Drug-Resistant Variants That Evolve During Nonsuppressive Therapy Persist in HIV-1–Infected Peripheral Blood Mononuclear Cells After Long-Term Highly Active Antiretroviral Therapy

Abstract

The aim of this study was to determine whether drug-resistant virus persists in peripheral blood mononuclear cells (PBMCs) after long-term suppression of virus replication. Proviral DNA was extracted from the PBMCs of 11 patients on long-term highly active antiretroviral therapy (HAART). Genotyping of the reverse transcriptase (RT) and protease gene of several proviral variants was performed using limiting dilution polymerase chain reaction and single-copy sequencing. All patients were on successful HAART for a mean period of 59 months but had a history of suboptimal therapy and genotypic drug resistance before. Comparison of the amino acid sequence of the RT and protease gene in the different proviral variants, with that of the plasma virus isolated before HAART treatment, revealed that the different drug-resistant viral variants that evolved during the process of gradually building up resistance were still detectable in the PBMCs in 10 of the 11 patients tested. The proportion of resistant variants was found to correlate with the time that the resistant variants had been able to replicate. These data clearly show that virus variants that are able to replicate for a certain period enter the latent reservoir and remain archived in the PBMCs for a very long period.

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

“…Most of these discordances may be attributed to differences in the selection of the samples used for the evaluation. While plasma RNA represents the population of short-lived actively replicating virus, viral DNA from infected cells is composed of a heterogeneous mix of DNA from acutely infected, actively virus-producing cells as well as quiescent cells that form the viral reservoir (13,27,40). Due to the limited capacities of current population-based sequencing genotyping methods, minor variants can remain undetectable (38).…”

“…Interruption of a failing regimen or a failing drug will result in an overgrowth of the drug-resistant variant by the more fit wild type (13,27,40). In the cellular reservoir on the other hand, the resistant strains will remain detectable for longer time.…”

Latent HIV-1 Can Be Reactivated by Cellular Superinfection in a Tat-Dependent Manner, Which Can Lead to the Emergence of Multidrug-Resistant Recombinant Viruses

“…Since viruses can be continually deposited into the latent reservoir during periods of low-level viremia or during treatment failure and can exit the latent reservoir when their host cell is activated (10), latency provides a means for the archiving and reemergence of sequences representing the history of a patient's quasispecies. When drugresistant viruses are present, they are also archived in the latent reservoir (11)(12)(13)(14)(15). Since viral rebound from latently infected cells occurs upon treatment interruption or treatment failure (16), previously existing drug-resistant viruses that are present in the latent reservoir preclude patients from being treated with that drug or drug combination in the future.…”

“…Recombination within individual patients has been documented in numerous studies (reviewed in reference 25), and recombination involving drug-resistant viruses provides a mechanism for the spread of drug resistance throughout a patient's quasispecies (28)(29)(30). Since the latent reservoir represents an archive of the history of a patient's quasispecies, including viruses with any previously existing drug resistance mutations (11)(12)(13)(14)(15), latent viruses represent an important source for the further generation of genetic diversity under selective pressure. This could occur following superinfection of a latently infected cell.…”

A Novel Assay Allows Genotyping of the Latent Reservoir for Human Immunodeficiency Virus Type 1 in the Resting CD4+T Cells of Viremic Patients

“…The latent HIV-1 population stored in resting CD4 ϩ T cells is a diverse collection of archival viral species exhibiting various degrees of divergence from the most recent common ancestor (37,44,51,55,67). Longitudinal studies have demonstrated a lack of temporal structure and evolutionary change in the viral species residing in this reservoir in most patients with undetectable plasma HIV-1 RNA measurements (3,26,41,52,55,77).…”