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

项目摘要 准确鉴定具有复制能力的HIV-1潜伏库为设计任何 治愈策略;然而，这是具有挑战性的，因为它的稀疏分布。HIV-1 DNA存在于 不同的形式和原病毒是遗传可变的。最近开发的全长个体前病毒 测序（FLIPS）试验允许鉴定基因完整且具有潜在复制能力的 HIV-1使用FLIPS，5%的前病毒被鉴定为基因完整并可能复制 在有效的长期（3-17年）抗逆转录病毒治疗（ART）的参与者的能力。此外，完好无损的 前病毒在效应记忆CD 4 + T细胞中富集，并且发现多个完整的前病毒在 相同，表明细胞增殖在维持潜伏的HIV-1储库中的作用。 我们已经报道了HIV-1附加型2-长末端重复环状DNA（2-LTR环），这可能是死亡的， 在体内病毒复制周期结束，在ART开始后持续相对高水平至少3年 在原发性或慢性HIV-1感染期间含有整合酶链转移抑制剂（Integrase strand transfer inhibitor，CITI）。自INSTI以来 现在所有指南都建议将其作为初始治疗方案的基本组成部分，大多数感染者 启动当代ART可能携带持续水平的2-LTR环。因此，有必要澄清 在开始含INSTI的ART的个体中是否存在遗传上完整的HIV-1 2-LTR环， 因为它们的持续存在会干扰基因完整性的准确量化，并且很可能 可复制储层。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个月内在CD 4 + T细胞中含有遗传上完整的HIV-1基因组吗？ 几年的INSTI含有艺术？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)基因完整的、有复制能力的前病毒形式如何在CD 4 + T细胞亚群中分布， 开始含INSTI的ART后？在治疗的前3年，这种分布是否发生变化？ (3)HIV-1复制是否发生在含INSTI的ART的前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-1 DNA的第一个特征，它准确地解释了线性/前病毒形式和2- LTR周期开始治疗后与INSTI含有艺术，这是现在的标准治疗，在许多 辖区遗传上完整的2-LTR环的持续存在将导致对潜在的 甚至当采用接近全长测序技术时，也是可复制的潜伏HIV储库。这 这也将是对遗传上完整的前病毒和2-LTR环景观的首次纵向评估， 这些在早期（0-3年）治疗期间在CD 4 + T细胞亚群中如何演变。这将告知潜在的 治疗策略的目标。最后，它将阐明是否正在进行的病毒复制保持潜伏水库 在治疗的最初几个月，或者这是否是由细胞增殖驱动的，即使在这些早期时间点。

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.