Microbiology & Immunology - Theses
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ItemA universal prophylactic vaccine against Hepatitis C Virus( 2023-03)Discovered in 1975, hepatitis C virus (HCV) is a ribonucleic acid (RNA) virus which exhibits an immense degree of genetic diversity that has hindered the development of a broadly protective vaccine to control the virus. HCV remains a global public health problem despite the development of effective antiviral treatment resulting in 95% cure rates. There are an estimated 57 million people infected with HCV globally, with an additional 1.4 million new infections diagnosed every year. Clinical presentation can range from mild disease to liver cirrhosis and cancer. As transmission is via blood-to-blood contact, most infections are contracted through intravenous drug-use, non-sterile surgical equipment, or unscreened blood transfusions and transplants. The people most often affected are already among the most vulnerable in society and the presence of a chronic viral infection presents an added burden. Currently, there is no vaccine available to prevent primary infection and developing a universal vaccine that generates a strong immune response is imperative for protective immunity. Evidence from individuals who spontaneously resolve HCV infection shows the involvement of both humoral and cellular responses in HCV resolution. This indicates that a highly effective vaccine for HCV will require the rapid induction of broad and potent neutralising antibodies (NAb) and a highly functional and a broadly reactive cellular response. The work in this thesis presents new HCV candidates to improve the breadth and specificity of the humoral response as well as a vaccine candidate that co-delivers, stimulates and induces both humoral and cellular responses. Glycoprotein E2 of HCV is the major target of the NAb response and harbours 3 variable domains. These domains are known to generate type specific antibody responses and occlude NAb epitopes. Previous work generated a recombinant soluble protein of E2 in which the variable domains have been removed (E2D123) and this antigen was shown to elicit a broadly neutralising antibody (bNAb) response in guinea pigs when using a high molecular weight species of the protein (E2D123HMW). The work in this thesis employs E2D123 and explores two vaccine delivery platforms to enhance the immune response generated by this immunogen. Viral vector vaccines were employed and engineered to deliver the E2D123 sequence, in addition to conserved T cell epitopes within the HCV genome. Immunisation studies were performed in which small animals were vaccinated with viral vector vaccines or recombinant protein vaccines or a combination of both and the immune responses characterised. The results show that a bivalent viral vector vaccine encoding both T cell epitopes and the E2D123 sequence stimulates multiple components of the immune system, inducing broad and potent humoral and cellular immune responses against multiple genotypes of E2. In addition, the work in this thesis establishes and characterises a virus-like particle (VLP) vaccine which displays multiple units of a monomeric form of E2D123 on the surface of a duck hepatitis B virus (DHBV) particle. The immunogenicity of these VLPs was analysed in guinea pigs and the results demonstrated the induction of high titres of antibodies reactive to native E2 with the capacity to inhibit E2 binding cellular receptor CD81. Additionally, NAbs capable of neutralising 7 genotypes of HCV were generated. Collectively, this thesis presents novel and promising vaccine candidates for HCV which contributes to our understanding of HCV infection and immunity and next-generation vaccine development.
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ItemHarnessing unconventional T-cells for vaccines and immunotherapies in pre-clinical animal models( 2023-03)Unconventional T-cells represent a heterogenous population of CD3+ T-cells that recognise protein and non-protein antigens through MHC-unrestricted mechanisms. Unconventional T-cells also undergo cytokine- or surface receptor-mediated activation independent of T-cell receptor ligation, a characteristic that is commonly associated with innate immune cells and permits rapid responses to stimuli. These cells have diverse effector responses following activation, including direct cytolytic activity against target cells, proinflammatory cytokine production to mediate other immune responses, and antigen presentation to conventional CD4+ and CD8+ T-cells. Several unconventional T-cell subsets have also been explored as immunotherapeutics, in part due to our ability to readily expand them pharmacologically, with some expressing highly conserved public T-cell receptors. Current knowledge gaps with unconventional T-cell immunotherapies includes our understanding of the frequency and phenotype of therapeutic cells in different tissue compartments, and how this is impacted by changes in pharmacological expansion protocols. Additionally, several unconventional T-cell subsets can augment conventional T- and B-cell responses associated with humoral immunity. However, the contribution of these unconventional T-cell populations to conventional adaptive immune responses against protein vaccines or viral infection is not well understood. The overarching aim of this thesis is to characterize unconventional T-cell in the context of immunotherapeutics and during vaccine-elicited immune responses, in pre-clinical animal models. Vgamma9Vdelta2 T-cells are a subset of unconventional T-cells that recognises endogenous and exogenous phosphoantigens and have garnered significant interest for immunotherapies to treat cancer and infectious diseases. While studies in pre-clinical animal models have shown promise, the clinical efficacy with Vgamma9Vdelta2 T-cell therapy has been limited. In Chapter 2, we characterised the Vgamma9Vdelta2 T-cell population at steady-state and following in vivo pharmacological expansion in pigtail macaques. We found the tissue distribution of pharmacologically expanded Vgamma9Vdelta2 T-cells changed based on the antigen administration route. Additionally, our pharmacological expansion protocol drove marked CCR6 downregulation and granzyme B upregulation in expanded Vgamma9Vdelta2 T-cells. Our results highlight how changes to pharmacological expansion protocols can alter the phenotype and tissue distribution of the expanded cell population, which is important to consider as this will likely impact therapeutic efficacy. Lymph nodes are a critical site of adaptive immune responses and the generation of antigen specific Tfh and BGC cells following vaccination. However, vaccine draining lymph node identification to study these responses is hindered by anatomical variations in lymphatic drainage between individuals, and lymph nodes being arranged in clusters with only a subset draining the vaccine site. To improve the identification of vaccine draining lymph nodes in preclinical animal models, we developed a vaccine strategy to label draining lymph nodes with tracking dyes (Chapter 3). We show that protein vaccines co-formulated with tattoo ink accurately labels vaccine draining lymph nodes in both mice and nonhuman primates. Ink-containing lymph nodes had higher frequencies of antigen specific BGC and Tfh cells compared to lymph nodes without ink. Furthermore, the ink coformulation was compatible with flow cytometry-based assays and did not alter the vaccine immune response serologically or at the B- and T-cell level. Unconventional T-cells are capable of humoral immune responses through multiple mechanisms including conventional antigen presentation, co-stimulatory signalling to Tfh cells, and providing both cognate and non-cognate B-cell support. Whether different unconventional T-cell subpopulations significantly contribute to the humoral immune response following vaccination or viral infection has not been well established. In Chapter, 4, we evaluated the contribution of unconventional T-cells to conventional adaptive immune responses elicited by vaccines or influenza infection, using transgenic mice that individually lack gamma delta T-cells, MAIT cells, and NKT cells. We found transgenic animals had comparable serological, Tfh, and BGC responses following immunisation with clinically-relevant vaccine formulation or subclinical influenza infection. Our findings indicate these unconventional T-cell subpopulations are not individually essential for mounting a robust humoral immune response to protein vaccines or viral infection. These results also raise questions about compensation between these unconventional T-cell populations, or a potential lack of unconventional T-cell recruitment to vaccine- or viral-mediated immune responses. Collectively, we evaluated unconventional T-cells in the context of immunotherapies and conventional humoral immune responses, in preclinical animal models. Better animal model characterisation will likely improve clinical translatability of candidate vaccines and therapeutics. Future utilisation of and improvements to the labelling techniques described here will help interrogate vaccine responses in regional draining lymph nodes in preclinical animal models. Our findings also raise important questions about modifying in vivo Vgamma9Vdelta2 T-cells treatment protocols to improve therapeutic cell delivery to target tissues, and modifying vaccine formulations to better recruit different unconventional T-cell populations as part of the humoral immune response.
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ItemTargeting the untargetable: Eliminating HIV latency using nanoparticle delivery systems( 2023-04)T cells form an important therapeutic target for the development of immunotherapies or the treatment of T cell-implicated pathologies. Specifically, CD4 T cells are subject to human immunodeficiency virus (HIV) infection. Whilst treatment with antiretroviral therapy (ART) successfully represses the viral load to undetectable levels, HIV treatment is lifelong, posing a medical, social and financial burden to those 38.5 million people currently living with HIV globally. The ability of HIV to establish a reservoir of latently infected cells is the foremost barrier to finding a cure for HIV. One approach towards eliminating HIV latency is the reactivation of viral transcription and subsequent elimination of infected cells through immune-mediated clearance or viral-mediated cytotoxicity. However, this approach so far has suffered from a lack of potency and dose-limiting toxicities due to the use of compounds that affect both host and viral transcription and the inability to specifically target the infected cells. One solution involves the use of nanoparticles for the targeted delivery of existing therapeutics or to advance the development of HIV-specific mRNA-based therapeutics including CRISPR-Cas. However, the generally low rate of endocytosis in CD4+ T cells forms a challenge to efficient nanoparticle-based drug delivery to CD4+ T cells in vitro and in vivo. This thesis describes our efforts towards rationally designing a nanoparticle platform capable of delivering HIV latency-reversing therapeutics to CD4+ T cells with high efficiency. We first established a methodology to improve the assessment of nanoparticle performance in vitro through the generation of absolutely quantitative, comparable data on nanoparticle-cell interactions. We then used this methodology to screen for nanoparticle designs with enhanced uptake kinetics in CD4+ T cells in vitro, using a novel, high-throughput assay to quantify nanoparticle internalization over time. We found that targeting sub-100 nm nanoparticles to T cell surface receptors undergoing rapid receptor cycling can be exploited to actively trigger nanoparticle uptake through receptor-mediated endocytosis and identified CD2 and CD7 as potent candidate receptors for future in vivo T cell targeting. We next aimed to use translate these findings to lipid nanoparticles, a well-established platform for the delivery of nucleic acid-based therapeutics. We investigated whether lipid nanoparticles could be used to deliver a next-generation latency-reversing agent based on CRISPR activation, which specifically targets the HIV proviral genome without affecting host-cell transcription. We identified a novel lipid nanoparticle formulation that can efficiently transfect T cell lines as well as resting CD4+ T cells. We showed that this lipid nanoparticle can co-encapsulate the three RNA components of the CRISPR activation system and induce strong latency reversal in a cell line model for HIV latency. We finally presented preliminary evidence that targeting lipid nanoparticles to rapidly cycling surface receptors increases mRNA delivery, further supporting our findings that targeting receptor-mediated endocytosis could be a viable strategy to increase nanoparticle-mediated drug delivery to traditionally hard-to-transfect T cells. These findings provide a compelling justification for the further assessment of CRISPR activation lipid nanoparticles for the elimination of the latent HIV reservoir, and more broadly contribute to the development of T cell-targeted nanomedicine for HIV and beyond.
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ItemFunctional antibody immunity in SARS-CoV-2 infection and vaccination( 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.
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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.