Doherty Institute - Theses
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ItemFalling down the cascade: the gaps in care delivery for people living with chronic hepatitis B in Australia( 2017)In Australia, an estimated 239,000 people were living with chronic hepatitis B (CHB) in 2016. Most of the affected community were born overseas and acquired their infection early in life, in countries with high and intermediate prevalence of hepatitis B. The mortality attributable to CHB is from both liver cancer (hepatocellular carcinoma) and cirrhosis. Hepatocellular carcinoma has poor five-year survival, increasing incidence globally, and was projected to become the sixth most common cause of cancer death in Australia in 2016. The first “Global Hepatitis Health Sector Strategy” was signed in 2016 by all the member states of the World Health Assembly including Australia, with the aim of eliminating viral hepatitis, including hepatitis B, as a public health concern by 2030. The five studies presented in this thesis use the cascade of care framework to approach different aspects of the health system response to chronic hepatitis B in Australia. The studies have used different data sources and methodologies to measure the cascade and explore factors associated with the delivery of care. The first study presents an analysis of national data from 2012. It proposed, for the first time, a cascade of care for chronic hepatitis B in Australia that included a novel “enrolled in care” indicator. The second study presents findings from a multicentre retrospective study of adherence to antiviral therapy for chronic hepatitis B from 2010-2013. The study measured the proportion of people adherent to treatment in tertiary settings and analysed the demographic and health system factors associated with poor adherence. The third study analysed the association of a pharmacy-based adherence measure (the medication possession ratio) with viral outcomes using a time-to-event analysis for favourable and unfavourable viral outcomes. The fourth study presents findings from a retrospective analysis of primary care data in a community health centre that received external support from a tertiary service to improve the delivery of guideline-based care for chronic hepatitis B, including surveillance for hepatocellular carcinoma. This study evaluated four and a half years of data focusing on hepatocellular carcinoma surveillance participation and adherence. The fifth study presents findings from a qualitative study: semi-structured interviews of African-Australians living with chronic hepatitis B. This study explored participants’ understanding of health risks associated with hepatitis B, including their perceptions of their risk of developing hepatocellular carcinoma. Findings from this thesis have shown that few people living with chronic hepatitis B in Australia were enrolled in care. It provided the first multicentre estimates of the adherence of people on antiviral therapy for chronic hepatitis B in our health system (using medication possession ratio as the measure of adherence) and that factors associated with poor adherence were younger age and poor continuity of clinician. In a further study, the association between medication possession ratio and unfavourable viral outcomes was demonstrated for the first time. This analysis found that there was no true cut-off point or threshold to define adherence and the risk of poor outcomes. Rather, there was an increasing hazard ratio for unfavourable events with decreasing medication possession ratio. The findings also include results from a study that demonstrated hepatocellular carcinoma surveillance in a tertiary-supported general practice – with both participation and adherence to six-monthly scans with supported recall and reminder systems – is hard to achieve. Finally, the fifth study presented as part of this thesis found that African-Australians living with chronic hepatitis B perceived and experienced significant risks to social and emotional wellbeing from the shock of diagnosis, fear of infectiousness, and discrimination from telling others about their illness, as well as physical or liver-related problems. The results from this thesis have informed the development of the current Australian cascade of care for chronic hepatitis B and provided insights into the challenges of delivering health services to people living with chronic hepatitis B. These findings have led to recommendations for further development of the cascade at a national and regional level, and the need for further research and evaluation of the health system response to chronic hepatitis B. The work presented demonstrates that Australia has failed to meet the targets of the 2014-2017 National Strategy and needs to rapidly improve essential elements of the cascade, including increasing the proportion diagnosed and enrolled in care, to reach the targets of elimination of chronic hepatitis B as a public health concern by 2030.
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ItemInvestigating alternative strategies for the prevention of pneumococcal colonisation and disease( 2017)Streptococcus pneumoniae (the pneumococcus) is an important respiratory pathogen that colonises the human nasopharynx. While generally asymptomatic, colonisation is a prerequisite for disease. Infants are exposed to pneumococci soon after birth, carry the bacterium more often compared to older children and adults, and suffer the greatest burden of pneumococcal disease. Pneumococcal conjugate vaccines (PCVs) target the bacterium’s polysaccharide capsule. PCV use has dramatically reduced pneumococcal carriage and invasive disease caused by 10 – 13 of the > 90 antigenically-distinct pneumococcal capsule types (serotypes). However, this reduction has been coupled with an increased prevalence of non-vaccine-type pneumococci within vaccinated populations. Consequently, overall pneumococcal carriage rates have remained largely unchanged. In addition, PCV efficacy against pneumococcal pneumonia and otitis media (OM) is not comparable to that observed for invasive disease. This thesis aims to investigate how two alternative strategies to PCVs – probiotics and a pneumococcal whole cell vaccine – affect pneumococcal colonisation and disease. The probiotic bacterium Streptococcus salivarius is a commensal of the human respiratory tract that can inhibit pneumococcal adherence to epithelial cells in vitro. Here, a pharyngeal epithelial cell model was used to investigate how pre- administration of S. salivarius inhibits pneumococcal adherence. The megaplasmids of S. salivarius strains K12 and M18, which harbour known bacteriocins, were found not to be required to inhibit pneumococcal adherence in vitro, despite mediating the inhibition of pneumococcal growth on solid agar. Further experiments testing K12 and two pneumococcal isolates (serotypes 19F and 6A) suggest that K12 employs more than one mechanism to inhibit pneumococcal adherence. A significant inverse correlation was observed between K12 adherence and 19F adherence, and no inhibition was observed when K12-epithelial cell contact was prevented using a transwell system, suggesting some form of direct competitive inhibition by intact bacteria was taking place. In contrast, K12 inhibited 6A in the absence of epithelial cell contact, and no correlation was observed between K12 and 6A adherence, suggesting other, possibly secreted factor mechanisms were associated with inhibition of 6A adherence. The pneumococcal whole cell vaccine (WCV) consists of killed, unencapsulated pneumococci. In mice, WCV induces serotype-independent protection against colonisation and invasive pneumococcal disease via TH17 and antibody-mediated immunity, respectively. This study used infant mouse models to investigate the effect of WCV in two clinically relevant settings: influenza A virus (IAV)-induced OM, and in the presence of established pneumococcal colonisation (“therapeutic” vaccination). A single sub-cutaneous dose of WCV did not prevent the development of IAV-induced pneumococcal OM, but did significantly reduce the density of pneumococci in the middle ears of mice six days after IAV co-infection. Experiments performed in immune-deficient mice suggest that both CD4+ T cells and antibodies were required for this effect. When administered to colonised mice, a single dose of therapeutic WCV significantly reduced pneumococcal colonisation density in what was apparently an antibody-mediated manner. Prior colonisation enhanced the systemic WCV- specific IgG response, but not splenic IL-17A responses. Significant reductions in pneumococcal density were also observed in the nasopharynx and middle ears of therapeutically-vaccinated mice after IAV co-infection. Overall, this thesis contributes to our knowledge of probiotic mechanisms inhibiting pneumococcal adherence, and provides the first evidence for the therapeutic use of WCV. Therapeutic strategies to reduce disease severity would be in particular useful in low-income settings where infants more often experience early, high-density pneumococcal carriage and the highest burden of pneumococcal disease.
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ItemCirculating and local IFN-g-mediated immunity to Salmonella Typhimurium infections to mice( 2017)Bacterial infections that are chiefly caused by food or water contaminated with Salmonella enterica lead to major health-threatening problems worldwide. In developing countries, where sanitation conditions and health services are less than ideal, the risk of Salmonella enterica infections is often increased, particularly where immunosuppressing infections co-exist, such as HIV/AIDS and malaria. Immunodeficient individuals are more susceptible to opportunistic infections with invasive Non-Typhoidal Salmonella (iNTS) strains, including Salmonella enterica serovar Typhimurium (S. Typhimurium). In endemic areas, mass immunisation against enteric fever (i.e. S. Typhi) is one measure used to assist in disease control, notably where the isolation of multidrug-resistant S. Typhi strains is more frequent. To date, there has been no vaccine licensed for iNTS. An animal model of iNTS, using nrampse/se mice (i.e. C57BL/6) infected with S. Typhimurium, has provided valuable information about protective immunity to this systemic disease. Past studies using this mouse model suggests a crucial role of interferon-gamma (IFN- γ) produced by CD4+ T cells in the clearance of invasive forms of the disease, with other IFN-γ-producing subsets playing secondary roles. However, a systematic characterisation of the IFN-γ-producing cells in organs primarily infected by S. Typhimurium is absent from the literature. Although the cellular requirements for protective immunity against primary and secondary infections with S. Typhimurium in the murine model are well defined, this dissertation attempted to systematically assess the protective roles of IFN- γ-producing cells from Salmonella-infected organs during primary and secondary infections. Particular emphasis was given to the use of transgenic mice, adoptive transfer of lymphocytes, and antibody-based depletion to define the contributions of specific subsets to immunity against lethal S. Typhimurium infection. This study, which used an IFN- γ- reporter (eYFP) mouse, describes the formation of IFN-γ-competent T cells after primary infection with live-attenuated S. Typhimurium strains, estimated by the increased numbers of T cells in the spleen and liver, with high transcriptional activity of the Ifng gene, which correlated with the appearance of protective immunity to lethal, secondary challenge. This study also suggested there was traffic of IFN- + T cells from the spleen to the liver through the blood after the formation of the ‘plateau’ phase of the primary infection. Adoptive protection via the transfer of immune cells to naïve animals has proven to be a valuable approach to dissect immunological mechanisms of protection. This Thesis rearticulates that it is very difficult to protect naïve immunocompetent (C57BL/6) and lymphocyte-deficient (RAG2-/- γChain-/-) recipient mice via adoptive transfer of memory splenocytes alone from immunised mice. Conversely, splenocytes transferred from early after the initiation of the primary infection (i.e. week 2) could adoptively protect naïve RAG2-/-γChain-/- recipients from highly virulent S. Typhimurium challenge. Antibody mediated depletion assays demonstrated that the protection provided by splenocytes from 2-week immunised mice is dependent on IFN-γ-producing CD4+ Thy1.2+ cells, but not from CD8+ or NK1.1+ cells. The IFN-γ-eYFP mouse showed that CD4+ T cells account for the majority of the total IFN-γ + cells in the spleen two weeks after primary infection; the highest transcriptional activity of the Ifng gene as a marker for cytokine (i.e. IFN- γ) production was seen at this time point. Donor CD4+ T cells accumulated in the liver of naïve recipients by 24 hours post-adoptive transfer, although they redistributed to other organs after this time point. High proliferative rates, measured by loss of dye staining, were also observed in transferred IFN-γ+ CD4+ T cells in the spleen and liver of recipients, collectively, suggesting that protective Th1 cell-mediated immunity is present in the spleen early after the primary Salmonella infection, but not after clearance of the bacterium. The absence of protective T cell-mediated immunity to iNTS in the spleen after clearance of primary infection was investigated. IFN-γ-eYFP mice infected with, and subsequently cleared from, attenuated S. Typhimurium strains demonstrated increased accumulation of IFN-γ + T cells in the liver than in the spleen, which reflected enhanced local immunity in the liver. However, adoptive transfer of liver leucocytes from immunised mice failed to confer protection to RAG2-/- γChain-/- recipients, which was consistent with a failure of donor Th1 cells to survive in the new host. Phenotypical analysis showed that conventional Th1 cells in the liver express several surface markers consistent with liverresident memory T cells, including the survival signal molecules P2X7 and ARTC2. Specific inhibition of ARTC2 enzyme enabled adoptive transfer of immunity by Th1 cells from the liver, but not from the spleen, to naïve recipients challenged with lethal S. Typhimurium, which was consistent with the survival of liver Th1 cells in the new host. This novel adoptive transfer protocol also demonstrated that liver CD69+ Th1 cells preferentially relocated to the liver after transfer, suggesting strong affinity for the organ. In conclusion, this study has provided systematic analysis of IFN-γ-competent cells in secondary and non-lymphoid organs after primary infections with S. Typhimurium strains, which correlated with the appearance of protective immunity to secondary lethal challenge. New adoptive transfer protocols demonstrated that protective immunity mediated by IFN-γ-producing CD4+ T cells is present in the spleen early after primary infections. This capability declines during the vaccine clearance phase and exists in the liver at ‘memory’ stage. Furthermore, some Th1 cells in the liver were protective and showed phenotypical and functional features of tissue-resident memory T cells.
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ItemTranscriptional and epigenetic signatures of memory CD8+ T cell differentiation( 2017)Immunological memory is a hallmark feature of the adaptive immune system, providing superior protection against re-infection with previously encountered pathogens. Memory in CD8+ T cells is characterised by rapid effector function and expansion in response to challenge, resulting in efficient control of intracellular pathogens. The rapid effector function of memory cytotoxic T lymphocytes (CTL) is the outcome of differentiation; a process whereby transcriptional and epigenetic mechanisms reinforce changes to the constellation of active genes. Memory differentiation occurs in response to signals encountered during naive cell priming; these include antigen, costimulation, inflammation and CD4+ T cell help. Much has been learned in recent years about the transcriptional and epigenetic signatures of memory and effector CTL, and of the signals that promote memory development. However, an integrated understanding of how signals required for memory formation control CTL differentiation remains elusive. This thesis addresses this gap in our understanding by using models of memory-signal deficiency to probe the transcriptional and epigenetic responses to these signals. This thesis demonstrated that CD28 costimulation during in vitro antigen stimulation activates a higher proportion of CD8+ T cells to produce the cytokines interferon γ (IFNγ), tumor necrosis factor α (TNFα) and interleukin 2 (IL-2), and found also that increased Ifng messenger RNA (mRNA) production coincided with enrichment of trimethylation on lysine 4 of histone 3 (H3K4me3) at the Ifng locus. This suggested that the augmented IFNγ production induced by CD28 is regulated through deposition of H3K4me3, but given that this enrichment was transient, also suggests that additional signals are required to maintain it in effector and memory CTL. An unexpected finding was that high strength costimulation delayed the rate of cell division, which may indicate inhibition of division induced by costimulation in combination with high doses of antigen stimulation. And interestingly, whilst the overall effect of CD28 costimulation on transcription of CD8+ T cells early after activation was to amplify the changes induced by T cell receptor (TCR) signal, costimulation also suppressed some TCR-induced changes as well as uniquely modulating other genes. The net effect of CD28 costimulation is thus an augmentation of the effector transcriptional program, as well as modulation of some genes that control the balance between effector and memory fate decisions. A specific mutation to the transmembrane portion of the TCRβ chain has been reported to disrupt NFκB signalling and memory formation in CD8+ T cells. To investigate the regulatory consequences of this disruption, retrogenic mice expressing this mutation in the OT-I TCR were generated. In comparison to the wildtype OT-I TCRβ, the mutated TCRβ exhibited a reduced association with CD3, and mutant retrogenic cells had a lower per-cell expression of the TCR. Surprisingly however, after adoptive transfer into wildtype mice followed by influenza X31-OVA infection, memory formation appeared normal in the mutant retrogenic cells, with regards to both population size and function. Given that this mutation has been reported not to adversely affect memory formation in response to low affinity ligands, such as self-peptide-MHC, markers of homeostatic expansion in the naive retrogenic CD8+ T cells were evaluated. Compared to transgenic OT-I cells, naive retrogenic cells contained a higher proportion with markers of homeostatic expansion, namely CD44 and Ly6C. In spite of selecting only naive retrogenic cells without markers of homeostatic expansion, memory formation in mutant retrogenic cells remained normal. Thus, the TCRβ mutation does not disrupt memory formation in all model systems. Finally, this thesis re-iterated the finding that CD8+ T cell memory, but not primary responses to influenza infection are dependent upon help from CD4+ T cells. Whilst the absence of help did not diminish function of CTL in terms of cytokine and granzyme B production, it reduced the size of the memory and secondary responses and promoted a terminally differentiated phenotype. Overall, the effect of a lack of help on transcription in effector was CTL small, but included upregulation of anergy signature genes, and downregulation of naive and late memory signatures, suggestive of T cell dysfunction. Moreover, largely overlapping sets of genes were marked by H3K4me3 deposition in the absence of help, whereas those that exhibited broad H3K4me3 regions included genes dysregulated during both anergy and exhaustion. Meanwhile, more genes in unhelped cells exhibited H3K27me3 deposition within promoters, including those involved in differentiation and apoptosis. Together these findings demonstrated an overlap in the molecular regulation of the helpless phenotype with anergy and exhaustion, but also demonstrate a broad similarity in the regulation of helped and unhelped effector cells, indicating the importance of extending these studies into early effector and memory phases. Together, the findings in this thesis illustrate the impact of integrating multiple activation signals on the molecular regulation of CTL differentiation, and demonstrate that both chromatin and transcriptional regulation underlie this process.
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ItemThe clinical application of whole-genome sequencing to the surveillance, understanding, and control of bacterial infectious diseases in Australia( 2017)The scientific advance of whole-genome sequencing (WGS) has revolutionised the field of microbiology, with the ability to examine, characterise and compare organisms at a DNA sequence level. In recent years, the improving costs and workflows to sequence bacteria have resulted in WGS emerging as a powerful technology with the potential to enhance clinical and public health microbiology. However, at the commencement of this PhD, experience in clinical and public health institutions was limited, with only a few small-scale proof-of-concept applications in Australia. This thesis explores three different applications of WGS in a public health microbiology reference laboratory. For national surveillance of Listeria monocytogenes, a foodborne pathogen of public health significance, WGS provided superior resolution to current typing methods used to affirm potential outbreaks of Listeria. Additionally, typing results could be predicted in silico from the WGS data for backwards compatibility. The implementation of routine, prospective WGS in a national surveillance system for Listeria was feasible, though three important issues for future implementation were identified. Firstly, for comparing genomes of the same strain type, the use of a closely related reference genome produced a more accurate and higher resolution phylogeny than using a distantly related reference genome. Secondly, WGS identified nucleotide-level differences between isolates previously assumed to be "isogenic". Thirdly, the optimal method to define an outbreak with the genomic data was unclear. These issues were further explored in using WGS to understand sexual transmission of Neisseria gonorrhoeae between men who have sex with men (MSM), and the increasing rate of antimicrobial resistance (AMR) among Neisseria gonorrhoeae isolates. For 33 of 34 MSM couples infected with gonorrhoea, the same strain of Neisseria gonorrhoeae was found in both partners, and within individual MSM infected at multiple anatomical sites. However, subtle nucleotide differences were again observed between "within-host" and "within-partnership" isolates. Genetic determinants of AMR were able to be identified from the WGS data, with the absence of differences between within-host and within-partnership isolates suggesting that increasing AMR among gonococcal isolates is driven by transmission from sexual partners, and controlling transmission of gonorrhoea is critical to limiting the spread of AMR. Following this, the use of real-time WGS was investigated to assist with the state-wide control of another group of resistant pathogens, KPC-producing Enterobacteriaceae, in Victoria, Australia. Due to the complexity of the outbreak, combined genomic and epidemiological analyses provided greater insight into the outbreak than the data from each approach separately. These analyses defined distinct transmission networks arising from multiple importations into Victoria in separate healthcare facilities, allowing more targeted public health interventions. Additional insights into the outbreak were gained through long-read sequencing to track interspecies plasmid transfer, multiple colony selection for sequencing to capture within-host diversity, and Bayesian evolutionary and transmission modelling to estimate the transmission tree, informing public health responses to the outbreak. These studies provide a framework for the implementation of WGS in public health microbiology in Australia, with the tools developed and datasets derived through this work adding to the resources available to assist future clinical applications of WGS.
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ItemAn evaluation of charged Pam2Cys-based lipopeptides as novel adjuvants for subunit-based vaccines( 2017)The use of subunit antigens in modern vaccines generally requires the addition of an adjuvant due to the lack of immunostimulatory features that would otherwise allow them to induce strong immune responses. A problem facing the use of classical adjuvants such as Alum is that it is not known for inducing effective Th-1 responses that are important for clearing viral and intracellular bacterial infection. There is a shortage of adjuvants that are appropriate for use in animals including humans and as a consequence there is a wealth of research being carried out to discover and design novel and safe ways of delivering vaccines that will induce strong antibody- and/or cell-based immune responses. The TLR2 agonist-based adjuvant R4Pam2Cys is a synthetic adjuvant with a branched structure that has been shown to enhance humoral and also CD8+ T cell responses when formulated with soluble protein antigens. The studies presented in this thesis describe experiments that were designed to improve the economies of chemical synthesis, with a view to improving manufacturing conditions, and to better understand the role played by geometry of the final Pam2Cys-based adjuvant in its in vitro and in vivo biological activities. The results demonstrate that the efficiency of synthesis process could be improved without affecting biological activity by solid phase assembly of the lipid moiety prior to assembly of the R4 moiety. This also saved consumption, and hence costs, of reagents. The branched structure of the peptide portion of R4Pam2Cys was shown to confer resistance to trypsin hydrolysis and to play an important role in providing optimum binding to protein antigens. Finally R4Pam2Cys was compared with the clinically approved and commercially available hepatitis B virus (HBV) vaccine in which Alum is used to improve immunogenicity of the HBV antigen HBsAg. Clear immunological benefits were apparent using R4Pam2Cys over Alum; not only were better antibody titres obtained but robust CD8+ T cell responses were elicited by HBsAg-R4Pam2Cys complexes whereas the commercially available vaccine elicited few if any CD8+ T cells. The use of R4Pam2Cys also resulted in an improvement of protective efficacy in an animal model against HBV infection when used in prophylactic and therapeutic settings. These are promising findings and encourage the lipopeptide’s future application in development of an HBV therapeutic vaccine.
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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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ItemInteraction of mouse norovirus (MNV) with the cellular immune response of host cells( 2017)Human noroviruses (HuNoV) cause the majority of non-bacterial gastroenteritis cases worldwide and generate an economic burden of 60 billion USD every year. Noroviruses are highly infectious and predominantly cause issues in closed environments such as cruise ships, hospitals and nursing homes. Due to the lack of a tissue culture or small animal model, HuNoV research has been impaired and so far no drug treatment or vaccine is available. Despite recent advances in the field and the successful replication of HuNoV in B cells and human intestinal organoids, models of HuNoV replication in vitro still remain to be established. Fortunately, murine norovirus (MNV) was discovered in 2003 and has since been used as a model system to investigate NoV infections. In this study we show that MNV infection reduces the surface expression of MHC class I proteins. The reduction in MHC class I levels on the cell surface is based on reduced intracellular levels of the protein. We reveal that MHC class I transcription is not reduced during MNV infection, implying that either MHC class I translation is affected or MHC class I proteins are degraded during MNV infections. We were able to partially rescue the surface expression of MHC class I proteins on MNV infected cells with MG132, a proteasome inhibitor. These findings indicate that MNV interferes with the MHC class I pathway in either directly degrading the protein or targeting it for the degradation pathway within the cell. Furthermore, we identified MNV NS3 as the viral protein which is essential and sufficient for the MHC class I surface reduction when separately expressed in cells. Additionally, we investigated the effect of MNV on cytokine secretion. The secretion of the cytokines IFNβ and TNFα is significantly reduced in MNV infected cells, which is not due to a down regulation of cytokine mRNA transcription. Analysis of the intracellular expression of cytokines and host cell translation in general, revealed a continuous decrease in global host cell translation in MNV infected cells. The translational shutdown seems to be induced by the dsRNA-sensitive regulator PKR. PKR becomes phosphorylated and phosphorylates the translation initiation factor eIF2α, impeding host cell translation. Whilst the translation of host proteins is stalled, viral proteins are still able to be translated due to a cap-independent mechanism. Furthermore, we interrogated the interaction of MNV with the microtubules and the microtubule-associated protein GEF-H1. We discovered an interaction of GEF-H1 with the viral protein MNV NS3, which leads to changes in the expression levels and location of GEF-H1 within the cell and prevents the formation of GEF-H1 induced microtubule fibres. This indicates a potential interference of MNV NS3 with GEF-H1, which has been proposed to play a major role in the immune detection of viral replication. Despite various approaches to identify a similar role of GEF-H1 during MNV infection, we have so far not been able to support the proposed function of the protein. Considering the multiple roles of GEFs like GEF-H1, it is possible that MNV and specifically NS3 acts on a different GEF-H1-regulated pathway during MNV infection.
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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.