1 research outputs found
Inter-Laboratory Reproducibility of Inducible HIV-1 Reservoir Quantification by TILDA
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