An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection

Abstract

Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4+ T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size.

HIV-1 subtypes and latent reservoirs

“…Subsequently, subtype-specific assays have emerged [39]. A variant assay, called U-TILDA, attempted to circumvent the challenges of viral genetic diversity and amplify the various HIV-1 genetic subtypes with uniform efficiency [40 ▪ ,41]. Using a panel of viral molecular clones representing eight HIV-1 subtypes, a droplet-digital PCR identified that gag and pol regions, compared to ltr , are better suited to quantify HIV DNA [42].…”