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Abstract

Malaria is still a disease of global health significance. Its causative agent, Plasmodium, has a complex lifecycle alternating between the female Anopheles mosquito and the vertebrate host. The blood stage of the infection is thought to cause the clinical symptoms of the disease and is also targeted by the humoral immune response of the host. Natural acquired immunity to malaria is largely mediated by immunoglobulins and is reflected both by a lower prevalence of infection with age and lower rates of disease. Previous studies have generally focussed on understanding the protective role of naturally acquired functional antibodies targeting the mature infected erythrocyte (mIE) and highly immunogenic merozoite. However, the ring-infected erythrocyte (rIE), has hardly been explored to date in the context of naturally acquired protective immunity. As it is the stage of the infection that is predominantly found in the peripheral circulation, its immunological significance is uncertain. This thesis was aimed at contributing to this knowledge gap by investigating the role and significance of rIEs in protective immunity. Firstly, to investigate the immunogenicity of rIEs, I tested whether they were recognised by malaria-immune antibodies using immunofluorescence and flow cytometry. I confirmed that malaria-immune antibodies do bind to rIEs using both laboratory-adapted (lab) and field isolates. This binding was strongly correlated with that directed against mIEs and merozoites, both of which have been shown to be important targets of protective immunity. Malaria-immune antibody binding to rIEs had no direct inhibitory effect on parasite survival and maturation. In separate assays, I tested whether rIEs were retained in the spleen independently of antibody using a microsphiltration assay. As has been previously reported, rIEs were retained in the spleen and I demonstrated for the first time that the retention rate was comparable between lab and field parasite isolates of the same age, suggesting that this may be a mechanism of parasite clearance that contributes to the low parasite densities observed in malaria endemic areas. Secondly, to investigate the likely physiological relevance of antibody binding to rIEs during P. falciparum infection, I tested whether the in vitro opsonic phagocytosis of rIEs predicted the outcome of infection in a controlled human malaria infection (CHMI) study. I established the flow cytometry based opsonic phagocytosis assay (OPA) of rIEs in our laboratory. I showed for the first time, to the best of my knowledge, that opsonic phagocytosis of rIEs was significantly associated with the outcome of infection (lower parasite densities and the need for treatment) in the CHMI study. Interestingly, phagocytosis of uninfected erythrocytes (uEs) in the ring culture was higher in untreated as compared to the treated individuals, suggesting a potential contribution to anaemia, as others have reported. In addition, I developed competition OPAs and used stage-specific spent media, to demonstrate that merozoite specific antibodies mediated the observed opsonic phagocytosis of the ring culture cells and uEs incubated in spent media respectively. Finally, to investigate the specific parasite antigens on the surface of ring culture cells and uEs incubated in spent media, I employed surface trypsinization, followed by mass spectrometry of supernatants. This is the first surface proteomics study to be conducted on rIEs and in this single study I was able to identify parasite proteins on the surface of rIEs and uEs that had previously been identified singly, in a piecemeal fashion (EBA140, EBA175, RAP1, RAP2, RhopH1, RhopH2, RhopH3 and MCP1), and ENO which I showed for the first time. These proteins have been speculated to be transferred by the merozoite to the surface of the newly invaded erythrocyte during invasion and have also been shown to be correlates of protective immunity, with the exception of MCP1, whose role in parasite clearance and protective immunity has not yet been explored. This thesis shows for the first time that antibody mediated clearance of rIEs in vitro, is correlated with the outcome of P. falciparum infection in a CHMI study and was mediated by anti-merozoite antibodies. This study also demonstrates that there are shared parasite targets between merozoites and rIEs. I conclude that rIEs are not immunologically inert and are targeted by antibodies leading to parasite clearance through mechanisms such as phagocytosis. This activity was significantly associated with lower parasite densities and resistance against clinical symptoms in a human malaria challenge study suggesting that it contributes to protective immunity. The enhanced phagocytosis of uEs in protected individuals suggest a potential detrimental impact of anaemia that requires further investigation.