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....