Microbiology & Immunology - Theses

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    Triplex forming oligonucleotides to silence the HIV reservoir
    Liu, Haoming ( 2023-10)
    The eradication of HIV remains a formidable challenge due to the virus's ability to establish latent reservoirs within host cells, evading antiretroviral therapy (ART). The overarching objective of this thesis was to evaluate the potential of Triplex-Forming Oligonucleotides (TFOs) as an innovative approach to inhibit HIV transcription from latency and to ascertain efficient nuclear delivery mechanisms, thereby contributing to the 'Block and Lock' strategy aimed at maintaining viral suppression without continuous ART. Our seminal findings underscore the ability of TFOs to inhibit HIV transcription, as evidenced in models such as the HEK 293T plasmid transfection and a latently infected cell line. However, challenges including limited nuclear penetration and potential binding impediments due to chromatin structure were observed. Encouragingly, encapsulation of TFOs within lipid nanoparticles demonstrated a moderate reduction in HIV transcription in the latently infected cell line, albeit optimization in delivery and stability being imperative. Distinct from previous studies targeting the LTR region, our investigation expanded to TFOs targeting the HIV gag, envelope, and pol genes, unveiling a high degree of conservation in target sequences, and thus broadening the target range of TFOs. This novel approach substantiates that non-LTR targeting TFOs can also significantly inhibit HIV virion production in vitro, offering a promising avenue to overcome the variability challenges associated with LTR region targeting employed by other gene modification tools. Despite the encouraging in vitro outcomes, the study acknowledges inherent limitations predominantly centered around the in vitro nature of the experiments and the need for further optimization in TFO delivery. Our work explored two nanoparticle formulations, gold nanoparticles and lipid nanoparticles, for their potential in conveying TFOs to target cells. Particularly, lipid nanoparticles emerged as a promising candidate for delivering 'Block and Lock' strategies, inspired by their recent success in mRNA COVID-19 vaccine technology. Through a systematic investigation, this thesis delineates a promising pathway towards HIV transcriptional inhibition using TFOs and underscores the significance of optimizing nanoparticle-mediated TFO delivery. The insights garnered from this work lay a robust foundation for future in vivo and clinical investigations, steering closer towards the long-term goal of an cure for HIV.
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    Obesity impairs virus-specific memory CD8+ T cell signalling and function
    Horvatic, Helena ( 2023-03)
    The main function of the immune system is to protect the host against invading pathogens. Immunological memory is formed after a primary infection and serves to provide protection in a fast and vigorous manner upon secondary encounter with the same pathogen. Under obesity conditions, however, protective immunity is impaired, constituting a significant risk factor for high incidence and severity of re-infections. Indeed, during the current COVID-19 pandemic, obesity has been recognized as a major risk factor for adverse clinical outcomes. Impaired immunity observed in obese individuals has been attributed to a dysfunction in CD8+ T cells, which are essential for the elimination and sterile clearance of viral infections. Yet, the underlying mechanisms of the immune-compromised status of obese individuals remain poorly understood. Using mouse models of diet-induced obesity and lymphocytic choriomeningitis virus infection, we showed increased morbidity and mortality of obese mice after re-infection, recapitulating the clinical situation in humans. Obese mice failed to mount protective immunity and showed a profound loss of virus-specific memory CD8+ T cells in the spleen and liver. This phenotype was associated with a compromised proliferation capacity and reduced ability to produce the effector cytokines IFNg and TNFa. Furthermore, our data revealed a severe decline specifically within hepatic tissue-resident memory T cell pool, which positively correlated with the body weight and contributed to the severe symptoms observed in obese mice. Additionally, we observed enhanced accumulation of IgA+, IL-10 producing, and PD-L1+ B cells in the liver of obese mice, and their absence was associated with normal numbers of hepatic tissue-resident memory T cells. Notably, genetic ablation of IL-10 production by B cells did not reconstitute the memory response, indicating that IgA+ B cells do not exert a suppressive effect towards the virus-specific CD8+ T cells via IL-10. Furthermore, our findings show that the impaired memory response in obese mice is due to T-cell intrinsic mechanisms driven by long-term exposure of virus-specific memory CD8+ T cells to the inflammatory environment, rather than by affecting their development and recruitment. Molecular analysis revealed transcriptional reprogramming of memory CD8+ T cells under obesity conditions, resulting in impaired T-cell receptor signaling. The obesity-inflicted changes in memory CD8+ T cells were highlighted by the impairment of Ca2+ influx upon CD8+ T cell stimulation in vitro. Collectively, our findings indicate that virus-specific memory CD8+ T cells exposed to an obese environment lose their ability to confer protection. The future intention of this project is to identify key molecules within the Ca2+ signalling pathway that could be therapeutically modulated to improve the ability of virus-specific memory CD8+ T cells to provide protection and to improve the immune response of obese individuals to re-infections.
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    Targeting the HBV pregenomic RNA and viral mRNAs using the CRISPR-Cas13b system and determining the impact of HBV splice variants Sp3 and Sp9 on wildtype HBV replication
    McCoullough, Laura Claire ( 2023-10)
    Chronic hepatitis B infection, caused by the hepatitis B virus (HBV), affects over 296 million people globally and almost a million people die annually because of hepatitis B associated complications such as liver disease and hepatocellular carcinoma (HCC). Upon infection, the relaxed circular DNA genome is released into the nucleus where it is converted into the covalently closed circular DNA (cccDNA) minichromosome. This is then transcribed into the HBV pregenomic RNA (pgRNA), which is the HBV replication intermediate, and the viral mRNAs, which together with the pgRNA encode the HBV proteins essential for replication. The pgRNA may also be spliced to produce smaller RNA forms, some of which impact HBV replication. There is global consensus that new HBV treatments are urgently required that target multiple stages of the HBV replication cycle to increase rates of functional HBV cure, defined as loss of HBV surface antigen, which is rarely achieved using current HBV therapies. The HBV pgRNA and viral mRNAs represent a novel target for new HBV treatments. Here, the bacterial CRISPR-Cas13b system, which targets RNA, was repurposed to target the HBV pgRNA, spliced RNAs and viral mRNAs to reduce HBV replication and protein expression, in pursuit of developing a potential new therapy for chronic HBV infection. This was the first study to repurpose CRISPR-Cas13b to target the HBV RNAs. CRISPR-Cas13b was first optimised to target the HBV RNAs using an in vitro transfection system. The efficacy of CRISPR-Cas13b targeting HBV was then tested using additional in vitro models and in an in vivo HBV model. A major barrier to developing more HBV treatments is that the HBV replication cycle and host/viral factors that influence HBV replication are not completely understood. One such aspect is the role of HBV splice variants, which are shorter DNA genomes, produced after reverse transcription of shorter RNA forms derived by splicing of the HBV pgRNA. The role of HBV splice variants in HBV replication and pathogenesis remains largely unknown, however an increased proportion of splice variants in patient sera has been associated with the development of liver disease and HCC. In addition, different HBV splice variants and their encoded novel fusion proteins have different effects on wildtype HBV replication in vitro. Here, the impact of two common but poorly characterised HBV splice variants, Sp3 and Sp9, on wildtype HBV replication and the impact of HBV splice variants on the host cell kinome was explored in vitro, to further investigate the role of HBV splice variants in HBV replication and pathogenesis, which may provide additional targets for new novel treatments.
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    Dissecting the role of gd T cells in T cell priming for liver stage immunity
    Le, Shirley ( 2023-11)
    Liver resident memory T cells (TRM) are poised for protection against repeat infection and rapidly form a robust defence against tissue-specific insults such as liver stage malaria. A direct correlation between liver stage immunity and gd T cells has been observed both in mice (Zaidi et al. 2017) and in humans (Seder et al. 2013; Ishizuka et al. 2016), but the precise molecular mechanisms by which these gd T cells exert their protective effect are yet to be defined. In mice, intravenous injection with radiation-attenuated sporozoites (RAS) confers sterile protection against challenge with live sporozoites. This protection is mediated by responding antigen-specific CD8+ and CD4+ T cells that migrate to the liver and form resident-memory T cells (TRM). In the absence of gd T cells, protective CD8+ liver TRM are not generated, leaving mice susceptible to reinfection. Using Plasmodium-specific T cells as a readout for effective immunity, we determined that IL-4 is important for the accumulation of CD8+ and CD4+ T cells. By utilising complex in vivo systems including mixed-bone marrow chimeras and adoptive transfer of gd T cells, we revealed that gd T cell-derived IL-4 is crucial for the expansion of antigen-specific CD8+ T cells. In addition, in vivo neutralisation of IL-12 or IFN-g confirmed a partial dependency for these cytokines, despite their traditionally opposing function to IL-4. Given IL-4, IFN-g and IL-12 all have a clear role in CD8+ T cell priming following RAS vaccination, we hypothesised that IL-4 and IFN-g synergise to enhance cDC1 activity. These findings led to our development of a novel model to reconstitute cDC1-deficient mice using CRISPR-edited primary dendritic cells. This model enabled the investigation of the mechanism by which gd T cell derived IL-4 leads to DC activation and therefore effective CD8+ T cell expansion for memory development. Collectively, this project has shown a significant role for IL-4 in the priming of malaria-specific CD8+ T cells and demonstrates a novel pathway for collaboration between gd T cells, cDC1s, and CD8+ T cells, revealing the potential for harnessing gd T cells in vaccination strategies against malaria.
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    Antibody-mediated passive immunity against Helicobacter pylori
    Gorrell, Rebecca Jane. (University of Melbourne, 2008)
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    Induction of immune responses by lipopeptide vaccines
    Lau, Yuk Fai. (University of Melbourne, 2006)
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    Control of pulmonary immunity by physical exercise
    Michla, Marcel ( 2023-03)
    Sedentary lifestyles combined with high caloric nutrition are widely known to severely contribute to the rise of metabolic diseases like diabetes, atherosclerosis, or obesity in recent years. While endurance training has been shown to induce the secretion of adipokines, and so-called myokines (muscle-derived cytokines), the impact of physical exercise on the host immune response in the context of bacterial or viral infections remains largely unknown. Here, we aimed to investigate the impact of voluntary wheel running (VWR), mimicking an active lifestyle, on the pulmonary immune system and to which extent a lack of exercise might affect the severity of pneumonia induced by bacterial L. longbeachae or viral influenza A virus (IAV) infection. We observed that VWR enhanced stamina to exercise and reduced visceral adipose tissue. Moreover, VWR induced the expression of myokines and lipolysis-associated genes and decreased the number of circulating monocytes. Notably, neither acute nor long-term (2 and 8 weeks, respectively) physical exercise significantly affected the abundance or metabolism of pulmonary immune cells in healthy mice. However, upon infection with L. longbeachae, acute physical training reduced pathogen burden, dampened anorexia-induced weight loss, and decreased the recruitment of neutrophils and monocytes to the airways. Additionally, pro-inflammatory cytokines associated with bacterial clearance, including IFN-g & TNFa increased in the lungs of exercised mice. Notably, VWR enhanced the potential to produce TNFa in both alveolar macrophages and infiltrating monocytes early and late in infection with L. longbeachae. Furthermore, in running mice we found increased mitochondrial and glucose dependency in myeloid cells, crucial for the inhibition of pathogen replication. Our results also show that CD4+ T cells from trained animals display reduced IFN-g production, suggesting that exercise may predominantly boost the innate immune response. In contrast, during IAV infection, we observed significantly decreased numbers of activated type 1 helper T (Th1) cells in running mice, critical for viral clearance. However, the frequency of tetramer+ CD4+ and CD8+ T cells was increased, indicating higher antigen specificity of the immune response. Additionally, we found higher viral RNA content in lung tissue from exercising mice, and elevated production of pro-inflammatory cytokines including type I interferons. Moreover, we found higher expression of interferon-signalling genes in the pulmonary tissue of exercising mice. VWR increased the gene expression of Ifng in CD44+ CD4+ T cells in running mice, suggesting an enhanced capacity of CD4+ T cells to produce IFN-g. Notably, VWR increased the expression of tissue-residency markers on CD8+ T cells. Taken together, our results suggest that VWR might have opposing effects on pulmonary immunity during infection. Hence, we conclude that acute physical exercise might enhance protection against bacterial invasion (L. longbeachae) by specifically boosting the innate immune response. In contrast, VWR reduces Th1-mediated anti-viral responses and increases the pulmonary viral RNA content, suggesting that physical exercise might enhance the susceptibility to IAV infection. However, by boosting the adaptive IFN-g-mediated anti-viral response, antigen-specificity, and increasing tissue residency, exercise may enhance the defence against secondary viral infections.
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    The Development, Homeostasis, and Function of Unconventional T Cells
    Xu, Calvin ( 2023-09)
    Unconventional T cells detect non-peptide antigens presented by MHC class-I-like molecules, such as MR1 and CD1. MR1-reactive T cells, CD1-reactive T cells, and gamma-delta T cells represent three broad unconventional T-cell lineages which collectively are abundant and play key roles in the immune response. The frequencies of these cells vary widely between individuals, and the factors that govern their numbers and diverse effector functions are not well-understood. This thesis investigates the mechanisms that controls the development, homeostasis, and function of unconventional T cells and their subsets in the thymus and peripheral tissues. In chapter 3, MR1 and group 1 CD1 (CD1a, CD1b, and CD1c) tetramers were used to isolate and characterise rare unconventional T-cell populations in the human thymus. Using tetramer-mediated enrichment, thymic MR1-reactive T cells were found to comprise Valpha7.2+ and diverse Valpha7.2- subsets that differed in phenotype, binding to antigen-loaded MR1 tetramers, TCR repertoire diversity, frequency, and post-natal expansion. Whilst tetramer-mediated enrichment allowed for isolation of thymic CD1a- and CD1b-reactive T cells, blockade of CD36 expressed on thymocytes was additionally needed in order to detect CD1c-reactive T cells. Group 1 CD1-reactive T cells were highly rare after enrichment and generally resembled other CD4+ thymocytes. These findings highlight diverse MR1 and CD1-reactive T cell subsets in the thymus and forms the basis for understanding their intrathymic generation and developmental pathways. The expansion of MAIT cells in NKT- and gamma-delta T cell-deficient mice was investigated in chapter 4. Although MAIT cells were highly elevated in mice deficient in both NKT and gamma-delta cells, they phenotypically and functionally resembled their counterparts in wildtype mice. Mechanistically, this MAIT cell expansion was due to: 1) increased rearrangement of the MAIT cell TCR alpha chain within developing Tcrd-/- thymocytes, and 2) a higher capacity of peripheral MAIT cells to proliferate in the absence of NKT and gamma-delta cells. Overall, this chapter provides evidence for a shared niche in which MAIT, NKT, and gamma-delta T cells reside and compete for common homeostatic factors, revealing a novel interplay between their steady-state frequencies. In chapter 5, the regulation of peripheral unconventional T cells by the purinergic P2RX7 receptor was examined. Human unconventional T cells expressed P2RX7, whereas their mouse T-bet+, but not RORgt+, counterparts highly co-expressed the ADP-ribosyltransferase, ARTC2, and P2RX7. P2RX7 activation in response to ATP induced death of both mouse and human unconventional T cells ex vivo. Mouse T-bet+ unconventional T cells were highly susceptible to the effects of ARTC2-dependent P2RX7 activation in response to nicotinamide adenine dinucleotide, which resulted in their cell death ex vivo and depletion in vivo. By blocking ARTC2 or P2RX7, this chapter demonstrated the existence of IFN-gamma/IL-4 co-producing unconventional T cells, including MAIT and other non-MAIT/NKT alpha-beta T cell populations, which were selectively regulated by ARTC2-dependent P2RX7 activation. In contrast, this axis did not affect IL-17-producing unconventional T-cell subsets. These findings reveal a unique mechanism that controls the number and functional diversity of unconventional T cells via the selective regulation of their T-bet+ and IFN-gamma/IL-4 co-producing subsets. Overall, this thesis has examined the presence of diverse unconventional T cells in the human thymus, their regulation within a homeostatic niche, and their modulation by P2RX7 activation. Whilst unconventional T cells are collectively abundant, understanding their development and homeostasis will provide insight into why their frequencies vary widely in humans and how their numbers can be finely-controlled. The homeostatic relationships between unconventional T cells and how their functional subsets are regulated will be important considerations in the targeting of one or more unconventional T-cell populations within the immune response. Given their production of functionally-opposing cytokines, findings in this thesis will guide the development of future immunotherapies that leverage the abundance and potent effector functions of unconventional T cells in treating disease.