Concurrent measures of total and integrated HIV DNA monitor reservoirs and ongoing replication in eradication trials

Abstract

Objectives: Interest in targeting HIV reservoirs is fueling trials that may decrease reservoir size and/or induce viral replication. Therefore, we aimed to develop strategies to sensitively measure changes in these parameters in patients on and off antiretroviral therapy (ART). Achieving these goals may help evaluate the effects of future clinical trials.

Design: To determine the relationship between measurements of total and integrated HIV DNA and their role as markers of reservoir size and ongoing replication, these parameters were measured during the first year of ART, during long-term effective ART, and during a clinical trial aimed at targeting reservoirs.

Methods: Total and integrated HIV DNA were measured in patient samples using quantitative PCR techniques. CD4(+)T cell counts and plasma viremia were also monitored.

Results: Unintegrated HIV DNA became undetectable during the first year of ART. Total and integrated HIV DNA levels were generally equal in well controlled patients on ART, and low-level plasma viremia correlated best with integration measures. Finally, patients who controlled plasma viremia (<400 copies/ml) during interferon-α monotherapy exhibited a decrease in the level of integrated but not total HIV DNA and a rise in the ratio of total to integrated HIV DNA over time.

Conclusion: Our findings suggest that appearance of unintegrated HIV DNA reflects residual HIV expression and de-novo reverse transcription, providing insight into the mechanism by which interferon-α reduces the HIV reservoir. We conclude that concurrent measurements of total and integrated HIV DNA provide information regarding reservoir size and ongoing replication in trials targeting HIV.

Figures

Figure 1. The Ratio of Total to Integrated HIV DNA in patients on ART

a: The Ratio of Total to Integrated HIV DNA Approaches 1 Within One Year of ART Initiation. The top bar graphs depict the total and integrated HIV DNA in PBMC samples from 2 HIV+ patients during the first year of ART. Samples were collected at time 0 (Off-ART), and following 6 months, and 12 months of ART. Black bars indicate the copies of total HIV DNA per µl and gray bars indicate the copies of integrated HIV DNA per µl. The numbers in the graph indicate the ratio of total to integrated HIV DNA at each time point. A ratio greater than 2.1 indicates the presence of unintegrated HIV DNA; marked with an asterisk (*). The bottom line graphs depict the CD4+ T cell count per µl at each time-point tested. b: Measurements of Total and Integrated HIV DNA in Longitudinal samples from 4 patients on ART. PBMC from patients who maintained good control of plasma viremia were obtained for concurrent measurements of total and integrated HIV DNA. Patients had maintained viremia at <50 copies per mL of plasma when monitored every three months for more than one year prior to inclusion in the study. The CD4+ T cell count is plotted against the right y-axis for each patient. Asterisks (*) denote samples in which the ratio of total to integrated HIV DNA is greater than 2.1, indicating the presence of unintegrated HIV DNA. Viral load remained below 50 copies/ml blood in all patients three of four patients. In one patient who had one episode of viremia (823 copies/mL) on 1/03 (V) two samples exhibited a ratio of total to integrated HIV DNA >2.1 (*). Arrows denote two of the time points (4/7/03 and 6/30/03) at which viremia was undetectable after the one viremic episode.

Figure 2. Patients with higher levels of both total and integrated HIV DNA have statistically higher low level plasma viremia in a cross-sectional cohort of patients on ART

Total and integrated HIV DNA was measured in CD4+ T cells from patients on ART. Residual plasma viremia was measured by ultra-sensitive assays to levels between 1 and 50 copies/mL. Higher plasma viremia levels also correlated with higher levels of integrated HIV DNA, but not total HIV DNA. The ratio of total to integrated HIV DNA was not statistically different in the presence and absence of viremia.

Figure 3. Total and Integrated HIV DNA in patients from IFN-α trial

PBMC from patients that maintained low levels (

Figure 4. Changes in residual viremia mimic changes in the ratio of total to integrated HIV DNA in pateints responding to IFN-α

The number of patients with and without a ratio of total to integrated HIV DNA greater than 2.1 is shown for each time-point (a). The increase in the number of patients with detectable unintegrated HIV DNA corresponded to changes in plasma viremia as measured by ultra-sensitive methods at similar time-points (b).