Abstract
The presence of a stable HIV-1 reservoir persisting over time despite effective antiretroviral suppression therapy precludes a cure for HIV-1. Characterizing and quantifying this residual reservoir is considered an essential prerequisite to develop and validate curative strategies. However, a sensitive, reproducible, cost-effective, and easily executable test is still needed. The quantitative viral outgrowth assay is considered the gold standard approach to quantify the reservoir in HIV-1-infected patients on suppressive ART, but it has several limitations. An alternative method to quantify the viral reservoir following the reactivation of latent HIV-1 provirus detects multiply-spliced tat/rev RNA (msRNA) molecules by real-time PCR [tat/rev induced limiting dilution assay (TILDA)]. This article provides a perspective overview of the clinical relevance, various applications, recent advancements of TILDA, and how the assay has contributed to our understanding of the HIV-1 reservoir.
Highlights
Over the past decade, multiple interventions to reduce or eliminate the latent HIV-1 reservoir have been proposed
A panel of latent HIV-1 reservoir assays have been recommended for use in HIV cure intervention trials
The quantitative viral outgrowth assay (QVOA), currently considered the reference assay for quantifying the replication-competent HIV-1 reservoir (Eriksson et al, 2013), quantifies the number of resting CD4+ T cells releasing infectious virus after in vitro stimulation
Summary
Multiple interventions to reduce or eliminate the latent HIV-1 reservoir have been proposed. Due to posttranscription blocks in RNA processing (Yukl et al, 2018), not all cells producing tat/rev msRNA transcripts will yield infectious virus (Hong et al, 2018), and quantifying these cells overestimates the replication-competent HIV-1 reservoir size Despite these discrepancies and limitations, TILDA is a wellestablished approach to quantify the inducible HIV-1 reservoir as a proxy for replication competence, and a technically feasible alternative to QVOA for routine execution in large scale HIV cure intervention trials [requires less blood, shorter assay turnaround time (2 days), and medium throughput]. The assay is very robust, as demonstrated in multiple studies to test intraand inter-laboratory reproducibility, which is important for the cross-validation of results in multi-center, multi-laboratory clinical trials
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