Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human Repeat Sequences.

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called -5LTR PCR, which utilises novel primer combinations. We tested the -5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free -gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1-12) and 14 (5-26) with the -gag and -5LTR, respectively. The ratios between -gag vs -5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2-5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. -5LTR assay sensitivity was also determined using an "integrated standard"; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.