Abstract
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 Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-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 Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1–12) and 14 (5–26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-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. Alu-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.
Highlights
IntroductionWithin the B cell follicle have recently been shown to be enriched in replication competent provirus [7,8,9,10,11]
This year, Artesi M. et al presented a novel approach called Pooled CRISPR Inverse PCR sequencing (PCIP-seq), which allows for the simultaneous identification of the integration site and tracking of clone abundance while sequencing the provirus inserted at that position [49]
Unlike total human immunodeficiency virus type-1 (HIV-1) DNA quantification methods, measurements of proviral HIV-1 DNA can be more challenging, because they rely on the amplification of the junction between human genome and HIV using an HIV-1 specific primer and a primer that anneals to human Alu sequences that are interspersed within the human genome at about 5 kb distance from each other [4,52,63,64,65,66,67]
Summary
Within the B cell follicle have recently been shown to be enriched in replication competent provirus [7,8,9,10,11]. A high proportion of regulatory T cells (Treg) and Th17 cells has been shown to harbour HIV-1 provirus [12,13,14,15,16]. CD32+ T cells have been identified as a reservoir of proviral DNA [17,18]; the contribution of these cells to the viral reservoir remains controversial [19,20,21,22].
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