Doctoral Degrees (Medical Virology)
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Browsing Doctoral Degrees (Medical Virology) by Subject "Antiretroviral agents"
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- ItemCharacterization of the persisting HIV-1 reservoir in early treated children on long-term suppressive ARV regimens(Stellenbosch : Stellenbosch University, 2019-12) Katusiime, Mary Grace Kato; Van Zyl, Gert Uves; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.Introduction: The major barrier to curing HIV-1 infection is a latent reservoir of long-lived, replication-competent proviruses that persists despite suppressive antiretroviral therapy (ART). Initiating ART during acute infection limits the development of phylogenetically diverse reservoirs. Novel approaches for reservoir elimination are emerging, providing hope for a cure. Perinatally infected, early-treated children, are likely good candidates for cure interventions as they have low immune activation states and a low proportion of central memory T-cells. We studied the post-CHER cohort of perinatally infected children, who initiated ART during acute infection and are in long-term follow-up. To inform cure interventions, we aimed to: (i) quantify latently infected cells and describe longitudinal genetic diversity, (ii) describe mechanisms that enable long-term reservoir persistence, (iii) describe the extent to which early therapy shapes the proviral landscape. Methods: In Aim I, we used a sensitive quantitative PCR assay for HIV-1 cell associated DNA (iCAD), followed by cell-associated DNA-single genome sequencing (CAD-SGS) of 1200bp in HIV-1 gag-pol to investigate genetic evolution in the reservoir during long-term ART. We performed 3 tests for evolution: (i) average pairwise distance (APD) for intra-patient viral population diversity, (ii) panmixia for probability of shifts in viral population structure, (iii) maximum likelihood (ML) root-to-tip distances to detect emergence of new viral populations. In Aim II, we performed integration site analysis (ISA) on samples from close to therapy initiation (baseline) and after 6-9 years on ART to investigate clonal expansion as a mechanism for reservoir persistence despite early, suppressive therapy. In Aim III, near full length- proviral amplification and sequencing (NFL-PAS) was performed to determine the proportion of genetically intact vs defective proviruses after 6-9 years on ART. Findings: We found low iCAD levels (median iCAD:22.45cp/106) in 16 children who initiated ART within the first 18 months of life. No significant changes in intra-patient proviral diversity, shifts in viral population structure or emergence of new viral populations were detected in children who were fully suppressed on ART, suggesting that ART prevents ongoing replication that replenishes the reservoir. ISA detected expanded clones in baseline samples of 6 children treated as early as 2 months of age, suggesting that infected cells begin clonally expanding before ART. Furthermore, there was a significant increase in the proportion of expanded clones after several years. A total of 738 NFL amplicons were generated from 9 children. Of these, 72.9% had large internal deletions, 23.7% were hypermutated, 1.4% had small internal deletions, and 1% had deletions in the gag-leader region. Intact proviruses were detected at a frequency of 1%. This study showed that early therapy and long-term suppression in children leads to limited reservoir size and genetic diversity, factors that are favourable for cure interventions. The reservoir appears to be maintained by clonal expansion that begins before therapy is initiated. Although a large proportion of proviral DNA in long-term suppressed children is defective, genetically intact variants persist and likely form part of expanded clones. This suggests the need for novel approaches that target HIV reservoirs by reducing proliferation of cells that harbour replication-competent proviruses.
- 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.
- 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.
- ItemThe role of clonally expanded HIV-1 infected cells in maintaining HIV reservoirs in adults and children on antiretroviral treatment(2020-12) Botha, Johannes Christiaan; van Zyl, Gert U; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: The HIV-1 pandemic remains a major public health dilemma. Treatment with combination antiretroviral therapy (cART) can, in the majority of patients, sufficiently suppress HIV viral replication. However, cART is not a cure as it does not affect long-lived reservoirs. HIV proviruses are harboured in the genome of long-lived CD4 helper cells that are maintained by having long half-lives and by being replenished through cellular proliferation. HIV infected cells that proliferate can be identified as clonal populations by all having the same integration site. A small proportion of proviruses are intact from where HIV rebounds after therapy interruption and is the barrier to achieving cure or remission. Therefore, many molecular assays have been designed to characterise HIV proviruses, with a focus on intact proviruses. From this the proviral landscape is estimated to consist of ~2% intact proviruses and up to 98% defective proviruses comprising deletions and hypermutations. However, highly deleted proviruses such as solo-long terminal repeats (LTRs) remain largely uncharacterised. Recently, it has become apparent that some proviral clones are able to express infectious HIV and cause clonal viraemia in patients on cART, which are not indicative of ongoing cycles of viral replication. This has further confirmed the importance of proviral clones in maintaining the HIV reservoir. Subsequently, aspects of proviral clones were investigated in this study. A novel assay capable of characterising severely deleted proviruses by targeting the unique integration sites was developed. The characterisation of proviral clones in paediatric patients with this novel assay revealed that severely deleted solo-LTR proviruses may be more prevalent than previously expected. Further investigation of these solo-LTR proviral clones was performed with another novel assay capable of quantifying proviruses based on unique integration sites. The longitudinal waxing and waning of solo-LTR proviral clones could be observed. Although solo-LTR proviruses do not contribute to the true reservoir, it suggests that current proviral landscape proportions require adjustment to account for all HIV integration events in cells. Clonal viraemia was studied in HIV positive adult patients on long term cART presenting with non-suppressible HIV viraemia. Three clusters of monotypic plasma viraemia and cell associated DNA HIV sequences were identified in one patient. These monotypic proviruses were shown to be replication competent by viral outgrowth. This provides evidence that clonally proliferated cells harbour at least a proportion of intact proviruses and therefore constitute an important component of the true HIV reservoir. Two additional patients were identified with uncommon cases of sustained viraemia. Firstly, a probable large cell clone harbouring non-infectious virus was found to leak proviral nucleic acid into plasma, resulting in apparent treatment failure. Secondly, possible clonal viremia was observed in a patient with very slow decaying viral load, despite objective evidence of adherence and initial undetectability of treatment-relevant drug resistance over a period of 2 years. We conclude that clonal viraemia may be underreported, and that cases like these show that clonal viraemia should be considered as an explanation of non-suppressed viraemia or apparent therapy failure in the management of HIV infected patients on cART.