Microbiology & Immunology - Theses
Now showing items 1-12 of 196
Discovery of the cells that express the antigen-presenting molecule MR1 in vivo
Major histocompatibility complex class I-related protein 1(MR1) is a monomorphic antigen-presenting molecule that is highly conserved across animal species. It presents vitamin B-related metabolite antigens, produced by a broad range of bacteria and yeast, to mucosal-associated invariant T cells (MAIT cells). This induces the activation of inflammatory and cytolytic MAIT cells to resolve microbial infections. The MR1-MAIT cell axis has been implicated in immunity against a range of major bacterial pathogens primarily in mucosal tissues. MR1 is also essential for the development and expansion of MAIT cells and can trigger anti-cancer responses. MR1 is thought to be expressed at very low levels ubiquitously in many cell types, but due to the difficult nature of detecting MR1, this has not been systematically investigated. Importantly, it is not known if the expression of MR1 varies among cell types in vivo or if it changes during pathological conditions. This project aims to address these unknowns by using a novel genetically altered mouse model that reveals the expression of MR1 by a fluorescent reporter. The fidelity of this model to report MR1-expressing cells has been validated by several means including quantitative real-time polymerase chain reaction (qPCR) and surface detection of MR1 after exposure to MR1 metabolite ligands. By employing the model, a range of expression levels of MR1 in diverse cell types with different tissue distributions in mice have been revealed. Overall, tissue-resident macrophages in the lungs and peritoneal cavity (PerC) had the highest MR1 expression. Factors that could influence MR1 expression in the healthy steady state were investigated. It was found that MR1 expression increased in mice with age up to 7 months, while there was no difference seen between the sexes. Bone marrow (BM) chimeras were used to reveal that MR1 expression in tissue-resident macrophages was not restricted to those originating from the embryonic precursors, but also in BM-derived macrophages. Then intriguingly, MR1 expression was not elevated in any cell type during pulmonary infection with Legionella longbeachae, but on the contrary, it was significantly downregulated in alveolar macrophages (AMs). Overall, this work reveals that MR1 has a cell type- and tissue-restricted expression profile in vivo, with tissue-resident macrophages expressing the highest levels, indicating that these cells may be the most potent MR1 antigen-presenting cells in vivo. Lung and peritoneal macrophages are instructed to express MR1 from the local tissue environment during their differentiation rather than from their precursor origins, and infection rapidly switches off its expression. This suggests that these innate MR1-presenting cells are already equipped with MR1 prior to infections, in order to rapidly activate MAIT cells upon microbial metabolite detection.
Nucleic acid sensing in CD4 T cells during HIV-1 and other viral infections
Viruses are small intracellular parasites and use the host cell's biosynthesis machinery to replicate and spread. Therefore, viral particles incorporate structures that are similar to the ones that naturally occur within host cells. A major mechanism to identify viral entry and replication within an infected cell is the recognition of viral nucleic acids. Sensors of the innate immune system can detect foreign nucleic acids by their unusual subcellular localisation or modification or both. Once innate immune sensors are activated, they induce distinct signalling cascades which modulate cellular responses to invading pathogens like viruses. In this thesis, we studied the role of RNA sensors RIG-I (retinoic acid-inducible gene 1) and MDA5 (melanoma differentiation-associated protein 5) in CD4 T cells during infections with SeV (Sendai virus) or HIV-1 (Human Immunodeficiency virus 1). HIV-1 is the causative agent for the acquired immunodeficiency syndrome (AIDS). Globally, more than 30 million people are living with HIV 1 and hundreds of thousands of people are newly infected every year. Today, HIV-1 infection is a chronic and manageable disease. The progression to AIDS is prevented by ART (antiretroviral therapy) which inhibits viral replication but is unable to clear the latent viral reservoir - inactive HIV-1 proviruses within long-lived subsets of immune cells. These latent viruses are not detected by innate and adaptive immunity. Furthermore, HIV-1 manipulates cellular restriction factors and sensors of viral infection to evade immune recognition. This highlights the demand for new approaches to restore innate immune sensing during latency reversal to allow the specific killing of infected cells to the clearance of the latent reservoir. We first studied the RIG-I signalling pathway in human CD4 T cells, the main reservoir for HIV-1 infection in vivo. Using SeV, a specific activator of RIG-I, and a cell-based type-I interferon reporter assay we showed that the RIG-I signalling pathway was functional in activated CD4 T cells. In resting CD4 T cells, we did not detect the release of type-I IFNs and used next generation sequencing (NGS) to verify the expression of members of the RIG-I signalling pathway. A typical type I IFN signature was observed in resting CD4 T cells following the stimulation of RIG I with SeV. These data also showed the downregulation of pathways relevant for T cell activation. We next evaluated how the activation of the RIG-I signalling pathway affects the biology of CD4 T cells. RIG-I activation diminished proliferation, metabolic activity and release of effector cytokine IFN in CD4 T cells. RIG-I and MDA5 are potential sensors for HIV-1 and their role during HIV-1 infection is not fully understood to date. We discovered that HIV-1 protease (PR) directly degrades RIG-I and MDA5 independently of other cellular factors. We showed this by co-expression of HIV-1 PR and RIG-I or MDA5 in HEK293T cells and in an in vitro assay using purified recombinant HIV-1 PR and RIG-I or MDA5 proteins. The degradation of RIG-I and MDA5 by HIV-1 PR sequestrated the sensing of stimulatory RNAs in an in vitro reporter assay. These data indicate that the degradation of RIG I and MDA5 is a potential immune evasion mechanism for HIV-1 which could be exploited in novel HIV-1 cure approaches. Furthermore, we generated RIG-I and MDA5 knockouts in primary human CD4 T cells and Jurkat cells and performed initial characterisations of those cell lines. Knockout cell lines will be useful in future studies on the role of RIG-I and MDA5 during HIV-1 infection.
Identifying factors regulating production and expression of HBV cccDNA and the minichromosome using in vitro and in vivo models of HBV replication
Chronic Hepatitis B (CHB) affects more 250 million people globally, causing significant morbidity and mortality from associated sequelae. The etiological agent, hepatitis B virus (HBV) contributes to the majority of cirrhosis and hepatocellular carcinoma (HCC) cases, resulting in nearly a million deaths annually. Carriage of the virus (HBsAg positivity) is associated with elevated risk of developing these liver complications, so while available nucleos(t)ide analogue (NUC/NA) therapy can effectively control viral replication, viral clearance to the point of ‘functional cure’ is required to significantly lower the risk. Moreover, development of a cure remains paramount as treatment is typically life-long, and cessation of therapy can lead to reactivation of the virus via the nuclear reservoir of a viral genome template known as the covalently closed circular DNA (cccDNA) minichromosome. Unaffected by conventional therapies, cccDNA is the required template for the synthesis of all viral transcripts, including greater-than-genome transcript pregenomic RNA (pgRNA), which in turn, is the template for reverse transcription to form the relaxed circular DNA (rcDNA) genome of progeny virions. Despite immense efforts, much remains to be elucidated about the virus and its replication cycle, largely due to the fact that the HBV research field is restricted by a severe lack of suitable/appropriate models for investigating all stages of the viral cycle, with the cccDNA minichromosome being particularly difficult to interrogate. This PhD project addressed this knowledge gap by characterising a monomeric/genome-length based transfection system both in vitro and in vivo, and investigating its utility as a novel model enabling investigation of cccDNA-like molecules. The model was utilised to further explore the impact of different/specific viral-host protein interactions on viral replication. Specifically, this was centred on the viral HBx and cellular DDB1 proteins, as the interaction between these two was recently demonstrated to facilitate viral transcription following viral infection. Finally, a broad proteomics approach involving microarray screening was applied to identify novel host factors/mechanisms associated with HBV replication. HBV interacts with many cellular proteins and factors, redirecting the associated processes to benefit viral replication and propagation. While some of these cellular components have been described previously, likely many more are yet to be discovered, and this project aimed to identify additional potential therapeutic targets that could ultimately contribute to HBV cure.
IFITMs & Immunity to Respiratory Viruses in the Ferret Model
Respiratory virus outbreaks pose a major threat to human health, with many of these zoonotic threats having potential for global pandemics. In order to gain a greater understanding of these viruses, considerable research into the immune responses elicited against these pathogens occurs in the ferret model for human disease. This PhD focussed on using this model to assess two of these pandemic threats in the ferret model, whilst gaining valuable insight into the immune mechanics at work within this understudied animal model. An infection study in the ferret model was used to assess the cellular immune response against A/Anhui/1/2013(H7N9) avian influenza virus in Chapter 3. This study found ferrets to have variable disease outcomes towards H7N9 infections, with severe disease marked by time dependent increases in two pro-inflammatory cytokines, IL-6 and IFNg. Unlike other studies investigating severe avian influenza, this study did not see any marked decreases in T cells other than an initial, transient lymphopenia, though there was evidence for T cell trafficking to the site of infection. The study also provided insight into the maturation and trafficking of antigen-presenting cells, giving new insight into immune cell dynamics in the ferret model. Chapter 4 took what was learned in the H7N9 study and applied it on a larger scale, using the ferret model to assess the immune response to an ongoing pandemic virus, SARS-CoV-2. This study primarily aimed to assess the immune response to a vaccine candidate against this virus, the AstraZeneca ChAdOx1-nCoV-2019 adenoviral-vectored vaccine. A prime- boost regime was found to be more effective at producing an immune response following SARS-CoV-2 infection than one dose of the vaccine, with the prime-boost ferrets producing greater B cell responses in the blood and tissues. Two doses of the vaccine also elicited a greater CD8+ T cell responses in the blood. As such, this study helps to guide the future application and administration of the vaccine to help overcome the effects of this wide-spreading virus. In order to gain greater insight into the immune genes that play a role in the protection against respiratory viruses, Chapter 5 sought to identify and classify and important family of interferon-stimulated genes, the Interferon-Inducible Transmembrane (IFITM) protein family, in the ferret model. The IFITM locus was found in the ferret to be of high similarity to the loci previously classified in other species, with this work having a particular focus on IFITM1, IFITM2, and IFITM3 due to their intrinsic anti-viral activity in other species. These genes were found to be expressed at a basal level across multiple tissues in the ferret, whilst also being highly upregulated in the presence of both IFNa and influenza virus. These results, along with sequence alignment confirmation, identify these genes as interferon-stimulated genes of the IFITM family, filling in an important piece of missing information for the ferret model. Overall, this thesis furthers our knowledge of the ferret model and the immune responses that occur within, providing a framework for future studies aiming to assess the immunology of respiratory viruses and their vaccine candidates.
Modelling host-pathogen interactions with the Salmonella Typhimurium effector protein, SopF
Characterisation of bacterial effector proteins is a growing research field, not only in the context of bacterial pathogenesis but also as novel molecular tools. During infection, many effector proteins are translocated by bacterial secretion systems into host cells to exert an “effect” on specific signalling pathways or host cell processes. Often these pathways are related to trafficking of intracellular compartments or the immune response. The overall result of these events is to provide an intracellular niche that is favourable to the invading bacteria, thereby lessening the chances of immune detection and increasing bacterial dissemination. Hence, both intracellular bacteria and their translocated effector proteins are a valuable source of information to advance molecular cell biology. This Thesis is presented in chronological order from the beginnings of effector protein characterisation to the utilisation of it as a novel molecular tool. The effector protein, SopF, is translocated by one of two Type III Secretion Systems from the bacterium Salmonella Typhimurium. S. Typhimurium can replicate freely in the cytosol of epithelial cells or reside in Salmonella-containing vacuoles (SCV). SopF was found to be a membrane-targeted effector protein, specifically localising to the SCV during infection. In particular, SopF maintained the vacuolar proportion of S. Typhimurium at early timepoints of infection. Towards the end of this study, we focus on components of the host autophagy pathway. Autophagy is an important cellular mechanism that degrades unwanted intracellular material to be recycled. Xenophagy is an arm of autophagy that targets foreign material, such as invading pathogens, for degradation. During our study, independent publications had shown that SopF is a xenophagic inhibitor and is capable of inhibiting recruitment of a key autophagy protein, ATG16L1. Using this information, we confirmed that SopF-dependent ATG16L1 inhibition was required for the effector to maintain the vacuolar residence of S. Typhimurium. This confirmed that xenophagic inhibition was a key requirement in continuing the intravacuolar niche of S. Typhimurium. Xenophagy, and thus components of autophagy, can be unfavourable to most invading pathogens. However, Coxiella burnetii is a bacterial pathogen that uses autophagy to its advantage. C. burnetii replicates in spacious, lysosome-like vacuoles termed Coxiella-containing vacuoles (CCV). Biogenesis of the large CCV requires fusion with autophagosomes, of which ATG16L1 is a key regulator. We confirmed that ATG16L1 is required for CCV biogenesis and C. burnetii replication. Furthermore, we used SopF to study this requirement further by inhibiting xenophagy during C. burnetii infection. The effect of ATG16L1 depletion was greater than that seen with xenophagy inhibition by SopF, which confirmed that SopF was a useful tool to separate the impact of autophagy- or xenophagy-targeted pathways. Together, the data in this Thesis contribute to the characterisation of a novel molecular tool that can be used to study host-pathogen interactions and host cell biology.
The dual role of cellular prion protein in colorectal cancer
Cancer is a major public health problem, being the second cause of death amongst non-communicable diseases worldwide. In Australia, cancer is currently the leading cause of death, which demonstrates the relevance of cancer research for public health. Amongst all types of cancer, colorectal cancer has the third highest incidence and has the second highest mortality in Australia. The high mortality is a consequence of the asymptomatic characteristic of colorectal cancer. This leads to later staging diagnosis when disease is already in the metastatic phase, decreasing survival significantly. In order to overcome the late diagnosis, cancer research has been focused on understanding the development and progression of this disease to identify potential biomarkers for diagnosis and new targets for treatment. Originally studied in the context of neurodegenerative disease, PrPc is a highly conserved membrane protein in mammals, that has been associated with a myriad of functions. In the context of cancer, PrPc has been correlated with disease progression and lower survival in gastric, pancreatic and colorectal cancer as well as multidrug resistance in gastric and breast cancer. In this project, PrPc expression and processing were investigated to determine the role of this protein in colorectal cancer. During cancer development, PrPc was found to have a preventative role particularly when cancer formation is driven by an inflammatory process. However, during cancer progression, PrPc expression and processing increased invasiveness and decreased necrosis promoting a malignant phenotype that could contribute to metastasis and consequently decrease patient survival. Together the results show that PrPc has a dual role in colorectal cancer, an anti-cancer role during development and a promoter role during progression.
Understanding immune responses to severe influenza disease
Respiratory viral infections caused by Influenza A viruses (IAVs) are responsible for annual seasonal epidemics that cause mild, severe or fatal pulmonary disease and estimated to result in 243,000-640,000 deaths globally every year. Severe infections can result in lung tissue damage, acute respiratory distress syndrome (ARDS) as well as secondary bacterial pneumonia, all of which are detrimental to the host and can potentially lead to a fatal disease outcome. Many of those at high-risk of developing severe disease include children, the elderly and immunocompromised individuals. Although current influenza vaccines are the best way to combat the seasonal influenza epidemics, the vaccines need to be updated annually to match strains predicted to circulate in the upcoming influenza season. Frequent mutations in the virus can lead to strain mismatches, which can affect the protective efficacy of these vaccines and result in significant increases in infection rates, putting those who are most vulnerable at risk for developing severe disease. The aim of this PhD thesis was to investigate host factors and mechanisms associated with severe influenza disease to provide insights into identification and prediction of severe disease outcomes. Co-expression of surface CD38 and MHC-II on CD8+ T cells is recognized as a classical hallmark of activation in many acute and chronic viral infection settings. However, high and prolonged CD38+MHC-II+ expression can be also associated with severe and fatal disease outcomes. Our previous studies have found that the persistence of CD38+MHC-II+ CD8+ T cells is associated with impaired IFN-gamma production in H7N9-infected patients with fatal outcomes. In Chapter III, we sought to investigate the mechanisms underpinning the expression of CD38+MHC-II+ markers using wild type and transgenic mouse models of influenza infection. We provided evidence that CD38+MHC-II+ CD8+ T cells are recruited early to site of infection during severe influenza A virus infection and can be activated in an antigen-driven as well as bystander manner. Our adoptive transfer experiments also demonstrated that MHC-II molecules were not expressed intrinsically by CD38+CD8+ T cells, but acquired from antigen presenting cells by trogocytosis, and the CD38+MHC-II+ phenotype was crucial for optimizing T cell recall ability. In Chapter IV, we investigated disease signatures during the early phases of infection to identify features associated with clinical outcomes. While comparable viral titres were observed between H7N9 patients who survived or succumbed to infection, there was a trend of higher level of inflammation responses in those who died. Our transcriptome analyses of blood samples from fatal and recovered H7N9 patients found that the gene Olah, encoding for a fatty acid hydrolase, was expressed more than 80-fold higher in fatal H7N9 patients compared to those that recovered at early days after disease onset. We established an Olah-overexpressing A549 cell line, which allowed us to develop a qPCR platform to screen Olah expression in human tissues and cells. Our analyses of Olah distribution across different anatomical sites found Olah expression in human lung, spleen and PBMCs, with the most prominent expression detected in CD14+ cells within the human lungs. Subsequently in Chapter V, we generated and established OLAH knockout (OLAH KO) mice to further investigate the role of Olah during severe influenza A virus infection. Compared to wild-type mice, OLAH KO mice displayed milder disease symptoms, less body weight loss and were protected from severe and fatal disease outcomes following IAV infection. In addition, OLAH KO lungs exhibited lower viral titres, less tissue damage and had significantly reduced levels of inflammatory cytokines and infiltrating innate immune cells, indicating the importance of Olah in controlling early excessive host responses and disease severity. These effects were associated with reduced lipid droplets in virus-infected epithelial cells lining the bronchioles, thus defining a role for OLAH in driving lipid metabolism during IAV disease. Overall, the findings from this PhD thesis highlight the importance of CD38+MHC-II+ CD8+ T cells during severe influenza A virus infection and identified the mechanisms underlying severe IAV disease. We also identified Olah as a potential biomarker of severe disease and demonstrated its role in modulating early host immune responses via the formation of lipid droplets and defined its impact on disease outcomes. Overall, data generated in this PhD thesis advance our understanding of factors underlying fatal clinical outcomes and provide insights into the development of novel treatment strategies and biomarkers against severe influenza disease.
Mucosal-associated invariant T (MAIT) cells and their function in bacterial infection
Mucosal-associated invariant T (MAIT) cells are a subset of innate-like alpha/beta T cells that recognize riboflavin metabolites presented by the monomorphic major histocompatibility complex (MHC) class I related protein-1 (MR1). The most potent antigen known to date is 5-(2-oxopropylidineamino)-6-D- ribitylaminouracil (5-OP-RU). MAIT cells are abundant in mucosal tissues and blood in humans. Upon bacterial infection, MAIT cells expand rapidly, with production of cytokines, including interferon-gamma (IFN gamma), tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-17 (IL-17), and cytotoxic granzymes. Their phenotype indicates that they play an important role in immunity. Previous studies showed a protective role in local infections, involving a single organ or tissue, but the mechanisms of MAIT cell-mediated protection in systemic infections are not fully understood. This study aimed to elucidate MAIT cell activation, protective role in primary infection and potential for a MAIT cell-based systemic vaccination to protect against Francisella tularensis live vaccine strain (LVS) and Legionella longbeachae. F. tularensis is a gram-negative intracellular bacterium which can cause systemic infection, in mice and humans. In this study, F. tularensis LVS was used to induce systemic infection in C57BL/6 (wild type) mice and in Mr1 -/- mice, which lack MAIT cells. A combination of CpGcombo (fused oligos for CpG-B and CpG-P) and synthetic 5-OP-RU antigen was used to vaccinate mice. Bacterial load, survival rate, and MAIT cell response kinetics and post-infection cytokine profiles were examined. MAIT cells expanded systemically and showed Th1-like cellular profile after infection with F. tularensis LVS. In several organs, C57BL/6 mice showed better control of bacterial burden compared with Mr1 -/- mice. Vaccination of MAIT cells with CpGcombo plus 5-OP-RU, but not CpGcombo alone, protected mice from infections by an otherwise lethal dose of F. tularensis LVS and L. longbeachae. Thus, MAIT cells displayed a protective role against systemic infections and potential to be boosted to protect against local and systemic infections. This study also showed that post infection, MAIT and non-MAIT alpha/beta-T cells manifest different contraction kinetics, indicating that these two groups could be regulated by different viability mechanisms. Specifically, the in vivo data illustrated that loss of receptor-interacting serine/threonine-protein kinase 3 (RIPK3, a key regulator of apoptosis and necroptosis), but not mixed-lineage kinase domain like pseudokinase (MLKL, a signaling molecule involved in necroptosis), preferentially increased MAIT cell abundance in a cell-intrinsic manner. In summary, the results in this thesis demonstrated that MAIT cells are critical in immune protection against systemic F. tularensis LVS infection, and are long lived with a differently regulated cell death and survival. These findings may inform the future development of vaccination strategies targeting MAIT cells.
Nanoparticle interactions with the immune system
Vaccination has been an incredibly successful public health intervention, saving the lives of 2-3 million people each year. Despite this success, we still lack effective vaccines for many infectious diseases including HIV, tuberculosis and malaria. Nanoparticles (ordered structures within the range of 10-1000nm) have great potential to supplement traditional vaccines based upon pathogen subunits, or killed or attenuated microorganisms, as demonstrated by the successful licensure of virus-like particle vaccines for human papillomavirus and liposomal mRNA vaccines for SARS-CoV2. However, the immunological mechanisms that explain the potent immunity of nanoparticle vaccines and the factors dictating their interaction with the immune system are poorly defined. This thesis studies how nanoparticle characteristics affect their interaction with the immune system with a view to improving vaccine strategies. First, the contribution of the protein corona on the association of engineered nanoparticles with primary human blood cells was assessed. The association of high protein binding (high-fouling) mesoporous silica (MS) particles and low-fouling zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) particles with human white blood cells was assessed by flow cytometry in the presence or absence of plasma proteins. The effect of precoating nanoparticles with serum albumin, IgG and complement protein C1q was also assessed. The differential association of low and high-fouling nanoparticles was found to be largely a consequence of the de novo formed, not pre-adsorbed, biomolecular corona. Specifically, an enrichment of complement proteins within the corona resulted in an increased association with B cells. Second, the immune mechanisms that give rise to the improved immunogenicity of a prototypic nanoparticle vaccine were investigated. Humoral immune responses to a self-assembling protein nanoparticle vaccine for influenza (HA-ferritin) were contrasted to a subunit influenza vaccine (soluble HA) in mouse and non-human primate models. Antibody titres and protective efficacy of the vaccines were compared followed by a detailed study of lymph node germinal centre B cell and T follicular helper cell responses. Vaccination of C57BL/6 mice with HA-ferritin nanoparticles elicited higher serum IgG titres and greater protection against experimental influenza challenge compared with soluble HA vaccination. Within the antigen-draining lymph nodes, germinal centre reactions were expanded and persistent following HA-ferritin vaccination. This augmented humoral immunity was not driven by ferritin-specific T follicular helper cells but rather driven by expanded antigen colocalization with follicular dendritic cells. However, this immune enhancement did not translate from mice to pigtail macaques where antibody titres and lymph node immunity following HA-ferritin nanoparticle vaccination were comparable to soluble HA protein vaccination. And thirdly, we explored innate immune activation by HA-ferritin and soluble HA in mice. This was achieved through in vitro assessment of antigen glycosylation and complement activation and in vivo through serum IgM titres and cell trafficking to the lymph nodes following vaccination. HA-ferritin vaccination of mice was found to elicit an early enhancement of antigen-specific serum IgM however in vitro complement activation was not detected. Trafficking of immune cells to the lymph nodes was found to be influenced by antigen glycan composition in conjunction with purification methods. The findings of this thesis suggest that nanoparticle interaction with the immune system is driven by the complex interplay of nanoparticle physiochemical properties, antigen glycosylation, corona formation and pattern-recognition receptors of innate immune cells. Further improvements in understanding the relationship between these features and how they may differ between animal species will speed the rational design of next-generation nanoparticle vaccines against diverse pathogens.
Latent tuberculosis infection and reactivation in Australia: defining the risk and planning the response
Tuberculosis (TB) elimination is now an aspirational goal in many low-incidence settings,1 born from an adaptation of the World Health Organization End TB Strategy, which states that low-incidence countries must progress towards eliminating TB as a public health problem. The TB incidence in Australia declined significantly during the twentieth century, but progress towards TB elimination has been influenced by high levels of migration from high-incidence countries. Most TB cases now occur among overseas born residents, and are attributed to reactivation of latent TB infection (LTBI) acquired overseas. LTBI is asymptomatic and not infectious, but those with LTBI can be treated to reduce their future risk of TB reactivation. However, there is uncertainty regarding the prevalence and distribution of LTBI in the Australian population and the risk that LTBI poses for future TB reactivation. Therefore, the effectiveness of LTBI screening and treatment strategies is also uncertain. The aims of this work were to: evaluate aspects of existing TB management in Australia; better understand and define the risk of LTBI and TB reactivation; and determine whether additional LTBI screening and treatment strategies for overseas-born residents could be cost-effective and advance Australia towards TB elimination. The thesis begins by reviewing current TB management in Victoria in relation to two factors identified as having an important impact on patient care: the private sector and gender. Observed disparities in the care received in the private versus public sector appeared to be primarily due to differing case-mix, and the small disparities in TB management and outcomes seen by gender were consistent with the possibility that males present with more severe disease, rather than any inequity in TB management. With this knowledge, Chapter 3 begins to explore the potential for additional LTBI screening and treatment strategies. Global annual risk of TB infection estimates were applied to Australian census data to estimate the LTBI prevalence in Australia, and identify those groups at the highest risk of LTBI. Two chapters are then devoted to improving our understanding of the risk of TB reactivation in the years and decades following infection and quantifying this risk. Chapter 4 presents a systematic review of the existing evidence on this topic and revealed that significant gaps remain in our understanding of reactivation risks in the years after infection. Chapter 5 combines the LTBI estimates from Chapter 3 with Australian TB case data to reveal that reactivation risks in overseas-born Australians largely decrease with increasing time from migration, with additional possible increases in risk during youth and in the elderly. In Chapter 6 the findings from previous chapters are applied in a cost-effectiveness analysis of additional LTBI screening and treatment strategies in recent Australian migrants. Modelled strategies prevented between 2.3 and 16.9% of future TB cases in each migrant cohort. However, because the majority of recent migrants are young adults with relatively low LTBI prevalence, low TB mortality risks and high emigration risks, strategies were unlikely to be cost-effective to the Australia health system unless most of the strategy cost was borne by migrants through pre-migration screening. Furthermore, application of even small quality of life decrements associated with LTBI treatment suggested these strategies would cause more ill-health than they prevent. This thesis draws on both historical and new evidence to improve our understanding of the risk that LTBI poses in the years after infection, and the management of that risk. Additional LTBI screening and treatment strategies cannot currently offer a cost-effective pathway to TB elimination in Australia. However, should any new research come to light, or new tools or treatments for LTBI become available into the future, the model presented in this thesis will be able to reassess this. In their absence, existing TB control strategies in Australia have ensured that TB incidence has remained stable despite decades of immigration from high-incidence countries, and these strategies should be maintained. Additionally, it is important to remember that the origin of the TB burden in Australia predominantly lies in infection that occurs overseas. Therefore, for as long as we continue to live in an interconnected, globalized world, the most effective current pathway to TB elimination, in both high and low-incidence settings, lies in supporting TB control in high-incidence countries. TB researchers in Australia and other low incidence settings should ensure that the research, messages and guidelines they champion address this need.
Recognition of Butyrophilin-family Members by gamma-delta T Cells
Human gamma-delta (gd) T cells can respond rapidly with cytotoxic responses to pathogenic infections and malignant cells. Although they are found at low numbers in peripheral blood, these might expand and constitute up to half of the total circulating T cells after infection. Most systemic gd T cells expressed a recombined heterodimeric T-cell receptor (TCR) with genes of the variable (V) g9 and d2 loci, termed Vg9Vd2 T cells. Opposed to conventional ab T cells that recognise processed peptides on major histocompatibility complex (MHC) class I and II and elicit delayed adaptive responses, gd T cells are innate-like T cells that recognise non-peptidic antigens akin to mucosal-associated invariant T (MAIT) or MHC-like lipid-presenting molecule (CD1)-restricted T cells which recognise vitamin-B derivates or lipids, respectively. The most interesting feature of Vg9Vd2 T cells is that they recognise phosphorylated antigens (aka phosphoantigens) which are essential for life including bacteria and particularly abundant in cancer cells. Little is known how gd T cells recognise phosphoantigens, even though, a few putative ligands have been described. Butyrophilins (BTNs) are a family of transmembrane molecules capable of regulating the immune activity of innate-like gd T cells. They dimerise to constitute complexes, some of which may interact with germline-encoding regions of the gd T cell receptors in murine or human species. For instance, mouse butyrophilin-like (BTNL) molecules shape the Vg7+ gd T-cell compartment in the intestine, while human BTNL counterparts selectively activate Vg4+ gd T cells of the colon. Here, tetrameric gdTCR clonotype probes are used for a genetic screen to identify a previously unknown molecular ligand essential for recognition of phosphorylated antigens by gd T cells. This screen identifies BTN2A1 and subsequent experiments elucidate this protein contacts with the Vg9 domain irrespective of the Vd recombined segment and is essential to confer reactivity to phosphoantigens together with BTN3A1. Thus, BTN2A1 and BTN3A1 constitute a functional complex of which we found both intracellular domains are critically important in maintaining an active conformation. Whereas internal BTN3A1 PRY/SPRY (B30.2) motif senses the antigen, the BTN2A1 intracellular domain appears fundamental to retain association of the complex. Mutagenic alanine screens reveal a dual-ligand binding site for the Vg9Vd2 TCR, where conserved residues of the Vg9 domain bind to BTN2A1 and several residues located at the complementarity-determining regions (CDRs) might react to a putative molecule of the BTN3A family. Thus, this work proposes a phosphoantigen-reactive gd T cells recognise a dual-ligand binding complex where BTN2A1 contacts the Vg9 domain and BTN3A1 is a phosphoantigen sensor molecule that plausibly induces the molecular switch necessary to induce immune responses. Lastly, the influence of tumour infiltrated phosphoantigen-reactive gd T cells in renal carcinogenic tumour patient-derived organoids (PDO) samples is examined and their relevance in healing disease assessed in comparison to previous clinical studies. These results are of vital importance to better understand the potential of gd T cells in prospective medical applications.
Using multiparameter imaging to understand HIV persistence in tissue in people living with HIV on antiretroviral therapy
I would like to take time now to thank the people that significantly contributed to me reaching this far. Herewith, being so grounded in my faith, I would like to start by thanking God, for being the source of my strength and peace. Secondly, I would like to thank my supervisors Paul Cameron and Sharon Lewin. Paul, thank you for your invaluable insight in formulating the research questions. At first, it was really hard for me to see you retire towards the end of my PhD, as I missed the option, I had of just popping in your office to ask you a question. Nevertheless, thank God for technology as we found a way to make it work. Therefore, I’m grateful to you for providing me with the opportunity to call you whenever I needed your help (including the weekends), not many students have that option. Also, thank you for asking me critical questions and pushing me to resolve issues on my own but also helping me when needed. Although it was time-consuming some days, I am grateful as it made me the independent person that I am today. I also appreciate you supporting me whenever I would tell you about a meeting or conference that I think might benefit me or my work. I am very appreciative of my second supervisor Sharon for allowing me to join her lab after leaving the Department of Medicine at the Austin Hospital to join the Department of Microbiology and Immunology at the Peter Doherty Institute. This was a huge move for me, but it has always been my dream to work on HIV-1/AIDS, so thank you for allowing that transition to happen so smoothly. Your insightful feedback pushed me to sharpen my thinking and brought my work to a higher level. Also, thank you for finding a way to collaborate with my former lab at the Austin Hospital and with other experts in this field such as Jake Estes, who have significantly helped with my PhD. As a somewhat shy person, this meant I was out of my comfort zone and that I would have to meet new people or be in a new working environment. Looking back, I can say that I have grown so much and became independent, and as a result, I was able to foster and maintain collaborations. I would also like to thank Sharon for her support when my health wasn’t optimal, this contributed to my recovery and my eagerness in returning to the lab and finishing. Empathy goes a long way! Sharon, I want to thank you for your support and for all the opportunities I was given to further my research. I would like to also thank Vanessa Evans for co-supervising me after Paul’s retirement. I appreciate you travelling to The Austin Hospital to meet me and your input in my research. You have been also so supportive, thanks for also being a listening ear when I needed it. I would also like to thank Thomas Rasmussen for assisting me towards the end with helping me to organize my results and proof-reading my thesis. I have always been amazed at how quickly you would get back to me, and for that, I am truly appreciative of. I am very thankful for our lab manager Ajantha Solomon and our research manager Jasminka Sterjovski (Peter Doherty Institute). Aj, thanks for assisting me and being so supportive when I was working at the Austin, I will miss our little walks. Jas, you have been so supportive and understanding. I will never forget when the Hurricane destroyed 90% of my island back home and I was unable to contact my family for a few weeks. This was one of the scariest moments in my life, but you were there every day checking to see if I was okay and offering me to go for a walk in the park. I will forever be appreciative of your kindness, empathy and support, you are one of a kind! Special thanks to my collaborators at Olivia Newton-John Cancer Centre Research Institute, Austin Hospital, Australia. Marzena Walkiewicz (my lab aunty), thanks for teaching me all there is to know about immunohistochemistry and for all the delicious treats. I would also like to thank Dani for training me on the Vectra imaging platform and Johnathon Cebon and Katherine Woods for being so graceful when I decided to switch to HIV-1 research and welcoming me back for collaboration, it felt as if I have never left! I would also like to thank my collaborators Claire Deleage and Jake Estes (Frederick National Laboratories for Cancer Research, USA), thank you for picking me up every morning and teaching me RNA/DNAscope and imaging. On the same note, I would also like to thank my other collaborators, Gustavo Reyesteren and Perla Del-Rio (Instituto Nacional de Enfermedades Respiratoriras, Mexico), Michael Gonzales, Samuel Thomson (Pathology Department, The Royal Melbourne Hospital, Australia) and Metta Jena and Rejhan Idrizi (Peter MacCallum Cancer Centre, Australia). In addition, I am greatly appreciative of the immunomodulation group (Peter Doherty Institute), who contributed to my research, particularly Judy and former member Renee, who has trained me so quickly on getting PC3 certified and on the in vitro model of Latency. I also owe gratitude to our research assistants, especially Caroline and former research assistant Surekha for always following up on my orders and contacting me if there were any issues. I would also like to thank present and former members of the lab Simin, Hao, Sandy and Jenny. To my parents, O’Neil Richardson and Julienne van der Leeuw-Richardson, thank you for the encouragement, sacrifices made and for permitting me to leave my Island at a young age to pursue my dreams. To my big sister Nyakomi, thanks for always being my hype man and believing in me, and to my other siblings, Glenroy and Abigail, my intelligent nephew Amaziah and friends overseas and the ones I’ve made in Australia (too numerous to mention), thank you for supporting me in every aspect during the completion of my PhD. After completing my masters, I was fortunate to work in my hometown St. Maarten (which has the largest amount of people living with HIV-1/AIDS in the Dutch Caribbean islands) at the HIV-1/AIDS foundation, interviewing patients suffering in secret. This has also contributed to me pursuing a PhD in this field. Therefore, I would like to acknowledge those living with HIV-1, particularly in places where stigma and discrimination are at the highest. I’ve learned that people will forget what you said, people will forget what you did, but people will never forget how you made them feel.' M. Angelou Thank you for all the positivity and support throughout this journey.