School of Pathology (ETDs)
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Item Equitable access to vaccines: exploring the role of accessability, acceptability, affordabilityand availability with a focus on COVID-19(University of the Witwatersrand, Johannesburg, 2024) Schwalbe, Nina; Cutland, ClareThe coronavirus disease (COVID-19) crisis brought to light many challenges, including “vaccine equity”. In other words, it raised the question: was the distribution of vaccines “fair”? While, on the one hand, there have been unprecedented advances in the science and technologies associated with vaccines, including extraordinary speed and scale-up of manufacturing, there were also significant barriers related to rollout and reaching those most at risk of severe COVID-19. These challenges have disproportionately affected low- and middle-income countries and low-income populations in high-income countries. Building on evidence from other vaccine preventable diseases, this thesis describes the challenges and opportunities concerning vaccine access with a focus on production and distribution (the “supply side”). It explores access using a “4A's” framework to conceptualise the components of access to medicines: affordability, availability, acceptability, and accessibility. The research identifies a range of access policy levers across the end-to-end process of vaccine research, development, and rollout (affordability, acceptability, accessibility, acceptability); reviews these levers as they apply to vaccine manufacturing (affordability, availability); explores the lever of financial incentives to increase coverage (acceptability); and explores the potential of using precision public health to improve vaccine impact by targeting vaccine distribution to groups most risk (accessibility). This thesis identifies several policy and program interventions ranging from regulatory harmonisation and intellectual property sharing, to using precision public health to target the delivery of vaccines to those most at risk. It also shows that while financial incentives may help, governments cannot “buy” coverage. It proposes that in future, vaccine development and deployment should start and end with a “4A’s” strategy and provides practical recommendations on how that can be achievedItem The development and value assessment of an integrated cardiovascular disease risk score in an African setting(University of the Witwatersrand, Johannesburg, 2024) Kamp, Michelle; Ramsay, Michѐle; Lewis, Cathryn; Pain, OliverCardiovascular diseases (CVD) are a significant health threat in Africa and are a leading cause of mortality and morbidity in the region. Risk stratification is the preferred approach to disease prevention but is challenging to apply due to scarce data and current scores not being validated in African populations. CVD risk is influenced by genetic and environmental factors, with the latter mostly considered in risk calculators. Precision medicine (PM) aims to tailor medical diagnostics and therapeutics by leveraging the genetics, environment, and lifestyle of individual patients. Polygenic scores (PGS) can quantify an individual’s genetic burden for CVD and improve the predictive value of risk tools when included with conventional risk factors. PGS for European ancestry populations are reaching the point where they may be useful in clinical care. However, transferability of European-derived PGS to African populations is limited and may hamper application in clinical practice. The overarching aim of the study was to develop and evaluate an integrated CVD risk score incorporating both genetic and non-genetic factors for continental African populations. This comprised of developing ancestry-aligned PGS for various cardiometabolic traits, and then deriving, evaluating, and comparing the predictive utility of genetic, non-genetic, and integrated (genetic + non-genetic) CVD risk prediction models using elastic net regression with nested 10-fold cross validation. Furthermore, we aimed to contextualise the potential utility of such PM-based tools in Africa through an assessment of the current landscape of translational genomics on the continent and by gauging clinician’s perceptions on implementing the derived CVD-risk stratification tool in South Africa’s public health setting. This study demonstrated the potential of genetic information to enhance disease risk stratification among continental African populations. Results revealed PGS provide both independent and complementary information in predicting dyslipidaemia, hypertension, and obesity - integrated scores improved prediction by at least 2.5% compared to models consisting of non-genetic factors alone. The study also highlights the challenges limiting the advancement of PM in Africa – 1. the paucity of genetic and phenotypic data within continental African populations limits the development and validation of robust and clinically useful models. This challenge is exacerbated by Euro-centric genomic and computational tools and echoes the call for more inclusive methodological approaches. 2. Despite positive perceptions of PM, there is an urgent need to address complex structural and operational barriers, including insufficient funding, lack of political will, healthcare infrastructure deficits, and training and education gapsItem Differential Gene Expression in Exfoliation Syndrome and Exfoliation Glaucoma in the Conjunctiva of Black South Africans(University of the Witwatersrand, Johannesburg, 2024) Hulley, Michaella RobynGlaucoma is a heterogeneous group of clinically and genetically complex optic neuropathies. The most prevalent identifiable secondary cause of glaucoma is the ocular manifestation of exfoliation syndrome (XFS), known as exfoliation glaucoma (XFG), a severe form of glaucoma characterized by rapid progression. While XFS is systemic, XFG is defined by fibrillar extracellular matrix (ECM) deposits in the eye. XFG has a recognised genetic aetiology, specifically with contributions from LOXL1 variants, however the molecular pathogenesis remains unclear. Conjunctival cells have not previously been used for whole transcriptome sequencing, and gene expression studies on XFG are lacking, particularly in South Africa. The aim of this study was to validate the use of conjunctival cells for whole transcriptome sequencing and interrogate the gene expression profiles of individuals with XFS and XFG. Conjunctival cells were collected from 19 cases and 15 control participants for RNA extraction, cDNA library construction and sequencing. Differential gene expression was assessed between cells from cases and controls and 81 genes were found to be differentially expressed, with 80 upregulated in cases. Interestingly, LOXL1, which has shown contradictory expression results in previous studies, was not identified as differentially expressed. The most significant overexpression was of the CCN2 gene, which encodes a matricellular protein that interacts with structural ECM molecules. A potential novel pathway involving megakaryocytes and the activation of neutrophils was, however, identified. Megakaryocytes and neutrophils are both involved with interstitial fibrosis, with neutrophils also responding to chemotactic signals to induce inflammation. The inflammatory pathway has long been associated with XFS, XFG and other fibrotic disorders, such as Alzheimer's disease and rheumatoid arthritis. Genes involved in the inflammatory process were overexpressed in XFG, including TGF-β, IL-1β, IL- 33, EGR1 and EGR3. EGR1 and EGR3 are regulated by fibrotic signals and therefore play an important role in fibrosis, which may contribute to XFS fibrillar deposits. They are also regulated by TGF-β, thus supporting a complex interplay of the inflammatory process and fibrosis in XFS pathogenesisItem The role of small genetic variants in the aetiology of developmental disorders in South Africa - a whole exome sequencing study(University of the Witwatersrand, Johannesburg, 2024) Molatoli, Mhlekazi Cathrine; Lombard, ZanéDevelopmental disorders (DD) are a diverse group of chronic conditions characterized by significant limitations to both mental and physical development. Genetic variants have been identified as the underlying aetiology in about 40-50% of DD cases. Whole exome sequencing (WES) is the recommended first-line genetic test in this group of patients and is associated with diagnostic yields of 16-45%. However, in South Africa and other resource-poor settings, karyotype testing and MLPA analysis (offering low diagnostic rates of 3% and ~9% respectively) are still being utilized for genetic testing. Thus, a higher proportion of patients remain with unexplained DD due to the limitations of these diagnostic tools and limited genetic services. The main challenge facing the clinical implementation of WES in African settings is the complex data analysis and interpretation associated with the large amount of variant data produced. This is especially challenging as African ancestry individuals have been demonstrated to have a high level of genetic diversity resulting in a higher number of novel variants reported compared to European ancestry individuals. This study seeks to investigate whether the clinical utility of WES can be replicated in an African setting. Additionally, we seek to make recommendations for variant filtering and prioritization, thus making the process of WES data analysis for DD patients more efficient. To achieve these, WES was performed in 117 patients with unexplained DD and their 180 unrelated parents. Variant data was filtered and prioritized using two in-house semi-automated pipelines. The first pipeline, prioritized variants overlapping known DD genes, as identified using the G2P-DDG2P bioinformatics analysis tool. The second pipeline identified de novo variants in trio families using the trio-dnm bioinformatics analysis tool. Sanger sequencing was used to validate low-quality prioritized variants prior to in-house interpretation and curation, and all subsequently identified putative disease-causing variants prior to reporting. Of the 117 patients from 115 families analysed, a positive molecular diagnosis was achieved for 29 families, resulting in a diagnostic yield of 25.2% (29/115). Leveraging currently available DD data, our findings demonstrate the diagnostic and clinical utility of WES which resulted in recommendations for improving patient clinical management and surveillance. This study has also developed and made recommendations for variant filtering and prioritization strategies, which can be implemented in both research and diagnostic settings to streamline and aid in the identification of putative disease-causing variants in DD patientsItem Defining Fc-mediated Functions in People Living with HIV during Respiratory Viral Infection and Vaccination(University of the Witwatersrand, Johannesburg, 2024) Motsoeneng, Boitumelo Madika; Moore, PennyThere are approximately 39 million people living with human immunodeficiency virus (HIV) (PLWH) worldwide. Furthering our understanding of humoral immune responses to respiratory viral infection and vaccination in PLWH is essential for reducing the burden of these diseases, in high HIV prevalence settings, and informing vaccine implementation in this population. Influenza virus hemagglutinin (HA) stalk-specific antibodies have been associated with protection and shown to mediate Fc-mediated functions. This thesis describes HA stalk-specific antibody- dependent cellular phagocytosis (ADCP), cellular cytotoxicity (ADCC) and complement deposition (ADCD) between PLWH and people without HIV (PWOH) following immunization with a seasonal trivalent inactivated influenza vaccine (TIV). Irrespective of HIV status, ADCD was boosted while ADCC was not. ADCP was only enhanced in PWOH. The coordination of these functions differed by HIV status. Additionally, differences in the regulation of these HA stalk Fc responses by HIV infection was reported. Furthermore, ADCC was not associated with protection in this study. Pre- existing ADCP reduced the risk of influenza virus infection while TIV-induced ADCD provided protection against influenza-illness. Overall, PLWH have unique responses to TIV and HA stalk- specific ADCD correlated with protection following TIV. For SARS-CoV-2, antiretroviral treatment (ART)-naïve PLWH had reduced humoral responses to respiratory infection. The infecting variants D614G and Beta differentially triggered ADCC, ADCD and antibody-dependent cellular trogocytosis (ADCT). Regarding the kinetics, PLWH infected with D614G had delayed neutralization and ADCP while Beta infection delayed ADCT, regardless of HIV status. PLWH showed improved coordination between immune responses following respiratory infection. ChAdOx-1 nCoV-19 vaccination differed from infection in that PLWH had delayed IgG binding while neutralization and ADCP were not delayed, and ADCC was substantially enhanced than in PWOH. In conclusion, despite the delayed and differential kinetics, PLWH on ART developed equivalent responses to PWOH, supporting the rapid rollout of ART and SARS-CoV-2 vaccines to PLWH. This thesis highlights the need to include high-risk groups with different responses in future vaccination trials and also supports the assessment of novel correlates of protection for future vaccines. Overall, this thesis provided insights into the mechanisms required for protection against severe respiratory diseases and improved our understanding of vaccine-induced immunity in PLWHItem Understanding SARS-CoV-2 vaccine hesitancy among pregnant women in Soweto, South Africa: A qualitative study(University of the Witwatersrand, Johannesburg, 2024) Zungu, Zwile; Myburgh, NellieThe study focused on understanding SARS-CoV-2 vaccine hesitancy among pregnant women in Soweto, South Africa. Pregnant women are at a greater risk of experiencing COVID-19 complications during pregnancy if infected with the SARS-CoV-2 virus. Vaccination uptake remains low in the population at large. This is a qualitative exploratory study approach using key-informant interviews. A total of sixteen key informant interviews with vaccinated pregnant women, unvaccinated pregnant women, healthcare workers and alternative healers were conducted. This study took place in Soweto townships, South Africa. Thematic qualitative analysis was used to construct themes in NVivo, where the gathered data was reviewed and analysed. The study found that pregnant women experience barriers and motivations that determine their decision to get vaccinated against COVID-19. Motivators to vaccinate health concerns, monetary benefit and structural motivators such as employment, travelling and education. Barriers included vaccine related fears were the main reason for poor vaccine uptake. The lack of knowledge, healthcare system barriers, misinformation, and lack of trust in the government were some reasons for vaccine hesitancy. The study's findings show that pregnant women's decisions to get vaccinated are significantly influenced by several barriers, perceptions and the motivators they haveItem Characterisation of the genetic variation in pharmacogenes involved in anti-tuberculosis drug metabolism across African populations(University of the Witwatersrand, Johannesburg, 2024) Malinga, Thandeka Vuyiswa Bongiwe; Twesigomwe, David; Othman, HoucemeddineTuberculosis (TB) is a major health burden in Africa. Although TB is treatable, anti-TB drugs are associated with adverse drug reactions (ADRs) which are partly attributed to pharmacogenetic variation. The distribution of star alleles (haplotypes) influencing anti-TB drug metabolism, is unknown in many African populations. This presents challenges in implementing genotype-guided therapy in Africa to decrease the occurrence of ADRs and enhance the efficacy of anti-TB drugs. Therefore, this study aimed to characterise the distribution of star alleles in genes that are involved in anti-TB drug metabolism (mainly isoniazid), namely CYP2E1, NAT1, NAT2, GSTM1 and GSTT1, across diverse African populations. We used 794 high-depth whole genome sequence datasets representative of eight Sub-Saharan African (SSA) population groups. Data sources included the 1000 Genomes Project and H3Africa AWi-Gen. CYP2E1, NAT1, NAT2, GSTM1 and GSTT1 star alleles were called from the WGS data using StellarPGx. Subsequently, novel star alleles were analysed, and their allele defining variants were annotated using the Ensembl Variant Effect Predictor. We present the distribution of both common and rare star alleles influencing anti-TB drug metabolism across various SSA populations, in comparison to other global populations. Various key star alleles were identified in the SSA study populations at relatively high frequencies including NAT1*10, GSTT1*0 (>50%), GSTM1*0 (49%), and NAT2*5B (21%). Additionally, we predicted varying phenotypic proportions for NAT1 and NAT2 (acetylation) and the GST enzymes (detoxification activity) between SSA and other global populations. Fifty potentially novel haplotypes were identified computationally across the five genes. This study provides insight into the distribution of star alleles in genes relevant to isoniazid metabolism across various African populations. The high number of potentially novel star alleles exemplifies the need for pharmacogenomics studies in the African context. Overall, our analysis provides a foundation for implementing pharmacogenetic testing in Africa to reduce the risk of ADRs related to TB treatment.Item Investigating the genetic cause of oculocutaneous albinism type 2 in individuals of African descent through exome sequencing(University of the Witwatersrand, Johannesburg, 2024) Mmbi, Phophi Mmapole; Ngcungcu, Thandiswa G.Oculocutaneous Albinism type 2 (OCA2) is a hypopigmentation disorder caused by variants in the OCA2 gene. A 2.7kb intragenic deletion is known to be the common variant that causes OCA2 in individuals of African descent. This variant accounts for 78% of OCA2-causing variant in the Southern African population. The diagnostic utility for individuals who tested either negative or heterozygous for the common 2.7kb deletion remained unsolved. This study reports on the identification of the NM 000275.3:c.1503+5G>A variant found in 3/8 (37.5%) non-2.7kb deletion OCA2 chromosomes within a small sample of OCA2 patients from the Southern African population, strongly suggesting the possibility of a second common variant, pending a larger screening study. This variant has been previously described in this population, but new variant interpretation tools have now permitted its reclassification. Furthermore, limitations encountered during the interpretation of data step in this study highlight the importance of informative clinical and phenotypic data for improved and sensible interpretation of genetic results.Item Characterising the combined eects of cytochrome P450 missense variants within the star allele nomenclature(University of the Witwatersrand, Johannesburg, 2024) Khoza, Nhlamulo; Othman, Houcemeddine; Hazelhurst, ScottGenetic variations in Cytochrome P450 (CYP) enzymes shape drug responses. However, many CYP haplotypes (star alleles) lack functional annotations, posing a barrier to under- standing drug metabolism comprehensively. To address this, our study investigates combined missense variant eects on CYP enzyme structures, analyzing 261 variants across 91 CYP haplotypes. We utilized Normal Mode Analysis (NMA; FoldX and ENCoM) to explore variant impact on protein stability. Subsequently, we conducted Molecular Dynamics (MD) simulations on key alleles, CYP2D6*2 and CYP2D6*17, to reveal star allele impact on protein dynamics. Integrating NMA and MD, we uncover the interactions that collectively shape the conformation and attributes of CYP enzymes. Notably, our investigation highlights significant deviations between wild-type and CYP2D6*17 -encoded proteins in the F/G loop region, pivotal for CYP functionality. Additionally, we compare NMA results with CYP2C9 and CYP2C19 Deep Mutational Scanning (DMS) results, identifying 65% concordance. Furthermore, our NMA predictions show 80% concordance with commonly used Variant Eect Predictor tools. This alignment underscores our approach’s reliability in predicting variant eects. Our study illuminates missense variants’ nuanced impact on CYP protein structures, significant for personalized medicine and drug response prediction, as accurate drug response predictions hinge on a comprehensive understanding of CYP missense variants. Moreover, our study highlights multi-scalemodelling potential for interpreting CYP missense variants, especially in star alleles. The synergy of NMA, MD simulation, and assays like DMS is invaluable, each with distinct strengths and limitations. This research deepens our understanding of the complexity of CYP metabolism profiles, providing insights into the functional assessment of CYP star alleles and missense variants with unknown functional classifications.Item Characterisation of the murine immune response to self-amplifying mRNA sequences encoding Hepatitis B virus surface proteins(University of the Witwatersrand, Johannesburg, 2024) Samudh, Nazia; Bloom, KristieVaccination against Hepatitis B virus (HBV) remains the most effective means of preventing infection. However, approximately 10% of vaccinated individuals fail to develop neutralising antibodies necessitating the development of improved vaccines which target the more immunogenic large HBV surface antigen (L-HBsAg) and can elicit cell-mediated immunity. Although Africa bears a high burden of HBV infections, placing many individuals at risk of contracting the disease, we rely on imported vaccines for prophylactic vaccination programmes. The COVID-19 pandemic was a stark reminder of Africa’s vaccine dependence and since then great interest has been generated in establishing vaccine manufacturing capabilities on the African continent. Herein, we explored the Alphavirus-based self-amplifying RNA (saRNA) vaccine platform to produce dose-sparing HBV vaccines which could contribute to vaccine independence. saRNAs encoding reporter proteins, small HBV surface antigen (S-HBsAg) or L-HBsAg were synthesised by optimised in vitro transcription. Expression of reporter proteins from saRNAs was achieved even at low concentrations and was observed for extended periods of time in vitro. saRNAs encoding S-HBsAg were able to trigger the interferon response in a dose-response manner in vitro, however, this did not hamper antigen expression. Expression of L-HBsAg was achieved but restricted to the intracellular space and will require sequence modification to facilitate secretion. In vivo delivery of saRNAs by electroporation or commercially available cationic liposomes was found to be unsuccessful, and further optimisation of in vivo saRNA delivery is required before determining the prophylactic potential of candidate vaccines. This preliminary study has produced promising results demonstrating the dose-sparing properties and self-adjuvanting nature of the saRNA vaccine platform