Doctoral Degrees (Medical Virology)
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Browsing Doctoral Degrees (Medical Virology) by browse.metadata.advisor "Neogi, Ujjwal"
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- ItemFrom 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.
- ItemMultidisciplinary viral analyses in people living with HIV-1C and receiving second-line combination antiretroviral therapy (cART) in South Africa(Stellenbosch : Stellenbosch University, 2019-12) Obasa, Adetayo Emmanuel; Jacobs, Graeme Brendon; Neogi, Ujjwal; Kamalendra, Singh; Cloete, Ruben; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical VirologyENGLISH ABSTRACT: The use of combination Antiretroviral Therapy (cART) has grown since its first introduction into the South African public sector. cART has significantly reduced the mortality rate caused by human immunodeficiency virus (HIV) in both high- and low-to-middle-income countries. The development of drug resistance has challenged the outcome of cART. This has led to the introduction of Integrase (IN) strand transfer inhibitors (InSTIs) as part of the first-line cART regimen. Due to their superior efficacy and high genetic barrier, this class of drugs was previously reserved as salvage therapy. The World Health Organization (WHO) supports InSTIs as first-line regimen non-nucleoside reverse transcriptase inhibitors (NNRTIs) particularly in regions where pre-treatment drug resistance to NNRTIs reaches 10%. Therefore, this study aimed to (i) to investigate the prevalence of InSTI mutations in treatment-naïve and treatment-experienced PLHIV using genotypic assays, which included Sanger sequencing, next-generation sequencing (NGS) and molecular modelling; (ii) analysed Long Terminal Repeats (LTR) to identify transcription factor binding sites. Chapter 2: Ninety-one (n = 91) treatment-naïve patients were obtained before the start of antiretroviral treatment in South Africa. Furthermore, we included 314 South African patient sequences obtained from the Los Alamos National Library database (www.lanl.gov). The IN gene ~ 900 base pairs [bps] was amplified and sequenced using conventional DNA Sanger sequencing. Homology structure was generated using the cryoEM structure of HIV-1B IN intasome (PDB file 5U1C) using ‘Prime’ of Schrodinger Suit. Chapter 3: Ninety-six (n = 96) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained for further analyses. We performed conventional DNA Sanger sequencing to analyse the complete pol gene (~3011bps) and sequences were analysed using the Stanford HIV drug resistance database to assess genotypic resistance associated mutations (RAMs). Chapter 4: Fifty-six (n = 56) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained. We performed a high-throughput (HT) sequence analyses on the complete pol gene using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV drug resistance database. Chapter 5 and 6: We performed in-silico analyses on diverse HIV-1 subtypes based on 8114 sequences. These included treatment naïve and downloaded sequences from the HIV Los Alamos National Library Database (www.lanl.gov). Homology derived molecular models of HIV-1 IN tetramers from different subtypes were generated using cryoEM structure of the HIV-1B IN intasome. Chapter 7: Fifty-six (n = 56) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained. We performed Sanger sequencing to analyse the LTR gene (~ 474 bps) followed by bioinformatics analyses to identify transcription factor binding sites.The data indicates that in South Africa, the prevalence of RAMs against InSTIs is low and InSTIs can be used as a potential viable salvage therapy option and/or first-line regimen. Molecular modelling was done for IN structural analyses, which revealed how naturally occurring polymorphisms might affect structural stabilities, viral DNA binding and drug-binding propensity. This study represents a true baseline InSTI resistance rate as the treatment-naïve patients were obtained before the cART introduction. We propose GRT for people living with HIV (PLHIV) before treatment initiation and we recommend continued InSTIs drug resistance monitoring when introduced on a larger scale in South African.