*Electronic Theses and Dissertations (PhDs)

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    Defining the development of gp120-gp41 interface directed broadly neutralizing antibodies in HIV-1 infection
    (2024) Hlatshwayo, Vincent Nkosinathi
    A prophylactic HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) against conserved HIV-1 envelope epitopes such as the gp120-gp41 interface which includes the FP. The isolation of gp120-gp41 interface-, and FP-directed bNAbs from chronically HIVinfected donors has made this epitope an appealing vaccine target. Moreover, promising preclinical immunogenicity animal studies have shown the possibility of eliciting such responses in animals. However, little is known about the population prevalence or kinetics of gp120-gp41 interface responses, including FP-directed responses. Lastly, few FP-directed antibodies have been isolated from people living with HIV (PLWH), limiting our understanding of common developmental pathways that can be explored for vaccine purposes. Here, we first assessed the prevalence of bNAbs in participants previously enrolled in the CAPRISA 004 Tenofovir gel trial (CAP004). We show that in this cohort, only 12% of individuals developed breadth at three years post-infection, and that high viral load and low CD4 count were associated with bNAb development, as previously reported. ELISA screening showed that only 13% of individuals developed FP-directed responses at three years postinfection. Of the 13% (n=9), only two donors had broad plasma responses, including donor CAP312, whose plasma exhibited 64% neutralization breadth at three years post-infection. In CAP312, FP binding and heterologous neutralization against a multi-clade 22 virus panel appeared simultaneously, within one year (~ 50 weeks post-infection). Taken together, our findings suggest that gp120-gp41 interface- and FP-directed responses are infrequently elicited during infection. As few FP-specific bNAbs have been isolated to date, little is known about their shared features that could be exploited for eliciting FP-specific bNAbs by vaccination. We next isolated three FP-specific mAbs from donor CAP312 at three years post-infection; AIRU-F8, AIRU-G9 and AIRU-G4 with 64, 45 and 5% breadth, respectively. We showed that our mAbs, and previously isolated bNAbs PGT151 and ACS202 share gene usage and have a similar unusually long CDRH3, as a result of a conserved motif inherited from the germline IGHJ6*02 gene. Furthermore, we showed that CAP312 mAbs have even longer CDRH3s compared to PGT151 and ACS202 despite this shared motif. We also showed, using point mutants and glycan mutants that our FP-specific mAbs have a unique neutralization profile compared to published mAbs. Overall, these results suggest that FP-specific mAbs share structural and genetic features that could be explored further for lineage vaccine development. Lastly, we delineated the ontogeny of the gp120-gp41 interface-directed nAb CAP248-2B by tracing the evolutionary pathways utilising longitudinal samples from 11-281 weeks postinfection (wpi). CAP248-2B interacts with the HIV-1 Env trimer through a long light chain CDRL3 that inserts into the viral membrane, and a heavy chain CDRH1 32ED33 motif that interacts with gp41. We showed that the unusually long light chain CDRL3 and the heavy chain 32ED33 motif are functionally redundant against heterologous viruses. We also showed that affinity maturation mutations in this lineage selected a lineage with limited heterologous neutralization breadth. In summary, these findings support further research into gp120-gp41- and FP-specific responses in multiple cohorts. Furthermore, future studies should aim to elucidate mechanisms governing the development of these responses so that productive developmental pathways can be identified and exploited for vaccine design.
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    The changing face of thrombotic thrombocytopenic purpura: the pathophysiological role of endotheliitis and complement activation in the development of human immunodeficiency virus associated thrombotic thrombocytopenic purpura
    (2024) Louw, Susan
    Introduction- Human Immunodeficiency virus (HIV) is a described risk factor for secondary thrombotic thrombocytopenic purpura (TTP) (HIV-TTP). The pathogenesis of this thrombotic microangiopathy (TMA) is however still unclear. The micro-thrombotic process in TTP is related to excess ultra-large von Willebrand Factor (ULVWF) multimers produced by the endothelial cells. Autoantibodies to the VWF cleaving protease, adisintegrin-and-metalloproteinase-with-thrombospondin-motifs 13 (ADAMTS-13), are postulated to be pivotal in initiating HIV-TTP. Inflammation and complement activation with resultant endothelial dysfunction and excessive release of ULVWF multimers have been implicated in other forms of TMA. These pathophysiological processes were investigated to assess the contribution to the development of HIV-TTP. Methods- Data were collected from patients presenting with HIV-TTP in an observational cohort study to delineate the routine presenting laboratory parameters and treatment outcomes. The published literature was reviewed to ascertain the documented prevalence, postulated pathogenesis and treatment outcomes of patients with HIV-TTP. An investigational study was performed in patients (n=35) presenting with HIV-TTP. In this study, patient samples were analysed for levels of endothelial activation markers (soluble intracellular adhesion molecule [sICAM] and soluble vascular adhesion molecule [sVCAM]), inflammatory cytokines (tumour necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]), and complement components C3 and 4 and complement Factor H (CFH), an inhibitor of the complement pathway. Published studies were also reviewed to define the baseline biomarker levels of endothelial dysfunction and coagulation activation in people living with HIV (PLWH) without TTP to serve as points of reference. Data were collected from 2 patient cohorts with HIV infection with either disseminated intravascular coagulation (DIC) or with HIV-TTP to assess the utility of scoring systems. Results- In contrast to published literature which suggests that the prevalence of HIV-TTP is declining, this TMA is prevalent in South African PLWH in Johannesburg with heterogeneous clinical and laboratory presentation. Conventional scoring systems specifically the PLASMIC (reduced platelet count, red blood cell haemolysis, absence of cancer, no history of transplantation, reduced red blood cell volume, preservation of the international normalised ratio and creatinine) score, developed for detection of other acquired forms of TTP, performed inconsistently in the 35 patients assessed with considerable overlap with other TMAs, notably disseminated intravascular coagulation (DIC). ADAMTS-13 levels were undetectable in all patients and all patients had anti-ADAMTS-13 antibodies. The clinical presentation was, however, atypical. To delineate intermediate pathophysiological markers, the complement system, proinflammatory system and coagulation system, as well as markers of endothelial activation were analysed before and after therapeutic plasma exchange (TPE). Complement components C3 and -4 were consistently at the lower limit of the normal reference range in HIV-TTP patients at presentation. The complement regulatory protein, complement Factor H, was increased. Patients with HIV-TTP had significantly increased levels of proinflammatory cytokines when compared with published results in PLWH without comorbidities. Endothelial activation markers, sICAM and sVCAM, were also significantly increased in the HIV-TTP cohort. Importantly, D-dimer levels were also raised in this cohort of patients. Conclusions- HIV-TTP remains a cause of HIV-related morbidity and mortality in South African patients. This study reports the findings on 35 PLWH who presented to hospitals in Johannesburg with suspected TTP. The clinical presentation was inconsistent with other secondary forms of TTP i.e., minimal evidence of systemic organ dysfunction. All patients presented with schistocytosis, evidence of haemolysis and thrombocytopaenia and all had low ADAMTS-13 activity levels at presentation with detectable antiADAMTS-13 antibodies. Biomarkers of endothelial dysfunction, proinflammatory cytokine levels and markers of coagulation were all significantly increased suggesting that the pathophysiology involves complementary proinflammatory pathways which directly impact secretion of VWF from a compromised endothelium. Activation of the coagulation system, as reflected by increased D-dimer levels, specifically suggests that there may be overlap in the pathophysiology of HIV-TTP and HIV-associated DIC. A potential strategy for differentiation of these disorders with modification of scoring systems is suggested. This study provides compelling evidence of the role of the endothelium in HIV-TTP. Future directions will include validation of the biomarkers described here in more extensive cohorts as well as investigation of these biomarkers in management of these patients.
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    Prognostic influence of myc aberrations and other clinicopathological factors of aggressive b-cell non-Hodgkin lymphomas
    (2024) Pather, Sugeshnee
    Introduction: Up to 30% of cancers in Africa are linked to infectious agents and in the context of the aggressive B-cell non-Hodgkin lymphomas (NHL), human immunodeficiency virus (HIV) infection is an established risk factor. Aggressive B-cell NHLs are frequently confirmed at the Chris Hani Baragwanath Academic Hospital due to the high seroprevalence of HIV infection. Therefore, an array of clinicopathological characteristics of these tumours was evaluated with the aim of identifying poor prognostic factors. Materials and methods: HIV-associated plasmablastic lymphoma (PBL), HIV-associated diffuse large B-cell lymphoma (DLBCL) and DLBCL, not otherwise specified (NOS) were included from October 2013 to June 2017. Formalin-fixed paraffin-embedded tissue sections were subjected to c-MYC immunohistochemistry (IHC), dual-colour chromogenic and fluorescence in situ hybridisation (CISH and FISH) for assessment of MYC gene rearrangement and MYC gene copy number enumeration. Thereafter, the clinicopathological characteristics were explored. Results: The study included 67 PBL patients and 63/64 (98%) were HIV-seropositive. HIVassociated PBL was typified by a mean age of 41 (standard deviation [SD] ± 10.1) years and a 54% female predominance. The patients received combination antiretroviral therapy (c-ART) prior to, or shortly after, the lymphoma diagnosis was confirmed. The median CD4 count was 170 (interquartile range [IQR] 249) cells/mm3 and 37% of the patients had CD4 counts y (P=0.02). Expression of c-MYC protein (i.e., ≥40%) occurred in 81% of PBL. Epstein-Barr virus (EBV) latent infection was detected in 90% of these tumours by utilising CISH. MYC gene aberrations included MYC rearrangement (70%) and a low-level increase in MYC gene copy numbers (43%). Concurrent MYC rearrangement with increased MYC gene copy numbers (49%) were also detected. In addition, there was low-level polysomy of chromosome (C) 8 (6%). MYC aberrations in HIV PBLs were significantly associated with a SS appearance (P=0.01), monomorphic morphology (P=0.03), c-MYC protein expression (P=0.03) and mortality (P=0.03). The median overall survival (OS) for HIV PBL was 75 days (95% CI 14–136). MYC aberrations in HIV PBL did not significantly influence the median OS [MYC+ 65 days (95% CI 0–143 days) and MYC- 71 days (95% CI 3–139 days), P=0.61] There were 22 HIV seronegative DLBCL, NOS patients (19%) with a mean age of 57 (SD ±16.7) years and a 59% male predominance. There were 93 patients (81%) with HIV-associated DLBCL, typified by a mean age of 42 (SD ±10.8) years and a 55% male predominance. The HIV seropositive patients were significantly younger at the time of presentation with lymphoma (P <0.01). c-ART was commenced prior to, or shortly after, the lymphoma diagnosis was confirmed. The median CD4 count was 162 (IQR 215) cells/mm3 and 33% of the patients had CD4 counts <100 cells/mm3 . The median viral load was 217 (IQR 182 981) copies/mL and 30% of the patients had a lower than detectable limit viral load. An advanced stage of lymphoma, i.e., stage III-IV, at presentation occurred in 87% of the patients. HIV DLBCL demonstrated a germinal centre (GC) and non-germinal centre (NGC) immunophenotypic cell of origin (COO) in 53% and 47%, respectively. Expression of c-MYC protein occurred in 58% of the HIV DLBCLs and this was significantly associated with a SS appearance (P=0.04) and high tumour proliferation indices, i.e., Ki-67 ≥90%, (P<0.01). Double expression of c-MYC and BCL2 proteins were significantly associated with the NGC COO immunophenotype (P<0.01). MYC aberrations included a low-level increase of MYC gene copy numbers (57%) and MYC rearrangements (12%). Infrequently, C8 polysomy, MYC gene clusters and concurrent MYC rearrangement with increased MYC gene copies were also identified in HIV DLBCL. The median OS was 228 days (95% CI 54–402) and 825 days (95% CI 309–1341) in the HIV seropositive and seronegative DLBCL groups, respectively (P=0.08). Compared with the HIV seronegative DLBCL group, an inferior median OS outcome occurred in the HIV seropositive group when the CD4 counts were <100 cells/mm3 (P=0.04) and when the Internal Prognostic Index (IPI) was 3–5 (P=0.01). MYC aberrations did not significantly influence the median OS [MYC+ DLBCL 155 days (95% CI 0–356) and MYC- DLBCL 154 days (95% CI 53–256), P=0.67]. In the multivariate regression analysis, the presence of concomitant infections negatively impacted the overall survival (hazard ratio 4.01 [95% CI 1.86–12.20], P=0.02).
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    Variation in the LBP-CD14-TLR4-LY96 gene complex and consequences of microbial translocation in HIV-1 infected black South Africans
    (2024) Mncube, Sizanani
    Persistent immune activation and inflammation in people living with HIV-1 (PLWH) has been associated with higher morbidity and mortality, even in individuals on antiretroviral therapy (ART). Microbial translocation, among other factors, has been identified as a major driver of persistent immune activation. A subgroup of PLWH collectively known as HIV-1 controllers can naturally control the HIV-1 infection without the use of ART. Little is known about the extent and the role of microbial translocation/immune activation in African HIV-1 controllers. Translocated lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls elicits innate immune responses through the activation of the toll-like receptor 4 (TLR4) in a complex pathway, which requires the use of cluster of differentiation 14 (CD14), LPS binding protein, and Lymphocyte antigen 96 (LY96) also known as Myeloid differentiation factor 2 (MD-2). Although numerous studies have reported associations of expression levels of the LPS recognition and signalling molecules as well as variants in the genes encoding for these molecules, with the risk and severity of various inflammatory, autoimmune, and infectious diseases, such studies are limited in African populations. Given the large genetic diversity in African populations, characterisation of both the constitutive expression levels and genetic variation in these molecules is essential to understanding HIV-1 infection in the populations most affected by the AIDS epidemic. We quantified constitutive expression of cell surface TLR4 and CD14 (mCD14) on monocytes and neutrophils using flow cytometry and quantified plasma levels of soluble CD14, LBP, and MD-2 using commercially available ELISA kits in two ethnically divergent South African populations [healthy HIV-1 uninfected black (n=17) and white (n=21) individuals]. Furthermore, the influence of sex and age on the expression levels of these molecules was also investigated. We found higher LBP plasma levels in black South Africans compared to white South Africans (p<0.0001), however these two populations did not differ significantly in expression levels of CD14 (mCD14 and sCD14), TLR4, or MD-2. Sex differences in the TLR4 expression levels, with higher TLR4 on total monocytes (p=0.016) and CD14+ 1CD16- (p=0.009) and CD14+CD16+ (p=0.009) subsets of monocytes in females compared to males were observed in the white South African population but not in the black South African population. Significant population and sex-specific negative correlations between age and CD14 expression on monocytes, monocyte subsets and neutrophils, and TLR4 expression on neutrophils were observed. In addition, we found that there is differential regulation of TLR4 expression on monocytes and neutrophils between black and white South Africans post stimulation with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Together, thesefindings suggest that population differences in plasma levels of LBP, and population-specific sex differences in TLR4 expression, are likely to differentially impact TLR4 functionality. Using whole genome sequencing data (WGS), we next sought to fully describe the genetic variation and linkage disequilibrium (LD) patterns in the LBP, CD14, TLR4, and LY96 genes in HIV-1 uninfected black South Africans (n=87, SA controls), and compared the representation of the variants to select populations from the 1000 Genomes Project. Our results revealed that the representation of genetic variants and LD patterns across these genes in the SA black population more closely mirrored those of representative African subpopulations (Yoruba in Ibadan, Nigeria, and Luhya from Webuye, Kenya) than the European and Asian populations. These findings emphasize that there are vast genetic differences in African populations compared to non-African populations, which could differentially affect gene regulation and associations with various diseases. Several novel variants and putative haplotypes were identified in the SA black population which, upon verification in future studies, will serve to add to understanding the genetic diversity in this population group. Using WGS data, we also assessed the representation of the LBP, CD14, TLR4 and LY96 gene variants in a cohort of black South African ART-naïve HIV-1 controllers (n=39) comprised of elite controllers (n=21), viraemic controllers (n=6), and high viral load long-term nonprogressors (n=12), relative to the SA controls. Only one CD14 5’ flanking region SNP (rs186291587) showed a significant difference in minor allele frequency (MAF) representation in elite controllers when compared to SA controls (p=0.024; OR=13.3, CI: 1.3 – 131.4). The representation of several TLR4 variants showed significant differences when HIV-1 controllers were compared to SA controls and the most significant differences were predominantly found in comparison to the HVL LTNPs - the most significant difference observed was overrepresentation of two SNPs in complete LD (r2=1), a newly identified intronic variant (TLR4 NI-2), and a 3’ flanking region SNP (rs113017335) in HVL LTNPs compared to SA controls (p=0.006; OR=24.71, CI: 2.46-248.51). The representation of several LBP variants also differed between HIV-1 controllers and SA controls, here predominantly when viraemic controllers were compared to SA controls. Minor allele frequency overrepresentation of the LBP intronic SNP (rs1250247980) in the total group of HIV-1 controllers (p=0.003), and viraemic controllers (p=0.0002), relative to the SA controls, was the most significant difference observed. Furthermore, differences in the representation of LY96 variants were observed when the total group of HIV-1 controllers, elite controllers and HVL LTNPs were compared to SA controls - the most significant difference observed was the MAF and heterozygosity overrepresentation of an intronic SNP (rs149605245) in elite controllers compared to SA controls (MAF: p=0.007; heterozygosity: p=0.007). These results suggest a potential role of the LPS recognition and signalling molecules in natural HIV-1 control. Lastly, in ART-naïve black South African elite controllers (n=44), HVL LTNPs (n=12), progressors (24), and in HIV-1 uninfected controls (HUCs, n=17), we measured and compared plasma levels of select innate immune molecules that are considered markers of microbial translocation and gut damage (LBP, sCD14, REG3α), or are important in interacting with TLR4 (MD-2). We found no differences between groups in plasma levels of LBP and MD-2. However, sCD14 was significantly higher in progressors compared to all groups (HUCs, p=0.0001; ECs, p0.05). Marked sexspecific differences in REG3α levels were evident, with females having significantly higher levels compared to males in all groups (HUCs and ECs, p=0.0001; ECs, p<0.0001; HVL LTNPs, p=0.0005), with no differences between HIV-1 uninfected controls, elite controllers and HVL LTNPs. Plasma levels of REG3α were unexpectedly significantly lower in progressors compared to elite controllers (p=0.007) and HVL LTNPs (p=0.018), however similar to HIV-1 uninfected controls (p>0.05). Marked sexspecific differences in REG3α levels were evident, with females having significantly higher levels compared to males in all groups (HUCs and ECs, p<0.0001; HVL LTNPs, p=0.036; progressors, p=0.005). Our data suggests that in black South Africans, REG3α plasma levels are not a reliable marker of gut damage, and that increased levels in elite controllers and HVL LTNPs might contribute to protection from excessive systemic activation in the presence of microbial translocation, consistent with reduced monocyte activation in these groups. Progressors, on the other hand, appear to have an inability to produce REG3α while having substantial monocyte activation. Our findings highlight the importance of sex differences, and that studies conducted in populations of different ethnic backgrounds are often not directly comparable Overall, findings presented in this thesis contribute to the understanding of the baseline expression levels and the genetic diversity in the LBP, CD14, TLR4, and LY96 gene complex in the black South African population, and the representation of these variants in black South African HIV-1 controllers. This thesis also highlights the importance of taking ethnicity, sex, and age into consideration when exploring measures that quantify biological parameters. Understanding of the molecules important in the TLR4 signalling pathway can help elucidate approaches that could contribute to curbing immune activation in the context of HIV-1 infection, as well as other diseases.
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    The association of the menopause transition with cardiometabolic diseases and their risk factors in african and other low- and middle-income countries
    (2024) Chikwati, Raylton Prosper
    Background: The changes in risk factors of cardiometabolic disease (CMD) over women’s midlife remain understudied in sub-Saharan African (SSA) and other lowand middle-income countries (LMICs). It is unclear whether these changes are driven by the menopause transition (MT) or chronological aging. LMICs including those from SSA have a high population growth of midlife women, and a high prevalence of CMD and human immunodeficiency virus (HIV). The aim of this study was therefore to investigate the MT and its association with CMD risk factor levels in SSA women and women from other LMICs and whether any association is modified by HIV. Methods: In this study, a meta-analysis of data on menopausal differences in CMD risk from LMICs was performed. Secondly, analysis of cross-sectional data embedded within a multi-centre study in five different sites across four SSA countries namely: South Africa (Dikgale and Soweto), East Africa (Nairobi, Kenya) and West Africa (Nanoro, Burkina Faso, and Navrongo, Ghana) was done. Thirdly, analysis oflongitudinal cardiometabolic and sex hormone data from women in Burkina Faso and Ghana was performed, as they passed from pre- to peri- or pre- to post- or early to late postmenopause, at two time points. Lastly, analysis of cross-sectional data from pre- and postmenopausal women in the HIV prevalent regions of Kenya and South Africa was done. Results: The meta-analysis showed that compared to premenopause, the postmenopausal stage was associated with higher metabolic syndrome (OR=1.18 (95% CI 1.11–1.27)), and higher triglycerides (OR=1.16 (95% CI 1.11–1.21)), elevated blood glucose (OR=1.21 (95% CI 1.15–1.28)), hypertension (OR=1.10 (95% 1.04- 1.16)) and higher waist circumference (OR=1.16 (95% CI 1.08–1.25)). However, BMI(OR=1.05 (95% CI 0.96–1.14)), high-density lipoprotein cholesterol (OR=0.71 (95% CI 0.19–1.22)) and carotid intima media thickness (OR=1.09 (95% CI 0.87–1.36)) did not differ between these groups. In the cross-sectional analyses, combined West African populations demonstrated that postmenopausal women had a larger waist circumference (β = 1.28 (95 % CI: 0.58; 1.98) cm), log subcutaneous fat (β =0.15 (0.10; 0.19)), diastolic (β = 3.04 (1.47; 4.62) mm Hg) and log systolic (β = 0.04 (0.02; 0.06)) blood pressure, log carotid intima media thickness (β = 0.03 (0.01; 0.06)), lowdensity lipoprotein cholesterol (β = 0.14 (0.04; 0.23) mmol/L) and log triglyceride (β= 0.10 (0.04; 0.16)) levels than premenopausal women. No such differences were observed in the South and East African women. In the longitudinal analyses, the sexhormones estradiol, sex hormone binding globulin, testosterone, and dehydroepiandrosterone decreased significantly but follicle stimulating hormone increased during the MT. Furthermore, androgens mediated minor changes in the levels of CMD risk factors whereas these changes were more strongly influenced by the study site. In the last cross-sectional analysis, age at menopause in women living with HIV was lower than in those living without HIV. However, there were no differences in CMD risk factor levels between menopausal groups and this was not affected by HIV status. Conclusions: These findings suggest that there is need for a context-specific interventions to lower the risks of CMD in midlife women from SSA populations and other LMICs. Furthermore, this study provides insights into the biology of the menopause in a SSA population by showing minor associations between sex hormones and CMD risk factors but stronger associations with environmental factors. Lastly, HIV does not seem to modulate the menopausal differences on CMD risk factors, but it is associated with a lower age at menopause.
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    The characterization of Mycobacterium tuberculosis amidase-like proteins
    (2024) Matlhabe, Ofentse
    Tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis, is a significant global threat to human health. The emergence of drug resistant M. tuberculosis necessitates the identification of new drug targets for the development of new, shorter regimens. The peptidoglycan (PG) core of the M. tuberculosis cell wall is a potential source of drug targets because it is unique to bacteria and plays a vital role in a multitude of cellular processes and host-mediated immune responses. PG is constantly remodelled by PG synthases and hydrolases in response to external stimuli. This research focuses on N-acetylmuramyl-Lalanine amidases (amidases), PG hydrolases that are implicated in bacterial growth, cell division, virulence and antibiotic tolerance. More specifically, this PhD aims to characterize the M. tuberculosis Ami1 (Rv3717) homologue and to highlight its potential as a drug target. Genotypic characterization of a previously generated M. tuberculosis mutant strain lacking ami1 (H37Δami1S) was conducted prior phenotypic assessments. The deletion of ami1 had no significant effect on growth rate and cell division in standard 7H9 media. In contrast, the growth rate of H37Δami1S was significantly reduced when grown in Sauton’s minimal media with (1%) or without glycerol as a carbon source. We then surmised that Ami1 possibly plays a role in intracellular survival, where host-derived carbon sources support bacterial growth. The survival of H37Δami1S was reduced in IFN-γ stimulated U937 macrophages. H37Δami1S displayed increased susceptibility to rifampicin when assessed by broth microdilution. This observation was credited to a weakened, more permeable cell wall. Consistent with this, H37Δami1S exhibited an increased ethidium bromide uptake. Subsequently, we hypothesized that H37Δami1S may display alterations in antibiotic tolerance/persistence. In a 7-day time-course experiment, H37Δami1S displayed increased susceptibility to vancomycin, ethionamide and isoniazid as evidenced by declining bacterial survival. We interrogated the isoniazid-associated phenotype further, by assessing the transcription of all three amidase-encoding genes. Only ami1 was induced following exposure to isoniazid whereas the expression of ami3 and ami4 remained at basal levels. The regulation of the ami1 gene was explored further through bioinformatics, which revealed two putative transcriptional regulators predicted to bind upstream of ami1, namely Rv1423 and Rv1776c. Protein homology modelling detected HTH DNA binding domains in both proteins. These proteins were then cloned for recombinant expression in the pET29a+ system for purification. Rv1776c was successfully expressed and purified. Electrophoretic mobility shift assays yielded preliminary data that suggested that Rv1776c binds the promoter region of the ami1 gene. Attempts to optimize binding were unsuccessful. To further evaluate the role of Rv1776c and Rv1423 in regulating ami1 gene expression, we over-expressed the regulators, using the tetracycline operator, and assessed effect on cell wall stability, via an ethidium bromide diffusion assay. Over-expression of Rv1776c was not achieved despite increasing concentrations of anhydrotetracycline, suggesting possible downstream regulation of Rv1776c; however, over-expression of Rv1423 was achieved. An increase in ethidium bromide uptake was observed in strains over-expressing Rv1776c and Rv1423. Increasing anhydrotetracycline concentrations in both strains resulted in marginal decreases in the transcription of ami1. Overall, this study has demonstrated that Ami1 plays a vital role in how M. tuberculosis utilizes a carbon source during normal growth and survival in vitro. Moreover, the transcription of ami1 is specifically and directly responsive to isoniazid exposure, possibly via two transcriptional repressors. This work therefore supports further characterization and development of Ami1 as novel drug target in M. tuberculosis.
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    Peptidoglycan amidation enzymes in Mycobacterium tuberculosis are new drug targets for Tuberculosis treatment and development of a novel TB vaccine
    (2024) Shaku, Moagi Tube
    Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) playing an essential role for maintenance of cell wall integrity and tolerance of osmotic pressure. In previous work, it was demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. In this thesis work, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. In Mycobacterium smegmatis, an experimental surrogate for Mycobacterium tuberculosis (Mtb), we confirm the essentiality of Dglutamate amidation in PG cross-linking by labeling cells with synthetic fluorescent PG probes that require amidated side chains for stable incorporation. We also use CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation in other bacterial species, and demonstrate that these genes are essential for mycobacterial growth. We show that MurT-GFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria and that these enzymes interact to form the PG amidation complex. Furthermore, time-lapse microscopic analysis of MurT-GFP localization in fluorescent D-amino acid (FDAA) - labeled mycobacterial cells during growth demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during cell wall remodeling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and β-lactam antibiotics. Cell growth cessation was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, the first component of this work in M. smegmatis demonstrates the importance of D-glutamate amidation in mycobacterial PG precursors. We further exploit this enzyme complex in the second part of the dissertation to develop a novel CRISPRi-based recombinant BCG (rBCG) vaccine named rBCG::iE-DAP. This rBCG vaccine is based on the depletion of MurT-GatD, which results in reduced PG side chain amidation. As amidation masks detection of PG by the NOD1 pathogen recognition receptor, we postulate that this novel recombinant BCG will facilitate enhanced immunity against TB disease. As expected, the recombinant BCG induced increased immune activation and protection against Mtb infection in a mouse model of TB infection. This work highlights the MurT-GatD complex as a novel drug target and as a target for development of a next generation TB vaccine targeting innate immune responses against Mtb infection.
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    Molecular profiling of colorectal cancer within South Africa
    (2024) McCabe, Michelle
    There is a global requirement to characterize colorectal cancer (CRC) by molecular subtyping within subpopulations for the assignment of relevant therapies to improve prediction outcomes. Previous CRC studies conducted within South Africa (SA) have mainly been epidemiological, and subtyping limited to immunohistochemistry (IHC) protein expression analysis. The conclusions from these reports provided limited insight and lacked supportive molecular investigations in the development of CRC specifically within our population. CRC develops through 3 main molecular pathways; i.e. (1) Chromosomal instability (CIN) (75- 85%), (2) Microsatellite Instability (MSI) (~15%), and (3) CpG Island Methylator Phenotype (CIMP) (15-20%) pathway. This study descriptively analyses histopathological and molecular information of CRC patients in a 5-year study cohort (2011-2015), by assessing MSI status to ascertain unique features associated with different population groups, particularly within the African population. This study showed a large proportion (37%) of African CRC patients present with early disease onset (<50 years) in comparison to other ethnic group (OEG) patients (15%). Molecular characterization of CRC revealed MSI CRC within African patients occurred at an increased frequency compared to other ethnic groups (15% vs 8%), lacked a BRAFV600E mutation, and the dominant deficient (d) mismatch repair (MMR) profile was dMSH2 and dMSH6, suggesting hereditary Lynch Syndrome (LS) as the dominant pathway of disease development. These results are unique, as international findings demonstrate, 75% of MSI-CRC are sporadic, associated with dMLH1 and BRAFV00E mutations. OEG SA patients however were mostly associated with MLH1 and BRAFV600E mutations, and therefore follow the more wellestablished sporadic MSI pathway. Further insight gained through universal MSI screening was the ability to differentiate MSI-H versus MSS and MSI-L CRC. MSS/MSI-L CRC categorized by tumour site (left versus right) and ethnicity revealed unique histopathological features associated with left-sided CRC (LCC) in African patients compared to OEG patients. In addition, a higher proportion of MSI-L LCC was seen in African patients associated with more advanced disease stage and unique molecular and histopathological features. These findings suggest MSI CRC found in African patients is predominantly of a hereditary form, and further variant screening analysis to determine causative germline pathogenic variants are required. MSS CRC particularly within the left colon, was associated with unique histopathological features in the African population group, suggesting an alternative carcinogenic pathway of development when compared to OEG patients. MSI-L CRC also illustrated unique features at this site in African patients, and suggest this to be a completely separate molecular subtype. Deeper molecular characterization by next generation sequencing, including somatic and germline cancer gene screening is required to provide more insight into the different molecular subtypes and pathways in the development of CRC within the SA. This research will therefore direct better clinical management strategies, improving diagnosis, genetic counselling and testing strategies, prognosis, treatment outcomes, survival, and the overall burden associated with the disease within SA.
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    Development of a multi-disease targeted next generation sequencing panel to study genetic aetiology of rasopathies
    (2024) Mudau, Maria Mabyalwa
    With Next Generation Sequencing (NGS), technologies it is now possible to screen a large number of genes simultaneously through massively parallel sequencing, significantly reducing costs and time generally associated with mutation screening. After an informal survey of the diagnostic needs of the clinicians in the Division of Human Genetics, National Health Laboratory Service (NHLS), it was established that the aetiology of genetic disorders called facial dysostoses, RASopathies and Cohesinopathies (FRASC) could be understood better using NGS. These are developmental disorders that are phenotypically different and are commonly referred to our genetic clinic, currently there is limited genetic testing for these conditions in South Africa. A NGS panel targeting genes associated with the FRASC disorders was designed and optimised. Upon successful optimisation the panel was then utilised to sequence samples from patients presenting with features suggestive of RASopathies. An overall detection rate of 56.6% (34/60) was obtained for all RASopathy patients sequenced in the current study. Detection rate of 46.7% (7/15) for neurofibromatosis type 1 (NF1) patients and 60% (27/45) for patients with non-NF1 RASopathies was obtained. No clinically significant variants were identified in 26 of the 60 patients (43.3%), two of the 26 had a VUS in the MAP2K1 gene. Seven patients of the panel negatives were successfully sequenced using whole exome sequencing (WES), one patient had a pathogenic variant and the rest had variants of uncertain significance identified. This is the first report profiling mutations and clinical features in patients with RASopathies as a whole in South Africa, compared to a study focusing on NS patients only. The detection rates obtained were comparable to other international studies using NGS, except for detection rate of LZTR1 and BRAF1 gene variants observed in Noonan syndrome patients. The (35/60) 58.3% (panel and WES) of patients with a disease causing variant identified in this study now have a molecular confirmation that they previously did not have. Our results show that a targeted panel could be an effective first-line diagnostic testing for RASopathies in SA. The development of the multi-disease targeted panel in the current study has contributed to the design of the 500 gene inherited disease NGS targeted panel (IDP) that is currently being utilised in our facility for diagnostic testing of various monogenic disorders.
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    Biomarkers to predict Tuberculosis treatment response
    (2024) Boshielo, Itumeleng
    Tuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis (Mtb). Despite the implementation of multifaceted TB prevention and control efforts, a significant number of people still die from TB. Consistent with this, an uptick in TB-related mortality was recently noted, which has been ascribed to the negative effects of Coronavirus disease-2019 (COVID-19) on TB programs. The complex life cycle of Mtb is largely due to the use of immune evasion mechanisms to establish initial infection, remain dormant in the host, and reactivate pathogenicity under favourable circumstances. The prolonged TB treatment regimen is necessitated by the slow response of bacterial populations to standard TB chemotherapy, a phenomenon that may be caused by persistent, drug-tolerant bacteria. Scientific literature has provided evidence for these types of bacterial populations in the form of Differentially Culturable Tubercle Bacilli (DCTB). It has been demonstrated that DCTB represent drug tolerant bacteria that appear to be cleared at slower rate than organisms detected by routine culture methods. However, it remains unclear if DCTB populations elicit different immune responses when compared to their conventionally culturable counterparts. Herein, we address this question by optimizing a laboratory model for the generation of DCTB in vitro and test the capacity of clinical isolates of Mtb from Lineage 2 (Beijing) and Lineage 4 (LAM) to adopt the DCTB state. Using the Most probable number (MPN) assay, in the presence of culture filtrate (CF) as a source of growth factors to resuscitate DCTB, and colony forming units, the amount of DCTB in our model was quantified. As demonstrated by the limited growth on agar plates and increased growth in liquid media supplemented with CF from an axenic culture of Mtb, our findings demonstrated that carbon starvation was able to generate DCTB from clinical Mtb strains. After generating these populations, we stimulated whole blood with DCTB and conventionally culturable populations and report on the stimulation of a select set of cytokines (IFN-γ, IL-4, IL-5, IL-6, IL-12p70 and TNF-α) using a Bead Array Multiplex Immunoassay. In comparison to H37Rv-DCTB and LAM-DCTB, Beijing-DCTB induced significantly reduced levels of IL-5 and TNF-α. When comparing cytokine production between culturable and DCTB populations, within a single strain, we noted that LAMDCTB was delayed in the production of IFN-γ whilst Beijing-DCTB was not able to induce production of this cytokine when compared to conventionally culturable counterparts. These data suggest that shifting to a non-replicating DCTB state does indeed affect the ability of clinical isolates to induce immune responses. Based on these observations, we next set out to determine if DCTB affects immune responses during treatment of Mtb infected individuals. In prior work, using a prospective observational cohort, we demonstrated a substantive heterogeneity in clearance of DCTB in individuals with drug susceptible TB. We were able to classify these response patterns into three broad groups including (I) participants who were able to clear DCTB within the first two weeks of treatment (treatment-responsive); (II) those with delayed ability to clear these organisms (delayed-responsive) and (III) a group of individuals where DCTB did not change substantively during treatment (non-responders). Given these stark differences in treatment response patterns, we hypothesized that the immune responses associated with these patterns would be substantively different. In the second component of this work, we set out identify immune biomarkers that predict an effective response of DCTB to TB treatment. To quantify cytokines, chemokines and growth factors in plasma from these groups, we used a 65-plex Luminex assay, with a broad selection of targets. Statistically significant differences between these groups were analysed using the Kruskal-Wallis test with Dunn’s multiple comparisons, with p<0.05 was considered as statistically significant. When compared to patients who had TB and HIV co-infection, the number of cytokines that may possibly be used to report on the effectiveness of TB treatment was significantly higher in Mtbonly infected patients. This suggests that HIV infection significantly reduces the number of cytokines that can be used to report on TB treatment response. The ROC analysis of I-TAC, G-CSF and VEGFA showed that these cytokines have a significant discriminatory power to distinguish treatmentresponsive and non-responsive patients from HCs using DCTB as the measure of treatment response. No unifying cytokine signature that predicted DCTB response in all groups was identified. Together, our results indicate that some inflammatory markers are elevated in individuals with TB that rapidly clear bacteria during treatment. Given that these responses are based on DCTB, which represent drug tolerant populations, these select cytokines may be useful in evaluating the effectiveness of novel shorter TB treatment regimens.
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    Exploring the interplay of chemokine receptors ccr5 and cxcr6 in mechanisms of natural control in HIV-1- infected black South Africans
    (2024) Koor, Gemma Whitney
    In sub-Saharan Africa, HIV-1 is a significant cause of morbidity and mortality. However, research remains primarily focused on North American and European population groups, who have remarkably different genetic backgrounds to individuals from sub-Saharan Africa. HIV1 controllers represent a model of HIV-1 functional cure, with some individuals able to control viral replication, and some able to sustain immune function in the presence of high viral loads, both in the absence of antiretroviral therapy (ART). The chemokine receptors CCR5 and CXCR4 are the major coreceptors HIV-1 utilises to enter cells. The use of alternative coreceptors, such as the CXCR6 coreceptor, is thought to contribute to the lower pathogenicity exhibited by the HIV-2 and SIVsmm strains. Building on previous work conducted in our research unit on these two coreceptors in South African populations, this thesis firstly describes CCR5 genetic variants that associate with HIV-1 control or risk of progressive infection in black South Africans, and then explores constitutive expression levels of CCR5 and CXCR6 on various peripheral blood immune cell subsets in the absence of HIV-1 infection in ethnically divergent population groups. The effect of sex, age, and select CCR5 and CXCR6 single nucleotide polymorphisms (SNPs) on expression levels of these two receptors was also investigated. The CCR5 5’UTR and 3’UTR regions were PCR-amplified and sequenced from genomic DNA extracted from 145 ART-naive black South African individuals living with HIV-1 (71 HIV-1 controllers – 23 elite controllers, 37 viraemic controllers, 11 high viral load long-term nonprogressors and 74 progressors). Findings confirmed results from other studies in showing that the CCR5 HHE haplotype is deleterious for HIV-1 disease progression, and the HHA haplotype and HHA/HHC genotype associated with protection from HIV-1 disease progression. Novel haplotypes were characterised, both in the 3’UTR and spanning the CCR5 5’UTR and 3’UTR. Overall, findings suggest that two CCR5 promoter SNPs (-2459 G>A and -2135 T>C) and one CCR5 3’UTR SNP (+2919 T>G) may be key functional variants with regards to HIV-1 control in black South Africans. To gain further insight into the constitutive expression of CCR5 and CXCR6 on peripheral blood immune cells and explore the relationship between select genetic variants and expression, immunophenotyping by flow cytometry was conducted using whole blood from age- and sex-matched ethnically distinct South African HIV-uninfected individuals (17 black, 21 white). Expression levels of CCR5 and CXCR6 were assessed on CD4+ and CD8+ T cells, B cells, monocytes and NK cells, and their respective subsets. The effects of age and sex on expression levels of these two receptors was also investigated. Population-specific differences with regards to CCR5 expression on all cell types, except for B cells, were evident. Generally, black South Africans exhibited a lower expression level of CCR5 compared to white South Africans. CXCR6 expression only differed with regards to percentage of CXCR6-expressing cells, not CXCR6 density (numbers of cell surface receptors). Black individuals had a lower percentage of CXCR6-expressing CD8+ T cell subsets (naïve and effector memory) and a higher percentage of CXCR6-expressing CD14+CD16+ monocytes compared to white individuals. Overall, we found significant population-specific differences in expression levels of both CCR5 and CXCR6, multiple associations with cell activation (as measured by HLADR expression) and CCR5 and CXCR6 expression, and CCR5 and CXCR6 expression was positively significantly correlated on multiple cell subsets. Furthermore, both sex and age influenced CCR5 and CXCR6 expression, however results varied widely across the two population groups studied. Sex differences were only evident in white individuals; predominantly CXCR6 expression was increased in males compared to females. Age associations with CCR5 and CXCR6 expression were also primarily found in white individuals. Four CCR5-related SNPs that are associated with HIV-1 control in this or other studies (rs553615728 -4223 C>T SNP, rs1799987 −2459 G>A SNP, rs746492 +2919 T>G SNP and rs1015164 G>A SNP) were assessed for their potential association with CCR5 expression levels. The +2919 TG genotype significantly associated with a higher percentage of CCR5- expressing total CD8+ T cells, transitional memory and terminally differentiated CD8+ T cells compared to the GG genotype. The +2919 GG genotype associated with a lower percentage of CCR5-expressing B cells compared to the TT and TG+TT genotypes, however, only in white South Africans. The +2919 TG and TG+TT genotypes associated with significantly higher CCR5 density on all CD8+ T cell subsets, except for naïve CD8+ T cells, when compared to the GG genotype. When evaluating two CXCR6 genetic variants previously associated with HIV-1 viraemic control (rs2234355 G>A and rs2234358 G>T) in relation to CXCR6 expression, possession of the rs2234355 SNP GA genotype associated with lower CXCR6 expression on select CD4+ and CD8+ T cell subsets as well as on B cells, while possession of the rs2234358 SNP TT genotype associated with higher CXCR6 expression on multiple cell types, primarily in white South Africans. Possession of the -358TT/+355GA genotype combination associated with lower CXCR6 expression on select subsets of CD4+ T cells and monocytes. In summary, this study provides information on genetic variation in the CCR5 gene in a South African context, describes genetic variants associating with HIV-1 control in black South Africans, adds novel insight into constitutive CCR5 and CXCR6 expression levels on CD4+ and CD8+ T cells, B cells, monocytes and NK cells in HIV-1-uninfected black and white South Africans, and describes the potential associations of select genetic variants and expression. Black and white individuals differed in their baseline expression levels of CCR5 or CXCR6, which was partly driven by host genetic factors that were explored. This work highlights the importance of considering effects of ethnicity, age, and sex in any studies addressing any immune molecules in relation to differential HIV-1 outcomes of infection susceptibility/protection, disease progression, or HIV-1 virological control on antiretroviral therapy. Although conducted on small numbers of individuals, these variables clearly influenced constitutive expression of CCR5 and CXCR6, and further population-specific studies are warranted to gain further insights. Findings from this study have implications for risk of acquisition of HIV-1 infection and for disease progression in people living with HIV-1. Understanding the role of these molecules is important for informing strategies for both HIV1 prevention and HIV cure.
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    Synthetic cytology image generation to augment teaching and quality assurance in pathology
    (2024) McAlpine, Ewen David
    INTRODUCTION- Urine cytology offers rapid and relatively inexpensive screening for the detection of high-grade urothelial neoplasia in patients with haematuria. In our setting of a public sector laboratory in South Africa, however, there is a paucity of such specimens with which to train cytotechnologists and cytopathologists. Advancements in Generative Adversarial Networks present a potential solution to this problem by allowing for the generation of synthetic urine cytology images to supplement teaching and training. We illustrate an end-to-end machine learning model – from dataset creation to testing synthetic images in pathology personnel – to assess this technology in a real-world setting. METHODS- Two hundred and fourteen urine cytology slides were digitised and processed to construct a morphologically balanced dataset containing examples of benign, atypical and malignant urine cytology images. This dataset was used to train a StyleGAN3 model to generate synthetic urine cytology images. These synthetically generated images were then tested in a group of pathology personnel – both pathologists and trainees – to assess whether a difference between real and synthetic urine cytology images exists. Diagnostic error rate and subject image assessment were tested. RESULTS- StyleGAN3 was able to generate a wide morphological diversity of realistically appearing benign, atypical and malignant urine cytology images. When testing how these synthetic images were perceived by pathology personnel, there was no significant difference in diagnostic error rate, subjective image quality or inclusion of synthetic images in a cytology teaching set. DISCUSSION This work presents a proof-of-concept illustration of the feasibility of the use of synthetic cytology images to supplement pathology teaching when real examples may be difficult to obtain. Furthermore, this work presents important insights into the dynamics of pathology dataset creation and discusses the use of synthetic data in health education and the ethical and legal issues that arise with the use of synthetic patient data. CONCLUSION- Our work demonstrates that realistic, morphologically diverse urine cytology images can be generated using existing GANs technology and that human observers find such synthetic data visually acceptable. Additionally, our data indicate that there is no significant difference in synthetic data in terms of subjective image quality or diagnostic classification as determined by pathology personnel.
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    The role of the 20-hydroxyecdysone (20E) signaling pathway in modulating Anopheles arabiensis reproduction, gut microbiome and anti-bacterial immunity
    (2024) Etouman, Elodie Ekoka
    Anopheles arabiensis is one of the main vectors of malaria in South Africa. Due to its resistance to the current insecticides used in the country, novel methods are being explored to eradicate its existence in South Africa. In this thesis, it was demonstrated that the 20-hydroyecdysone (20E) signaling pathway could be a good target for the insecticides manufactured to kill the mosquito. In fact, impairing the 20E signaling pathway with RNA interference was proven to affect the reproductive capacity, the immune responses and the gut microbiome of the mosquito, all of which are important for its vectorial capacity. From these results it is anticipated that insecticides such as methoxyfenozide or halofenozide, which target the 20E signaling pathway, could be used in the country to eradicate the mosquito species.