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Item Engineering bispecific antibodies targeting HIV-1 subtype C(University of the Witwatersrand, Johannesburg, 2024) Moshoette, Tumelo; Killick, MarkThe introduction of antiretroviral therapy has revolutionised the management of HIV-1 infection, effectively reducing morbidity and restoring life expectancy of HIV-1 patients. Continual improvements into the tolerability, increasing the genetic resistance barrier and reduction in antiretroviral drug dosing frequency have improved patient adherence and outcomes, but nonetheless require life-long therapy. By contrast, the development of an effective HIV-1 prophylactic vaccine or practical cure strategy remains elusive. Ongoing development of novel therapies and preventative strategies will be crucial in bringing an end to the global HIV-1 pandemic. Several immune-based therapeutic and preventative strategies using combinations of isolated HIV-1 broadly neutralising antibodies are currently undergoing clinical development. However, the high cost of combination immunotherapy/immunoprophylaxis in comparison to antiretroviral drugs remains a major obstacle in wide-spread clinical implementation, especially in resource-limited settings like Africa. Specifically engineered antibodies comprising dual- (bispecific) or tri-paratopes (trispecific) have been developed as an alternative to combination-based therapy. This study aimed to design and evaluate novel bispecific neutralising antibodies that outperform (i.e., exhibit improved breadth and potency) combinations of antibodies in preventing HIV-1 infection in vitro. A range of bispecific constructs were designed and incorporated the knob-in-a-hole and CrossMab mutations to promote preferential assembly of heterologous heavy and light antibody polypeptide chains into a native-like IgG configuration. Antibody sequences used in the bispecific constructs included known broadly neutralizing antibodies against HIV-1 as well as xvi ibalizumab (iMab), which targets human CD4. The bispecific antibodies and parental monoclonal antibodies were produced by transient transfection of HEK293T cells and purified by protein-A affinity chromatography followed by size-exclusion chromatography. Purified antibodies were evaluated by SDS-PAGE under reducing and non-reducing conditions and confirmed heterologous assembly of the dual heavy and light chain constituents in the bispecific antibody configurations. The bispecificity of the antibodies was confirmed by binding ELISA to HIV-1 Env and two-domain/four- domain human CD4 immobilized proteins. A diverse panel of 21 pseudoviruses inclusive of the global HIV-1 panel, was used to evaluate the breadth and potency of the bispecific antibodies in an in vitro HIV-1 neutralization assay, and compared to their monoclonal parental antibodies, alone or in combination, and selected bispecific antibodies such as 10E08-iMab and PG9-iMab. We successfully engineered and characterised two novel bispecific antibodies, iMab- Cap256 (Chapter 2; Publication 1) and iMab-N6 (Chapter 3; Publication 2). Our data demonstrate that both antibodies exhibited greater breadth over their respective parental antibodies when evaluated individually and in combination. Impressively, iMab-Cap256 exhibited remarkable potency over its parental antibodies (on average 4× and 59× more potent than CAP25 and iMab, respectively). Although iMab-N6 showed no enhancement in potency over its parental antibodies, it is calculated to have an exceptional IC80 coverage at < 1 μg/ml of 90% of global circulating HIV-1 strains, versus 86% and 48% for the N6 and iMab parental antibodies, respectively. These findings are significant as an IC80 < 1μg/ml may predict real-world clinical outcomes in protecting against HIV-1 acquisition in humans. In summary, the described bispecific antibodies may be particularly important in the context of HIV-1 xvii subtype C infection, which predominates global infections, and requires uniquely tailored treatment and prevention strategies to overcome the resource-limited challenges facing these endemic regions. These novel bispecific antibodies show great potential for further clinical development given their breadth and potency, but understandably require further testing and development before entering human clinical trials.Item Inactivating hepatitis B virus replication using obligate heterodimeric TALEN- encoding mRNA(University of the Witwatersrand, Johannesburg, 2024) Smith, Tiffany Shenay; Arbuthno, PatrickThe hepatitis B virus (HBV) poses a significant global health challenge, as chronic infection can lead to cirrhosis and hepatocellular carcinoma, resulting in over 1 million deaths annually. While vaccines exist, they have limitations, particularly in addressing chronic infections or non- responders. Current treatments, including nucleoside/nucleotide analogues and pegylated interferon-α, are inadequate for curing chronic HBV infections because they cannot eliminate covalently closed circular DNA (cccDNA) from infected cell nuclei. cccDNA persists as a viral gene transcription template, sustaining chronic infection and allowing viral reactivation upon treatment cessation. Obligate heterodimeric transcription activator-like effector nucleases (TALENs) have emerged as a promising tool for targeting HBV. These TALENs feature a DNA- binding domain and a second-generation FokI nuclease domain that enable precise DNA cleavage exclusively when heterodimeric TALENs are in close proximity on opposite DNA strands, preventing homodimeric cleavage and minimising off-target effects. This cleavage activates non- homologous end-joining DNA repair, inducing mutations at double-strand break sites. To assess the clinical potential of TALENs targeting HBV core and surface open reading frames, TALEN- encoding mRNA was synthesised by in vitro transcription using T7 RNA polymerase and three distinct cap analogues: anti-reverse cap, post-transcriptional Vaccinia cap, and CleanCap® AG. Immunocytochemistry was first performed in liver-derived cells to detect the haemagglutinin tag located within the TALEN construct. This experiment confirmed that optimal expression of Core and Surface TALENs was achieved using CleanCap® AG. TALEN efficacy was then tested in three in vitro models: Huh7 cells with HBV replication-competent plasmid, HepG2-hNTCP cells infected with HBV, and Huh7 cells transduced with adeno-associated virus type 2 (AAV2) containing HBV genome sequences from subgenotype D3. Surface TALENs reduced HBsAg Page | vii levels by 40%, 30%, and 90% across the models, with no observed cytotoxicity. In vivo studies using lipid nanoparticle-encapsulated Core and Surface TALEN-encoding mRNA in a transgenic HBV murine model showed a 63% reduction in HBsAg and over 99% reduction in viral particle equivalents, with no liver toxicity or significant inflammatory response. While Sanger sequencing did not detect targeted mutations with sufficient sensitivity, next-generation sequencing could offer more detailed insights as observed data indicate a significant difference in viral fitness between mock and TALEN-treated mice. This study represents the first successful production of obligate heterodimeric TALENs as mRNA. It highlights their potential as a therapeutic approach for chronic HBV infection by targeting and permanently inactivating HBV production.Item Molecular Characterization of Group B Streptococcus (GBS) and Its Association with the Vaginal Microbiome Among Pregnant Women from Low–Middle Income Countries(University of the Witwatersrand, Johannesburg, 2024) Matuvhunye, Takudzwa; Madhi , Shabir A.; Kwatra , GauravIntroduction: Recto-vaginal colonization by Group B streptococcus (GBS) in expectant women is a risk factor for invasive GBS disease in newborns and may predispose to stillbirths and preterm births. The focus of the research included: i. Determine the genetic composition of colonizing GBS sequence types (STs) and clonal complexes (CCs) in pregnant women and their newborns from six sub-Saharan African and two Southeast Asian low and middle-income countries (LMICs); ii. Determine the prevalence of concurrent colonization by more than one GBS serotypes using microarray; iii. Investigate the association between vaginal GBS colonization and the vaginal microbiome in the pregnant women from LMICs. Methods: Group B streptococcus isolates cultured from rectum or vagina of pregnant women (n=816) and skin surface or throat mucosal colonizing isolates from their newborns (n=628) immediately following delivery were whole genome sequenced using next generation sequencing platform (Illumina HiSeq 2000; Illumina, USA) and analyzed using bio-informatic tools, including FastQC for quality control and the short-read sequence typing tool (SRST) to determine the capsular serotypes, sequence types and antimicrobial resistance genes (AMR). Vaginal swabs from pregnant women from Kenya, Ethiopia, Mali, Bangladesh, Nigeria and South Africa were analyzed for vaginal colonization by more than one GBS serotype using microarray, and the vaginal microbiome was investigated using open array quantitative polymerase chain reaction (qPCR) methods. Results: A total of 53 STs in pregnant women and 51 STs in newborns were identified, including five new described STs. There was high concordance at the serotype (90%; 547/608), ST (88%; 533/608) and CC level (89%; 537/608) between the maternal and newborn isolates. Consequently, further detailed whole genome sequencing analyses focused on the maternal colonizing isolates. The CC23 was the most common lineage in India (47.4%; 65/137), Mozambique (40.0%; 44/110) and Kenya (29.1%; 32/110). The lineage CC17 was common in Mozambique (27.3%; 30/110), Kenya (28.2%; 31/110) and South Africa (20%; 21/105) but absent in Southeast Asian countries. The clonal complex 255 (3.4%; 3/89), CC889 (13.5%; 12/889), and CC1212 (4.5%; 4/89) were detected exclusively in Bangladesh, albeit at low frequency. There was within serotype geographic variability in relation to ST and CC between countries. Notably, serotype III GBS isolates were variably associated with the hypervirulent CC17, including high prevalence in Mozambique (97%; 30/31), Kenya (89%; 31/35) and South Africa (77%; 17/22), modest prevalence in Mali (45%; 9/20), Nigeria (35%; 6/17) and Ethiopia (33.3%; 2/6); whilst it was not identified in any of eleven serotype III isolates from the Southeast Asian countries. All serotype III isolates (66.9%; 95/142) positive for the hypervirulent A gene (hvgA gene) were associated with CC17. A rare genotype CC934 (0.2%; 2/816) which also includes the hvgA gene, was identified in Ethiopia. The prevalence of any alpha-like family genes (alp1, alp2/3, alphaC, and rib) ranged from 80.9% (72/89) in Bangladesh to 100% (110/110) in Kenya. The erythromycin resistance gene was modestly prevalent in Bangladesh (31.5%; 8/89) and Mozambique (20.9%; 23/110), whilst the prevalence thereof was less than 5% in Ethiopia, India, Nigeria, Mali, and Kenya. Analysis of the vaginal microbiome was done using open array qPCR-based method, which was targeted to identify 52 organisms. Investigation of the vaginal microbiome was done of vaginal swabs in women from Kenya, Ethiopia and South Africa. Four community state types (CSTs) were identified, including CST I dominated by lactobacillus crispatus (3.9%; n=11/282), CST III (Lactobacillus inners dominant, 7.1%; n=20/282), CST IV (dominated by diverse anerobic bacteria, 87.2%; n=246/282), and CST V (Lactobacillus jensenii dominant, 1.8%; n=5/282). The prevalence of GBS colonization was commonly associated with CST IV-A (56.4%; n=22/39), less common in CST III (30%; n=6/20) but absent in women with CST V (n=5). Colonization with Veillonella parvula (adjusted odds ratio, 2.89; 95% confidence interval, 1.53-5.43), Staphylococcus aureus (aOR, 12.3; 95% CI, 5.31-28.24) and Candida albicans (aOR, 3.98; 95% CI, 2.33-6.79) were positively associated with vaginal GBS colonization, whilst no association was evident for other organisms. Among women with GBS vaginal colonization, more than one serotype was detected using microarray in 10% (n=72/702) of women and ranged from 3.7% (n=2/54) in Bangladesh to 14.4% (n=20/139) in Mali. Conclusion: Differences in the prevalence of GBS STs and CCs between low and middle-income countries of Southeast Asia and sub-Saharan Africa may account for the regional differences in the burden of invasive GBS disease between countries. In particular, the low prevalence of CC17 associated serotype III isolates among Southeast Asian countries (India and Bangladesh), and the high prevalence of CC17 serotype III isolates from African countries (including South Africa, Mozambique, Nigeria, and Kenya) may be contributing to the reported differences in the incidence of early-onset GBS disease between the sub-Saharan African and Southeast Asia countries. Vaginal colonization by more than one GBS serotype is low in pregnant women from LMICs. The association between vaginal GBS colonization and other bacteria colonizing the vagina in pregnant women, suggest that the maternal vaginal microbiome may also influence the risk of invasive GBS disease in their newborns.Item Antifungal Activity of Synthetic Peptides Targeting Apoptosis in Candida auris(University of the Witwatersrand, Johannesburg, 2024) Shaban, Siham; Ahmad, Aijaz; Patel, MrudulaBackground: Nosocomial infections caused by the multidrug-resistant (MDR) yeast Candida auris pose a significant healthcare threat. This pathogen presents a major clinical challenge due to its resistance profile, virulence factors, including biofilm formation, and the overexpression of efflux pumps. Current antifungal treatments are often limited in efficacy against MDR fungal pathogens, particularly C. auris. Therefore, novel strategies to address these challenges are urgently needed. Apoptosis, a form of programmed cell death, represents a potential target for novel antifungal approaches. Furthermore, strategies targeting virulence and efflux pump activity could be a valuable approach to combat C. auris infections. Human antimicrobial peptides (AMPs) hold promises due to their broad-spectrum activity, including activity against various fungi. Thus, this study investigated the potential of selected human AMPs against C. auris and the role of apoptosis induction in their antimicrobial activity. These peptides were also evaluated for their ability to inhibit virulence and efflux pump in C. auris. Methods: Antifungal efficacy of seven distinct human AMPs, including HNP-1-3, hBD-1-3 and His 5, against ten C. auris clinical isolates, was determined adhering to the established CLSI guidelines for antifungal susceptibility testing. The antifungal activity of the AMPs was further assessed via time-kill curves and cell viability assays. The potential synergy with established antifungals (fluconazole, amphotericin B, and caspofungin) was explored using combination assay. To understand their antifungal mechanism, propidium iodide staining and confocal imaging were employed to evaluate their effects on C. auris membrane permeability. The three most active AMPs (HNP-1, hBD-3, and His 5) were further investigated for their ability to induce apoptosis in two C. auris isolates. Specific markers for apoptosis, including phosphatidylserine externalization (FITC Annexin-V/PI assay) and DNA fragmentation (TUNEL assay), were employed. Additionally, mitochondrial membrane potential (JC-10 staining), cytochrome c release (spectrophotometry), and reactive oxygen species (ROS) production (H2DCFDA staining) were assessed to understand potential intracellular triggers of apoptosis. Furthermore, the study investigated the impact of the most active AMPs on C. auris virulence factors and efflux pumps. Adherence and proteinase activity were assessed using adherence assays and bovine serum albumin (BSA) plates, respectively. The effects on biofilm formation, maturation and structure were evaluated using the MTT (3-(4, 5-dimethylthiazolyl- v 2)-2, 5-diphenyltetrazolium bromide) reduction test and confocal microscopy. The influence on efflux pump activity was determined by measuring rhodamine-6-G accumulation and efflux. Reverse transcription quantitative real-time PCR (RT-qPCR) was employed to investigate the effects on the expression of virulence and efflux pump-related genes. Finally, their safety was evaluated through a haemolytic assay. Results: All examined AMPs exhibited fungicidal activity against C. auris, with hBD-3 demonstrating the most potent effect. Time-kill curves confirmed the fungicidal activity of all tested peptides and viability assays demonstrated a significant decrease in viable cells upon exposure to AMPs. Notably, AMPs displayed synergistic interactions with conventional antifungals, particularly caspofungin. Mechanistically, AMPs disrupted C. auris membrane permeability, correlating with growth inhibition. Furthermore, FITC Annexin-V/PI staining and TUNEL assays demonstrated that HNP-1 and hBD-3 induced both early and late apoptosis in C. auris, while His 5 primarily caused necrosis. HNP-1 and hBD-3 triggered significant mitochondrial membrane depolarization and cytochrome c release. In contrast, His 5 displayed minimal impact on mitochondrial function. All peptides significantly increased ROS production. In addition, subinhibitory concentrations of HNP-1, hBD-3, and His 5 significantly reduced C. auris adherence and proteinase activity. All three peptides effectively inhibited biofilm formation and reduced the density of established biofilms. They also impaired efflux pump activity and downregulated genes associated with virulence and efflux pumps. Furthermore, haemolytic assays confirmed the safety of these peptides towards mammalian red blood cells. Conclusion: These findings highlight the potential of human AMPs as broad-spectrum antifungal agents against C. auris. Their ability to target multiple mechanisms, including direct killing, apoptosis induction, virulence inhibition and efflux pump reversal, suggests a multifaceted approach to combat C. auris infections. Additionally, the synergistic interaction with conventional antifungals suggests potential for combination therapy. Overall, this study findings provide a strong rationale for further investigation and development of human AMPs as novel antifungal agents against this MDR pathogen.Item Designing and evaluating the utility of a panel of de novo mutation enriched genes for diagnosing South African patients with developmental delay(University of the Witwatersrand, Johannesburg, 2024) Nevondwe, Livhuhani; Krause, Amanda; Carstens, NadiaDevelopmental disorders (DDs), are diverse and life-altering conditions, with a higher prevalence in sub-Saharan Africa. Approximately 50% of DDs have a genetic basis, but many patients remain undiagnosed due to limitations in current testing methods. Whole exome sequencing (WES) and whole genome sequencing (WGS) are recommended due to their high diagnostic rates, but their routine use is impractical in many low- and middle-income countries (LMICs), including South Africa. Thus, cost-effective alternatives are needed. Targeted gene panels, despite declining global use, might still be relevant in LMICs. This study explored a targeted de novo mutation (DNM)-enriched gene panel in two cohorts: a well-phenotyped group and a group with developmental delay but less phenotypic detail. The analysis included WES on samples from 96 patients in each cohort, followed by a virtual DNM-enriched gene panel analysis. Results showed that 15% of the well-phenotyped cohort and 5% of the unphenotyped cohort had causal variants, which aligns with global diagnostic yields for targeted gene panels in DDs. Among these variants, 47% were de novo mutations, 5% were maternally inherited, and 47% had indeterminate inheritance due to the absence of parental samples. Significant clinical interventions were identified for the majority of patients. Approximately 50% of the causative variants were novel, thus expanding the mutation profile of DDs. Additionally, we identified key factors influencing diagnostic yield, such as the availability of phenotypic data and the use of trio analysis. These findings provide valuable insights into the implementation of routine genetic testing in LMICs, highlighting the importance of comprehensive clinical data submission, trio sequencing and/or analysis, and the evaluation of copy number variants in the diagnostic workup of individuals with DDs. We developed a DNM-enriched gene panel as a starting point for creating practical, resource- efficient diagnostic strategies for DDs. We conclude that this approach would be beneficial for the diagnostic evaluation of DDs and developmental delays in resource-constrained settings.Item Heat Shock Factor (HSF) Expression and Its Effect on Life Table Parameters in the Main African Malaria Vector Anopheles funestus(University of the Witwatersrand, Johannesburg, 2024) Bloch, Nerissa Wendy; Koekemoer, LizetteAnopheles funestus is a major malaria vector, which is predominantly responsible for malaria transmission in Africa and this vector needs to be controlled due to the devastation of the disease. Temperatures worldwide are rising steadily, which can affect mosquito distribution and behaviour. Heat shock factor (HSF), a transcription factor, is crucial in these physiological and behavioral processes. This is because HSF has a high degree of functional variability and is temperature dependent, enabling mosquitoes to survive the heat by repairing damaged proteins in the cell. This study aimed to understand the biological role of HSF in the African malaria vector An. funestus with regard to HSF transcript abundance and its effect on life table parameters. In this work, the HSF gene was the target of RNA interference (RNAi). RNAi was implemented using short interfering RNA (siRNA) that was previously synthesised and was performed to understand the role of HSF in An. funestus. HSF is a poorly understood gene, yet it has been found to have a variety of functions within insects and, more specifically, mosquitoes. Understanding the role of HSF in An. funestus is important to advance knowledge in this understudied vector. HSF transcript abundance was measured using real-time quantitative PCR (qPCR) throughout the four life stages in An. funestus in order to assess the average expression levels of HSF at 25°C. The HSF transcript abundance was significantly lower during the egg and pupal stages in comparison to the larval and adult stages. Inhibition, via siRNA, of the HSF transcript was subsequently used to understand its biological role in An. funestus. A system was designed to deliver the siRNA to the adult using the ingestion of sugar water. This was the chosen delivery system due to its ease of use and the potential to replicate this method in the wild because of the sugar-feeding behaviour of the adult mosquitoes. A known reference gene, RPL19, was used to evaluate the efficiency of vii a “siRNA-sugar feeding delivery system” in An. funestus. Results showed this would be an effective system for evaluating transcript inhibition or knockdown. Different concentrations of HSF-siRNA were utilised, 10 ng/μl, 20 ng/μl and 30 ng/μl at three different temperatures of 20°C, 25°C and 30°C, to evaluate the impact of different concentrations and temperatures on the fold change of HSF transcript abundance after siRNA treatments. A 20 ng/μl siRNA concentration at 25°C reduced the HSF transcript abundance. Therefore, these conditions were selected to study the phenotypic effect of HSF-siRNA on longevity and reproduction in An. funestus. Longevity, fertility, fecundity, and time-to-hatch were all reduced when mosquitoes were treated with HSF- siRNA. In addtion, the impact of decreased survivability in males was more prominent than in females. In conclusion, HSF might regulate vector development and needs to be investigated in the future. Furthermore, this study confirms the pleiotropic effect of HSF in An. funestus.Item Determining the risk profile for chronic kidney disease (CKD) in rural South Africans using genetic risk scores and protein markers(University of the Witwatersrand, Johannesburg, 2024) Govender, Melanie Ann; Ramsay, MichèleChronic kidney disease (CKD) is a global public health concern, with disproportionate morbidity and mortality in low- and middle-income regions, including Sub-Saharan Africa. Recent advancements in multi-omics approaches have explored disease risk indicators and contributed to the understanding of the pathophysiology of CKD in high-income populations. The overall aim of this research was to assess and understand CKD in a Sub- Saharan population using genetic risk models for kidney disease and to evaluate the proteomic profile of individuals with hypertension-associated albuminuria, with a view to detecting indicators of CKD and disease progression in a Sub-Saharan cohort. The first objective was a scoping review that was undertaken to evaluate existing literature for potential biomarkers for CKD and to identify gaps in literature. Key literature gaps identified included the lack of studies that focus on HT in the context of kidney disease and only one study on African individuals residing in Africa. In this work, two research studies were developed based on existing data from the African Research Kidney Disease (ARK) study, a well characterised population-based cohort study of black individuals from Agincourt in the rural Mpumalanga Province, South Africa. The second objective was a genomics study which aimed to examine the potential of using summary statistics from three discovery datasets to assess the predictive accuracy of polygenic scores (PGSs) for CKD and kidney function markers. Limited transferability was observed, explaining <1% of the variability in kidney disease markers in this African cohort. A PGS model derived from the transethnic cohort for estimated glomerular filtration rate (eGFR) explained the highest variability (0.8%) in African individuals and was significantly associated with HT (P<0.001), diabetes (P=0.007), and HIV (P=0.001). The third objective was a proteomics study which aimed to compare proteomic profiles of cases with both HT and albuminuria to controls (neither condition) to identify proteins and pathways involved in hypertension- associated albuminuria. Pathways including immune system (q=1.4x10-45) and innate immune system (q=1.1x10-32) were linked with hypertension-associated albuminuria. Proteins including angiotensinogen, apolipoprotein L1, and uromodulin had the highest disease scores (76–100% confidence). A machine learning approach was able to identify a set of 20 proteins that contributed to classifying disease status (ie, hypertension-associated albuminuria). Page | VI The assessment of PGSs for kidney function markers contribute to understanding of CKD genetic risk prediction in Africa, while the proteomics research added new knowledge to understanding the role of proteins and pathways involved in hypertension-associated albuminuria in Africa. This research addresses the gap of a lack of ‘omics research in resident African populations. It also contributes to the understanding of risk prediction for CKD and identifies potential proteomic markers for hypertension-associated albuminuria that may inform the development of personalised treatment strategies in Africa.Item The Utility of Clinical Exome Sequencing as a First-Tier Diagnostic Tool in Critically Ill Infants in South Africa(University of the Witwatersrand, Johannesburg, 2024) Campell, Lisa; Carstens , Nadia; Krause, AmandaGenetic disorders are significant contributors to infant mortality, morbidity, hospitalisation, and the need for intensive care globally. The identification and diagnosis of genetic disorders in ill infants is challenging due to their indistinct, and often atypical disease presentations. Diagnosis is traditionally driven by a differential clinical diagnosis; however, broad genotype-first approaches are now the recommended strategy in ill infants. The use of NGS-based testing, including gene panels, whole exome sequencing and whole genome sequencing, has successfully been utilised to diagnose genetic disorders in ill infants and has begun to be implemented in global settings; however, representation of low- and middle-income countries is lacking. Local infrastructure, capacity and expertise significantly affect successful implementation in these contexts. We therefore aimed to investigate the utility and implementation of singleton virtual exome sequencing panels in a cohort of 32 ill infants in the South African State healthcare system, providing the first investigations into the use of NGS in a neonatal intensive care unit setting in Africa. Three virtual panels were used to analyse exome sequencing data: a curated panel of genes implicated in neonatal-onset conditions and enriched for variants in South African populations; a ClinVar panel; and the Developmental Disorders Genotype-to- Phenotype (DDG2P) panel. A diagnostic yield of 22% was achieved across the three virtual panels, providing a definitive molecular diagnosis in seven ill infants. These infants experienced changes in their clinical management as a result of this diagnosis, including the initiation of palliative care, familial screening and prenatal testing for future pregnancies. The adaptation of recommended implementation strategies was necessary in the South African context to address shortages in resources, infrastructure and bioinformatics capacity through the use of gene panels instead of whole exome sequencing and the use of locally available technologies; shortages in the trained genetics workforce through the reliance on primary care clinicians for referrals; and through the singleton sequencing approach to address the unavailability of parents for trio-sequencing and the additional cost factors. This pilot study demonstrated the utility of NGS for the diagnosis and management of ill infants in the South African State healthcare system and explored the challenges in implementing NGS technologies in resource-limited settings. The implementation strategy explored through this research provides a baseline from which advanced NGS diagnostic v strategies can be developed and from which scaled up investigations of utility in the South African State setting can commence.Item Molecular epidemiology and characteristics of immune adaptations across the SARS-CoV-2 spike glycoproteins from Gauteng, South Africa, 2020 to 2022(University of the Witwatersrand, Johannesburg, 2024) Subramoney, KathleenThe SARS-CoV-2 global pandemic has been fueled by several variants of concern (VOC) that have gained more efficient transmission or immune evasion properties over time. To better understand the diversity and evolutionary characteristics of SARS-CoV-2 lineages in South Africa we described the analysed the SARS-CoV-2 lineages and VOCs circulating during 2020 to 2022, as well the impact of the S protein and its potential to act as a candidate vaccine. The first objective of this study was to rapidly identify emerging VOCs based on key SARS- CoV-2 S protein mutations. The second objective was to describe the impact of intra-host immune adaptations on the evolution of SARS-CoV-2 S protein genes among individuals with SARS-CoV-2 infections. Thirdly, by timing the emergence SARS-CoV-2 dominant variants we aimed to unravel the significance and abundance of low-frequency lineages that emerged during five COVID-19 waves in South Africa. The final objective was to assess if accounting for diversity among SARS-CoV-2 S protein’s improved predicted epitope coverage of a derived immunogen. Single nucleotide polymorphism (SNP) PCR-based genotyping assays targeting specific mutations were used to detect VOCs that circulated in 2021. The allele frequencies (AF) as determined by SNP PCR analysis and variant calling from FASTQ reads using galaxy.eu were performed to describe intra-host SARS-CoV-2 S protein variants. Whole genome sequencing was performed to identify and analyse SARS-CoV-2 strains circulating in South Africa from 2020 to 2022 and detect low-frequency lineages. Mosaic vaccine suite tools were used to design an optimal S protein construct from sequences generated in this study. The construct was further tested for antigenicity, toxicity, N- and O-linked glycosylation sites and CTL predictions. A combination of P681R and L452R SNPs were detected in 73.6% (538/731) of the samples classified as Delta, while N501Y and del69/70 SNPs were detected in 3.6% (26/731) of samples classified as Alpha. The detection of the del69/70 and K417N coupled with SGTF is efficient to exclude Alpha and Beta variants and rapidly detect Omicron BA.1. SNP assays detected 5.3% of cases with Delta that displayed heterogeneity at delY144, E484Q, N501Y and P681H. However, heterogeneity was confirmed by sequencing only for the E484Q and Characterisation of SARS-CoV-2 Page 9 of 155 delY144 mutations. Variant calling from FASTQ reads identified intra-host diversity in the S protein among 9% of cases that were infected with Beta, Delta, Omicron BA.1, BA.2.15, and BA.4 lineages. Heterogeneity was primarily identified at positions 19 (1.4%) with T19IR 371 (92.3%) with S371FP, and 484 (1.9%) with E484AK, E484AQ and E484KQ. In 2020, 24 lineages were detected, with B.1 (3%; 8/278), B.1.1 (16%; 45/278), B.1.1.348 (3%; 8/278), B.1.1.52 (5%; 13/278), C.1 (13%; 37/278) and C.2 (2%; 6/278) circulating during the first wave. Beta dominating the second wave of infection in 2020. B.1 and B.1.1 continued to circulate at low frequencies in 2021 and B.1.1 re-emerged in 2022. Beta was outcompeted by Delta in 2021, which was thereafter outcompeted by Omicron sub-lineages during the 4th and 5th waves in 2022. Several significant mutations (del69-70, delY144, E484K, N501Y and D614G) identified in VOCs were also detected in low-frequency lineages. During the 5 waves of infection, B.1 and C.1/ C.2 lineages co-circulated with a dominant VOC. Following our findings of co-circulation of VOCs and other lineages and evidence of quasispecies we investigated if accounting for diversity of SARS-CoV-2 strains would render an improved S immunogen. The optimal mosaic S protein generated had predicted CTL epitope coverage of ~95% to 98% and was classified as an antigen based on a prediction score of 0.47. Reverse translation was used to generate the novel S gene for the expression construct SC2M2. The NTD and RBD regions were non-toxic, and the derived novel S protein comprised 10 additional N-linked glycosylation sites and 4 O-linked glycosylation sites when compared to the Wuhan Hu-1 strain. Our study findings have shown that (i) rapid detection of emerging VOCs was possible using SNP genotyping assays, and can be used by low to middle income countries to detect Alpha, Beta, Delta and Omicron BA.1; (ii) heterogeneity within the S protein encourages escape from neutralising antibodies and the evolution of SARS-CoV-2, which may contribute to the ongoing emergence of new variants associated with continued outbreaks globally; (iii) low frequency lineages that share mutations with VOCs could lead to convergence and recombination events that result in the next novel lineages or variants that may further increase transmissibility, infectivity and escape immunity; and lastly (iv) the novel S expression construct designed, based on previous and currently circulating VOCs and lineages, could potentially be used to develop improved SARS-CoV-2 vaccines.Item Prevalence and Molecular Epidemiology of Bordetella pertussis Infection in South Africa(University of the Witwatersrand, Johannesburg, 2024) Moosa, Fahima; Wolter, Nicole; du Plessis, MignonPertussis remains a public health concern in South Africa, with increases in cases and outbreaks in recent years. We determined the incidence, transmission dynamics, serological attack rates and molecular epidemiology of B. pertussis in South Africa. Data from a longitudinal study enrolling individuals each year in 2016–2018 from two communities were used. Nasopharyngeal swabs were collected from participants twice-weekly and tested by real-time PCR. Serum was collected at 8 time points and tested using the anti-pertussis toxin IgG ELISA kit. Whole genome sequencing was performed on all available cultures (n=32) sourced from three additional surveillance programs between 2015–2019. Data were described and analyzed using univariate and multivariable regression models. Among 1684 participants, the incidence of B. pertussis was 0.21 (95% confidence interval 0.17–0.25) per 100 person-weeks. The mean duration of infection was 12 days (±standard deviation 19.1). Transmission of infection was more likely to occur from male index cases [adjusted odd ratio 12.20 (95%CI 1.57–94.96)], and individuals with ≥7 day’s infection duration [aOR 24.80 (95%CI 2.74–224.30)]. B. pertussis seroprevalence ranged from 1.8% to 5.2% across eight blood draws. The serological attack rate was 5.8% (87/1509), which was similar to the PCR attack rate (6.2%, 94/1509) (p=0.64). PCR-positive individuals aged 5–18 years (vs 19-44, aOR 6.8, 95% CI 1.3-35.1) and with episode duration of ≥7 days (vs <7 days, aOR 13.3, 95% vi CI 3.4-51.1) were more likely to seroconvert. For all individuals that seroconverted, the ≥4-fold rise in anti-PT IgG titer was detected by the next blood draw (mean: 2.9 months (range 3 weeks – 5.9 months). Using genome data, all isolates were identified as the globally-disseminated sequence type 2 and harbored the pertussis toxin promoter ptxP3. The dominant genotype was ptxP3-ptxA1-ptxB2-prn2-fimH2 (31/32, 96.9%), with no pertactin-deficient or other mutations in vaccine antigen genes identified. Within the community, despite a high incidence of B. pertussis, there was an overall low seroprevalence. Our data highlighted that increases in cases in South Africa are not likely due to evolutionary changes in the genome but potentially waning immunity due to the use of acellular vaccines and/or population immunity gaps.