Accurately defining the distribution of the genetically intact and potentially replication-competent HIV-1 reservoir during the first 3 years of integrase inhibitor containing antiretroviral therapy

项目总结 准确识别具有复制能力的HIV-1潜伏库为设计任何 治愈策略；然而，这是具有挑战性的，因为它的分布稀少。此外，HIV-1DNA存在于 不同的形式和前病毒在遗传上是不同的。最近开发的全长个人前病毒 测序(FLOPS)分析允许鉴定基因完整的和潜在的复制能力 HIV-1。使用Flips，5%的前病毒被鉴定为基因完整并有可能复制 能胜任长期(3-17年)有效抗逆转录病毒治疗(ART)的参与者。此外，完好无损 前病毒富含效应记忆的CD4+T细胞，多个完整的前病毒被发现 完全相同，表明细胞增殖在维持潜伏的HIV-1储存库中发挥了作用。 我们已经报道了HIV-1的2个长末端重复环状DNA(2-LTR环)，它们可能已经死亡 在体内病毒复制周期结束后，在ART开始后至少3年内保持相对较高的水平 在原发或慢性HIV-1感染期间含有整合酶链转移抑制物(INSTI)。自INSTIS以来 现在所有指南都建议将其作为初始方案的基本组成部分，大多数感染者 发起当代艺术可能会带来持久的2-ltr圈子。因此，重要的是要澄清 在开始含有INSTI的抗逆转录病毒疗法的患者中，是否存在基因完整的HIV-1 2-LTR环， 因为他们的持久力会干扰基因完整的基因的准确量化 具有复制能力的储集层。因此，我们将在拟议的研究中回答三个重要问题： (1)持续存在的HIV-1 2-LTR环在前3个月中是否在CD4+T细胞中包含基因完整的HIV-1基因组 多年的含有INSTI的艺术？如果是，与基因完整的线性/前病毒HIV-1相比，处于什么水平？ (2)基因完好、复制能力强的前病毒形式是如何分布在CD4+T细胞亚群中的 在启动含INSTI的抗逆转录病毒治疗之后？这种分布在治疗的前3年中有变化吗？ (3)HIV-1复制是否发生在含INSTI的抗逆转录病毒药物治疗的头3年，2-LTR和 整合的病毒序列相互关联，并随着时间的推移而进化？ 这将是HIV-1DNA的第一个特征，它准确地解释了线性/前病毒形式和2- 在开始使用含有INSTI的抗逆转录病毒药物治疗后，LTR循环，这现在是许多人的标准护理 司法管辖区。遗传上完整的2-LTR环的持续将导致对潜在的 即使使用了近乎全长的测序技术，也可以复制能力较强的潜伏艾滋病毒储存库。这 也将是第一次纵向评估遗传完整的前病毒和2-LTR圈和 在早期(0-3年)治疗期间，这些细胞是如何在CD4+T细胞亚群中进化的。这将通知潜在的 以治愈策略为目标。最后，它将澄清正在进行的病毒复制是否保持着潜伏的储存库 在治疗的头几个月，或者即使在这些早期时间点，这是否由细胞增殖驱动。

PROJECT SUMMARY Accurate identification of the replication-competent HIV-1 latent reservoir provides a basis for the design of any cure strategy; however, this is challenging because of its sparse distribution. Further, HIV-1 DNA exists in different forms and proviruses are genetically variable. The recently developed Full-Length Individual Proviral Sequencing (FLIPS) assay allows for the identification of genetically intact and potentially replication-competent HIV-1. Using FLIPS, 5% of the proviruses were identified as genetically intact and potentially replication competent in participants on effective long-term (3-17 years) antiretroviral therapy (ART). In addition, intact proviruses were enriched in effector memory CD4+ T cells, and multiple intact proviruses were found to be identical, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir. We have reported that HIV-1 episomal 2-long terminal repeat circular DNA (2-LTR circles), which are likely dead ends in the viral replication cycle in vivo, persist at relatively high levels for at least 3 years after initiation of ART containing an integrase strand transfer inhibitor (INSTI) during primary or chronic HIV-1 infection. Since INSTIs are now recommended in all guidelines as essential components of initial regimens, most infected individuals initiating contemporary ART may carry persistent levels of 2-LTR circles. It is therefore important to clarify whether genetically intact HIV-1 2-LTR circles are present in individuals who commenced INSTI-containing ART, because their persistence can interfere with the accurate quantification of the genetically intact and most likely replication-competent reservoir. We will therefore answer three important questions in the proposed study: (1) Do persisting HIV-1 2-LTR circles contain genetically intact HIV-1 genomes in CD4+ T cells during the first 3 years of INSTI-containing ART? If yes, at what level compare to genetically intact linear/proviral HIV-1? (2) How do genetically intact, replication-competent proviral forms distribute in CD4+ T cell subsets prior to and following initiation of INSTI-containing ART? Does this distribution change during the first 3 years of therapy? (3) Is HIV-1 replication occurring during the first 3 years of INSTI-containing ART and how do 2-LTR and integrated viral sequences relate to each other and evolve over time? This will be the first characterization of HIV-1 DNA, that accurately accounts for the linear/proviral forms and 2- LTR circles following initiation of treatment with INSTI-containing ART, which is now standard of care in many jurisdictions. Persistence of genetically intact 2-LTR circles will cause an overestimation of the potentially replication-competent latent HIV reservoir even when near full-length sequencing techniques are employed. This will also be the first longitudinal assessment of the landscape of genetically intact provirus and 2-LTR circles and how these evolve within CD4+ T cell subsets during early (0-3 years) therapy. This will inform the potential targeting of cure strategies. Finally, it will clarify whether ongoing viral replication maintains the latent reservoir during the first months of therapy, or whether this is driven by cellular proliferation even at these early time points.