Background

Latent reservoir (LR) poses a significant obstacle to HIV-1 disease management. Despite successful antiretroviral therapy (ART), low-level viral replication persists, attributed to the existence of a transcriptionally silent virus (Chun et al., 1995, 1997; Finzi et al., 1997; Siliciano et al., 2003). Precise quantitation of the size of this latent reservoir before and after the medical intervention is critical for 'cure' or therapeutic research.

A variety of assay formats have been developed to characterize HIV-1 latent reservoirs (Falcinelli et al., 2019). These assays differ from one another in terms of the viral product they measure, complexity, and sensitivity, among other attributes. DNA PCR-based assays offer the technically simplest experimental format. However, such assays quantify total proviral DNA, regardless of replication competence of the integrated viruses, thus, overestimating the overall viral reservoir size (Massanella and Richman, 2016). The highly popular quantitative viral outgrowth assay (QVOA) measures the number of infectious viral units per million CD4 T-cells (Siliciano and Siliciano, 2005). The QVOA, considered to be the gold standard technique, suffers from several limitations, including the complex experimental design, and the need for a large quantity of blood (120–180 mL). Additionally, since only a fraction of replication-competent proviruses is activated under any experimental condition, QVOA tends to underestimate the size of the viral reservoir.

Among a horde of assays available to characterize HIV-1 latent reservoirs, a strategy that aims to detect the frequency of cells producing viral transcripts offers the most powerful platform. The Tat/rev induced limiting dilution assay (TILDA) can measure cells producing multiply-spliced viral transcripts following cell activation (Procopio et al., 2015). The TILDA format offers a realistic approximation of the size of the replication-competent latent reservoir, as it detects HIV-1 transcripts, not proviral DNA. Importantly, TILDA exploits the power of PCR amplification, thus offering a highly sensitive experimental format. Additionally, unlike QVOA, TILDA requires a significantly smaller volume of a clinical sample (15–20 mL). Despite these considerable technical merits, TILDA is highly susceptible to the genetic variation of several HIV-1 viral subtypes. Therefore, the several TILDA platforms reported thus far are limited by the ability to target a specific HIV-1 genetic family, or even only a proportion of viral strains of a subtype (Bertoldi et al., 2020; Leyre et al., 2020). We recently reported a novel TILDA assay format labelled as U-TILDA ('U' for universal), that can target all the major genetic subtypes of HIV-1 (A, B, C, D, and AE subtypes) with high sensitivity and comparable diagnostic efficiency (Mehta et al., 2021). In contrast to subtype-specific TILDA formats, in U-TILDA, the primer pairs and the probe target the highly conserved exons of HIV-1, thus significantly broadening the breadth of detection of the assay.

Here, we describe the detailed protocol of U-TILDA using stored PBMC of an HIV-1 seropositive subject. The CD4 T-cells, negatively enriched using paramagnetic beads, and activated with PMA and ionomycin for 12 h, or left without activation for control, were serially diluted. One step qRT-PCR was performed in sixteen replica wells for each cell dilution. The viral transcripts were converted into cDNA using HIV-1-specific RT primers, without RNA purification. This was followed by the first-round of PCR amplification in the same reaction vial. Diluted PCR reaction content was then used as the template for the second-round of the nested-PCR, in a real-time PCR format. Finally, the Ct values of the replicate wells were converted to the frequency of transcription-competent viral events using a free web tool.