Impact of Interventions on Clonally Expanded Proviruses and Their RNA Expression

背景：已提出的治疗HIV的干预措施包括使用HDAC抑制剂等药物来激活潜伏期前病毒，诱导细胞内RNA的表达，希望这种表达将导致病毒的产生，并通过病毒细胞毒性或靶向细胞毒性T淋巴细胞(CTL)反应杀死感染细胞。然而，我们与北卡罗来纳大学的David MarGolis博士合作进行的先前的研究表明，单剂量或多剂量使用HDAC抑制剂voinostat治疗的参与者对减少水库的影响有限(Archin等人，J.Infect。迪什。210：728-735,2014；Archin等，自然，487：482-485,2012)，包括没有通过艾滋病毒单拷贝分析(SCA)测量体内病毒产量的增加。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，其中一些研究是由匹兹堡大学由John Mellors博士指导进行的(Cillo等人，PNAS 111：7078-7083,2014；魏等人，PLoS pathog。10：e1004071,2014)。虽然这些有效的抑制剂被认为在诱导前病毒表达方面更有效，但目前尚不清楚诱导细胞杀伤所需的前病毒表达水平。此外，通过比较来自相同个体的未诱导和诱导的PBMC，我们将确定哪些抑制剂能够在扩大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的药物)，哪些只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。在进行HDAC抑制剂的体外研究的同时，我们还计划评估体内对扩增克隆的治疗性干预的效果。我们将使用通过PITT合同从HDAC抑制剂罗米迪辛研究(A5315协议)、广中和抗体VRC-01研究(A5342协议)、低剂量甲氨蝶呤研究(A5314协议)以及其他完成后获得的样本，通过与Frank Maldarelli博士和Stephen Hughes博士(艾滋病毒动力学和复制计划)合作对治疗前、治疗期间和治疗后收集的特定克隆进行整合位点分析(ISA)和定量数字液滴聚合酶链式反应(DdPCR)。_成就：除了我们对伏立诺对接受抗逆转录病毒治疗(ART)的个体中HIV病毒血症水平的影响的研究做出贡献外(Gay等人，AIDS Res.Hum。逆转录病毒34：111-122,2018)，我们还参与了一项早期ART干预研究(Henrich等人，PLoS Med。14(11)：e1002417,2017)，这表明在感染的早期启动ART显著延迟了病毒反弹的时间。与这一发现一致，我们使用艾滋病临床试验组(A5142方案)的样本进行了另一项研究，表明在启动ART时艾滋病毒基因多样性较高，可能与出现耐药性和ART失败有关(Kearney等人，PLoS One 13(1)：E0190438,2018)。我们的研究得出的结论是，早期开始抗逆转录病毒治疗将减少HIV储备库的大小，并限制病毒学失败。_我们目前的研究主要集中在抗PD-1对体内HIV储存库的影响上。与NIAID的Eli Boritz博士合作，我们从接受抗PD-1注射的HIV-1感染患者身上获得样本，以确定这种检查点抑制物在抗逆转录病毒治疗期间对HIV感染细胞水平的影响。使用我们的Flip-SGS分析(在Zia BC 011681项目中描述)，我们正在鉴定在抗PD-1治疗前后获得的感染细胞中整合到宿主基因组的位置和前病毒结构。

BACKGROUND: Proposed curative interventions for HIV have 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., J. Infect. Dis. 210:728-735, 2014; Archin et al., Nature 487:482-485, 2012), 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. Furthermore, 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 will use samples accessible through the Pitt contract from the single-dose infusion of the HDAC inhibitor romidepsin study (protocol A5315), the broadly neutralizing antibody VRC-01 study (protocol A5342), the low-dose methotrexate study (protocol A5314), and others as they are completed, by performing the integration sites assay (ISA) and quantitative digital droplet PCR (ddPCR) of specific clones on samples collected before, during, and after treatment in collaboration with Drs. Frank Maldarelli and Stephen Hughes (HIV Dynamics and Replication Program). ____ACCOMPLISHMENTS: In addition to our contribution to studies on the effect of vorinostat on HIV viremia levels in individuals treated with antiretroviral therapy (ART) (Gay et al., AIDS Res. Hum. Retroviruses 34:111-122, 2018), we also participated in an early ART intervention study (Henrich et al., PLoS Med. 14(11):e1002417, 2017) that showed that initiating ART in very early infection significantly delayed the time to viral rebound. Consistent with this finding, we conducted another study using samples from the AIDS clinical trial group (protocol A5142) showing that higher HIV genetic diversity at the time of initiating ART may be associated with the emergence of drug resistance and ART failure (Kearney et al., PLoS ONE 13(1):e0190438, 2018). Our studies lead to the conclusion that early ART initiation will reduce the size of the HIV reservoir and limit virologic failure. ____Our current research is focused on the effect of anti-PD-1 on the HIV reservoir in vivo. In collaboration with Dr. Eli Boritz at NIAID, 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. Using our FLIP-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.