Abstract
Substantial efforts to eliminate or reduce latent HIV-1 reservoirs are underway in clinical trials and have created a critical demand for sensitive, accurate, and reproducible tools to evaluate the efficacy of these strategies. Alternative reservoir quantification assays have been developed to circumvent limitations of the quantitative viral outgrowth assay. One such assay is tat/rev induced limiting dilution assay (TILDA), which measures the frequency of CD4+ T cells harboring inducible latent HIV-1 provirus. We modified pre-amplification reagents and conditions (TILDA v2.0) to improve assay execution and first internally validated assay performance using CD4+ T cells obtained from cART-suppressed HIV-1-infected individuals. Detection of tat/rev multiply spliced RNA was not altered by modifying pre-amplification conditions, confirming the robustness of the assay, and supporting the technique’s amenability to limited modifications to ensure better implementation for routine use in clinical studies of latent HIV-1 reservoirs. Furthermore, we cross-validated results of TILDA v2.0 and the original assay performed in two separate laboratories using samples from 15 HIV-1-infected individuals. TILDA and TILDA v2.0 showed a strong correlation (Lin’s Concordance Correlation Coefficient = 0.86). The low inter-laboratory variability between TILDAs performed at different institutes further supports use of TILDA for reservoir quantitation in multi-center interventional HIV-1 Cure trials.
Highlights
Novel experimental approaches designed to reduce or eliminate long-lived HumanImmunodeficiency Virus type 1 (HIV-1) reservoirs are increasingly being tested in clinical trials, creating a demand for sensitive, accurate and reproducible assays to evaluate their potential effect on the size of the latent reservoir [1]
tat/rev induced limiting dilution assay (TILDA) [17] is a widely adopted method used to measure latent HumanImmunodeficiency Virus type 1 (HIV-1) reservoirs in different groups of people living with HIV-1
Due to its low sample requirement and short turn-around time compared to quantitative viral outgrowth assay (QVOA), TILDA is an appropriate alternative for quantifying the replication-competent
Summary
Novel experimental approaches designed to reduce or eliminate long-lived HumanImmunodeficiency Virus type 1 (HIV-1) reservoirs are increasingly being tested in clinical trials, creating a demand for sensitive, accurate and reproducible assays to evaluate their potential effect on the size of the latent reservoir [1]. Alternative methods are under development to circumvent these limitations in estimating HIV-1 reservoir size These include assays that quantify the amount of latently infected cells with inducible provirus (transcription-competent) or the number of cells capable of expressing viral proteins (translation-competent) [11,12,13,14,15,16]. These techniques require large blood draws to get a sufficient amount of input cells (10–15 million CD4+ T cells) [12], which may not always be available in interventional clinical studies. The assay has a broad dynamic range (up to 3 logs), an inter-assay coefficient of variation (CV) of 21%, and a limit of detection (LOD) of 1.4 inducible reservoir cells per million CD4+ T cells [17]
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