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High-Throughput Assay Platform for Quantifying Latent HIV Reservoirs

Background
One of the most significant hurdles to overcome in evaluating strategies to cure HIV infection is the lack of a simple method for quantifying changes in the size of the latent reservoir of replication-competent HIV in resting CD4+ memory T cells in individuals on highly effective antiretroviral therapy. Most of the HIV DNA in these cells represents defective virus; less than 0.01% of highly purified resting CD4 cells harbor replication-competent provirus.  As a result, PCR-based methods tend to over-estimate the size of the reservoir and do not correlate with the number of cells producing functional virus in a viral outgrowth assay. However, viral outgrowth assays are labor-intensive and require large volumes of blood.
Project Goal
The goal of this project is to design a high-throughput assay platform that can be used to reproducibly quantify changes in the size of the replication-competent latent HIV reservoir in resting CD4+ memory T cells isolated from individuals on highly effective antiretroviral therapy. Applicants must provide a plan for validating the assay by demonstrating correlation with quantitative viral outgrowth assays (QVOA) and/or functional non-induced HIV proviruses using cells isolated from virally suppressed HIV+ individuals on optimized antiretroviral therapy.
Phase 1 activities
?       Development of technologies for detecting replication-competent latent proviruses
?       Validation of detection methods using standardized controls
?       Optimization of sensitivity to detect low-frequency latently infected cells
?       Demonstration of correlation with replication-competent provirus vs. defective provirus
Phase 2 activities
?       Further optimization of the assay platform technology and validation of assay reproducibility
?       Increased throughput
?       Comparison of assay to other methods published in the literature
?       Testing of clinical samples from diverse cohorts of HIV+ individuals with varying levels of residual viral reservoirs
?       Comparison of blood vs. tissue samples from virally suppressed individuals
?       Modification of assay to detect latent HIV in humanized mouse models and latent SIV in nonhuman primate models in the context of optimized antiretroviral therapy
 
?       Use of assay to demonstrate changes in the size of the latent HIV/SIV reservoir in response to an intervention

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