Microbiology & Immunology - Theses
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ItemFc-effector functions and plasma IgA in viral pandemics (HIV-1 and SARS-CoV-2)( 2023)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - the causative agent of the COVID-19 pandemic- has caused over 6 million deaths globally since late 2019. Mutations in the receptor binding domain (RBD) of SARS-CoV-2 have resulted in the emergence of variants of concern (VOC). Following infection and/or vaccination SARS-CoV-2-specific antibodies are generated with neutralising and/or Fc functional properties. Neutralising antibodies can protect from subsequent infections. While Fc-effector functions, including antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent cellular cytotoxicity (ADCC) are important for control and resolution of many infectious diseases including SARS-CoV-2. Notably, the neutralising antibody response wanes rapidly following infection with SARS-CoV-2, however, the durability of antibody mediated Fc-effector functions including ADCP remains largely unknown. To investigate the SARS-CoV-2 Fc functional antibody response, we developed two in vitro cell based assays to assess SARS-CoV-2 specific phagocytosis and cell association (trogocytosis) over time in mild-moderate convalescent COVID-19 individuals (Chapter 2). Interestingly, we observed evidence of SARS-CoV-2 specific trogocytosis occurring during the cell association assay using confocal microscopy. We demonstrate that the SARS-CoV-2 Fc functional antibody response, specifically ADCP and cell association, were more durable than the neutralizing antibody response. All COVID-19 individuals retained detectable antibody mediated phagocytosis and cell association at ~4 months post symptom onset, while 30% of the cohort lost detectable neutralization. Therefore, highlighting the potential importance of Fc-effector functions in long-term immunity from SARS-CoV-2 reinfection. The importance of IgG antibodies for protection and control of SARS-CoV-2 has been extensively reported. However, other antibody isotypes including IgA have been poorly characterized. We aimed to examine the functional contributions of plasma IgA to neutralisation and Fc-effector functions following SARS-CoV-2 infection (Chapter 3). Using a multiplex surrogate neutralisation assay, we assessed the neutralising capacity of IgA and IgG depleted plasma and purified antibody fractions against ancestral SARS-CoV-2 Spike receptor binding domain (RBD) and RBDs with common single amino acid mutations. Notably, more than 60% of the cohort showed significantly reduced neutralising capacity following IgA depletion (p = 0.0001). Furthermore, 30% of the cohort induced stronger IgA-mediated neutralization than IgG when purified antibody fractions were tested at equivalent concentrations. Moreover, convalescent purified IgA and IgG recognized similar RBD mutations and showed comparable neutralisation of RBD mutants. Depletion of IgG significantly reduced Fc-effector functions (ADCP and cell association) of convalescent plasma, in contrast no change was observed with depletion of IgA. We demonstrate that plasma IgA has the capacity to neutralize ancestral SARS-CoV-2 RBD, however, IgA contributes minimally to SARS-CoV-2 plasma Fc-effector function. Overall, neutralizing IgA and duel functional IgG contributes to the COVID-19 antibody response after infection. A constellation of RBD mutations have resulted in enhanced transmission and/or immune escape of SARS-CoV-2 circulating strains, giving rise to new variants of concern (VOCs). Mutations within the RBD can reduce antibody recognition, leading to reduced neutralising potency and potentially altering vaccine efficacy. However, the impact of RBD mutations on Fc-effector functions following vaccination remains unknown. We examined the capacity for SARS-CoV-2 Pfizer (BNT162b2) vaccine (2 weeks post second dose) and infection induced antibodies to mediate Fc-effector functions against SARS-CoV-2 VOCs (Chapter 4). We measured IgG binding to RBDs and engagement of RBD specific antibodies with Fc gamma receptors (FcyRs) via multiplex for 6 historical VOCs (Alpha, Beta, Gamma, Delta, Kappa and Omicron BA.2). Notably, FcyRIIa and FcyRIIIa engagement was significantly reduced for the VOCs Beta, Gamma, and Omicron BA.2. Furthermore, we confirmed that reduced FcyR engagement to RBD mutants resulted in reduced cellular Fc-effector functions, via a novel competitive SARS-CoV-2 duplex ADCP assay. This novel SARS-CoV-2 ADCP duplex assay enables assessment of the functional capacity of the same pool of antibodies to two different SARS-CoV-2 variants in a competitive high throughput assay. Taken together, we successfully optimised a novel SARS-CoV-2 ADCP assay and show that mutations within the SARS-CoV-2 RBD may have consequences on the Fc functional capacity of vaccine induced antibodies. The human immunodeficiency virus (HIV-1) is the causative agent of the acquired immune deficiency syndrome (AIDS) epidemic which has resulted in an estimated 40.1 million deaths globally since the early 1980s. Antibodies including IgG and IgA can recognise HIV-1 to elicit antiviral functions such as neutralisation and Fc effector functions. Plasma IgA can engage with the Fc alpha receptor (FcaR) to activate Fc-effector functions including phagocytosis. However, IgA can also mediate inhibition of Fc effector functions via FcaR and potentially interfere with protective antibody functions during viral infections. Notably, elevated IgA levels were associated with reduced vaccine efficacy and inhibited ADCC in the RV144 HIV-1 human vaccine trial. We investigated the Fc functional contributions of plasma IgA to HIV-1 phagocytosis during early and chronic HIV-1 infection (Chapter 5). We depleted IgA from plasma at two early and one chronic HIV-1 timepoint and assessed the IgA functional contribution. Notably, depletion of IgA at early timepoints resulted in significantly reduced ADNP (p < 0.05), suggesting IgA contributes to HIV-1 phagocytosis during early infection. However, depletion of IgA during chronic HIV-1 plasma enhanced ADNP, suggesting the IgA at this timepoint interferes with Fc-effector functions. Furthermore, we also observed IgA mediated inhibition of ADCP for various HIV-1 mAbs with purified IgA from people living with or without HIV-1, although the magnitude of inhibition is heterogenous amongst mAbs. We suggest this inhibitory effect is at least partially mediated via the FcaRI. However, this is a preliminary study and future studies are essential to investigate this phenomenon in greater depth with a larger cohort. Overall, our studies highlight the importance of Fc-effector functions and the complexity of the functional IgA response during SARS-CoV-2 and HIV-1 infections. Plasma IgA can induce potent neutralisation and contribute to ADNP during acute/ early infections. However, plasma IgA may interfere with Fc-effector functions during chronic HIV-1. Future studies should investigate the effect of IgA on other Fc-effector functions such as trogocytosis, complement activation and ADCC in different acute and chronic viral infections.
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ItemGenerating an effective T cell-based influenza vaccine( 2023)Protective immunity against influenza virus is heavily dependent on humoral and cellular immune responses. Current influenza vaccines predominantly utilise antibody immunity, but as this is strain-specific, it leaves the population vulnerable to antigenic drift by influenza virus and importantly fails to protect against novel pandemic strains. CD8+ T cell immunity, on the other hand, due to its ability to recognise highly conserved antigenic determinants of influenza virus, enables the possibility of broadly protective universal immunity. In particular, CD8+ T cells that reside within the lung called tissue-resident memory T cells (TRM) are the responsible subset mediating cross-protection against influenza virus. However, current vaccines either do not or poorly generate CD8+ T cell responses. Therefore, this PhD thesis investigated the capacity of a novel T cell-based vaccine candidate to elicit lung CD8+ TRM and critical parameters required for the optimal induction of cross-protective influenza-specific lung CD8+ TRM. We investigated the cellular immune response evoked following a single-cycle replication-incompetent influenza vaccine candidate called S-FLU. Intranasal S-FLU immunisation generated lung CD8+ T cells and CD8+ TRM of reduced magnitude and functional avidity relative to natural influenza virus infection controls. Interestingly, the limited inflammatory profile of S-FLU immunisation conferred a clonally diverse CD8+ T cell and TRM profile in the lung. As a result, a greater propensity of these cells cross-reacted against a naturally occurring variant and prevented the development of T cell escape mutants. Our findings suggest the inflammatory milieu of a vaccine is an important consideration as this may influence the T cell receptor repertoire, resulting in downstream alterations in the cross-reactivity and capacity to subvert viral variants. Vaccine studies investigating protective efficacy must take into consideration pre- existing influenza-specific immunity generated by prior infections and the annual vaccination regime that occur over the course of an individual’s lifetime. Using a panel of live attenuated influenza virus vaccine candidates (cold-adapted and single-cycle), we next investigated the capacity of live attenuated influenza vaccines to elicit lung CD8+ TRM responses in the face of pre-existing immunity against the vaccine backbone. We determined that pre-existing antibodies specific for the vaccine backbone inhibited CD8+ T cell priming and therefore memory CD8+ T cell development and lung CD8+ TRM populations. Importantly, high dose vaccination could mitigate the impairment in CD8+ T cell priming, for which the resultant lung CD8+ TRM were protective against heterologous influenza virus challenge. Influenza infection can result in a transient depot of antigen long after viral clearance that influences influenza-specific CD8+ T cell responses, but it is unclear how this antigenic stimulation impacts the local cognate antigen-requisite lung CD8+ TRM compartment. Our studies suggest that residual antigen persistence is likely applicable to only certain epitopes. Furthermore, persistence of residual antigen activated naive CD8+ T cells that then formed CD8+ TRM populations in the lung, however these cells exhibited reduced polyfunctionality and longevity. Our results thus imply that lung CD8+ TRM generated from residual antigen following influenza viral clearance are unlikely to meaningfully participate in protection against re-infection with influenza virus. Overall, we show vaccines that evoke lung CD8+ T cells and CD8+ TRM of broad repertoire diversity are valuable against influenza virus variants and that this local immunity may be compromised in hosts with pre-existing humoral immunity against the vaccine backbone. As such, our work uncovered several insights in the optimal implementation of T cell-based vaccines aiming to induce universal protective immunity against influenza virus.
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ItemThe molecular basis for Zn(II) import via the solute-binding protein AdcAII of Streptococcus pneumoniae( 2022)The metal ion zinc is required by all forms of life. The Gram-positive bacterial pathogen Streptococcus pneumoniae scavenges this essential metal ion from human tissues to facilitate colonisation and mediate disease. To achieve this, S. pneumoniae employs the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII, which acquire zinc from the extracellular milieu. Although both SBPs are required for full virulence of S. pneumoniae, AdcAII has a greater role during the early stages of infection. However, the molecular details of how AdcAII acquires zinc ions remain poorly defined. This can be attributed to the inability of crystallographic approaches to determine a high-resolution structure of metal-free AdcAII. To address the lack of structural and mechanistic information for this SBP, a mutational approach was taken, wherein each of the four zinc-coordinating residues of AdcAII were mutated to systematically untether regions of the protein from zinc coordination and observe the impact on SBP conformation. Structural analyses of the AdcAII variant proteins revealed that the protein undergoes highly localised rearrangements upon interaction with a zinc ion. A combination of in vitro metal-binding assays, affinity determination experiments, and phenotypic studies of S. pneumoniae strains harbouring mutant adcAII alleles showed how protein conformational changes are coupled to each zinc-coordinating residue at the metal-binding site. These analyses also revealed that, in stark contrast to AdcA, AdcAII was permissive for interacting with other metal ions. Intriguingly, the impact of mutant adcAII alleles on the growth of S. pneumoniae did not directly correlate with changes in SBP affinity for zinc. Instead, they were more closely aligned with the degree of structural perturbation observed in mutant AdcAII proteins. Taken together, these data suggest that the closed, zinc-bound conformation of AdcAII is important for efficacious uptake of the metal by S. pneumoniae. The conformational dynamics of AdcAII were further characterised through biophysical and computational approaches, establishing a foundation for future investigations of zinc acquisition and release by the SBP. Finally, the role of AdcAII in pneumococcal growth and fitness, and, by extension, the effects of zinc deprivation on the biology of the pathogen, were explored using strains of S. pneumoniae encoding mutant alleles of adcAII. The physiological defects of these strains provided insight into how zinc influences numerous pathways in S. pneumoniae, including cell division and virulence. Building on this work, the importance of pneumococcal zinc uptake for localised and systemic disease was investigated in murine models of infection. These studies highlighted the importance of SBP affinity and conformational changes in achieving pneumococcal zinc homeostasis. Collectively, this work delivers a putative mechanistic model for zinc-binding in AdcAII and provides new insights into how prokaryotic SBPs facilitate efficacious selective recruitment of essential metal ions. The findings presented in this thesis significantly advance our understanding of the strategies utilised by S. pneumoniae to overcome zinc limitation and cause bacterial disease.
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ItemGenerating CD8+ liver-resident memory T cell immunity against malaria( 2022)Liver resident memory CD8+ T (Trm) cells are attractive vaccine targets for malaria (Plasmodium) liver-stage immunity and can be effectively generated by glycolipid-peptide (GLP) vaccines. To gain insight into underlying mechanisms, we examined the requirements for priming, differentiation, long-term maintenance, and secondary boosting of liver Trm cells. We found that type I conventional dendritic cells (cDC1) were essential for priming CD8+ T cell responses, during which exposure to IL-4, most likely provided by activated type I natural killer T (NKT) cells, enhanced liver Trm cell formation. In addition, optimal generation of liver Trm cells required exposure to a combination of vaccine-derived inflammatory and antigenic signals post-priming, with antigen recognition being associated with enhanced Trm cell longevity. After primary immunisation with GLP vaccines, boosting of liver Trm cells could be achieved with the same GLP vaccine but a substantial delay was required for optimal boosting. This appeared to be due to NKT cell anergy post-priming as NKT cell-independent heterologous boosting could be achieved much earlier. Overall, our study revealed that the generation of liver Trm cells by GLP vaccination is IL-4 and cDC1 dependent, with longevity increased by post-priming antigenic signals and homologous boosting influenced by NKT cell recovery. Like many other malaria subunit vaccines, however, the utility of GLP vaccines is somewhat limited by the scarcity of protective CD8+ T cell epitopes. This issue is particularly prominent in the context of rodent P. berghei ANKA (PbA) infection of B6 mice, an extensively studied model of malaria. Using a combination of mass-spectrometry and in-silico approaches, we generated a library of 400 PbA-derived MHC I-restricted epitopes, from which we identified 4 immunogenic candidates that each reproducibly stimulated CD8+ T cells after pre-erythrocytic and blood-stage infections of B6 mice. Further characterisation of one of these peptide candidates, Db163, revealed cross-reactivity with a known immunogenic, but non-protective peptide PbA GAP5040-48. Targeting two additional epitopes, Db100 and Db177, by GLP vaccines induced substantial CD8+ liver Trm cells but these responses lacked protective efficacy against sporozoite challenge. The fourth epitope is derived from the PbA X, a predominantly late liver-stage antigen. Promisingly, this epitope could be targeted by a GLP vaccine to evoke liver Trm cell-mediated immunity against malaria in B6 mice. This protective immunity was remarkably long-lived with liver Trm cells persisting for at least 210 days. Furthermore, we demonstrated that X-specific liver Trm cells could execute a protective immune response cooperatively with those specific for PbA TRAP130-138, leading to improved sterile immunity even against high-dose sporozoite challenges. Lastly, the discovery of two novel HLA-A 02:01-restricted epitopes within the P. falciparum X proteins provides a future opportunity to dissect their usefulness as human vaccine candidates. Overall, this thesis provides novel mechanistic insights to maximise liver Trm cell formation and longevity after vaccination. Additionally, this thesis identifies novel antigenic targets of liver Trm cells that could be exploited for vaccination to induce immunity against malaria.
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ItemInterleukin-1 Is Unique in Its Ability to Modulate PD-L1 and PD-L2 Expression by Mo-DCs( 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.
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ItemProgrammed cell death during norovirus infection and the development of a reverse genetic tool to study viral replication( 2022)Human norovirus (HuNoV) is the leading cause of acute gastroenteritis worldwide with over 680 million cases each year resulting in an estimated $65 billion in associated costs and over 200,000 deaths. Despite the significance of this pathogen to global health, challenges in culturing noroviruses have significantly impeded our understanding of how these viruses infect, cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important component of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication and facilitate clearance and inflammation. In these studies, we characterised PCD during murine norovirus (MNV) infection of bone marrow-derived macrophages and determined that a dramatic reduction in cell viability correlated with the exponential release of infectious virus. We showed, genetically in both immortalized and primary macrophages, and chemically, that MNV-induced cell death and replication occurs independent of the essential effectors of pyroptosis: caspase-1, caspase-11 and gasdermin D; necroptosis: RIPK1, RIPK3 and MLKL; and extrinsic apoptosis: caspase-8. Intriguingly we observed that during infection with MNV the initiators of necroptosis, namely RIPK1 and RIPK3, were reduced in abundance as the infection progressed. Further analysis revealed that MNV infection promoted the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. Overall, these observations confirmed that MNV infection promoted the induction of intrinsic apoptosis facilitating viral replication and release. Additional investigations revealed a previously unrecognised mechanism of apoptosis induction by MNV. We showed that either infection or over-expression of the viral protein NS3 was sufficient to induce apoptosis through repression of host cell translation and reduction of pro-survival BCL-2 family proteins including MCL-1. Treatment with the proteosome inhibitor MG-132 or the caspase inhibitor QVD protected cells against apoptosis but only MG-132 prevented MCL-1 loss and apoptosis induction. Furthermore, through generation of truncation mutants and tripartite alanine mutants we mapped the region of NS3 responsible for translational shut-off and apoptosis and identified key residues between amino acids 67-100 of the protein. Additionally, we found that HuNoV homologue of NS3 also induced translation repression and apoptosis, signifying a conserved function and possible antiviral target for potential drug design. Lastly, we describe the development of a novel reverse genetics tool to study noroviruses. Through application of circular polymerase extension reaction (CPER), we optimised the system for propagation of MNV and demonstrate that it could be used to isolate HuNoV directly from clinical material. Further advancing the approach, we designed and constructed multiple recombinant, chimeric and reporter-tagged viruses. While many of these failed to yield infectious virus due to replication constraints, NS3-mutant viruses could be recovered offering a powerful reagent to further our investigations into MNV-induced apoptosis. Together we describe the benefits and immense potential of CPER for NoVs. Overall, this research has contributed to the understanding of virus-host interactions and has provided a molecular and biochemical mechanism that results in MNV-induced translational control and virus release. Incorporating our new CPER approach we can interrogate not only the functional capacity of the MNV NS3, but provide an application to pave the way in the recovery of HuNoV directly from patient samples.
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ItemT cell responses to virus infection in the intestinal mucosa( 2022)The intestines are a major reservoir for a range of immune cells, of which CD8+ T cells are key in the elimination of viral infections. The structure of the small and large intestines differs significantly, potentially providing specific tissue niches with different capacities to support immune responses. The structural architecture of the intestines is maintained by a network of stromal cells that contribute to tissue homeostasis and communication with immune cells. Tissue-resident memory T cells (TRM) form following infection and reside in organs such as the gut without recirculating, providing accelerated protective immunity against subsequent infections. In the small intestinal epithelium, most TRM have been characterised by CD103 and CD69 expression in the context of acute infection, where their formation requires the cytokine TGF-beta. However, different infections can modulate TRM formation in different ways, and less is known about the dynamics of T cells in the large intestine. Further, how the stromal cell network influences immune responses in the intestines has not been established. In this thesis, acute and chronic strains of Lymphocytic Choriomeningitis Virus (LCMV) were utilised to characterise T cell and stromal cell populations in the mouse intestine. The antigen-specific CD8+ T cell population underwent a significantly larger contraction in the large versus small intestine after acute LCMV infection. There were low numbers of TRM cells in the large intestine compared to the small intestine, resulting in part from limiting TGF-beta signals in the large intestine. Comparatively, during chronic LCMV infection, LCMV-specific T cells remained in high numbers in both the small and large intestine. The stromal cell network differed significantly between the small and large intestine and was altered during chronic infection. Memory T cells in the large intestine after acute LCMV infection differed phenotypically to those in the small intestine, which may be due to local conversion of circulating memory T cells to TRM. Overall, the work in this thesis has highlighted differential capacities of the small and large intestines to support the formation and maintenance of immune memory after acute viral infection.
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ItemUnderstanding the early events in influenza A virus infection of human airway macrophages compared to epithelial cells( 2022)Airway epithelial cells (AEC) and airway macrophages (AM) represent the major cellular targets for infection by different respiratory viruses. These include influenza A virus (IAV), a member of the Orthomyxoviridae family, and parainfluenza virus type-3 (PIV-3), a member of the Paramyxoviridae family. Virus-infected cells rapidly induce interferons (IFNs) which in turn activate hundreds of interferon-stimulated genes (ISGs) in infected cells and in neighbouring uninfected cells. The family of interferon-induced transmembrane (IFITM) proteins are ISGs that have shown broad antiviral activity against different virus families by interfering with virus entry. In this thesis, we evaluated the antiviral activity of human IFITM1, IFITM2 and IFITM3 against IAV and PIV-3, two respiratory viruses with distinct entry requirements. Using a doxycycline (DOX)-inducible IFITM overexpression system we found that all three IFITM proteins limited IAV infection to varying degrees, with IFITM3 having the strongest effect. Our studies confirmed that IFITM proteins display antiviral activity early, but not late, in the IAV replication cycle. None of the IFITM proteins modulated PIV-3 replication at either early or late-stages in the replication cycle. Different approaches were explored for their utility to modulate IFITM1, IFITM2 or IFITM3 expression to assess potential antiviral activity against IAV and PIV-3. Herein, we highlight caveats associated with constitutive IFITM protein overexpression as well as using CRISPR/Cas9 gene editing techniques to knockout endogenous IFITM expression. Observations herein also demonstrated that entry of IAV into epithelial cells by endocytosis conferred sensitivity to restriction by endosome-localized IFITM2 and IFITM3. Indeed, key steps in the IAV replication cycle from cellular entry through to exit are relatively well defined in epithelial cells. However, comparatively less is known about all stages of the viral life cycle during IAV infection of macrophages (M), particularly for primary human AM, where previous studies have largely been performed using mouse M. We investigated IAV entry into primary human M (human monocyte-derived macrophages (MDM) and airway macrophages (AM)) by defining the role of sialic acid (SIA) and C-type lectin receptors (CLRs) in determining susceptibility to the early stages of infection. Our results showed differential expression of SIA between MDM isolated from peripheral blood and AM obtained from bronchoalveolar lavage (BAL). MDM expressed both, alpha2,3- and alpha2,6-linked SIA, while AM expressed predominantly alpha2,3-linked SIA. AM were less susceptible to infection by IAV strains that preferentially recognised alpha2,6-linked SIA. Further, we found that although MDM and AM both expressed the CLR macrophage mannose receptor (MMR), it appeared that CLRs played a more prominent role in facilitating IAV infection of AM compared to MDM. We also systematically compared primary MDM and AM isolated from the same donor to show that both M types were susceptible to the early stages of IAV infection, but only MDM supported (limited) productive virus replication. Infection of AM by IAV resulted in strictly abortive replication for two different IAV subtypes, where newly-synthesised infectious virions were not released. Together, these findings make a substantial contribution to our overall understanding of IAV infection and replication in primary human M.
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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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ItemNo Preview AvailableThe molecular and cellular basis of antigen recognition by CD1a-restricted T cells( 2022)In contrast to conventional T cells that recognise peptide antigens presented by MHC molecules, a group of “unconventional” T cells recognise lipid antigens presented by MHC-like CD1 family members, CD1a, CD1b, CD1c and CD1d. Studies suggest that CD1a-restricted T cells comprise a unique subset in human blood that recognise CD1a-lipid complexes and play a unique functional role in skin immunity. While they comprise a decent proportion of T cells compared to CD1d-restricted, natural killer T (NKT) cells, they remain relatively less well-understood. This thesis describes the phenotypic characterisation of CD1a-restricted T cells in human tissues directly ex vivo. Phenotypic analyses and single cell RNA-sequencing of CD1a-restricted T cells revealed that they are distinct from other CD1-restricted T cells. They did not express typical innate-like markers such as CD161, IL-18R, and PLZF, which are expressed by NKT cells, distinguishing them as a unique population of unconventional T cells. This thesis also elucidates how T cell receptors (TCRs) interact with CD1a-lipid complexes. Profiling the TCR repertoire of CD1a-restricted T cells, demonstrated that while diverse, there is a bias towards TCR variable genes that endow optimal TCR configurations to interact with CD1a and lipid antigens. Experiments with CD1a mutant cell lines revealed that individual TCRs bind at various sites across the entire binding cleft of CD1a, which likely increases the diversity of lipid antigens that can be recognised by CD1a-restricted T cells. Indeed, these T cells were observed to recognise numerous lipid antigens including self-lipids and dideoxymycobactin (DDM), a lipid antigen derived from Mycobacterium tuberculosis, with some CD1a-restricted TCRs even displaying cross-reactivity to lipids with distinct chemical structures. Reagents were developed as tools to study lipid-reactive T cells in macaques, especially for non-human primate models of disease. A suite of CD1 tetramers were generated to isolate CD1-restricted T cells in pig-tailed macaques and for preliminary enumeration and phenotypic analysis of CD1-restricted T cell subsets in macaque tissues. Lastly, tetramers were used to investigate CD1a-restricted T cells in human skin. Populations of lipid-reactive T cells and gd-T cells were isolated for phenotypic analysis and TCR sequencing, thus demonstrating that they may play a role in healthy skin. C12-15 alkyl-benzoate, a common oil in dermatological products, was identified as a novel CD1a antigen, suggesting a role for CD1a-restricted T cells in allergic dermatitis. These studies provide insight into the functional properties of CD1a-restricted T cells and their molecular interactions with CD1a-lipids. Collectively, they represent a step forward in characterising CD1a-restricted T cells and provide a greater understanding of their role in the immune system.