Doctoral Degrees (Medical Virology)
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Browsing Doctoral Degrees (Medical Virology) by browse.metadata.advisor "Jacobs, Graeme Brendon"
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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.
- ItemHepatitis B virus mother-to-child transmission in Namibia: transmission dynamics and possibilities for elimination(Stellenbosch : Stellenbosch University, 2019-12) Tamandjou Tchuem, Cynthia Raissa; Andersson, Monique I.; Preiser, Wolfgang; Wiysonge, Charles S.; Jacobs, Graeme Brendon; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: Introduction: Hepatitis B virus (HBV) remains endemic in sub-Saharan Africa (SSA). While the roll-out of pediatric HBV immunization from six weeks of age has had an impact on horizontal transmission of the virus, mother-to-child transmission (MTCT) has been identified as the driver of the current HBV epidemic in the region. Given the high likelihood of developing chronic HBV infection (CHB) if the infection is acquired during infancy, preventing HBV MTCT in SSA is vital. Where MTCT occurs, it is essential to identify the HBV-infected children for appropriate management, especially in the context of HIV. Current antiretroviral therapy (ART) for the management of HIV infection includes tenofovir for children ≥ 10 years old. Children below the age of 10 years are treated with lamivudine. However, many of the HIV/HBV co-infected children are left on lamivudine treatment for more than ten years and are at risk of developing HBV drug resistance and uncontrolled HBV infection. Uncontrolled HBV infection is a known factor for increased risk of severe liver damage. Aim: This research project aimed to (1) assess the molecular character of HBV and the liver health of HIV/HBV co-infected children who have been on long-term lamivudine treatment, (2) to determine the feasibility of a screen-treat-vaccinate intervention to prevent HBV MTCT, and (3) to measure the costs and health outcomes of combined prophylactic measures against HBV MTCT, in Namibia. Methodology: Three sub-studies were conducted as part of this research project, to answer each of its aims. The first sub-study involved HIV/HBV co-infected children and adolescents below the age of 18 years old, and who have been exposed to lamivudine. Venous blood samples were collected from these children for HBV serological testing (HBsAg, HBeAg, anti-HBe and anti-HBc total) using Murex ELISA assays. Dried blood spots (DBS) samples were used for HBV DNA levels measurement and genotyping. HBV DNA measurements were completed using the automated AmpliPrep/COBAS TaqMan HBV test V2.0. Genotyping and mutation analyses were performed using online tools. Liver health was assessed through AST platelet ratio index (APRI). An APRI score > 0.5 was considered a sign of liver fibrosis. Mothers attending with these children and adolescents were also enrolled in the study, to determine the role of HBV MTCT in these pediatric HBV infections. DBS were collected from these mothers for HBV molecular characterization as well. The second sub-study focused on pregnant women attending antenatal clinics (ANCs) in Windhoek. These women were recruited following informed consent and screened for HBV using the Alere DetermineTM HBsAg rapid test. HBsAg positive pregnant women were tested for further HBV serological markers (HBeAg, anti-HBe, anti-HBc IgM), and HBV viral loads were measured to determine the risk of MTCT. Positive mothers at high risk of MTCT were reviewed for antiviral prophylaxis and offered treatment where necessary. HBV-exposed babies were immunized as per Namibian guidelines, and followed-up to determine the rate of MTCT. The feasibility of offering routine antenatal HBV rapid testing was assessed quantitatively and qualitatively. The former involved determining the diagnostic accuracy of the rapid test used for HBsAg screening, and the latter focused on the perceptions of this antenatal care service by healthcare workers (HCWs). In the third sub-study, the costs and health outcomes of four interventions against HBV MTCT were assessed through a cost-effectiveness analysis. The interventions included: (1) universal birth dose (BD) vaccination, (2) BD vaccination and HBIG, (3) BD vaccination, HBIG, and maternal antiviral prophylaxis informed by sequential HBV viral load testing, and (4) BD vaccination, HBIG, and maternal antiviral prophylaxis informed by sequential HBeAg testing. All resources including consumables, HCW’s time, building space and facilities (and their quantity) were measured and valued to determine the unit costs of HBsAg screening at the ANCs, providing antenatal treatment and administering pediatric immunoprophylaxis. Health outcomes were measured in terms of the number of pediatric HBV infections averted. The incremental cost-effectiveness ratios (ICERs) of these interventions were calculated and were used to compare each intervention to the previous less expensive one. Results: Fifteen HIV/HBV co-infected children/adolescents and six mothers attending with the children were enrolled in the first sub-study. Ten serum samples obtained from Windhoek were further tested for HBV serological markers; seven were HBeAg positive/anti-HBe negative (7/10; 70%), three were HBeAg negative (3/10; 30%), and all were reactive for anti-HBc (total) (10/10; 100%). Among HBeAg negatives, one was anti-HBe negative and two were anti-HBe positive. Eight of the fifteen children (8/15; 53.3%) were HBV DNA positive. The viral strains were grouped with genotype E (6/8; 75%) and genotype D3 (2/8; 25%) and harbored lamivudine drug-associated resistance variants and immune escape mutants. Liver health was assessed in nine children: five with detectable levels of HBV DNA and four with undetectable levels of HBV DNA. An abnormal APRI score of 0.713, was detected in one HBV DNA positive child (1/9; 11.1%). In the second sub-study an HBsAg seroprevalence of 5.4% was observed among 515 (28/515) pregnant women enrolled at ANCs in Windhoek. Three pregnant women (3/28; 10.7%) were positive for HBeAg; of whom one was HIV/HBV co-infected and the other two were HBV mono-infected. The two (2/28; 7.14%) HBV mono-infected/HBeAg-positive patients presented with viral load > 105 IU/ml, the study cut-off for antenatal treatment to prevent HBV MTCT; one received antiviral prophylaxis with tenofovir, the other was offered prophylaxis but did not receive it. Postpartum, 25 of the 28 HBV-exposed babies (25/28; 89.3%) were traced and followed-up to determine their HBV status and the rate of HBV MTCT. Fourteen (14/25; 56%) were males, and eleven were females (11/25; 44%). All babies had been vaccinated against HBV at birth, and 15 (15/25; 60%) had received hepatitis B immunoglobulin (HBIG). The 25 babies were tested for HBsAg at a median age of seven weeks (Range: 5.57 weeks – 20.29 weeks). All were non-reactive for HBsAg, including both babies born to the highly viremic women. With regards to the feasibility of HBV rapid testing as part of antenatal care services, the DetermineTM HBsAg rapid test had a 100% diagnostic sensitivity and specificity. HCWs found the test simple to use and showed a preference for rapid testing over laboratory testing for routine antenatal screening of HBV. They believed that this method would improve early diagnosis and treatment of HBV of pregnant women. In the cost-effectiveness analysis conducted in the third sub-study, a preventive strategy with universal BD vaccination alone was the cheapest option but was less effective. Adding HBIG to BD vaccination, and providing maternal antiviral prophylaxis was the most effective and the most costly strategy. The strategy that includes antiviral prophylaxis with sequential HBeAg testing added to BD vaccination and HBIG had an ICER of US$6 262.42 per infection averted, in comparison to the strategy including BD vaccination and HBIG only. The BD vaccination and HBIG strategy had an ICER of US$4 550.34/ pediatric HBV infection averted, in comparison to BD vaccination alone. These ICERs were highly sensitive to the prevalence of highly infectious pregnant women, the cost of the HBeAg test, and the effectiveness of each strategy for preventing MTCT in highly infectious pregnant women. Conclusion: Results from this research project reemphasized the issue of pediatric CHB, especially in HIV/HBV co-infected children. The data described in this study also showed that elimination of MTCT of HBV in Namibia is achievable, through routine antenatal HBsAg screening, treating pregnant women at high risk of MTCT, and providing HBV vaccination from birth. Screening using rapid testing was found cheap, and a feasible alternative for detecting HBV infection in pregnant women. The costs and health benefits of implementing antenatal antiviral prophylaxis and HBIG presented in the study provide background data for further assessment of the value for money of these interventions in SSA, and to explore alternatives excluding HBIG for HBV PMTCT.
- ItemInvestigating the structural impact of hiv-1 integrase natural occurring polymorphisms and novel mutations identified among group m subtypes circulating in sub-Saharan Africa(Stellenbosch : Stellenbosch University, 2020-12) Mikasi, Sello Given; Jacobs, Graeme Brendon; Cloete, Ruben; Van Zyl, Gert Uves; Engelbrecht, Susan; Ikomey, George Mondinde; Kasang, Christa; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.Introduction HIV/AIDS remains a major health concern worldwide, with sub-Saharan Africa (SSA) carrying the largest burden.HIV is characterised by extremely high genetic diversity, with all the major groups and subtypes circulating in SSA. Combination antiretroviral therapy (cART) have substantially reduced HIV related deaths, but this is counteracted by the development of HIVdrug resistance, caused by certain drug resistance-associated mutations (RAMS). Integrase (IN) strand transferase inhibitors (INSTIs), the newest class of antiretroviral drugs,has a high genetic barrier and can be used in individuals that previously exhibited resistance to other classes of drugs. The World Health Organisation (WHO) approved the use of Dolutegravir (DTG) as part of first-line cART. Methods This is a descriptive experimental design study, which aimed to identify IN natural occurring polymorphisms (NOP) among different HIV-1 group M subtypes and Drug resistance mutations within the HIV-1 pol gene fragment of INSTI naïve patients from South Africa (SA) and Cameroon (CR), using the Stanford University genotypic resistance interpretation algorithm. Structural computational methods that included; homology modelling, molecular docking, molecular dynamics simulations and interaction analysis was performed to understand the structural impact of mutations from diverse HIV-1 subtypes on DTG drug binding. ResultsWe observed low-level RAMs against INSTIs in SA (2.2%) and CR sequences (5.4%). Through Fisher’sexact test we noted that the two NOPs occurred: VI72I and R269K, with p-values ≤0. 05, were statistically enriched. The impact of having these mutations are yet to be fully understood. Through molecular modelling and stability predictions, we observed a destabilizing effect of the known G140S mutant on the HIV-1C IN protein structure and simulation analysis showed that it affected structural stability and flexibility of the protein structure. Interactions analysis of different drug binding conformations to different HIV-1 IN subtypes reported differences in the number of binding interactions to different HIV-1 IN subtypes, but we did not observe any significant differences in binding affinity for each INSTIs. This implies no significant alteration to the binding site in the wild type IN, which may not prevent INSTIs drug binding. In addition, all accessory mutations that resulted in a change in the number of interactions encompassing residues were found within the stable alpha-helix secondary structure element and not in close proximity to the drug active site.ConclusionThe study data indicate that RAMS against INSTIs remain low both in SA and in CR.Subtype C in SA and CRF02_AG in CR continues to be the driving force ofthe epidemic. We further reported on the impact of various NOPs on drug susceptibility. The analyses suggested that NOPs does not have an impact on IN protein structure and stability,and does not affect drug binding in the WT IN, but the known mutation G140S affect DTG binding. The study support recommendations made by the WHO to use DTG as part of salvage therapy in patients with RAM’s and accessory mutations. Data obtained from thisstudy can help to tailor effective treatment strategies in the African population, where diverse HIV subtypes circulate.
- 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.