Research Article: Cellular Levels of HIV Unspliced RNA from Patients on Combination Antiretroviral Therapy with Undetectable Plasma Viremia Predict the Therapy Outcome

Date Published: December 31, 2009

Publisher: Public Library of Science

Author(s): Alexander O. Pasternak, Suzanne Jurriaans, Margreet Bakker, Jan M. Prins, Ben Berkhout, Vladimir V. Lukashov, Fatah Kashanchi. http://doi.org/10.1371/journal.pone.0008490

Abstract: Combination antiretroviral therapy (cART), the standard of care for HIV-1 infection, is considered to be successful when plasma viremia remains below the detection limit of commercial assays. Yet, cART fails in a substantial proportion of patients after the apparent success. No laboratory markers are known that are predictive of cART outcome in initial responders during the period of undetectable plasma viremia.

Partial Text: Combination antiretroviral therapy (cART), the standard of care for human immunodeficiency virus type 1 (HIV-1) infection [1]–[3], is an exemplary success of modern medicine, as it has resulted in a dramatic decrease in HIV-related morbidity and mortality [4], [5]. Since the introduction of cART, the response to therapy has dramatically improved, with currently most patients reaching HIV RNA levels in plasma that are below the limit of detection of modern commercial assays (<50 copies/ml). Despite these encouraging results, failure of ART is still a common problem. In the Netherlands, the annual proportion of previously therapy-naïve patients who experience failure increased from 6% to 9% in the period between 1997 and 2007 (F. de Wolf. The HIV epidemic in the Netherlands. The 2nd Netherlands Conference on HIV Pathogenesis, Prevention and Treatment, 25 November 2008, Amsterdam, The Netherlands). Cellular HIV-1 load (prDNA, usRNA, and msRNA) was quantified in PBMC at baseline and at multiple time points during the period of undetectable HIV-1 plasma viremia on cART (“the eclipse phase”) in patients who were successfully treated with cART (“successes”) and those who experienced VR on cART after achieving undetectable HIV-1 load in plasma (“failures”). UsRNA and prDNA were detected and quantified in all baseline PBMC samples and in 94% of the under-therapy PBMC samples. MsRNA could be detected and quantified in 83% of the baseline samples, but only in 16% of the under-therapy samples (Table S1), precluding any quantitative comparison of msRNA levels under cART between the patient groups. To account for the possible effects of mismatches in the primer and probe binding regions on the efficiency of real-time PCR, all quantified amounts of prDNA and usRNA in the baseline and under-therapy PBMC samples were normalized to the individual mismatch-related quantification errors (see Methods and Figure S1). The main clinical objective of cART is suppression of HIV-1 plasma viremia to below the lowest existing detection limit of commercial assays. In most patients on cART, this objective is achieved, and therefore plasma viremia in these patients cannot be informative about the subsequent outcome of therapy. Hence, additional markers have to be identified that are associated with therapy outcome in patients with fully suppressed plasma viremia. In this study, we demonstrated that the level of HIV-1 usRNA in PBMC from such patients is predictive of subsequent VR. To the best of our knowledge, this is the first report showing that a viral parameter–measured in a patient on cART during the period of undetectable plasma viremia–is predictive of the outcome of therapy. Several reports have indicated the presence of HIV-1 mRNA in PBMC from patients on cART [28]–[34]. However, the mere presence of intracellular viral RNA does not necessarily imply residual virus replication, and may instead reflect production of virus from stable reservoirs without new replication cycles. In contrast, our observation that patients with higher levels of usRNA in PBMC were more prone to failing cART might link higher cellular HIV-1 RNA load in these patients with virus replication under therapy, which, in turn, resulted in selection of drug-resistance mutations. Acquired drug-resistance mutations were indeed observed in all plasma samples sequenced after failure (data not shown). It remains to be investigated whether drug-resistance mutations can indeed be detected in PBMC of these patients during the eclipse phase. Low-level plasma viremia was recently detected by ultrasensitive assays in most patients on cART [15], [24]–[26]. Whether this residual viremia reflects ongoing virus replication despite therapy, or the production of virus from stable reservoirs without new replication cycles, is controversial [15], [23], [26], [35], [36]. The possible association of the residual plasma viremia with the outcome of cART and its correlation with the levels of usRNA in PBMC remain to be studied. Source: http://doi.org/10.1371/journal.pone.0008490

 

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