Lung produced surfactant protein D (SP-D) is essential for both homeostasis and as an innate immune opsonin. In the project presented here, we aimed to translate data recently published by our group, which demonstrated that SP-D contributes to protection against murine parasitic nematode infections, to human work. In the first part of this study, we determined whether individuals exposed to helminths have altered serum SP-D in comparison to unexposed individuals, through analysis (ELISA and Western Blot) of bio banked samples in 2 clinical cohorts from South Africa. Secondly, we aimed to identify if SP-D influences the magnitude of anti-nematode responses in human immune cells (type 2 innate lymphoid cells, monocytes and macrophages) through in vitro cell work and flow cytometry. Our findings indicated an association between serum SP-D and exposure to helminths that have a lung migration stage as part of their life cycle (Ascaris spp and Toxocara spp). Furthermore, in vitro analysis demonstrated that human immune cells primed with SP-D might have an altered response to helminth antigen. These findings point toward the need for further investigation into the novel role of SP-D in the control of human helminth infections in the context of immune physiology, as a biomarker and eventually treatment option.

South Africa recently experienced a Listeriosis outbreak, which was responsible for over 180 deaths, caused by an intracellular, rod-shaped bacilli called Listeria monocytogenes (LM). LM can infect both phagocytic and non-phagocytic cell types and induces its uptake by expressing internalin A and B, then secretes listeriolysin O (LLO), a virulence factor forming pores on the phagosome membrane to escape into the cytosol. Macrophages can phagocytose invading pathogens and induce innate inflammatory responses. Production of cytokines and eicosanoids by antigen presenting cells activates the adaptive immunity. Eicosanoids (epoxyeicosatreinoic acids, prostanoids and leukotrienes) are generated from metabolites of 20-carbon chained polyunsaturated fatty acids and arachidonic acid. Leukotrienes (LTs) are generated from 5- lipoxygenase-metabolism of arachidonic acid to LTB4 and cysteinyl LTs (cysLTs). CysLTs are pro-inflammatory lipids that have pathobiological functions in asthma. CysLTs function through three G-protein coupled receptors (CysLTR1, CysLTR2 and GPR99). The CysLTR1 and its ligands function has been well elucidated in asthmatic and allergic responses however, its role in bacterial infections is unknown. The aim of our study was to elucidate the role of CysLTR1 on disease progression in mice and macrophages infected with LM. In this study, we showed that CysLTR1 mRNA expression is upregulated by LM infection in WT macrophages and mice. Mice deficient of CysLTR1 had no defects at homeostasis. During time kinetic experiments with LM, CysLTR1 knockout mice displayed increased neutrophil recruitment and decreased lymphocyte cells at 3dpi, however, bacterial burdens were comparable to wild-type mice. In addition, macrophages deficient of CysLTR1 have no effect on the intracellular growth of LM. In conclusion, CysLTR1 signalling plays a role in lymphoid cell activation and neutrophilic recruitment during early LM infection, however, further studies are required to better understand the role of CysLTR1 during inflammatory responses.

Cysteinyl leukotrienes (cysLTs) are potent inflammatory lipid mediators that play a major role in the pathophysiology of inflammatory diseases. They signal primarily through cysteinyl leukotriene receptor-1 (cysLTR1) and have been reported to drive Th2 immune responses. Initiation and amplification of robust Th2 immune responses is crucial for conferring protective immunity to helminth (Schistosoma mansoni and Nippostrongylus brasiliensis) infection in mice. The role played by cysLTs in the development of protective immune responses to helminth infections is not well documented. Hence in the present study, we investigated the role of cysLTs during helminth infection using cysteinyl leukotriene receptor-1 deficient (cysLTR1-/- ) mice. Under steady state conditions, young naïve cysLTR1-/- mice did not reveal any significant alteration of the cellular, tissue and phenotypic profile although we did observe expansion of central memory T cells (Tcm) in secondary lymphoid organs in cysLTR1-/- as compared to wildtype mice. Primary infection with N. brasiliensis indicated increased worm burden in cysLTR1-/- mice at day 7 post infection and a delay in the resolution of infection by day 9 post infection when compared to wild type mice. Furthermore, we observed reduced Th2 immune responses as well as impaired contractility of the small intestine, which are key features required for protective immunity to N. brasiliensis infection. Furthermore, recall of memory responses to N. brasiliensis was abrogated in cysLTR1 -/- mice, with higher numbers of adult worms recovered at day 5 post re-infection in cysLTR1-/- mice comparison with wild type mice. Additionally, cysLTR1-/- mice exhibited impaired production of IL-13 in the lungs and draining lymph nodes compared with wildtype mice. Finally, there was reduced recruitment of effector CD4+ T cells and central memory CD4+ T cells in the lungs of cysLTR1 deficient mice compared to control mice. Taken together, these data demonstrated an essential role played by cysLTR1 in clearance and resolution of N. brasiliensis infection. CysLTR1-/- mice survived acute S. mansoni infection similarly to wildtype mice. In addition, cysLTR1-/- mice displayed reduced granulomatous inflammation and reduced cellular responses in the liver compared with wildtype mice. Further analysis revealed reduced gut fibrosis but cytokine production, immune cell recruitment in the gut and both type 1 and type 2 antibodies were found to be comparable between wildtype and knockout mice, demonstrating that cysLTs signaling through cysLTR1 contribute to granuloma formation in the liver. Similar to acute schistosomiasis, cysLTR1-/- mice were not susceptible to chronic schistosomiasis and indicated by prolonged host survival. This increased host survival observed in cysLTR1-/- mice was associated with reduced granulomatous inflammation, reduced fibrosis and hepatocellular damage, impaired production of IL-4 in the liver, and reduced intracellular secretion of IL4 by CD4+ T cells and ILC2s in cysLTR1-/- mice compared with wildtype mice. Furthermore, we observed reduced granulomatous inflammation in the lungs of chronically infected cysLTR1-/- mice despite the heightened Th2 immune response in the lungs. Collectively, these data revealed that disruption of cysLTR1 leads to reduced granulomatous inflammation and reduced production of IL-4 in the liver during chronic schistosomiasis. In conclusion, the current study demonstrated both positive and negative roles for cysLTs signaling through cysLTR1 during different helminth infection models. Absence of cysLTR1 during N. brasiliensis leads to delayed expulsion of adult worms and impaired recall of memory responses, indicating that cysteinyl leukotriene signaling via cysLTR1 is essential for orchestrating host protective responses. On the other hand, signaling via cysLTR1 appears to be dispensable for the development of host protective responses during acute schistosomiasis in mice. However, mice deficient of cysLTR1 had reduced liver pathology during chronic schistosomiasis, suggesting that inhibition of this receptor could be a potential therapy for reducing granulomatous liver pathology.

There are so many people who I would like to thank. If it takes a village to raise a child, it certainly takes a city to train a scientist. Firstly, I would like to thank my supervisors, A/Prof Reto Guler, Prof Frank Brombacher and Dr Mumin Ozturk for allowing me to further my studies and allowing me to work on several interesting projects. Specifically, I would like to take this opportunity to thank A/Prof Reto Guler for his insightful patient advice, training and ever willingness to talk about my work. His brilliant example has made me a better scientist. Further, I would also like to thank Dr Mumin Ozturk for his constant, patient mentorship, help, advice and friendship. His influence, guidance and example have affected me more than he'll ever know. Lastly, but certainly not least, I would like Professor Bavesh Kana and for all his advice and support. I would also like to express my gratitude to my labmates from the Brombacher group. All the conversations, laughs, celebrations and commiserations have made this journey undeniably easier. In particular, I would like to thank Shelby-Sara Jones for her constant willingness to help with lab work whilst chatting about everything under the sun. To my friends and family, there are no words to express my unending gratitude. Without their love and support along the way, I would never have gotten to this stage in my life. To my parents and sister, I would like to say a huge thank you for their constant support and love during my academic career so far. You guys have been wonderful. A huge thank you to Daniela de Almeida and the French's for their support and love from afar– you guys have been great. I would like to say a special thank you to Dustin Fischer who has always been there for a beer and good old-fashioned rant. I can only hope that my friendship and advice have been even the smallest bit as helpful to him as he has been to me during our long trek through academia. To the Cunniffes, thank you for all your support down this long road and truly making me feel like one of the family. Last but by no means least, I would like to thank my partner, Teagan Cunniffe, whose effortless grace, wit, humour, friendship and constant love have been the single greatest gifts I have ever received. I look forward to our adventures to come. Thank you for being there every step of the way and keeping me sane - This dissertation is dedicated to you.

Includes abstract. / Includes bibliographical references.

Includes bibliographical references. / Studying prospective immune correlates of risk of TB disease following BCG vaccination is an important first step towards determining correlates of protection against TB, which can be identified only in a placebo-controlled randomized controlled trial (RCT) of an effective vaccine. To study correlates of risk of TB disease, we collected and stored blood from healthy 10-week old infants vaccinated with BCG at birth. During two years of follow up, infants who developed lung TB were defined as cases, while those who did not develop TB disease were defined as controls. We measured Th1/Th17 cytokine production by BCG-specific T cells, release of pro- and anti-inflammatory mediators, cytotoxic T cell potential and proliferation in response to BCG as potential correlates of risk of TB disease but none of these outcomes were different between cases and controls. However, transcriptional profiling of PBMC revealed two clusters of infants and interestingly, the gene expression profiles from cases and controls in the two clusters were in opposite directions. Based on this, we hypothesised that analysing the two clusters of infants separately will allow discovery of correlates of risk of TB, which were absent when clustering was not taken into account.

BACKGROUND: Each year over 4 million infants die from infections, of which many are vaccinepreventable. Young infants respond poorly to vaccines, but the basis of reduced immunity is controversial. We hypothesized that myeloid-derived suppressor cells (MDSC) that might be induced during gestation, would persist at birth leading to active suppression of infant-immune responses. OBJECTIVE: We evaluated the ontogeny of MDSC and the effect of MDSC on vaccine immunogenicity during early life in South African infants and mothers, and in HIVexposed uninfected (HEU) infants and HIV+ mothers. METHODS: HIV-infected and uninfected mothers and their infants were recruited from Khayelitsha, Cape Town and followed-up for one year. In whole PBMC and after MDSC (CD15+) depleted, we measured BCG, Hepatitis B, Tetanus toxoid and Bordetella pertussis vaccine-specific CD4+ T cell proliferation by CFSE and IFN-γ responses using ELISpot assay.

Maternal microbiota during pregnancy, as well as maternal disease state, may impact offspring gut bacterial colonisation. Here, we explore the impact of maternal antibiotics during gestation and/or nursing on offspring gut microbiota. Further, we investigate the effect of preconception helminth infections on maternal and infant gut microbiota. For maternal antibiotic experiments, dams were fed vancomycin, polymyxin B, or both, in drinking water during gestation, nursing or gestation plus nursing, and their offspring microbiota analysed at 14 days of life, alongside immunity in the spleens. Offspring born to vancomycin treated mothers had significantly higher relative abundance of Proteobacteria and Tenericutes while maternal oral polymyxin B led to significantly lower abundance of Proteobacteria and Deferribacteres in infants. Maternal oral vancomycin led to significant reduction in proportions of infant central memory CD4+ T cells (CD4+CD44hiCD62Lhi) regardless of antibiotic timing. Effector memory CD4+ T cells were significantly lower in pups born to dams treated with polymyxin B while nursing while proportions of central memory CD4 T cells were significantly increased in gestation only or gestation plus nursing pups. In addition, oral vancomycin in dams during nursing resulted in significantly reduced proportions of both total and follicular B cells in offspring born to antibiotic treated dams. Pups born to Vancomycin treated mothers had a significant delay in growth when infected with Respiratory Syncytial Virus (RSV). On the other hand, pups born to mothers treated with Polymyxin B during gestation or gestation plus nursing were susceptible to Nippostrongylus brasiliensis (Nb) infection. In the second study, we infected female BALB/c mice with 500Nb L3 three weeks prior to mating and examined the effect of preconception helminth infection on offspring microbiota and immunity. Preconception Nb infections led to alterations of maternal gut microbiota during pregnancy. In addition, we observed dramatic differences in offspring microbiota in pups born to previously helminth infected dams. Coriobacteriaceae were predominant in pups born to previously Nb infected dams when compared to uninfected dams. Overall, manipulation of maternal microbiota during gestation or lactation profoundly impacts offspring growth, intestinal microbiota and immunity to RSV and helminths.

Parasitic worms have the ability to modulate the hosts immune response to promote host control of the infection and also parasite survival in the host. Helminth infections classically induce a potent Th2-biased and regulatory immune imprint. This immune response also influences unrelated inflammatory processes in the host. Studies have shown helminth infections have bystander influences on unrelated conditions such as allergy and autoimmunity. Additionally, helminth infections can alter susceptibility to other infections. In this thesis, we investigate the systemic influences of murine nematode Nippostrongylus brasiliensis infection on host immunity in colonized and non-colonized tissues, and the implications of these effects on susceptibility to subsequent herpesvirus infections in vivo. We show that prior N. brasiliensis infection enhanced control of acute respiratory murid gammaherpesvirus (MuHV-4) infection, with an increase in viral-specific CD8+ T cells in colonized lung tissue. Enhanced effector cytokine responses by cytotoxic T cells were also observed with prior helminth exposure. Conversely, despite enhanced primary control, prior helminth exposure was associated with earlier and heightened genital reactivation of MuHV4. This demonstrates differences in local bystander and systemic effects of helminth exposure on the host, and on unrelated viral infections. We also show that N. brasiliensis infection, which transits the respiratory and gastrointestinal tracts, also systemically influences immunity in the female genital tract (FGT) in vivo. Here, helminth infection induced Th2-type immunity in the FGT, namely increased tissue IL-4, IL-5 and long-lasting eosinophilia. We further demonstrated that systemic influences of N. brasiliensis infection results in exacerbated genital pathology and inflammation, following subsequent intravaginal herpes simplex virus type II (HSV-2) infection. Increased HSV-2 pathology with prior helminth exposure was associated with diminished innate anti-viral immunity, increased IL-33, ILC2 and IL-5 responses, as well as significant eosinophilia. Interestingly, abolition of canonical Th2 immune signalling by the lack of IL-4Rα expression, enhanced innate anti-viral defences and provided protection from HSV-2 pathology. However, N. brasiliensis-induced exacerbation of HSV-2 illness was IL-4Rαindependent, associated with significant genital eosinophilia. Furthermore, antibody-depletion of eosinophils ameliorated nematode-exacerbated HSV-2 pathology, suggesting that nematode-induced genital eosinophilia mediates increased HSV-2 pathology in coinfected mice. We have therefore shown that helminth infections can induce local and systemic bystander immunity to lymphoid and myeloid immune compartments, which alters susceptibility to subsequent herpesvirus infections.

INTRODUCTION Since 2008, 43 renal transplants have been performed from HIV positive deceased donors to HIV positive recipients with renal failure predominantly due to HIV-associated Nephropathy (HIVAN). Recipients received Anti-thymocyte globulin (ATG) induction therapy and maintenance immunosuppression. Despite transplantation across Human Leukocyte Antigen (HLA) mismatches, there were few rejection events in the first year post-transplant (PT). Recipient CD4 counts did not decrease and HIV viral load also remained undetectable at one year PT. To gain insight into immune homeostatic mechanisms after ATG induction, immunosuppression and transplantation, a subset of 10 transplant recipients were investigated. This dissertation examined levels of peripheral inflammatory and regulatory cytokines. A polychromatic flow cytometry panel was also developed to measure the phenotypic T cell proportions of T regulatory cells (Tregs) in the blood circulation. METHODOLOGY A multiplexed Luminex assay was used to measure the concentrations of 67 inflammatory and regulatory plasma cytokines immediately pre-transplant, at 1, 3, 6 and 12 weeks PT. Two separate manufacturers of Luminex panels were used and a series of statistical analyses were employed to identify intra- and interplate variation. Firstly, data was cleaned up by excluding analytes for which >90% of measured values were outside of the observable range of the standard curve. Secondly, the measurable values were assessed for differences between replicates (intra-plate variation). A Bland-Altman plot was used to identify and exclude highly divergent replicates of the same sample. Thirdly, a Paired Ttest/Wilcoxon Signed Rank Test was used to investigate differences between inter-plate controls (inter-plate variation). Fold change from baseline was calculated for all values to correct for inter-plate variability. After correcting for variability, fold change trends in all included analytes were examined for each recipient. Trends in recipients with rejection events (rejectors) and recipients without rejection events (non-rejectors) were also compared. Fold change from baseline was assessed to identify single analytes that differed over time using a Paired T-test/Wilcoxon Signed Rank Test. A hierarchical clustering analysis (HCA) was used to identify groups of analytes for which fold change may have been significantly influenced by age, sex, baseline CD4 count at transplantation (TP), number of HLA matches, or time. A mixed effect generalised linear model (MEGLM) was constructed to calculate differences between participants for each cytokine. A polychromatic flow cytometry panel was devised to measure Treg CD4+ T cells consisting of the following antibodies: CD3-BV650, CD4-PE/Cy5.5, CD8- HorizonV500, CD27-PE/Cy5, CD45RA-PerCP/Cy5.5, CD25-BV421, CD127- PE/CF594, and FoxP3-Alexa647. Antibody concentrations in the panel were optimised by titrating each marker on resting, or Phytohaemagglutinin (PHA) stimulated, peripheral blood mononuclear cells (PBMCs). The median fluorescent intensity (MFI) of the positive and negative populations for each marker was used to calculate the signal:noise ratio for all titrating volumes. The optimal volumes were determined by the highest signal:noise ratio for each marker. RESULTS In all recipients, when compared to baseline, IL-2, IFN-α2 and IFN-γ were significantly decreased at 12-weeks PT. IL-35 was significantly decreased at weeks 1, 3, 6 and 12 PT, whilst IL-10 increased significantly at 1-week PT in 5 recipients. Hierarchical clustering showed no association with analyte fold changes due to age, sex, CD4 count, or number of HLA matches. It did show a decrease over time in IL-35, IFN-γ, IL-20, IL-28A, and IL-11 for all 10 recipients. A mixed-effects generalized model (MEGLM) was used to identify analytes with variable concentrations between recipients. It showed that the concentrations of IL-28A, IL-6Rα, IL-6Rβ, sTNFR1, Pentraxin-3 and IFN-γ varied the most between recipients. IL-35 and TNFSF12 were shown to vary the least between recipients. These data suggest heterogeneity in the highly variable analytes, none of which were shown to differ over time. The heterogeneity is likely due to genetic diversity, history of opportunistic infections and relevant prophylaxis. The concentrations of IL-35 did not vary between recipients, but it was shown to decline over time in all recipients. This data suggests a consistent decline in the concentration of IL-35 in all recipients over the first 12-weeks PT. An 8-colour Regulatory T cell (Treg) flow cytometry panel was designed based on the Luminex results and optimised to phenotype peripheral T-cell subsets. It distinguished between different T-cell phenotypes, namely naïve and memory, CD4 or CD8, and activated and regulatory T cells. Antibody titrations identified the optimal volume of each marker to use in a combination cocktail. Due to time constraints, the panel was not used on patient material, but the panel and its optimisation has been described in detail. CONCLUSIONS Statistical investigations into the Luminex results identified variability between replicates and between plates. These differences needed to be accounted for before combining data between plates and kits to arrive at biological conclusions. By using stringent analysis, this dissertation shows that multiplex data is highly variable and a series of statistical approaches should be employed to avoid including erroneous data. A decrease in both inflammatory and regulatory proteins was shown in the 12 weeks after transplantation and ATG induction. The transient increase in IL-10 suggested the induction of effector T-cells to become IL-10 producing Tregs (Tr1), known to occur in response to ATG induction. Combined with the consistent decline in IL-35 in all recipients, these results suggested that there was preferential secretion of IL-10 over IL-35 in some patients early after transplantation.