Medical Biology - Theses

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Studies of Plasmodium-Iron Interactions

Clucas, Danielle Bridget ( 2023-09)

Background Iron deficiency anaemia and malaria co-exist across sub-Saharan Africa where they disproportionately affect young children and pregnant women. Iron supplementation is recommended to treat anaemia, but there are concerns regarding its safety and the potential protection afforded by iron deficiency. An interaction between host iron status and malaria risk has been hypothesised for decades, but confounding factors mean that conclusions are difficult to draw from field studies alone. In this thesis the complex interaction between Plasmodium and iron is investigated using clinical, pre-clinical and in vitro studies. Methods The effect of host iron deficiency on the risk of Plasmodium falciparum parasitaemia by PCR was assessed in a cohort of 711 anaemic Malawian pregnant women, and the risk of intravenous iron supplementation on subsequent risk of parasitaemia explored. These findings were dissected using Plasmodium berghei infection in C57Bl/6 mouse models of iron deficiency (Tmprss6 knockout (Tmprss6-KO)) and iron overload (inducible hepcidin knockout (iHamp-KO)). Tmprss6 knockdown (Tmprss6-KD) was used to assess the phenotype in Tmprss6-KO mice and to explore Tmprss6 as a druggable target; achieved through treatment of wild-type C57Bl/6 mice with siTMP, a GalNAc conjugate targeting Tmprss6. The effect of iron restriction on the parasite was investigated through iron chelation of in vitro P. falciparum cultures, followed by transcriptomic and proteomic analysis. A role of post-transcriptional regulation in the response to iron chelation was further explored. In the setting of known post transcriptional regulation of cellular iron in other organisms via the iron responsive element (IRE)/Iron regulatory protein (IRP) system, and with an IRP-like protein described in P. falciparum, the role of PfIRP was explored through the generation and characterisation of PfIRP-KO parasites. Results Among anaemic Malawian pregnant women iron deficiency was associated with a 53% reduced risk of P. falciparum parasitaemia (Adjusted risk ratio 0.47, 95% confidence interval (0.34, 0.60), p<0.0001), with this finding robust to varied definitions of iron deficiency. Intravenous iron did not increase the subsequent risk of P. falciparum parasitaemia. These findings were supported by the mouse models. Tmprss6-KO mice had improved survival when infected with P. berghei. Conversely, iHamp-KO mice exhibited decreased liver stage infection but an unaltered course in the blood stage of infection. Tmprss6-KD did not replicate the KO phenotype; the disease course was not changed in siTMP treated mice. In vitro, iron chelation inhibited parasite growth and induced substantial changes in the transcriptome and proteome of P. falciparum. Differential expression of genes and proteins involved in key processes in the parasite’s lifecycle, and with plausible links to iron were identified, as was a possible role for post-transcriptional regulation. Investigating this showed PfIRP-KO parasites were more susceptible to iron chelation. Transcriptomic and proteomic analysis identified proteins that might be regulated in an IRE/IRP-like manner. Conclusions This work adds to the current understanding of the complex interaction between Plasmodium and iron. Taken together, these data support iron deficiency being protective against P. falciparum infection. In vitro studies highlighted genes of potential further interest in P. falciparum iron homeostasis and support an iron related role for PfIRP.
The Parasite Genetic and Host Immunological Determinants of Immune Escape in Plasmodium falciparum Malaria

Naung, Myo ( 2022-12)

Abstract Human malaria remains a major global public health problem with an estimated 241 million clinical cases and 627,000 deaths in 2020, expected to increase in future years. Highly effective vaccines are urgently needed to progress the control and elimination of the disease. There are dozens of candidates in development, however only one vaccine (RTS, S) targeting the most virulent human malaria parasite, Plasmodium falciparum, has reached Phase 4 implementation trials with 50% efficacy that is short-lived and strain specific. As WHO has outlined a goal for malaria vaccines with a 75% efficacy against clinical malaria in all malaria-endemic countries by 2030, novel approaches are needed to increase efficacy. The limited efficacy of malaria vaccines to date has been in part attributed to the extreme diversity of parasite antigens being developed as ‘subunit’ vaccines, with only one or two randomly selected allelic variants as the basis for inducing immune responses. Antigen diversity has evolved as a means for malaria parasites to evade host immune responses - a process known as an immune escape. Pinpointing specific antigen polymorphisms that drive immune escape would help to prioritise antigens and alleles for inclusion in vaccine formulations. In my Ph.D. project, I investigated the hypothesis that specific polymorphisms in leading P. falciparum vaccine candidates are associated with immune escape. To test this hypothesis, I first analysed the publicly available MalariaGEN genome sequence data to catalogue the global genetic diversity of the genes encoding 25 leading P. falciparum vaccine candidate antigens. Predicted regions of immune selection were identified on both the linear gene sequence and the 3-dimensional protein structure. We then focused on two cohorts of children from malaria endemic regions of PNG conducted during moderate and high transmission periods. We analysed samples from 758 children, conducting multiplexed high-throughput assays on serum samples to measure IgG responses against 27 antigens, and targeted amplicon sequencing of 38 parasite antigen genes in sequentially collected samples from each child to measure the rate of allelic turnover for each antigen. The analysis identified critical immune escape genes and their specific polymorphisms that contribute to immune escape. The relationship between measures of genetic diversity and immune selection in the global data, and the antibody response in the children identifies antigens driving immune escape and those where diversity did not appear to contribute to immune escape. This research provides a vital framework for the prioritization of vaccine candidate antigens and a ‘serotype classification system’ to identify immune escape polymorphisms and for evaluating strain specific efficacy during vaccine trials.
Understanding how malaria-induced T-bet expression impacts the development of protective immunity to infection

Pietrzak, Halina Mary ( 2022)

Malaria is a globally significant parasitic disease infecting millions of people annually. Clinical immunity to infection takes years of frequent exposure to develop and only partially protects the host against clinical symptoms, with individuals in endemic areas often developing chronic, asymptomatic infections. These observations suggest defects in the generation, maintenance, or effector capacity of immune memory induced in response to infection. Antibody responses are a critical component of clinical immunity to malaria. Recent work from our group demonstrated that inflammatory pathways contributing to the development of clinical malaria episodes play a negative role in the induction of humoral immunity. IFN-g produced in response to acute malaria infection was found to upregulate the expression of transcription factor T-bet in T follicular helper cells (Tfh), the key T cell subset required to provide help to B cells for the induction of protective antibody responses to infection. T-bet expression in Tfh cells impairs their normal differentiation and compromises downstream humoral responses to acute infection. The contribution of T-bet expression to the development of Tfh memory cells in malaria is unknown. To investigate this, the Tfh memory cell compartment was examined using PBMC samples from human P. vivax patients, and a murine model of severe malaria infection. Together, these analyses involving flow cytometry, adoptive transfer, and RNA-sequencing approaches revealed that the T-bet influences the composition and development of the Tfh memory cell compartment in malaria. Specifically, the main results from this investigation revealed that T-bet expression in CD4+ T cells impairs the development of Tfh central memory (TfhCM) cells which are an important compartment that support and bolster long-lived memory responses. This data provides evidence that malaria-induced inflammation negatively impacts the development of memory populations required for an efficient response to malaria, thus restraining a potent immune response to re-infection.
Discovery of antimalarials with novel mechanisms of action

Bailey, Brodie ( 2022)

Today, despite an annual investment of $3 billion from governments, NGOs and pharmaceutical companies, malaria remains one of the most devastating parasitic diseases in human history. Of great concern is the emergence of resistance to all currently available antimalarial treatments, particularly the front-line artemisinin combination therapies. Therefore, an urgent need has arisen towards the development of antimalarials with novel mechanism of action to combat the rise of resistance. To discover new antimalarial chemotypes a high-throughput screen of the Janssen Jumpstarter library against asexual stage P. falciparum was undertaken and uncovered the novel 2-(N-phenyl carboxamide) triazolopyrimidine scaffold. This thesis describes the optimisation and mechanistic characterisation of the 2-(N-phenyl carboxamide) triazolopyrimidine antimalarial class. In Chapter 2, the optimisation and phenotypic characterisation of the triazolopyrimidine series was conducted. In the optimisation process, the structure activity relationship was defined and delivered analogues with EC50 values below 100 nM against the asexual stage parasite. Phenotypic characterisation determined this class exhibited a slow to moderate rate of kill and arrested asexual stage development at the trophozoite stage. Equipotent activity against P. falciparum multi-drug resistant field strains and moderately reduced activity in P. knowlesi indicated a distinct mechanism of action to clinically relevant antimalarials and a conserved target, albeit with potential species differentiation. In Chapter 3, the mechanism of action of triazolopyrimidine series was explored using chemoproteomic and chemogenomic techniques. Chemical probes were synthesised for use in affinity pulldown and fluorescent imaging. However, these probes were unable to clearly identify the molecular target of the chemical series. Resistance selection was undertaken and whole genome sequencing of resistant clones identified amplifications and non-synonymous point mutations in a putative mitochondrial carrier protein (PF3D7_0407500) of unknown function. Metabolomic analysis of drug treated parasites indicated disruptions in pyrimidine metabolism and depletion of pantothenate metabolites. The unique metabolic signature reveals a potential multifaceted and indirect effect on the function of the electron transport chain and CoA pathways of the mitochondria. Finally, Chapter 4 describes research towards the validation of the putative mitochondrial carrier protein (PF3D7_0407500) as the molecular target of the triazolopyrimidine series. Genetic validation of the target is described in which we attempt to introduce the resistance conferring mutations into wild-type parasites. Another component of the validation focuses on the characterisation of the indirect effect of the triazolopyrimidine series on the function of the mitochondria. Pantothenate uptake experiments show that a frontrunner triazolopyrimidine analogue blocks the flux of this metabolite into the parasite. To definitively confirm the function of the carrier protein, recombinant expression systems were trialed unsuccessfully to enable biochemical substrate screening and structural studies. Together this research describes the exploration of a novel antimalarial class with a distinct mechanism of action. Future research will aim to genetically validate the carrier protein and further mechanistically characterise its involvement in mitochondria function and development of the malaria parasite. Using these compounds as tools, we have uncovered a novel carrier protein that has a unique function that could represent a novel druggable target for future antimalarial development.
Signaling pathways in apicomplexan parasites

Wilde, Mary Louise ( 2022)

The phylum Apicomplexa contains several parasitic species of medical and agricultural importance, including Plasmodium falciparum and Toxoplasma gondii. Apicomplexan parasites have complex life cycles involving several morphological stages across multiple hosts, and rely on extensive signaling networks to sense their environment, transduce external signals and coordinate appropriate responses. Currently, there are few specific and effective therapeutics to treat parasitic infections and great efforts have been made to understand the signaling pathways and downstream post-translational modifications that regulate crucial parasite life cycle transitions. In the current literature (reviewed in Chapter 1), phosphorylation and the upstream pathways that mediate this post-translational modification is well described in apicomplexan parasites, while ubiquitination is less well understood due to a lack of extensive characterisation of the ubiquitination machinery. Intracellular Ca2+ signaling and the downstream activity of Ca2+-dependent protein kinases (CDPKs) is paramount to parasite motility, and therefore facilitates host cell invasion and egress. In recent years the function of cyclic nucleotide signaling in regulation of Ca2+ mobilisation and motility has come to light. cGMP signaling and its downstream effector kinase, protein kinase G (PKG) is crucial for activating parasite motility, while cAMP signaling via protein kinase A (PKA) has been shown to be a negative regulator of motility in Toxoplasma tachyzoites. PKA has been implicated in Plasmodium merozoite invasion as the kinase responsible for phosphorylation of the key invasion ligand apical membrane antigen 1 (AMA1), however it is unclear how Plasmodium PKA functions in motility. Chapter 3 explores the function of PKA in Plasmodium falciparum through conditional knockdown of the PKA catalytic subunit (PfPKAc) in asexual stage parasites. I show this kinase to be essential in merozoite invasion of erythrocytes, and provide further evidence that PfPKAc is responsible for phosphorylation of AMA1. In Chapter 4, I perform a comprehensive characterisation of the OTU deubiquitinase (DUB) complement in Toxoplasma. OTU DUBs play important regulatory functions in eukaryotic cells, and through bioinformatic analysis I show that OTU DUBs are expanded in Toxoplasma which suggests a functional significance of OTU DUB function in these parasites. I perform a comprehensive biochemical characterisation of OTU DUBs in Toxoplasma. I identify activities against both ubiquitin and NEDD8-based substrates, and reveal ubiquitin linkage preferences Lys6, Lys11, Lys48 and Lys63-linked chain types. I also show that accessory domains of Toxoplasma OTU DUBs are important for regulating OTU domain function and impose linkage preferences. Utilising the auxin-inducible degron (AID) to generate knockdown parasite lines, I also identify TgOTUD6B is important for Toxoplasma growth. This dissertation explores several aspects of post-translational modifications in apicomplexan parasites and provides new insights into PKA function in Plasmodium, and the OTU deubiquitinase family in Toxoplasma.
Identifying and Characterising Exported Hepatic Effector Proteins of the Human Malaria Parasite Plasmodium falciparum

Krol, Jelte Martinus Maria ( 2022)

The parasitic disease malaria is caused by symptomatic infection of Plasmodium parasites during their infection in erythrocytes. Plasmodium falciparum, the most lethal parasite that infects humans, must first undergo replication during the asymptomatic liver stage, which makes it a primary target for prophylactic intervention. Our current understanding of P. falciparum liver stage biology is limited, particularly around the parasite’s ability to subvert host innate defences and exploit host nutrients. This is due to the limited models for liver stage research as P. falciparum replicates in the hepatocyte for 7 days forming tens of thousands of merozoites in an enclosed parasitophorous vacuole. Merozoites in turn infect erythrocytes upon liver stage egress and subsequent release into the blood stream. By infecting an erythrocyte, the parasite forms a parasitophorous vacuole and exports proteins across this vacuole membrane into the host, leading to molecular changes causing malaria-associated pathology. Many parasitic proteins are targeted for export by a pentameric amino-acid motif known as the PEXEL (RxLxE/D/Q), that is proteolytically cleaved in the ER by the aspartyl protease plasmepsin V. Matured effector proteins, as well as PEXEL-negative exported proteins, are translocated into the host-cytoplasm by the PTEX complex. The process of protein export has been widely studied during blood stage infection, but little is known about this process during liver stage. Important questions are whether the export pathway is active and important during the liver stage of the parasite life cycle and whether effector proteins can be identified. In this PhD. we are aiming to address these questions by attempting to identify and characterise novel liver stage exported proteins using two approaches. First, an agnostic approach involving proximity ligation proteomics has been hypothesised to identify liver-stage proteins in subcellular compartments of the parasite and host cell. Second, PEXEL-searching has identified 20 effector candidates of which some have been visualised utilising epitope tagged parasites and polyclonal antibodies. Some effectors have been functionally characterised using rapamycin-mediated conditional gene excision to disrupt expression of respective genes during the P. falciparum life cycle. In vitro sporozoite invasion and traversal assays as well as in vivo liver stage experiments utilising chimeric mice with engrafted human hepatocytes were used to demonstrate the role of these proteins during liver stage. Future identification of novel P. falciparum liver stage exported proteins may provide new drug and vaccine targets for preventive therapeutics targeting the pre-erythrocytic stage of infection by malaria parasites that infect humans.
Molecular mechanisms of liver infection by the human malaria parasite Plasmodium falciparum

Verzier, Lisa Helene ( 2021)

Malaria is the disease caused by Plasmodium parasites. The parasite infects the red blood cells giving rise to symptoms but it musts first infect the liver to reach the blood. Blocking liver infection would prevent both malaria disease and onward transmission as well as stimulate immunity. However, little is known about parasite-host interactions during liver infection of Plasmodium falciparum, the species responsible for the most lethal form of malaria in humans, as its pre-erythrocytic stages are challenging to study. Plasmodium sporozoites are injected in the dermis by the bite of an infected mosquito. They make their way from the skin to the bloodstream and finally the liver, where they invade and replicate within a hepatocyte. The sporozoite’s journey from the skin to the host liver is enabled by a remarkable process called cell traversal that allows parasites to migrate and penetrate deeper into host tissues by entering and then rupturing host cells. Little is known about the key molecular interactions involved in this mechanism especially with respect to the host cell. There is a lack of knowledge about the importance of host factors and proteins involved in sporozoite infectivity. A deeper understanding of cell traversal and hepatocyte invasion could lead to novel interventions. This work aimed to identify key proteins involved in cell traversal and hepatocyte invasion by P. falciparum. A robust sporozoite production protocol was initially established to ensure the feasibility of the project. Host factors involved in cell traversal were systematically investigated using a whole genome CRISPR/Cas9 knock-out screen. The unbiased screen was enabled by the design of a new positive selection cell traversal assay that kills traversed hepatocytes, permitting the enrichment of traversal-resistant cells. Validation of more than one hundred curated hits identified several human genes involved in infection by other pathogens that are putative proteins involved in P. falciparum cell traversal. Finally, antibodies targeting different regions of the most abundant P. falciparum sporozoite surface protein — the circumsporozoite protein (CSP) — were characterised for their inhibition potential. To do so, an improved method allowing both cell traversal and hepatocyte invasion by P. falciparum sporozoite to be quantified by flow cytometry was established before inhibition assays were performed. Different inhibition profiles were identified, highlighting a role for the N-terminus of CSP in hepatocyte invasion. Identifying essential factors and parasite-host interactions during this first step of the malaria parasite lifecycle will provide more insight into support of a prophylactic treatment for malaria.
Development of antibody therapeutics for deadly infectious diseases: malaria and COVID-19

Chan, Li Jin ( 2021)

Monoclonal antibodies are one of the most powerful drugs used in the treatment of human diseases, particularly for autoimmune diseases and cancer. The development of therapeutic antibodies to combat infectious diseases is expanding, with successful treatments available against viral diseases such as COVID-19, Ebola virus, HIV and RSV. The use of monoclonal antibodies complements vaccines by providing immediate protection, preventing disease in immunocompromised people and in containing emerging disease outbreaks. In this thesis, I aimed to characterise neutralizing antibodies against two of the deadliest infectious diseases that affect global populations, malaria and COVID-19. The growth and replication of both these pathogens are critically dependent on their entry into host cells. Neutralizing antibodies that block pathogen entry into host cells can prevent infection and reduce severe disease. Plasmodium vivax is the most widespread relapsing human malaria, which invades reticulocytes through the critical interaction between P. vivax reticulocyte binding protein 2b (PvRBP2b) and human Transferrin receptor 1 (TfR1). I identified TfR1 residues that are critical for complex formation with PvRBP2b. In addition, I characterised naturally acquired human monoclonal antibodies to PvRBP2b, and using structural biology, revealed the epitopes of eight high affinity inhibitory antibodies that block complex formation through different mechanisms. The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which utilizes its spike protein to engage human angiotensin-converting enzyme 2 (ACE2) for host cell entry. I performed deep mutational scanning on a lead neutralising nanobody against SARS-CoV-2 to improve its potency, stability and affinity. These structural studies increase our understanding of host-pathogen interactions and the antibodies that block them, which can inform the development of antibody therapeutics or vaccines.
Low birthweight and infant growth among children in Papua New Guinea - effect of malaria and other infectious diseases during childhood

Ome-Kaius, Maria ( 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.
Plasmodium vivax naturally acquired immunity: patterns and influences

Liu, Shih-Jung ( 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.

Medical Biology - Theses

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