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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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ItemMolecular mechanisms of liver infection by the human malaria parasite Plasmodium falciparum( 2021)Malaria is the disease caused by Plasmodium parasites. The parasite infects the red blood cells giving rise to symptoms but it musts first infect the liver to reach the blood. Blocking liver infection would prevent both malaria disease and onward transmission as well as stimulate immunity. However, little is known about parasite-host interactions during liver infection of Plasmodium falciparum, the species responsible for the most lethal form of malaria in humans, as its pre-erythrocytic stages are challenging to study. Plasmodium sporozoites are injected in the dermis by the bite of an infected mosquito. They make their way from the skin to the bloodstream and finally the liver, where they invade and replicate within a hepatocyte. The sporozoite’s journey from the skin to the host liver is enabled by a remarkable process called cell traversal that allows parasites to migrate and penetrate deeper into host tissues by entering and then rupturing host cells. Little is known about the key molecular interactions involved in this mechanism especially with respect to the host cell. There is a lack of knowledge about the importance of host factors and proteins involved in sporozoite infectivity. A deeper understanding of cell traversal and hepatocyte invasion could lead to novel interventions. This work aimed to identify key proteins involved in cell traversal and hepatocyte invasion by P. falciparum. A robust sporozoite production protocol was initially established to ensure the feasibility of the project. Host factors involved in cell traversal were systematically investigated using a whole genome CRISPR/Cas9 knock-out screen. The unbiased screen was enabled by the design of a new positive selection cell traversal assay that kills traversed hepatocytes, permitting the enrichment of traversal-resistant cells. Validation of more than one hundred curated hits identified several human genes involved in infection by other pathogens that are putative proteins involved in P. falciparum cell traversal. Finally, antibodies targeting different regions of the most abundant P. falciparum sporozoite surface protein — the circumsporozoite protein (CSP) — were characterised for their inhibition potential. To do so, an improved method allowing both cell traversal and hepatocyte invasion by P. falciparum sporozoite to be quantified by flow cytometry was established before inhibition assays were performed. Different inhibition profiles were identified, highlighting a role for the N-terminus of CSP in hepatocyte invasion. Identifying essential factors and parasite-host interactions during this first step of the malaria parasite lifecycle will provide more insight into support of a prophylactic treatment for malaria.