Medical Biology - Theses
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ItemLow birthweight and infant growth among children in Papua New Guinea - effect of malaria and other infectious diseases during childhood( 2020)Globally, young children continue to die or fail to thrive from treatable and preventable causes including low birthweight (LBW), childhood undernutrition (wasting, stunting, underweight) and infectious diseases. Reducing the burden of these are an essential pillar of global child health and survival targets. These global trends are reflected in Papua New Guinea (PNG), a resource constrained setting that continues to observe high rates of illness and death in young children with LBW, childhood undernutrition and infectious diseases (especially malaria, pneumonia and diarrhoea) continuing to be the leading causes. Improving child health and survival in PNG requires evidence about the risk factors for LBW, sub-optimal growth and infectious diseases, as well as the inter-relatedness of risk factors, that can inform policies and identify appropriate interventions and strategies. This thesis aims to address critical knowledge gaps in this area and provide insights to inform interventions and strategies aimed at reducing low birthweight, childhood undernutrition and malaria in young children in PNG and globally. LBW is caused by a multitude of factors which are often inter-related and with that, it is often difficult to distinguish between independently direct and indirect effects. Moreover, current interventions targeted at preventing LBW generally assume a single dominant cause overlooking the inter-relatedness of risk factors and the possibility of factors exerting joint effects on LBW. These are difficult to establish with widely used standard statistical methods and have therefore been rarely investigated. Using structural equation modelling, we showed intermittent preventive treatment of malaria during pregnancy with sulphadoxine-pyrimethamine (SP) plus azithromycin (AZ) to be independently directly associated with reduced probability of LBW. Unexpectedly, anaemia at enrolment was also directly associated with reduced probability of LBW. Maternal undernutrition at enrolment was independently directly associated with increased probability of LBW. No significant indirect associations between risk factors and LBW were established. After birth, children in lowlands PNG experience high rates of malaria, pneumonia and diarrhoea alongside undernutrition. Infections and undernutrition are believed to have a bi-directional relationship and whilst the effect of undernutrition on risks of illness and death is well known, little is known about the effects of malaria, pneumonia and diarrhoea on growth faltering, particularly among PNG children. By using multivariable regression and distributed lag models for data analysis, we observed malaria pneumonia and diarrhoea to have a differential impact on child growth. The effect of acute malaria on child growth was observed to be long-term while pneumonia and diarrhoea had short-term effects lasting up to 3 months. Of these three diseases, malaria was once ranked the top leading infectious cause of childhood morbidities and mortality in PNG, especially in lowlands PNG. The nationwide scale-up of malaria control interventions significantly reduced overall malaria transmission between 2008 and 2014 but a detailed understanding of the impact of this changing transmission on the epidemiology and risk profile of malaria infections and disease due to the two main species in young children was lacking. By analysing three consecutive longitudinal child cohorts (1-5-year-old children) conducted over the period of improved control (2013), we observed a differential impact of improved control on P. falciparum and P. vivax. Additionally, we showed that with declining malaria transmission, burden of malaria infections and illness were highly spatially localised to areas that had the highest burden prior to scale-up, highlighting potential hotspots of transmission. Collectively, the findings from this thesis provide important insights for improving child health in PNG and globally.
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ItemPlasmodium vivax naturally acquired immunity: patterns and influences( 2020)Malaria is caused by Plasmodium parasites and Plasmodium vivax is the dominant Plasmodium spp. in low-transmission regions outside of Africa. Due to the unique biological characteristics of this parasite, such regions often feature asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. Serology is a promising tool for monitoring transmission levels, estimating past and recent exposure and identifying populations at risk of infections. However, due to key gaps in our knowledge of naturally acquired antibody responses to P. vivax, the full potential of serology has not yet been reached. This thesis aimed to establish antibody kinetics against a large panel of P. vivax antigens following infections in western Thailand, and investigate the factors potentially associated with the acquisition and development of antibody responses. A multiplexed bead-based assay was first established and antibody measurements against more than 50 antigens were taken in P. vivax-infected individuals from western Thailand following symptomatic and asymptomatic infections. I found that most P. vivax antigens followed a highly similar post-infection kinetic pattern in the absence of any boosting infections. The magnitude and longevity of antibody responses varied between antigens, antibody subclasses and subtypes. An assay quantifying the antigen-specific memory B cell responses was established and verified to determine the role of memory B cells on antibody kinetics for future experiments. Lastly, I reported that the genetic diversity of an antigen sequence had a significant impact on antigen-specific antibody responses, and such impact increased in individuals with more past exposure and mature immunity. The findings presented in this thesis provide novel insights into naturally acquired immunity development to P. vivax and support the decision of taking genetic diversity of antigen sequences into consideration for the development of highly efficacious sero-surveillance tools and vaccines.
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ItemHost-Parasite Interactions in the Pathogenesis of Severe Plasmodium falciparum Malaria( 2020)Severe Plasmodium falciparum malaria has been attributed to cytoadhesion and sequestration of infected erythrocytes (IEs) to microvascular endothelium, and rosetting of IEs with other non-infected erythrocytes. These mechanisms are mediated by the interaction between variant surface antigens (VSAs) on the IEs and host receptors on the endothelial cells and erythrocytes. There are at least three VSAs important for this process, which are P. falciparum Erythrocyte Membrane Protein-1 (PfEMP-1), Repeat Interspersed Family protein (RIFIN), and Subtelomeric Variant Open Reading Frame (STEVOR). This study aimed to investigate whether the polymorphisms in the host genome that encode VSA receptors may influence the pathogenesis and mediate protection against severe malaria. Secondly, as PfEMP1, RIFIN, and STEVOR have been shown to mediate rosetting, the potential co-expression among these three VSAs were investigated by comparing their expression on the erythrocytes infected by rosetting and non-rosetting P. falciparum parasites in two different host environments based on ABO blood types (A+ vs O+ erythrocytes). Thirdly, the development of antibody against rosetting-mediating VSAs were screened in longitudinal cohort children from Papua New Guinea, and determined whether they were associated with protection against severe malaria. For the host genetic aspects, this study focused on investigating the role of the polymorphisms on the Endothelial Protein C Receptor (EPCR) encoding PROCR gene and the ABO blood types, which were shown to be associated with severe malaria, in determining the protection against severe malaria in a cohort of very young children from Papua New Guinea (PNG). In children with the PROCR rs867186 polymorphism, there was a risk-reduction trend for severe malaria incidence but it was not significant which was likely due to small number severe malaria cases (n=24). However, the ABO blood group was not found to be associated with protection against severe and clinical malaria. In addition, the significantly higher levels of antibodies to rosetting-associated than EPCR binding PfEMP-1-CIDR domains in children carrying the PROCR polymorphism suggested the preference towards parasites expressing non-EPCR binding VSAs, such as rosetting mediating VSAs. The in vitro cultures for rosetting parasites in A+ and O+ erythrocytes showed distinct patterns of upregulated genes where more VSAs consisting mainly PfEMP-1 and RIFIN were seen in A+ than O+ rosetting parasites, and consequently a higher rosetting rate in the former. This is consistent with previous studies showing that individuals with O blood type tended to have low rosetting rate and were protected from severe malaria. The antibody detection using the sera from the PNG cohort children against rosetting associated RIFIN and STEVOR identified in this study showed a consistent pattern indicating the role of ABO blood group as well as the PROCR polymorphism in determining the selection of VSA subtypes. In conclusion, this study is the first to link host genetic polymorphisms with differential exposure to malaria antigens and highlights the importance of considering the diverse environment in which natural infections occur. This study has provided a better insight into the complex host-parasite interactions during P. falciparum pathogenesis, which is crucial to form a basis to further develop the most effective approach to interrupt this process. Future studies are mainly directed to validate the findings using a study population with more severe disease cases and higher level of immunity, as well as replicating the in vitro rosetting study in different parasite isolates to confirm whether VSA expression is conserved across different P. falciparum rosetting parasite strains.
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ItemAspartic proteases and their potential for transmission blocking strategies( 2019)Sexual stage development in Plasmodium spp. is essential for transmission through the mosquito and to the human host. It represents objects to study a broad range of biological processes, including stage conversion and parasite/host co-adaptation. After the bloodmeal, male and female gametes emerge from intracellular gametocytes and zygote formation follows fertilization. Ookinetes develop from the zygote and traverse through the midgut epithelial cell layer to the basal lamina side of outer wall and develop into oocysts, the only parasite developmental stage that grows extracellularly and this growth and development creates thousands of sporozoites. Once fully developed and egressed, these sporozoites are released into the mosquito hemocoel and they migrate to the salivary gland ready to infect next mammalian host and continue their life cycle. This sexual stage also represents a major bottleneck during the life cycle of Plasmodium as, in mosquito midgut, parasites have to persevere for up to 24 hours outside host cell, exposed themselves to various risk factors such as components of human immune system included within bloodmeal, natural midgut microbial flora in mosquito midgut, and mosquito innate immune system. This exposure can lead up to an approximate 300-fold decrease in parasite survivability during the transmission to mosquito. Due to this unique feature, sexual stage is prime target for transmission blocking intervention strategies aimed to inhibit spread of the disease by the mosquito. Protease enzymes are essential during many steps of malaria parasite development in the blood and transmission stages and an important group of these enzymes are the plasmepsins, of which there are 10 in Plasmodium acting at various points through the life cycle. So far, only 4 plasmepsins are identified to be involved in critical processes and required for transmission. Firstly, plasmepsin VI is highly expressed during sexual stages and was previously shown to be involved in sporozoite development in P. berghei. Secondly, plasmepsin VIII is expressed in mature sporozoite and responsible for sporozoite motility in P. berghei. Finally, PMIX and X are found to be essential in both blood and mosquito stages, making them stand out as promising drug targets. In this study, we attempted to determine the biological functions of plasmepsin VI, IX, and X during transmission of malaria parasites. We found that plasmepsin VI is required for transmission of P. falciparum and might plays an important role in sporozoite egress process instead of sporozoite development as observed in P. berghei. We also found that our dual inhibitor that target both plasmepsin IX and X is able to block the transmission of P. falciparum to mosquito while another antimalaria compound that target only plasmepsin X is enough to block transmission of P. berghei from mouse to mosquito suggesting that both plasmepsin IX and X are essential for transmission. Taken together, our data has identified 3 plasmepsins that play important roles in sexual stage of malaria parasites and more works are needed in order to determine the mechanism of action of these 3 proteases.
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ItemMolecular mechanism of cell traversal by Plasmodium falciparum( 2016)Malaria is an infectious mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Each year, malaria affects over 200 million people, causing considerable morbidity and mortality. A central feature of the virulence of malaria parasites is the ability of the liver-infective form of the parasite, known as sporozoites, to migrate from the mosquito bite site in the skin through host tissues to the target organ, the liver. The ability of sporozoites to traverse through different host cell types is crucial for the establishment and development of parasites within the mammalian host. Over the past decade, our understanding of traversal has become clearer through important studies using rodent models of malaria, such as P. berghei and P. yoelii. However, it remains unclear how these findings apply to malaria parasite species that infect humans, such as P. falciparum and P. vivax. Furthermore, proteins involved in the process, as well as a step-wise molecular model of it, remain unknown. In order to address these questions, the work presented in this thesis utilises molecular genetics and cellular biology to investigate the role of proteins in the traversal mechanism. Overall, this study has identified a novel role for two well-known proteins, Apical Membrane Antigen 1 (AMA1) and Merozoite Apical Erythrocyte Binding Ligand (MAEBL), in the traversal process. Furthermore, this study has validated the role Sporozoite Protein Essential for Cell Traversal (SPECT) and Perforin-Like Protein 1 (PLP1) in P. falciparum sporozoites, which are two proteins that previously have been identified as playing a crucial role in traversal using rodent models of malaria. Using mice engrafted with human hepatocytes, this study also demonstrated the importance of traversal for P. falciparum sporozoites to establish infection of human hepatocytes in vivo. Together, these findings provide the first molecular understanding of cell traversal by P. falciparum and give valuable insights into the complexity of traversal and allowed the formation of a basic molecular model for this process.
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ItemNaturally acquired humoral responses to Plasmodium vivax and Plasmodium falciparum: identification of antigenic targets to inform rational biomarker and vaccine development( 2016)Malaria is an infectious disease caused by Plasmodium spp. parasites, transmitted by the bite of infected Anopheles mosquitoes. Among the five species that can cause disease in humans, P. falciparum and P. vivax are responsible for the majority of the cases and deaths. Due to increased political commitment and funding, the last decades have experienced a dramatic reduction in the burden of malaria, with several countries now attempting to permanently eliminate this disease. Achieving the goal of malaria elimination would be greatly facilitated by the development of biomarkers that can identify the remaining populations at-risk, as well as an effective vaccine. However, while it is clear that individuals living in endemic areas become gradually protected against malaria disease, the targets and mechanisms underlying the acquisition of natural immunity are complex and still poorly understood, hindering the development of such tools. This thesis aimed to investigate comprehensive panel of P. vivax and P. falciparum proteins as targets of natural immunity in Asia Pacific populations, and how this information can be used to inform rational vaccine and biomarker development. Strong associations of antibody responses to both novel and known P. vivax antigens with protection against clinical malaria were identified, as well as optimal antigenic combinations with predicted protective efficacy above 90%. By comparing humoral responses to P. vivax and P. falciparum, this thesis shows that early immune responses are markers of exposure and thus increased risk, whereas prolonged exposure and higher antibody titers are required to achieved clinical protection. The findings of this study support the development of a highly efficacious multicomponent malaria vaccine, and the use of serology as a surveillance tool.
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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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ItemActin regulation in Plasmodium falciparum: towards understanding the elusive nature of malarial actin filaments( 2015)Malaria disease, caused by the unicellular parasites from the genus Plasmodium, is a major cause of morbidity and mortality in many developing countries throughout the world. While there have been many improvements in intervention strategies in recent years, parasite resistance to front-line therapeutics is on the rise, highlighting the need for new and improved treatments and vaccines. To this end, a greater understanding of the biological mechanisms underpinning the disease will be crucial in the push towards malaria eradication. Across the malaria life cycle the parasite must traverse tissues and invade host cells in order to establish an infection and replicate. A conserved acto-myosin motor, anchored at the parasite periphery, generates the requisite force to drive the parasite forward, facilitating both invasion and motility. The actin at the heart of this motor is extremely divergent, forming filaments that are highly dynamic and unstable. Tightly controlled regulation of malaria actin is therefore necessary to direct the formation and disassembly of filaments in an appropriate spatio-temporal manner. However, malaria parasites possess a markedly reduced repertoire of actin regulators, of which coronin is one of the only predicted filament regulators. Much of the current literature surrounding Plasmodium actin biology relies on the production of actin from recombinant sources. In this study I investigate the various published methods for purifying recombinant malaria actin, and determine that the unusual characteristics previously reported for this actin are likely artifacts driven by incomplete protein folding in heterologous expression systems. This finding lead to the identification of the key actin folding chaperonin CCT in the Plasmodium genome, an essential protein complex required for producing native, functional actin in the cell. In parallel, characterization of the filament regulator, coronin, revealed its critical role in the organization of actin filaments. Using in vitro observations from recombinant Plasmodium falciparum coronin (PfCoronin), I have demonstrated that PfCoronin binds to actin filaments and bundles them together in parallel arrays. Furthermore, in vivo observations revealed PfCoronin to be located at the periphery of the parasite, consistent with the pellicular space in which the actin-myosin motor is housed. This localization is likely mediated by peripheral interactions with PI(4,5)P2 at the plasma membrane. These data identify PfCoronin as a potentially key regulator of actin filament recruitment and bundling at the cell cortex of motile Plasmodium parasites. Taken together, the identification of Plasmodium CCT and the characterization of PfCoronin have opened up new avenues for further development of these as potential drug targets, with the eventual aim of potentially crippling the motile malaria parasite and halting the progression of disease.