Medical Biology - Theses
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ItemIntestinal Microfold Cells Orchestrate Microbe- Immune Interactions( 2022)Microbiota–immune cell interactions play a vital role in defenses against potentially harmful external organisms such as viruses and bacteria, and environmental agents including food. Microfold (M) cells are specialized cells within the epithelium of the intestines that sample the gut contents and pass them to the local guardians – a complex array of immune cells. Once harmful invaders are detected by immune cells, they swing into action to fight the infection. M cells provide the pivotal link between the gut lumen and the immune cell network, positioned to rapidly orchestrate appropriate immune responses. Exactly how M cells orchestrate these events, however, is not clear. Despite their critical function, to date few specific tools have existed to study intestinal M cells, the molecular mechanisms that regulate their generation, or how they drive mucosal immunity. To overcome this gap, we have generated novel gene modified mouse strains to allow us to visualize M cells and tease apart their behavior. I discovered that M cells are present along the entire intestinal tract, and not just localized to the Peyer’s patch as previously thought. Analysis of gut epithelial cells at different sites along the gut using single cell RNA sequencing revealed tissue-specific heterogeneity allowing us to define distinct gene signatures for M cells based on their location. These molecular blueprints identify distinct maturation programs that reflect local environmental cues shaped by the ingested material and the microbiota. Collectively, these features shape the delicate network of immune cells and show how the body can regulate gut region-specific diseases.
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ItemInvestigating the Genetic Causes of Primary Immunodeficiency and Autoimmunity( 2019)Primary Immunodeficiencies (PIDs) are a heterogeneous collection of several hundred disorders, that have in common deficient or dysregulated immunity. This leads to an increased susceptibility to infections, autoimmune disease, or to uncontrolled inflammation; in some PIDs there may be features of all three components. Understanding the pathogenesis of PIDs has led to insights into the immune system broadly, and this knowledge has aided improvements in the treatment of many more common infective and inflammatory diseases. Many PIDs are monogenic, and advances in technologies that enable the interrogation of the genetic basis of these conditions, have led to a steep increase in the number of diseases now recognized as PIDs. In this project we aimed to establish a cohort of individuals with the most common form of PID, Predominantly Antibody Deficiency (PAD). We sought to characterize the diagnostic and clinical features of these individuals, and apply genomic sequencing methods for more precise diagnoses, as well as to identify new genetic etiologies of PAD. One of the most striking findings regarding the clinical features of the patient cohort, was the morbidity associated with PADs. In particular we observed very frequent complications of immune dysregulation, that manifest as autoimmune disease, particularly affecting the haematological system as cytopenias, or gastrointestinal tract as enteropathy, or malignancies. These complications are challenging to manage in the setting of PID, hence understanding the mechanism of disease is crucial to improving outcomes. In a group of PAD patients we identified monogenic causes of disease, that led to some individuals receiving precision treatments. In the realm of gene discovery, we identified several novel genetic variants, two in genes that had not previously been recognized as disease-causing in PAD. The first, NFKB1, is now recognized as the most common genetic etiology of PAD. The importance of NF-kappaB signaling and its regulation was further highlighted by the discovery of other disease-causing variants in the NF-kappaB pathway. The effect of NFKB2 mutations on the immune response was investigated in a kindred with two sisters affected by PAD, who demonstrated disparate clinical and immunological features. Further studies were performed in a mouse model of NF-kappaB2 deficiency, whereby B cell and CD8+ T cell behaviours were studied in a quantitative manner, and revealed the cell-type specific effect of loss of this component on the adaptive immune response. Finally, an entirely new monogenic PID was identified. In a young man with a clinical diagnosis of PAD, complicated by autoimmune disease, a homozygous, frameshift mutation in NFKBID was identified. The mutation was demonstrated to cause loss of expression of IkappaBNS, an inhibitor of the NF-kappaB pathway, and with that, dysregulated NF-kappaB signaling. The finding of a second patient with PAD and compound heterozygous variants in NFKBID has added further evidence that this is a bona-fide novel PID.
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ItemManipulation of apoptotic signaling promotes cell-mediated immunity and death of infected cells during chronic viral infection( 2015)Overwhelming chronic viral infections produced by human immunodeficiency virus (HIV), Hepatitis B and C viruses (HBV and HCV respectively) subvert immunity to enable their persistence. These viruses maintain high levels of viraemia and antigaemia that in part drives immunodeficiency during infection. Cytotoxic CD8+ T cells are critical effectors of the immune system that promote the death of infected cells. However, CD8+ T cells gradually loss the ability to secrete effector cytokines, proliferate, kill infected cells and dramatically decline in number as chronic viral infections persist, a phenomena commonly termed ‘exhaustion’. A number of groups have tried to reverse the ‘exhausted’ T cell phenotype with cytokine therapy or antagonism of inhibitory receptors, however the proteins driving the loss of effector CD8+ T cells responding to the infection are largely unknown. Two programmed death pathways, intrinsic and extrinsic apoptosis, are critical for immune homeostasis before, during and after short-lived acute infections. Bim, a pro-apoptotic protein of the intrinsic apoptotic pathway plays a prominent role during all stages of immune homeostasis. Considering it’s indispensable immunoregulatory role, I wanted to examine if Bim regulated CD8+ T cell attrition during chronic viral infection. To test this I used the well characterised lymphocytic choriomeningitis virus (LCMV) murine model of chronic viral infection. I infected Bim knock out mice and mice with cell specific deletion of Bim to show a significant increase in the number of antigen-specific CD8+ T cells in the absence of Bim. Furthermore, equivalent rescue of effector cell numbers were seen with the loss of Bak and Bax, the critical gatekeepers of intrinsic apoptosis. Therefore Bim appears to be the sole intrinsic apoptotic protagonist. Cells rescued from apoptosis retained their ability to kill and to secrete cytokine, despite high expression of the inhibitory receptor PD-1. In spite of increased numbers of functional antigen-specific cells, no mice cleared chronic LCMV infection. However, the data is the first to identify Bim as the sole therapeutic target necessary to inhibit intrinsic apoptotic signaling and rescue functional effector cells during chronic viral infection. Having uncovered a critical effector protein controlling CD8+ T cell fate, I then examined the utility of promoting Fas/FasL mediated apoptotic death to promote viral clearance during chronic LCMV and HBV infection. FasLΔs/Δs mice possess a mutation in the extracellular cleavage domain of FasL. I found FasLΔs/Δs mice demonstrated enhanced FasL expression during chronic LCMV infection, that led to rapid control of a semi-acute strain of LCMV and a consistent trend toward accelerated control of HBV viraemia, within our murine model of chronic HBV infection. In addition, I found lpr-/- mice, which lack Fas receptor expression, fail to control HBV viraemia, thereby uncovering a previously unappreciated role for Fas signaling in the control of HBV infection. Lastly, I tested the ability of clinically relevant cancer therapeutics to sensitize infected cells to death within the LCMV and HBV chronic infection models. I found a strong synergism between the FasLΔs/Δs mice and the SMAC mimetic compound Birinapant in promoting HBV control and clearance of viraemia. Manipulation of cellular death pathways with cancer therapeutics represents a novel reimagining of this class of drugs and has important implications for future therapies against infectious diseases. In summary, to combat chronic viral infections I have promoted effector cell survival through Bim antagonism, enhanced cell-mediated cytolysis through FasL/Fas signaling within infected cells and further potentiated FasL/Fas sensitivity with SMAC mimetic treatment. Together, the data described herein has uncovered three avenues for the development of novel therapeutic interventions against chronic viral infection.
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ItemMerozoite antigens of Plasmodium falciparum elicit strain transcending opsonising immunity( 2015)Despite progress towards reducing the global burden, malaria continues to cause approximately 200 million cases and 600,000 deaths annually (World Health Organization, 2014). Although several malaria vaccines are currently in clinical trials, no advanced vaccine candidate has yet demonstrated sufficient efficacy to be a stand-alone vaccine against the highly variant Plasmodium falciparum parasite. Development of effective vaccine strategies requires knowledge of the essential mechanisms for protective immunity and robust assays to serve as correlates of protective immunity. However, exactly which antibody functions are necessary to control parasitemia and clinical symptoms during natural infection remains unclear. The merozoite represents an attractive vaccine target, as antibodies to numerous merozoite antigens have been associated with protective immunity in human cohort studies. This thesis aimed to investigate the importance of merozoite opsonising antibodies for immunity to malaria. Opsonising antibodies, and the Fc Receptor-mediated functions these antibodies elicit, have been poorly studied in malaria partly due to limitations of in vitro assays. Therefore, in this thesis a merozoite phagocytosis assay was developed and validated (Chapter 3), and robust and reproducible phagocytosis responses from THP-1 cells were observed. This assay was then used to measure merozoite opsonisation in a longitudinal study of semi-children from Papua New Guinea (PNG), and phagocytosis responses were demonstrated to correlate with protection from clinical disease and high-density parasitemia (Chapter 4). Due to the highly diverse nature of P. falciparum merozoites, it was important to assess whether merozoite opsonisation involved strain-specific or strain-transcending specificities (Chapter 5). Highly consistent opsonisation and associations with immunity were observed across a panel of common laboratory strains and PNG parasites adapted to growth in vitro. Through use of transgenic parasite lines, the absence of MSP3, MSP6, MSPDBL1 or MSP1-19 was not observed to impact the overall level of merozoite phagocytosis. By depleting antibody reactivity to 3D7 merozoites, opsonisation of merozoites from PNG strains also declined, suggestive of conserved antigenic targets across parasite strains. The findings in this thesis have demonstrated the importance of opsonising antibodies and their associated phagocytic responses for protective immunity to malaria. Robust, reproducible and well-validated assays are a priority to aid pre-clinical and clinical malaria vaccine development. The consistent responses and protective associations provide strong support for merozoite opsonisation as a robust correlate of protective immunity in malaria endemic populations. As the majority of merozoite opsonising antibodies were strain-transcending, uncovering these conserved domains within merozoite surface antigens may yield important novel vaccine candidates with which to tackle this deadly disease.
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ItemTargets of antibodies to the surface of Plasmodium falciparum-infected erythrocytes and protective immunity to human malaria( 2012)Effective clinical immunity that protects against symptomatic malaria in humans develops gradually after repeated exposure to Plasmodium falciparum. Naturally acquired antibodies targeting antigens expressed on the surface of infected erythrocytes (IE) represent an important component of protective immunity against malaria. During intra-erythrocytic development, P. falciparum dramatically remodels the host erythrocyte membrane through the export of novel parasite proteins. Among these are antigens expressed on the IE surface, known as variant surface antigens (VSA), that include PfEMP1, RIFIN, STEVOR, SURFIN proteins and possibly others such as PfMC2TM and modified host band 3. These antigens are highly polymorphic and some are known to undergo clonal antigenic variation for immune evasion. Numerous studies have reported that individuals living in malaria endemic regions were capable of agglutinating P. falciparum-IEs suggesting the recognition of VSAs expressed on the IE surface. Longitudinal studies further suggested that anti-VSA antibodies were associated with protection from P. falciparum malaria. Previous studies have only been able to measure the acquired antibody response towards all VSAs expressed on the IE surface, due to a lack of tools to dissect the antibody responses to individual VSAs. Although studies have also evaluated antibodies to recombinant proteins, it has been difficult to directly quantify the contribution of each native VSA to the overall antibody response to the IE surface. The aims of this thesis were to quantify the significance of VSAs as targets of naturally acquired antibodies, with a particular focus on P. falciparum erythrocyte membrane protein 1 (PfEMP1) and evaluate the importance of naturally acquired antibodies to PfEMP1 and other antigens that afford clinical protection from symptomatic P. falciparum malaria. Novel approaches using transgenic P. falciparum with inhibited PfEMP1 expression have enabled the quantification of PfEMP1 relative to other VSAs as a target of acquired antibodies. This was achieved by the transfection of parasites with a construct that encodes a var gene promoter without a downstream var gene (vpkd; presented in Chapter 3) thus resulting in a PfEMP1-deficient line and the transfection of parasites with a construct that has a deletion of the pfsbp1 gene required for PfEMP1 trafficking (SBP1KO; presented in Chapter 4). These approaches were then applied to human studies in Kenya and Papua New Guinea (PNG) and comparisons between parental and PfEMP1-deficient transgenic parasites allowed for the determination of antibodies specific to PfEMP1. The functional significance of naturally acquired antibodies was determined using assays that specifically measure antibody-mediated phagocytosis of IEs by undifferentiated monocytes. In addition, the clinical importance of PfEMP1-specific antibody responses was further investigated in a longitudinal cohort study with PNG school children. Characterisation of the transgenic vpkd parasites demonstrated reduced var gene transcription by Northern blot analyses and the absence of PfEMP1 proteins by Western blot analyses of IE membrane extracts, thus suggesting that PfEMP1 expression was inhibited in these parasites. In addition, characterisation of the SBP1KO parasites confirmed the absence of PfSBP1 protein in Western blot analyses and immunofluorescence microscopy of pigmented trophozoite IEs. However, other VSAs such as RIFIN and STEVOR, and other IE membrane proteins such as PfEMP3 were still expressed by the transgenic vpkd and SBP1KO parasites. Furthermore, transmission electron microscopy of pigmented trophozoite IEs confirmed the presence of knob structures on the IE surface of the vpkd parasites, similar to parental parasites. These findings suggest that despite the inhibition of PfEMP1, other IE membrane proteins and knob assembly occurred normally in the transgenic vpkd and SBP1KO parasites. Among malaria-exposed children and adults from Kenya and PNG, IgG binding to the surface of erythrocytes infected with the transgenic vpkd parasites was substantially reduced compared to parental. This suggests that majority of the acquired antibody response to the IE surface was predominantly directed towards PfEMP1, while other VSAs appear to play a minor role in relation to immunity. These key findings were confirmed with two genetically different parasite lines, 3D7 and E8B. Furthermore, using sera from children, adults and pregnant women available from Kenya or PNG, IgG binding to the surface of erythrocytes infected with the transgenic SBP1KO parasites was also markedly reduced compared to parental, suggesting that antibodies primarily targeted major antigens expressed on the IE surface that are dependent on PfSBP1 for trafficking. Currently, only PfEMP1 is known to be trafficked by PfSBP1 and this study has demonstrated that other VSAs such as RIFIN and STEVOR proteins remain expressed by the SBP1KO parasites. Comparing the antibody responses between PNG adults and children demonstrated that IgG binding to the vpkd and SBP1KO parasites was substantially reduced in both groups, suggesting that both adults and children had a great proportion of PfEMP1-specific antibodies. Evaluating the effect of trypsin treatment of IEs on antibody binding showed that most serum samples targeted trypsin sensitive epitopes expressed on the IE surface, consistent with PfEMP1 being the major target of antibodies. However, some samples appeared to target trypsin resistant epitopes on the IE surface of the vpkd and SBP1KO parasites. This study provides major new evidence that PfEMP1 is the dominant target of naturally acquired antibodies to the IE surface. In assays that specifically measure antibody-mediated phagocytosis by undifferentiated monocytes, the level of opsonic phagocytosis activity was greatly reduced in the transgenic vpkd parasites compared to parental. These results suggest that PfEMP1-specific antibodies are essential to promote IE opsonisation for phagocytosis by monocytes, an important mechanism in parasite clearance. Thus, these finding provide further evidence that PfEMP1 represents the major target of functional antibodies. Some measurable level of opsonic phagocytosis activity was still detected with the transgenic vpkd parasites although the level of IgG binding to these parasites were extremely low, suggesting that antibodies to non-PfEMP1 antigens may also function to opsonize IEs for phagocytic clearance. The clinical importance of antibodies to PfEMP1 and other VSAs was further evaluated in a longitudinal study conducted with school children from Madang, PNG. Antibodies to the 3D7 parental and 3D7-PfEMP1 (reflected in the difference between IgG binding to 3D7 parental versus 3D7vpkd) were associated with a significantly reduced risk of symptomatic P. falciparum malaria whereas antibodies to 3D7vpkd (reflecting antibodies to non- PfEMP1 antigens) were not associated with protective immunity. Children with antibodies to other isolates such as E8B and XIE-ICAM also had a reduction in malaria risk, however these associations were not statistically significant. It should be noted that there was insufficient statistical power in the current study to detect differences in small effect sizes and weak associations. The protective association with 3D7 observed in the current study of PNG school children complements a longitudinal study conducted with children in Chonyi, Kilifi (J. Chan and K. Howell et al 2012, J Clin Invest, in press) whereby antibodies to 3D7 parental and 3D7-PfEMP1 were associated with protection but antibodies to 3D7vpkd were not. Therefore, these findings indicate that PfEMP1 represents a major target of naturally acquired antibodies that are associated with protective immunity. However, these studies do not exclude an important role for other VSAs as targets of protective antibodies and further studies are essential to understand their significance as antibody targets and their association with protection from malaria. The results presented in this thesis provide major new evidence that among the VSA families present on the surface of P. falciparum-IEs, PfEMP1 represents the dominant target of naturally acquired human antibodies and antibodies to PfEMP1 contribute to protective immunity against malaria. Novel approaches using PfEMP1-deficient transgenic parasites performed in this study offered a unique insight to determine the relative contribution of PfEMP1 and other VSAs to the overall antibody response to the IE surface. Therefore, the work presented in this thesis enhances the understanding of humoral immunity to malaria and will aid the development of vaccines against malaria.