Doherty Institute - Theses
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ItemThe interface of nanotechnology and the human immune system( 2017)Harnessing nanomaterials for the benefit of human health has the potential to improve drug delivery, vaccination and diagnostic imaging. However, a greater understanding of the interaction between nanomaterials and the human immune system is required to improve the clinical translation of nanomedicines. Knowledge of the bio-nano interface has arisen largely from studies in cell lines and rodent models, and our poor understanding of bio-nano interactions in primary human systems remains a key knowledge gap in the development of clinical applications of nanomedicine. This thesis uses novel nano-engineered materials to characterise how material properties influence biological outcomes in primary human samples. By investigating the human bio-nano interface, this research has the potential to improve the rational design of novel nanomedicines. Blood is the first tissue encountered by nanomedicines following intravenous administration – the most common delivery method in the clinic. Therefore, human blood was used as both a source of primary blood cells to examine cell association, targeting and activation, and of plasma for the formation of complex biomolecular coronas. Flow cytometry and confocal microscopy were employed to characterise the role of key physicochemical properties of nanoparticles: charge, reactive surface chemistry, and targeting with antibodies and antibody fragments. A range of nano- engineered particles were developed including caveospheres, hyperbranched polymers (HBP), star polymers and pure PEG particles. Attempts were also made to determine how the biomolecular corona formed in human blood influences the observed bio-nano interactions. Using antibody-capture caveosphere nanoparticles, CD4+ and CD20+ human immune cells could be targeted within mixed cell populations following antibody- functionalisation. Moreover, functionalisation with anti-CCR5 antibodies enabled nanoparticle internalisation into HIV-tropic, non-phagocytic CD4+ T cells, a key hurdle in the delivery of nanoparticle-based anti-HIV therapeutics. Nanoparticle charge defined clear patterns of HBP association with blood cells. These patterns varied for nanoparticles of different material and size, and were not defined by the plasma biomolecular corona that forms in blood. Follow up studies demonstrated cationic, but not anionic or neutral, HBPs activated the myeloid subset of dendritic cells – an important cell target for vaccine applications. The effect of surface chemistry was examined using star polymers. Engineering thiol-reactive pyridyl disulfides onto star polymers directed their association with cancer cell lines, platelets (without activating them) and distinct immune cells subsets. Further studies using preclinical polymer vaccine nanoparticles demonstrated clear differences in blood phagocyte clearance based on brush vs. linear architectures of PEG. Lastly, immunologically stealth particles were functionalised with bispecific antibodies to evaluate cell targeting in the presence of complex biomolecular coronas and the impact of targeting moieties on particle stealth properties. Targeted stealth particles demonstrate potential for the targeted delivery of therapeutics or imaging agents in the presence of plasma coronas, with high specificity and only minimal disruption to particle stealth properties. Phagocytic uptake of PEG particles was dependent on the plasma biomolecular corona. Taken together, these findings further our understanding of the interactions between nano-engineered materials and the human immune system. Ultimately, the development of comprehensive human bio-nano principles will contribute to the rational design of novel nanomedicines.
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ItemInvestigating the interactions between dendritic cells, T cells and B cells mediated by targeting Clec9A( 2016)Dendritic cells (DC) are endowed with an array of receptors that can be exploited for immunotherapy. Targeted delivery of antigen to CD8α+ DCs via Clec9A in vivo induces versatile immune responses, most notably potent thymus-dependent humoral responses even in the absence of adjuvant. However, the basis of the immunogenicity of Clec9A-targeted antigen remains incompletely understood. This thesis describes the complex interactions between CD8α+ DCs and T and B cells mediated by Clec9A to promote and/or regulate immunity. Characterization of CD4+ T cells responding to Clec9A-targeted antigens revealed that they had the phenotype, localization pattern and effector functions consistent with T follicular helper cells (TFH) that provide B cell help. Furthermore, targeting Clec9A primed long-lived memory CD4+ T cells capable of robust secondary TFH responses, even in the absence of adjuvant. Thus, in the steady-state Clec9A-targeted CD8α+ dendritic cells are capable of stimulating CD4+ T cells to promote the development of fully polarized TFH cells. Strikingly, Clec9A was also found to mediate direct interactions between CD8α+ DCs and B cells. B cells were rapidly activated through recognition of native antigen presented on the surface of CD8α+ DCs upon Clec9A-targeted immunization. Direct activation of B cells by CD8α+ DCs was critical for optimal Clec9A-mediated antibody responses as it enabled B cells to effectively acquire help from cognate CD4+ T cells at the T/B borders within the spleen and lymph nodes. Thus, the effective triad of interactions mediated by Clec9A drives potent antibody responses in the steady-state. Unlike TFH and B cells that were potently activated in the steady-state, cross-priming of cytotoxic lymphocytes (CTLs) by Clec9A-targeted antigen required co-administration of adjuvant. In contrast to B cells, Clec9A-mediated primary CTL responses were impaired by the presence of CD4+ T cells. Clec9A-mediated MHC II-restricted presentation favoured the expansion of pre-existing Foxp3+ regulatory T cells (Tregs) in the steady-state, which presumably impaired non-Tregs capacity to activate CD8α+ DCs. Collectively, the data presented in this thesis reveal the versatile capacity of CD8α+ DCs to interact with various cell types to promote immunity/tolerance and reinforces the notion that targeting Clec9A in vivo is a promising strategy to exploit for immunotherapy.
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ItemUsing dendritic cell receptors to enhance immunity( 2017)Dendritic cells (DCs) are the most potent initiators of immune responses, being highly specialised for the uptake and presentation of antigens (Ag) to activate T cells. Their priming potential can be harnessed to generate stronger immune responses by targeting Ag to DCs via monoclonal antibodies (mAbs) specific for DC-expressed surface receptors. This thesis builds upon the concept of targeting DCs in two main ways: firstly, by investigating a novel method of targeting adjuvant to DCs, and secondly, by investigating how DC-targeting constructs can be used to prime and boost responses. It was considered whether not only Ag, but also adjuvants could be targeted to DCs to improve their efficacy. A recent finding that the DC receptor DEC-205 can bind to and mediate the immunostimulatory effects of CpG oligonucleotide (ODN) adjuvants led to the hypothesis that CpG ODNs could be targeted to DCs via DEC-205 in order to enhance their potency. The interaction between DEC-205 and CpG ODNs was further characterised to determine the molecular properties of ODNs required for binding. This information was then used to enhance the DEC-205 binding capacity of a particular CpG ODN that normally only weakly binds DEC-205. Enhanced DEC-205 binding was found to significantly improve the stimulatory capacity of this ODN, demonstrating that targeting adjuvant to DCs could be a viable method to improve adjuvant potency. Another receptor, CD14, has also been reported to bind CpG ODNs, so the potential for CD14 to act in synergy with DEC-205 was investigated. However, CD14 was not observed to mediate the uptake or stimulatory effects of CpG ODNs. The identification of natural ligands of DEC-205 is critical for understanding its physiological function. Although ODNs are synthetic molecules, their binding to DEC-205 may signify that DEC-205 is capable of binding other types of DNA that structurally resemble ODNs. A panel of biological DNA samples was screened for DEC-205 binding. While none of the DNA samples were observed to bind DEC-205, some DNA samples were found to bind another receptor, RAGE, suggesting a role for RAGE as a detector of both pathogenic and self-DNA. Most vaccines must be administered more than once, or “boosted”, to achieve optimal efficacy, and DC-targeted vaccines should be no exception. However, our data suggested that simply administering the same DC-targeting construct twice does not effectively boost the response. This was due to interference from the primary antibody response, which can cross-react with and neutralise a subsequently administered boosting construct. To overcome this issue, the efficacy of various heterologous prime-boost strategies designed to reduce the reactivity of the primary response against the boosting construct was assessed. Ultimately, a combination of anti-Clec9A and anti-XCR1 targeting constructs was found to induce the least cross-reactivity and strongest response after boosting. These findings contribute to the development of better adjuvants and immunisation strategies that optimise the efficacy of DC-targeted vaccines. More broadly, they also highlight the value of understanding the underlying biological mechanisms that drive immune responses, which can then be applied to the rational design of more effective vaccines.