Characterisation of the Function and Vaccine Potential of Surface Exposed Antigens Required for Malaria Parasite Invasion of Human Host Cells
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(Thesis)
Date
2021
Authors
Henshall, Isabelle Grace
Editors
Advisors
Wilson, Danny
Beeson, James
Paton, James
Beeson, James
Paton, James
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Thesis
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Abstract
With over 200 million cases annually, malaria remains a significant global health problem. Infection with Plasmodium parasites causes malaria with P. falciparum and P. vivax the most clinically important species. Essential for long-term malaria control is the development of a highly effective vaccine. A key target for vaccine development is the exposed merozoite stage which invades red blood cells (RBCs) and is the first stage in disease-causing blood stage growth. Using transgenic parasite lines this thesis investigates function and antibody interactions of surface exposed merozoite antigens, P. falciparum Merozoite Surface Protein (MSP) 2 and P. vivax MSP3H. PfMSP2 antibodies are associated with protection and Combination B, a vaccine featuring PfMSP2, reduced parasite density by 62% in clinical trials, underscoring the vaccine potential of PfMSP2. This thesis explored the hypothesis that PfMSP2 is an essential merozoite protein mediating merozoite RBC interactions that can be targeted by protective antibodies. Phylogenetic analysis found MSP2 was likely present in ancestral Plasmodium, while CRISPR cas9 gene knockouts of PfMSP2 in two different malaria isolates shows that this protein is not essential for P. falciparum growth in vitro, debunking long-held assumptions about PfMSP2. An initial screen using PfMSP2 knockout lines showed PfMSP2 antibodies contributed little to direct inhibition of P. falciparum by sera from malaria immune individuals. These antibodies may contribute to complement inhibition, consistent with published evidence that shows PfMSP2 antibodies can act through complement and cell-mediated mechanisms. Inhibitors of merozoite and RBC interactions, where PfMSP2 was postulated to function, did not show additive entryblocking activity with PfMSP2 knockout. However, PfMSP2 knockout was found to sensitise merozoites to inhibition from antibodies targeting PfMSP1 and PfAMA1. Although PfRON2 peptide mimics had contrary result. Overall, these data suggest PfMSP2 may act as an immune shield protecting key proteins from the inhibitory effects of protective antibodies. Study of P. vivax MSPs has been hindered by the lack of long-term in vitro culture of P. vivax. P. knowlesi, a primate Plasmodium closely related to P. vivax, can be cultured in vitro long-term. Combined with gene-editing techniques P. knowlesi provides a platform to conduct in vitro studies of P. vivax MSPs. The overarching study aim was to generate chimeric P. knowlesi expressing PvMSP3H, a promising vaccine candidate, to facilitate in-depth molecular characterisation. As little is known about PkMSP3s this thesis first examined endogenous PkMSP3 proteins. All endogenous PkMSP3s were shown to be dispensable, with simultaneous deletion of up to three endogenous proteins possible. Attempts to generate a line devoid of endogenous PkMSP3s failed, suggesting that blood stage growth requires a minimum of one functional PkMSP3. Initial strategies to introduce PvMSP3H also failed, with alternate approaches now being explored. Together this work provides insights into the minimal compliment of PkMSP3s needed for parasite growth, tools for the study of Pk/PvMSP3 function and immunogenicity and lays the groundwork for the generation of PvMSP3H expressing P. knowlesi. Overall, this thesis demonstrated the utility of transgenic parasite lines in studies of MSP function and vaccine potential for two different clinically relevant Plasmodium species.
School/Discipline
School of Biological Sciences : Molecular and Biomedical Science
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2021
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