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

条形码项目：追踪早期幼稚水库的策略

• 批准号: 10762820
• 负责人: Una T O'Doherty
• 金额: $ 82.89万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10762820
• 关键词: Acute; Antigens; Bar Codes; CD4 Positive T Lymphocytes; CD8B1 gene; Cells; Cessation of life; Chronic; Clonality; Coculture Techniques; Cohort Analysis; Data; Early treatment; Exons; Exposure to; Failure; Frequencies; Future; Goals; HIV; Half-Life; Individual; Infection; Infectious Agent; Inflammatory; Link; Maintenance; Measurement; Measures; Memory; Methods; Modeling; Phenotype; Play; Population; Positioning Attribute; Pre-Clinical Model; Preclinical Testing; Proliferating; Property; Provirus Integration; Proviruses; Resistance; Role; Sample Size; Sampling; System; T memory cell; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Tropism; Viral; Virus; Work; cohort; design; experience; experimental study; immune clearance; in vivo; innovation; insight; integration site; mathematical model; memory CD4 T lymphocyte; novel; novel strategies; pharmacologic; response; time use

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.

然而，新的证据表明，记忆 CD4+ T 细胞被认为是 HIV 的主要储存库。 具体而言，测序研究表明幼稚 CD4+ T 细胞具有重要作用。 在慢性进展者中对完整的艾滋病毒库有很大贡献，但在精英中实际上不存在 虽然初始水库很小，但它可能会对整个水库产生巨大的影响，因为我们 根据初步的克隆追踪研究，找到原始储存库重新填充记忆。 长期目标是剖析原始储存库对急性感染个体中整体储存库的作用 在主要的潜伏期模型中，我们将首先确定急性感染的程度和频率。 在这一人群中，幼稚病毒库尚未得到充分研究，但作为早期 ART 的启动。 由于变得更加频繁，有必要了解对整个水库的影响。 注意到原始储层可能是整体储层衰减的良好预测器。前提：底层。 该假设认为，原始水库具有独特的能力，可以提供长期相对安全的水源。 由于它们对免疫清除的相对抵抗力，它们成为原病毒的港湾，而记忆库则更多 容易表达 HIV 并经历 CD8 清除 目的：我们将确定感染者的贡献。 幼稚 T 细胞对急性治疗个体的储库动态进行研究 (Aim1)，并提出了一种新颖的离体模型 测试 naïve 贡献机制背后的基本假设（目标 2）。 为了实现这两个目标，我们将测量储存库的大小、多样性、克隆性、原病毒方向及其随时间的动态变化 为了限制避免稀释，我们开发了原病毒测序和新型整合位点测序。 利用 Primer-ID 将条形码原病毒序列连接到其整合位点的方法。 这种独特的条形码追踪方法使我们能够追踪单个受感染的幼稚 T 细胞的命运。 揭示了原始储层的独特特性，与其重新激活潜力以及其对 我们模型的一个关键创新是能够跟踪同源抗原、LRA 和 CD8。 在 Aim3 中，我们使用受限感染生成的数据。 （目标2）使用数学模型剖析幼稚和记忆感染的作用，然后比较 初始储存库对来自 Aim1 的急性治疗个体的 HIV 动态的影响我们设想了这项工作。 将表明，幼稚感染对于强大的储存库至关重要，并将最终提供有用的 我们将评估持久性和未来治疗机制的临床前模型。 幼稚型与记忆库上的各种 LRA 和 CD8，以提供更彻底的机制 这些实验将提供对潜伏期逆转失败的深入了解。 并最终揭示了变相针对该水库的新方法。