Medicine (RMH) - Theses
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ItemThe role of neutrophils in immunity to malaria( 2023-07)The global malaria burden continues to remain high with most deaths occurring in young children. Understanding the protective immune mechanisms against malaria is imperative for the development of a highly efficacious vaccine since RTS,S and R21 which are recommended malaria vaccines, have modest efficacy and are limited to young children. Recent reports indicate that neutrophils may potentially play a key role in protective immunity against malaria via antibody-mediated interactions with Fc-gamma receptors (FcgR) expressed on their surface. Additionally, complement proteins may promote parasite clearance via interaction with antibodies. This thesis presents new findings on antibody interactions with complement (C1q) and FcgRs on neutrophils in immunity against severe malaria. It further presents new information on neutrophil phenotypes in response to naturally acquired malaria as well as phenotypes and transcription profiles in controlled human malaria infections (CHMI), and in vitro models. First, cytophilic IgG1 and IgG3 antibody subclasses, and antibodies that can fix C1q, or engage FcgRs to induce phagocytic clearance of merozoites were investigated. Additionally, opsonic phagocytosis of merozoites by neutrophils was quantified. These investigations were carried out on samples from young Papua New Guinean (PNG) children (n = 383) presenting with severe malaria versus uncomplicated malaria. In children presenting with severe malaria, cytophilic antibodies, antibodies that can fix C1q, or engage FcgRs against whole merozoites and neutrophil phagocytosis of merozoites opsonized by serum antibodies were lower than children with uncomplicated malaria. There was a strong association between high antibody responses to merozoites that fix complement or bind FcgRs, and reduced odds of severe disease. Second, cryopreserved neutrophils from children and adults in PNG, were phenotyped by examining receptors relevant to functions and malaria immunity. Neutrophils from children infected with P. vivax malaria (n = 37) had increased CD66b expression compared to uninfected children. Increased statistical power by combining uninfected children (n = 29) and uninfected adults (n = 35) into one group and comparing them with P. vivax and P. falciparum (n = 25) infected children further highlighted increased CD64 expression on neutrophils from malaria infected children compared to uninfected group. Neutrophils from uninfected children had distinct phenotypic differences from uninfected adults. There were also some differences identified in neutrophil phenotypes in males compared to females infected with P. falciparum. In addition, neutrophil phenotypes from uninfected adults and uninfected children varied substantially from neutrophils from malaria-naive adult Australian residents (n = 11), revealing increased CD64, CD54, and CD66b, and decreased CD62L, CD16, and CD32a expression, although CD32a expression in malaria-exposed children did not differ from malaria-naive adults. Neutrophil phenotype changes relevant to effector function and immunity to malaria model were assessed at multiple time points in a controlled human experimental P. falciparum infection trial. The study included 8 healthy Australian adult participants who were treated at day 8 of malaria infection. Neutrophil phenotypic changes occurred as early as day 4, prior to substantial blood-stage parasitemia. There were significant and substantial changes in neutrophil phenotypes following treatment to clear parasitemia and some changes persisted weeks post-treatment. To further understand neutrophil responses to malaria, phenotype changes in response to direct interaction with merozoites in an in vitro model were examined. There were no phenotype changes after neutrophils were incubated with unopsonized merozoites or with merozoites opsonized with serum from malaria-naive donors. In contrast, distinct changes in phenotypes were observed after incubation of neutrophils with merozoites opsonized by naturally-acquired antibodies from children. These changes included upregulated CD66b and downregulated CD64, CD32a, CD16, and CD62L expression. Finally, transcriptional analysis of neutrophil gene sets relevant to functions and clearance of malaria parasites were explored among samples from a controlled P. falciparum experimental infection trial in healthy Australian adults (n = 12) and using in vitro models. CHMI data revealed upregulation of gene sets including phagocytosis pathways and cytokine mediated signalling pathways. In vitro transcriptional analysis of neutrophils incubated with merozoites opsonized by purified IgG from malaria-exposed children showed upregulated gene sets including respiratory burst and cytokine activity compared to neutrophils incubated with merozoites opsonized with IgG from malaria-naive individuals. However, gene set enrichment analysis did not reveal any significant change in phagocytosis gene sets. The data generated in this thesis advances our knowledge on antibody mechanisms involved in immunity to malaria and provides new knowledge on the phenotypes and functions of neutrophils in response to malaria. Given that emerging data are increasingly indicating an important role for neutrophils in naturally-acquired and vaccine-induced immunity, this knowledge will be valuable for informing the development of more efficacious malaria vaccines.
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ItemDissecting the antibody responses in pregnancy malaria( 2023-09)Abstract Malaria remains a major public health problem in tropical and subtropical areas of the world. It is a leading cause of morbidity and mortality in these areas, especially in young children and pregnant women. Pregnant women, despite lifetime malaria exposure, have increased susceptibility to Plasmodium falciparum malaria. This is due to the ability of parasites to sequester in the placenta by expressing VAR2CSA, a P. falciparum erythrocyte membrane protein 1 (PfEMP1) that is upregulated during pregnancy. With repeated exposure, pregnant women develop antibodies to VAR2CSA that are associated with reduced adhesion of infected erythrocytes (IEs) to the placenta, which may improve pregnancy outcomes. However, the development and function of these antibodies may depend on a combination of factors, including transmission intensity, cross species immunity, and maternal genetics. This PhD thesis aims to dissect diverse aspects of antibody responses to malaria in pregnancy and factors that may affect acquisition and functions of these antibodies. Aim 1 in this thesis examined antibody responses to P. falciparum and P. vivax antigens in pregnant women residing in low malaria transmission areas in the Brazilian Amazon region (chapter 4 and 5). The total and opsonic IgG antibody levels to pregnancy specific whole parasite surface antigens on CS2-IEs (CS2VSA), individual recombinant VAR2CSA domains and to other malaria antigens were assessed in the plasma of 408 Brazilian pregnant women using various assay formats including flow cytometry, enzyme linked immune assay (ELISA), and multiplex assays. Antibodies to CS2VSA were generally low but were higher in currently infected women and women with multiple P. falciparum episodes over pregnancy. Many women (21%-69%) had antibodies against each individual VAR2CSA DBL domain, and antibodies to DBLs correlated with each other, but not with antibody to CS2VSA or history of infection. We did not find a significant association between antibodies to CS2VSA with pregnancy outcomes. However, antibodies to individual VAR2CSA DBLs and certain P. falciparum merozoite antigens were significantly associated with lower odds of maternal anaemia (OR range 0.38-0.54, p<0.038). Aim 2 explored whether exposure to P. vivax antigens could lead to the production of cross-reactive antibodies to VAR2CSA. The acquisition of antibodies specific to VAR2CSA is generally assumed to occur through exposure to P. falciparum during pregnancy. More recent evidence has suggested that individuals exposed to P. vivax can also acquire antibodies to VAR2CSA. However, this evidence is based on antibodies to recombinant proteins and may be an artifact of the protein expression system. Therefore, in chapter 6 of this thesis, we investigated a hypothesis that P. vivax exposure does not result in antibodies that bind to VAR2CSA on the surface of the IEs and the acquisition of antibodies to VAR2CSA is restricted to pregnancy. The study cohort included P. vivax only infected primigravid women, P. falciparum infected multigravid women, uninfected pregnant women, men, and children from Brazil. When antibodies were measured to surface antigens on P. falciparum IEs, the P. vivax infected women showed significantly lower levels of antibodies to whole parasite CS2VSA in comparison to P. falciparum-infected women (p<0.0001). Additionally, the levels of CS2VSA-specific IgG in primigravid women infected with P. vivax was not significantly different compared to antibody levels in plasma samples from uninfected women. However, it was observed that pregnant women regardless of infection status and gravidity exhibited comparable levels of IgG response to recombinant VAR2CSA DBL3 expressed in P. pastoris and VAR2CSA DBL5 expressed in Chinese hamster ovary (CHO) cells. Intriguingly, men and children also exhibited antibodies against the recombinant VAR2CSA DBL3 domain at levels similar to those observed in pregnant women. In summary, antibodies to CS2VSA did not appear to result from exposure to P. vivax. Nonetheless, antibodies to individual recombinant VAR2CSA domains are not restricted to pregnancy or exposure to P. falciparum. Aim 3 investigated the effect of allotypic variations on the transplacental transfer and functions of malaria-specific IgG3 antibodies. Antibody allotypes are polymorphisms in the constant regions of immunoglobulin heavy and light chains. IgG3 allotypic variations can have a significant impact on associated Fc-mediated effector functions and transplacental transfer of malaria specific antibodies. We identified a novel IgG3 allotype in a cohort of pregnant women from PNG which has a histidine-to-glutamine replacement at residue 433 (IgG3-Q433). The maternal IgG3 gene was sequenced in a total 558 women to identify the IgG3-Q433 polymorphism. A total of 298 pairs of maternal and cord serum samples were analysed to investigate the effect of this allotypic variation on transplacental transfer of IgG3 antibodies. Mother to cord transfer ratios were calculated. We used four VAR2CSA DBL3-specific monoclonal antibodies with different heavy chain constant regions (IGHG1*01, IGHG3*01, IGHG3*30 and IGHG3*30-H435R) to assess the effect of IgG3-Q433 polymorphism on the FC-mediated effector functions, including complement fixation and antibody dependent cellular phagocytosis. In brief, the novel IgG3 variant displayed enhanced ability to induce phagocytosis by neutrophils and THP-1 cells, but it did not show an increased ability to activate the complement system. Additionally, there was no significant difference in antibody transfer from mother to baby between the novel variant and common IgG3 and IgG1 allotypes (Chapter 7). In summary, this thesis analysed diverse aspects of antibody responses to malaria in pregnancy and investigated factors that may affect acquisition and Fc- mediated effector functions of these antibodies. It explored whether these antibodies confer protection against malaria associated pregnancy outcomes or just serve as markers of prior exposure. Furthermore, this thesis investigated the effects of IgG3 allotypic variations on the transplacental transfer of antibodies and the associated Fc-mediated functions. Continued research in this area will add to our understanding of pregnancy associated malaria and potentially reveal novel therapy and vaccine strategies to combat this disease.
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ItemThe role of antibody, complement, and innate immune cells in protective immunity to malaria( 2022)Malaria continues to cause high morbidity and mortality worldwide. In Plasmodium falciparum malaria, the membrane of infected erythrocytes (IEs) is modified to express the parasite-derived protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is the main antigenic target contributing to protective immunity through antibody development. Naturally acquired antibodies are key to protection from malaria that can inhibit parasite growth and neutralise parasites, but sterile immunity is never achieved. Therefore, it is important to dissect antibody features that best correlate with protective immunity. These antibody features are driven by the antibody constant (Fc) region that could generate protective immune responses against malaria. My thesis aimed to 1) dissect the role of antibody-mediated complement activation in immunity to Plasmodium falciparum infection, 2) understand in vitro the role of antibody-dependent phagocytosis (ADP) by phagocytic cells in peripheral whole blood in parasite clearance, and 3) assess the role of ADP of IEs by neutrophils and monocytes as a clinical correlate of protection against severe malaria in children and placental malaria in pregnant women from malaria-endemic areas. For this purpose, for the first time, we developed high throughput plate-based assays to simultaneously measure ADP of P. falciparum-IEs by peripheral blood leukocytes using prediluted whole blood. We also used the developed whole blood phagocytosis assays to assess clinical correlates of protection from placental malaria in pregnant women and severe malaria in young children from malaria-endemic regions and infected with P. falciparum. Our results suggested that opsonic antibody levels directed against P. falciparum-IEs promoted antibody-opsonic phagocytosis of IEs by neutrophils and monocytes in a malaria exposure-specific manner. We also showed that opsonising antibodies that promoted ADP of IEs in whole blood mainly targeted PfEMP1 on IEs, although antibodies also target secondary antigens on the IE surface, such as the most abundant band 3 proteins. Next, we showed that antibody-complement interactions are an integral immune mechanism contributing to enhanced phagocytic clearance of P. falciparum-IEs by neutrophils and monocytes from peripheral whole blood. However, complement did not induce antibody-mediated lysis in the presence of plasma from malaria-exposed pregnant women. In malaria-infected pregnant women, opsonising antibody levels against placental binding-IEs were associated with protection against placental malaria. But the same opsonising antibodies that recognised the recombinant DBL-3 domain of VAR2CSA were not protective against placental malaria. Thus, we suggest that opsonising antibody levels that indicate protection against placental malaria are conformation-specific. Last, our results demonstrated that in young children, opsonising antibody levels against EPCR-binding P. falciparum-IEs that promoted ADP by both neutrophils and monocytes were not associated with recovery from severe malaria. Our results suggested that whole blood assays could be exploited to understand antibody and complement interactions during P. falciparum infection that could be translated to low-resource settings to identify antibody correlates of protection in future vaccine design.
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ItemCharacterisation of bromodomain proteins in the malaria parasite Plasmodium falciparum( 2020)Plasmodium falciparum is a protozoan parasite that is responsible for the most severe and fatal form of human malaria. In addition to the canonical histones, the parasite possesses divergent and Apicomplexa-specific histone variants. The N-terminal tails of histones in P. falciparum are extensively acetylated and carry unique acetylation marks. P. falciparum relies on this network of histone post-translational modifications and interactive, chromatin-associated proteins to modulate its gene expression. Integral to this process are bromodomain (BRD) histone reader proteins which interact with acetylated lysine. The rapid emergence and development of human BRD inhibitors for the treatment of many diseases prompted our project to study P. falciparum BRD proteins (PfBDPs) and assess their value as potential, novel, antimalarial therapies. There are eight bromodomain proteins in P. falciparum. P. falciparum bromodomain protein 1 (PfBDP1) is critical for the coordinated regulation of invasion genes and is essential for parasite survival. In addition, a piggyBac transposon mutagenesis forward-genetic screen in P. falciparum showed that PfBDP2, PfGCN5 and PfTAF2 are essential for the parasite during the blood, asexual life cycle. Recent studies showed that PfBDPs can interact with histone acetylations and transcription factors as well as other chromatin-associated proteins to form a network of histone reader complexes. This thesis investigates the functions of PfBDP4 and PfBDP3 and their importance to parasite survival. Conditional disruption of PfBDP4 caused a growth defect from which an essential role in the blood stage, asexual life cycle was inferred. Chromatin immunoprecipitation sequencing (ChIP-seq) indicated that PfBDP4 was enriched upstream of highly expressed genes, especially genes that are important for parasite invasion. In addition, PfBDP4 shares genomic localisation with an Apicomplexa-specific transcription factor AP2-I and the BRD protein PfBDP1, confirming their interaction in a complex as observed in previous studies. PfBDP4 was also enriched upstream of genes that are involved in metabolic pathways such as proteolysis and phosphorylation which demonstrates that PfBDP4 likely regulates multiple critical pathways of the parasite. In contrast, parasite growth was not inhibited by knockdown of PfBDP3, which is consistent with previous studies. In trophozoites, PfBDP3 was enriched in heterochromatin or compact chromatin, where genes are generally silenced. As PfBDP3 has two BRDs, PfBDP3 may act as a scaffold for proteins that are associated with heterochromatin. Finally, hit compounds from collaborators biochemical screens of recombinant PfBDP BRDs were tested for P. falciparum in vitro growth inhibition. Potent hit compounds were identified. These confirmed hits should now be tested for specificity and selectivity to confirm that they are acting on-target and do not cause cytotoxic effects in human cells. Overall, this study showed that PfBDP4 is associated with active promoters of select processes and its BRD is essential for the parasite survival while PfBDP3 is associated with heterochromatin and is non-essential during the asexual life cycle. This work also highlighted PfBDPs as novel drug targets in P. falciparum and demonstrated the feasibility of identifying potent PfBDP inhibitors as candidates for future investigation.