Microbiology & Immunology - Theses
Now showing items 1-12 of 168
Neuroimmune responses in viral infection
The regulation of the immune responses is important in maintaining good health. Interactions between the nervous and immune systems are increasingly studied and widely appreciated to be influential in orchestrating immune responses. T cells express adrenergic receptors (AR) that enable them to respond to neurotransmitters produced by the sympathetic nervous system (SNS), noradrenaline (NA) and adrenaline, inducing downstream signalling and modulating cell functions, although whether this is stimulatory or inhibitory in T cell antiviral responses is unclear. In this thesis, I examine the effects of SNS in various models of viral infection through chemical sympathectomy and treatment with AR agonists. Modulation of sympathetic signals in systemic infections with LCMV had minor influences on T cell responses but resulted in increased viral loads. Notably, the infection results in a loss of tyrosine hydroxylase (TH) positive sympathetic fibres in the spleen as early as day 3 post infection and is reflected by decreased NA splenic NA. The immune response may play a role with interferon g partially contributing to the depletion. Additionally, this thesis also investigates the capability of long-lasting resident memory T cell (TRM) responses in the highly innervated, immune-privileged cornea. Using a model of herpes infection of the cornea, I showed that T cells are effectively recruited to the cornea with a small heterogenous population able to persist following cessation of immune responses. These cells express CD69 and CD103, canonical markers of tissue residency, to varying degrees. Persistence, but not recruitment, of these cells is dependent on antigen availability at the cornea. These memory cells are capable of responding to secondary encounters with antigen. Moreover, circulating memory cells are also able to infiltrate the immune-privileged cornea more efficiently following infection. Together, these results highlight the important nuances in the regulation of immune responses by the nervous system.
The dynamics of the B cell response during influenza A virus infection
Although vaccination remains the most effective method of managing influenza epidemics, there is still much that remains to be characterized about humoral immunity against the varying contexts in which influenza infection can occur. With the continuous subversion of humoral immunity by seasonal influenza through antigenic drift and the potential of zoonotic influenza viruses adapting and spreading through human populations through antigenic shift, improving our understanding of B cell immunity against different types of influenza infection could provide important insights into improving management of epidemics and vaccine formulations. In order to understand B cell responses during influenza infection, the well-characterized C57BL/6 mouse model was used to investigate and compare humoral responses in the context of different influenza infection histories. Markers that identified specific B cell subsets such as germinal centre (GC) B cells and plasmablasts were analysed by flow cytometry paired with influenza virus-specific B cell ELISPOT assays to investigate strain-specific antibody secreting responses within the same experiment. As the surface glycoprotein HA is thought to be the immunodominant response for B cell responses against influenza virus, the prediction is that the greater the antigenic differences between the HA of the first and second infecting strains, the more primary-like the response to the second strain would be. Primary and homologous secondary B cell responses in the mediastinal lymph node (MLN) and spleen were first characterized using this model to establish baseline responses against influenza virus before heterosubtypic infection was studied through infection of mice with H1N1A/Puerto Rico/8/34 (PR8) virus followed by H3N2 A/Udorn/305/72 (Udorn) virus 7 weeks later. Unexpectedly, a secondary-like plasmablast, GC B cell and Udorn-specific antibody secreting cells was observed during heterosubtypic infection, with earlier and higher magnitude B cell responses. These findings suggested a possible role for cross-subtype T cell memory in modulating B cell responses. The effect of antigenic drift on the B cell responses during influenza infection was then analysed with the same model. Mice were infected with H3N2 strains isolated between 1972 and 1979, representing different antigenic distance from a virus isolated in 1982 (Ph82). Seven weeks post infection mice were reinfected with Ph82 and the B cell response over the course of infection examined. It was found that infection of strains up to 10 years apart appeared to induce a secondary-like B cell response in the secondary lymphoid organs when compared to baseline primary and secondary responses against Ph82 virus. Prior infection with any H3N2 strain also resulted in minimal viral replication during the secondary challenge when compared to primary infection groups. However, data from both primary antibody inhibition and HA-specific B cell responses appears to suggest a narrower threshold of recognition, around a maximum of 3 years drift before serum and HA-specific responses cease to bind with other strains. Taken together, secondary-like B cell responses in both heterosubtypic and drift models of infection and in the case of drift responses, irrespective of reactivity of HA-specific B cells, appear to refute the hypothesis that virus-specific B cell responses would reflect antigenic relatedness between the HA of the infecting strains. Overall, data from this study identifies the diversity of the overall B cell response against influenza infection in the context of prior exposure to strains of different antigenic properties. Further study into the reactivity of these B cells against different influenza virus components and the role of memory T cells in the observed responses may provide important insights into the nature of host immunity against the ever-shifting target of influenza virus.
Understanding the mechanisms utilised by Coxiella burnetii for intracellular replication within the host phagolysosome
The human pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which may lead to chronic disease in a small percentage of infected individuals. C. burnetii is a unique Gram negative intracellular bacterium which replicates within a host cell lysosome derived vacuole, termed the Coxiella containing vacuole (CCV). Currently, the exact mechanisms which allow C. burnetii to replicate within this normally hostile compartment are unknown. In order to understand how C. burnetii survives within this intracellular niche, this research investigated carbon metabolism of both intracellular and axenically cultivated bacteria, using steady state metabolic profiling and 13C stable isotope labelling. Both C. burnetii populations were shown to assimilate exogenous [13C]glutamate and [13C]glucose, with concomitant labelling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [13C]glucose utilisation but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters that may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the Galleria mellonella insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [13C]glucose labelling approach, this study identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13C label incorporation. Despite maintaining a relatively small genome, C. burnetii have retained seemingly redundant strategies to obtain glucose. This suggests that glucose may be an important metabolite for C. burnetii. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected. In addition, this thesis also investigated the novel and unique C. burnetii protein CBU2072, a small, 18.3 kDa protein, which has subsequently been named essential for intracellular replication A (EirA), as loss of this protein prevents replication of C. burnetii within the CCV. Intracellular replication of the EirA mutant can be restored during co infection of the same vacuole with C. burnetii wild type, which is analogous to the phenotype observed for mutants of the Dot/Icm type 4B secretion system in C. burnetii. EirA localises to the C. burnetii inner membrane, and absence of this protein leads to the loss of Dot/Icm effector translocation. These data together contribute important understanding of the unique mechanisms involved in C. burnetii pathogenesis within the host phagolysosome.
The molecular and cellular basis of antigen recognition by unconventional T cells
Unconventional T cells are evolutionarily conserved populations of T cells, many of which recognise non-peptide antigens in the context of monomorphic antigen presentation molecules related to the major histocompatibility complex (MHC). These MHC related molecules, known as CD1 and MR1 present lipid and small metabolite antigens respectively. Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that are highly abundant in humans and recognise the potent riboflavin biosynthesis derived metabolite 5-OP-RU presented by MR1. The majority of MAIT cells in the peripheral blood of humans express the CD8 co-receptor, a molecule also expressed by other T cells that contributes to antigen recognition and T cell activation. In contrast to other T cells, a role for CD8 on MAIT cells has not been formally tested. The first chapter of results in this thesis examines whether CD8 is able to bind MR1, and if CD8 is important for the activation of MAIT cells and other MR1-reactive T cells. The data revealed that CD8 binds directly to MR1, in a manner concordant with MHC class I, and that this interaction is important for the functional response of MAIT cells. Specifically, both isoforms of CD8 (CD8aa and CD8ab) bound to MR1 tetramers and the CD8-MR1 interaction could be abrogated by mutating MR1 in the putative CD8 binding site. The effects of the CD8-MR1 interaction were examined on primary MAIT cells, where MR1 tetramers bound more strongly to CD8+ compared CD8- MAIT cell subsets. Importantly, mutating MR1 tetramers reduced CD8+ MAIT cell engagement to CD8- MAIT cell levels. To determine the effect of the CD8-MR1 interaction on function, primary MAIT cells were activated in the presence of wild type or mutant MR1. In line with the importance of CD8 on MR1 recognition, the cytokine secretion by CD8+ MAIT cells was decreased in the absence of CD8 binding to MR1. Furthermore, the data here establishes that CD8 is vital for the recognition of MR1 presenting less potent, lower affinity antigens such as folate derivatives. Similarly, low affinity MR1-antigen recognition and cytokine secretion by other MR1-reactive T cells was completely abrogated in the absence of CD8 binding. Thus, CD8 is an important co-receptor for the function of MAIT cells and may expand the diversity of ligands recognised by MAIT and other MR1-reactive T cells. CD1b-restricted T cells are a subset of unconventional T cells in humans that recognise lipid antigens derived from endogenous and microbial sources that are presented by CD1b. Comparatively little is known about the biology of CD1b-restricted T cells, particularly autoreactive CD1b-restricted T cells that recognise endogenous lipid antigens such as phospholipids. The second chapter of results examines autoreactive TCR recognition of CD1b, including the breadth of permissive endogenous lipid antigens that are bound by mammalian CD1b. Autoreactive CD1b-restricted T cells were identified from healthy blood donors using CD1b tetramers presenting heterogeneous mammalian lipids and the autoreactive T cells expressed distinct T cell receptors (TCRs) which were cloned to generate autoreactive T cell lines. Different CD1b restricted T cell lines exhibited altered staining reactivity with CD1b tetramers loaded with different mammalian phospholipid antigens and strikingly, some autoreactive T cell lines recognised CD1b in an antigen independent manner. An activation assay using the autoreactive T cell lines cocultured with a series of mutant CD1b expressing cell lines revealed several CD1b binding ‘hotspots’ along the a1 and a2 helices that were critical for TCR-mediated activation. Using soluble proteins, autoreactive TCR permissive ligands were isolated from mammalian CD1b by separating ternary TCR-CD1b-lipid complexes from binary CD1b-lipid complexes that did not bind to the TCR using size-exclusion chromatography. Mass spectra analysis of the fractionated proteins revealed an abundance of phospholipids, particularly phosphatidylcholine as permissive CD1b lipid antigens. These data suggest autoreactive TCR antigen-specificity is more diverse than previously described and that these TCRs may adopt novel docking modes onto CD1b to recognise distinct endogenous antigens. In the final chapter of results, the phenotype and functional characteristics of M. tuberculosis-reactive CD1b-restricted T cells was investigated. Glucose monomycolate (GMM) is a cell wall lipid expressed by pathogenic Mycobacteria, including M. tuberculosis, that is presented by CD1b to T cells. GMM-reactive T cells were first isolated directly ex vivo from the peripheral blood of latent M. tuberculosis infected donors using CD1b tetramers. Herein, an optimised CD1b tetramer enrichment was developed to isolate very small frequencies of GMM-reactive T cells from healthy donor blood directly ex vivo. In contrast to M. tuberculosis infected patients, GMM-reactive T cells from healthy donors expressed a diverse TCR repertoire. GMM-reactive T cells were exclusively CD4+CD8- and most cells expressed the memory marker CD45RO. Similar to other unconventional T cells, GMM-reactive T cells from healthy donors expressed the transcription factor promyelocytic leukaemia zinc finger (PLZF). In vitro stimulation revealed GMM-reactive T cells secrete both TNF and IFNg, similar to MAIT cells. To characterise these cells further, single-cell transcriptomic analysis was performed on GMM-reactive T cells isolated using CD1b-GMM tetramers. GMM-reactive T cell expressed unique transcriptomic signatures that were accurately distinguishable from the transcriptomes of natural killer T (NKT) cells and CD4+ T cells, indicating that these cells may be functionally distinct from other unconventional and conventional T cells populations. In summary, these data describe a role for the CD8 co-receptor on MAIT and other MR1-reactive T cells and expand on the limited knowledge of CD1b-restricted T cells in healthy blood in regard to TCR repertoire, antigen-specificity and phenotype.
Amoebae as a host for Legionella replication
Legionella pneumophila (L. pneumophila) is a gram-negative bacterium found ubiquitously in natural water sources where it replicates within amoebae, such as Acanthamoeba castellanii (A. castellanii). The evolution in amoebal hosts allows L. pneumophila to ‘accidentally’ infect human lungs after inhalation of aerosols containing L. pneumophila. In both human macrophages and amoebae, L. pneumophila replicates intracellularly within a vacuole known as the Legionella containing vacuole (LCV) that avoids fusion with the endocytic pathway. Fundamental to this process is the translocation of over 330 proteins by L. pneumophila into host cells by the Dot/Icm type 4 secretion system. To date, only a subset of Dot/Icm effector proteins has been characterised. These proteins are involved in manipulating host cellular processes such as ER vesicle recruitment, post-translational modifications and host cell survival. The majority of L. pneumophila effector proteins remain uncharacterised due to functional redundancy of effector proteins and complex effector protein regulation. In addition, despite the ecological and evolutionary significance of the intra-A. castellanii stage of the L. pneumophila life cycle, very little is known about this host-pathogen interaction. In this study, we constructed in-frame markerless mutants in L. pneumophila 130b according to genomic regions enriched for putative Dot/Icm effector genes and revealed host-specific genomic regions that are required for optimal L. pneumophila replication in A. castellanii. Specifically, by identifying a putative glutamate transporter necessary for replication in A. castellanii, but not in macrophages, this study suggests an aspect of L. pneumophila metabolism adaptation in different hosts. A. castellanii presents two life stages: active trophozoites and dormant cysts. Trophozoites act as reservoirs for L. pneumophila and are hijacked by L. pneumophila for bacterial replication. Cysts are resistant to anti-microbial treatments and were previously thought to facilitate L. pneumophila persistence and dissemination. However, in this study, we showed that the infection of A. castellanii with L. pneumophila led to inhibition of the transition from trophozoites to cysts in a Dot/Icm dependent manner. This inhibition partially required the gene letA, which encodes the regulator of two-component system LetAS. Pathogenic amoeba cysts prose a human health problem. This study provides a biological model for anti-cyst research and provides clues into critical encystment pathways. L. pneumophila can interfere with a broad range of host cellular pathways to establish the replication niche. Here, we investigate A. castellanii transcriptional response with L. pneumophila infection by RNA sequencing analysis. As a result, A. castellanii genes associated with mitosis, DNA replication and cell wall synthesis were significantly downregulated at late infection phase, which might contribute to the inhibition of A. castellanii proliferation and encystment. Compared with transcriptional profile in macrophage, L. pneumophila displayed different regulation on host eEF1a expression and genes associated with ATP production. Furthermore, a gene encoding sirtuin family protein, Sir6f, was upregulated during L. pneumophila infection, and silencing of sir6f led to decreased bacterial replication, suggesting that Sir6f is a host factor that facilitates L. pneumophila replication in A. castellanii. Evolution of L. pneumophila in amoebal hosts is hypothesized to allow its adaptations to survive in macrophages. Overall, this study highlights a number of strategies utilised by L. pneumophila during A. castellanii infection, including host-specific virulence factors, inhibition of A. castellanii encystment, affect host transcriptome and hijack of host factors. These microbial strategies will add valuable insights and provide potential targets regarding the development of new mechanisms for L. pneumophila control and prevention.
Molecular studies of Staphylococcus epidermidis
Staphylococcus epidermidis is a conspicuous member of the human microbiome, widely present on human skin. The shift in modern medicine towards invasive procedures has favoured its emergence as a significant nosocomial pathogen, especially in the setting of prosthetic devices. Despite being the most genetically diverse of the staphylococcal species, with 989 S. epidermidis multilocus sequence types characterised thus far, a single lineage, ST2, accounts for most clinical disease. Unfortunately, existing typing schemes lack the sensitivity to offer any epidemiological insights beyond this. Furthermore, the absence of dedicated molecular tools coupled with the genetically intractable nature of S. epidermidis has hindered the study of its molecular genetics, pathogenesis and treatment. This thesis utilised new genome sequencing technologies to provide a modern perspective on the global molecular epidemiology of S. epidermidis with a focus on antimicrobial resistance. The project began with the functional analysis of the first complete genome of a multidrug resistant ST2 S. epidermidis. Then extended globally, to uncover the previously unrecognised international spread of three multidrug-resistant, hospital-adapted lineages of S. epidermidis (two ST2 and one ST23) that emerged in recent decades. Acquisition of a dual D471E plus I527M RpoB substitution was common to all three lineages and demonstrated to have become fixed in the populations. Analysis of isolates from 96 institutions in 24 countries identified the RpoB D471E plus I527M combination as the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Furthermore, these substitutions were found to occur almost exclusively in isolates from the ST2 and ST23 lineages. By breaching lineage-specific DNA methylation restriction modification (RM) barriers, then performing site-specific mutagenesis, these rpoB mutations were shown to confer both rifampicin resistance and reduced susceptibility to the glycopeptide antibiotics vancomycin and teicoplanin. Importantly, these findings suggest that current clinical practice has contributed to the generation of rifampicin and vancomycin resistance and spread of the three multidrug-resistant S. epidermidis lineages, warranting the review of staphylococcal treatment guidelines. The final work in this thesis is the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis. Contrary to current assumptions, analyses revealed that the type I RM systems in S. epidermidis are not lineage specific like Staphylococcus aureus, instead demonstrating considerable diversity even within a single genetic lineage. This diversity can be attributed to marked differences in the gene arrangement, chromosomal location and movement of type I RM systems in S. epidermidis compared to S. aureus. Findings indicated that genetic manipulation of S. epidermidis must be tailored to each strain of interest. Using Escherichia coli plasmid artificial modification to express S. epidermidis hsdMS, the restriction barriers in S. epidermidis were readily overcome, with electroporation efficiencies equivalent to modification-deficient mutants. Through functional experiments, the use of genomic data to predict both the activity of type I RM systems and the potential for a strain to be electroporation proficient were demonstrated, and an efficient approach to the genetic manipulation of any S. epidermidis strain of interest, including those that have hitherto been intractable is presented. Collectively, the works in this thesis offer new insights into the molecular epidemiology, resistance mechanisms, treatment recommendations and genetic manipulation of S. epidermidis. The method outlined for the genetic manipulation of any given S. epidermidis isolate should prove valuable in assisting future molecular studies and advancing knowledge about this significant nosocomial pathogen.
Understanding influenza B humoral immunity to improve vaccine design
Annual influenza epidemics cause significant morbidity and mortality globally. Although influenza B virus (IBV) is responsible for approximately 25% of the global influenza burden, it remains understudied compared to influenza A virus (IAV). Current influenza vaccines elicit mostly strain- specific antibody responses, so vaccine efficacy is dramatically reduced when mutated variants dominate in circulation. Improved IBV vaccines require a better understanding of humoral immunity against IBV in order to inform improved vaccine design. In this thesis, we examined the human IBV-specific humoral response following influenza vaccination and investigated the potential utility of ferritin nanoparticles and IBV HA stem antigens to induce broader protective immune responses. IBV-specific antibody responses have been described in previous studies, but the knowledge we have regarding the specificities, protection and epitopes of cross-reactive antibodies remains limited. Using a flow cytometry-based approach, we delineated different B cell populations with either single-lineage or cross-lineage specificities from samples collected following seasonal influenza immunization clinical trials. Both neutralizing and non-neutralizing antibodies protected mice from lethal challenges with IBV, but protection by non-neutralizing antibodies was non-sterile and dependent on Fc-effector functions. We also localized neutralizing epitopes of both lineage-specific and cross-lineage antibodies on IBV HA by sequencing viral escape mutants. The comprehensive information we gathered from this study may guide future efforts to design broadly protective IBV vaccines. Nanoparticles as a novel vaccine carrier system has drawn increasing attention over the past decade. The self-assembling ferritin nanoparticles loaded with IAV HA have been proven to induce robust and broad humoral response in mice and ferrets. We investigated the feasibility and protective potential of displaying IBV HA on ferritin nanoparticles. The results demonstrated that ferritin nanoparticles significantly boosted the immunogenicity of IBV HA. Furthermore, we found that co-loading HAs of both IBV lineages led to antibody responses against both lineages and conferred similar level of protection in comparison of mixing two monovalent nanoparticles. Overall, ferritin nanoparticles presenting IBV HA, either monovalent or multivalent, may provide some immunological advantages over the conventional influenza vaccines, hence are promising foundations to build improved IBV vaccines upon. The highly conserved stem domain of HA has long been considered as a major target for generating cross-reactive antibody response. Due partially to the difficulty in preparing stable IBV stem proteins, the protective potential of the IBV stem is poorly understood. Despite problematic expression and/or misfolding, IBV stem proteins we generated were immunogenic in mice and elicited robust cross-lineage antibody responses. Mice vaccinated with IBV stem proteins were partially protected from simultaneous challenge with IBV of both antigenic lineages. The broad protection of IBV stem demonstrated by these experiments suggests that it is a promising candidate for universal IBV vaccines. In summary, this thesis improves the understanding of humoral immunity against IBV both by elucidating important aspects of HA-specific antibodies and exploring different approaches to improve current influenza vaccines. Our findings add to the field of IBV research and inform development of better IBV vaccines.
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.