Contribution of Gag and Protease to HIV-1 phenotypic drug resistance in pediatric patients failing Protease inhibitor-based therapy

Jennifer Giandhari, Adriaan E. Basson, Katherine Sutherland, Chris M. Parry, Patricia Cane, Ashraf Coovadia, Louise Kuhn, Gillian Hunt, Lynn Morris*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicative in vitro assay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistanceassociated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of the gag gene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.

Original languageEnglish
Pages (from-to)2248-2256
Number of pages9
JournalAntimicrobial Agents and Chemotherapy
Volume60
Issue number4
DOIs
Publication statusPublished - Apr 2016

Bibliographical note

Funding Information:
We thank Elaine J. Abrams and Renate Strehlau for access to the specimens. We acknowledge Didier Trono (EPFL, Lausanne, Switzerland) for the pMDG plasmid and Nigel Temperton (Medway School of Pharmacy, Universities of Kent and Greenwich, United Kingdom) for the pCSFLW plasmid carrying the luciferase gene. LPV and RTV were supplied by the National Institutes of Health (NIH) AIDS Reagent and Reference Program. We thank Public Health London (formerly the Health Protection Agency) for providing J. Giandhari with training on the protease phenotypic assay. We thank the South African Medical Research Council (MRC) Self-Initiated Research Program, the Poliomyelitis Research Foundation (PRF), the KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH), and the Faculty of Health Sciences Research Council of the University of the Witwatersrand (FRC) for funding this study. J. Giandhari was funded by bursaries from the PRF and the National Research Foundation (NRF) of South Africa. J. Giandhari received funding from the Health Protection Agency Global Health Fund for the training that she received on the protease phenotypic assay. The parent study that collected these specimens was funded by Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) grant HD 47177.

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