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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ItemStrategies for the elicitation of broadly neutralising antibodies to the HIV-1 envelope protein( 2017)An effective prophylactic vaccine for HIV will likely require the elicitation of neutralising antibodies directed towards the Envelope protein (Env) of HIV. In particular, broadly neutralising antibodies (bNAbs) capable of mediating neutralisation against a wide variety of HIV strains would be desirable. bNAbs frequently contain a large degree of affinity maturation, required for the development of their neutralisation breadth, thus the induction of affinity maturation during vaccination may be crucial for the elicitation of bNAbs. This thesis aimed to investigate strategies to enhance bNAb elicitation, in particular, to enhance the affinity maturation of anti-Env antibodies. This was attempted by immune targeting of Env to Clec9A and CR2, which have previously been shown to enhance affinity maturation. Targeting to CR2 was achieved by fusing Env to its ligand, C3d, although this was found to be ineffective at enhancing immunogenicity with the soluble protein constructs assessed. Targeting Clec9A was initially investigated using an anti-Clec9A scFv fused to Env, however when this was found to be unable to bind cell-surface Clec9A, the targeting domain was re-engineered as an anti-Clec9A scFab. While cell-surface Clec9A targeting was achieved successfully, this did not alter the parameters of Env immunogenicity measured. This may have been impacted by the immunodominance of the targeting domains, which future studies will need to address. The conserved epitopes of bNAbs are often poorly exposed, and this contributes to the difficulty in eliciting antibodies against these sites, which are often outcompeted by higher affinity interactions directed towards variable regions of Env. Therefore a novel mutation, ΔN, was investigated for its ability to enhance the exposure of bNAb epitopes in soluble Env constructs. The introduction of the ΔN mutation into SOSIP constructs of the AD8 Env strain enhanced the exposure of the epitopes for multiple bNAb specificities. An immunogenicity study in guinea pigs revealed that AD8 ΔN SOSIP elicited significantly higher titres of antibodies able to block the binding of bNAbs whose epitope exposure was enhanced in this protein. By contrast, ΔN-mediated epitope enhancement and preferential bNAb-like antibody elicitation was not observed with a BG505 strain SOSIP immunogen. Thus, the redirection of the immune response to produce bNAb-like specificities by ΔN appears to correlate with its ability to enhance bNAb epitope exposure in the SOSIP immunogen. The majority of bNAbs are extensively mutated such that most Env strains cannot bind to their precursor antibodies, thus identification of Env immunogens able to bind bNAb precursors is required. A panel of Envs isolated early during infection were screened for interaction with multiple bNAb precursors. This screen identified an Env strain, SC45, able to mediate low binding of the precursors of multiple bNAbs when it is expressed in a membrane-bound form. Expression of soluble SOSIP SC45 abrogates the binding to bNAb precursors, however this protein displays favourable biophysical characteristics desirable in a vaccine immunogen. The introduction of the ΔN mutation into SC45 SOSIP results in a large enhancement in PGT121 epitope exposure, and SC45 SOSIP ΔN is, therefore, a highly promising vaccine candidate.
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ItemDevelopment of virus-like particles as immunogens for HIV-1 envelope glycoprotein( 2017)A prophylactic vaccine eliciting broadly neutralising antibody (bNAb) responses against HIV-1 envelope glycoprotein (Env) would be optimal to prevent HIV-1 transmission. Replication incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure to the immune system to elicit humoral responses against Env. VLP-associated Env better resembles the viral membrane-bound Env encountered by the immune system during HIV-1 infection than recombinant forms of the glycoprotein. This is likely to be critical for induction of bNAb responses. As Env is highly glycosylated, the expression of VLPs bearing a native N-linked glycosylation profile is also important, especially since many known monoclonal bNAbs incorporate N-linked glycans (N-glycans) into their epitopes. The glycosylation profile of Env is heterogeneous with both populations of typical mammalian N-glycans (complex) and under-processed forms (high-mannose). Furthermore, this profile differs depending on the format of Env used, with virus-associated Env bearing predominantly high-mannose N-glycans whereas recombinant Env is decorated with a greater proportion of complex N-glycans. Here, the viral and expression system factors potentially influencing the differing glycosylation profile were investigated. Recombinant AD8 strain gp120 Env was found to bear a greater proportion of high-mannose N-glycans than when expressed on a viral membrane. The virus-associated Env glycosylation was not influenced by the presence of HIV-1 accessory proteins nor the cell-culture conditions during virus expression. Comparison of the glycosylation profile of recombinant and virus-associated Env using the AD8 and JR-CSF strains, suggested that distinct N-glycan profiles may not be universally conserved for all HIV-1 isolates, although further analysis on a wider range of Env strains is required to confirm this observation. An existing single-plasmid VLP expression vector, based upon DNA T cell vaccine plasmids that were proven safe in human trials, was optimised to maximise Env incorporation and particle budding. The unmodified expression cassette generated VLPs with incomplete protease-mediated cleavage of group specific antigen (Gag) and were irregularly sized. The introduction of alternative mutations that completely removed the reverse transcriptase domain, but preserved most other safety mutations, enabled efficient production of protease-processed, mature-form VLPs (mVLPs). Trimeric Env that presented multiple bNAb epitopes was incorporated into mVLPs, which were capable of viral fusion activity at a level approaching that of wild-type virions. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). The presentation of bNAb epitopes on the HA-TMCT-modified Env was retained, with the exception of some membrane-proximal epitopes. The mVLP-associated Env was stabilised via the introduction of a trimerisation point mutation and disulfide bonds between Env subunits (SOSIP), which improved the presentation of quaternary bNAb epitopes and diminished the exposure of poorly neutralising antibody sites. Vaccination with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralisation activity. These mVLPs are potentially useful immunogens for eliciting neutralising antibody responses that target native Env epitopes on fully-infectious HIV-1 virions.