A detailed characterization of drug resistance during darunavir/ritonavir monotherapy highlights a high barrier to the emergence of resistance mutations in protease but identifies alternative pathways of resistance

Background: Maintenance monotherapy with ritonavir-boosted darunavir has yielded variable outcomes and is not recommended. Trial samples offer valuable opportunities for detailed studies. We analysed samples from a 48 week trial in Cameroon to obtain a detailed characterization of drug resistance. Methods: Following failure of NNRTI-based therapy and virological suppression on PI-based therapy, participants were randomized to ritonavir-boosted darunavir (n=81) or tenofovir disoproxil fumarate/lamivudine+ritonavir-boosted lopinavir (n=39). At study entry, PBMC-derived HIV-1 DNA underwent bulk Protease and Reverse Transcriptase (RT) sequencing. At virological rebound (confirmed or last available HIV-1 RNA≥60 copies/mL), plasma HIV-1 RNA underwent ultradeep Protease and RT sequencing and bulk Gag-Protease sequencing. The site-directed mutant T375A (p2/p7) was characterized phenotypically using a single-cycle assay. Results: NRTI and NNRTI resistance-associated mutations (RAMs) were detected in 52/90 (57.8%) and 53/90 (58.9%) HIV-1 DNA samples, respectively. Prevalence in rebound HIV-1 RNA (ritonavir-boosted darunavir, n=21; ritonavir-boosted lopinavir, n=2) was 9/23 (39.1%) and 10/23 (43.5%), respectively, with most RAMs detected at frequencies ≥15%. The resistance patterns of paired HIV-1 DNA and RNA sequences were partially consistent. No darunavir RAMs were found. Among eight participants experiencing virological rebound on ritonavir-boosted darunavir (n=12 samples), all had Gag mutations associated with PI exposure, including T375N, T375A (p2/p7), K436R (p7/p1) and substitutions in p17, p24, p2 and p6. T375A conferred 10-fold darunavir resistance and increased replication capacity. Conclusions: The study highlights the high resistance barrier of ritonavir-boosted darunavir while identifying alternative pathways of resistance through Gag substitutions. During virological suppression, resistance patterns in HIV-1 DNA reflect treatment history, but due to technical and biological considerations, cautious interpretation is warranted.