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Item The role of regulatory T cells in adults in South Africa with active tuberculosis(2010-01-28T10:11:24Z) Mayne, Elizabeth SarahIntroduction: Regulatory T cells (Tregs) are increasingly being recognized as key immunological players in immunosuppression and have been seen to be permissive for certain infections. Aim: This study aimed to elucidate the role that Tregs play in symptomatic infection with Mycobacterium tuberculosis (TB), both with and without co-infection with human immunodeficiency virus type 1 (HIV 1) by quantification of these cells at ex vivo. It was then attempted to characterise the behaviour of FoxP3 positive cells in culture with stimulation. Methods: Peripheral blood mononuclear cells were purified from uninfected controls, patients with active TB, patients with HIV infection and patients with HIV infection and active TB. The frequencies of Tregs were assessed by flow cytometry at ex vivo and again after four days of culture with stimulation with anti-CD3, Purified protein derivative, tetanus toxoid and HIV peptide superpools (gag and nef). These frequencies were compared between the four groups of patients. The ability of Tregs and effector T cells to proliferate was also assessed. Interferon-γ secretion was used as a measure of effector T cell response to stimulation. vi Results: Frequencies of Tregs were significantly reduced in patients with active TB as compared with HIV infected patients and uninfected controls. Co-infected individuals showed a broad range of frequencies which were not significantly different from controls. These frequencies remained stable in culture with the exception of those individuals infected with HIV who showed a decline in the frequency of those cells expressing FoxP3 over the period. Cells expressing FoxP3 were not anergic and responded to stimulation. HIV specific proteins, in addition, resulted in specific effects on the Tregs with a positive interferon response to gag correlating with increased Treg frequencies and FoxP3 expression in CD4+ T cells correlated with the proliferative response of CD4+ T cells to Nef in HIV infected individuals. Conclusions: This study shows significant differences of frequencies of FoxP3 positive producing cells in the peripheral blood at ex vivo in patients with active TB. The function of these cells in this population is uncertain and further functional data and long-term clinical follow-up is required. In addition, the frequencies of these cells remained constant over time and showed proliferative response to stimuli (most notably CD3) suggesting that these cells may be generated in the periphery.Item Assessing the propensity of drug resistant tuberculosis to enter and exit the differentially culturable state(2024) Nonkula, BomikaziTuberculosis (TB), one of the oldest and most contagious infectious diseases, continues to be a global health concern. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC) which comprises of several species. These species are further subdivided into strains based on subtle genetic differences. The success of M. tuberculosis as a pathogen can be attributed to its ability to survive various stresses by adopting different growth states. Previous studies have shown that sputum from TB infected patients harbours a large proportion of drug-tolerant bacteria that are unable to form colonies on agar plates but can grow in liquid media. This population of organisms, termed differentially culturable tubercle bacilli (DCTB), could be resuscitated to grow by supplementing liquid media with cell free culture filtrates from axenic cultures of wild type M. tuberculosis H37Rv or mutant H37Rv lacking all five resuscitation promoting factors (Rpfs). Laboratory models that induce this differentially culturable state are critical for studying the physiology and metabolism of these bacteria in order to develop new TB diagnostic tests. In this study, five Beijing and five LAM drug resistant strains of M. tuberculosis were selected and used to robustly generate DCTB through an in vitro stress model using carbon starvation. The most probable number (MPN) assay and colony forming units were used to determine the amount of DCTB. Furthermore, the phenotype of these cells was studied using microscopy as well as metabolic probes that target the peptidoglycan (PG) component of the bacterial cell wall. Our findings demonstrated that applying the carbon starvation model to clinical M. tuberculosis strains (Beijing and LAM) resulted in robust levels of DCTB, as evidenced by limited growth on agar plates and enhanced growth in liquid media supplemented with culture filtrate from LAM and Beijing strains. Comparison of cell length between carbon starved cells to those grown in routine laboratory media suggested that DCTB appeared to be non-replicating and significantly shorter. The metabolic activity of the starved cultures was restored when they were supplemented with H37Rv, LAM and Beijing culture filtrate. Our results also demonstrated that Beijing strains had a higher propensity to produce DCTB compared to LAM strains and that the supplementation with Beijing culture filtrate resuscitated more DCTB. Collectively, our findings allow for the advancement of experimental systems that enable further investigation of DCTB and the properties of the Beijing strain that facilitate better adoption of the differentially culturable state.Item Peptidoglycan amidation enzymes in Mycobacterium tuberculosis are new drug targets for Tuberculosis treatment and development of a novel TB vaccine(2024) Shaku, Moagi TubeMycobacteria 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.Item Machine Learning on biochemical data for the prediction of mutation presence in suspected Familial Hypercholesterolaemia(2024) Hesse, ReinhardtBackground Familial hypercholesterolemia (FH) is a common monogenic disorder and, if not diagnosed and treated early, results in premature atherosclerotic cardiovascular disease. Most individuals with FH are undiagnosed due to limitations in current screening and diagnostic approaches, but the advent of machine learning (ML) offers a new prospect to identify these individuals. Our objective was to create a ML model from basic lipid profile data with better screening performance than low-density lipoprotein cholesterol (LDL-C) cut-off levels and diagnostic performance comparable to the Dutch Lipid Clinic Network (DLCN) criteria. Methods The ML model was developed using a combination of logistic regression, deep learning and random forest classification and was trained on a 70% split of an internal dataset consisting of 555 individuals clinically suspected of having FH. The performance of the model, as well as that of the LDL-C cut-off and DLCN criteria, were assessed on both the internal 30% testing dataset and a high prevalence external dataset by comparing the area under the receiver operator characteristic (AUROC) curves. All three methodologies were measured against the gold standard of FH diagnosis by mutation identification. Furthermore, the ML model was also tested on two lower prevalence datasets derived from the same external dataset. Results The ML model achieved an AUROC curve of 0.711 on the high prevalence external dataset (n=1376; FH prevalence=64%), which was superior to that of the LDL-C cut off alone (AUROC=0.642) and comparable to that of the DLCN criteria (AUROC=0.705). The model performed even better when tested on the medium prevalence (n=2655; FH prevalence=20%) and low prevalence (n=1616; FH prevalence=1%) datasets, with AUROC curve values of 0.801 and 0.856 respectively. Conclusions Despite the absence of clinical information, the ML model was better at correctly identifying genetically confirmed FH in a cohort of individuals suspected of having FH than the LDL-C cut-off tool and comparable to the DLCN criteria. The same ML model performed even better when tested on two cohorts with lower FH prevalence. The application of ML is therefore a promising tool in both the screening for, and diagnosis of, individuals with FH.Item Molecular epidemiology of M and E protein coding genes from South African SARS-CoV-2 strains, 2020 to 2021(2024) Marsden, FabianSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the current pathogen causing the COVID-19 pandemic across the world. While vaccines that elicit anti-SARS-CoV2 antibodies have been developed and licenced, there is a reduced protection against variants of concern (VOCs) such as Beta, Delta and Omicron. This is due to the mutations within the spike (S) protein which is the antigen targeted by most vaccines. Other potential vaccine targets include the structural proteins namely the membrane (M) and envelope (E) proteins of SARSCoV-2 which are more conserved. In this study we aimed to determine the extent of genetic diversity in the M and E protein genes from South African SARS-CoV-2 strains and its impact on predicted B and T cell epitopes. M and E gene sequences were extracted from South African SARS-CoV-2 genomes obtained from the Global Initiative on Sharing All Influenza Data (GISAID) database for the period 01March 2020 to 31 December 2021. Maximum-likelihood phylogenetic tree analysis shows that among South African E gene sequences only the Omicron VOC sequences form a distinct cluster. Similarly, the Omicron M gene sequences also form a distinct cluster compared to the Wuhan reference strain, Beta and Delta sequences. The predicted T cell and B cell epitopes of M and E proteins were identified with specific regions that have shown to have identical regions in both the variants and the reference strain, this shows the conserved nature of the M and E genes. SARS-CoV-2 are shown to have varying antigenic probabilities for M and E proteins from each of the variants considered as probable antigens. The allergenicity and toxicity of the M and E proteins was assessed in the context of potential vaccine development with certain peptides of each shown to have toxic properties. The predicted B and T cell epitopes show that despite the presence of mutations in the VOCs’ derived protein sequences, there is a common epitopic region that is shared between the reference and the variants. There is a strong 9-mer coverage by the natural sequences despite some non-coverage due to non-silent mutations. The results from the epitope predication and HLA typing shows the conserved nature of the M and E proteins which highlights the potential use for the development of vaccines.Item Maternal vaccination in South Africa: timing and completeness(2024) Bourne, JuliaMaternal immunisation is an invaluable public health measure that protects not only the mother, but also the foetus and new-born infant against a host of diseases; and is recommended by both the World Health Organisation (WHO) and South African national health authorities. Pregnancy induces a heightened state of immune system vulnerability, leaving women more susceptible to severe influenza outcomes, whilst neonatal tetanus has a fatality rate of between 80-100% in the absence of medical intervention. Maternal immunisation against influenza and tetanus has been successfully utilised as a public health strategy across the globe to uphold maternal and neonatal health. Maintaining coverage is imperative for both diseases as influenza strains change seasonally and tetanus cannot be eliminated, highlighting the importance of continued maternal immunisation. This study aimed to describe the uptake of both influenza and tetanus vaccinations during pregnancy, the completion of the tetanus vaccination schedule and the timing of both influenza and tetanus immunisation within South African antenatal care facilities. In addition, this study described clinical and demographic factors affecting maternal immunisation uptake. Clinical and demographic data were collected in a parent study and were retrospectively analysed in this study using the statistical software Stata. Influenza vaccination uptake within the sampled population was found to be 16.62% (806/4851). The odds of influenza vaccination were significantly higher in women aged 21-30 years, and women with six or more ANC visits. Metro East Cape Town site in the Western Cape outperformed Gauteng sites, with significantly increased odds of influenza vaccination amongst women frequenting that site. Appropriate influenza immunisation: defined as immunisation occurring during the period of either 01/04/2017-31/07/2017 or 01/04/2018-30/06/2018, occurred in 74.86% (530/708) of the cohort. Women who were alcohol users were significantly more likely to receive an influenza vaccine – yet this may be explained by the Metro East site which had the higher influenza coverage having the highest prevalence of alcohol use during pregnancy. Of 7105 women, 7031 (98.96%) received at least one dose of tetanus toxoid vaccine (TTV). Of these women, 39.24% (2759) received one dose; 51.06% (3590) received two doses and 9.70% (682) received the recommended three doses of TTV in their index pregnancy. Tetanus schedule completion was significantly more likely in women ≤20 years, and those who presented for their booking antenatal care (ANC) visit in the first trimester. In addition, women with more than three visits had an increased likelihood of TTV schedule completion. The odds of TTV schedule completion were decreased by negatively parity and gravidity, values over one and less than six, and greater than one respectively. Women with hypertension were significantly less likely to receive three TTV doses compared to women without hypertension. Julia Bourne MSc(Vaccinology) Research Report: Version 2.0 v Tetanus immunisation schedule adherence prevalence was 0.60% (4/670) in women with three doses and 90.34% (3209/3552) in women with two. Improvements may be made in South African maternal immunisation coverage, with this study supporting the idea of targeted educational campaigns and a revision of the maternal immunisation schedule to include the tetanus, diphtheria & acellular pertussis vaccine instead of the tetanus toxoid vaccine.Item Evaluation of the genetic and metabolic determinants of postprandial glucose variability in Black South Africans(2024) Masango, BontleThis study aimed to determine the metabolic and genetic factors that account for the variation in postprandial glucose (PPG) in Black South Africans (SA). The study included 794 participants from the middle-aged Sowetan Cohort (MASC). PPG was calculated using the integrated area under the curve (iAUC) in response to an oral glucose tolerance test. Principal component analysis was applied to 31 metabolic factors to generate clusters represented by principal component variables. Polygenic risk scores (PRS) were computed for each participant using variants and weights from a validated African type 2 diabetes PRS. Linear regression models were used to evaluate the variance. The PRS did not contribute to the PPG variability in men or women. Central fat, serum lipids, and liver enzymes explained 10.8% of PPG variability in women. In men, peripheral fat, serum lipids, liver enzymes, and steroid hormones explained 10.6% of PPG variability. Our work has identified metabolic factors that predict PPG variability in Black SA.Item The use of insecticide treated eave ribbons as a protection tool against pyrethroid-resistant populations of mosquitoes that transmit malaria and dengue fever(2024) Shirima, Ruth SeverinVector control methods such as insecticide treated nets (ITNs) and indoor residual spraying (IRS) have been successful in preventing mosquito-borne diseases like malaria and dengue. However, these methods face challenges including insecticide resistance, high costs, logistical difficulties, low adoption rates, and limited durability. Therefore, there is a need for simpler and more affordable interventions that can be used on a large scale in disease-endemic communities to supplement current approaches. This study evaluated the effectiveness of using insecticide-treated eave ribbons as a potential tool for complementing the current vector control methods. Eave ribbons are pieces of hessian fabric that can be placed around the eave spaces of poorly constructed houses to kill or repel mosquitoes. Laboratory cone bioassays were conducted to assess the efficacy of eave ribbons treated with the organophosphate, pirimiphos-methyl, for killing the malaria vectors, Anopheles funestus and Anopheles arabiensis, and the dengue vector, Aedes aegypti, under varying exposure durations and insecticide doses. In addition, a semi-field experiment was done to assess the efficacy of eave ribbons treated with pirimiphosmethyl against the malaria vectors. Indoor and outdoor biting was assessed by the number of mosquitoes captured indoors in window exit traps and outdoors by human landing catches, respectively. Mortality of recaptured mosquitoes was recorded after 24, 48, and 72 hours. The findings revealed that treated eave ribbons resulted in higher mosquito mortality than the untreated ribbons, but the impact increased with increased exposure duration or dose. The semi-field study indicated moderate levels of bite prevention and mortality of the mosquitoes. At the doses of 1 g a.i./m2 and 2 g a.i./m2 pirimiphos-methyl, there was no significant protection against An. arabiensis, but at the dose of 4 g a.i./m2 pirimiphos-methyl, there was only significant protection against outdoor biting An. arabiensis, but not An. funestus. In conclusion, while insecticide-treated eave ribbons may have potential for controlling malaria and dengue vectors, further research is needed to validate their efficacy in field settings and to identify suitable insecticides or insecticide combinations that are safe and effective.Item Impact of donor CYP3A5 genotype on pharmacokinetics of tacrolimus in South African paediatric liver transplant patients(2024) Wheeler, CaitlinTacrolimus is characterised by a narrow therapeutic target range and wide interpatient variability. Pharmacogenetic research attributes CYP3A5 single nucleotide polymorphisms to the variable inter-patient tacrolimus concentration dose ratios (CDR). In this study, we compared the mean tacrolimus CDR in paediatric liver transplant recipients and their living liver donors’ CYP3A5 rs776746 T>C genotypes(*1/*1, *1/*3 and *3/*3), accounting for donor and recipient characteristics. The graft-to-recipient weight ratio and the CYP3A5 donor genotypes were found to be independent factors significantly impacting the mean tacrolimus CDR. Donor CYP3A5 expressors (*1/*1 and *1/*3) were shown to have significantly lower recipient tacrolimus CDRs, therefore, higher dosages would be required in comparison to CYP3A5 non-expressors to reach the same therapeutic target range. The potential implementation of a stratified medicine dosage algorithm, combining living liver donor CYP3A5 genotyping with the calculation of the graft-to-recipient weight ratio, may predict the optimal tacrolimus dosage schedules for liver transplant recipients.Item The role of the 20-hydroxyecdysone (20E) signaling pathway in modulating Anopheles arabiensis reproduction, gut microbiome and anti-bacterial immunity(2024) Etouman, Elodie EkokaAnopheles 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.Item Characterisation of RSV fusion proteins from South African patients with RSV disease, 2019 to 2020(2024) Mabilo, PrinceRespiratory syncytial virus (RSV) is classified into subtypes A (RSV-A) and B (RSV-B), which are classified into different genotypes based on genetic variability of the G surface glycoprotein gene. The F surface protein gene is more conserved however variability in signal peptide, transmembrane domain, and antigenic sites have been reported. The study was conducted in the Virology laboratory, Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), South Africa. Study participants included patients of all ages from whom respiratory samples were submitted for respiratory viruses diagnoses from 2019 to 2020. The complete RSV F genes were amplified, library prep was preformed using the Nextera DNA prep kits and sequenced using amplicon-based next generation sequencing on the Illumina MiSeq sequencing platform. The Genome Detective Virus tool v2.27 was used to assemble sequencing reads and MEGA X was used for phylogenetic analysis. N-linked glycosylation and amino acid sequence variation was assessed. The overall prevalence of RSV was 5.8% (101/1 734). Seventy (69.3%; 70/101) RSV-positive samples were available for genetic characterisation of the F protein gene and thirty-one (30.7%; 31/101) were excluded due to insufficient sample volume. Only RSV-A strains were identified (91.2%; 31/34). Twenty three of thirty-one (74.2%) of the RSV F gene sequences from 2019 to 2020 clustered together with bootstrap values ranging from 64% to 99% and were NA1-like. A N-glycosylation site at position 120 gained by South African strains from 2018 is retained in strains from this study. This N-glycosylation site is present in approximately 25.8% of RSV strains from this study. The diversity of RSV-A F proteins was low, with amino acid variations observed at 30/571 (5.3%) sites. Ten mutations were detected in 4/6 antigenic sites (I, II, IV and V), with frequencies ranging from 0.3 to 100%. Antigenic changes seen exclusively among South African strains are: Y33H (0.3%) and V384T (7.3%) at site I and S275F (0.3%) at site II. Seven mutations associated with escape of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocyte (CTL) were predicted in seven epitopes. Overall amino acid mutation frequency for 2019 to 2020 RSV-A F genes is similar to that reported for South African strains from 2018 (3.3% to 6.7%). For the first time in South Africa we detected the S275F mutation which causes palivizumab resistance.Item Prognostic influence of myc aberrations and other clinicopathological factors of aggressive b-cell non-Hodgkin lymphomas(2024) Pather, SugeshneeIntroduction: 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).Item Exploring the interaction of host genetics and the gut microbiome in obesity in an African population(2024) Schnell, Samantha SusanObesity is a highly prevalent health concern that is on the rise in Sub-Saharan Africa. Even though genetic variation and gut microbiota have been implicated in the development of obesity independently, the interactions between these factors have not been previously explored in a South African cohort. This study aimed to identify possible associations between host genomes, body mass index as a measure of obesity, and gut microbiota composition (in the form of V3-V4 16S rRNA sequencing) in a female African cohort. Polygenic risk scores predictive of body mass index in this cohort were generated to categorise the participants into high- and low-risk groups. Subsequently, several statistical analyses were performed comparing gut microbiota between these groups. High-risk participants with high body mass index had associations identified with increased abundances of Prevotella_9 and VadinBE97. In contrast, the low polygenic risk and low body mass index sub-group was associated with greater Bacteroides levels. This study acknowledges the plausible interactions between these factors in an African cohort.Item Evaluating the clinical utility of a SNP-based microarray platform: a comparative study(2024) Mayisela, Minenhle PenielDevelopmental disorders make up a majority of cases seen in genetic clinics at the National Health Laboratory Service (NHLS). Chromosomal microarray analysis (CMA) is the first line testing for individuals with developmental disorders and congenital anomalies. This study compared the clinical utility of single nucleotide polymorphism (SNP) - based array (CytoScan® Optima array) and a comparative genomic hybridization (CGH) array (SurePrint G3 Unrestricted CGH ISCA v2, 8x60 kb microarray) in diagnosis of developmental disorders. This was done by looking at the differences in copy number variant calls, segment size differences and gene content between the two microarrays. The cost to run each type of microarray test for diagnosis was also compared. The copy number variant calls made by the two platforms were comparable. The SNP-based array did provide additional information that would be useful in molecular diagnosis. The final recommendation was for the CGH array and the SNP-based array to be used interchangeably based on clinical requests at the Division of Human GeneticsItem Off-label evaluation of alternative specimen types: Cobas® plasma separation card for HIV viral load and dried blood spots for COVID-19 serology testing(2024) Mampa, Thabiso MmammitsiPlasma is the preferred specimen for HIV viral load (VL) monitoring and COVID-19 serology testing but poses a challenge in resource-limited settings due to the need for venous blood, skilled phlebotomy, and cold storage for specimen integrity. In this study dried blood spots and novel plasma separation devices (PSC, HSSE, and VLPlasma) versus plasma were investigated as alternative specimen types. The plasma separation devices (PSD) were compared to DBS to determine if eliminating cellassociated nucleic acids could improve HIV VL performance. Paired PSD (n=72), DBS (n=72) and plasma (n=72) were prepared from HIV positive residual whole blood. Similarly, paired PSC, DBS (n=91) and plasma (n=91) were prepared from HIV positive prospective whole blood to assess PSC as an alternative specimen for use on the Abbott m2000. The eluates were processed on the GeneXpert (residual blood), Abbott m2000 (residual and prospective blood) and Roche cobas® 68/8800 (prospective blood). Using plasma as reference, residual blood: DBS outperformed PSC, HSSE and VLPlasma in terms of accuracy 91.8%, compared to 87.8%, 79.1% and 75%. Prospective blood: PSC had improved performance over DBS in terms of sensitivity (92.2% and 87.1%), specificity (65% and 61.9%), and accuracy (86.9% and 80.7%). Additionally, the performance of DBS was evaluated for COVID-19 serology testing in 45 PCR-confirmed, COVID-19 positive individuals by preparing laboratory paired DBS-plasma samples. DBS were eluted using two diluents followed by manual ELISA and results compared to reference plasma testing. DBS-PBS and DBS-manufacturer’s diluent showed the same accuracy (93.6%). Kappa values (0.817 and 0.845) and sensitivity (100% and 91.4%) were similar, but DBS-PBS showed low specificity (75%) compared to DBS-diluent (100%). Off-Label use of the cobas® PSC for HIV VL and DBS for COVID-19 serology testing provides expanded options for testing in resource-limited settings. Further evaluation on capillary blood and automated laboratory workflow optimisation would still be required prior to scaled implementation.Item The characterization of Mycobacterium tuberculosis amidase-like proteins(2024) Matlhabe, OfentseTuberculosis (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.Item Defining the development of gp120-gp41 interface directed broadly neutralizing antibodies in HIV-1 infection(2024) Hlatshwayo, Vincent NkosinathiA 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.Item Variation in the LBP-CD14-TLR4-LY96 gene complex and consequences of microbial translocation in HIV-1 infected black South Africans(2024) Mncube, SizananiPersistent 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.Item Molecular profiling of colorectal cancer within South Africa(2024) McCabe, MichelleThere 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.Item Exploring the interplay of chemokine receptors ccr5 and cxcr6 in mechanisms of natural control in HIV-1- infected black South Africans(2024) Koor, Gemma WhitneyIn 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.