Investigation of persistent HIV immune stimulation in lymphoid tissues during therapy as a cause of sustained immune activation

抗逆转录病毒疗法(ART)充分抑制HIV复制，将血浆病毒载量(PVL)降低到 检测极限，但免疫激活(IA)未正常化，IA标志物-IL-6，TNF， 转化生长因子、C反应蛋白(CRP)和D-二聚体持续升高与高血压风险增加相关 心血管疾病，内皮功能障碍和凝血异常，肺高压，间质 肾炎，非艾滋病相关恶性肿瘤的发展，以及中枢神经系统的异常。多种机制 已经提出了解释抗逆转录病毒治疗下IA持续存在的原因，包括微生物易位和疱疹 病毒感染(如单纯疱疹病毒、巨细胞病毒、EB病毒)。然而，我们在这里提出，艾滋病毒本身是导致 持续的IA，因为在接受抗逆转录病毒治疗期间，淋巴组织(LT)中正在产生病毒。在本修订版中 提议，我们有一个特定的目标来检验两个假设：1)在ART期间持续的IA是由 再激活潜伏感染细胞中病毒粒子的产生和/或病毒抗原的表达 持续的低水平病毒复制；以及2)抗逆转录病毒治疗期间持续的低水平病毒复制是 LT中不能完全抑制复制的抗逆转录病毒(ARV)药物的细胞内浓度(IC) 病毒生产。对于我们的第一个假设，我们将寻找持续性IA和 通过在单细胞水平上鉴定：1)产生病毒的CD4T细胞，在LT中产生病毒/抗原表达 缺乏激活和增殖标记的细胞可以维持低水平的 病毒产生；2)B细胞滤泡中的T滤泡辅助细胞(TFH)，最近被证明是一种 病毒持续存在的独立蓄水池；以及3)重新激活潜伏感染的T细胞，产生病毒或p24。 我们建议通过我们的验证和高度敏感的原位杂交(ISH)来研究IA的这些驱动因素 方法对LT中的病毒(V)RNA+和病毒产生细胞进行检测、表型和定位； 敏感的高通量“基于诱导转录测序的包膜检测”(EDITS)试验；以及 一种广谱中和抗体(BNab)方法，用于浓缩HIV包膜(ENV)+细胞。对于假设的 病毒驱动因素与IA的相关性，我们将使用标准的基于流量的抗体分析和 对LT单核细胞进行单细胞转录分析，以确定与 内务部。我们的第二个假设将通过量化急性呼吸道合胞病毒的细胞内浓度(IC)在LT和 确定ARV-IC之间的关系和检测三种可能的病毒驱动因素的频率 内务部。确定艾滋病毒本身是IA的原因将表明未来的发展将完全抑制病毒 LT油藏的生产在减少IA和相关病理方面都有好处，并且作为一种基本的 艾滋病毒治疗战略的组成部分。

Antiretroviral therapy (ART) sufficiently suppresses HIV replication to reduce plasma viral load (pVL) below the limit of detection, but immune activation (IA) is not normalized and the elevated levels of IA markers- IL-6, TNF, TGFß, C reactive protein (CRP), and D-Dimer remain elevated are associated with increased risk for cardiovascular disease, endothelial disfunction and clotting abnormalities, pulmonary hypertension, interstitial nephritis, development of non-AIDS associated malignancies, and CNS abnormalities. Multiple mechanisms have been proposed to explain the persistence of IA under ART including microbial translocation and herpes virus infections (e.g., HSV, CMV, EBV). However, here we propose that HIV itself is a major cause of persistent IA because of ongoing virus production in lymphoid tissues (LT) while on ART. In this revised proposal, we have one specific aim that tests two hypotheses: 1) Sustained IA during ART is driven by production of virions and/or expression of viral antigens in reactivated latently infected cells with or without persistent low-level virus replication; and 2) that persistent low-level virus replication during ART is the result of intracellular concentrations (IC) of antiretroviral (ARV) drugs in LT that do not completely inhibit replication and virus production. For our first hypothesis we will seek direct evidence of correlations between persistent IA and virus production/antigen expression in LT by identification, at the single cell level, of: 1) virus-producing CD4 T cells lacking markers of activation and proliferation that we have previously shown can sustain low levels of virus production; 2) T follicular helper cells (Tfh) in B cell follicles that have recently been shown to be an independent reservoir for viral persistence; and 3) reactivated latently infected T cells producing virus or p24. We propose investigations of these drivers of IA by our validated and highly sensitive in situ hybridization (ISH) methods to detect, phenotype and locate virus (v) RNA+ and virus-producing cells in LT; by a validated highly sensitive high throughput “envelope detection by induced transcription-based sequencing” (EDITS) assay; and a broadly neutralizing antibody (bNab) method to enrich for HIV-envelope (ENV)+ cells. For the hypothesized correlations of the virus drivers with IA, we will measure IA with standard flow-based antibody assays and with single cell transcriptomic analysis of LT mononuclear cells to identify unique gene signatures associated with IA. Our second hypothesis will be tested by quantification of ARV-intracellular concentrations (ICs) in LT and determine the relationships among ARV-ICs and the frequency of detecting the three putative virus drivers of IA. Establishing that HIV itself is a cause of IA would point future developments to fully suppress virus production in the LT reservoir with benefits both in reducing IA and associated pathologies and as an essential component of HIV Cure Strategies.