Medical Biology - Theses
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ItemDevelopment of antibody therapeutics for deadly infectious diseases: malaria and COVID-19( 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.
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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.