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ItemThe Parasite Genetic and Host Immunological Determinants of Immune Escape in Plasmodium falciparum Malaria( 2022-12)Abstract Human malaria remains a major global public health problem with an estimated 241 million clinical cases and 627,000 deaths in 2020, expected to increase in future years. Highly effective vaccines are urgently needed to progress the control and elimination of the disease. There are dozens of candidates in development, however only one vaccine (RTS, S) targeting the most virulent human malaria parasite, Plasmodium falciparum, has reached Phase 4 implementation trials with 50% efficacy that is short-lived and strain specific. As WHO has outlined a goal for malaria vaccines with a 75% efficacy against clinical malaria in all malaria-endemic countries by 2030, novel approaches are needed to increase efficacy. The limited efficacy of malaria vaccines to date has been in part attributed to the extreme diversity of parasite antigens being developed as ‘subunit’ vaccines, with only one or two randomly selected allelic variants as the basis for inducing immune responses. Antigen diversity has evolved as a means for malaria parasites to evade host immune responses - a process known as an immune escape. Pinpointing specific antigen polymorphisms that drive immune escape would help to prioritise antigens and alleles for inclusion in vaccine formulations. In my Ph.D. project, I investigated the hypothesis that specific polymorphisms in leading P. falciparum vaccine candidates are associated with immune escape. To test this hypothesis, I first analysed the publicly available MalariaGEN genome sequence data to catalogue the global genetic diversity of the genes encoding 25 leading P. falciparum vaccine candidate antigens. Predicted regions of immune selection were identified on both the linear gene sequence and the 3-dimensional protein structure. We then focused on two cohorts of children from malaria endemic regions of PNG conducted during moderate and high transmission periods. We analysed samples from 758 children, conducting multiplexed high-throughput assays on serum samples to measure IgG responses against 27 antigens, and targeted amplicon sequencing of 38 parasite antigen genes in sequentially collected samples from each child to measure the rate of allelic turnover for each antigen. The analysis identified critical immune escape genes and their specific polymorphisms that contribute to immune escape. The relationship between measures of genetic diversity and immune selection in the global data, and the antibody response in the children identifies antigens driving immune escape and those where diversity did not appear to contribute to immune escape. This research provides a vital framework for the prioritization of vaccine candidate antigens and a ‘serotype classification system’ to identify immune escape polymorphisms and for evaluating strain specific efficacy during vaccine trials.
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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.