Impact of Interventions on Clonally Expanded Proviruses and Their RNA Expression

背景技术：已提出的HIV的治疗性干预包括用激活潜伏原病毒的试剂（如HDAC抑制剂）治疗，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）应答杀死感染的细胞。然而，我们与大卫马戈利斯博士（北卡罗来纳州大学）合作的先前研究显示，在用单剂量或多剂量HDAC抑制剂伏立诺他治疗的参与者中，对储库减少的作用仅有限（Archin等人，J. Infect. 210：728-735，2014; Archin等人，Nature 487：482-485，2012），包括缺乏通过HIV单拷贝测定（SCA）测量的体内病毒产生增加。在体外研究中，已经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John Mellors博士指导的匹兹堡大学进行的（Cillo等人，PNAS 111：7078-7083; Wei等人，PLoS Pathog. 10：e1004071，2014）。尽管这些有效的抑制剂被认为在诱导前病毒表达方面更有效，但目前尚不知道什么水平的前病毒表达是诱导细胞杀伤所必需的。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重新激活扩增细胞克隆中的潜伏前病毒（在诱导之前不表达HIV RNA的那些），哪些抑制剂仅增加在诱导之前已经以低水平表达的前病毒的表达，以及这些影响在前病毒中的异质性。在对HDAC抑制剂进行体外研究的同时，我们还计划评估治疗性干预对体内扩增克隆的疗效。我们将使用来自HDAC抑制剂罗米地辛研究的单剂量输注的Pitt合同可获得的样本（方案A5315），广泛中和抗体VRC-01研究（方案A5342），低剂量甲氨蝶呤研究（方案A5314），以及其他完成的方法，通过对之前收集的样品进行特定克隆的伊萨和定量数字液滴PCR（ddPCR），在治疗期间和治疗后与Frank Maldarelli博士和Stephen Hughes博士（HIV动力学和复制计划）合作。_成就：我们目前的研究重点是抗PD-1对体内HIV储库的影响。我们与NIAID的Eli Boritz博士合作，从接受抗PD-1输注的HIV-1感染患者中获得样本，以确定这种检查点抑制剂对ART期间HIV感染细胞水平的影响。（见项目ZIA BC 011681），我们正在表征整合到宿主基因组中的位点和在用抗PD-1治疗之前和之后获得的感染细胞中的前病毒结构。到目前为止，我们已经证明了具有缺陷前病毒的感染细胞响应于抗PD-1的扩增。我们目前正在评估抗PD-1对具有完整前病毒的细胞的影响。_使用我们的方法，我们还证明了暴露前预防在体内阻断具有部分抗逆转录病毒抗性的变体的传播方面的失败（Spinelli等人，临床感染Dis.，出版，2020年），我们的第二个这样的报告（科恩等。例如，Lancet HIV 6：e43-e50，2019）。我们打算对这些人进行随访，以确定非常早期治疗对HIV储库和感染细胞克隆扩增的影响。我们未来的研究还包括表征体内感染细胞克隆的动态，以便我们能够准确评估新干预措施对此类细胞克隆的影响。

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; 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 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: 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. 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. ____Using our methods, we also demonstrated the failure of pre-exposure prophylaxis to block transmission in vivo of variants with partial antiretroviral resistance (Spinelli et al., Clin. Infect. Dis., in press, 2020), our second such report (Cohen et. al., Lancet HIV 6: e43-e50, 2019). We intend to follow up with these individuals to determine the effect of very early treatment on the HIV reservoir and on the clonal expansion of infected cells. Our future studies also include characterizing the dynamics of infected cell clones in vivo so that we can accurately assess the effect of new interventions on such cell clones.