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

• Main Authors: Kavita Mehta, Yuvrajsinh Gohil, Swarnima Mishra, Anish D’silva, Afzal Amanullah, Deepak Selvam, Neelam Pargain, Narendra Nala, G. N. Sanjeeva, Udaykumar Ranga
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2021-08-01
• Series: Frontiers in Immunology
• Subjects: latent reservoir; HIV latency; HIV-1; TILDA; transcriptional silence
• Online Access: https://www.frontiersin.org/articles/10.3389/fimmu.2021.715644/full

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.