Development of a next-generation quantitative viral outgrowth assay (QVOA) for the standardized measurement of the HIV-1 latent reservoir

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Project Summary/Abstract Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that infects CD4+ T cells of the immune system. If left untreated, HIV-1 infected individuals will progress to AIDS and may ultimately die as a result. Combination antiretroviral therapy is extremely effective at stopping the replication of HIV-1 in infected individuals. Despite the success of this therapy at suppressing HIV-1 replication to clinically undetectable levels, antiretroviral therapy is not curative. This is due to the persistence of HIV-1 in a silent, or latent, state within a subset of CD4+ T cells known as resting memory CD4+ T cells. In this latent state, these infected cells are not targeted by antiretroviral drugs and cannot be eliminated by the immune system. In HIV-1 infected individuals, latently infected CD4+ T cells are found at extremely low frequencies (~1 per million resting memory CD4+ T cells). However, this population of latently infected cells is very stable, demanding that HIV-1 infected individuals remain on antiretroviral therapy indefinitely. Therefore, this population of latently infected CD4+ T cells is the main barrier to curing HIV-1 infection. Developing strategies to eliminate latently infected cells is a major focus of the NIH, NIAID, and the HIV-1 research field. To demonstrate the efficacy of therapeutics targeting the latent reservoir, we must be able to measure the frequency of latently infected cells using rapid and accurate assays that can be scaled for widespread clinical use. The currently accepted gold-standard assay for measuring latent HIV-1 is the quantitative viral outgrowth assay developed by the Siliciano laboratory in the mid-1990s. Due to the complexity and high resource requirements of the QVOA, this assay can only be performed in a small number of research laboratories. Accelevir Diagnostics, LLC is developing a new quantitative viral outgrowth assay with improved precision, reproducibility, and scalability. Broadly, this proposal aims to optimize assay conditions and perform key assay validation studies, including laboratory automation studies. The goal of this proposal is to develop an optimized commercial prototype, with accompanying standard operating procedures, and set the stage for rapid analytical validation and market entry. !