title: Identifying Merozoite Targets of Protective Immunity Against <i>Plasmodium falciparum</i> Malaria
Creators: name: Kamuyu, Gathoni
Publisher: name: The Open University
Year: 2017

description: The observation that individuals living in malaria endemic regions who are repeatedly infected with <i>P. falciparum</i> can acquire immunity, first to severe, then to uncomplicated clinical episodes of malaria, and finally to high parasite densities, provides hope that a vaccine is achievable. Immunoglobulins have been identified as a key component of naturally acquired immunity and identifying the targets and mechanisms by which this protection is achieved is a clear research priority. To date, only a small proportion of the parasite proteome has been evaluated in the context of naturally acquired immunity. This thesis was aimed at contributing to this knowledge gap by identifying novel potential antigen targets of protective antibodies and validating these in samples from Tanzanian adults. <br><br> First, to identify merozoite antigens that were immunogenic, I resolved proteins extracted from <i>P. falciparum</i> merozoites by two-dimensional gel electrophoresis and tested these for reactivity with immunoglobulins from malaria immune adults. Immunoreactive proteins were then identified by mass-spectrometry. In complementary studies, purified <i>P. falciparum</i> merozoites were treated with proteolytic enzymes to release proteins localised on the surface of merozoites, which were subsequently identified by mass-spectrometry. Using stringent criteria, where I combined the data obtained from 2D-immunoblots and surface-trypsinization experiments with bioinformatics prediction (for the presence of a signal peptide and/or transmembrane domain), I narrowed down to 222 potential merozoite vaccine targets. These included known surface and/or immunogenic proteins such as the 6-cysteine proteins (Pf12, Pf38, Pf41), MSP-1, 3, 7, 9, 10, GLURP, AMA1, GAMA, MTRAP, LSA3 and RhopH3 as well as many unstudied proteins. From the set of unstudied proteins, I prioritised 27 antigens for immunoprofiling and identified 19 antigens that are targets of naturally acquired antibodies and potential novel vaccine candidates. <br><br> Next, using a cohort of adults living in a village in Tanzania that experiences hyperendemic malaria transmission throughout the year, I examined antibody responses to the novel potential vaccine candidates to test whether they were correlated with protective immunity. I began by identifying a panel of antigens that were immunogenic and elicited a stable antibody response in adults. Subsequently, I identified six antigens that were individually associated with protection from clinical episodes of malaria. Individuals who became ill during the follow-up period had significantly lower levels of these antibodies compared to those who did not. These antigens were the pantothenate transporter (PfPAT), a putative amino acid transporter (PF3D7_0629500), PF3D7_0830500, PF3D7_1025300, PF3D7_1345100 and PF3D7_1401600. In addition, the breadth of antibody responses to the tested antigens was associated with protection from clinical malaria. Finally, four of these six antigens strongly correlated with protective effector functions. Antibody responses to PfPAT were strongly correlated to both the ability to fix soluble factor C1q (C1q-fixation) to merozoites as well as with their interaction with neutrophils to release reactive species (ADRB). In addition, antibody responses to the putative amino acid transporter, PF3D7_1345100 and PF3D7_1401600 were strongly associated with C1q-fixation ability. Recruitment of the soluble factor C1q onto the surface of merozoite results in lysis via the classical complement pathway. The release of reactive oxygen species by neutrophils is thought to be toxic to the intra-erythrocytic stages of the parasites and is associated with parasite clearance. <br><br> This thesis shows that 19 antigens, some of which have been studied for the first time in this work, are targets of naturally acquired antibody responses. Six of these antigens appeared to be associated with protective immunity to malaria in adults and correlated strongly with immune effector mechanisms that are thought to be important for parasite clearance. These findings provide a set of antigens that warrant further evaluation for inclusion into the vaccine pre-clinical development pipeline.