(P28) Investigating HIV-1 resistance patterns to protease inhibitors in HIV-1 infected patients failing second-line therapy in South Africa
Duncan T. Njenda1(1, 2,), Given Mikasi(2), Adetayo E. Obasa(2), Ujjwal Neogi(1), Graeme B. Jacobs(2), Susan Engelbrecht(2 )
1. Division of Clinical Microbiology, Department of Laboratory Medicine, ANA Futura, campus Flemingsberg, Karolinska Institutet, Sweden 2. Division of Medical Virology, Department of Pathology, Faculty of Medicine and Health sciences, Tygerberg campus, Stellenbosch University, Cape Town South Africa
HIV -1 subtype C dominates about half of world’s HIV infected population and up to 90% of new infections that occur in Low and middle income countries. Second-line treatment regimen in low and middle-income countries often contains a protease inhibitor(PI) as part of the Antiretroviral (ARV) regimen used to tread first-line failure HIV-1 patients. Regardless of optimal treatment and drug adherence, patients who fail treatment often do not have major PI associated mutations in their viral sequences implying that HIV-1 develops resistance to PIs in a mechanism that is unclear. The Gag and protease genes of HIV are hypothesized to contain natural occurring nucleotide polymorphisms that could account for the mechanism of resistance. However, very few studies have been able to give a correlation between genotypic sequencing and phenotypic data to support this hypothesis. Using patient samples of second-line therapy failure, we investigate the cause of virological failure using genotypic and phenotypic methods.
Materials and Methods
Patient samples from a cohort of 105 HIV-1 subtype C infected patients failing PI-based therapy from South Africa were used to isolate viral RNA that was then reverse transcribed into cDNA and amplified to yield Gag-protease amplicons. Amplicons were Sanger sequenced and additional sequencing was done on the Gridion™ using Oxford Nanopore sequencing technology (ONT). Sequence results were analyzed for cleavage and non-cleavage site mutations in the gag gene as well as protease drug resistant mutations in the protease gene. Sequences were systematically selected for recombinant cloning into an infectious vector backbone and used in a two–round PI-based drug susceptibility assay to assess drug impact of mutations.
Gag – protease gene was successfully amplified in 55/105 (52,4%) patient samples. Resistance algorithms from the Stanford Database identified M36I, I54V, L76V and V82A protease mutations in the 3 patients giving a prevalence of 0.05%. 27/55 (49%) samples were selected for Oxford Nanopore technology(ONT) sequencing and 15 were successfully sequenced. ONT sequencing data showed 95% good correlation with Sanger sequencing data. Overall sequence data identified 50 non-cleavage site mutations in the Gag that occurred with more than 80% frequency. Five out of the successfully amplified patient-derived viral sequences in both ONT and Sanger sequence sets were cloned to generate recombinant viruses that contained enriched mutations in the gag gene. Preliminary results for two round PI-Dug sensitivity assay results indicated reduced drug susceptibility for clones containing gag non- cleavage site mutations when compared to the wild type control.
HIV-1 gag non–cleavage site mutations affect the efficacy of PI-based treatment in HIV-1 subtype C infected patients. Genotypic tests should be modified to include the Gag gene in investigating protease-inhibitor based therapy failure