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. 2020 May 19;18(1):204.
doi: 10.1186/s12967-020-02368-y.

A comparative analysis of unintegrated HIV-1 DNA measurement as a potential biomarker of the cellular reservoir in the blood of patients controlling and non-controlling viral replication

Chiara Orlandi  1 , Benedetta Canovari  2 , Federica Bozzano  3 , Francesco Marras  4 , Zeno Pasquini  2   5 , Francesco Barchiesi  2   5 , Andrea De Maria  4   6 , Mauro Magnani  1 , Anna Casabianca  7
Affiliations

A comparative analysis of unintegrated HIV-1 DNA measurement as a potential biomarker of the cellular reservoir in the blood of patients controlling and non-controlling viral replication

Chiara Orlandi et al. J Transl Med. .

Abstract

Background: 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). 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.

Methods: A 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.

Results: The 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).

Conclusions: The 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.

Keywords: 2-LTR circles; Aviremic patients; Blood cells; HIC; HIV-1; Reservoir; Total HIV DNA; Unintegrated HIV DNA; Viremic patients; qPCR.

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Conflict of interest statement

There are no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1
Real-time PCR strategy for the quantification of various HIV-1 DNA species. Approximate locations of forward and reverse primers used for the amplification of both total HIV-1 DNA (integrated and unintegrated species) and unintegrated forms (PBS primers indicated in black) and for the selective amplification of 2-LTR circles (2-LTR primers indicated in grey) are shown. The table shows the legend and sequences of primers used. In the U-assay, the QIAamp plasmid Mini Kit (Qiagen) was used to separate the low molecular weight DNA (LMW, consisting of unintegrated HIV DNA species) from the high molecular weight DNA (HMW, containing the integrated proviral HIV DNA). In the 2-LTR assay, the elongation time of 35 s, is not compatible with a reliable amplification of non-specific products. The HIV-1 DNA genome is not drawn to scale
Fig. 2
Fig. 2
Sensitivity differences between the U-assay and 2-LTR assay for unintegrated HIV DNA detection. a Quantification of unintegrated HIV DNA and 2-LTR circles in blood samples of Off-ART patients (n = 12), viremic (n = 14) and aviremic (n = 29) On-ART patients, and untreated infected controller patients (HIC, n = 20). The dotted line indicates the limit of quantification (QL, 2 copies). Detectable values below the QL are depicted by empty symbols. b Proportion of samples from the four groups of HIV-1 patients, with quantifiable (colored bars) or detectable (white bars) levels of unintegrated HIV DNA (black) and 2-LTR circles (gray). The number of samples from each group quantified or detected below the QL by each assay is indicated. The frequency with which the levels of unintegrated viral DNA were quantified was higher using the U-assay than the 2-LTR assay (overall p < 0.0001, Fisher’s exact test)
Fig. 3
Fig. 3
Quantification of total and unintegrated HIV DNA in blood samples from HIV-1 infected patients. Levels of a total HIV DNA, b unintegrated HIV DNA and c 2-LTR circles were measured in Off-ART patients (n = 12), viremic (n = 14) and aviremic (n = 29) On-ART patients, and untreated infected controller patients (HIC, n = 20). Each DNA sample was tested using three 0.5-μg replicates and three (or up to five) 1.0-μg replicates for a total of up to 6.5 µg (~ 106 WBC). The number of copies was reported per µg of cellular DNA (left) and per 104 CD4+ (right) converting the value relative to 1 µg of cellular DNA for the CD4+ count and the proportion of CD4+ T cells in each subject. Lines represent the median and 25th to 75th percentiles. The Kruskal–Wallis test and Dunn’s multiple comparison test were used for comparisons, only p < 0.05 are reported; *p < 0.05; **p < 0.01; ***p < 0.001. d Correlations between the levels of total HIV DNA, unintegrated HIV DNA and 2-LTR circles expressed as copy number per µg of cellular DNA or per 104 CD4+ T cells in the four different groups of HIV-1 patients. Correlations were assessed by Spearman’s rank-correlation coefficient. The correlation coefficients and p-values are shown in each plot
Fig. 4
Fig. 4
Composition of HIV DNA in blood samples from HIV-1 infected patients. Percentages of a uDNA among total HIV DNA, b 2-LTR circles among total HIV DNA and c 2-LTR circles among the totality of uDNA forms in Off-ART patients (n = 12), viremic (n = 14) and aviremic (n = 29) On-ART patients, and untreated infected controller patients (HIC, n = 20). Each bar within a group represents the datum from a single sample. The percentages refer to data expressed as HIV DNA copies per µg of DNA. The percentages of uDNA or 2-LTR circles in total HIV DNA and of 2-LTR circles in uDNA were reduced to 0% when uDNA or 2-LTR circle copy numbers were < QL of the assays. When both uDNA and 2-LTR circle copy numbers were < QL, the fractions are reported as non-determinable (ND). d The percentages from the above a-b panels are presented as mean ± SEM. Subtracting uDNA and 2-LTR circles from total HIV DNA, the remaining fraction is constituted by integrated HIV DNA (black dotted line). e The percentages from the above c panel are presented as mean ± SEM. Subtracting the 2-LTR circle DNA fraction from uDNA, the remaining fraction is constituted by the sum of 1-LTR circles, 2-LTR circles with anomalous junction sequence, rearranged circles and linear HIV DNA (red dotted line). The Kruskal–Wallis test and Dunn’s multiple comparison test were used for comparisons among groups of patients (black asterisks), the Wilcoxon signed rank test was used for matched samples (red asterisks), only p < 0.05 are reported; *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 5
Fig. 5
Correlations between the levels of the different forms of HIV DNA in blood samples from HIV-1 infected patients. Samples were obtained from Off-ART patients (n = 12), viremic (n = 14) and aviremic (n = 29) On-ART patients, and untreated infected controller patients (HIC, n = 20). Correlations were assessed by Spearman’s rank-correlation coefficient. The correlation coefficients and p-values are shown in each plot; *p < 0.05; **p < 0.01; ***p < 0.001
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