Browsing by Author "Njenda, Duncan Tazvinzwa"

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    From bedside to bench and back : future options for antiretroviral drugs in non-B HIV-1 subtypes
    (Stellenbosch : Stellenbosch University, 2020-12) Njenda, Duncan Tazvinzwa; Engelbrecht, Susan; Jacobs, Graeme Brendon; Neogi, Ujjwal; Sonnerborg, Anders; Spetz, Anna-Lena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
    Introduction: HIV-1 drug resistance remains a burden in low- and middle-income countries (LMIC). Regardless of the advances in antiretroviral (ARV) therapy, there is an increase in the trend of acquired and pre-treatment drug resistance mutations (DRM) in LMIC affected by HIV-1 non-B subtypes. The overall aim of this thesis is to understand the potential subtype-specific differences in viral fitness and drug susceptibility against the drugs that target different stages of viral replication that includes, protease-mediated cleavage and maturation (Paper I), reverse transcription (Paper II) and integration (Paper III). Methods:The thesis presents cross-sectional drug resistance data from predominantly HIV-1 non-B subtypes. It also includes virological drug sensitivity and enzyme-based in vitro assay results of recombinant viruses derived from predominantly HIV-1 non-B and few HIV-1B infected patients. The following areas were investigated: a) the role that HIV-1 subtype C (HIV-1C) gag-p6 gene could play in response to proteaseInhibitors PIs in the absence of known PI primary mutations (Paper I). b) Ex vivo potency of the newer drug 4’-Ethynyl-2’-Fluoro-2’ deoxyadenosine (EFdA),first and second generation non-nucleoside reverse transcriptase inhibitors (NNRTIs)and Tenofovir alafenamide(TAF) against diverse HIV-1 subtypes (Paper III). c)Ex vivo potency of Integrase strand transfer inhibitors (INSTIs) against diverse HIV-1subtypes as well as genotypic resistance data comparing INSTI naïve and INSTI-experienced patients. Results: In paper I, the study showed an increase in viral fitness for HIV-1C viruses carrying the PYxE insertion in gag-p6 when compared to the wild-type (WT) HIV-1C viruses. Furthermore, some PYxE-carrying viruses had low sensitivity to LPV and (TAF) when tested in drug sensitivity assays. Clinical data analysis showed a higher pre-therapy viral load and a decrease in CD4+ T-cell counts for patients harboring PYxE-carrying viruses when compared to WT. Paper II. demonstrated that EFdA has a high inhibitory potential independent of HIV-1 subtype and high antiviral activity against resistant viruses. However, HIV-1C viruses had a significantly reduced susceptibility to NNRTIs, specifically Rilpivirine and Etravirine. In paper III, the drug susceptibility of INSTIs results indicated that INSTIs such as Dolutegravir (DRV), Bictegravir (BIC) and Cabotegravir (CAB) inhibited non-B subtypes significantly as compared to HIV-1B subtypes. Conclusion: Inferences can be made from the results to suggests that subtype-specific differences play an essential role in influencing the ARV susceptibility which could further impact the treatment efficacy in sub-optimal adherence. To reduce the trend of increasing DRMs in non-B HIV-1 subtypes which are mainly dominating in LMICs, adherence support and viral load monitoring should be prioritized. A rapid adaptation of INSTIs and newer drugs that have long-acting potential is encouraged. However, pre-clinical studies and clinical trials that are mainly restricted to the HIV-1B enrolled patients, should be inclusive of non-HIV-1B infected patients before the massive roll-out of INSTIs and newer drugs continues in non-HIV-1B dominated settings.
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    Investigating the fitness benefit of reverse transcriptase (RT) mutation A62V when co-occurring with M184V and K65R in HIV-1 subtype C
    (Stellenbosch : Stellenbosch University, 2016-03) Njenda, Duncan Tazvinzwa; Van Zyl, Gert Uves; Engelbrecht, Susan; Jacobs, Graeme Brendon; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
    ENGLISH ABSTRACT: Background and Aims Tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) or emtricitabine (FTC) combined with efavirenz is the predominant first-line antiretroviral regimen in the Southern African region. Resistance to TDF and 3TC/FTC is largely through the occurrence of the drug resistance mutations (DRMs) K65R and M184V, respectively. Preliminary data from a large laboratory-based dataset of HIV drug resistance that showed a high prevalence of these mutations in patients who received the TDF regimen also revealed a significant co-occurrence of A62V with M184V and K65R. The aim of this study was to investigate the functional interaction and effect on viral fitness that A62V has when it co-occurs with M184V and K65R reverse transcriptase mutations in HIV-1 Subtype C. Materials and Methods Using Infusion™ cloning and site-directed mutagenesis techniques, eight full-length genome infectious clones containing the HIV-1 subtype C polymerase gene were synthesised having all combinations of DRMs - A62V, M184V and K65R, either being present or absent. The mutations in these constructs were verified by sequencing. The constructs were transfected into 293T cells for virion production and harvested virus was infected in the TZM-bl cell line in head to head growth competition experiments and assayed for growth kinetics using an allele-specific quantitative real-time polymerase chain reaction (PCR) assay. Results The growth competition experiment between two viruses (A62V+K65R+M184V vs K65R+M184V) evaluated by taking the mean of 3 biological replicates in the assay in the absence of antiretroviral drugs, revealed that A62V mutation has no significant impact on fitness (Wilcoxon signed rank test p-value = 0.56). The overall coefficient of variation (CV) in the experiment was 12.8% indicating the high reproducibility of the growth competition assay using real-time PCR measurement of relative growth. Conclusion and recommendations A62V mutation has no effect on fitness when it co-occurs with M184V and K65R. The co-occurrence with M184V and K65R remains unexplained and might be due to an effect on TDF resistance in combination with K65R.This requires investigation in future studies as TDF regimens are part of 1st line therapy in many Sub-Saharan countries in the treatment of HIV-1. Finally, the cloning and mutagenesis techniques used coupled with a very sensitive and reproducible real-time quantitative PCR assay provide an efficient system for detection of mutation fitness interactions and can be used in any future work to study HIV mutation fitness interactions.