Microbiology & Immunology - Theses
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Fc-functional antibody immunity to HIV: the role of neutrophils and IgA
A safe and effective prophylactic vaccine against HIV-1 is an essential component to limit the HIV-1 epidemic. The RV144 HIV vaccine efficacy trial has highlighted the importance of generating Fc functional antibodies to prevent the further spread of HIV infection. Fc functional antibody responses have also been shown to correlate with delayed HIV disease progression. Despite the intensification of interest in Fc-mediated responses to HIV infection, there has been limited research focused on the Fc functional capacity of neutrophils, which are a key innate immune cell at mucosal surfaces and in the blood. The majority of Fc-effector studies in HIV focus upon examining NK cells and/or monocytes responses, while other effector cells such as neutrophils remain understudied. NK cells lack the FcalphaR and cannot mediate any IgA-dependent Fc-mediated effector responses therefore, other immune cells like neutrophil are necessary for IgA to be studied. Neutrophils are highly functional innate effector cells with the potential to induce both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis. In chapter 2, methods were optimized to evaluate antibody-dependent neutrophil phagocytosis (ADNP) and neutrophil-mediated rapid fluorometric antibody-dependent cellular cytotoxicity (RFADCC) effector responses, using freshly isolated primary human neutrophils from blood. In vitro, neutrophil-mediated RFADCC responses peaked at 4 hours, which was faster than primary NK cells or monocyte mediated responses. There was a large spectrum of responses of both ADNP and neutrophil-mediated RFADCC responses across a cohort of 41 viremic antiretroviral-therapy naive HIV positive subjects. ADNP and RFADCC responses correlated well with each other, suggesting that they measure overlapping functions. The viral load of the patients inversely correlated with the ADNP responses, suggesting that these antibody-mediated neutrophil-based assays could prove useful in dissecting HIV-specific immunity. The role that IgA plays in active HIV infection remains controversial, with some reports of HIV-specific IgA being able to inhibit HIV infection and potentially being protective. Chapter 3 investigated if HIV progression was influenced by HIV-specific ADNP and neutrophil-mediated RFADCC responses and the effects of IgA on these responses. It was shown that, although neutrophil-mediated RFADCC responses were higher in the plasma of subjects who controlled their viremia levels (viremic controllers), IgA from both viremic controllers and viremic non-controllers inhibited both ADNP and neutrophil-mediated RFADCC responses similarly. The IgG mediated ADNP responses from both viremic controller and viremic non-controllers were broadly inhibited by both autologous HIV positive IgA and HIV negative pooled purified IgA. The IgA inhibition was able to be blocked by pretreating neutrophils with an Fc alpha receptor (FcalphaR) blocking antibody. This suggests that IgA inhibition of ADNP responses can be mediated by 2 mechanisms; 1) antigen dependent, FcalphaR independent and 2) antigen independent, FcalphaR dependent. The RV144 vaccine trial has generated interest in Fc functional antibodies and in the role that HIV-specific IgA can play during HIV vaccination strategies and in HIV infection. The RV144 vaccine induced IgG antibodies that were able to mediate ADCC responses. However, the vaccine efficacy was reduced in the presence of high concentrations of HIV-specific IgA. Monoclonal IgA that was isolated from the plasma of the RV144 vaccinees was able to block the potentially protective IgG antibodies from binding similar epitopes, thus preventing ADCC responses with NK cells. This indicates there was epitope competition between IgA and IgG antibodies in the RV144 vaccine trial. NK cells lack the FcalphaR and cannot mediate any IgA-dependent Fc-mediated effector responses. Chapter 4 assessed plasma samples from the RV144 vaccine trial for their ability to induce neutrophil-mediated responses and if IgA was able to inhibit these responses. IgG from the RV144 vaccinees was able to induce modest HIV-specific ADNP and neutrophil-mediated RFADCC responses. Plasma IgA from the vaccinees was able to inhibit ADNP responses but not neutrophil-mediated RFADCC responses. Using pooled IgG from the vaccinees, it was shown that pooled purified IgA from vaccinees, pooled purified IgA from HIV positive donors and pooled purified HIV negative IgA were able to inhibit the IgG mediated ADNP responses. Overall, this thesis shows that neutrophils can mediate HIV-specific antibody-dependent phagocytosis and neutrophil-mediated RFADCC responses. HIV-specific IgG mediated neutrophil responses, induced by either infection or vaccination, can be inhibited by plasma IgA in an antigen dependent mechanism and an antigen independent mechanism that is a FcalphaR dependent mechanism. The inhibitory effects of IgA may assist in understanding HIV pathogenesis and improving future HIV vaccine designs.
Developing vaccines to prevent mycobacterial infections
Tuberculosis (TB) is a leading cause of disease by an infectious agent. It is the most common mycobacterial disease of humans, followed by leprosy and Buruli ulcer. TB is caused by infection with Mycobacterium tuberculosis (MTB). TB affects people in every part of the world, predominantly throughout Asia (especially India and China) and Africa. Roughly one-quarter of the world’s population is latently infected with MTB. Asymptomatically infected people carry an approximate 10% risk of developing active disease. In 2018, there were an estimated 10 million new cases of TB and 1.45 million associated deaths. TB is treatable however it requires six months of combination antibiotic therapy. Buruli ulcer (BU), is a neglected tropical disease and has been reported in more than 30 countries world-wide but the dominant endemic foci of this disease occur in rural regions of West Africa. In the past five years BU cases have increased dramatically in South-East Australia, near Melbourne. BU is caused by infection of subcutaneous tissue with Mycobacterium ulcerans, typically presenting as deep and extensively ulcerated skin lesions. MTB and M. ulcerans are closely related mycobacterial species, therefore a vaccine against one of these bacteria might induce cross-protection against the other. The Mycobacterium bovis ‘bacille Calmette-Guerin’ (BCG) vaccine, a live-attenuated whole cell vaccine against MTB, is widely used. It is most effective in children below two years of age and against disseminated TB but efficacy wanes after about 10-15 years. In adults, BCG is between 0-80% effective. There is no effective vaccine against any mycobacterial disease and immune correlates of protection for mycobacterial vaccines are not well defined. This thesis sought to address these knowledge gaps and explored the development of different vaccines to protect against TB and BU. The vaccines were tested in murine infection models and the types of immune responses induced by each vaccine were measured. Where a vaccine was able to elicit robust immune responses, the animals were then challenged with the mycobacterial pathogen to assess protective efficacy. The first chapter is an introduction to this thesis and includes a literature review of TB and BU, their respective causative agents, immune responses to infection, and recent vaccine developments. This chapter introduces the key concepts and motivations for this thesis. The second chapter describes the development of a protein-based vaccine against TB. The vaccines utilised MTB-specific proteins ESAT-6 and Ag85B in conjunction with lipopeptide adjuvant R4Pam2Cys in C57BL/6 mice. The vaccines were not capable of generating measurable interferon (IFN)-gamma responses from CD4+ T cells recovered from the spleen or from the lungs, which have been shown to be crucial to the control of TB. The vaccines were however able to induce high protein-specific antibody titres against Ag85B. These vaccines were then modified with M. ulcerans-specific proteins to try and develop a vaccine against M. ulcerans. The third chapter focusses on the development of two protein-based vaccines against two highly expressed M. ulcerans cell wall-associated proteins, MUL_3720 and Hsp18. These proteins were bound to lipopeptide adjuvant R4Pam2Cys and their ability to generate a strong antibody response was measured in BALB/c and C57BL/6 mice. M. ulcerans is predominantly an extracellular pathogen and a strong antibody response against M. ulcerans could play a role in prevention of infection. Both MUL_3720 and Hsp18 in conjunction with R4Pam2Cys were capable of generating strong protein-specific antibody responses in both mouse strains. These antibody responses remained augmented after subcutaneous challenge with M. ulcerans on the mouse tail, however strong antibody responses did not correlate to protection. All vaccinated mice succumbed to infection 40 days after M. ulcerans infection. This suggests that these proteins were not suitable vaccine candidates. There was also no difference in protection between vaccinated mice and mice vaccinated with BCG. The BCG vaccine is not wholly protective against M. ulcerans but previous studies have shown that the vaccine delays the onset of disease. The lack of difference in this study may be due to the high bacterial challenge dose and suggested the need for a different animal model of infection. The fourth chapter describes the development of a vaccine targeting the mycolactone biosynthesis pathway. Mycolactone is a polyketide toxin and is the main virulence factor of M. ulcerans. Mycolactone affects the host immune response, causing immune cells to display modulated or decreased cell function which enables bacteria to evade immune responses. Prior to the creation of a new vaccine formulation, a new murine model of infection was established to reflect a more realistic, lower pathogen challenge dose. In this new murine model, mouse tails were coated in engineered bioluminescent M. ulcerans and the contaminated skin was subcutaneously pierced with a sterile needle to replicate trauma-induced introduction of bacteria into the subcutaneous tissue. The bioluminescent bacteria enabled the visualisation and quantification of bacterial load over time using an in vivo imaging system (IVIS). Once a new murine challenge model was established, this chapter assessed the efficacy of a new vaccine formulation comprising a protein domain, enoyl reductase (ER). The ER functional domain is required for the biosynthesis of mycolactone. Recombinant ER protein was coupled to R4Pam2Cys and BALB/c mice were vaccinated and boosted. This vaccine provided comparable protection against BU compared to the BCG vaccine. Additionally, this vaccine was statistically more protective than no vaccination. Analysis of systemic cytokine responses suggest that control of disease correlates to the level of inflammatory cytokines found in the spleen compared to the draining lymph node (site of infection). The immune responses correlating to protection from a BCG vaccine differed to the responses generated by ER+R4Pam2Cys. This study indicates that protection against BU may be achievable by different immune responses. This study also suggests that the highly conserved mycolactone biosynthesis pathway may be an effective target for a vaccine. However, understanding the immune correlates of protection requires much further study. In conclusion, this thesis demonstrated that MTB proteins in conjunction with the chosen adjuvant (R4Pam2Cys) do not elicit immune responses, in particular IFN-gamma responses, that are typically required to protect against TB in a murine model. M. ulcerans proteins, Hsp18 and MUL_3720 also using the R4Pam2Cys adjuvant, did not induce protection against BU in a murine challenge model. However, vaccine-induced protection was observed by incorporating the M. ulcerans mycolactone ER functional domain with R4Pam2Cys and a murine model more reflective of a natural M. ulcerans infectious dose. These experiments highlighted the potential for an effective vaccine that targets the mycolactone biosynthesis pathway. This work also demonstrated that protection against M. ulcerans might be achieved via different combinations of immune responses. An effective vaccine against M. ulcerans will likely have useful lessons for developing vaccines protective against MTB (and vice-versa), whether through cross protection or by using vaccines as tools to probe and measure the host immune responses required for control or protection against infection.
Characterisation of the putative cysteine protease effectors OspD2 and OspD3 from Shigella species
Diarrheal disease caused by bacterial pathogens continues to be a major public health concern worldwide due to significant increases in mortality annually. Members of the Shigella genus contribute significantly to bacterial diarrheal incidences worldwide. Shigella is a Gram negative facultative anaerobe that belongs to the family Enterobacteriaceae. They are considered highly infectious as only 10-100 organisms are required to cause disease. Like many other Gram-negative gut pathogens, Shigella utilizes a type III secretion system (T3SS) during infection to translocate bacterial effector proteins into host cells which interfere with host signaling pathways to benefit their survival. The exact function of many T3 effector proteins remains unknown. However recently, the T3SS effector EspL from enteropathogenic Escherichia coli (EPEC), was shown to contain a cysteine protease catalytic motif that targets and degrades the host RHIM domain containing proteins, RIPK1, RIPK3, TRIF and DAI, hence blocking inflammation and necroptotic cell death during infection. Homologues of EspL are also found in Shigella, namely: OspD2 and OspD3. Although previously labelled as Shigella toxins, the exact function of these effectors is yet to be elucidated. The primary aim of my study is to characterize the role of OspD2 and OspD3 and to determine their host cell targets. Overexpression experiments with OspD2 or OspD3 and the RHIM family of proteins suggest that OspD3, but not OspD2 targets and cleaves the RHIM family of proteins. We also showed OspD3 blocking both inflammation and necroptotic cell death in NF-kB dependent luciferase assays and cell viability assays respectively. However, this was not seen upon infection of HeLa 299s with wildtype and mutant Shigella strains. This therefore led to further investigations for the identification of other host cell targets of OspD2 and OspD3 via mass spectrometry. Several unique host cell targets for OspD2 and OspD3 were identified from mass spectrometry and an enrichment analysis of the hits, suggest the involvement of these proteins in anti-viral defense, particularly the Type I IFN signaling pathway. Elucidating the roles of these effectors in the interferon signaling pathways will be important in understanding the roles between bacteria pathogens and interferon as unlike viral infections, these are severely understudied.Further analysis via overexpression studies showed that OspD2 and OspD3 may be working cooperatively in cleaving IRF3/IRF7 and IRF9 during Type I IFN signaling and hence blocking the pathway. Furthermore, we also demonstrated by infection of HeLa 299s that OspD2 cleaves IRF3, however cleavage of IRF9 by OspD3 was not seen. In summary, we have performed characterization of two cysteine protease effectors of Shigella and their host cell targets. This work creates the platform for understanding how these effectors function and how this knowledge may be used as valuable tools for subsequent investigation of host cell defense mechanism.
Identifying novel compounds to eliminate latent HIV infection
There are currently an estimated 36.9 million people living with human immunodeficiency virus (HIV) (PLWH) worldwide. In the past few decades, the advent of antiretroviral therapy (ART) has significantly reduced the number of deaths associated with this virus. However, ART is not curative. The persistence of HIV latently infected CD4+ T-cells presents the major barrier towards a cure for HIV. Latently infected T-cells are formed when the virus integrates into the host genome of infected cells without ensuing productive infection. Due to these latently infected cells, viral gene expression and production infection rebounds from the integrated viral DNA if ART is ceased. Thus, ART must currently be taken life-long, posing a tremendous economic burden. The “shock and kill” approach is an extensively studied cure strategy that involves the use of pharmacological agents termed latency reversing agents (LRAs) to reactivate or “shock” the latent virus to express viral RNA and proteins. Following the reactivation of latently infected cells, the production of HIV proteins and viral particles was proposed to result in the elimination of these cells through immune-mediated clearance or cytopathic events. Results from clinical trials that involve a single LRA to reactivate latently infected cells in PLWH have not yielded any significant impact on the HIV DNA reservoir. This can be attributed to a number of different reasons that include the potency of the LRAs to reactivate latency, the failure to elicit an effective immune response and the inhibition of T-cell clearance by cytopathic viral proteins. There is clearly a need for more potent LRAs as well as novel strategies that will result in the clearance of these latently infected cells once reactivated. In this thesis, we investigate several novel pro-apoptotic compounds in isolation as well as in combination with LRAs to clear latently infected cells. We have also developed two new methods in which to study the effects of LRAs and pro-apoptotic drugs on latently infected cells. Dual-fluorescent reporter viruses have proven to be useful tools in studying latent HIV infection in vitro. Here we have modified a dual-fluorescent reporter HIV aiming to improve its functional characteristics in a pre-activation model of HIV latency. The new virus termed, DuoAdvance, contains two fluorescent viral reporters: a latent GFP reporter driven by elongation factor 1-alpha (EF1-alpha) and a productive E2 Crimson reporter driven by the HIV long terminal repeat (LTR) (Chapter 2). Using DuoAdvance, we demonstrate that DuoAdvance can successfully infected Jurkat T-cell lines. In a pre-activation model of HIV latency in primary resting CD4+ T-cells, DuoAdvance infection resulted in little to no latent GFP expression. Subsequent analysis of the GFP negative population of cells revealed DuoAdvance infection can result in the production of latently infected cells carrying latent provirus but the expression of the GFP latency reporter was perturbed. Due to the partial expression of this GFP latent reporter in primary resting T-cells, DuoAdvance is limited to use in dividing T-cell lines and potentially a post-activation model of HIV latency using activated CD4+ T-cells, where better expression of the GFP latency and E2 Crimson reporters were seen. Latency reversing agents can reactivate latent HIV but the effects on decreasing HIV DNA in PLWH has been less encouraging. In this thesis, we examine the effects of different pro-apoptotic drugs combined with different LRAs on decreasing HIV DNA in cultures of CD4+ T-cells from PLWH on ART ex vivo. Here we tested a number of LRAs together with several phosphoinositide-3 kinase (PI3K) inhibitors: IPI-443, IPI-3063 and wortmannin, as well as a B-cell lymphoma-2 (Bcl-2) inhibitor venetoclax as our pro-apoptotic drugs. The LRA romidepsin combined with all pro-apoptotic drugs resulted in synergistic decreases in the levels of integrated HIV DNA in the PLWH CD4+ T-cells ex vivo (Chapter 3). Additionally, several other LRA and pro-apoptotic combinations also decreased integrated HIV DNA in CD4+ T-cells ex vivo. All drugs were able to induce HIV viral transcription. Interestingly, we show that the pro-apoptotic drugs alone also led to an increase in HIV transcription and a decrease in HIV DNA. These data demonstrated the select combinations of pro-apoptotic drugs and LRAs together or pro-apoptotic drugs alone can result in a decrease in HIV integrated DNA in CD4+ T-cells from PLWH on ART ex vivo. However, we were unable to distinguish if there was selective death of the reactivated latently infected cells with minimal impacts on uninfected T-cells also in the cell cultures. In order to explore this, we developed a new approach to detect selective cell death (Chapter 4). This method involves the use of PrimeFlow, a HIV RNA in situ hybridisation method combined with branched-DNA technology, together with a cell death stain and analysis of stained cells using flow cytometry. Using this approach, we were able to demonstrate selective cell death in ACH2 T-cell lines treated with a combination of the PMA LRA, and venetoclax or IPI-443 PI3K inhibitor pro-apoptotic drug in a latently infected T-cell line. However, due to the elaborate staining procedure and large cell loss from the multi-step staining procedure, further investigation is required to move this staining approach into testing these drugs upon inducing the selective death of latently infected CD4+ T-cells from PLWH ex vivo. In summary, we have developed two new methods to investigate the effects of LRAs and/or pro-apoptotic drugs on HIV latency. Although further work is required to optimise these methods for use of the novel DuoAdvance fluorescent reporter virus with primary resting CD4+ T-cells for drug testing, or for use of the novel PrimeFlow assay to study the selective impact of these drugs upon latently infected CD4+ T-cell samples from PLWH ex vivo. Most importantly, our work demonstrates novel combinations of pro-apoptotic drug and LRA combinations that can decrease HIV integrated DNA in cultures of CD4+ T-cells from PLWH on ART ex vivo. This has important therapeutic implications for using these drug combinations to deplete latently infected cells in PLWH on ART and additional studies that investigate these combinations in a clinical setting is warranted. In conclusion, our work demonstrates that latency reversal combined with a drug-based strategy to promote apoptosis can eliminate HIV latently infected CD4+ T-cells from PLWH on ART ex vivo and thus this approach holds important potential to lead to HIV remission off ART in PLWH.
Evolutionary Dynamics of Successful Clones of Methicillin-Resistant Staphylococcus aureus in Australia and New Zealand
Staphylococcus aureus is a common cause of bacterial infections in humans and a leading nosocomial pathogen, associated with significant morbidity, mortality and economic impact. A cornerstone in the evolution of staphylococcal lineages that infect humans has been their remarkable ability to rapidly and efficiently develop or acquire mechanisms of antimicrobial resistance, impacting effective disease management, prevention, and eradication. Improving these measures or developing novel approaches requires a comprehensive understanding of the infecting agent and while S. aureus has been extensively studied there remains considerable gaps in our knowledge surrounding this pathogen, especially concerning population dynamics. What drives the emergence and/or persistence of certain staphylococcal lineages? What evolutionary pathways and molecular mechanisms are being utilised and under what circumstances? What environmental and host factors have the greatness influence on bacterial population adaptation? And ultimately, what are the consequence of these population level changes and what impact do these have on staphylococcal disease? This thesis represents three projects undertaken to strengthen our understanding of the evolutionary dynamics surrounding staphylococcal population adaptation, using a combination of comparative genomics and detailed phenotypic profiling to provide insight into antibiotic-resistant lineages of S. aureus that circulate in Australia and New Zealand. The first project investigated the long-term persistence of the globally disseminated, multidrug resistant hospital associated methicillin-resistant S. aureus (MRSA) lineage ST239 in Australia. From this work, it has been identified that the ST239 MRSA population circulating in Australia represents not one, but two genetically distinct clades; the previously unrecognised introduction of the Asian-Australian clade followed by successful local expansion in the state of Victoria has contributed to the persistence of ST239 by supplementing the diminishing population size of the local clone, the Australian clade. The ability of the Asian-Australian clade to spread following its introduction is owed to the reduced susceptibility this population developed against anti MRSA antibiotics, namely the glycopeptides and daptomycin, prior to its importation. This same phenotype has convergently emerged in the local Australian clade. However, this adaption has come at a cost as both clades were found to have reduced replicative fitness and impaired pathogenic potential, the latter occurring through loss of functionality or reduced expression of a staphylococcal global virulence regulatory system, the accessory gene regulator. The second project considered a different evolutionary circumstance; the rapid emergence of a novel MRSA lineage in the New Zealand community. This region has a high incidence of staphylococcal skin and soft tissue infection and over the last two decades has seen a significant shift in the local molecular epidemiology of S. aureus, with the emergence of multiple fusidic acid resistant clones. This work has focused on the predominant MRSA lineage, an ST5 clone locally referred to as AK3. Genomic investigation of this lineage has found that AK3 represents a single phylogenetic clade that has arisen through local population expansion, having emerged from a resident fusidic acid susceptible methicillin-susceptible S. aureus upon the acquisition of a novel chimeric SCCmecIVa-fusC II mobile element harbouring the fusidic acid resistance determinant fusC. This phenomenon was not restricted to ST5, with the other newly emerged lineages having acquired fusC via structurally distinct SCC and SCCmec elements. Indicating that, the unregulated use of fusidic acid in the region has supported the emergence and expansion of these novel lineages and is potentially contributing to the high local incidence of staphylococcal infection. Mobile elements can greatly influence staphylococcal populations, therefore the third project focused on exploring the evolution of the multidrug resistant plasmid family pSK1 and the role it has played in augmenting antimicrobial resistance and biocide tolerance in the ST239 MRSA population in Australia. Modelling the evolutionary history of this plasmid identified that it emerged during the late 1970s and over the following four decades has undergone significant structural change, involving a combination of chromosomal integration, transposon loss/gain, structural inversion and deletion events. When aligned to a phylogenetic model of the ST239 population, it became apparent that these changes in plasmid configuration represented a clear pathway of step wise adaptation; the shortened, chromosomally integrated plasmid structural variants having emerged on multiple occasions in a convergent manner. Further, these changes correlated with the development of enhanced tolerance against chlorhexidine, an important finding as it implicates biocide use as a factor potentially influencing MRSA evolution. Collectively, this work enhances our understanding of how antibiotic-resistant lineages of S. aureus evolve and adapt; exemplifying the evolutionary pathways facilitating adaptation in staphylococcal populations and the circumstances under which they are used. Further, it provides detailed insight into two highly successful lineages of MRSA. This knowledge can be exploited to improve measures that reduce the burden of antibiotic-resistant staphylococcal infection. Importantly, this work enhances and reinforces our awareness about the consequences of antimicrobial overuse and misuse.
MAIT cell diversity, function and impact on dendritic cells
T cells represent an important component of the immune system. Whilst early studies were largely focused on the role of conventional CD8+ and CD4+ T cells that recognize peptide-antigens in association with MCH molecules, more recently, T cells that recognize other types of antigens have been described. Mucosal associated invariant T (MAIT) cells are such a cell population and belong to the broad family known as ‘unconventional’ T cells, due to their non-peptidic antigen recognition characteristics. MAIT cells are defined by their recognition of microbial vitamin B2 metabolites presented by MHC related protein 1 (MR1). Upon antigen recognition they immediately display effector functions, like secreting cytokines and expression of cytotoxic proteins. Whilst the majority of MAIT cell studies have focused on the role of MAIT cells to bacterial infections, however their function within the immune system and interaction with other immune cells is still unknown. This thesis focuses on the role that MAIT cell activation has on other immune cells like dendritic cells (DCs) and other T cells. Furthermore, the full potential of MR1-recognition by other T cell subsets was also examined, revealing that MR1-reactive T cells may extend beyond what is currently describe as MAIT cells. The first chapter of this thesis investigates the role of MAIT cell activation on DCs in an in vivo mouse model. MAIT cells were activated by intratracheal injection of the activating MAIT cell antigen 5-amino-6-D-ribitulaminouracil/ methylglyoxal (5-A-RU/MeG). This activation of MAIT cells led to migration of DCs from the lung to the mediastinal lymph node (medLN) as well as DC maturation in an MR1-dependent manner. Furthermore, production of the chemokines CCL17 and CCL22 was induced by MAIT cell activation, which suggests that MAIT cells are able to modulate the immune system far more than previously thought. The possible role of MAIT cell induced DC maturation on initiation of a CD8+ T cell response is analyzed within the second result chapter. No enhanced antigen-specific CD8+ T cell response to the model antigen ovalbumin (OVA) was observed by additional MAIT cell activation. Besides MAIT cells, recently more MR1-reactive T cells were identified. By using antigen-loaded MR1 tetramers, a population of FOXP3+ T-bet+ T cells was identified in human thymus that can bind to MR1 tetramers. In the third chapter this FOXP3+ T-bet+ T cell population was further characterized by analysis of their phenotype as well as their TCR usage. The results in this chapter will serve as a basis for further investigation of the diversity of MR1-recognition within the T cell pool. In conclusion, this thesis reveals a new role of MAIT cells that may be used to manipulate their functions to treat different diseases like autoimmune diseases or cancer. Moreover, the knowledge of MR1-reactive T cell diversity is extended including a potential regulatory role of MR1-reactive T cells and MAIT cells. In summary, this thesis extends the current knowledge of MAIT cell biology.
Role of the chemokines CCL17 and CCL22 in the immune defence against Salmonella infection
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.
Genomic approaches to stop the spread of bacterial infectious diseases
The advent of affordable whole genome sequencing has been a disruptive force that is changing the biological sciences, with public health microbiology being no exception. As microbial genomics is a new frontier for public health microbiology, a substantial amount of development and validation research is required. Genomic-based investigations in response to bacterial public health problems are gaining traction, but optimal approaches to bioinformatics and data analysis are still being developed. In order to help address these needs, this thesis investigated the utility of microbial genomics for Australian public health microbiology through three case studies of three distinct bacterial pathogens. Hospital adapted pathogens pose a significant health threat through their ability to rapidly emerge with adaptive phenotypes that permit them to circumvent infection control practices. In the first results chapter, comparative bacterial population genomics was used to characterise and better understand the genomic changes that have accompanied and driven the emergence of a novel and highly hospital associated lineage of Enterococcus faecium called ST796. These analyses revealed that the novel strain belonged to a highly hospital adapted lineage and shared a close evolutionary origin with another newly emerged hospital associated lineage of E. faecium. Complete assembly and analysis of a representative ST796 genome identified several key genomic events that were distinctive in the newly emerged lineage. Investigations of bacterial disease outbreaks caused by environmental pathogens have traditionally relied upon insights delivered through epidemiology supported by pathogen genotyping methods. Genotyping can be critical for identifying the source of an outbreak, but methods vary in their resolving power and genotyping data can be difficult to compare between investigations. In the second results chapter, a genomics-based machine learning approach was developed and used to predict the source of clinical Legionella pneumophila isolates using genomic patterns obtained prospectively through environmental sampling of bacteria. A proof-of-concept method was developed for assigning outbreak sources without using phylogenetic trees. Notably too, this study demonstrated phylogenetics can be misleading when assessing L. pneumophila environmental origins. The predictive statistical genomic method presented here has utility as a powerful tool for use alongside conventional L. pneumophila disease outbreak epidemiological investigations. Understanding a pathogen’s mode of transmission and infectious reservoir is a key goal in order to control the spread of disease, as such knowledge permits deployment of evidence-based strategies to manage outbreaks and prevent future occurrences. In the third results chapter, genomics was used to conduct high-resolution, micro-epidemiology population analyses on clinical isolates of another environmental bacterial pathogen, Mycobacterium ulcerans, the cause of Buruli ulcer. Bacterial population genomics and phylogeographic modeling suggested a westward migration of the pathogen across south east Australia that aligned well with the disease epidemiology. In addition, demographic models inferred from the genomic data provided a plausible explanation for a recent increase in cases of Buruli ulcer in south-eastern Australia. These insights demonstrate that M. ulcerans can be introduced to a new environment and then expands, rather than the awakening of a quiescent pathogen reservoir. Such discoveries inform our understanding of Buruli ulcer transmission and control. Collectively, these studies further demonstrate that microbial genomics significantly improves our understanding of the transmission dynamics behind clinically important pathogens over standard epidemiological investigations. This research demonstrates the advantages of the implementation of microbial genomics in public health microbiology.
The regulation of skin-homing receptors and its implications on T helper cells following localized Herpes Simplex Virus infection
Effective cellular immunity relies not only on the appropriate differentiation of antigen-specific T cells directed toward particular pathogens but also on the targeted migration of these cells to the sites of infection. E-selectin ligand (ESL) is a homing receptor widely recognized for its role in supporting the migration of leukocytes to the skin particularly during non-specific inflammation. However, the role of ESL in the migration of CD4+ T cells to the skin during viral infection and its regulation of expression on these cells are less understood. This thesis aimed to explore these aspects by utilizing an in vivo mouse model of localized skin infection with herpes simplex virus-1 (HSV-1) as well as a detailed investigation of in vitro polarized CD4+ T cells. Following epicutaneous but not intranasal HSV-1 infection, adoptively transferred transgenic HSV-specific CD4+ T cells induce the expression of ESL particularly in the infected skin. Similarly, genetic ablation of Fut7, a gene required for the generation of ESL, significantly decreased the capacity of CD4+ T cells to enter the skin of HSV-infected mice highlighting the importance of this homing receptor. While epicutaneous HSV infection of C57BL/6 mice predominantly induces a T helper type 1 (Th1) biased CD4+ T cell response, the data herein proved that the induction of ESL was not strictly related to Th1 differentiation of the responding HSV-specific CD4+ T cells. Interestingly CD4+ T cells deficient in the Th1 master regulator T-bet displayed a preference toward Th17 differentiation upon HSV-1 skin infection, yet still expressed high levels of ESL and there was no evidence of impaired migration to the infected skin. Further supporting this finding, in vitro analyses subsequently demonstrated the expression of ESL on both in vitro differentiated Th1 and Th17 cells but not Th2 cells. The glycosyltransferase core-2-beta-1,6-glucosaminyltransferase-I (C2GlcNAcT-I), which contributes to the synthesis of ESL epitope, appeared to be the primary determinant of the differential ESL expression between these Th subsets. As the differentiation of Th subsets and the induction of ESL in the draining lymph nodes in vivo both required TCR signaling and were evident prior to egress from the draining lymph nodes, the relationship between priming signals and ESL expression was assessed in vitro. The data indicated that both TCR signaling and the presence of exogenous cytokines were important for the induction and/or regulation of ESL expression. While TCR signaling was required for ESL expression, this signal alone was not sufficient. Rather the balance of cytokines, particularly of TGF-beta 1 and IL-4, had a marked impact on levels of ESL expression, with TGF-beta 1 acting to induce and IL-4 to suppress ESL expression on activated CD4+ T cells. Finally, this thesis investigated the temporal regulation of ESL induction during T cell priming and demonstrated that while the addition of TGF-beta 1 early during CD4+ T cell priming skewed the differentiation of Th subsets, the addition of this cytokine between 48-72 hours of T cell activation allowed for significant induction of ESL with only minimal interference on Th subset differentiation. Hence, the signals required for Th differentiation and ESL expression might actually occur in a consecutive manner to allow different Th subsets to express ESL and access the skin.
Characterisation of extracellular vesicles released upon NLRP3 activation and their impact on bystander cells
Inflammasomes are cytosolic multi-protein complexes that assemble upon detection of danger signals of infectious or sterile origin. Activation of the NOD-like receptor NLRP3 results in activation of caspase-1, which subsequently leads to cleavage and secretion of IL-1beta and IL-18, cytokines crucial for inflammation. In addition, NLRP3 activation has also been demonstrated to result in the secretion of extracellular vesicles (EVs). EVs are membraneous compartments, present in almost all body fluids, that derive either directly from the cell membrane (apoptotic bodies and ectosomes) or from multi-vesicular bodies (exosomes). They carry a set of proteins and RNAs distinct from their cell of origin and have been reported to be important mediators of cell-to-cell communication. Within this thesis I show that EVs can be isolated from conditioned tissue culture media most efficiently using a size exclusion chromatography-based protocol. This protocol enriches three distinct EV fractions: large EVs, intermediately sized EVs and small EVs. To investigate the impact of NLRP3 inflammasome activators on all EV subpopulations, THP-1 macrophages were treated with various NLRP3 inflammasome activators or different TLR ligands. All NLRP3 activators are capable of inducing EV secretion, which is primarily NLRP3- and caspase-1-dependent. TLR ligands also induce EV secretion, but in an NLRP3- and caspase-1-independent way. Comparisons of the RNA content of intermediately sized and small EVs show vast differences between the two EV subpopulations, suggesting a regulated loading of cargo into EVs. However, within the same EV subpopulation different NLRP3 activators cause the secretion of a very conserved RNA content, here defined as an EV-associated NLRP3 signature, which is divergent from the RNA content of TLR- induced EVs. Since activation of the NLRP3 inflammasome occurs in various inflammatory diseases, such as gout, atherosclerosis, type II diabetes and mutation-associated auto-inflammatory syndromes, this data may have clinical relevance for biomarker discovery. Additionally, transfer of NLRP3-induced EVs causes the induction of an interferon signature in EV recipient cells. Since interferons are known to counter-regulate inflammasome responses generally and I show here that NLRP3-induced EVs can dampen inflammasome responses in unprimed recipient cells specifically, the EV-induced interferon signature may provide an endogenous mechanism to prevent the induction of a systemic hyper-inflammatory state upon NLRP3 activation.
The role of T cell help in shaping Dendritic cell function
CD8+ T cell priming depends on antigen presentation by dendritic cells (DCs) and their capacity to communicate contextual cues associated with antigen acquisition. DCs often also require additional signals from helper CD4+ T cells, which upon mediation via CD40-CD40L further modulate the communication of contextual cue to the responding CD8+T cells. The present study was designed to explore the kinetics and molecular mechanisms underpinning this helper-dependent modulation of DC function. To address this, we employed an in vitro system of bone marrow (BM)-derived equivalents of CD8+ DCs (eCD8+ DCs) and we assessed the role of different CD40 signalling components in driving their IFN-aA-induced cytokine and chemokine responses by using flow cytometry, mass spectrometry-based proteomics, real time PCR and RNA sequencing. This brought to light remarkable and distinct patterns of gene regulation through which CD4+ T cells triggered CD40 and thereby amplified the capacity of IFN-aA to induce or downregulate a broad range of genes. We also observed an unexpected pattern of gene regulation: some genes required both T cell help and IFN-aA stimulations but could not be induced by ‘help’ or IFN-a alone. By varying the exposure time, we further discovered that eCD8+ DCs required 1-2 hours of IFN-aA to become responsive to CD40 triggering. Once this pre-activated state was achieved, CD40 stimulation rapidly amplified responses with remarkably fast kinetics. Combining proteomics and RNA sequencing data presented in this thesis suggests a complex interplay between the IFN-aA signalling pathway involving IRFs transcription factors and the NF-kB signalling pathway. These findings not only reveal new insights into how T cell help adjusts the responsiveness of DC to innate stimuli, but also reveal that this can occur with remarkable speed, which aligns with in vivo imaging studies describing very brief interactions between eCD8+ DCs and CD4+ T cells during CD8+ T cell priming.
Modelling melanoma control by immunotherapy and tissue-resident memory T cells using CRISPR/Cas9-based approaches
In recent years, immunotherapy has demonstrated remarkable efficacy in the treatment of metastatic melanoma due to the development of T cell-based therapies such as checkpoint inhibitors or adoptive T cell transfer therapy (ACT) directed against defined antigens. However, tumours frequently relapse from therapy by diverse acquired resistance mechanisms. Currently, it is not well understood how the choice of target antigen influences resistance mechanisms to antigen-specific immunotherapies. A better understanding of tumour recognition by the immune system is of utmost importance to further improve currently used immunotherapies. Therefore, we established CRISPR-assisted insertion of epitopes (CRISPitope), a technique that fuses a model CD8+ T cell epitope, human gp100, to endogenous gene products. We applied CRISPitope to murine melanoma cells to tag the endogenous melanosomal protein, TYRP1, and the oncogenic protein, CDK4R24C, with the same model epitope, rendering them targetable by the same TCR-transgenic T cells. This defined experimental setting enabled us to investigate how the choice of the targeted gene product impacts on therapy outcome and immune evasion mechanisms. Using experimental mouse models, we could identify different escape mechanisms to gp100-specific immunotherapy in TYRP1 versus CDK4R24C melanomas. Resistance to ACT targeting TYRP1 was mainly caused by permanent antigen loss, accompanied by a non-inflamed microenvironment, or reversible downregulation of the antigen associated with melanoma phenotype switching. In contrast, CDK4R24C melanomas escaping ACT displayed antigen persistence and were associated with an IFN-rich inflamed tumour microenvironment. In CDK4R24C melanomas IFN-driven feedback inhibition by negative immune-checkpoint molecules promoted resistance to ACT despite persistent antigen expression. Applying CRISPitope to syngeneic mouse models, we could show that target antigen choice can influence ACT resistance mechanisms, phenotype and immune contexture of melanomas in response to antigen-specific immunotherapies. Thus, our work could help to better understand acquired resistance and optimise personalised cancer immunotherapy. Furthermore, we aimed to apply this platform to a model of melanoma immune surveillance by TRM cells in order to understand the importance of cognate antigen expression and presentation for long-term tumour control by CD8+ tissue-resident memory T cells (TRM). To address this question, we used a modified CRISPitope-approach, called SWITCHitope, to generate melanoma cell lines that express a floxed model antigen under the control of an endogenous promoter and a Tamoxifen-inducible Cre-recombinase. We could confirm successful Tamoxifen-inducible depletion of the model antigen in melanoma cells in vitro and in vivo. Moreover, we showed that antigen-depleted melanoma cells have significantly reduced potential to activate TCR-transgenic T cells in vitro. Using a transplantable epicutaneous melanoma inoculation technique, we could demonstrate that SWITCHitope-engineered melanoma cells can prime naïve T cells, recruit them into the skin and induce T cell differentiation towards a TRM phenotype. Our approach enables us to investigate the importance of antigen expression and presentation for TRM melanoma control. This work will help to better understand the interplay between tumour cells and TRM cells and thereby advance clinical translation.