Microbiology & Immunology - Theses
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ItemRole of the chemokines CCL17 and CCL22 in the immune defence against Salmonella infection( 2019)The chemokines CCL17 and CCL22 are both ligands of the chemokine receptor CCR4, which is expressed on dendritic cells (DC) and a variety of different effector T cells including regulatory T cells (Treg). Both chemokines are mainly produced by DC, but also by macrophages. CCL17 promotes numerous inflammatory and allergic diseases, whereas CCL22 is rather associated with an immunosuppressive milieu. These differential roles are reflected by preferential recruitment of distinct subsets of T cells to site of inflammation. While CCL17 facilitates chemotaxis of effector T cells and supports DC-T cell interactions as well as DC migration towards CCR7-ligands, CCL22 induces chemotaxis of Treg cells. In addition, CCL22 signalling induces a more rapid desensitisation and internalisation of CCR4 than CCL17, suggesting biased agonism of CCL17 and CCL22. The functionality of CCL17 and CCL22 should, therefore, be considered in combination as well as individually in the context of immune-related diseases. The role of CCL17 and CCL22 in infectious diseases has not been well understood. The central hypothesis was that CCL17 and CCL22 play important but potentially different roles during bacterial infection. This was modelled using a well-studied bacterial pathogen, Salmonella enterica serovar Typhimurium (STM). It was hypothesised that CCL17 expression may direct the migration of STM-infected DC from the gut to draining lymph nodes a key bottleneck in early infection that controls bacterial dissemination to systemic sites. It was further hypothesised that CCL22 may play a role in immune regulation through the induction of Treg cells. These regulatory cells may have downstream effects on Th1 responses, which are critical for the control of Salmonella infection. In the first part of the thesis, the role of CCL17+ DC in the transmission of STM was investigated. Histological analysis of CCL17 reporter mice revealed that CCL17-expressing cells co-localised with Salmonella in the dome area of Peyer’s patches (PP). Further, CCL17-expressing DC contributed to dissemination of STM from PP to the mesenteric lymph nodes (mLN). Within the mLN, STM were found within CCL17+ DC as well as in other DC, monocytes and macrophages. Analysis of the STM+ DC subpopulations revealed that all DC subsets carried STM, but the CD103+ CD11b- DC could be identified as the main STM-containing population. STM infection triggered upregulation of CCL17 expression in specific intestinal DC subsets in a tissue-specific manner. Interestingly, the CD103+ DC subsets upregulated CCL17 in the PP, whereas CD103- DC subsets upregulated CCL17 in the mLN. In the second part of this thesis, the role of CCL17 and CCL22 in the induction of antigen-specific CD4+ T cell responses was investigated. CCL17/CCLL22 double-deficient, CCL17- and CCL22 single-deficient, and wild type mice were analysed after live-attenuated STM TAS2010 vaccination, vaccination/challenge and in steady-state. Mice deficient in both chemokines, CCL22 and CCL17, demonstrated a reduction of effector Treg cells. This promoted an enhanced STM-specific Th1 immune response characterised by an expansion of Th1 T cells, resulting in a more favourable effector Treg/activated Tconv ratio and a significantly improved vaccine efficacy to challenge with virulent Salmonella. In conclusion, the work presented within this thesis showed the contribution of CCL17+ DC in the dissemination of STM and identified CCL22 as a potential target to improve vaccine approaches.
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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.
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ItemIncreasing the efficacy of subunit vaccines with synthetic adjuvants( 2016)The results of experiments in which two different vaccine delivery systems, demonstrating the safety, economy of dose and the ability to induce antibody and CD8+ T cell responses using model antigen and real world antigens are described in this thesis. A chitosan polymer-based depot system was shown to induce a long-lasting antibody-mediated immune response that was maintained for at least one year following inoculation with a single dose of vaccine. Furthermore, the vaccine was able to facilitate antigen and adjuvant dose-sparing effects. A cationically charged vaccine delivery system, R4Pam2Cys and its PEGylated form, R4Pam2Cys-PEG were also investigated. PEGylation of R4Pam2Cys was shown to reduce the size of vaccine particulates and enhanced the expansion of primary antigen-specific CD8+ T cells. The PEGylation of R4Pam2Cys provided a vaccine candidate which suppressed tumour growth and improved survival time. Finally, the efficacy of a whole inactivated influenza virus (WIV) preparation formulated with R4Pam2Cys was shown to induce influenza-specific antibody and also cross-protective CD8+ T cells which protected against homologues and heterologous influenza virus challenge. A dose-sparing effect was also observed using WIV which could be of great assistances during pandemic outbreaks when vaccines are often in short supply. The findings described in this work highlight the importance of the use of appropriate vaccine delivery systems to activate appropriate arms of the immune response.
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ItemSalmonella Typhimurium metabolism in the murine host and importance to virulence( 2014)The bacterial pathogen Salmonella enterica is responsible for considerable global morbidity and mortality, being the cause of several enteric and systemic diseases, including typhoid fever. Non- typhoidal Salmonella (NTS) serovars such as Typhimurium cause gastroenteritis in immunocompetent individuals, but in the context of HIV-co-infection can cause invasive disease with high fatality rates. Successive generations of antimicrobials have become ineffective against S. enterica pathogens due to the widespread development of resistance, and new drugs are critically needed. In addition, vaccines against typhoid fever have sub-optimal efficacy and no human NTS vaccines are currently available. Although the in vitro metabolism of S. enterica is relatively well defined, little is known about the specific nutrients that the pathogen consumes in its hosts, and the metabolic mechanisms by which S. enterica utilise these nutrients. Therefore, the central aim of this study was to investigate and characterise S. enterica serovar Typhimurium metabolism in the murine host. Attention was focused on two areas of S. Typhimurium metabolism which have been speculated to contribute to bacterial virulence: methylglyoxal detoxification and central carbon (sugar) catabolism. It was anticipated that this study may contribute to better defining the metabolic requirements of S. Typhimurium during infection and disease, and present opportunities for the identification of potential drug targets and metabolically-attenuated strains amenable to use as live vaccines. Several studies have ventured that methylglyoxal detoxification mechanisms are required for survival of bacterial pathogens in the mammalian host, but this hypothesis has not been thoroughly tested. In the current study, although S. Typhimurium mutants defective in the glutathione-dependent glyoxalase system (GDGS) or Kef-mediated potassium efflux were highly sensitive to methylglyoxal, they were not attenuated for intracellular replication and growth in mice, suggesting that these methylglyoxal detoxification mechanisms are not required for S.Typhimurium pathogenesis in the mammalian host and are not suitable targets for antimicrobial therapy against S. Typhimurium disease. While others have reported that the Embden-Meyerhof pathway (EMP) and the ability to utilise glucose are required for S. Typhimurium virulence in mice, the importance of other sugars and carbon catabolic pathways to S. enterica virulence is unclear. In this study, S. Typhimurium mutants blocked in several sugar catabolic pathways including the Entner-Doudoroff pathway (EDP) were found not to be attenuated for intracellular growth or fulminant infection of mice, demonstrating that gluconate, glucuronate, galacturonate are not essential carbon sources for S. Typhimurium in vivo. However, evidence was presented which suggested that the ability to utilise gluconate and glucose is required for optimal shedding of the pathogen in the murine faeces, revealing potential strategies for reducing the faecal-oral transmission of S. Typhimurium. EMP/EDP double mutants showed greater attenuation in mice than a EMP mutant, suggesting that the EMP mutant utilises the EDP in order to facilitate it’s modest growth in vivo. These findings demonstrated the functional redundancy of S. Typhimurium metabolism and suggested that combinational drug therapies targeting several bacterial pathways concurrently might be a viable option for treating S. Typhimurium disease. The S. Typhimurium EMP/EDP mutant TAS2010 was found to provide increased, long-term protection from virulent infection in a murine typhoid vaccination model than the prototypical aro-negative vaccine strain BRD509. Given that the lack of effective vaccines against S. enterica pathogens can be largely attributed to an insufficient understanding of the host immune response to the pathogen, the immunological response to the TAS2010 vaccine strain was characterised in mice to understand the mechanisms responsible for the increased protective capabilities of this strain. In comparison to BRD509, TAS2010 was found to replicate to higher numbers in murine organs and induce an increased immune response in the form of increased interferon-gamma secretion by splenic CD4+ T cells. Evidence was presented which suggested that the protection provided by TAS2010 is less reliant on T cells and more dependent on a CD4-CD8-Thy1+ lymphocyte subset, probably Thy1+ NK cells. In conclusion, this study has enhanced the understanding of S. Typhimurium metabolism in the murine host and introduced a live vaccine strain with improved protective and immunogenic properties.
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ItemHepatitis C virus envelope glycoprotein E2: isolation and characterisation of a functional core domain( 2010)Hepatitis C virus (HCV) is a major indicator of liver disease and over 200 million chronic infections are estimated worldwide. No vaccine is available and current treatments report limited efficacy. A strong neutralizing antibody response is a key determinant in clearance of acute HCV infection although often appears delayed as broadly neutralizing antibodies do not appear until after chronic infection is established. Recent studies suggest that HCV evades neutralization through the accumulation of immune escape variants within a highly variable, immunodominant sequence of the viral envelope glycoprotein E2. The E1 and E2 envelope glycoproteins are transmembrane proteins that are embedded in the viral membrane and mediate virus attachment and entry into liver cells. The E2 glycoprotein has been shown to interact with the cellular receptors scavenger receptor class B type 1 (SRB1) and CD81 during virus entry and is a major target for neutralizing antibodies. There is currently no high-resolution structure of the HCV E2 glycoprotein to further understand its mechanism of viral entry or immune evasion. However, a soluble E2 ectodomain fragment has been identified that can be efficiently secreted from cells and displays CD81 receptor-binding function. The E2 ectodomain has three discrete variable regions interspersed between conserved CD81-binding motifs: the immunodominant hypervariable region 1 (HVR1), hypervariable region 2 (HVR2) and the intergenotypic variable region (igVR). In this study, simultaneous substitution of these variable regions (VRs) with short, flexible linker motifs within diverse E2 ectodomain sequences was described with the retention of both native folding and CD81 binding function. This indicated that the E2 VRs are excluded from the functional core domain of the glycoprotein. The conserved E2 core domain, lacking all three VRs, was further shown to elicit higher titers of broadly neutralizing antibodies than the unmodified E2 ectodomain suggesting that the VRs occlude conserved neutralization-sensitive epitopes within the underlying core domain. The E2 VRs were demonstrated to modulate CD81 binding in a manner consistent with solvent-exposed structures and illustrated a potential mechanism by which the VRs could mediate immune evasion during acute infection. Therefore, the conserved E2 core domain was proposed to represent a novel and improved antigen for redirecting the immune response towards conserved, neutralization-sensitive epitopes within the E2 glycoprotein. Further biochemical and functional characterization of the E2 core domain is also presented towards optimization of this antigen as viable candidate for future vaccine trials.