Microbiology & Immunology - Theses
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Melanoma immunosurveillance by CD4+ T cells
The immune system can recognise and control cancer cells in a process termed cancer immunosurveillance. There is increasing evidence that CD4+ T cells play an important role in melanoma immunosurveillance but considerable debate surrounds the underlying anti-tumoral mechanisms. This project thus sought to unravel the role of CD4+ T cell responses to melanoma using a transplantable orthotopic murine melanoma model in conjunction with newly generated genetically modified B16 melanoma cell lines. Remarkably, adoptive transfer of naive or activated antigen-specific CD4+ T cells was highly protective against the development of melanoma. In addition to a classical “helper” function, CD4+ T cells acted as peripheral anti-tumoral effector cells whereby they migrated into the skin, differentiated into Th1 cells and mediated local suppression of tumor development. Accordingly, we provide evidence that CD4+ T cells can directly kill melanoma cells in vitro through several cytotoxic pathways, including TNF superfamily signalling via TNF and FasL as well as perforin-dependent cell lysis. Finally, we investigated the role of MHC-II expression by melanoma on the antitumoral function of CD4+ T cells. Whilst MHC-II expression by melanoma cells promoted CD4+ T cell infiltration into the primary tumor site it was dispensable for control mediated by CD4+ T cells. This suggested an important role for indirect display of MHC-II-restricted epitopes by antigen-presenting cells within the tumor microenvironment. This was supported by visualization of melanoma-specific CD4+ T cells in the tumor microenvironment using two-photon microscopy, where activated CD4+ T cells appeared to interact with melanoma cells via intermediary cells, presumably professional antigen-presenting cells. Finally, we observed a reduction in metastatic lesions in the tumor-draining lymph node in mice challenged with MHC-II deficient melanoma cells. These data suggest that MHC-II may play context-dependent roles in control of primary tumors and lymph node metastases by CD4+ T cells. In summary, this study demonstrates the important role of CD4+ T cells in melanoma immunosurveillance and provides important insights into underlying antitumoral mechanisms.
Generation of protective immunity against severe influenza virus infection in Indigenous Australians
Morbidity and mortality rates from seasonal and pandemic influenza virus infections occur disproportionately in high-risk groups, including Indigenous people globally. Although adaptive immunity is essential for combating viral infections, it is largely understudied in Indigenous populations, including Aboriginal Australians. To rationally design a protective vaccine against both seasonal and pandemic influenza viruses for both Indigenous and non-Indigenous people, in-depth knowledge about B cell and T cell responses is needed. Therefore, the aim of this PhD thesis was to identify and characterise CD8+ T cell responses restricted by HLA alleles dominant in Indigenous Australians as well as understand CD4+ T cell, B cell and humoral responses to the inactivated influenza vaccine in Indigenous and non-Indigenous Australians. The data generated in this PhD thesis provide key insights into a rational design of a cross-protective influenza vaccine. The HLA-A*24:02 allele was associated with a higher mortality during the 2009 influenza pandemic and is highly expressed in Indigenous populations globally. In Chapter 3, we identified novel influenza A and B virus (IAV and IBV, respectively) CD8+ T cell targets presented by HLA-A*24:02 and characterised their immunogenicity in HLA-A*24:02 transgenic mice, and HLA-A*24:02+ Indigenous and non-Indigenous individuals. We discovered the first IBV-derived CD8+ T cell epitopes presented by HLA-A*24:02. A total of 6 novel immunogenic IBV epitopes were identified, with one epitope, A24/PB2550-558 being cross-reactive between IBV and the IAV variant, making it especially important for a cross-protective universal vaccine. We defined epitope-specific CD8+ T cells ex vivo in the blood of healthy Indigenous, non-Indigenous and acutely-infected donors. These newly identified epitope-specific CD8+ T cells were readily detected during acute infection, mainly of an effector memory phenotype and expressed activation markers such as PD-1, CD38, HLA-DR and CD71, thus highlighting their involvement during influenza virus infection. Sub-cutaneous vaccination of transgenic mice with three immunogenic IBV peptides resulted in reduced viral titres, reduced pro-inflammatory cytokines MIP-1a, MIP-1b and RANTES in the lung as well as reduced weight loss in comparison to unvaccinated mice. These data highlight the importance of identifying novel CD8+ T cell epitopes for their potential use as a universal influenza vaccine in the context of a highly prevalent HLA allele in Indigenous people globally. Indigenous Australians express a unique HLA class I profile which includes high frequencies of HLA-A*24:02, A*11:01, A*34:01, B*13:01 and B*15:21. Except for HLA-A*24:02 and HLA-A*11:01, no influenza virus-derived epitopes have been identified for the Indigenous-associated HLAs. In Chapter 4, we generated single HLA-expressing cell lines to use as antigen-presenting cells for identifying and characterising CD8+ T cell responses towards the Indigenous-associated HLAs. We optimised an influenza virus infection assay, instead of using peptide pool cultures, to stimulate and expand rare influenza-specific CD8+ T cells, and thus allow sufficient numbers for the sequential dissection of single epitope candidates. As such, we identified the first IAV and IBV epitopes presented by HLA-B*13:01. While responses to IAV were dominated by the highly variable B13/NP404-412 epitope, IBV-responses were directed towards a variety of epitopes, including the dominant and highly conserved B13/HA371-379 and B13/HA427-435 peptides. We sequenced IAV- and IBV-specific CD8+ T cell receptors and identified unique TCR signatures between the different epitopes. While B13/NP404-412-specific CD8+ T cells showed a limited bias for the expression of TRBV19 with a public TCR, B13/HA371-379-specific CD8+ T cells almost uniquely expressed TRAV3 paired with a variety of different TCRb-chains. The developed tools proved to be an efficient method to identify and characterise IAV and IBV epitopes presented by Indigenous-associated HLAs to further develop a universal influenza vaccine that can protect Indigenous Australians. Current influenza inactivated vaccine strategies delivered intramuscularly induce mainly B cell and antibody responses. Responses to the influenza vaccine are studied extensively but not fully understood in high-risk populations. Despite the strong recommendations for annual influenza vaccination, Indigenous Australians are completely underrepresented in studies that analyse vaccine responses. In Chapter 5, we defined cellular and humoral responses to the inactivated influenza vaccine in Indigenous Australians vaccinated between 2016 and 2018, and compared these responses to non-Indigenous Australians. We identified robust antibody responses to the vaccine that were comparable to non-Indigenous donors and were cross-reactive with viruses circulating more than 10 years ago. Antibody responses on day 23+ post vaccination correlated with acute activation of circulating T follicular helper cells type 1 (cTFH1) in line with previous studies. System serology of selected Indigenous and non-Indigenous donors revealed significant reduction of the contribution of IgG3 to influenza-specific antibodies in Indigenous Australians, which correlated with a higher frequency of the G3m21 allotype. The generated data are important to support vaccine recommendations for Indigenous Australians, but also highlight the need to improve influenza vaccinations by harnessing the protective capacity of CD8+ T cells in future vaccine designs. Overall, this PhD thesis provides highly important knowledge of T and B cell immunity to IAV and IBV in Indigenous Australians. The findings of this PhD thesis provide key insights into the development of a universal influenza vaccine that also protects Indigenous Australians, one of the high-risk groups of developing severe influenza disease.
Understanding human B cell and antibody responses against seasonal influenza viruses
Vaccination is the best available means to reduce the burden of seasonal influenza. However, current influenza vaccines need to be updated frequently to keep up with evolution among circulating viruses. Antigenic evolution, otherwise termed drift, is most rapid among A/H3N2 viruses, and the A/H3N2 component of vaccines is frequently updated. Despite this, influenza vaccine effectiveness against the A/H3N2 subtype has been poor in recent years, especially among previously vaccinated individuals. Protection induced by inactivated influenza vaccines is largely mediated by B cells and antibodies reactive against the head of the hemagglutinin (HA) protein, with help from T follicular helper cells. The cellular and molecular mechanisms that underlie the attenuating effects of prior vaccination and existing immunity are largely undefined. It has been suggested that existing antibodies clear or mask antigen, or that memory B cells induced by prior exposures competitively dominate responses so that B cells and antibodies become focused on epitopes that are shared between prior and prevailing vaccine strains. The aim of the work presented in this PhD thesis was to examine the impact of pre-existing immune responses induced by prior infection with different A/H3N2 strains on influenza vaccine immunogenicity. In depth antibody as well as B cell assessments were performed to understand the impact of existing antibodies and memory B cells following vaccination and provide insights into the design of new vaccine strategies. As a lead up to the ex vivo analysis of B cells from vaccinees, we first sought to understand how human naive versus memory B cells differentiate in vitro. Experiments were conducted in Chapter 3 to compare the stimuli required for their differentiation into plasmablasts, and subsequently understand how they change phenotypically once stimulated. Specifically, sorted human naive and memory B cells from healthy individuals were stimulated in vitro to induce differentiation into plasmablasts. Data obtained in this PhD thesis showed that stimulation with the Toll-like receptor (TLR) 7/8 agonist R848 in the presence of monocytes induced the highest activation of both naive and memory B cells. Conversely, stimulation with the TLR9 agonist CpG or with R848 in the absence of monocytes induced little to no differentiation of naive B cells but were able to stimulate memory B. cell differentiation. Despite robust differentiation into antibody secreting plasmablasts, naive-derived B cells remained phenotypically distinct from memory-derived B cells up to day 6 after in vitro activation, with differential expression of CD27, CD38 and CD20. This work resulted in a first-author publication in Clin Transl Immunol, 2019. The focus of Chapters 4 and 5 was to understand how prior influenza virus infection affects antibody and B cell responses to influenza vaccination. To address this question, vaccine responses were investigated in a unique influenza vaccine-naive cohort in Viet Nam, that had been monitored for both clinical and asymptomatic influenza virus infection for more than 9 years. In 2016, twenty-eight participants without documented A/H3N2 virus infection (since 2007) and 72 participants who had been infected with A/H3N2 viruses, belonging to a range of genetic clades, received an inactivated trivalent influenza vaccine containing an A/Hong Kong/4801/2014-like (H3N2) antigen. This work investigated whether influenza vaccination induced naive B cell responses specific for new epitopes or largely recalled B cells specific for conserved epitopes, common to the vaccine A/H3N2 component and prior infecting strains. Hemagglutination inhibition antibody titres were measured in pre- and serial post-vaccination sera against 40 A/H3N2 viruses spanning 1968-2018 to understand how the titre and cross-reactivity of antibodies against the HA head evolve. B cells were assessed by flow cytometry using a panel of phenotypic markers in addition to recombinant HA probes representing the vaccine and recently infecting strains (A/Perth/16/2009, A/Victoria/361/2011 and A/Switzerland/9715293/2013). Participants who had at least one pre-vaccination A/H3N2 virus infection had on average 2 to 3-fold higher vaccine-specific antibody titres, steeper titre rises in the weeks following vaccination (mean peak on day 14), and less titre decay by days 21 and 280 compared to participants without prior infection. Moreover, participants with prior infection exhibited greater and better-maintained titre rises against viruses that circulated a year after vaccination, indicating that prior infection extends the strain coverage of antibodies induced by vaccination. Notably, A/H3N2 viruses that circulated 275-340 days after vaccination caused illness in only 1.4% of participants with infection prior to vaccination and in 14% of participants without prior infection. This suggests that vaccine effectiveness can be enhanced by pre-existing immunity. However, it was also clear that the range of strains against which antibodies were induced was dictated by the strain with which participants were previously infected, indicating that vaccination may simply recall rather than update antibody-mediated immunity. HA-probe reactive B cell frequencies and activation status increased substantially after vaccination. The greatest increases in HA probe-reactive B cells were detected among participants who had recent prior infection, with the majority of B cells exhibiting cross-reactivity with prior strains. A modest but significant increase in the frequency of B cells that reacted with the HA of the vaccine strain, but not of past strains, could be detected in participants who lacked prior infection. The phenotype of vaccine HA single-positive B cells, including increased IgM expression, indicated that they may have been naive-derived B cells. Vaccination induced B cells that preferentially reacted with the HA of A/Perth/16/2009 and/or A/Victoria/361/2011 viruses, but not A/Switzerland/9715293/2013 viruses, among participants who had prior A/Perth/16/2009-like virus infection. However, B cells induced by vaccination in participants who had prior A/Switzerland/9715293/2013-like virus infection were equally cross-reactive with HA of all tested viruses. These results support the inference that immune responses to standard inactivated influenza vaccines are dominated and shaped by recalled memory B cells with limited activation of naive B cells to update immunity. Overall, this PhD thesis investigated how pre-existing immunity induced by documented influenza virus infection affected the humoral response to seasonal influenza vaccines in healthy adults. This work provides new insights into the capacity of influenza vaccines to stimulate naive B cells, which may be limited due to memory B cell dominance and to a lack of sufficient stimulation to activate naive B cells. This knowledge could be used to design new vaccine strategies and improve influenza vaccine-induced protection.
Dynamics and control of T follicular helper cell-dependent and -independent responses to influenza virus infection and immunization
Seasonal influenza viruses circulate globally and cause recurrent disease in humans. Worldwide, annual epidemics are estimated to cause 1 billion infections, with 3 to 5 million cases of severe illness and 290,000 to 650,000 deaths. Influenza viruses undergo rapid antigenic evolution allowing mutant viruses to escape from host immune responses acquired to parental virus strains. Current seasonal influenza vaccines are effective when vaccine strains are matched with circulating strains. However, there is little to no cross-protection against antigenic variants, emerging pandemic or zoonotic outbreak strains. There is therefore tremendous interest in the development of novel universal vaccines which induce potent, broad and durable antibody responses against most or all influenza viruses. T follicular helper cells are crucial for the generation of high affinity antibodies and the maintenance of B cell memory. But relatively little is known about Tfh in important animal models of influenza. Insights gained from the study of Tfh cell responses will facilitate the design of next generation vaccines against influenza. In this thesis, we first developed an activation-induced marker assay for the identification of antigen specific Tfh cells in mice after influenza virus infection and hemagglutinin protein immunization. We showed that the AIM assay was robust and sensitive for the detection of murine Ag specific Tfh cells by quantifying the upregulation of surface CD154 or CD25 OX40 following either HA peptide pool or whole HA protein stimulation for 18 hours. This murine AIM assay makes it feasible to delineate Ag specific Tfh cells in mice without the need for transgenic mice or MHC II tetramers restricted to specific epitopes. Importantly, Ag specific Tfh cells can be sorted for TCR sequencing or adoptive transfer since AIM assay is a live cell assay. Ferrets are a well established animal model for influenza research and are widely used to investigate the pathogenesis and transmission of influenza viruses and preclinically evaluate the efficacy of influenza vaccines. However, little is known about ferret Tfh cells due to the lack of ferret reactive immunological reagents. To enable the study of ferret Tfh cells, we screened commercial markers of Tfh cells, antiBCL6, CXCR5 and PD1 antibodies, and found two anti-BCL6 antibodies had cross reactivity with lymph node cells from influenza infected ferrets. We also developed two murine monoclonal antibodies against ferret CXCR5 and PD1 using a single B cell PCR based method. We were able to clearly identify Tfh cells in LNs from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will facilitate the assessment of vaccine induced Tfh responses in the ferret model and the design of novel vaccines against influenza infection. HA stem is an attractive target for the development of universal influenza vaccines due to its relatively conserved feature. However, HA stem is poorly immunogenic when administered alone in a soluble form. Immunogen multimerization can enhance the immunogenicity of poor immunogens even in the absence of the help of T cells, which serves as an alternative pathway to improve the immunogenicity of stem without the dependence on Tfh responses. We showed that chemically coupling a peptide derived from the head domain of PR8 HA, P35, with the weakly immunogenic HA stem protein caused aggregation of the HA stem which significantly enhanced stem specific B cell responses independent of Tfh cell help in mice. P35 conjugation represents a new pathway to boost stem specific antibody responses without introducing exotic carrier proteins which will elicit anti carrier responses. Collectively, we investigated Tfh responses to influenza virus infection and immunization in mice and ferrets and explored the effects of immunogen multimerization on humoral immunity in the context limiting Tfh responses to HA stem. An increased understanding of Tfh dependent and independent mechanisms to enhance humoral immune responses will assist developing novel vaccines to prevent the infection of influenza and other viruses.
The biofilm formation and iron acquisition systems of Klebsiella pneumoniae
Klebsiella pneumoniae is an opportunistic bacterial pathogen and a common cause of healthcare-associated infections. Due to the emergence of antimicrobial-resistant and hypervirulent strains, K. pneumoniae is recognised as a major public health threat. The ability of some K. pneumoniae strains to form biofilms increases this concern, particularly in healthcare settings where bacteria can colonise surfaces of indwelling devices. Biofilms can mediate more effective host colonisation and bacterial cells within biofilms are often more resistant to antimicrobial treatments. Given the importance of biofilms in enhancing virulence and complicating treatment regimens, greater understanding of biofilm formation among Klebsiella isolates is required. This dissertation revealed that the K. pneumoniae clinical isolates AJ094, AJ097 and AJ218 exhibited different biofilm formation capabilities, and their biofilms had variable responses to DNase and proteinase treatment, suggesting qualitative differences. The early-stage biofilms formed by AJ094 and AJ218 could be destabilised by DNase, suggesting that the presence of extracellular DNA in the biofilm matrix was important for biofilm development. Dispersal of AJ218 biofilms by proteinase treatment indicated the importance of protein components such as fimbriae in maintaining the biofilm. Proteomic analysis revealed that cells were more metabolically active in the planktonic compared to the biofilm state, and differential expression of certain proteins suggested physiological variation between planktonic and biofilm cells. Proteomic experiments also showed that type 3 fimbrial proteins were expressed at higher levels in the biofilm state, particularly in the AJ218 strain, which is known to form biofilms via type 3 fimbriae. Some proteins expressed in the biofilm state were involved in metal ion uptake and AJ094, AJ097 and AJ218 were all shown to require iron supplementation for optimal growth and biofilm formation in minimal media. Mutation of an enterobactin-mediated iron acquisition gene in AJ094 significantly reduced biofilms under iron limiting conditions compared to iron-replete conditions. On the other hand, the siderophore yersiniabactin was shown to be less important than enterobactin for supporting in vitro biofilm formation and growth. Highly invasive Klebsiella strains often carry genes for yersiniabactin synthesis and, compared to enterobactin, this siderophore is less well-understood. A transposon mutagenesis approach was employed in this study to identify novel regulators and efflux systems for yersiniabactin. The study was successful in identifying known yersiniabactin-related factors, including genes located outside of the yersiniabactin operon that were directly or indirectly related to yersiniabactin metabolism and transport. During construction of the transposon mutant library, a spontaneous 70-kb chromosomal deletion occurred in the parent strain. The deletion mutant exhibited reduced siderophore activity when associated with a transposon insertion within a hypothetical gene (orf684). However, when the 70-kb region was present, inactivation of the orf684 gene no longer caused reduced siderophore activity. This study has improved our understanding of biofilm composition and the requirement of iron and siderophores in the formation of K. pneumoniae biofilms. Further research into fundamental characteristics of iron, siderophores and matrix components in regard to bacterial growth and biofilm formation may lead to the development of novel drugs or preventive strategies to reduce the burden of bacterial infection.
Characterisation of Neutralising and Functional Antibody Responses to Different HIV-1 Env Vaccines in Bovines
Two main challenges have impeded the development of an effective HIV-1 envelope (Env) vaccine, with antibodies eliciting neutralisation of virions as well as Fc-effector functions, such as antibody-dependent cytotoxicity (ADCC), phagocytosis (ADP) or complement deposition (ADCD). On one hand, designing the right Env vaccine to elicit humoral or cellular protection has been challenging and, to date, SOSIP-Env trimers which are covalently constrained in the closed, pre-fusion conformation are the best vaccine candidate over uncleaved (Unc), open-structured trimers. On the other hand, eliciting heterologous neutralising antibodies in several animal models (including humans) has been difficult. Cows nevertheless produce unique antibodies with long CDRH3 regions, capable of accessing neutralising epitopes beneath the glycan shield, inaccessible for other animals. We tested how differences in clade and/or structure of HIV-1 Env vaccines affect the neutralising activity and Fc-effector functions of antibodies elicited, using recombinant trimers of clades A (KNH1,BG505), B (AD8, PSC89) and C (MW), which exposed either an open structure (Unc gp140) or a closed structure (SOSIP gp140). KNH1/BG505 SOSIP gp140 vaccine elicited the best neutralising IgGs against heterologous tier-2 pseudoviruses with high potency and breadth. While AD8 Unc gp140 also induced neutralisation, it was against only tier-1 pseudoviruses. Nevertheless, it was the only vaccine able to elicit IgGs that engaged CD32 (FcgRIIa), induced phagocytosis and complement-activation. The different antibody profile observed with both vaccines was explained by the Env immunogen structure, as KNH1/BG505 SOSIP gp140 induced mostly IgGs targeting the V1/V2 loop, whereas AD8 Unc gp140 induced antibodies targeting CD4-binding site and CD4-induced epitopes. In addition, analysis of IgG repertoires from animals of KNH1/BG505 SOSIP 100 and AD8 Unc 500 groups showed that KNH1/BG505 SOSIP gp140 induced higher rates of somatic hypermutation in germline genes compared to AD8 Unc gp140, with each animal presenting a unique antibody profile, and with germline antibodies already presenting high affinity towards HIV-1 Env trimers, as high levels of affinity maturation were not required to obtain antibodies with high neutralising activity. Overall, the results in this work show that open structured trimers elicit antibodies which highly activate antibody-effector functions, while SOSIP trimers focus antibody responses to concealed neutralising epitopes. The high neutralising responses observed in bovines against HIV-1 Env are due to antibodies which do not need high levels of somatic hypermutations and, in particular for KNH1/BG505 SOSIP, this antigen induced high levels of affinity maturation, probably favouring the improvement of both binding and neutralisation. Our study suggests that an effective vaccine regimen may include both uncleaved gp140 and SOSIP gp140, in order to target epitopes required for antibody-dependent effector functions as well as neutralisation, or a new trimeric structure with flexibility in the gp120-gp41 interface, exposing both epitopes involved in Fc-effector functions as well as neutralising ones.
Inducing immunity to liver stage malaria through endogenous tissue resident memory cells
Tissue resident memory CD8 T (TRM) cells provide effective tissue surveillance and can respond rapidly to infection due to their strategic location. Within the liver, TRM cells can induce effective protection against liver-stage Plasmodium infection. Recently, members from our group identified a highly immunogenic peptide (named Pb 1) within the putative 60S ribosomal protein L6 of P. berghei ANKA. Experiments conducted and presented in this thesis aimed to assess the suitability of Pb 1 for the induction of endogenous liver TRM cells that confer sterilizing protection in B6 mice. To this end, a series of different immunisation strategies targeting the Pb 1 epitope were implemented and specific CD8 T cell responses were assessed. Results revealed that the number of naive specific CD8 T cell precursors for the Pb 1 epitope was very large. Substantial expansion and formation of specific liver TRM cells was achieved by two different immunisation strategies: i) Single injection with Clec9A mAb plus adjuvant and ii) Prime and trap, both targeting the Pb 1 epitope. While mice vaccinated with Clec9A mAb developed partial protection, almost all mice vaccinated with prime-and-trap targeting Pb 1 were sterilely protected against liver stage challenge. Inflammation favours the formation TRM cells and adjuvants can affect their numbers. Accordingly, a second focus of this thesis sought to investigate how to enhance liver TRM cell formation by using TLR and RIG I like receptors agonists as adjuvants. For this, eight different agonists were assessed for the generation of liver TRM cells induced by Clec9A targeted immunisation with the Pb 1 epitope. Data from this screen showed that CpG based adjuvants were most effective at inducing the formation of TRM cells in the livers of vaccinated mice and that the transfection reagent DOTAP enhanced this effect. Based on this understanding, we then investigated the potential of CpG and its encapsulation in DOTAP to improve TRM cell generation by other vaccination strategies. Surprisingly, these studies revealed that CpG based adjuvants did not improve liver TRM cell generation by vaccination with radiation attenuated sporozoites. The basis for this outcome is discussed. Altogether, these findings provide insights into elements that favour the generation of protective liver TRM cells; information that can be used for the design of TRM cell based subunit vaccines against Plasmodium infection.
Legionella pneumophila: from amoeba to macrophage metabolism
Legionella pneumophila is an aquatic bacterium that has emerged as an accidental human pathogen. Within the aquatic environment, L. pneumophila has evolved virulence factors to survive predation by environmental amoebae. These virulence factors are hypothesised to allow the adaptation of the bacteria to replicate in human alveolar macrophages. During infection, L. pneumophila forms a replicative vacuole termed the Legionella-containing vacuole (LCV) that sustains the bacterial intracellular replication. Establishment of the LCV requires the Dot/Icm type IV secretion system (T4SS), that secretes over 330 bacterial proteins termed effectors into the infected host cell in order to manipulate host processes and facilitate bacterial replication. Despite their central role in LCV biogenesis, to date most effector proteins remain uncharacterised. Therefore, to aid in the characterisation of Dot/Icm effector proteins, in this study, we generated large genomic region mutants. To date, we have created nine genomic deletion mutants (A-I), as well as two multiple-region deletion mutants (FGHI and DFGHI) in L. pneumophila, resulting in the deletion of 68 effector genes and 138 non-effector genes. These mutants were then used to identify the genomic regions important for bacterial replication in vitro and in vivo. Despite the loss of up to 42 effector-encoding genes, all mutants can replicate efficiently in THP1 macrophages. Meanwhile, in the protozoan host, at least six mutants showed a severe replication defect. Interestingly, in the mouse model, four mutants displayed an unexpected increase in bacterial burden, while one mutant showed a reduction in bacterial replication. Surprisingly, two of the mutants showing an increase in bacterial load in the mouse model were unable to replicate in Acanthamoeba castellanii. Together, these highlight the difference in requirements to survive in different hosts. This also suggests that the large effector repertoire of the Dot/Icm T4SS effectors likely evolved to enable an intracellular lifestyle in a diverse range of hosts. Finally, the mutants were also used to identify a Dot/Icm effector protein that degraded host GAPDH mRNA. RNA sequencing of infected cells revealed that L. pneumophila downregulated multiple host glycolytic mRNAs which depended on a particular Dot/Icm effector. Taken together, this project has used mutants carrying large genomic deletions to identify genetic regions important for bacterial replication, as well as those manipulating host immune defence.
Functional and cellular heterogeneity of the myeloid cell system
Cells of the myeloid lineage form the innate part of the immune system and are characterized by a high level of functional plasticity, which is required to address the diverse set of functions of these mononuclear cells. Monocytes, Macrophages and dendritic cells (DC) are collectively categorized as the mononuclear phagocyte system (MPS), to highlight their functional equipment that specializes them to the phagocytosis of pathogens as a starting point to elicit an immune response. Besides this role, cells of the MPS are also involved in a wide variety of homeostatic functions including early development and regulation of physiological processes. However, the multitude of mechanisms required to acquire this functional plasticity remains poorly understood. The work that has been performed in the scope of this dissertation aimed to advance current knowledge of the causes and consequences of functional and cellular plasticity of the myeloid immune system. High-dimensional characterization of the effects of a Western diet on myeloid immune cell progenitor cells revealed a long-term transcriptional and epigenetic reprogramming of the myeloid cell compartment. The formation of an innate immune memory in myeloid progenitor cells leads to lasting inflammatory priming of monocytes, which may directly contribute to the progression of myeloid cell-associated diseases. In addition, single-cell RNA-seq elucidated unreported cellular heterogeneity of the monocyte and dendritic cell compartment in human peripheral blood. A combination of phenotypic and transcriptional analyzes resulted in a precise categorization of the human DC compartment consisting of pDCs, cDC1, two cDC2 subsets, and a deeply characterized preDC subset. Furthermore, a universal strategy for the integration of cellular atlases was conceptualized and applied to establish a consensus map of the human DC and monocyte cell space. This thesis provides mechanistic insights into the cellular composition of myeloid cells and their functional plasticity, which will form the foundation for further investigations into the dynamic changes of the immune cell compartment during diseases and will be critically relevant for designing effective treatments for a wide variety of pathologies linked to myeloid cells.
Investigating the ability of mammalian Membrane Associated RING-CH proteins to modulate respiratory virus infections
Respiratory viruses generally infect epithelial cells lining the upper and lower airways, however subsets of airway immune cells are also susceptible to infection. Of interest, airway epithelial cells (AEC) and airway macrophages (AM) are both susceptible to influenza virus infection, but only AEC support productive virus replication. Constitutive expression and/or induction of intracellular host proteins to limit or block virus replication is a well-known antiviral mechanism, however few such “restriction factors” have been well characterized and the antiviral activity of many putative restriction factors against respiratory viruses has not been reported. A recent RNA-seq study performed in our laboratory compared gene expression between AEC and AM in the presence or absence of influenza infection, allowing us to identify protein families with differential expression between these two cell types in steady-state and/or following virus infection. We hypothesized that amongst genes differentially expressed between AEC and AM would be those that represent novel restriction factors and these might contribute to the abortive versus productive phenotype observed in AM and AEC, respectively, in regard to influenza replication. One of the protein families identified were the membrane-associated RING-CH (MARCH) family proteins. MARCH proteins are E3 ubiquitin ligases involved in the final step of the ubiquitination process. Amongst their functions, MARCH proteins modulate host immune responses by virtue of their ability to control the turnover of multiple immune molecules, including major histocompatibility complex (MHC) proteins. More recent evidence indicates that they can also modulate virus infection, acting as restriction factors against viruses such as human immunodeficiency virus (HIV)-I, or as proviral factors for viruses such as hepatitis C virus (HCV). Based on these findings, this thesis aimed to investigate the ability of MARCH family proteins to modulate infections caused by influenza and other respiratory viruses. First, we generated stable cell lines with doxycycline (DOX)-inducible expression of several human MARCH proteins (MARCH1/2/3/5/6/7/8/9) and evaluated their ability to modulate either the early or late stages of infections caused by influenza A viruses (IAV), respiratory syncytial virus (RSV) or human metapneumovirus (hMPV). Although none of the MARCH proteins tested affected the early stages of IAV infection (as measured by flow cytometry for newly-synthesized viral proteins), inducible expression of MARCH8 was associated with a significant reduction in titres of infectious IAV released from infected cells. Moreover, MARCH8 expression was also associated with a reduced percentage of RSV-infected cells, consistent with its ability to restrict at an early stage of the RSV replication cycle, although this was not explored further in this thesis. We did not observe any significant differences in the ability of any MARCH protein tested to modulate early or late stages of hMPV infection. Subsequent studies have focussed on understanding MARCH8-mediated restriction of influenza virus infections. Next, we characterized the antiviral activity of MARCH8 against influenza viruses. MARCH8 was shown to mediate antiviral activity against a range of influenza viruses relevant to human health, including A/H1N1, A/H1N1pdm09 and A/H3N2 viruses, as well as against an influenza B virus. We demonstrated that expression of MARCH8, but not the closely related MARCH1, was associated with a reduction in the specific infectivity of virus particles released from IAV-infected cells. Moreover, virus particles released from IAV-infected cells in the presence of MARCH8 exhibited altered protein composition and virion morphology, and MARCH8 was incorporated into the nascent virions. Of interest, MARCH8 expression did not alter cell-surface expression of the viral proteins HA, NA and M2, indicating no major defect in their synthesis and transport during viral replication. Overall, these findings are consistent with MARCH8 acting to block a step late in the virus replication cycle, possibly during virus assembly and/or budding from the cell surface. Studies described in this thesis also attempted to determine features of MARCH8 and IAV relevant to MARCH8-mediated restriction of IAV. We used site-directed mutagenesis and reverse genetics to modify lysine residues in the cytoplasmic tails of the viral hemagglutinin (HA), neuraminidase (NA) and matrix protein (M)2 proteins, either alone or in combination. However, none of these substitutions were sufficient to overcome MARCH8-mediated restriction of IAV. We also modified several lysine residues in the viral M1 protein but again did not identify specific residues to abrogate MARCH8-mediated restriction. Currently, the identity of the particular viral and/or host proteins targeted by MARCH8 which result in inhibition of late stage IAV replication are not known. To study MARCH8 itself, we generated mutants lacking E3 ligase activity and confirmed that this was essential for its anti-IAV activity. Furthermore, given that MARCH8, but not the closely related MARCH1, mediated potent anti-IAV activity against some strains of IAV, we generated cell lines with DOX-inducible expression of MARCH1-MARCH8 chimeric proteins in an effort to determine particular domains of MARCH8 critical to its anti-IAV activity. Chimeric proteins contained one or more domains/regions of MARCH1 substituted into the MARCH8 backbone. While all chimeric proteins retained activity against a common target protein (CD86), all of them also retained ability to restrict IAV replication. Based on these studies, we were unable to identify critical domains of MARCH8 that were essential for its antiviral activity against IAV. Finally, we used a MARCH8 knockout (KO) mouse to evaluate the role of endogenous MARCH8 in modulating IAV infection in vivo. Following intranasal infection, MARCH8 KO mice exhibited enhanced weight loss and viral replication in the lungs at day 5 post- infection, although no major differences in virus titres or virus clearance were observed at later time-points. Moreover, we did not observe major differences in soluble inflammatory mediators or inflammatory cells at day 7 or 10 post-infection. These findings are consistent with a role for endogenous MARCH8 in controlling the early stages of IAV infection in vivo but suggest that it does not have a major impact on virus clearance or the development of adaptive immunity in this model. Of interest, siRNA knockdown of endogenous MARCH8 in a human epithelial cell line also resulted in a modest, but significant, reduction in titres of IAV released from infected cells in vitro. Together, the studies presented in this thesis describe and characterize the antiviral activity of MARCH8 against influenza viruses. In addition, they provide preliminary data to indicate that MARCH8 can also mediate antiviral activity against the pneumovirus RSV, albeit by a distinct mechanism. Overall, these findings contribute to a growing body of evidence that MARCH8 plays an important role in modulating infections caused by a range of different viruses with relevance to human health.
Coxiella burnetii control of the host transcription factors TFEB and TFE3
Coxiella burnetii, the etiological agent of the zoonotic disease Q fever, is an obligate Gram-negative intracellular bacterial pathogen that replicates inside the lysosome-derived CCV (Coxiella-containing vacuole) within mammalian hosts. The CCV maintains the degradative and acidic nature of the host lysosome despite C. burnetii directing the massive expansion of this compartment to accommodate the replicating pathogen. To establish this unique replicative niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS). This T4SS translocates approximately 150 effectors into the host cell to modulate various cellular processes. To date, the functional role of very few of these effectors have been defined. Given the CCV’s origins it is not surprising that C. burnetii infection increases host autophagy and lysosome biogenesis. To investigate this at the protein level, we employed an elegant SILAC based proteome analysis of human cells infected with C. burnetii. This validated that many proteins involved in these processes are increased in abundance during infection. This prompted us to examine the role of the human transcription factor EB (TFEB) and its close homologue TFE3 during C. burnetii infection. TFEB is a master transcription regulator directly controlling the expression of a network of genes responsible for autophagy and lysosome biogenesis. 3 day’s post-infection with C. burnetii, TFEB/TFE3 is activated as demonstrated by TFEB/TFE3 trafficking from the cytoplasm into the nucleus. The nuclear translocation of TFEB/TFE3 appears to be controlled by C. burnetii as blocking bacterial translation with chloramphenicol leads to TFEB/TFE3 movement back into the cytoplasm. siRNA silencing of tfeb and tfe3 additionally demonstrated their contribution towards the intracellular success of C. burnetii. Interestingly, these host factors did not contribute to the replication of C. burnetii but in the absence of TFEB and TFE3 the CCV did not undergo its typical massive expansion. This research was able to demonstrate that C. burnetii induced activation of TFEB/TFE3 was dependent on the Dot/Icm T4SS thus we hypothesized that an effector(s) of this system may manipulate TFEB/TFE3. An unbiased visual screen was conducted to identify effectors that influence this process and two putative C. burnetii effector proteins, namely CBU1701 and CBU2016, were identified. We demonstrated that ectopic expression of these proteins leads to nuclear localisation of TFEB. Subsequent characterisation of the impact of CBU1701 and CBU2016, demonstrated that they influence the host proteome in similar ways but with surprisingly little impact on TFEB- regulated proteins. These results indicated that nuclear localisation of TFEB in response to CBU1701 and CBU2016 may be uncoupled from activation of this host transcription factor. In addition to characterising the impact of these effector proteins on host cellular function, we set out to understand the role they play in intracellular replication and virulence of C. burnetii. Genetic manipulation of this pathogen is in its infancy and remains technically challenging however this study reports the successful production of multiple mutant strains. Initial characterisation experiments demonstrate that CBU1701 and CBU2016 likely make important contributions to the establishment of the C. burnetii intracellular niche. Overall, the research carried out in this thesis has worked towards elucidating the contribution of TFEB and TFE3 to C. burnetii infection and developing an understanding of the molecular players in this process. Significant tool development and foundational findings reported here will pave the way to a deeper understanding of the interplay between intracellular bacterial pathogens and the host response to infection. Additionally, using C. burnetii effector proteins as a novel biological toolbox may uncover important insights that will impact our understanding of a range of human molecular pathways that impact human health.