Microbiology & Immunology - Theses

Permanent URI for this collection

Search Results

Now showing 1 - 10 of 23
  • Item
    Thumbnail Image
    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.
  • Item
    Thumbnail Image
    Functional antibody immunity in SARS-CoV-2 infection and vaccination
    Haycroft, Ebene Regina ( 2023-01)
    Since the emergence of SARS-CoV-2 in late-2019, the virus has caused substantial mortality in humans, with the confirmed death-toll exceeding 6.5 million lives and causing over-600 million confirmed cases. The introduction of COVID-19 vaccines has been instrumental in reducing the risk of hospitalisation and death globally. However, with the rise of novel variants, SARS-CoV-2 continues to cause high levels of infections, and thus the impact of the COVID-19 pandemic remains far from over. Antibodies play crucial roles in the protective immunological response to SARS-CoV-2. As such, this MPhil thesis explores the antibody response to SARS-CoV-2 in the context of both infection and vaccination using high-throughput multiplexing technologies. We began by investigating the systemic (plasma) and mucosal (saliva) antibody responses during acute (first-fourteen days) infection in adults and children, shown in Chapter 3 of this thesis. We used a cohort of individuals recruited from household clusters of COVID-19 infections during the first waves of ancestral-strain SARS-CoV-2 in Melbourne, Australia. Using systems serology analysis, we found that households with higher rates of secondary transmission to household contacts display greater levels of virus in the nasopharynx; and correspondingly display enhanced systemic antibody responses, suggesting a relationship between viral load and seroconversion. Additionally, we observed differential antibody response induced during acute infection between adults and children. Levels of IgA antibodies to SARS-CoV-2 spike (S)- and nucleocapsid-proteins were substantially lower, notably in the saliva, of children compared to adults. We speculate this in part may reflect that adults have greater prior exposure to endemic human coronaviruses. Given little information is known regarding the mucosal response in children, future studies are needed to profile the mucosal response more extensively in this population. The emergence of variants of SARS-CoV-2 are a challenge to public health. Many variants harbor mutations in the RBD of the SARS-CoV-2 S, a dominant target for neutralising and non-neutralising antibodies. In Chapter 4, we comprehensively characterised the effect of RBD mutations, including mutations present in the Omicron (BA.2) variant, on ACE2-binding affinity, as well as the functional antibody response induced in BNT162b2-vaccinated recipients and mild-convalescent SARS-CoV-2 infected individuals. The RBD of variants of concern (VOCs) displayed greater affinity to ACE2 than ancestral SARS-CoV-2 RBD. Furthermore, using a surrogate neutralisation assay that examined 39-naturally occurring RBD mutations, we showed reduced capacity of plasma antibodies to block ACE2-binding to VOCs. Additionally, we show a reduced capacity by RBD-binding plasma antibodies to engage Fc-receptors (FcRs), suggesting the potential for RBD-binding antibodies to recruit Fc effector functions are compromised by mutations in the RBD. These findings highlight the capacity of viral variants with RBD mutations to subvert host antibody responses. Lastly, with the observation that S-binding antibodies are attenuated by mutations found in variants, it is thought enhancing the magnitude of the antibody response may be useful in offering protection against SARS-CoV-2. As such, in Chapter 5 we investigated the effect of administration of a novel priming vaccine strategy designed to augment antibody responses – Bacillus Calmette-Guerin (BCG) immunisation – prior to immunisation with two homologous doses of either COVID-19 vaccine BNT162b2 or ChadOx1 on the antibody responses to the SARS-CoV-2 S. No differences in the SARS-CoV-2 antibody levels between BCG- and non-BCG-vaccinated individuals was observed, suggesting this strategy may not substantially improve the efficacy of current COVID-19 vaccines. Further studies are needed to determine whether this reflects the time window (>1 year) between BCG-vaccination and COVID-19 vaccines, or whether BCG- priming is not a suitable option for augmenting COVID-19 antibody responses. In summary, our work provides greater insights into the antibody response to SARS-CoV-2, of which can be further explored in prospective work.
  • Item
    Thumbnail Image
    Interleukin-1 Is Unique in Its Ability to Modulate PD-L1 and PD-L2 Expression by Mo-DCs
    Gourley, Katherine Heather Aarons ( 2022)
    Expression of PD-1 ligands PD-L1 and PD-L2 on the surface of tumour and immune cells has led to the widespread success of checkpoint blockade immunotherapy, yet despite decades of research, knowledge of the underlying mechanisms tumour cells implement to avoid recognition by the immune system is still evolving. Research from our laboratory has validated that human Mo-DCs can increase surface expression of PD-L1 and PD-L2 in the presence of inflammatory stimuli. PD-L1 on APCs has been implicated in the conversion of conventional T cells into Tregs, however the role that PD-L2 may play in this system has not been explored. Furthermore, the mechanism by which tumours can elicit expression of PD-1 ligands on the surface of APCs, and the impact that this may have on infiltrating T cell phenotype and function is incompletely characterised. In this study, human Mo-DCs were generated and assessed for their ability to simultaneously upregulate PD-L1 and PD-L2 in response to stimulation with proinflammatory cytokines. It was discovered that IL-1 could elicit upregulation of both PD-1 ligands more effectively than TNF, and IFN-gamma could induce low levels of PD-L1 but was unable to modulate PD-L2 expression. Other members of the IL-1 superfamily did not have the same ability as IL-1, and it appeared that the cellular response was limited to Mo-DCs as lymphocytes and macrophages did not respond similarly. While attempting to reproduce these results in a more biologically relevant system, it was discovered that A375 melanoma cells were able to lose their ability to modulate PD-L1 and PD-L2 expression, however modification of the culture conditions to mimic features of the tumour microenvironment partially restored this function. Further analysis of the supernatants of tumour cell-lines resulted in the identification of an inhibitory factor which antagonised the IL-1beta-mediated PD-L1 and PD-L2 upregulation by Mo-DCs, and the efficacy of this factor could be modulated by culture conditions. Finally, CD4 T cells cultured with cytokine-stimulated Mo-DCs expressing PD-L1 and PD-L2 showed increased proliferation and expression of FOXP3, however it was not possible to determine whether differentiation into functional Tregs had occurred. Overall, this study demonstrated that pro-inflammatory cytokines such as IL-1 can have dual functions that contribute to immunoregulation on specific cell types. Additionally, tumour cells were shown to have the capacity to produce factors which can positively or negatively modulate the immune response, and the secretion of these factors can be impacted by extracellular conditions. We were also able to demonstrate that co-culture of cytokine stimulated Mo-DCs with CD4 T cells promoted proliferation and expression of regulatory transcription factor FOXP3 by some T cells, suggesting that differentiation and function of these cells could be modulated by Mo-DCs. These findings have helped improve understanding of the mechanisms by which tumour cells resist the immune response or immunotherapy, and further identification of upstream modulators of PD-L1 and PD-L2 expression within the TME has the potential to uncover novel immunoregulatory factors which when targeted may provide a therapeutic advantage.
  • Item
    Thumbnail Image
    Inactivation Mechanisms of Therapeutic CD8+ T Cells against a non-Hodgkin B-cell Lymphoma Mouse Model
    Dou, Zixuan ( 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.
  • Item
    Thumbnail Image
    Characterising novel cytotoxic T lymphocyte dysfunction by spatiotemporal analysis, and optimizing adoptive T cell therapy against cancer while preventing autoimmune side effect.
    Nakamura, Toshihiro ( 2022)
    Adoptive cell therapy (ACT) is an emerging strategy for cancer treatment. However, ACT is not always successful because of various factors. In ACT using cytotoxic T lymphocytes (CTLs), T cell dysfunction is one of the most significant causes of failure. Our laboratory has previously discovered a novel type of T cell dysfunction, which we named “stunning”. We used a mouse model of ACT in the context of B-cell lymphoma. Here, mice were injected with Eu-myc lymphoma cells expressing ovalbumin (OVA) followed by injection of OVA-specific CTLs (OT-I CTL). In this model, we found that the presence of a large tumour burden led to rapid deletion and inactivation of OT-I CTLs in an antigen-specific manner. We hypothesised that at early stage after ACT, one CTL encounters a large number of antigen-presenting tumour cells within a short period of time, and this multiple interaction leads to stunning. Therefore, we aimed to investigate (1) How tumour cells and CTLs localise in organs, (2) Exact timing when stunning occurs, (3) How frequently CTLs interact with tumour cells, and (4) How CTLs behave after encountering tumour. In order to obtain these questions, we utilise both static (confocal) and dynamic (intravital) imaging techniques in this research. Using confocal fluorescence imaging, we found that in the spleen and lymph nodes, the proportion of antigen-specific CTLs that localised in T cell zones was decreased under a higher tumour burden. We next investigated the motility of CTLs in the lymph nodes by intravital imaging and found that motility was decreased in the context of high tumour burden. Accordingly, we propose the like between spatiotemporal characteristics of T cells and their dysfunction, which might be a potential therapeutic target. In addition to T cell dysfunction, another shortcoming of ACT is the potential for autoimmunity. If adoptively transferred CTL has specificity to an antigen that is shared with self cells, self-tissues are also susceptible to the attack by CTLs. Several clinical trials of ACT resulted in severe self-tissue destruction. Thus, balancing anti-cancer CTL responses with prevention of autoimmunity is paramount to the success of ACT. Typically, tumour-associated antigens are highly expressed in tumour cells compared to normal untransformed cells. Therefore, we hypothesised T cells with a lower affinity for antigen may be better candidates for use in ACT expecting that the T cell would only recognise the antigens on the tumour but not the ones on healthy tissues. We utilised OT-3 CTLs which express a lower affinity TCR than that of the OT-I cells. After optimising the concentration of IL-2 and cellular density for in vitro T cell priming, we found that OT-3 CTLs were still able to kill targets as effectively as OT-I CTLs. This provides evidence that low-affinity T cells may act as candidates for ACT.
  • Item
    Thumbnail Image
    Discovery of the cells that express the antigen-presenting molecule MR1 in vivo
    Yan, Yuting ( 2021)
    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.
  • Item
  • Item
    Thumbnail Image
    Investigation of rare actinomycetes for novel antimicrobials
    Byrne, Janet ( 2020)
    Nocardia are a genus of ubiquitous environmental bacteria belonging to the phylum Actinobacteria. Genomics has revealed that Nocardia species are endowed with extensive and varied arrays of secondary metabolite biosynthetic gene clusters with the potential to produce natural products that have antibiotic properties. Furthermore, the abundance of such gene clusters within the Nocardia rivals that of Streptomyces, the signature genus among the Actinobacteria, owed to the fact that Streptomyces species have yielded many clinically used antibiotics. This project aimed to address the current antibiotic resistance crisis and the shortfall in new compounds within the drug discovery pipeline. A range of natural product discovery techniques were utilised amongst different Actinobacteria with a particular focus on a collection of species within the generally overlooked genus Nocardia. This study had three primary objectives, the first was to use a traditional, high-throughput, empirical screen of 169 pathogenic actinomycetes predominantly from the genus Nocardia. These isolates were screened for antibiotic activity on 19 distinct growth media against a panel of five highly prevalent, multidrug resistant pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium and Acinetobacter baumannii). Secondly, whole genome sequencing and bioinformatic interrogation of 100 Nocardia species was conducted to assess their genetic potential to biosynthesise natural products. This facilitated the selection of a single Nocardia isolate which possessed a non-ribosomal peptide synthetase locus that appeared to be unique amongst other Nocardia species. The locus was also transcriptionally silent. Bioengineering using promoter refactoring was employed to activate expression of this gene cluster, the product of which might have potential as a novel antimicrobial. Thirdly, by utilisation of liquid chromatography-mass spectrometry (LC-MS), bioinformatics and molecular networking, a metabolomic approach was employed to gain a global secondary metabolic footprint of ten predicted “biosynthetically talented" Nocardia species grown on five distinct media types. This project identified: (i) A Nocardia sp. with activity against multidrug resistant Acinetobacter baumannii. (ii) Two Streptomyces isolates (Streptomyces cacaoi and Streptomyces sp.) which exhibited antimicrobial activity against multidrug resistant Escherichia coli and Acinetobacter baumannii respectively. Secondary metabolite extracts from each of these producing isolates were investigated by LC-MS/MS and the resulting spectra was assessed for uniqueness through a dereplication data platform developed specifically for bacterial natural product identification. No hits for previously discovered metabolites were obtained suggesting that the antimicrobials discovered within this project appear to be unique and have potential as new drug leads for today’s ever-decreasing antibiotic discovery pipeline. (iii) Four distinct families of bioactive secondary metabolites that were produced by multiple Nocardia species following LC-MS/MS and molecular network analysis. The identified secondary metabolites were correlated with genome sequence data to identify their probable biosynthetic origin in Nocardia species.
  • Item
    Thumbnail Image
    The dynamics of the B cell response during influenza A virus infection
    Lee, Hoi Yee ( 2020)
    Although vaccination remains the most effective method of managing influenza epidemics, there is still much that remains to be characterized about humoral immunity against the varying contexts in which influenza infection can occur. With the continuous subversion of humoral immunity by seasonal influenza through antigenic drift and the potential of zoonotic influenza viruses adapting and spreading through human populations through antigenic shift, improving our understanding of B cell immunity against different types of influenza infection could provide important insights into improving management of epidemics and vaccine formulations. In order to understand B cell responses during influenza infection, the well-characterized C57BL/6 mouse model was used to investigate and compare humoral responses in the context of different influenza infection histories. Markers that identified specific B cell subsets such as germinal centre (GC) B cells and plasmablasts were analysed by flow cytometry paired with influenza virus-specific B cell ELISPOT assays to investigate strain-specific antibody secreting responses within the same experiment. As the surface glycoprotein HA is thought to be the immunodominant response for B cell responses against influenza virus, the prediction is that the greater the antigenic differences between the HA of the first and second infecting strains, the more primary-like the response to the second strain would be. Primary and homologous secondary B cell responses in the mediastinal lymph node (MLN) and spleen were first characterized using this model to establish baseline responses against influenza virus before heterosubtypic infection was studied through infection of mice with H1N1A/Puerto Rico/8/34 (PR8) virus followed by H3N2 A/Udorn/305/72 (Udorn) virus 7 weeks later. Unexpectedly, a secondary-like plasmablast, GC B cell and Udorn-specific antibody secreting cells was observed during heterosubtypic infection, with earlier and higher magnitude B cell responses. These findings suggested a possible role for cross-subtype T cell memory in modulating B cell responses. The effect of antigenic drift on the B cell responses during influenza infection was then analysed with the same model. Mice were infected with H3N2 strains isolated between 1972 and 1979, representing different antigenic distance from a virus isolated in 1982 (Ph82). Seven weeks post infection mice were reinfected with Ph82 and the B cell response over the course of infection examined. It was found that infection of strains up to 10 years apart appeared to induce a secondary-like B cell response in the secondary lymphoid organs when compared to baseline primary and secondary responses against Ph82 virus. Prior infection with any H3N2 strain also resulted in minimal viral replication during the secondary challenge when compared to primary infection groups. However, data from both primary antibody inhibition and HA-specific B cell responses appears to suggest a narrower threshold of recognition, around a maximum of 3 years drift before serum and HA-specific responses cease to bind with other strains. Taken together, secondary-like B cell responses in both heterosubtypic and drift models of infection and in the case of drift responses, irrespective of reactivity of HA-specific B cells, appear to refute the hypothesis that virus-specific B cell responses would reflect antigenic relatedness between the HA of the infecting strains. Overall, data from this study identifies the diversity of the overall B cell response against influenza infection in the context of prior exposure to strains of different antigenic properties. Further study into the reactivity of these B cells against different influenza virus components and the role of memory T cells in the observed responses may provide important insights into the nature of host immunity against the ever-shifting target of influenza virus.
  • Item
    Thumbnail Image
    Establishing two systems to study MAIT cell responses during bacterial infection
    Zhu, Tianyuan ( 2019)
    Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells, which are abundant in humans and also present in many mammals, including mice [1]. MAIT cells express semi-invariant T cell receptors (TCR) are activated by recognizing microbial vitamin B2 (riboflavin) metabolites presented on non-classical major histocompatibility complex (MHC) class I related protein 1 (MR1) [1-6]. Upon activation, MAIT cells can either directly kill infected cells or secrete IFN, IL-17, TNF and other functional molecules that enhance the anti-microbial activity of other immune cells [7, 8]. These characteristics suggest an important role played by MAIT cells in anti-bacterial immunity in mammals. Bacterial infections are major problems in clinical settings. While MAIT cells have shown antibacterial properties, clinically relevant in vivo animal models have not been extensively developed for further exploration of the mechanisms of MAIT cell protection. Using three clinically common bacteria; Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli, this study established bacterial peritonitis models in mice for MAIT cell research. The three bacteria were all able to produce antigens that stimulate MAIT cells in vitro. For mouse infection models, the optimal dose of K. pneumoniae for inducing productive but nonlethal infection was not established in this study. S. aureus and E. coli peritonitis incurred mild and robust MAIT cell responses in vivo, respectively. Further experiments with E. coli peritonitis showed that MAIT cells accumulated in the intraperitoneal cavity, the spleen and liver, which contributed to bacteria control. The finding suggests possible MAIT cell-based treatments in clinical conditions. While in vitro studies have shown that MAIT cells can be stimulated by various types of cells upon infection, there have been few investigations into how MAIT cells are activated in vivo. In the second project presented in this thesis, the contributions of antigen presenting cells (APCs) in initiating activation in MAIT cells was studied with Francisella tularensis infection. We found that APCs were burdened with bacteria in the early phase of infection. With an ex vivo cellular co-culture assay was specifically optimized for assessing MAIT cell activation induced by the F. tularensis burdened APCs, we found that alveolar macrophages contributed to the best activation of MAIT cells in an MR1 dependent manner. Other cell subsets: monocytes, neutrophils and dendritic cells were also capable of inducing a mild activation. In summary, the present study helped optimize two novel systems for MAIT cell research. With the ex vivo cellular co-culture system, this thesis contributes to understanding how APCs activate MAIT cells in vivo upon bacterial infection. With the in vivo peritonitis models, this study introduces clinically relevant bacteria to the current MAIT cell research in mice, and ultimately to inform and complement clinical research. It is hoped that the insights gained from this study could be of assistance to current research methods and to achieve a better understanding of MAIT cell responses during bacterial infection.