Microbiology & Immunology - Theses
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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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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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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.
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ItemRole of IFN-y induced genes in cell autonomous defence against Legionellae( 2021)L. pneumophila and L. longbeachae are ubiquitous environmental bacteria that can cause a severe pneumonia, known as Legionnaires’ Disease, when contaminated aerosols are inhaled by susceptible humans. The co-evolution of the bacteria with their environmental hosts has equipped the bacteria with the ability to subvert the cell intrinsic host defence mechanisms in human cells, thereby allowing the pathogens to survive and replicate within the lung macrophages. During infection, Legionella establishes an intracellular niche, known as the Legionella containing vacuole (LCV). The biogenesis of the LCV is dependent on the defective organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system, which translocates a large arsenal of bacterial effector proteins into the host cell. These effectors are known to modulate host metabolism and cell-autonomous defence, protect the integrity of the LCV and allowing the bacteria to acquire nutrients from the host to ensure intracellular survival and enable intracellular replication of the pathogen. It is known that during Legionella lung infection, the mammalian host mounts a robust inflammatory response, producing cytokines, such as TNF-alpha, IL-1-alpha, IL-6, IL-12 as well as type I interferons and IFN-gamma, which usually leads to the restriction of intracellular replication and culminates in the clearance of the infection. It was previously shown that IFN-gamma is crucial for host defence against Legionella in mice, since disruption of IFN-gamma signalling or IFN-gamma deficiency results in a high replication of Legionella in the lung as well as a failure to clear the infection from the host, despite the activity of other inflammatory cytokines. Exposure of cells to interferons (IFN), including IFN-gamma, results in the induction of a network of genes that combat infection, leading to so-called IFN-mediated cell-autonomous defence. This network is finely-tuned to balance efficient pathogen control while preventing collateral tissue damage. However, which interferon-induced genes and through what mechanism this strikingly potent restriction is mediated remains elusive for Legionella. In this study, we shed some light on the mode of action of the IFN-gamma induced host defence against Legionella. We identified a new mechanism of host defence mediated by interferon stimulated genes (ISGs), that results in the disruption of effector translocation into host cells by the Dot/Icm secretion system. We demonstrated that this mechanism is uniquely triggered by interferon signalling and is independent of well-known host defence mechanisms such as host cell death, direct bactericidal activities, inflammasome activation as well as proteasome and autophagy-mediated degradation. By utilising mRNA sequencing of IFN-gamma and type I interferon-stimulated macrophages, we identified possible factors that mediate this inhibition: ISG15 and PARPs. These proteins have not previously been implicated in Legionella host defence and represent a unique opportunity to increase our knowledge of interferon mediated cell-autonomous host defence. Currently, more than 65 Legionella species are known and roughly half of them have been clinically associated with infection, frequently in immune compromised patients. After L. pneumophila, L. longbeachae is the second most common causative agent of Legionnaire’s Disease worldwide and is the leading causative agent in Australia and south-east Asia. Despite this, knowledge about the pathogenesis of L. longbeachae is minimal. Therefore, during this study, we also aimed to provide new insights into the pathogenesis of L. longbeachae infection and characterise the impact of IFN-gamma on immune control. We observed unique features of L. longbeachae infection in comparison to L. pneumophila, such as the ability to survive within a wider range of lung phagocytes, dampening of the cytokine response of the host and translocation of effectors into all lung phagocytes tested. These unique features may enable L. longbeachae to subvert the host defence more efficiently than L. pneumophila and thus replicate to higher numbers. Furthermore, we were able to show that IFN-gamma is crucial for host defence against L. longbeachae in vivo, with neutrophils and monocyte derived cells dependent on IFN-gamma signalling to mediate their bactericidal properties. In addition, we were able to demonstrate that IFN-gamma stimulation restricts L. longbeachae Dot/Icm secretion system effector translocation into host cells. Overall, this study substantiates the importance of IFN-gamma in host defence against Legionella and supports the need to broaden research efforts to non-L. pneumophila species. Investigation and deeper understanding of critical host defence mechanisms can be used as a starting point to develop anti-infective agents against pathogens targeting the process of effector translocation or effector mediated manipulation of host function and cell-autonomous defence.
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ItemThe viral glycoproteins of the Hepaciviruses. Structural and functional studies to inform vaccine design( 2022)Hepatitis C virus (HCV) infection is a major global health burden, with an estimated 71 million people infected worldwide. Vaccine design for HCV is challenging for multiple reasons, including high sequence variability of the glycoprotein E2, as well as the lack of a small animal challenge model in which to test vaccine candidates. This thesis addresses aspects of both challenges. Glycoprotein E2 is present on the virion surface and is a major target of neutralizing antibodies which can prevent infection. The N-terminal hypervariable region 1, HVR1 (residues 384-411), of E2 is an immunodominant region within E2 and elicits neutralizing antibodies that are usually isolate specific. We previously identified a novel murine monoclonal antibody, MAb33, which binds to an unusual epitope bridging HVR1 and the adjacent target of broadly neutralizing antibodies referred to as epitope I (residues 412-423). MAb33 potently neutralizes genotype 1a viruses and can cross-neutralize 3 different HCV genotypes. This study defined the epitope of MAb33 to include residues within the E2 region 401-415 and resolved its structure in complex with its epitope. The epitope adopts an alpha-helical conformation with residues G406, A407 and N410 involved in direct polar interaction with the antibody. The helical structure of the epitope differs from the extended conformation of other E2 crystal structures that include this region, suggesting that it could be conformationally flexible. Sero-surveys of HCV positive individuals have identified significant reactivity to a peptide encompassing the MAb33 epitope, indicating a role for MAb33-like antibodies in natural infection. To address the need for an immune competent model for HCV vaccine development, a Rodent hepacivirus (RHV) was investigated as it shows close evolutionary relatedness and virological similarities with HCV and represents an important potential model of HCV pathogenesis and host responses. While the T cell response to RHV has been characterized, detailed studies characterizing the RHV E2 glycoprotein, the development of the humoral immune response after infection and in vaccination studies are lacking. This thesis characterized the antigenic and immunogenic properties of RHV E2. A minimal ectodomain expressed as a soluble protein was defined (residues 418-603), which has 4 sites for N-linked glycans and 12 cysteine residues. The development of the anti-E2 antibody response in outbred rats infected with RHV was analysed and the appearance of anti-E2 antibodies observed at 28 days post-infection. RHV E2 was assessed as a potential vaccine antigen in immunisation/challenge studies in rats. Rats received a E2 protein prime followed by a protein boost, or a combined vaccination of E2 protein and a simian adenovirus (ChAdOx1) encoding the non-structural proteins NS3-NS5B from RHV. Both groups failed to generate anti-E2 antibody prior to challenge at 6 weeks. Following challenge with RHV, anti-E2 antibodies appeared 14 days later, with most animals seroconverting by 28 days post challenge. These studies are the first of their kind to define a soluble ectodomain of the RHV E2 protein and explore the development of anti-E2 antibodies in infection.
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ItemIdentification and characterization of proteins and mechanisms involved in the uptake and traffic of vitamin B related antigens( 2021)Major histocompatibility complex, class I-related (MR1) presents Vitamin B-related antigens (VitBAg) at the cell surface to activate mucosal associated invariant T (MAIT) cells, directing homeostasis and immune responses. Although previous work has suggested endocytosis as a participant in MR1 presentation, how these antigens are captured by the cell is currently unknown. It is likely that MR1 ligands are uptake as metabolites for they have several structural similarities with molecules known to be transported through solute carrier (SLC) transporters. Here, we shown that flavins are pathway-specific inhibitors of MR1-5-OP-RU, and do not inhibit MR1-Ac-6-FP upregulation. We revealed that 5-OP-RU, ribityl lumazine (RL) and bacterial VitBAg, but not folate derived ligands, enter the cell through SLC52A family of riboflavin transporters, as their expression increases MR1 presentation and MAIT cell activation in a riboflavin modulated manner. In contrast, knock-outs models SLC52A family drastically reduce the incorporation of RLs but do not abolish the capacity to present 5-OP-RU through MR1. In fact, pathway specific inhibitors of MR1-5-OP-RU and MR1-Ac-6-FP extend to nucleosides, nucleobases and other drugs, arguing for the contribution of more SLC transporters in their uptake. Likewise, MR1 presentation during infection is increased by ligand-producing bacteria located in the cytosol, stating a cytosolic step to reach empty MR1 molecules. Finally, we showed that 5-OP-RU alters the metabolome of cells like LPS, leading to changes in their transcriptome profile. Our results unveil a new route for 5-OP-RU, RL and bacterial VitBAg uptake through SLC52A transporters, contributing to their capture and modulating MR1 presentation, together with a new potential role of 5-OP-RU as a pathogen-associated molecular patterns (PAMPs) molecule.
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ItemT cell response to an MHC-II restricted epitope of rodent malaria( 2021)Malaria is caused by different Plasmodium species that can infect a variety of animals including humans and rodents. The life cycle of these parasites is complex and includes a liver stage followed by a blood-stage in their vertebrate hosts. While the host’s immune response against each of these stages is incompletely understood, CD4 T cells are known to play an important role in immunity to Plasmodium infection during both stages. This project aims to examine the specific CD4 T cell response to a novel MHC II-restricted epitope in Plasmodium infection in C57BL/6 mice, and to characterise the protective capacity of these T cells. To this end, we made use of a recently generated TCR transgenic mouse line, termed PbT-II, which responds to a so far unknown Plasmodium derived epitope. In this project, the PbT-II epitope was identified as derived from heat shock protein 90, residues 484 to 496 (Hsp90484-496 or abbreviated DIY). Different priming methods, such as injection of an anti-Clec9A antibody attached to the Hsp90 epitope (aClec9A-DIY), infection with P. berghei ANKA (PbA) infected red blood cells (iRBCs) or immunisation with radiation attenuated PbA sporozoites (RAS), were used to characterise PbT-II memory cell formation. Results revealed the formation of memory PbT-II cells expressing surface markers associated with central memory T cells (TCM), effector memory T cells (TEM) and tissue resident memory T cells (TRM). Given the importance of tissue-resident memory T cells in peripheral immunity, mainly studied in CD8 T cells, we focused our study on the formation and function of CD4 TRM cells in the liver. Parabiosis studies using RAS vaccinated mice confirmed the liver residency of a CD69+ PbT-II cell population. Gene expression profile analysis revealed that these CD4 T cells expressed a core gene signature similar to that of CD8 resident memory T cells. Furthermore, differences in the gene expression profile of PbTII TRM cells generated via different protocols, suggested lineage specific effector mechanisms, such as IL-4 production or perforin expression, for subsets of CD4 TRM cells in the liver. As CD4 T cells can potentially act against both the liver and blood-stage of Plasmodium infection, we sought to investigate the protective potential of PbTII effector and memory cells for both of these stages. While none of the PbT-II priming methods resulted in a reduction of liver parasite burden upon sporozoite infection, mice injected with large numbers of in vitro polarized PbT-II Th1 or Th2 cells showed reduced parasitemia after PbA blood-stage infection. Surprisingly, most of these mice were protected from experimental cerebral malaria (ECM), although they were not able to clear PbA blood-stage infection.
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ItemNanoparticle interactions with the immune system( 2021)Vaccination has been an incredibly successful public health intervention, saving the lives of 2-3 million people each year. Despite this success, we still lack effective vaccines for many infectious diseases including HIV, tuberculosis and malaria. Nanoparticles (ordered structures within the range of 10-1000nm) have great potential to supplement traditional vaccines based upon pathogen subunits, or killed or attenuated microorganisms, as demonstrated by the successful licensure of virus-like particle vaccines for human papillomavirus and liposomal mRNA vaccines for SARS-CoV2. However, the immunological mechanisms that explain the potent immunity of nanoparticle vaccines and the factors dictating their interaction with the immune system are poorly defined. This thesis studies how nanoparticle characteristics affect their interaction with the immune system with a view to improving vaccine strategies. First, the contribution of the protein corona on the association of engineered nanoparticles with primary human blood cells was assessed. The association of high protein binding (high-fouling) mesoporous silica (MS) particles and low-fouling zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) particles with human white blood cells was assessed by flow cytometry in the presence or absence of plasma proteins. The effect of precoating nanoparticles with serum albumin, IgG and complement protein C1q was also assessed. The differential association of low and high-fouling nanoparticles was found to be largely a consequence of the de novo formed, not pre-adsorbed, biomolecular corona. Specifically, an enrichment of complement proteins within the corona resulted in an increased association with B cells. Second, the immune mechanisms that give rise to the improved immunogenicity of a prototypic nanoparticle vaccine were investigated. Humoral immune responses to a self-assembling protein nanoparticle vaccine for influenza (HA-ferritin) were contrasted to a subunit influenza vaccine (soluble HA) in mouse and non-human primate models. Antibody titres and protective efficacy of the vaccines were compared followed by a detailed study of lymph node germinal centre B cell and T follicular helper cell responses. Vaccination of C57BL/6 mice with HA-ferritin nanoparticles elicited higher serum IgG titres and greater protection against experimental influenza challenge compared with soluble HA vaccination. Within the antigen-draining lymph nodes, germinal centre reactions were expanded and persistent following HA-ferritin vaccination. This augmented humoral immunity was not driven by ferritin-specific T follicular helper cells but rather driven by expanded antigen colocalization with follicular dendritic cells. However, this immune enhancement did not translate from mice to pigtail macaques where antibody titres and lymph node immunity following HA-ferritin nanoparticle vaccination were comparable to soluble HA protein vaccination. And thirdly, we explored innate immune activation by HA-ferritin and soluble HA in mice. This was achieved through in vitro assessment of antigen glycosylation and complement activation and in vivo through serum IgM titres and cell trafficking to the lymph nodes following vaccination. HA-ferritin vaccination of mice was found to elicit an early enhancement of antigen-specific serum IgM however in vitro complement activation was not detected. Trafficking of immune cells to the lymph nodes was found to be influenced by antigen glycan composition in conjunction with purification methods. The findings of this thesis suggest that nanoparticle interaction with the immune system is driven by the complex interplay of nanoparticle physiochemical properties, antigen glycosylation, corona formation and pattern-recognition receptors of innate immune cells. Further improvements in understanding the relationship between these features and how they may differ between animal species will speed the rational design of next-generation nanoparticle vaccines against diverse pathogens.
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ItemAdvancing the ferret as an immunological model to study B-cell responses( 2020)Introduction Influenza is a clinically significant disease, causing 24000-62000 deaths alone in the United States during the 2019-2020 season. While annual vaccines are available, variable efficacies have been reported and annual updates are required due to antigenic drift. Ferrets are a useful model for studying human respiratory viruses and have been widely used to evaluate vaccines and transmission of influenza. Sera from ferrets infected with different influenza strains are used in HI assays as part of strain determination of seasonal influenza vaccines. A key limitation of the ferret model is the paucity of immunological reagents to characterise immune responses and a lack of knowledge regarding the ferret immune system. This PhD thesis aims to advance the ferret as an immunological model to study human respiratory viruses by developing methods and reagents which will enable in-depth interrogation of ferret B-cell responses. Methods While a draft copy of the ferret genome is available, immunoglobulin sequence information is not well-annotated. Hence, we first annotated the ferret genome with immunoglobulin variable, diversity, joining and constant chain genes by inferring homology using human and canine orthologs (Chapter 3). Novel PCR primers targeting 5’- leader, 3’- joining and 3’- constant chain immunoglobulin genes were derived, enabling the recovery of functional, paired heavy and light chain transcript sequences from single sorted ferret B-cells. Ferret immunoglobulin constant sequences were validated by RNA-seq, which enabled the development of ferret IgG expression plasmids. Using this technique, HA-specific B-cell responses were characterised for the first time in ferrets at the transcript level (Chapter 4). Candidate ferret mAbs were derived from the recovered sequences, expressed and screened for HA binding specificity and in-vitro influenza virus neutralisation activity. We noted poor recovery of ferret HA specific mAbs and subsequently sought to improve flow cytometric panels available for ferrets. We established a methodology using previously developed murine single-cell BCR sequencing methods to recover murine anti-ferret mAbs (Chapter 5). First, coding sequences of ferret B and NK-cell reagents were identified on the ferret genome and validated by sequence and structural comparisons with other mammalian homologs. C57BL/6 mice were subsequently immunised with these antigens and candidate mAbs were recovered for examination by ELISA and flow cytometry. Results Ferret variable, diversity, joining and constant chain coding genes were identified on the draft copy of the ferret genome and show good sequence similarity to human and canine variants. Our novel ferret immunoglobulin specific PCR primers enabled the recovery and characterisation of germline ferret immunoglobulin genes from single sorted ferret B-cells. RNA-seq validation of ferret immunoglobulin constant chain genes subsequently enabled the construction of ferret IgG/IgL expression plasmids. This facilitated the expression of chimeric human-ferret CR9114 IgG antibody retaining HA binding specificity. Subsequently, using previously developed trimeric HA probes, clonally expanded sequences were recovered from single sorted HA-specific B-cells derived from infected ferrets. Screening of candidate ferret monoclonal antibodies enabled the identification of two novel antibodies, belonging to the same clonal family showing HA binding specificities. Further examination by HAI and microneutralization assays revealed the ability of the mAbs to neutralise influenza virus in vitro. Viral escape mapping revealed binding epitope to previously reported Sa site of the HA head domain, showing proof of concept for mapping HA epitopes using these recombinant ferret mAbs. We next attempted to improve flow cytometric panels for ferrets which will enhance recovery of ferret immunoglobulin transcripts. As there are currently no mAbs targeting B and NK-cell markers in ferrets, we identified key markers for murine mAb development including CD19, IgD, CD138, NKp46 and LAMP-1. We identified candidate anti-ferret CD19 and IgD mAbs which bound to cognate recombinant antigens by ELISA, validating this method for generating anti-ferret mAbs to improve panels for flow cytometry and confocal microscopy. As the mAbs in this thesis lacked the capacity to resolve ferret cell populations by flow cytometry, we identified and discussed key steps in the process which will inform future use of this approach to develop anti-ferret mAb reagents. Conclusion The body of work presented in this thesis forms the proof of concept of studying antigen-specific B-cell responses at the mAb level in ferrets. Future improvements in tools developed in this thesis and future development of reagents will enable detailed interrogation of the ferret immune system.