Concurrent Measures Of Total And Integrated HIV DNA Monitor Reservoirs And Ongoing Replication In Eradication Trials

Recent clinical reports have rekindled an interest in developing a cure for HIV [1–3]. This renewed interest is fueling several studies aimed at targeting treatment resistant reservoirs in patients with HIV [4–7]. While these studies have already yielded interesting and informative results, more work is needed to determine the efficacy of new therapeutic approaches in clinical trials. In many studies, measuring changes in reservoir size and detecting the onset of viral replication would help evaluate the effects of each therapeutic approach, their efficacy in individual patients, and the mechanisms involved in effecting a cure. HIV persists in the cells of patients on antiretroviral therapy (ART), creating a reservoir that resurfaces if therapy is discontinued [8–3]. Currently, the infectious units per million (IUPM) assay remains the best accepted method to measure actual HIV reservoirs [14] but PCR measurements of either total or integrated cellular HIV DNA have been proposed as surrogate measures of reservoir size [6]. Although the IUPM assay is considered the gold-standard, this method requires large numbers of viable cells and has a high variance [14] making it difficult to reliably measure small differences in replication competent virus. Measurements of total and integrated HIV DNA require fewer cells and produce results with smaller errors [15, 16]. However, these methods overestimate reservoir size because they include proviurses that do not contribute to viremia. Furthermore, whether the proportion of replication incompetent DNA is constant over time is unclear. Total HIV DNA further overestimates reservoir size because it includes unintegrated HIV DNA species with variable half lives. Therefore, it remains unclear whether total or integrated DNA would be a better surrogate for measuring the HIV reservoir and how these two measures differ in specific clinical settings. During viral replication, most of the cell-associated HIV DNA is full-length, linear, and unintegrated [10]. Only a fraction of total HIV DNA is integrated and a smaller fraction still is capable of producing infectious virus [10]. After completion of reverse transcription, unintegrated HIV DNA accumulates in cells if integration does not occur. Since the majority of unintegrated HIV DNA may degrade and contributes little to viral production [17–20], measuring integrated HIV DNA could be a more reliable surrogate of replication-competent virus than measuring total HIV DNA. It has been previously reported that untreated HIV+ patients (progressors and elite-suppressors) exhibit significantly different levels of total compared to integrated HIV DNA in PBMC [10, 15, 16]. The difference between these measures in patients with ongoing replication [21, 22] denotes that there is detectable unintegrated HIV DNA and that total and integrated HIV DNA are not interchangeable in the presence of ongoing replication. However, since the majority of unintegrated HIV DNA appears to have a short half-life in vitro, it is reasonable to expect that most of the HIV DNA will be integrated over time when replication is completely halted [23]. Therefore, if effective ART controls ongoing replication, the levels of total and integrated HIV DNA should be similar on ART because the majority of unintegrated HIV DNA should disappear over time [23, 24]. Thus, measures of total and integrated HIV DNA have been suggested to be interchangeable surrogates for the reservoir size in PBMC in patients on ART [23, 25, 26]. However, therapies that target reservoirs may induce ongoing replication making it important to determine which measure is a more reliable marker for reservoir size and how often measurements of total and integrated HIV DNA are unequal in patients on ART. Here, we apply a unique, robust, and sensitive assay for measurements of total and integrated HIV DNA to patient samples from several cohorts in order to compare these surrogates for reservoir size in PBMC in different clinical settings. We evaluated samples from patients before and after ART initiation, samples from patients on effective ART (i.e. patients with < 50 copies/ml of plasma) longitudinally and cross-sectionally, and samples from a recent clinical trial aimed at targeting viral reservoirs. The results of the latter trial are reported in detail elsewhere (currently under review). Briefly, patients on stable ART with undetectable viremia (< 50 copies/ml of plasma) were treated with Peg-IFN-Alpha-2A (IFN-α) plus ART for 5 weeks followed by IFN-α monotherapy during a 12 week ART interruption. A fraction of patients in the study (9/20, 45%) maintained plasma viremia levels below 400 copies/mL while on IFN-α monotherapy for a period of 12 weeks after ART interruption and were termed “responders”. Previous attempts at structured treatment interruptions without concurrent immunotherapy had resulted in control of viremia at this level (<400 copies/ml) in less than 9% of patients, suggesting immunotherapy with IFN-α may have affected reservoir size [27–29]. Here, we studied the relationship between measures of total and integrated HIV DNA in the responders after IFN-α monotherapy and found a decrease in the levels of integrated but not total HIV DNA. Furthermore, both measures together suggested residual HIV expression and de novo entry and reverse transcription had occurred during IFN-α monotherapy, suggesting an immune mechanism for the reduction in reservoir size. Therefore, measuring multiple viral intermediates may also be useful in other clinical trials aimed at reducing reservoir size.