Microbiology & Immunology - Theses

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    Developing an image analysis toolbox to uncover dynamic immune cell interactions and behaviour
    Schienstock, Dominik ( 2023-09)
    Cell interactions are important for effective immune responses. Mapping these interactions in time and space is technically challenging and can be labour intensive. Here I present a multifunctional toolbox, termed Cecelia (Cell-cell interaction analyser), that allowed me to dissect spatiotemporal immune responses. I use this tool to dissect interactions between CD8+ T cells and dendritic cells (DCs) in the context of HSV and LCMV infection. I implemented the toolbox as a combination of R/shiny and python/napari that enables researchers to readily identify cell populations, behaviour and interactions in 2D or 3D static images and 3D timelapse images. To investigate the role of XCR1+ DCs during early T cell priming, I utilised the XCR1-DTR system to specifically deplete this DC subtype and applied a combination of flow cytometry as well as confocal and intravital two-photon microscopy during HSV and LCMV infection. I primarily utilise the generalist deep learning package Cellpose for cell segmentation and the HDF5-based file format Anndata to store and further process object information with, among others, Hidden Markov Models, Leiden clustering, flow cytometry gating and neighbour detection. XCR1+ DCs have been shown in several infection and tumour models to be instrumental for T cell priming. Using Cecelia to apply deep learning and advanced statistical approaches, I show that T cell priming is reduced in the absence of XCR1+ DCs, yet T cells were able to gather activation signals from other cells that resulted in defined areas of activation within the draining lymph node in the context of HSV infection. I further validated the broad utility of Cecelia for image analysis by identifying cell populations, interactions and stromal compartments from commonly used multiplex imaging techniques such as spectral unmixing, IBEX and imaging mass cytometry. I integrated the combination of different segmentation workflows to capture a variety of cell shapes, sizes and fluorescent signals within various imaging modalities. Cecelia is an open-source package that simplifies image analysis workflows that currently require complex or expensive combinations of software packages.
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    Understanding key correlates of severe respiratory virus infections during pregnancy
    Habel, Jennifer Rose ( 2024-01)
    Respiratory infections such as those caused by SARS-CoV-2 and influenza viruses are of major concern for human health. While most otherwise-healthy individuals do not succumb to infection, there are groups at increased risk of disease severity, including pregnant women. The mechanisms for severe respiratory infection during pregnancy are currently not well understood but could be related to the altered state of the immune system in pregnancy. To investigate this, 217 immunological parameters were assessed to characterise immune cell activation, SARS-CoV-2-specific antibodies, and inflammation in pregnant and non-pregnant women with COVID-19. Generation of SARS-CoV-2-specific antibodies was similar in pregnant and non-pregnant women, however systems serology identified distinct antibody and Fc-gamma-R-binding profiles between pregnant and non-pregnant women. Analysis of immune cell activation ex vivo revealed differences in NK cell and gamma/delta T cell activation dynamics in pregnant women. Healthy pregnant women displayed pre-activated NK cells and gamma/delta T cells, based on expression of HLA-DR and CD38, when compared to healthy non-pregnant women, and interestingly during COVID-19, remained phenotypically unchanged. Alternatively, non-pregnant women had typical NK and gamma/delta T cell activation profiles. Conventional CD4+ and CD8+ T cell and T follicular helper cell activation were similar between SARS-CoV-2-infected pregnant and non-pregnant women. Healthy pregnant women had higher levels of IL-8, IL-10 and IL-18, which remained elevated during acute and convalescent COVID-19. Key findings relating to altered NK cell and gamma/delta T cell activation in normal healthy pregnancy warranted further investigation into perturbations of innate immunity during pregnancy to determine whether they are a correlate of protection or disease severity. To identify mechanisms driving increased NK cell activation during pregnancy, >350 proteins were screened on NK cells from healthy pregnant and non-pregnant women. Differentially expressed proteins in NK cells during pregnancy related to cell activation, adhesion, and lipid metabolism. To determine whether these differences were also reflected at the transcriptional level, single-cell RNA sequencing was performed. Gene ontology analysis of 361 differentially expressed genes in NK cells from pregnant and non-pregnant women revealed an upregulation of genes involved in cell activation during pregnancy. Having identified pathways contributing to perturbed NK cell activation during pregnancy, functional assays will demonstrate their involvement in anti-viral immunity. CD8+ T cells play an important role during viral infections, and while total CD8+ T cell activation was similar between pregnant and non-pregnant women with COVID-19, it is unknown whether differences occur at the epitope-specific level. Given that SARS-CoV-2 emerged in 2020, little was known about the CD8+ T cell responses in COVID-19. Therefore, to first understand the CD8+ T cell response to SARS-CoV-2 and identify CD8+ T cell epitopes restricted by the HLA-A*02:01, PBMCs from HLA-A*02:01+ (A2) SARS-CoV-2 convalescent individuals were stimulated with SARS-CoV-2 peptides predicted to bind to HLA-A*02:01. This identified that the Spike269-277 (S269) peptide stimulated CD8+ T cell activation. The use of tetramers consisting of A2/S269 peptide-HLA conjugates allowed detection of A2/S269-specific CD8+ T cells in pre-pandemic and COVID-19 convalescent PBMC samples directly ex vivo. This demonstrated that pre-pandemic A2/S269-specific CD8+ T cells were low in frequency and had a naive phenotype, while COVID-19 convalescents had increased frequencies and a central memory-like phenotype, indicating antigen experience. Additionally, compared to other HLA-A*02:01-restrictied epitopes from influenza A virus (IAV) and Epstein-Barr virus (EBV), A2/S269-specific CD8+ T cell frequencies in convalescent individuals were low in frequency, likely reflecting the primary/single exposure to SARS-CoV-2. Having identified a SARS-CoV-2 CD8+ T cell epitope, and using known epitopes derived from IAV, respiratory syncytial virus, cytomegalovirus and EBV, antigen-specific CD8+ T cell immunity was assessed in HLA-A*02:01+ pregnant and non-pregnant women. This identified novel transcriptional and phenotypic profiles within CD8+ T cell populations in the peripheral blood and placenta in full-term pregnancies. Analysis of acute respiratory and chronic virus-specific CD8+ T cells showed that pregnancy can alter T cell states in an antigen-specific manner. Overall, the culmination of the studies presented in this thesis identified that pregnancy causes heightened NK cell and gamma/delta T cell activation in the normal healthy state, which may have negative consequences during respiratory virus infections. Furthermore, in-depth analysis of NK cell and CD8+ T cell immunity revealed unique phenotypic and transcriptional states during pregnancy which provides insight on potential immunotherapy targets for preventing severe respiratory infections during pregnancy.
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    Characterising the molecular drivers of group A Streptococcal evolution
    Jespersen, Magnus Ganer ( 2023-10)
    Streptococcus pyogenes is a human bacterial pathogen cable of causing multiple diseases, from self-limiting infections to lethal sepsis, and autoimmune complications. Serological and molecular studies show evidence of S. pyogenes exhibiting high epidemiological diversity. With whole genome sequencing approaches, S. pyogenes is now recognised as comprising hundreds of co-circulating and co-evolving genetic lineages, causing more than 500,000 deaths each year and over 700 million self- limiting infections worldwide. Genomic studies have largely focussed on clinically dominant S. pyogenes lineages from high-income settings. These studies elucidated evolutionary histories and reveal the role of mobile genetic elements and other horizontal genetic transfer types. Few efforts have examined S. pyogenes in global and diverse frameworks, where species wide molecular traits, evolutionary, and disease processes can be aligned and examined. To address questions of this thesis, new bioinformatic tools, Corekburra and Magphi, were devel- oped. These automate parts of genomic analyses and allow new ways of exploring pangenomes based on core gene synteny. With these, S. pyogenes has been interrogated with respect to chro- mosomal inversions, pangenome gene essentiality, and accessory gene content not related to mobile genetic elements. Examining 249 reference genomes, mechanisms causing chromosomal inversions were examined. Around half of the genomes carried evidence of inversions, with 23 unique inversion breakpoints identified. Prophages and insertion sequences were found to be associated with rare inversion breakpoints. However, an overrepresentation of insertion sequence subgroups, IS1239 and IS1548, were associated with inversions. A common cause of inversions was related to regions conserved in the S. pyogenes genome. These regions were quasi-symmetrically placed around the origin of replication and are a unique feature of the S. pyogenes genome, compared to five other well studied Streptococcal species. Markers of S. pyogenes adaptation and gene essentiality were determined by systematically re- analysing publicly available transposon mutant library studies within a species-wide pangenome framework. Gene essentiality for 3 S. pyogenes emm lineages and 9 conditions was catalogued. As predicted, core genes were found to be of greater essentiality, compared to accessory genes. However, lineage adaptation was observed with an accessory gene being highly essential across all lineages and conditions. Genes associated with infection were highlighted by calculating in vitro and in vivo essentiality scores, exposing 245 genes with greater essentiality under in vivo conditions. Examining accessory genes in a pangenome from 2083 diverse and globally sampled genomes identi- fied dynamics and genotypes underlying the S. pyogenes population structure. Across well-sampled regions of the world, prevalence of accessory genes was found to be highly correlated, contrasting the genomic lineage composition. In addition, nine accessory genes, not related to mobile genetic elements, were identified as statistically associated, separating the S. pyogenes population by geno- type. The genes are speculated to be linked with epidemiological observations of emm pattern, a surrogate for tissue tropism. This thesis provides insights into genomic differences in S. pyogenes, guiding future research. It highlights conserved mechanisms for chromosomal inversions potentially linked to phenotype switches, sheds light on lineage adaptations, and suggests a connection between genotype and tis- sue tropism. These findings can provide insights into the role of specific genes and loci shaping S. pyogenes behaviour.
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    Evaluation of novel strategies targeting the HBeAg on their ability to generate therapeutic anti-HBV responses
    Droungas, Yianni ( 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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    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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