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. 2016 Mar 3;12(3):e1005472.
doi: 10.1371/journal.ppat.1005472. eCollection 2016 Mar.

Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth

Maja Kiselinova  1   2 , Ward De Spiegelaere  1 , Maria Jose Buzon  2   3 , Eva Malatinkova  1 , Mathias Lichterfeld  2   3   4 , Linos Vandekerckhove  1
Affiliations

Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth

Maja Kiselinova et al. PLoS Pathog. .

Erratum in

Abstract

The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6) between time point 1 and 2; and median of 31 days (IQR: 28-36) between time point 2 and 3. Patients were median of 6 years (IQR: 3-12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2-8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication-competent virus in ART suppressed patients.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Workflow schematics.
Fig 2
Fig 2. Viral reservoir dynamics.
Long and short-term dynamics of cellular markers of HIV-1 persistence. A) unspliced HIV-1 RNA; B) multiply spliced HIV-1 RNA; C) Total HIV-1 DNA; and D) 2LTR circles. *Of note: all analysis was performed using non-parametric statistics.
Fig 3
Fig 3. Linear regression analysis between viral reservoir markers.
Associations between: A) Integrated HIV-1 DNA and total HIV-1 DNA; B) Integrated HIV-1 DNA and unspliced HIV-1 RNA; C) Integrated HIV-1 DNA and viral outgrowth assay; D) Total HIV-1 DNA and viral outgrowth assay; and E) Total HIV-1 DNA and viral outgrowth assay–outliers removed.
Fig 4
Fig 4. Bland Altman analysis.
Agreement for HIV-1 copies quantification between: A) Total HIV-1 DNA (IDNA) and IUPM, mean difference (log 10) and 95% Limits of agreement; B) Slope-intercept equation for total HIV-1 DNA and IUPM; C) Integrated HIV-1 DNA (DNA) and IUPM, mean difference (log 10) and 95% Limits of agreement; D) Slope-intercept equation for integrated HIV-1 DNA and IUPM; E) Total HIV-1 DNA (DNA) and integrated HIV-1 DNA (IDNA), mean difference (log 10) and 95% Limits of agreement; F) Slope-intercept equation for total and integrated HIV-1 DNA; G) Total HIV-1 DNA (DNA)–time point 1 and IUPM–time point 2, mean difference (log 10) and 95% Limits of agreement; and H) Slope-intercept equation for total HIV-1 DNA–time point 1 and IUPM–time point 2. *Of note: Open label symbol represent test results below the detection threshold for VOA.
Fig 5
Fig 5. Linear regression analysis between unspliced (us) HIV-1 RNA and restriction factors.
Associations between us HIV-1 RNA and: A) TRIM5α; B) SAMHD1; C) MX2; D) SLFN11; E) pSIP1; F) APOBEG3G; and G) PAF1.
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