Medical Biology - Theses
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ItemA conserved molecular mechanism of erythrocyte invasion by malaria parasites( 2023-10)Malaria is major global health burden causing over 240 million cases every year and leading to over 600,000 deaths mostly in pregnant women and children under the age of five. There is an urgent need to development novel therapeutic interventions for the control and elimination of malaria. During infection, malaria parasites must invade host erythrocytes in order to live within them. Invasion is a complex multi-step process that involves many molecular host-parasite interactions. In Plasmodium falciparum, the deadliest species of the parasite, the invasion protein PfRh5 assembles into a complex to bind its receptor basigin on the erythrocyte surface. Recent work has revealed two novel members of this complex, PfPTRAMP and PfCSS, form a heterodimeric platform for PfRipr, PfCyRPA, and PfRh5 binding. The PfPTRAMP-PfCSS-PfRipr-PfCyRPA-PfRh5 (PCRCR) complex, and its engagement with basigin, is essential for P. falciparum invasion. PfRh5 does not have an orthologue in all species of malaria, however PTRAMP, CSS and Ripr orthologues are present across the entire Plasmodium genus. This thesis sought to investigate these conserved proteins in other malaria species to further dissect the essentiality of these proteins for Plasmodium invasion more broadly. Orthologues of PTRAMP, CSS and Ripr from two important species of malaria, P. vivax and P. knowlesi, were investigated using recombinant expression and biophysical analysis. Assessment of complex formation shows a conserved assembly of the three proteins in both species, with similarities to P. falciparum. Structural determination of part of the complex revealed the basis of heterodimer formation between PTRAMP and CSS. Antibodies and nanobodies were produced and exhibit a high degree of cross-reactivity between species. A novel protein was identified that may bind to the complex and impart an erythrocyte binding function. The function of the complex and its components in invasion was confirmed using ex vivo invasion assays in Cambodian P. vivax field isolates. Taken together, this thesis shows that a three-membered complex consisting of PTRAMP, CSS and Ripr is conserved in three species of Plasmodium, likely forming a common invasion scaffold in all species of the genus, suggesting a conserved invasion mechanism with implications for cross-species vaccine development for the control of both P. vivax and P. falciparum malaria.
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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.