Impact of pre-amplification conditions on sensitivity of the tat/rev induced limiting dilution assay

Antiretroviral therapy (ART) can lower a patient’s HIV plasma viral load to an undetectable level, but following cessation of ART viremia rapidly rebounds. It has been shown that ART does not eliminate latent viruses sequestered into anatomical and cellular reservoirs. Therefore, in patients that have ceased ART, the following rebound in HIV viremia is caused by the activation of latent HIV reservoirs. A major issue in HIV cure research is the quantification of these latent HIV reservoirs. Various reservoir measurement methods exist, but the gold standard technique remains the culture-based quantitative viral outgrowth assay (QVOA). Recently, a new PCR-based assay, named the tat/rev induced limiting dilution assay (TILDA) was described which measures the frequency of inducible latently infected CD4⁺ T cells that actively produce multiply-spliced RNA coding for the Tat/Rev proteins. The objective of this study was to further optimize the assay by examining the influence of varied factors, such as the amount of products transferred from the pre-amplification step to the PCR reaction, storage of pre-amplification products prior to PCR runs, and the number of cells used, on the assay’s sensitivity and reproducibility. We also investigated whether the assay could be used to quantify HIV reservoirs in monocytes/macrophages.