Impact of Interventions on Clonally Expanded Proviruses and Their RNA Expression

背景：在抗逆转录病毒治疗（ART）期间，HIV的持续存在已被证明是由开始治疗前感染的细胞增殖驱动的，而不是由外周血或组织（如淋巴结或肠道）中持续的HIV复制周期驱动的（McManus等，J. clinin）。投资，129:4629-4642,2019；Bozzi et al., Sci。ad5:eaav5045, 2019)。然而，对于细胞增殖的驱动因素或它们在抗逆转录病毒治疗期间的选择性维持，或在没有抗逆转录病毒治疗的情况下使用旨在控制艾滋病毒的新干预措施治疗期间的选择性维持，人们知之甚少。在没有抗逆转录病毒治疗的情况下实现艾滋病毒缓解的新干预措施包括潜伏期逆转剂、广泛中和抗体、艾滋病毒原病毒的遗传修饰、增强对受感染细胞的免疫识别等等。我们目前的研究旨在了解抗逆转录病毒治疗期间细胞克隆的选择和持久性，抗逆转录病毒治疗前和治疗期间克隆增殖的驱动因素，以及新干预措施对感染细胞克隆扩增和抗逆转录病毒治疗期间持续存在的原病毒表达的影响。首先，我们研究了抗逆转录病毒治疗前细胞克隆中HIV整合位点的分布，并将其与抗逆转录病毒治疗多年后细胞克隆的分布进行了比较，以确定尽管接受治疗，但允许HIV持续存在的选择压力（Coffin et al., JCI Insight 4:e128432, 2019; Coffin et al., 2019）。中华医学杂志。17:e1009141,2021；王晓明，王晓明等。生物化学学报，21(2):568- 568,2016。我们发现持续抗逆转录病毒治疗的HIV前病毒更频繁地整合在宿主基因组中不高度表达的区域，并且它们通常以与宿主基因相反的方向整合。我们还发现，受感染的细胞克隆出现在急性感染中，并在抗逆转录病毒治疗中持续至少9年。我们现在正在研究潜伏保留剂对受感染细胞克隆的持久性和动力学以及受感染细胞克隆内HIV表达的影响。_____一种针对HIV的治疗性干预措施包括使用HDAC抑制剂等药物进行治疗，这些药物可以激活潜伏的前病毒，诱导细胞内RNA的表达，希望这种表达将导致病毒产生，并通过病毒细胞毒性或靶向细胞毒性t淋巴细胞（CTL）反应杀死受感染的细胞。然而，之前我们与David Margolis博士（北卡罗来纳大学）合作进行的研究显示，单剂量或多剂量的HDAC抑制剂vorinostat对参与者的储库减少作用有限（Archin et al., Nature 487:482-485, 2012; Archin et al.， J. infection）。Dis. 210:728-735, 2014)，包括通过HIV单拷贝试验（SCA）测量的体内病毒产量缺乏增加。在体外研究中，从更有效的抑制剂和HDAC抑制剂的组合中获得了更有希望的数据，其中一些是由匹兹堡大学John Mellors博士指导进行的（Cillo等人，PNAS 111:7078-7083, 2014； Wei等人，PLoS Pathog. 10:e1004071, 2014）。虽然这些有效的抑制剂被认为在诱导原病毒表达方面更有效，但目前尚不清楚什么水平的原病毒表达是诱导细胞杀伤所必需的。通过比较来自同一个体的未诱导的和诱导的pbmc，我们将确定哪些抑制剂能够在扩增的细胞克隆中重新激活潜伏的前病毒（那些在诱导前不表达HIV RNA的病毒），哪些抑制剂只能增加在诱导前已经低水平表达的前病毒的表达，以及这些作用在前病毒之间的异质性。在对HDAC抑制剂进行体外研究的同时，我们还计划评估治疗干预对体内扩展克隆的疗效。我们正在使用通过与NIAID的Eli Boritz博士合作获得的样本，这些样本来自单剂量抗pd -1输注前后，以测量对感染和未感染细胞克隆的克隆增殖的影响。____成就：我们目前的研究重点是抗pd -1对体内HIV储存库的影响。与Boritz博士合作，我们获得了接受抗pd -1输注的hiv -1感染患者的样本，以确定这种检查点抑制剂在抗逆转录病毒治疗期间对hiv感染细胞水平的影响（Perez-Rodrigo等人，Conference on Retroviruses and Opportunistic Infections, 2021）。利用我们的MDA-SGS检测（在ZIA BC 011681项目中描述），我们对抗pd -1治疗前后获得的感染细胞中整合宿主基因组的位点和前病毒结构进行了表征。到目前为止，我们已经证明了带有缺陷原病毒的感染细胞对抗pd -1的反应是扩增的。我们目前正在评估抗pd -1对完整原病毒细胞的影响。_____为了了解体内hiv感染细胞克隆的起源，我们调查了尚未启动ART的急性感染供体中细胞克隆的分布（Coffin等，JCI Insight 4:e128432, 2019）。然后，我们将这种分布与抗逆转录病毒治疗持续2-3年的细胞克隆进行了比较。我们发现，在急性感染中，感染的细胞克隆早在Fiebig III期就出现了，一些克隆在抗逆转录病毒治疗中持续了数年。____由于儿童的免疫系统与成人不同，包括更高比例的幼稚T细胞，据报道，幼稚T细胞对HIV感染的抵抗力更强，因此我们调查了儿童中感染细胞克隆的数量、出现时间和抗逆转录病毒治疗的持久性（Bale， Katusiime等人，mBio 12:e00568- 21,2021）。我们发现儿童中受感染的细胞克隆出现在早期感染中，并在抗逆转录病毒治疗中持续至少9年。我们还发现，在宿主基因的相反方向和不高度表达的基因中存在对原病毒的选择。____为了研究抗逆转录病毒治疗前和期间感染细胞克隆扩增的驱动因素，我们比较了从短期和长期抗逆转录病毒治疗供体中收集的离体感染PBMCs与体内感染PBMCs的整合位点谱（Coffin等人，PLoS Pathog. 17: e1009141,2021）。我们发现整合入癌基因只是感染细胞克隆扩增的次要驱动因素，这表明稳态和/或抗原驱动的克隆扩增是抗逆转录病毒治疗期间HIV持续存在的主要驱动因素。____我们开发了一种新技术来研究抗逆转录病毒治疗之前和期间感染细胞克隆的动态，其准确性和灵敏度高于整合位点测定（Brandt等人，Viruses 13:1235, 2021）。利用这一技术，我们发现带有完整或缺陷原病毒的克隆在处理过程中可以增加或减少大小。

BACKGROUND: The persistence of HIV during antiretroviral therapy (ART) has been shown to be driven by the proliferation of cells that were infected prior to initiating treatment and not from ongoing cycles of HIV replication in the peripheral blood or in tissues such as lymph nodes or gut (McManus et al., J. Clin. Invest. 129:4629-4642, 2019; Bozzi et al., Sci. Adv. 5:eaav5045, 2019). However, little is known about the drivers of cellular proliferation or about their selective maintenance during ART or during treatment with new interventions aimed at controlling HIV without ART. New interventions aimed at achieving HIV remission without ART include latency-reversing agents, broadly neutralizing antibodies, genetic modifications of HIV proviruses, enhancement of immune recognition of infected cells, and more. Our current studies are aimed at understanding the selection and persistence of cell clones during ART, the drivers of clonal proliferation before and during ART, and the effect of new interventions on clonal expansion of infected cells and on the expression of proviruses that persist during ART. Firstly, we investigated the distribution of HIV integration sites in cell clones prior to ART and compared it to the distribution of cell clones after years on ART to identify the selection pressures that allow HIV to persist despite treatment (Coffin et al., JCI Insight 4:e128432, 2019; Coffin et al,. PLoS Pathog. 17:e1009141, 2021; Bale, Katusiime et al., mBio 12:e00568-21, 2021). We found that HIV proviruses that persist on ART are more frequently integrated in regions of the host genome that are not highly expressed and that they are often integrated in the opposite orientation of the host gene. We also found that infected cell clones arise in acute infection and persist for at least 9 years on ART. We are now investigating the effect of the latency-reserving agents on the persistence and dynamics of infected cell clones and on the expression of HIV within infected cell clones. _____One proposed curative intervention for HIV has included treating with agents, such as HDAC inhibitors, that activate latent proviruses, inducing expression of intracellular RNA, in the hopes that such expression will result in viral production and killing of infected cells by viral cytotoxicity or targeted cytotoxic T-lymphocyte (CTL) response. However, previous studies, in which we collaborated with Dr. David Margolis (University of North Carolina), showed only a limited effect on reservoir reduction in participants treated with single or multiple doses of the HDAC inhibitor vorinostat (Archin et al., Nature 487:482-485, 2012; Archin et al., J. Infect. Dis. 210:728-735, 2014), including a lack of increased viral production in vivo measured by the HIV single-copy assay (SCA). More promising data have been obtained from more potent inhibitors and from combinations of HDAC inhibitors in in vitro studies, some of which were conducted by the University of Pittsburgh directed by Dr. John Mellors (Cillo et al., PNAS 111:7078-7083, 2014; Wei et al., PLoS Pathog. 10:e1004071, 2014). Although these potent inhibitors are believed to be more effective at inducing proviral expression, it is not currently known what levels of proviral expression are necessary to induce cell killing. By comparing uninduced and induced PBMCs from the same individuals, we will determine which inhibitors are capable of reactivating latent proviruses (those that are not expressing HIV RNA prior to induction) in expanded cell clones and which only increase expression of proviruses that were already expressing at low levels prior to the induction, and the heterogeneity of these effects among proviruses. While conducting the in vitro studies on the HDAC inhibitors, we also plan to assess the efficacy of curative interventions on expanded clones in vivo. We are using samples accessible through our collaboration with Dr. Eli Boritz at NIAID from before and after a single-dose infusion of anti-PD-1 to measure the effects on clonal proliferation of infected and uninfected cell clones. ____ACCOMPLISHMENTS: Our current research is focused on the effect of anti-PD-1 on the HIV reservoir in vivo. In collaboration with Dr. Boritz, we obtained samples from HIV-1-infected patients who received anti-PD-1 infusions to determine the effect of this checkpoint inhibitor on levels of HIV-infected cells during ART (Perez-Rodrigo et al., Conference on Retroviruses and Opportunistic Infections, 2021). Using our MDA-SGS assay (described in Project ZIA BC 011681), we are characterizing the sites of integration into the host genome and the proviral structure in infected cells obtained before and after treatment with anti-PD-1. Thus far, we have demonstrated the expansion of infected cells with defective proviruses in response to anti-PD-1. We are currently evaluating the effect of anti-PD-1 on cells with intact proviruses. _____To understand the origin of HIV-infected cell clones in vivo, we investigated the distribution of cell clones in donors in acute infection who had not yet initiated ART (Coffin et al., JCI Insight 4:e128432, 2019). We then compared this distribution to the cell clones that persisted for 2-3 years on ART. We found that infected cell clones emerged as early as Fiebig stage III in acute infection and that some clones persisted for years on ART. ____Because children have immune systems that are different from adults, including a much higher fraction of naive T cells that have been reported to be more resistant to HIV infection, we investigated the number of infected cells clones in children, the timing of their emergence, and their persistence on ART (Bale, Katusiime et al., mBio 12:e00568-21, 2021). We found that infected cell clones in children arise in early infection and persist for at least 9 years on ART. We also found that there is a selection for proviruses that are in the opposite orientation of the host gene and are in genes that are not highly expressed. ____To investigate the drivers of clonal expansion of infected cells before and during ART, we compared the integration site profile in ex vivo infected PBMCs to in vivo infected PBMCs collected from donors on short- and long-term ART (Coffin et al., PLoS Pathog. 17:e1009141, 2021). We found that integration into oncogenes is only a minor driver of clonal expansion of infected cells, suggesting that homeostatic and/or antigenic-driven clonal expansion are the main drivers for persistence of HIV during ART. ____We developed a new technology to investigate the dynamics of infected cell clones before and during ART with higher accuracy and sensitivity than the integration sites assay (Brandt et al., Viruses 13:1235, 2021). Using this technology, we showed that clones with intact or defective proviruses can increase or decrease in size during treatment.