Abstract
BackgroundThe persistence of HIV-1 in reservoir cells is one of the major obstacles to eradicating the virus in infected individuals receiving combination antiretroviral therapy (ART). HIV-1 persists in infected cells as a stable integrated genome and more labile unintegrated DNA (uDNA), which includes linear, 1-LTR and 2-LTR circular DNA. 2-LTR circle DNA, although less abundant, is considered a surrogate marker of recent infection events and is currently used instead of the other unintegrated species as a diagnostic tool. This pilot study aimed to investigate how to best achieve the measurement of uDNA.MethodsA comparative analysis of two qPCR-based methods (U-assay and 2-LTR assay) was performed on the blood of 12 ART-naïve, 14 viremic and 29 aviremic On-ART patients and 20 untreated spontaneous controllers (HIC), sampled at a single time point.ResultsThe U-assay, which quantified all unintegrated DNA species, showed greater sensitivity than the 2-LTR assay (up to 75%, p < 0.0001), especially in viremic subjects, in whom other forms, in addition to 2-LTR circles, may also accumulate due to active viral replication. Indeed, in aviremic On-ART samples, the U-assay unexpectedly measured uDNA in a higher proportion of samples (76%, 22/29) than the 2-LTR assay (41%, 12/29), (p = 0.0164). A trend towards lower uDNA levels was observed in aviremic vs viremic On-ART patients, reaching significance when we combined aviremic On-ART and HIC (controllers) vs Off-ART and viremic On-ART subjects (non-controllers) (p = 0.0003), whereas 2-LTR circle levels remained constant (p ≥ 0.2174). These data were supported by the high correlation found between uDNA and total DNA (r = 0.69, p < 0.001).ConclusionsThe great advantage of the U-assay is that, unlike the 2-LTR assay, it allows the accurate evaluation of the totality of uDNA that can still be measured even during successful ART when plasma viremia is below the cut-off of common clinical tests (< 50 copies/mL) and 2-LTR circles are more likely to be under the quantification limit. UDNA measurement in blood cells may be used as a biomarker to reveal a so far hidden or underestimated viral reservoir. The potential clinical relevance of uDNA quantification may lead to improvements in diagnostic methods to support clinical strategies.
Highlights
The persistence of HIV-1 in reservoir cells is one of the major obstacles to eradicating the virus in infected individuals receiving combination antiretroviral therapy (ART)
Sensitivity differences between the two versions of the assay for unintegrated DNA (uDNA) measurement To examine the sensitivity of the U-assay and 2-long terminal repeated (LTR) assay for uDNA quantification, we used blood samples from Off-ART, viremic On-ART, aviremic On-ART as well as HIVinfected controllers (HIC) patients (Fig. 2, Additional file 2: Table S2)
We found a lower rate of quantified samples using the 2-LTR assay: 25% (3/12), 29% (4/14) and 41% (12/29) for Off-ART, On-ART viremic and OnART aviremic patients, respectively
Summary
The persistence of HIV-1 in reservoir cells is one of the major obstacles to eradicating the virus in infected individuals receiving combination antiretroviral therapy (ART). The uDNA exists in linear and circular DNA forms The latter contain one long terminal repeated (LTR) copy (1-LTR) or two LTR copies (2-LTR) generated by ligation of both ends of the linear precursor by the host cell non-homologous end joining (NHEJ) pathway and other rearranged circular species [13,14,15]. Some of these forms derive from autointegration events or error-prone reverse transcription reactions leading, for instance, to 2-LTR circles harboring imperfect palindromic junctions in contrast to 2-LTR circles originating from NHEJ [16]. Recent data suggest that 2-LTR circle DNA may constitute a potential reserve supply of genomes for de novo integration [23]
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