In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix

Abstract

Background: Protease Inhibitors (PIs) are the second-and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-protease genotypic and phenotypic changes within six Nigerian patients who failed PI-based regimens without known drug resistance associated protease mutations in order to identify novel determinants of PI resistance.Methods: Target enrichment and NGS by Illumina Miseq were followed by haplotype reconstruction. Full length gag-protease regions were amplified from baseline (pre-PI) and virologic failure (VF) samples, sequenced and used to construct gag/protease pseudotyped viruses. Phylogenetic analysis was performed using maximum likelihood methods.Susceptibility to lopinavir (LPV) and darunavir (DRV) were measured using a single-cycle replication assay. Western blotting was used to analyse Gag cleavage.Results: In one of six participants (subtype CRF02_AG) we found 4-fold lower LPV susceptibility in viral clones during failure of second line treatment. A combination of four mutations (S126del, H127del, T122A and G123E) in p17 matrix of baseline virus generated a similar 4x decrease in susceptibility to LPV but not darunavir. These four amino acid changes were also able to confer LPV resistance to a subtype B gag-protease backbone. Western blotting did not demonstrate significant Gag cleavage differences between sensitive and resistant isolates. Resistant viruses had around 2-fold lower infectivity compared to sensitive clones in the absence of drug. NGS combined with haplotype reconstruction revealed resistant, less fit clones emerged from a minority population at baseline and thereafter persisted alongside sensitive fitter viruses. Conclusions:We have used a multi-pronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in p17 matrix, revealing the interplay between Gag associated resistance and fitness.

HIV-1 evolutionary dynamics under non-suppressive antiretroviral therapy

“…In the absence of major PI mutations, we first examined non-synonymous mutations across the whole genome (Figures 4-6), with a specific focus on pol (to identify known first and second line NRTI-associated mutations) and gag (given its known involvement in PI susceptibility). We and others have previously shown that gag mutations accumulate during non-suppressive PI therapy 34,35 .…”

Limited emergence of resistance to Integrase strand transfer inhibitors (INSTIs) in HIV-experienced patients failing dolutegravir-based antiretroviral therapy: Cross-sectional analysis from a Northeast Nigerian cohort

“…In a recent analysis of pooled evidence on virological and resistance outcomes following DTG failure in SSA region, there was an overall high rate of virological response to DTG; 88.5% (95% CI: 73.8-97.8) with the overall proportion of patients failing showing limited evidence of DRMS 19 over short periods of time. It is likely that prolonged virologic failure will select for DRM to components of ART regimens within the viral quasispecies as a result of intrahost evolution 20,21 .…”