AUTHOR=Mehta Kavita , Gohil Yuvrajsinh , Mishra Swarnima , D’silva Anish , Amanullah Afzal , Selvam Deepak , Pargain Neelam , Nala Narendra , Sanjeeva G. N. , Ranga Udaykumar TITLE=An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection JOURNAL=Frontiers in Immunology VOLUME=12 YEAR=2021 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.715644 DOI=10.3389/fimmu.2021.715644 ISSN=1664-3224 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.