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    Identification and characterisation of monoclonal antibodies against malaria
    Chen, Yijun ( 2018)
    Plasmodium falciparum and Plasmodium vivax are responsible for the majority of malaria-related deaths globally. The clinical symptoms of malaria are caused by the blood-stage infection. Malaria parasites invade erythrocytes using multiple parasite ligand-host receptor interactions. For P. vivax, its infection is strictly limited to human reticulocytes which are young erythrocytes. Recent work showed that P. vivax reticulocyte binding protein 2b (PvRBP2b) was identified to bind reticulocyte receptor transferrin receptor 1 (TfR1) to mediate the invasion into human reticulocytes. For P. falciparum, one of these ligand-receptor interaction is between P. falciparum reticulocyte binding protein-like homologue 4 (PfRh4) and the erythrocyte receptor complement receptor 1 (CR1). The PfRh4-CR1 invasion pathway is responsible for the majority of sialic acid-independent invasion. Anti-PvRBP2b mouse monoclonal antibodies obtained by hybridoma fusion method, anti-PfRh4 antibodies purified from either mouse or rabbit immunisation, or from immune individuals in Papua New Guinea inhibit the PvRBP2b-TfR1 and PfRh4-CR1 interaction respectively, and block parasite invasion. These results proved that PvRBP2b-TfR1 and PfRh4-CR1 interactions play critical roles in P. vivax and P. falciparum invasion respectively, and also suggest the potential application of anti-PvRBP2b and anti-PfRh4 monoclonal antibodies in parasite invasion research and malaria treatment. I aimed to isolate, express and characterise mouse monoclonal antibodies using the single B cell expression cloning method that combines antigen-specific B cell sorting and antibody gene cloning. I enriched B cells from PvRBP2b-immunised mice and sorted-PvRBP2b-specific B cells with B cell surface markers and fluorescent PvRBP2b. From a single B cell, I synthesised the respective cDNA and amplified the antibody genes with nested-PCR. The antibody chains were cloned into expression plasmids for expression in human embryonic cells followed by antibody purification. I characterised ten antibodies and identified three monoclonal antibodies that are able to inhibit PvRBP2b binding on reticulocytes. I also planned to use the same technology to isolate anti-PfRh4 human monoclonal antibodies from individuals in the P. falciparum endemic area. I was unable to adapt this technology because of the presence of CR1, the receptor for PfRh4, on human B cells. However, I optimised PfRh4 functional validation assays as a screen for antibody reactivity and functional inhibition for future researches. Overall, my work demonstrates the successful adaption of single B cell expression cloning techniques for isolation of antigen-specific monoclonal antibodies against P. vivax and P. falciparum targets, which will deepen our understanding of parasite invasion into erythrocytes and provide potential immunotherapy for malaria treatment.