The barcode project: a strategy to track the early naïve reservoir

记忆中的CD4+T细胞被认为是HIV的主要储存库；然而，新的证据表明 幼稚的CD4+T细胞的重要作用。具体来说，测序研究表明，天真的储集层 对慢性进展期患者完整的HIV储备库有很大贡献，但在精英人群中几乎没有 控制器。虽然天真水库很小，但它可能会对整个水库产生过大的影响，因为我们 根据初步的克隆追踪研究，寻找天真的储藏者重新填充了记忆。目标： 长期目标是在急性感染的个体中剖析单纯宿主在总体宿主中的作用。 在潜伏期的主要模型中。我们将首先确定幼稚感染的程度和频率 接受治疗的个体。在这群人中，对天真储油层的研究一直不足，但作为早期的艺术启蒙 随着开采变得更加频繁，了解其对整个储集层造成的后果是至关重要的。我们 假设天真的储集层可以很好地预测整个储集层的衰败。前提：基础 这一假设是这样一种想法，即天真的水库具有一种独特的能力，可以提供相对安全的长寿 由于前病毒对免疫清除的相对抵抗力，所以港口，而记忆库则更多 容易表达HIV，并经历CD8清除。目标：我们将确定感染者的贡献 急性治疗个体(Aim1)中幼稚T细胞对水库动力学的影响，并提出一种新的体外模型 测试天真贡献机制背后的潜在假设(AIM2)。设计和方法：In 这两个目标，我们将测量储集层的大小，多样性，克隆，前病毒的方向和它们随时间的动态 使用前病毒测序和一种新的整合位点测序。为了避免限制稀释，我们开发了 方法利用Primer-ID将条形码前病毒序列与其整合位点连接。我们的优化模型 具有条形码跟踪的独特功能使我们能够跟踪单个感染的幼稚T细胞的命运。这种方法 揭示了与其重新激活潜力相关的朴素储集层的独特属性以及它对 同源抗原、LRAs和CD8s。我们模型的一个关键创新是能够跟踪 扰乱水库的各种条件。在Aim3中，我们使用从限制性感染生成的数据 (AIM2)使用数学模型剖析幼稚和记忆感染的作用，然后比较 来自Aim1的急性治疗个体中幼稚病毒储备者对HIV动态的影响。我们设想这项工作 将表明幼稚感染是一个强大的储蓄者的基础，并最终将提供有用的 用于评估持久性机制和未来治疗的临床前模型。我们将评估这些措施的效果 天真与记忆库上的各种LRA和CD8，以提供更全面的机制 对朴素储集层的理解。这些实验将为潜伏期逆转的失败提供深入的见解 并最终揭示了变相瞄准这个水库的新方法。

Memory CD4+ T cells are believed to be the primary reservoir for HIV; however, emerging evidence suggests an important role for naïve CD4+ T cells. Specifically, sequencing studies suggest the naïve reservoir contributes substantially to the intact HIV reservoir in chronic progressors, but is practically absent in elite controllers. While the naïve reservoir is small, it may have outsized effects on the overall reservoir since we find the naïve reservoir repopulates the memory based on preliminary clone tracking studies. Objective: The long-term goal is to dissect the role of the naïve reservoir on the overall reservoir in acutely infected individuals and in a primary model of latency. We will first determine the extent and frequency of naïve infection in acutely treated individuals. The naïve reservoir has been understudied in this population, but as early ART initiation becomes more frequent, it is essential to understand the consequences on the overall reservoir. We hypothesize that the naïve reservoir may be a good predictor of overall reservoir decay. Premise: Underlying this hypothesis is the idea that the naïve reservoir has a unique ability to provide a long-lived relatively safe harbor for proviruses due to their relative resistance to immune clearance, while the memory reservoir is more prone to express HIV and to experience CD8 clearance. Aims: We will determine the contribution of infected naïve T cells to reservoir dynamics in acutely treated individuals (Aim1) and present a novel ex vivo model to test the underlying hypothesis behind the mechanism of naïve's contribution (Aim2). Design and Methods: In both aims, we will measure reservoir size, diversity, clonality, proviral orientation and their dynamics over time using proviral sequencing and a novel integration site sequencing. To avoid limiting dilution, we developed methods utilizing Primer-ID to link barcoded proviral sequences to their integration sites. Our optimized model with barcode tracking uniquely enables us to follow the fate of individual infected naïve T cells. This approach reveals distinct properties of the naïve reservoir related to its reactivation potential as well as its response to cognate antigen, LRAs and CD8s. A critical innovation of our model is the ability to track specific subsets under various conditions that perturb the reservoir. In Aim3, we use the data generated from restricted infections (Aim2) to dissect the role of naïve and memory infection using mathematical models and then compare the effect of the naïve reservoir on HIV dynamics in acutely treated individuals from Aim1. We envision this work will show that naïve infection is fundamental to a formidable reservoir and will ultimately provide a useful preclinical model for evaluating mechanisms of persistence and future therapies. We will evaluate the effect of various LRAs and CD8s on the naïve versus memory reservoir to provide a more thorough mechanistic understanding of the naïve reservoir. These experiments will provide insights into the failure of latency reversal and ultimately shed light on new approaches for targeting this reservoir in disguise.