Effect of Short-Term Antiretroviral Therapy Interruption on Levels of Integrated HIV DNA

Abstract

Analytic treatment interruption (ATI) studies are required to evaluate strategies aimed at achieving ART-free HIV remission, but the impact of ATI on the viral reservoir remains unclear. We validated a DNA size selection-based assay for measuring levels of integrated HIV DNA and applied it to assess the effects of short-term ATI on the HIV reservoir. Samples from participants from four AIDS Clinical Trials Group ATI studies were assayed for integrated HIV DNA levels. Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained for 12 participants with available samples pre-ATI and approximately 6 months after ART resumption. Four participants also had samples available during the ATI. The median duration of ATI was 12 weeks. Validation of the HIV integrated DNA size-exclusion (HIDE) assay was performed using samples spiked with unintegrated HIV DNA, HIV-infected cell lines, and participant PBMCs. The HIDE assay eliminated 99% of unintegrated HIV DNA species and strongly correlated with the established Alu-gag assay. For the majority of individuals, integrated DNA levels increased during ATI and subsequently declined upon ART resumption. There was no significant difference in the levels of integrated HIV DNA between the pre- and post-ATI time points, with a median ratio of post- to pre-ATI HIV DNA levels of 0.95. Using a new integrated HIV DNA assay, we found minimal change in the levels of integrated HIV DNA in participants who underwent an ATI, followed by 6 months of ART. This suggests that short-term ATI can be conducted without a significant impact on the levels of integrated proviral DNA in the peripheral blood.IMPORTANCE Interventions aimed at achieving sustained antiretroviral therapy (ART)-free HIV remission require treatment interruption trials to assess their efficacy. However, these trials are accompanied by safety concerns related to the expansion of the viral reservoir. We validated an assay that uses an automated DNA size-selection platform for quantifying levels of integrated HIV DNA and is less sample- and labor-intensive than current assays. Using stored samples from AIDS Clinical Trials Group studies, we found that short-term ART discontinuation had minimal impact on integrated HIV DNA levels after ART resumption, providing reassurance about the reservoir effects of short-term treatment interruption trials.

Keywords: HIV; assay; integrated DNA; reservoir; treatment interruption.

FIG 1

Viral loads and CD4⁺ counts. Blue lines depict viral loads, and red lines depict CD4⁺ cell counts. The ATI period is shaded gray, and on-ART data are shown as white.

FIG 2

HIDE assay validation. (a) DNA recovery percentage for each sample after 20-kb size selection on the BluePippin platform. (b) HIV DNA levels of uninfected DNA spiked with linear or circular HIV DNA before and after genomic DNA size selection. (c) HIV DNA levels in three cell lines (J-Lat, HIV-infected CEM, and two dilutions of 8E5) containing integrated, replication-deficient HIV provirus before and after size selection by HIDE assay. (d) Levels of total and size-selected HIV DNA in a representative participant (PID 4) at pre-ATI, during ATI, and post-ATI time points. (e) Correlation between integrated HIV DNA levels measured by the HIDE assay and by the Alu-gag assay for cell lines (triangles) and participant samples (circles).

FIG 3

Treatment interruption had minimal effect on levels of integrated HIV DNA in PBMCs. (a) Levels of integrated HIV DNA pre-ATI, during treatment interruption, and post-ATI in four participants. Participant numbers are written next to the corresponding pre-ATI data point. (b) Pre- and post-ATI levels of integrated HIV DNA for 12 participants. For samples that are below the LOD, the LOD for that sample is used (open circle). (c) Ratio of post-ATI to pre-ATI levels of integrated HIV DNA. An open circle represents whether either the pre- or post-ATI time point was below the LOD. The line represents the median value.