Background Bacillus Calmette-Guerin (BCG) remains the only licensed vaccine for use against tuberculosis (TB), however, it is poorly efficacious against pulmonary TB in adults. The poor efficacy has been attributed in part to coinfections with many other unrelated pathogens that overlap geographically with Mycobacterium tuberculosis (M. tuberculosis). In this study, we used a murine model to investigate the effect of Plasmodium species virulence and the timing of infection on BCG-induced immune responses and efficacy against M. tuberculosis both in vivo by aerosol challenge and ex vivo by Mycobacterial Growth Inhibition Assay (MGIA). Methods and Results To assess the impact of malaria parasite virulence on BCG, wild-type C57BL/6 mice were vaccinated intraperitoneally with BCG Pasteur and 6 weeks later, they were infected with either virulent Plasmodium berghei (P. berghei) or less-virulent Plasmodium chabaudi chabaudi AS (P. chabaudi) infected red blood cells with appropriate controls. The mice were euthanased at 7, 10, 17, 26 days after malaria infection for multiparameter flow cytometry analysis. We compared P. berghei and P. chabaudi, their effects on B cells, effector and memory T cells and the outcome on BCG-induced protective efficacy against M. tuberculosis H37Rv infection by subsequent aerosol challenge. P. berghei induced a significant decrease in the frequency of central memory T cell (CD44hiCD62Lhi), marginal zone B cell (B220+AA4.1-CD1dlo), and follicular B cell (B220+AA4.1-CD1dhi ) populations. In contrast, infection with the less virulent P. chabaudi induced depletion in the marginal zone B cells but not the follicular B cells or the central memory T cells. A strong effector T cell/effector memory T cell (CD44hiCD62Lo) response enhanced by BCG vaccination was observed in both species. The reduction in the central memory T cells observed during P. berghei infection was attributed to T cell apoptosis. It should be noted that the observed changes in T cell and B cell populations described above are relative proportions of these cells among splenocytes. Surprisingly, BCG-mediated protection against M. tuberculosis H37Rv by aerosol challenge was retained in both the virulent P. berghei and less virulent P. chabaudi species despite modulations in the immune responses. We also investigated the effect of malaria infection post BCG vaccination and malaria infection prior to BCG vaccination on the cytokine responses and the efficacy of BCG vaccine both in vivo and ex vivo. Splenocytes from wild type C57BL/6 mice infected with P. yoelii 17XNL 4 weeks post BCG vaccination were analysed 13 days after P. yoelii 17XNL infection. We compared the cytokine responses and growth inhibition by MGIA in four groups of 6 mice each: naïve, BCG, BCG-malaria, and malaria control. BCG Pasteur Aeras was used as a surrogate for M. tuberculosis in the MGIA. Restimulation with PPD-T induced a significant increase in both the proportion and absolute cell numbers of CD4+ IFNγ+ and CD4+ TNF+ T cells in the BCG vaccinated compared to the naïve control. No significant differences in the proportion and cell numbers of CD4+ IFNγ+ T cells were observed between the BCG and BCGmalaria groups restimulated with PPD-T. This indicates that the presence of malaria did not significantly hamper the IFNγ response to BCG. In contrast, a significant (p<0.05) reduction was detected in the proportion of CD4+TNF+ T cells in the BCG-malaria group compared to the BCG group following restimulation with PPD-T. P. yoelii 17XNL infection also led to significant production of IL-4 by CD4+ T cells in the groups that were infected with malaria with or without BCG vaccination when restimulated with PPD-T or P. yoelii 17XNL peptide pool. Minimal production of CD4+ IL-17 T cells and CD8+ T cells were observed when restimulated with PPD-T. MGIA revealed a significant decrease in net bacterial growth (0.2 log 10) in the BCG and BCG-malaria splenocyte cultures compared to the naïve and malaria control splenocyte cultures, indicating that BCG immunized mice were able to control the growth of mycobacteria in the presence of malaria. For P. yoelii 17XNL infection prior to BCG vaccination, wild type C57BL/6 mice (n=6-11) were infected with either P. yoelii 17XNL or P. chabaudi and after 13 (acute malaria) and 21 (cleared malaria) days or 7 (acute malaria) and 28 (cleared malaria) days respectively, mice were vaccinated with BCG. They were either sacrificed 6 weeks after BCG vaccination for cytokine analysis and MGIA or challenged with M. tuberculosis H37Rv by aerosol for bacterial growth determination. BCG vaccination caused a significant increase in the proportion of CD4+ IFN-γ + and CD4+TNF+ T cells compared to the naïve control when restimulated with PPD-T. However, we observed a significant reduction in the proportion of CD4+TNF+ but not CD4+ IFN-γ + T cell responses in the acute malaria-BCG group compared to the BCG vaccinated mice following in vitro restimulation with PPD-T. Interestingly, these cytokine levels were significantly elevated upon clearance of the P. yoelii 17XNL parasites. Therefore, acute malaria infection at the time of BCG vaccination induces a transient decrease in Th1 response which is restored once the parasite is cleared. An ex vivo MGIA assay on splenocytes showed a significant decrease (0.2 log reduction) in net bacterial growth in all BCG vaccinated groups except the acute malaria-BCG group, compared to the unvaccinated groups with or without malaria. Interestingly, a significant increase in net bacterial growth was observed in the acute malaria group compared to the BCG vaccinated group, indicating that acute P. yoelii 17XNL infection prior to BCG vaccination decreases the protective efficacy of BCG in the MGIA. In an in vivo aerosol challenge with M. tuberculosis, a significant decrease was observed in bacterial burden in the spleen (0.6 log reduction) and lungs (1 log reduction) of mice that were vaccinated during acute P. chabaudi infection, vaccinated when malaria had cleared or vaccinated in the absence of malarial infection. This was further confirmed by higher numbers of acid-fast bacilli observed in all unvaccinated groups compared to all vaccinated groups with or without malaria, implying BCG vaccine efficacy is maintained in an acute or cleared P. chabaudi infection prior to BCG vaccination. This discrepancy between the in vivo aerosol challenge and ex vivo growth inhibition assay may be attributed to the different Plasmodium species with different clinical characteristics used in the study or the different kinetics in the two assays used. Conclusions Therefore, malaria parasite virulence does not inhibit the ability of BCG to control the growth of M. tuberculosis in vivo despite alterations in the immune responses. Additionally, neither Plasmodium infection prior to BCG vaccination nor Plasmodium infection post BCG vaccination abolishes the efficacy of BCG against M. tuberculosis although a decrease in CD4+ Th1 cytokine responses and concomitant increase in bacilli burdens are observed in the group of acute Plasmodium infection prior to BCG vaccination.

Includes abstract. / Includes bibliographical references (leaves 93-109). / Nearly 130 years have elapsed since the discovery of Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, yet today it is estimated that 1 in every 3 of the worldʼs population is infected with this pathogen. In South Africa alone there were approximately 1000 new TB cases per 100 000 population in 2007, ranking the country second in incidence rate, globally. Hence research into new vaccine strategies to control the epidemic is vital. Current vaccines under development are prophylactic and designed to boost pre-existing immunity induced by the only licensed TB vaccine, BCG. A new approach is the development of a post-infection vaccine aimed at inducing an immune response that prevents progression to TB disease when administered to individuals latently infected with M.tb. This vaccine would have a dramatic impact on the worldwide TB burden. Our objective was to address 2 areas in TB vaccinology, firstly a novel postinfection TB vaccine strategy, and secondly, optimal measurement of vaccineinduced responses using a new immunological assay. The aim of the first study was to investigate human T cell responses to antigens that have been associated with M.tb latency. Rv2660 and Rv2659 were investigated, as these antigens are candidate antigens for a postinfection vaccine based on findings from in vitro models of M.tb suggesting preferential expression during latency in vivo. No information exists on the immune response to these antigens in M.tb infected or TB diseased individuals. Hence, we investigated the immune recognition of Rv2660 and Rv2659 in these 2 groups, and further characterised the nature of these antigen-specific T cell responses. We observed that (i) these antigens are significantly more likely to be recognised during M.tb infection compared with TB disease as shown by measurement of soluble IFN-γ in response to the specific antigens, (ii) M.tb infected persons had greater Rv2660- and Rv2659- specific CD4+ T cell proliferation and associated cytokine expression compared, with TB diseased persons. We propose that Rv2660 and Rv2659 may be candidates for incorporation into a post-infection vaccine.

Includes abstract. / Includes bibliographical references (leaves 101-111). / Interleukin-4 receptor alpha (IL-4Ra) signaling, mediated by the ligands IL-4 and IL- 13, is important for effective host protection during murine Nippostrongylus brasiliensis (N. brasiliensis) and Schistosoma mansoni (S. manson!) infection. Among other cell types, IL-4Ra responsive smooth muscle cells influence immunological responses and are needed for host protection during N. brasiliensis and S. mansoni infection.

Includes bibliographical references (leaves 75-91).

Lymphoblastic leukemia 1 (Lyl1) is a well-studied transcription factor known to exhibit oncogenic potential during various forms of leukemia. Since its discovery in 1989, many reports have been published describing its relationship with cancer as well as demonstrating its function during hematopoiesis. Lyl1 has been shown to serve a significant role during thymopoiesis by contributing to T-cell development. However, it has been recently reported that irrespective of its significance during T-cell development, mature comparable single positive T-cells are observed in mouse models. The use of murine models has been crucial in identifying potential targets for host-directed therapies (HDT) which has been shown to provide great potential in treating tuberculosis (TB). It is evident that Mycobacterium tuberculosis (Mtb), the causative agent for TB, is capable of developing resistance to various treatments that target the bacterium itself. Therefore, by designing therapies that directly target host factors could assist in circumventing Mtb resistance. By analyzing Mtb-infected bone marrow-derived macrophages (BMDM) that have been subjected to genome-wide transcriptional deep sequencing of total RNA using a single molecule sequencer in conjunction with the cap analysis gene expression (CAGE) technique, various differentially expressed genes were identified, including the oncogenic transcription factor, Lyl1. With the use of murine models, we investigated whether Lyl1 is important for various immunological responses at steady state, the regulation of Lyl1 in response to various immune stimulants including LPS and whether this transcription factor is relevant in bacterial infections including Listeria monocytogenes (Lm) and Mtb. The data in this thesis demonstrate comparable immunological responses, including cellular recruitment by means of flow cytometry and cytokine responses by means of ELISA, between naïve littermate control and Lyl1-deficient mice. Further evaluation of Lyl1 regulation revealed the influence of MAPk and NFκB signaling on Lyl1 expression upon LPS stimulation by significantly downregulating this transcription factor in immune stimulated macrophages. A role for Lyl1 during bacterial infections was observed in Lm-infected mice whereby Lyl1-/- mice succumbed earlier to listeriosis compared to the littermate controls. We further established a functional role for this transcription factor during Mtb infection in vitro and in vivo. The early surrender of Lyl1-deficient mice to Mtb HN878 infection, accompanied by increased bacterial burden during chronic Mtb infection, demonstrated enhanced susceptibility in the absence of Lyl1. We show that Lyl1-deficient host susceptibility is a consequence of enhanced inflammatory responses and increased bacterial growth. This is demonstrated by increased neutrophilic inflammation, pro-inflammatory cytokine and chemokine secretion along with a reduction in anti-inflammatory cytokine release during chronic Mtb infection. Here, we demonstrate the first non-leukemia role for Lyl1 by suggesting a role and requirement for this transcription factor during bacterial infections. Given the significant role during Mtb infection, our studies suggest the use of Lyl1 associated pathways as a potential HDT target for TB.

Southern Africa is disproportionately affected by HIV-1, with Botswana being among the most affected countries. The interindividual heterogeneity in susceptibility or resistance to HIV-1 and progression upon infection is attributable to, among other factors, host genetic variation. Characterisation of human genetic variations can contribute towards understanding the genetic aetiology of HIV-1 and foster development of novel preventive and treatment strategies against HIV-1. Despite the high burden of HIV-1 in Botswana, the population of Botswana is significantly underrepresentation in genomics studies of HIV-1. Furthermore, the bulk of previous genomics studies evaluated common human genetic variations, however, there is increasing evidence of the influence of rare variants in the outcome of diseases which may be uncovered by comprehensive complete and deep genome sequencing. This research aimed to characterise human genomic variations of Batswana in order to elucidate mutation burden, assess population structure and evaluate the role of these genomic variations in susceptibility to HIV-1 and progression through bioinformatics analyses. Whole genome sequences (WGS) of 265 HIV-1 positive and 125 were HIV-1 negative unrelated individuals from Botswana were computationally analysed. The sequences were mapped to the human reference genome GRCh38. Population joint variant calling was performed using Genome Analysis Tool Kit (GATK) and BCFTools. Variant characterisation was achieved by annotating the variants with a suite of databases in ANNOVAR. The genomic architecture of Botswana was assessed through principal component analysis and structure analysis and FST. Cumulative effects of rare variant sets on susceptibility to HIV-1 and progression (CD4+ T-cell decline) were determined with optimized Sequence Kernel Association Test (SKAT-O). Functional analysis of the prioritized variants was performed through gene-set enrichment using databases in GeneMANIA and Enrichr. Variant characterization revealed 24 damaging variants with the most damaging variants being ACTRT2 rs3795263, HOXD12 rs200302685, ABCB5 rs111647033, ATP8B4 rs77004004 and ABCC12 rs113496237. There was admixture of Khoe-San, Niger-Congo and European ancestries observed in the population of Botswana, however, there was no evidence of overall substructure among the HIV-1 positive/negative individuals of Botswana, indicating similar genetic exposure among HIV-1 samples. No variant set was significantly associated with susceptibility to HIV-1, while sets of novel rare-variants within the ANKRD39 (8.48 x 10- 8 ), LOC105378523 (7.45 x 10-7 ) and GTF3C3 (1.36 x 10-6 ) genes were significantly associated with HIV-1 progression. Functional analysis revealed that the variants affected several pathways including chemokine signalling, glycolysis, glycosylation, HIV-1 and host receptor glycoprotein biosynthesis, intracellular transport of molecules and transcription pathways. These findings highlight the significance of whole genome sequencing in pinpointing rare variants of clinical relevance. This PhD thesis unravelled novel genes and novel rare variants that are putatively linked to HIV-1 progression. The thesis contributes towards a deeper understanding of the host genetics HIV-1 and offers promise of population specific interventions against HIV-1.

Oculocerebrorenal or Lowe Syndrome (OMIM #309000) is an X-linked recessive condition characterized by a triad of congenital cataracts, proximal renal tubular dysfunction, and variable central nervous system involvement. Nearly all affected boys will be hemizygous for a pathogenic variant in the OCRL (NM_000276.4 c.2615delC) gene. We present a clinical and molecular characterization of an extended multiplex family of three affected boys with Lowe Syndrome and describe a novel variant, predicted to be pathogenic, in the OCRL gene. This is to the best of our knowledge the first description of its kind in South African patients and future research into more families with Lowe syndrome will be beneficial.

Includes abstract. / Includes bibliographical references. / Infectious diseases are endemic in Africa, especially tuberculosis (TB), malaria and human immunodefiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). Genomics research has the potential to improve the health of Africans through identification of genetic markers associated with either disease susceptibility or therapeutic drug response. This project was set to investigate the genetic correlates for drugs associated with mitochondrial toxicity that are used as part of HIV therapy, especially nucleoside reverse transcriptase inhibitors (NRTIs). Toxicity from NRTIs manifests through metabolic diseases such as peripheral neuropathy, lipodystrophy, lactic acidosis and hyperlactatemia but show interpatient variability. Studying African populations is likely to open the door for the population to benefit from novel diagnostic tools and drugs developed on the basis of pharmacogenomics knowledge. In an effort to contribute to this knowledge, the role of variation in mitochondrial DNA (mtDNA) and polymerase gamma (POL-γ) on how patients respond to stavudine-containing antiretroviral therapy (ART) among adult Malawian HIV/AIDS patients was investigated.

Includes abstract. / Includes bibliographical references. / This study investigates imprinting of the murine fetal immune system by maternal infection with the helminth Nippostrongylus brasiliensis (Nb) prior to pregnancy and its effect on control of the Salmonella enterica serovar typhimurium (STm) in offspring. We show that maternal Nb infection in BALB/c mice results in the transfer of Nb antigen (NES)-specific IgG1 in utero and through breastmilk, changes in lymphocyte populations and early germinal center formation in naive offspring. Maternal Nb infection does not interfere with control of STm in offspring in BALB/c mice, but may interfere with control of STm in C57BL/6 mice.

An earlier observation by E.H. Harley (supervisor of this thesis) of curious metabolic anomalies in the red cells of black rhinoceros, and in particular a high free tyrosine level, suggested that a range of unusual, but presumeably physiological, processes might be found in mammalian red blood cells. As a follow-up to this, low molecular weight metabolites were examined in a range of mammalian species, using HPLC-based methods to compare levels in red cells with plasma levels. A remarkable interspecies diversity in red cell HPLC profiles was observed, with the unprecedented accumulation of substances including tyrosine, tryptophan, urate, and urate riboside occuring within the red cells of some species. Whereas novel evolutionary adaptations may characterise most of these species-specific variations, the ability of red cells to produce urate is proposed to be an inducible feature common to the red cells of many, or possibly even all, mammalian species. A surprisingly high degree of intraspecies genetic heterogeneity was evident in tyrosine and urate levels within horse, and urate riboside levels within cow red cells. This was in contrast with the greater homogeneity seen in levels of these and other low molecular weight substances in red cells from the other species evaluated. The next phase of investigation addressed the potential function(s) of these soluble substances accumulating within the red cell, particularly relating to a role in antioxidant defense. Using in vitro antioxidant assays such as the 'oxygen radical absorbance' (ORAC) and 'ferrous ion oxidation-xylenol orange' (FOX) assays, results were obtained consistent with a role for these substances as endogenous red cell antioxidants against a variety of reactive species produced by pathophysiological processes in the body. The demonstration that haemoglobin is involved in facilitating some of this activity further substantiates the idea that the red cell may be playing a crucial role in maintaining circulatory redox balance, and hence protecting other tissues from oxidative damage. If indeed such low molecular weight substances contribute to systemic antioxidant activity in some mammalian species, then apart from the intrinsic interest of such unexpected biological phenomena, these findings could pave the way for a plethora of further investigations, geared towards potential clinical applications (eg. as biomarkers or therapeutic approaches) in human and/or veterinary conditions associated with oxidative stress.