Medical Biology - Theses
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ItemIdentifying and Characterising Exported Hepatic Effector Proteins of the Human Malaria Parasite Plasmodium falciparum( 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.
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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.