Microbiology & Immunology - Theses
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ItemNo Preview AvailableEvaluation of novel strategies targeting the HBeAg on their ability to generate therapeutic anti-HBV responses( 2023-10)The Hepatitis B virus (HBV) was discovered over 50 years ago, yet HBV infections remain a major contributor to global health morbidity. It is estimated that over 296 million people are living with chronic hepatitis B (CHB) infection worldwide, including 240,000 Australians. Despite the availability of prophylactic vaccines and suppressive antiviral treatments, life-long therapy is often required to control infection but rarely leads to curative outcomes. HBV causes a non-cytopathic infection which can remain undetected for over 30 years; however, the host’s immune system eventually elicits responses against viral HBV antigens and infected hepatocytes. These immune responses often lead to cirrhosis, liver disease and hepatocellular carcinoma, the most prevalent form of liver cancer. An estimated 820,000 deaths per year are attributed to CHB infection, making HBV a medically important pathogen. Unfortunately, HBV does not receive the same attention as other viral infections, and its insidious nature results in the majority of infected individuals being unaware of their status and contributing to its spread. Therefore, there is an urgent demand for the development of novel strategies aiming to reduce the current morbidity and mortality rates caused by HBV infections on a global scale. The Hepatitis B e antigen (HBeAg) is a secreted, accessory viral protein essential for the initial establishment of a chronic infection by modulating many of the host immune responses. The presence of HBeAg is generally associated with ongoing HBV replication and liver disease. Historically, the HBeAg has been used as a serum marker to determine disease progression and treatment recommendations, yet its kinetics vary among different genotypes. HBeAg seroconversion to anti-HBe antibodies may take 30 years or longer to occur, and it is a current treatment endpoint, associated with reduced HBV replication and disease control. However, none of the current treatments target the HBeAg directly. HBeAg seroconversion is often associated with precore and basal core mutations which introduce stop codons, leading to either production of truncated, non-functional HBeAg or lack of production altogether. Importantly, it is a preceding step to hepatitis B surface antigen (HBsAg) loss, the current definition of functional cure. The focus of this PhD project has been the promotion of anti-HBe antibody immune responses to induce suppression or seroconversion of HBeAg, investigated via 2 distinct approaches, either by treatment with bio-engineered virus-like particles (VLPs) with exposed HBe-epitopes (eAg-VLPs), or treatment with anti-HBe monoclonal antibody (mAb) immunotherapy. Other than being the structural component of the viral envelope, the HBsAg is able to self- assemble into smaller, non-infectious sub-viral VLPs. HBsAg VLPs make excellent platforms for the insertion and presentation of exposed foreign epitopes due to their repetitive structure, leading to robust immune responses. The main objective of this project focused on bio- engineered HBsAg VLPs to deliver exposed immunogenic HBeAg-specific epitopes at high antigenic density and drive HBeAg seroconversion in a mouse model of HBV persistence. Chapter 3 of this thesis characterised the anti-HBe antibody responses in cohort of HBeAg -ve CHB patients (St Vincent’s, Melbourne) demonstrating that the unique HBeAg-specific precore domain is indeed immunogenic in the majority of these patients. Thereafter, Chapter 3 focused on the design and production of chimeric eAg-VLPs, followed by the characterisation of their biochemical structural and functional capabilities to select the best candidates for subsequent in vivo studies. Subsequently, Chapter 4 utilised the most antigenic eAg-VLP candidates to investigate their immunogenic potential in an immune competent mouse strain and demonstrated that the induced anti-HBe antibodies against the selected HBe-epitope were able to bind against native HBeAg. Next, an investigation of the treatment outcomes of chimeric eAg-VLPs in a mouse model of HBV persistence was performed leading to significant rates of HBeAg seroconversion. Chimeric eAg-VLPs possibly represent suitable tools to promote and induce immune responses to clear important viral targets and facilitate functional cure outcomes. Finally, Chapter 5 investigated an alternative approach to successfully promote long-term, sustainable HBeAg suppression in a mouse model of HBV persistence via anti-HBe mAb immunotherapy. Overall, the HBeAg has been overlooked as a potential therapeutic target, but herein we demonstrated that targeted HBeAg seroconversion may assist to the development of alternative therapeutic approaches to contribute to the global scientific effort for the development of an HBV cure by 2030.
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ItemTriplex forming oligonucleotides to silence the HIV reservoir( 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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ItemObesity impairs virus-specific memory CD8+ T cell signalling and function( 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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ItemTargeting 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( 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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ItemDissecting the role of gd T cells in T cell priming for liver stage immunity( 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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ItemAntibody-mediated passive immunity against Helicobacter pylori(University of Melbourne, 2008)
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ItemThe role of T cells in the immunopathology of Helicobacter pylori infections in mice(University of Melbourne, 2007)
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ItemThe role of dendritic cells in CD8+T cell priming after epidermal HSV-1 infection(University of Melbourne, 2006)
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ItemInduction of immune responses by lipopeptide vaccines(University of Melbourne, 2006)
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ItemControl of pulmonary immunity by physical exercise( 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.