Medical Biology - Theses
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ItemComplement evasion mechanisms of the deadly human pathogen Plasmodium falciparum( 2016)The human complement system is a front-line defence system against invading pathogens. It has over 50 different protein components that are involved either in pathogen clearance or in the regulation of complement. The two main mechanisms of clearance are direct membrane lysis or opsonisation leading to enhanced phagocytosis. Despite the presence of this potent system, many pathogens thrive in human serum due to the evolution of complement evasion strategies. One common evasion strategy involves pathogens recruiting host regulators of complement activation to down- regulate complement attack on their surfaces. Merozoites, the invasive stage of malaria parasites are exposed to serum after egress from the host red blood cell. In this thesis, we examined whether merozoites recruit human regulators of complement activation to their surface to evade complement-mediated destruction. We found that merozoites recruit the human regulators Factor H, Factor H-like 1 and C1 esterase inhibitor to their surface. Factor H and Factor H-like 1 are recruited by an interaction between the merozoite surface protein Pf92, a member of the six cysteine family of merozoite surface proteins, and the complement control protein modules 5-6 of the Factor H and Factor H-like 1 proteins. When bound to the merozoite surface, Factor H and Factor H- like 1 retain cofactor activity, a key function that allows them to down-regulate the alternative pathway of complement activation. Deletion of the Pf92 gene resulted in a loss of Factor H and Factor H-like 1 recruitment and an increased susceptibility of merozoites to immune destruction. We also showed that C1 esterase inhibitor is recruited to the merozoite surface by an interaction between PfMSP3.1, a member of the MSP3 family of merozoite surface proteins, and the C1 esterase inhibitor serpin domain. Bound C1 esterase inhibitor retained the ability to complex with complement activating proteases C1s, MASP1 and MASP2, allowing it to down-regulate both the classical and lectin pathways of complement on the merozoite surface. Deletion of the PfMSP3.1 gene led to a loss of C1 esterase inhibitor recruitment and an increase in complement deposition on merozoites. However, this resulted in enhanced merozoite invasion in the presence of active complement rather than merozoite destruction. Overall, the ability of merozoites to sequester host complement regulators has important implications for the immune evasion strategy of malaria parasites amid a growing body of evidence for an important role of complement in protection.
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ItemInvestigation of the export pathway in Plasmodium parasites utilising small molecule inhibitors of plasmepsin V( 2016)The human malaria parasite Plasmodium falciparum exports several hundred proteins into the host cell erythrocyte that are involved in cellular remodelling and severe virulence. The majority of proteins exported to the erythrocyte possess a conserved N-terminal export motif termed the Plasmodium export element (PEXEL). In order for proteins to be exported, the PEXEL motif (RxLxE/Q/D) must be processed by an ER-resident aspartic protease called plasmepsin V. Plasmepsin V is conserved across all Plasmodium species, including the most virulent human parasites P. falciparum and P. vivax, and is essential for blood-stage parasite survival. Therefore, plasmepsin V is considered a prime target for the development of new antimalarial therapies. Transition-state peptidomimetics of the natural PEXEL substrate are the most potent documented inhibitors of plasmepsin V to date. One such inhibitor, WEHI-916, showed that plasmepsin V plays a crucial role in protein export and that this process is essential for parasite survival, confirming that plasmepsin V is an important antimalarial target in the asexual blood stages. WEHI-916 has high affinity for plasmepsin V (IC50 20 nM), but only has moderate potency in blocking P. falciparum growth (EC50 2.5 μM). The large disconnect between biochemical and parasite growth inhibition can be attributed to the mimetic’s peptide-like character, which are known to possess poor membrane permeability and are susceptible to proteolytic degradation. Here, two strategies are described to overcome the liabilities associated with peptide-like molecules. These strategies were applied to transition-state mimetics that are potent inhibitors of plasmepsin V, with the aim of improving their membrane permeability and proteolytic stability, in order to enhance their activity against Plasmodium parasites. The first strategy utilised to improve these properties involved N-methylation of the backbone amide bonds. The second approach that was undertaken explored the structure-activity relationship within the S3 pocket of plasmepsin V to find a suitable isostere to replace the highly polar PEXEL P3 arginine, with the aim to improve membrane permeability. To further explore the biological role of plasmepsin V across multiple stages of the Plasmodium lifecycle, preliminary studies were also preformed towards the generation of a novel fluorescent probe that would fluoresce only once irreversibly bound to plasmepsin V. The synthesis of these analogues and the outcome of these strategies are discussed.
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ItemPre-clinical evaluation of glycosylphosphatidylinositol as a potential multi-stage malaria vaccine( 2016)As part of the global effort to eliminate and eradicate Plasmodium malaria parasites, vaccine development has proven to be difficult. This is due in part to the high complexity of the Plasmodium parasite that consists of up to thirteen differentiated forms in the life cycle, and five different Plasmodium parasite species that can infect humans. Furthermore, proteins expressed by the parasite are capable of antigenic variation and exhibit high allelic diversity. For these reasons, vaccine candidates that only target single antigens are likely to be inadequately protective, making an effective subunit malaria vaccine difficult to achieve. In order to overcome the problem presented by variable and allelic diverse proteins presented by the malaria parasite, a novel carbohydrate target known as glycosylphosphatidylinositol (GPI) was selected as an attractive target for our vaccine development program. This is because parasite GPI is structurally conserved and it is found on most malaria parasite life forms, thus making it a potential multi-stage and pan-species target. Furthermore, GPI is also known as a parasite toxin that contributes to the pathogenesis of malaria clinical symptoms during infection. Hence, Plasmodium GPI seemed to be a compelling target for vaccine development. If successful, the vaccine targeting a conserved structure might be able to prevent both parasite infection and malarial disease. To test the above hypothesis, our lab had previously developed a synthetic GPI vaccine based on the structure of Plasmodium falciparum GPI and found that it prevented malarial disease in the Plasmodium berghei rodent malaria model. This finding suggested the need for further investigation on the potential of this vaccine in preventing malaria infection. In order to align our vaccine development goals with the target product profile (TPP) of a malaria vaccine required for the malaria eradication agenda, the studies undertaken for this PhD thesis aimed to test proof-of-concept of the efficacy of the synthetic GPI vaccine against Plasmodium sporozoites infection and parasite transmission in a rodent malaria model. The data provided in this dissertation have proven the possibility of targeting Plasmodium GPI as a multi-stage and pan-species antigen and have provided evidence to support future vaccine development towards the malaria eradication agenda.
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ItemThe molecular epidemiology of malaria in Solomon Islands( 2016)Historically, Solomon Islands in the Southwest Pacific has endured considerable P. falciparum and P. vivax burden. In the last 20 years, it has achieved 90% reduction in malaria cases through sustained, intensified malaria interventions (long lasting insecticide nets, indoor residual sprays and artemisinin-combination therapy) and is aiming for elimination by 2030. In 2012 and 2013, we conducted two cross-sectional surveys (study 1, all age, n=3501; study 2, age 0.5-12 years, n=1078) in Ngella, an area of low to moderate transmission. We aimed to investigate the natural reservoir and local epidemiology of P. vivax and P. falciparum. The contrast was striking. In the 2012 survey, only five clonal P. falciparum infections were identified from a single village and had the same msp2 genotpye. P. vivax prevalence was found to be moderately high (PCR, 13.4%), with predominantly afebrile, submicroscopic infections. The P. vivax infections displayed high genetic complexity (by genotyping with msp1F3 and MS16) and considerable spatial heterogeneity among and within different Ngella regions, and even at sub-village level with some households disproportionately harboring more infected co-inhabitants than others. In the 2013 study, a further seven P. falciparum infections were found in multiple locations, indicating that transmission of this species is continuing but at very low levels and infections are predominantly asymptomatic. To investigate the transmission scenario of the two species in more detail, we undertook population genetics analyses. We typed the five 2012 P. falciparum infections at 10 polymorphic microsatellite loci and 323 P. vivax infections at nine microsatellite loci. The five P. falciparum infections also clonal by this panel of 10 markers. Subsequent analyses of diversity (FST, GST, Jost’s D) and structure (Bayesian clustering) for P. vivax, revealed a genetically diverse population, but spatially fragmented, even among villages 6-15km apart. This indicates that whilst P. vivax may be more difficult to eliminate than P. falciparum, local parasite populations of both species have been affected by control interventions. A noteworthy epidemiological result from the 2012 survey was that living in a household with at least one other P. vivax carrier increased the risk of P. vivax infection, suggesting possible intra-household transmission. Subsequent analysis of genetic relatedness of P. vivax infections within households vs. among households indicated supported this hypothesis. Isolates from the same household were more genetically related than isolates from different households, and a high level of genetic kinship was retained among households located up to 100 meters of each other. Associations of P. vivax infection with human genetic factors known to confer protection against infection (α-thalassemia and Southeast Asian ovalocytosis, SAO) have been investigated in a second cross-sectional study conducted in 2013 in children aged 6 months to 12 years of age. SAO was not found in Ngella, whereas approximately a third of 1078 subjects were found to harbor the α-thalassemia alleles. The findings presented in this thesis will be discussed in the context of factors which may impact on follow-up elimination strategies in Solomon Islands, the Southwest Pacific and elsewhere in the endemic world where both P. falciparum and P. vivax are co-endemic.
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ItemMerozoite surface protein 1: insights into complex formation and function in erythrocyte invasion( 2016)The invasion of Plasmodium falciparum into host erythrocytes during the parasites’ asexual blood stage is a critical step in the perpetuation of symptomatic infection of malaria in the human host. Merozoites, the invasive form of the parasite, express a glycosylphosphatidylinositol-anchored 190 kDa Merozoite Surface Protein 1 (MSP1) on the surface. This abundant, essential protein exists in a large complex that includes other peripheral Merozoite Surface Proteins (MSPs). Together, these large macromolecular complexes are thought to mediate the initial stages of invasion. MSPs are of great interest to the field as they are exposed to the host immune system and also contribute directly to the invasion process. Therefore, there is a strong consideration for considering them as therapeutic targets. The majority of the work assessing these molecules as potential vaccine candidates has been performed with single MSP antigens and vaccine trials on these MSPs have shown variable results. A main concern arising from these trials is the fact that these antigens are often found in complex with other antigens and therefore have regions that are masked when found on the parasite surface. In order to address this, the work presented in this thesis utilises parasite-derived complexes to understand how peripheral MSPs: MSP3, MSP6, MSPDBL1, MSPDBL2 and MSP7 utilise MSP1 as a platform to be presented on the merozoite surface to form an array of complexes with different functional roles. In addition, multiple forms of erythrocyte binding complexes were found to have overlapping functions in invasion. Complexes that are involved in erythrocyte binding were characterised, where two components, MSPDBL1 and MSPDBL2 were shown to mediate erythrocyte binding directly. Overall, this study has identified and validated the presence of multiple Merozoite Surface Protein 1 complexes that are involved in mediating the interaction of the merozoite to receptors on the red blood cell surface, which is a vital process for successful invasion of parasites into host erythrocytes. Together, these findings have provided valuable insights into the complexity of MSP1 complexes and have contributed to the most complete model for the molecular arrangements that occur on the parasite surface to date.
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ItemCell-cell interactions during malaria parasite invasion of the human erythrocyte( 2015)Red blood cells are remarkably resilient, flexible and dynamic structures. These properties are required for their passage through small capillaries and are imparted by the cytoskeleton, a network of proteins that underlies and links to the cell membrane. To successfully invade the blood stage malaria parasite, called a merozoite, must induce rapid and drastic changes to the structure of the target erythrocyte, including the formation of a tight junction and a new cellular compartment, the parasitophorous vacuole. These key modifications involve the infolding of the red blood cell membrane, membrane fusion and fission events and the secretion of parasite proteins into the host. Although detailed cellular descriptions of merozoite invasion have been achieved over the past few decades, comparatively little is known about the molecular basis of how the host cell responds to parasite entry. In fact, in contrast to what is known about the invasion strategies of most other intracellular pathogens, the prevailing model of Apocomplexan invasion imagines a largely binary system within which an active parasite, driven by its acto-‐ myosin motor, invades a passive host cell. There is a growing body of evidence, however, that suggests that Apicomplexan host cells may not be as inactive as initially thought. Nonetheless, to date, there is no direct evidence for the notion that erythrocytes contribute actively to merozoite invasion. This PhD took at its starting point the hypothesise that to invade, merozoites interface with endogenous erythrocyte pathways that regulate membrane and cytoskeletal remodelling, and that the tight junction is a key structure that coordinates the this host-‐pathogen interaction during the brief moment of entry. To address this proposition, this PhD studied P. falciparum merozoite invasion using a combination of in silico bioinformatic screening, high-‐definition imaging, quantitative and high-‐throughput invasion inhibition assays and quantitative phospho-‐proteomics. Work presented in this thesis further elaborates the molecular architecture of the P. falciparum merozoite tight junction, outlines a model for the secretion of virulence factors by the parasite during entry, establishes that an active erythrocyte is a prerequisite for successful merozoite invasion and demonstrates, for the first time, that the red blood cell responds to early invasion events through the phosphorylation of components of its membrane and cytoskeleton. Taken together, these findings provide strong support for a shift in how we conceptualise invasion, from paradigm that focuses almost exclusively on the activity of the parasite towards one in which both the merozoite and the erythrocyte act cooperatively to achieve the requisite remodelling events that lead to successful intracellular infection. By further expounding the way in which the malaria merozoite orchestrates its interaction with its target red blood cell during invasion, and in particular shedding light on the potential host-‐cell contribution to this process, this work informs future endeavours aimed at the development of novel chemotherapeutic targets to stop invasion and hence prevent or treat malaria disease.
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ItemAnalysis of 6-cys proteins and calcium fluxes during erythrocyte invasion by Plasmodium falciparum parasites( 2015)Plasmodium parasites amplify their population within the human host by invading, growing and replicating within the body’s erythrocytes. When the population becomes high enough, the damage caused produces symptomatic malaria disease. To develop new drugs and vaccines against malaria it is therefore important to know as much possible about how parasites grow within the human host and particularly about how the extracellular merozoite stage invades erythrocytes, since this short-lived stage is highly vulnerable. This thesis provides new information from the most deadly human malaria pathogen P. falciparum, on the biochemical characteristics of a little known family of merozoite surface proteins which were thought to facilitate erythrocyte invasion as well revealing with unprecedented resolution, new details about how merozoites enter erythrocytes. P12, P38, P41, and P92 comprise a group of blood-stage merozoite surface proteins that belong to the 6-cys family and all except P41 are predicted to have membrane anchors. To functionally characterize the proteins, specific antibodies were made and were then employed to block merozoite invasion by interfering with the binding of 6-cys to erythrocytes. The effect of the antibodies was very weak and therefore not indicative of a major role for 6-cys in invasion. The antibodies were then used as localization probes and indicated that P12 and P41 were at the merozoite periphery with some concentrated towards the apex. In addition, the non-anchored P41 was held on the merozoite surface through heterodimerization with the membrane anchored P12. Despite the P12/P41 heterodimer being in prime position to bind erythrocytes during invasion no evidence for binding could be established. Characterisation of P92 was next conducted and revealed that like the P12/P41 heterodimer, it was tightly associated with the parasite membrane and later cleaved off possibly during invasion. On the other hand, P38 did not shed from the merozoite surface, and it was carried into the erythrocyte. P92 was strictly localised to the apical end of the merozoite while P38 displayed both apical and surface localisation. Similar to the P12/P41 heterodimer, P92 does not appear to bind erythrocytes. In a final attempt to derive a function for the blood stage 6-cys, their genes were individually knocked out but none of the mutants produced any defective growth or invasion phenotypes suggestive of function. To further study invasion, the morphology and kinetics of this process in P. falciparum merozoites was examined with high-speed live-cell microscopy. With greater temporal resolution, novel cellular actions of the merozoites were observed. For example, during the 7.5 s pre-invasion phase the merozoite deforms the erythrocyte plasma membrane multiple times whilst re-orientating. After a brief rest, the merozoite invaded over a ~17 s period forming a vacuole mainly from wrapping the erythrocyte’s membrane around itself. About 18.5 s after entry, the merozoite began spinning in a clockwise direction to possibly to help disconnect itself from the erythrocyte membrane. After spinning had commenced the host erythrocyte began to develop a spiculated appearance called echinocytosis. Suspecting that calcium influx into the erythrocyte during invasion might be responsible for the echinocytosis, the appearance of these fluxes was monitored during invasion by live cell imaging. These observations confirmed for the first time, that a calcium flux originated as an intense spot emanating from the area of contact between the merozoite and erythrocyte suggestive of pore formation between the cells. Further experiments with modified levels of calcium indicated the ion is required for efficient invasion and may play role in causing echinocytosis. Other work using the calcium flux as a visual marker indicated that pore formation coincided with the deployment of tight adhesive proteins from the merozoite that commit it to invasion. The live cell imaging work presented therefore sheds considerable light on many details of merozoite invasion that could inform future drug and vaccine development. Supplementary Videos: Video 1. High-speed time-lapse acquisition of 3D7 merozoite invading the erythrocyte (40 fps). Video 2. The 3D7 merozoite invading the BODIPY FL C12-sphingomyelin labelled erythrocyte (2 fps, 2× real speed). Video 3. The 3D7 merozoite invading the erythrocyte in the presence of Fluo-4 AM showing the punctate apical calcium and calcium influx in the infected erythrocyte (3 fps, 8× real speed) Video 4. Fluo-4-stained 3D7 parasite culture showing merozoites attempting to invade in the presence of R1 peptide (3 fps, 8× real speed). The punctate apical calcium and influx in the attached erythrocyte were detectable. Video 5. Fluo-4-stained 3D7 parasite culture showing merozoites attempting to invade in the presence of R1 peptide (variable speed). The echinocytotic erythrocyte had not recovered after ~20 min of recording. Video 6. CytD-treated 3D7 merozoite attempting to invade the Fluo-4 labelled erythrocyte (variable speed). The punctate apical calcium was visible but the calcium influx was difficult to observe. The echinocytotic erythrocyte had not recovered after ~20 min of recording.
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ItemB cell responses during severe malaria: the impact of inflammation on T follicular helper cell and germinal centre responses( 2015)Despite many advances in malaria control and elimination, infection by Plasmodium remains a significantly widespread cause of morbidity and mortality worldwide. Naturally acquired immunity to the parasite plays an important role in protection against malaria infection and the development of symptomatic disease. However, no evidence exists of sterile immunity to the disease and the development of sustained clinically protective antibody responses has been shown to require repeated infections. While many studies have focused on the complex nature of these responses against the antigenically diverse parasite, few have addressed the effect of malaria infection on the generation of memory B cell responses. A study of children in areas of high seasonal malaria transmission revealed a delay in malaria-specific MBC generation despite continual exposure to the parasite. In contrast, in a low transmission setting, lasting memory B cell responses were detected in adults following a single exposure to the parasite. These data indicate clinical malaria infections may hinder the generation and maintenance of malaria-specific memory B cell populations. Long-lived populations of B cells, including memory B cells and long-lived plasma cells, are generated during the germinal centre (GC) reaction in secondary lymphoid organs, such as the spleen. In support of the notion that clinical malaria episodes hinder the induction of humoral memory, histological studies revealed that human fatal malaria infections are accompanied by dramatic changes in splenic architecture, including impaired GC formation. The bulk of studies examining the induction of GC responses following malaria infection have made use of self-resolving infection models in mice. To specifically address the impact of severe malaria infections on these processes, the development of GC responses was assessed using the P. berghei ANKA model of severe malaria in comparison to immunisation with an equivalent antigenic load of attenuated parasites. This model permitted the uncoupling of the effects of severe malaria infection and parasite exposure, and demonstrated that severe malaria infections profoundly impede the correct generation of GC structures. Further, compared to immunised control animals, infected animals had reduced numbers of GC B cells. Critically, the excessive inflammatory processes caused by severe malaria infection directly impaired T follicular helper cell differentiation and lead to the preferential accumulation of Tfh precursors. As a consequence of impaired GC induction, memory responses were not efficiently generated following severe malaria. Collectively, the data presented in this thesis demonstrate a novel role for inflammation in the control of Tfh and GC responses and provide valuable insight into the mechanisms underlying inefficient B cell responses following clinical malaria infections in humans.
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ItemDissection of the coordinated events during Plasmodium falciparum infection of the human erythrocyte( 2013)Malaria disease continues to place significant social and economic burdens on the developing world. Of the Plasmodium parasites responsible for the disease, P. falciparum causes the most severe form and thus kills up to 1 million people each year. Unfortunately, recent years have seen rising signs of resistance to even our most successful drug-based therapies and a continued underperformance of promising vaccine prospects during clinical trials. This signals a need for continued research, particularly that focussed on providing new targets for therapy and on the development of methods to more effectively understand new and existing therapeutic approaches during their early stages of development. Invasion and subsequent remodelling of the erythrocyte by the merozoite form of the parasite mark two areas of particular interest. Indeed, both are critical for the establishment of symptomatic infection. Despite their importance and interest as therapeutic targets, study of the P. falciparum merozoite, erythrocyte invasion and early remodelling events have all been hampered by shortfalls in methodology. This has left much to be understood about this period of the lifecycle. Using recent advances in our ability to isolate free, viable, P. falciparum merozoites, I therefore develop methods to fix parasites at each step of, and in the minutes following, erythrocyte invasion. For the first time, this allows detailed imaging of these processes on a molecular level using various imaging platforms, including widefield deconvolution, ‘super-resolution’ three-dimensional structured illumination, and transmission electron microscopies, along with electron tomography. In particular, the application of cutting edge microscopy combined with sophisticated quantitative imaging analysis makes for a powerful investigative approach. Initially, these techniques are developed and used to investigate a number of processes that are critical for merozoite invasion: attachment, tight junction formation, surface protein shedding, actomyosin motor activation and organelle secretion. This study identifies interactions mediated by merozoite surface adhesins as the important initiator of subsequent invasion processes, which all follow without further checkpoints. It also points to the tight junction as a nexus that organises and directs these processes. I then dissect aspects of erythrocyte remodelling, providing previously lacking cellular evidence for the role of the Plasmodium translocon of exported proteins (PTEX) complex during protein export from the parasite. In particular, this study identifies key events that occur in the latter parts of invasion which are critical for subsequent protein export. This points to an important level of coordination between invasion and remodelling events that may be occurring up to 24 hours later. Together the contributions made during this PhD provide the most complete model for invasion and early parasite remodelling to date. The methods developed also provide an important platform from which others can develop our understanding of these critical events in the future.
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ItemTargets of antibodies to the surface of Plasmodium falciparum-infected erythrocytes and protective immunity to human malaria( 2012)Effective clinical immunity that protects against symptomatic malaria in humans develops gradually after repeated exposure to Plasmodium falciparum. Naturally acquired antibodies targeting antigens expressed on the surface of infected erythrocytes (IE) represent an important component of protective immunity against malaria. During intra-erythrocytic development, P. falciparum dramatically remodels the host erythrocyte membrane through the export of novel parasite proteins. Among these are antigens expressed on the IE surface, known as variant surface antigens (VSA), that include PfEMP1, RIFIN, STEVOR, SURFIN proteins and possibly others such as PfMC2TM and modified host band 3. These antigens are highly polymorphic and some are known to undergo clonal antigenic variation for immune evasion. Numerous studies have reported that individuals living in malaria endemic regions were capable of agglutinating P. falciparum-IEs suggesting the recognition of VSAs expressed on the IE surface. Longitudinal studies further suggested that anti-VSA antibodies were associated with protection from P. falciparum malaria. Previous studies have only been able to measure the acquired antibody response towards all VSAs expressed on the IE surface, due to a lack of tools to dissect the antibody responses to individual VSAs. Although studies have also evaluated antibodies to recombinant proteins, it has been difficult to directly quantify the contribution of each native VSA to the overall antibody response to the IE surface. The aims of this thesis were to quantify the significance of VSAs as targets of naturally acquired antibodies, with a particular focus on P. falciparum erythrocyte membrane protein 1 (PfEMP1) and evaluate the importance of naturally acquired antibodies to PfEMP1 and other antigens that afford clinical protection from symptomatic P. falciparum malaria. Novel approaches using transgenic P. falciparum with inhibited PfEMP1 expression have enabled the quantification of PfEMP1 relative to other VSAs as a target of acquired antibodies. This was achieved by the transfection of parasites with a construct that encodes a var gene promoter without a downstream var gene (vpkd; presented in Chapter 3) thus resulting in a PfEMP1-deficient line and the transfection of parasites with a construct that has a deletion of the pfsbp1 gene required for PfEMP1 trafficking (SBP1KO; presented in Chapter 4). These approaches were then applied to human studies in Kenya and Papua New Guinea (PNG) and comparisons between parental and PfEMP1-deficient transgenic parasites allowed for the determination of antibodies specific to PfEMP1. The functional significance of naturally acquired antibodies was determined using assays that specifically measure antibody-mediated phagocytosis of IEs by undifferentiated monocytes. In addition, the clinical importance of PfEMP1-specific antibody responses was further investigated in a longitudinal cohort study with PNG school children. Characterisation of the transgenic vpkd parasites demonstrated reduced var gene transcription by Northern blot analyses and the absence of PfEMP1 proteins by Western blot analyses of IE membrane extracts, thus suggesting that PfEMP1 expression was inhibited in these parasites. In addition, characterisation of the SBP1KO parasites confirmed the absence of PfSBP1 protein in Western blot analyses and immunofluorescence microscopy of pigmented trophozoite IEs. However, other VSAs such as RIFIN and STEVOR, and other IE membrane proteins such as PfEMP3 were still expressed by the transgenic vpkd and SBP1KO parasites. Furthermore, transmission electron microscopy of pigmented trophozoite IEs confirmed the presence of knob structures on the IE surface of the vpkd parasites, similar to parental parasites. These findings suggest that despite the inhibition of PfEMP1, other IE membrane proteins and knob assembly occurred normally in the transgenic vpkd and SBP1KO parasites. Among malaria-exposed children and adults from Kenya and PNG, IgG binding to the surface of erythrocytes infected with the transgenic vpkd parasites was substantially reduced compared to parental. This suggests that majority of the acquired antibody response to the IE surface was predominantly directed towards PfEMP1, while other VSAs appear to play a minor role in relation to immunity. These key findings were confirmed with two genetically different parasite lines, 3D7 and E8B. Furthermore, using sera from children, adults and pregnant women available from Kenya or PNG, IgG binding to the surface of erythrocytes infected with the transgenic SBP1KO parasites was also markedly reduced compared to parental, suggesting that antibodies primarily targeted major antigens expressed on the IE surface that are dependent on PfSBP1 for trafficking. Currently, only PfEMP1 is known to be trafficked by PfSBP1 and this study has demonstrated that other VSAs such as RIFIN and STEVOR proteins remain expressed by the SBP1KO parasites. Comparing the antibody responses between PNG adults and children demonstrated that IgG binding to the vpkd and SBP1KO parasites was substantially reduced in both groups, suggesting that both adults and children had a great proportion of PfEMP1-specific antibodies. Evaluating the effect of trypsin treatment of IEs on antibody binding showed that most serum samples targeted trypsin sensitive epitopes expressed on the IE surface, consistent with PfEMP1 being the major target of antibodies. However, some samples appeared to target trypsin resistant epitopes on the IE surface of the vpkd and SBP1KO parasites. This study provides major new evidence that PfEMP1 is the dominant target of naturally acquired antibodies to the IE surface. In assays that specifically measure antibody-mediated phagocytosis by undifferentiated monocytes, the level of opsonic phagocytosis activity was greatly reduced in the transgenic vpkd parasites compared to parental. These results suggest that PfEMP1-specific antibodies are essential to promote IE opsonisation for phagocytosis by monocytes, an important mechanism in parasite clearance. Thus, these finding provide further evidence that PfEMP1 represents the major target of functional antibodies. Some measurable level of opsonic phagocytosis activity was still detected with the transgenic vpkd parasites although the level of IgG binding to these parasites were extremely low, suggesting that antibodies to non-PfEMP1 antigens may also function to opsonize IEs for phagocytic clearance. The clinical importance of antibodies to PfEMP1 and other VSAs was further evaluated in a longitudinal study conducted with school children from Madang, PNG. Antibodies to the 3D7 parental and 3D7-PfEMP1 (reflected in the difference between IgG binding to 3D7 parental versus 3D7vpkd) were associated with a significantly reduced risk of symptomatic P. falciparum malaria whereas antibodies to 3D7vpkd (reflecting antibodies to non- PfEMP1 antigens) were not associated with protective immunity. Children with antibodies to other isolates such as E8B and XIE-ICAM also had a reduction in malaria risk, however these associations were not statistically significant. It should be noted that there was insufficient statistical power in the current study to detect differences in small effect sizes and weak associations. The protective association with 3D7 observed in the current study of PNG school children complements a longitudinal study conducted with children in Chonyi, Kilifi (J. Chan and K. Howell et al 2012, J Clin Invest, in press) whereby antibodies to 3D7 parental and 3D7-PfEMP1 were associated with protection but antibodies to 3D7vpkd were not. Therefore, these findings indicate that PfEMP1 represents a major target of naturally acquired antibodies that are associated with protective immunity. However, these studies do not exclude an important role for other VSAs as targets of protective antibodies and further studies are essential to understand their significance as antibody targets and their association with protection from malaria. The results presented in this thesis provide major new evidence that among the VSA families present on the surface of P. falciparum-IEs, PfEMP1 represents the dominant target of naturally acquired human antibodies and antibodies to PfEMP1 contribute to protective immunity against malaria. Novel approaches using PfEMP1-deficient transgenic parasites performed in this study offered a unique insight to determine the relative contribution of PfEMP1 and other VSAs to the overall antibody response to the IE surface. Therefore, the work presented in this thesis enhances the understanding of humoral immunity to malaria and will aid the development of vaccines against malaria.