Medical Biology - Theses
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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.
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ItemPlasmodium falciparum merozoite invasion mechanisms and inhibitors of invasion( 2012)Malaria threatens 40% of the global population resulting in approximately 225 million cases of disease and 800 000 deaths per year. Recently marked improvements in the implementation of control measures and increased use of artemisinin combination therapies (ACTs) have contributed to a reduction in malaria mortality and morbidity (World Health Organization, Global Malaria Programme, 2010). Furthermore the licensing and roll-out of the first malaria vaccine, RTS,S, is hoped to occur by 2015 (White, 2011; Agnandji et al., 2011). However, a sustained reduction in malaria burden and eradication of malaria seems unlikely with current control strategies alone. The largest malaria burden is caused by infection with P. falciparum parasites. All symptomatic illness occurs during asexual replication in the blood that is initiated when the merozoite form of the parasite invades red blood cells (RBCs). A limited understanding of merozoite invasion and immune mechanisms inhibiting invasion has hampered rational vaccine and drug development targeting this stage of the parasite life cycle. This thesis is based on the study of merozoite invasion of the RBC with a particular focus on the role of merozoite surface proteins in invasion and mechanisms of inhibition targeting these proteins. Part of the difficulty in studying P. falciparum merozoite invasion is due to the lack of efficient techniques to isolate merozoites that maintain invasive capacity. Chapter 3 describes the development of methods to isolate merozoites that maintain viability. Importantly, the method only requires basic laboratory equipment, therefore is accessible to both resource rich and developing laboratories. Highly synchronized cultures were treated with a cysteine protease inhibitor to block schizont rupture and then merozoites isolated via membrane filtration (Boyle et al., 2010a). Approximately 15% of isolated merozoites maintained viability and were able to successfully invade when incubated with RBCs. This allowed for the development of methods to fix merozoites during invasion for microscopy and invasion inhibition assays. Invasion of isolated merozoites was independent of serum components and the invasive half-life of merozoites was approximately 8 minutes. Merozoite isolation and invasion assays are now being used by a number of research groups and are a powerful technique to study merozoite invasion mechanisms (Riglar et al., 2011) and inhibitors of invasion. In Chapter 4, microscopy of invading merozoites is used to investigate the shedding of merozoite surface antigens during invasion. The initial steps of merozoite invasion are hypothesized to be mediated by merozoite surface proteins that contact with the RBC via weak receptor-ligand interactions. During invasion it is thought that merozoite surface proteins are cleaved and then shed from the merozoite to allow invasion to occur. This has been most clearly demonstrated for MSP1, with compounds that inhibit MSP1 cleavage and/or shedding also inhibiting invasion (Blackman et al., 1994; Singh et al., 2006; Woehlbier et al., 2010; Fleck et al., 2003; Blackman and Holder, 1992). Contrary to the current paradigm, it was found that merozoite surface proteins MSP2 and MSP4 were not shed from the merozoite surface during invasion and were instead carried into the RBC without apparent cleavage. Post invasion, MSP2 was rapidly degraded within a few minutes, whereas MSP4 was maintained for a number of hours. Interestingly, during invasion some MSP2 antibodies were found to be internalized into the RBC. Internalized antibodies were maintained for approximately 24 hours post invasion. This work establishes that there is differential cleavage and shedding of merozoite surface proteins during invasion and suggests that some merozoite surface proteins may have roles outside initial contact events. Chapter 5 investigates the mechanisms by which antibodies inhibit merozoite invasion in the presence of physiological relevant concentrations of complement-active serum. This work was possible due to capacity of isolated merozoites to invade in both the absence of serum and high serum concentrations. While the importance of IgG in mediating parasite clearance is well established (Sabchareon et al., 1991; McGregor, 1964b), the mechanisms of antibody function remain poorly understood. Naturally acquired antibodies from malaria-exposed individuals, as well as antibodies from vaccinated rabbits and humans had complement-dependent inhibition activity targeting merozoite invasion. The complement component C1q was required and appeared to be sufficient for complement-dependent inhibition. MSP1 and MSP2 were identified as targets of complement-dependent antibody mediated inhibition. Antibody mediated complement-dependent inhibition of invasion is a novel mechanism targeting merozoites that may be important in understanding protective immunity and for evaluating candidate merozoite vaccines. Finally, Chapter 6 explores the interaction of heparin with merozoite surface proteins and the potential of heparin-like-molecules (HLMs) as the basis for novel drug development. Heparin is a known inhibitor of merozoite invasion, and appears to act by inhibiting early contact events. Utilizing a heparin-binding assay with native merozoite proteins, heparin was shown to bind the processed fragment of MSP1, known as MSP1- 42. A panel of novel HLMs were screened for growth/invasion inhibition activity and a number of highly inhibitory compounds were identified. This work will be a basis for further studies to identify novel invasion inhibitors that may be used as the basis for drug development. The development of a method to isolate viable merozoites has allowed this thesis to explore a number of aspects of merozoite invasion mechanisms and inhibition of invasion. As well as increasing our understanding of P. falciparum merozoite biology and immunity to malaria, it is hoped that this work will contribute to the development of tools to combat malaria disease.
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ItemThe innate immune response and human severe malaria( 2011)Malaria remains a major cause of morbidity and mortality throughout the tropical world. The emergence of parasite strains resistant to chloroquine and other commonly used anti-malarial drugs provides an urgent call for vaccine development and novel drug discovery. The clinical manifestations of a P. falciparum infection can range from a mild febrile illness to life-threatening severe malaria, however the determinants of disease severity are not well understood. Although the early, innate immune response to malaria serves to limit parasite growth and reduce clinical manifestations, endogenous mediators produced in response to bioactive parasite products may contribute to pathology associated with malaria. The P. falciparum glycosylphosphatidylinositol (GPI) anchor has been described as a malarial toxin, capable of stimulating pro-inflammatory cytokine responses in vitro and in vivo. Vaccination with a synthetic form of the GPI glycan protects mice from experimental cerebral malaria and death. However attempts to investigate the bioactivity of GPI have been hindered by a lack of suitable reagents for its study. Monoclonal antibodies were raised to P. falciparum GPI and used as probes to investigate the role of GPI in parasite cell biology. The expression of free GPI in late schizonts, dynamic localization of GPI during merozoite invasion, and growth inhibition in the presence of an anti-GPI monoclonal antibody suggest that GPI may have a role in invasion of erythrocytes. Furthermore, the pan-species and multiple life stage reactivity of anti-GPI antibodies have important implications for vaccine development. The second part of this thesis describes a study of cellular cytokine and chemokine responses in a severe malaria case control study in Papua New Guinean children. Cytokines and chemokines produced by convalescent peripheral blood mononuclear cells (PBMC) in response to stimulation with parasitized red blood cells or Toll-like receptor 2 (TLR2) and TLR4 agonists were significantly associated with severe malaria. A more comprehensive understanding of the role of these endogenous mediators in malaria pathogenesis will aid in the development of effective vaccines and may provide the opportunity for novel interventions.
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ItemNovel membrane proteins of Plasmodium falciparum( 2011)Apicomplexan parasites such as Plasmodium spp. cause a multitude of illnesses through infection of both human and livestock hosts. Of the Plasmodium species that infect humans, P. falciparum is associated with a large proportion of symptomatic malarial disease, resulting in approximately 800,000 deaths and a further 250 million clinical cases annually (WHO World Malaria Report, 2010). Although there are many facets that contribute to the global health catastrophe caused by malaria, including social, geographical, economic and political; the biology of the parasite itself is a major contributing factor. P. falciparum has a complex lifecycle involving stages in both its mosquito vector and human host, within which it carries out its development in distinct phases in both the liver and blood stream. Symptomatic malaria is associated with blood-stage infection and it is investigations into this asexual life-stage that have formed the basis of my PhD research. In particular I have focused on investigating the functions of membrane proteins, particularly those residing in detergent resistant membranes. These membranes contain regions where the lipids are more ordered and enriched in proteins and are the sites of many important cellular activities such as signal transduction and membrane trafficking. Previous studies have shown that when detergent resistant membranes are recovered from the invasive merozoite stage of P. falciparum, many proteins from the parasite’s pellicle; a three-membrane layer that covers the parasite, are extracted also. Furthermore, proteins that reside in the parasitophorous vacuole, a membrane that envelops young intra-erythrocytic ring-stage parasites, also preferentially associate with the detergent resistant fraction. Within both the merozoite and ring stage parasites reside many proteins of unknown function and during my candidature I have studied several of these proteins. Initial studies presented here have focused on the characterisation of a novel family of Apicomplexan-specific proteins termed the GAPM family which have been identified within the detergent resistant membrane fraction of P. falciparum developing merozoites. It is shown here that the GAPM family of proteins is conserved across all sequenced Apicomplexa and associate into high molecular weight structures within the inner membrane complex of both P. falciparum and the related Toxoplasma gondii. The inner membrane complex forms the innermost pair of membranes of the parasite pellicle. The data presented here demonstrates that the GAPMs bind to both the actinmyosin host-cell invasion motor on the outside of the inner membrane complex and to the parasite cytoskeletal network on the inside of the complex, possibly linking the invasion motor and cytoskeleton together. It has long been recognized in the field that for the invasion motor to work and propel the merozoite into its red blood cell host that the motor must be attached to a solid scaffold such as the cytoskeleton. My functional characterization of the GAPMs has provided the first candidates for proteins that likely tether the parasite motor to the cytoskeleton and therefore shed light on important aspects of how the invasion machinery is assembled. The P. falciparum parasitophorous vacuole membrane is also enriched in detergent resistant membrane proteins and has been shown previously to contain the recently discovered PTEX complex that putatively helps export hundreds of proteins into the host human cell. Exported proteins help remodel the host and contribute greatly to parasite virulence and consequently there has been great interest in unraveling how the export process occurs. Investigations presented here have focused on the biochemical characterisation of the three major components of the PTEX complex, HSP101, PTEX150 and EXP2 in order to provide functional insight into PTEX’s trafficking role. Specifically these studies aimed to glean insight into the overall expression and formation of this important protein complex with particular focus on EXP2 and its putative role as a parasitophorous vacuole membrane-associated pore. Data presented here has revealed that the PTEX components are made in late schizonts/rings and prior to invasion are likely to reside within the dense granules prior to their secretion into the nascent parasitophorous vacuole during invasion. Within the parasitophorous vacuole, EXP2 is the most strongly membrane associated component and is present within this structure as a large oligomeric complex to which the remainder of the PTEX complex attaches. Overall, this data is consistent with EXP2 forming a membrane-associated oligomer, which anchors the remainder of the PTEX components, supporting the hypothesis that this protein is forming the putative membrane-associated pore. PTEX likely functions with several other proteins to establish an export pipeline starting within the parasite where exported proteins are first made and then cross the parasite into the vacuole, upon which time PTEX translocates the protein cargo into the red blood cell. To characterise other proteins possibly involved in export, in the last chapter of my thesis I have investigated a novel parasitophorous vacuole protein; PF14_0201, which was originally identified in the detergent resistant membrane proteome of P. falciparum rings. My studies provide a preliminary characterisation of this novel protein and investigate whether it is indeed a novel PTEX component as suggested by preliminary data. Although PF14_0201 is a resident parasitophorous vacuole protein that it is strongly membrane associated, likely via a GPI-anchor, no substantial interaction was detected between PF14_0201 and the PTEX components indicating that this protein is probably playing another role within the vacuole. Further possible functions of PF14_0201 were explored but proved uninformative. Overall my study of these three sets of detergent resistant membrane proteins, namely the GAPMs, PTEX and PF14_0201, have provided useful information about the formation and function of structures present within different phases of the P. falciparum asexual blood stage cell cycle.