Microbiology & Immunology - Theses
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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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ItemThe epidemiology of influenza in Northern Australia( 2021)The epidemiology of influenza in Northern Australia may be unique due to the tropical climate, large Indigenous population and wide dispersal of the population in the region. To mitigate the impact of the next influenza pandemic and seasonal influenza epidemics, it is important that the epidemiology of influenza in this area be better understood. Furthermore, estimates of the effectiveness of the influenza vaccine against hospitalisation in Northern Australia and comparisons of vaccine effectiveness in Indigenous and non-Indigenous Australians are lacking. This is despite the Australian Federal Government currently covering the cost of influenza vaccination for all Indigenous Australians aged over 6 months. Chapter 2 of this thesis consists of epidemiological analyses of notified influenza cases in the Northern Territory. Rates of influenza were higher for Indigenous Australians in all age groups through 2007-2016 with the disparity being largest for those in the 55-64 age bracket (rate ratio: 5.56; 95% CI: 4.71, 6.57). Bimodal peaks in influenza activity were seen in the Top End region of the Northern Territory in 3 out of the 10 years studied. Chapter 3 details the first ever use of phylogenetic methods to describe influenza activity in the Northern Territory. Influenza strains in the Northern Territory were shown to undergo regular extinction and are related to strains present in many diverse global regions. A mismatch was seen in the influenza vaccine strain and a circulating influenza B strain during an outbreak in late 2013-early 2014. Chapter 4 consisted of a case-control study employing a test-negative design to examine the effectiveness of the influenza vaccine against hospitalisation in the Northern Territory between 2009-2014 using 1075 cases and 3461 controls. Odds ratios for vaccination in each year were obtained from logistic regression models and meta-analysed using a random-effects model. Overall vaccine effectiveness was estimated at 32% (95% CI: -1%, 54%). Vaccine effectiveness was estimated at 40% (95% CI: -10%, 68%) for non-Indigenous Australians and 23% for Indigenous Australians (95% CI: -16%, 48%). Differences in vaccine effectiveness between Indigenous and non-Indigenous Australians were not statistically significant (p>0.15), but available sample size limited ability to detect a difference. This thesis highlights the burden of influenza upon the Indigenous population of the Northern Territory, the challenges that semi-annual influenza epidemics present in this region, ongoing cycles of importation and extinction of influenza viruses occurring in the Northern Territory and provides evidence that current vaccines have limited effectiveness against hospitalisation in this area. This thesis provides a framework for examining the impact of many infectious diseases in Northern Australia.
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ItemThe role of prion strain in disease progression and pathogenesis( 2021)Prion diseases are a family of invariably fatal, transmissible neurodegenerative diseases. The classic symptoms of prion disease are due to severe, rapid neurological decline hence by the time a diagnosis is made it is too late to commence therapeutic intervention as the patient will have already sustained significant impairments or even have succumbed to disease. Identifying prodromal markers of prion disease will enable diagnosis and treatment to be implemented prior to significant neurodegeneration. The highly variable clinical presentation of prion disease also confounds medical diagnosis and can delay the identification of these fatal, transmissible disorders. This variation has been attributed to the existence of prion strains. The lack of medically relevant models has hampered our understanding of prion strains. The current thesis characterises novel, medically relevant, mouse-adapted, human prion strains with a focus on identifying novel, prodromal disease signs. Human-derived sporadic Creutzfeldt–Jakob disease strains have undergone serial passaging to enable their adaptation to murine hosts. Three sporadic Creutzfeldt–Jakob disease strains, T1MM, T2MM and T3MM (Collinge classification system) were used to generate mouse-adapted models MU-01, MU-02 and MU-03 respectively. Characterisation of these strains in contrast to the mainstay murine-adapted prion strain of Australian researchers, M1000, was performed to enable comparison of the clinical profile, neuropathology and biochemical features of these strains. Several novel and potentially prodromal clinical signs were identified and underwent further investigation. The mouse-adapted prion strains exhibited high circulating cholesterol levels, dilated cardiomyopathy and increased appetites leading to weight gain. Weight gain was the first sign of disease observed in MU-03 inoculated mice and presented markedly earlier than classic signs of prion disease neurological decline. These findings led to the examination of the patient database held by the Australian National Creutzfeldt-Jakob Disease Registry to determine if these signs are phenotypical of human prion disease. There were significantly more prion disease cases who were identified as displaying overeating, weight gain and hypercholesterolemia when compared to non-prion cases afflicted with other neurological illnesses. The inverse was found when examining the presence of prion-like cardiomyopathy, which was significantly underrepresented in prion disease cases. This may reflect prion disease patients succumbing to prion-associated cardiomyopathy prior to fulfilling the clinical criteria for referral to the Australian National Creutzfeldt-Jakob Disease Registry. Rapid obesity of an unknown origin may be an early indication of prion disease and has the potential to aid early identification and treatment of prion disease prior to the classic phase of irreversible, rapid decline. High circulating cholesterol levels may prove to be supportive in a diagnosis of prion disease and the results of the current study support further investigation into the use of statin treatment in patients with prion disease. Further work is required to determine the prevalence and relevance of prion-associated cardiomyopathy in patients as well as any transmission risk it may pose during cardiac surgery. The newly, characterised human-derived, mouse-adapted prion strain models are well placed to be valuable resources for understanding these newly identified disease signs and enabling the development of strategies to improve outcomes for patients.
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ItemInactivation Mechanisms of Therapeutic CD8+ T Cells against a non-Hodgkin B-cell Lymphoma Mouse Model( 2021)Clinical and animal studies have demonstrated the capability of innate or adaptive components of the immune system to eliminate tumour cells. Cytotoxic CD8 T lymphocytes (CTLs) are the main population of the adaptive immune system involved in tumour cells elimination. In clinical studies, autologous tumour associated antigen (TAA)-specific CTLs have been adoptively transferred into patients to eliminate tumour cells expressing the cognate antigen, an approach known as Adoptive Cell Therapy (ACT). In many conditions, these CTLs are functionally impaired. Similar observations have been made in mouse models, including our own. We found that anti-ovalbumin (OVA) OT-I CTL injected into mice were capable of eliminating non-Hodgkin B cell lymphoma cells that express OVA as a model TAA. However, this ACT failed in mice harbouring a large tumour burden because many of the OT-I CTL were eliminated soon after ACT, and even though the surviving ones expanded, they remained functionally impaired. These observations recapitulate successful vs failed outcomes of ACT in the clinic. So far little is known about the extrinsic and intrinsic mechanisms that determine these outcomes. My results show that the mechanisms of CTL inactivation we observe are most likely physiological responses to large target cell burdens and provide experimental system to further understand the mechanisms of inactivation and approaches for the generation of more effective CTL for ACT.
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ItemNo Preview AvailableGlobal 3’-UTR Length Changes Form a Novel Layer of Regulation in the Macrophage Response to Interferon-Beta( 2021)Interferon signaling is one of the most important mechanisms shaping innate immune responses and needs to be tightly regulated to successfully fight infections and modulate immune responses while avoiding toxicity. Type I interferons (IFNs) have been shown to induce multiple transcriptional, translational and metabolic changes. The global response of murine and human macrophages to IFNb stimulation was characterized in this study using multi- omics strategies. These analyses gave insight into a complex regulatory network of transcripts, proteins and metabolites that results in global reprogramming of the cell. Post-transcriptional gene regulation is an important component of this network and is centered around 3’-untranslated regions (3’-UTRs), regions heavily targeted by miRNAs and harboring binding sites for many RNA-binding proteins. Poly-A-tail sequencing (PAT-seq) experiments revealed that many transcripts expressed shortened 3’-UTRs in response to IFNb, a result of changed alternative polyadenylation (APA) patterns. APA and changed 3’-UTR lengths are emerging fields of broad importance in physiological and pathological processes that are only starting to be explored. Differences in APA patterns and their regulation have not previously been studied in context of IFN. Recent publications have described a scaffold-like role for 3’-UTRs that facilitates the formation of different protein complexes depending on 3’-UTR length, which can affect localization and function. This unique regulatory mechanism was investigated for two IFNb-regulated transcripts with shortened 3’-UTRs, EIF4EBP2 and MAVS. 3’-UTR-dependent protein-protein interactions were identified by mass spectrometry using tagged overexpression constructs encoding the different transcript isoforms. This study describes a new aspect of IFN signaling and a novel layer of regulation through genes that are not part of the typical and well-characterized IFN transcriptional response. It shows how differential expression of distinct 3’-UTR transcript isoforms influences macrophage innate immune responses.
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ItemInvestigating the ability of RIG-I agonists to provide protection in mouse and ferret models of respiratory virus infection( 2021)Respiratory infections caused by influenza A virus (IAV) or respiratory syncytial virus (RSV) lead to substantial morbidity and mortality. Treatment options are limited and there is urgent need for the development of efficient therapeutic and prophylactic treatments. Pattern recognition receptors (PRRs) such as the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are part of the innate immune system. RIG-I can be activated by recognition of viral nucleic acids, leading to downstream activation of interferon-stimulated genes (ISGs) and restriction of viral replication. We have used synthetic RNA oligonucleotides to stimulate RIG-I to inhibit replication of respiratory viruses using in vitro and in vivo models of infection. Our in vitro approaches used airway cell lines from humans, mice and ferrets and investigated the effects of RIG-I agonist pre-treatment on subsequent infection with either IAV or RSV. Prophylactic RIG-I agonist treatment induced multiple ISGs and inhibited infection and growth of respiratory viruses in cell lines from each of the different species. In vivo, we utilised mouse and ferret models to study the antiviral potential of RIG-I agonists against IAV and RSV. In mice, we compared animals which do or do not express a functional Mx1 protein and found that a single prophylactic treatment with RIG-I agonist via the intravenous route resulted in ISG induction in the lungs and this correlated with reduced IAV replication. Of interest, these effects were particularly potent and long-lasting in mice expressing a functional Mx1 confirming an important role of Mx1 for RIG-I agonist-induced protection against IAV. In a mouse model of RSV, we found that a single prophylactic treatment with RIG-I agonist resulted in reduced replication of virus in the lung, as observed using bioluminescence imaging of luciferase-labelled RSV as well as plaque assay for infectious virus. Thus, our studies in mouse models indicate that a single pre-treatment with RIG-I agonists resulted in potent inhibition of two very different respiratory viruses. In ferrets, after establishing assays to monitor ISG induction in the blood and in airway tissues, we confirmed that a single intravenous injection of RIG-I agonist induced ISG induction in both peripheral blood mononuclear cells (PBMCs) and the lungs. Moreover, a single treatment prior to infection also resulted in reduced replication of both IAV and RSV in ferret lungs, although this treatment had only negligible effects on virus replication in the nasal tissues. A single treatment to animals with an established IAV infection also resulted in reduced virus titres in the lungs, suggesting its potential as a therapeutic antiviral agent. Myxoma (Mx) proteins are ISGs with potent antiviral effects against IAV. While human and mouse Mx proteins have been studied in detail, ferret Mx proteins have not been characterised. Therefore, we generated different experimental approaches to assess the induction of three endogenous ferret Mx (two splice variants of Mx1 as well as Mx2) in a ferret cell line, as well as in vitro overexpression systems to assess the cellular localisation and antiviral functions of each ferret Mx. Our findings indicate that each ferret Mx localises to the cytoplasm and that particular proteins exhibit antiviral functions against IAV, but not RSV. However, further studies are required to clearly define the antiviral activity of ferret Mx, since our preliminary results indicate that ferret Mx proteins display different antiviral activity following overexpression in human or in ferret cells. Together, studies described in this thesis demonstrate the potential of RIG-I agonists as antiviral treatments against diverse respiratory viruses both in vitro and in vivo and represent an important step towards the development of novel antiviral treatments in humans.
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ItemStudy of innate immune responses to neurotropic virus infection( 2021)Viral encephalomyelitis is one of the leading causes of morbidity and mortality associated with viral infections. The alphaviruses are an important group of viruses that cause disease in humans and animals. Experimental studies in rodents using Semliki Forest virus (SFV), a prototypical alphavirus, have shown that the outcome of brain infection is age dependent. For this infection and several others, maturation of the central nervous system is associated with less severe encephalitis. However, the age-dependent susceptibility mechanisms to virus encephalitis are still unclear. In this thesis, I examined the age-dependent susceptibility to SFV infection in immature and mature primary cortical neurons and the role of type I IFN in SFV infected neurons. Both immature and mature neurons could be infected with SFV, but infection was greater in immature neurons than in mature neurons. Relative to immature neurons, differentiated mature neurons showed reduced virus replication and virus production. Notably, in response to the SFV infection, mature neurons produced IFN-beta whereas immature neurons failed to produce IFN-beta. I then explored whether IFN-beta could protect primary neuronal cultures against SFV infection. Immature neurons responded to IFN-beta pre-treatment however, pre-treatment with IFN-beta in immature neurons sets up only a transient antiviral state which is lost as the infection progresses. In conclusion, mature neurons produce IFN-beta in response to SFV infection whereas, immature neurons do not. However, immature neurons can respond to IFN-beta transiently curtailing virus replication. I further investigated the role of type I IFN in SFV infected neurons, and the results demonstrated that in its absence, mature neurons are more susceptible to SFV infection. Immature neurons from both WT and IFNAR1-/- mice exhibited the same characteristics in terms of infection rates and viral loads. These findings show that the susceptibility of immature neurons to infection is independent of the type I IFN response. In another approach to investigate the mechanisms, including the difference in IFN responses, underlying the age-dependent differential susceptibility of neurons to SFV infection, we undertook a transcriptomic approach. The transcriptomic profile of immature neurons is very different from mature neurons. The differential gene expression analysis and subsequent pathway activation and upstream regulatory analysis confirmed that mature neurons are equipped to curtail infection and immature neurons lack the key innate immune mechanisms to respond to infection. Additionally, this thesis also investigated the capacity of lyssaviruses P protein to inhibit interferon induction. There is a diversity among different lyssaviruses in their ability to inhibit IFN induction. I characterised an important P protein sequence that strongly inhibits RIG-I signalling and IFN induction. Together, these results highlight the importance of type I IFN in neurotropic virus infection. The findings presented in this thesis have shown that neuronal maturation and innate immunity are linked. Collectively, maturation of neurons is associated with increased levels of components of the innate immune system, contributing to the development of a functioning innate immune system that has the ability to restrict SFV replication in mature neurons.