellular immune activation parameters & single copy assays._We are further characterizing the CD4 cell population by quantitating subsets of CD25+FoxP3+ (suppressor T reg) & IL-17+ (helper cells). Our hypothesis is that persistent viremia will be positively correlated with the relative frequency of FoxP3+ cells because these cells have immunosuppressive function, resulting in higher levels of HIV-1._We are also using our well-characterized group of patients with long-term suppressed viremia on ART to characterize soluble markers of inflammation as correlates of viremia. Levels of soluble markers, such as D-Dimer, IL-6, C-reactive protein all cause mortality in HIV infection, even after suppression on ART._There are no data correlating the relative levels of HIV viremia to predictive outcome markers. We will determine whether levels of soluble markers correlate with persistent viremia._We will also quantify the effects of immune responses on HIV genetic variation. Using single-genome sequencing (SGS) techniques, we will determine whether prolonged viral suppression & partial immune reconstitution result in selection for cells infected with HIV immune escape variants. These studies will provide the first fine-structure analysis of HIV populations during prolonged ART._In addition, we have initiated several new studies of HIV persistence. We are investigating HIV in plasma, PBMC, & cells derived from ileum & colon in infected individuals taking combination ART with suppressed 50 copies who are undergoing colonoscopy at the NIH Clinical Center. We have performed these colonoscopies in collaboration with J. Kovacs in the protocol "Virologic & Immunologic Evaluation of Lymph Node, Tonsillar & Intestinal Biopsies, & Bronchoalveolar Lavage Fluid" & have used a new sampling strategy that will yield useful information regarding the distribution of HIV-infected cells in the gastrointestinal tract. In a second study of HIV persistence, we are studying HIV from plasma & PBMC from patients with viral RNA suppressed on ART who undergo short antiretroviral discontinuation. Using SGS to investigate HIV from the earliest rebound viremia occurring within 7-14 days of discontinuation, we will identify a critical source of viremia.__In collaboration with S. Hughes (HIV DRP) , X. Wu (Leidos), J. Coffin (Tufts), M. Kearney (HIV DRP) & J. Mellors (University of Pittsburgh), we have investigated HIV integration sites in vivo, characterizing HIV from plasma & PBMC of patients. Drs. Hughes & Wu have completed analysis of integration sites in these individuals. These studies revealed that specific integration sites may be linked to clonal expansion of HIV-infected cells, suggesting a novel mechanism for HIV persistence.

抗逆转录病毒疗法（ART）会导致HIV-1感染患者明显抑制病毒血症。但是，尽管艺术引起的抑制作用，但治疗尚未治愈，并且可检测到的病毒血症和能力竞争性的HIV-1仍然存在。持续性病毒血症在治疗上的起源尚不确定。潜在的来源包括持续的HIV-1复制循环，长期寿命的被感染细胞的寿命储层，抗逆转录病毒剂渗透较差的避难所或这些可能性的组合。了解对艺术的病毒持久性的来源和机制对消除病毒的未来治疗方法和策略具有至关重要的意义。我们通过开发具有单拷贝敏感性的病毒血症（HIV RNA）的测定法和开发临床方案以确定艺术强化的影响，从而开始研究持续HIV的来源。这些研究和其他研究表明，药物加强后持续性病毒血症没有减少，这表明持续性病毒血症可能是长期感染细胞的长寿命储层的产物。其他人则报道了2-LTR圆圈的效用，是ART和RALTEGRAVIR加强期间这种圆形形式中持续艾滋病毒复制的指标。此外，对解剖储层的调查显示，与结肠和直肠相比，与结肠和直肠相比，在药物强化期间，与结肠和直肠相比，末端回肠的肠道相关淋巴机（GALT）的HIV RNA与DNA比较高。这样的详细分析证明了宿主病毒相互作用的复杂性，并突出了我们对抑制艺术期间持久性机制的理解的局限性。我们正在基于先前的研究基础，该研究使用敏感方法来量化和遗传表征血浆中的病毒，以调查细胞室中的HIV储量，现在正在扩大我们分析的范围，通过应用新的单单细胞方法，应用新的单细胞方法并隔离受感染及其在适应性疗法的cormune suppertion supplation and supplate for Supply for Supply forsempless forsements forsements suppersionss._to suptials.__持续的病毒血症。我们通过研究ART启动之前和之后的细胞免疫激活水平来启动这些研究。在治疗之前，细胞免疫激活标记（例如CD8+CD38+DR+细胞）的相对比例很高，但在启动ART后急剧下降。最终，HIV RNA水平和免疫激活水平稳定在持续的稳态，其时间范围大致相同。先前的研究人员在ART期间检测到持续的细胞免疫激活，但迄今为止的分析仅限于2 - 3年的ART。为了确定在达到稳态持续性病毒血症后是否仍会升高免疫激活，我们在ART和年龄，性别和种族匹配的未感染的对照中量化了7年的病毒血症患者的细胞免疫激活标记水平。即使在ART上使用7年后，也可以检测到适中但显着的细胞免疫激活（CD8+CD38+DR+细胞）。同时，我们量化了持续性病毒血症的水平。初步评估表明，持续性HIV RNA和CD8存储子集的水平（r = 0.51，p = 0.0004）和CD8+CD38+CD38+DR+免疫激活（r = 0.44，p = 0.003）之间存在很强的关联。这些数据表明，响应持续的病毒血症，存在广义的细胞激活本身可以驱动艾滋病毒的产生或激活。如果广义激活是持续性病毒血症的来源，则用刺激免疫系统并增加细胞激活的药物的治疗将导致潜在储层的HIV产生增加，然后在病毒血症中进行总体衰变。相反，如果免疫激活增加直接控制持续性病毒血症的水平，那么进一步的免疫激活可能会导致其衰减。使用HVIB翻译研究单元，我们将采用双重方法来定义CD4+ CD38+和CD8+ CD38+ CD38+细胞亚群的进一步特征，通过量化长期抑制患者的其他标记。我们的预测是，CD8的激活和增殖标记将与病毒血症相关，但CD4标记不会。如果是这样，我们将在引入艺术之前和之后确定细胞免疫激活与病毒血症之间的关键区别。通过定量CD25+ FOXP3+（抑制剂t reg）和IL-17+（辅助细胞）的亚群来进一步表征CD4细胞的种群。将炎症的可溶性标记表征为病毒血症的相关性。我们将确定可溶性标记的水平是否与持续性病毒血症相关。_我们还将量化免疫反应对HIV遗传变异的影响。使用单基因组测序（SGS）技术，我们将确定长时间的病毒抑制和部分免疫重建是否会导致选择感染HIV免疫逃避变体的细胞。这些研究将在延长的艺术期间对HIV种群进行首次精细结构分析。我们正在研究血浆，PBMC和细胞中的HIV，并在受感染的个体中衍生出源自Ileum＆Colon的细胞，并在NIH临床中心接受了50份正在接受结肠镜检查的被抑制的副本。我们已经与J. Kovacs合作进行了这些结肠镜检查，该协议是“淋巴结，扁桃体和肠活检的病毒和免疫学评估，以及Bronchoalveolal灌洗液，并使用了新的抽样策略，该策略将产生有关HIV感染细胞在气体障碍物中的HIV感染细胞分布的有用信息。在第二项HIV持久性研究中，我们正在研究抑制病毒RNA的患者血浆和PBMC的HIV，这些患者抑制了经历短暂的抗逆转录病毒中断的ART。使用SGS在中断的7-14天内从最早发生的反弹病毒血症调查HIV，我们将与S. Hughes（HIV DRP），X. Wu（Leidos），J。Coffin（Tufts），M. Kearney（HIV DRP）＆J。Mellors（J. Mellors（J. Mellors）（Pitsburgh）列为Pitsburgh的Viv viv nove viv novs novs inviv hy novs inverggh，我们将确定病毒血症的关键来源.__。来自患者血浆和PBMC的HIV。博士。 Hughes＆Wu已经完成了对这些人的集成站点的分析。这些研究表明，特定的整合位点可能与感染HIV感染细胞的克隆扩张有关，这表明了HIV持久性的新型机制。