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High-Throughput Assay Platform for Quantifying Latent HIV Reservoirs

Description:

Fast-Track proposals will be accepted Number of anticipated awards: 1-2 Budget (total costs):

Phase I: $300,000 for up to 1 year
Phase II: $2,000,000 for up to 3 years
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 I 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 II 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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