Medical Biology - Theses
Permanent URI for this collection
Search Results
1 - 10 of 13
-
ItemAspartic proteases and their potential for transmission blocking strategies( 2019)Sexual stage development in Plasmodium spp. is essential for transmission through the mosquito and to the human host. It represents objects to study a broad range of biological processes, including stage conversion and parasite/host co-adaptation. After the bloodmeal, male and female gametes emerge from intracellular gametocytes and zygote formation follows fertilization. Ookinetes develop from the zygote and traverse through the midgut epithelial cell layer to the basal lamina side of outer wall and develop into oocysts, the only parasite developmental stage that grows extracellularly and this growth and development creates thousands of sporozoites. Once fully developed and egressed, these sporozoites are released into the mosquito hemocoel and they migrate to the salivary gland ready to infect next mammalian host and continue their life cycle. This sexual stage also represents a major bottleneck during the life cycle of Plasmodium as, in mosquito midgut, parasites have to persevere for up to 24 hours outside host cell, exposed themselves to various risk factors such as components of human immune system included within bloodmeal, natural midgut microbial flora in mosquito midgut, and mosquito innate immune system. This exposure can lead up to an approximate 300-fold decrease in parasite survivability during the transmission to mosquito. Due to this unique feature, sexual stage is prime target for transmission blocking intervention strategies aimed to inhibit spread of the disease by the mosquito. Protease enzymes are essential during many steps of malaria parasite development in the blood and transmission stages and an important group of these enzymes are the plasmepsins, of which there are 10 in Plasmodium acting at various points through the life cycle. So far, only 4 plasmepsins are identified to be involved in critical processes and required for transmission. Firstly, plasmepsin VI is highly expressed during sexual stages and was previously shown to be involved in sporozoite development in P. berghei. Secondly, plasmepsin VIII is expressed in mature sporozoite and responsible for sporozoite motility in P. berghei. Finally, PMIX and X are found to be essential in both blood and mosquito stages, making them stand out as promising drug targets. In this study, we attempted to determine the biological functions of plasmepsin VI, IX, and X during transmission of malaria parasites. We found that plasmepsin VI is required for transmission of P. falciparum and might plays an important role in sporozoite egress process instead of sporozoite development as observed in P. berghei. We also found that our dual inhibitor that target both plasmepsin IX and X is able to block the transmission of P. falciparum to mosquito while another antimalaria compound that target only plasmepsin X is enough to block transmission of P. berghei from mouse to mosquito suggesting that both plasmepsin IX and X are essential for transmission. Taken together, our data has identified 3 plasmepsins that play important roles in sexual stage of malaria parasites and more works are needed in order to determine the mechanism of action of these 3 proteases.
-
ItemCharacterisation of the Plasmodium aspartyl proteases DNA-damage inducible protein 1 (DDI1) and Plasmepsin VII (PMVII)( 2019)Plasmodium falciparum resistance to artemisinin-(ART) based combination therapies (ACTs) and other antimalarials poses a major threat to malaria control and elimination. Current efforts are aimed towards identifying potent antimalarials which inhibit multiple stages of the parasite lifecycle or discovering novel drug targets which may help overcome ART-resistance. This work aimed to characterise two aspartyl proteases of P. falciparum which may hold promise as antimalarial targets. One strategy recently proposed to overcome ART-resistance is the synergistic use of a parasite-selective proteasome inhibitor to sensitise ART-resistant parasites to artemisinin. Therefore, development of an inhibitor targeting a parasite-specific protein involved in the P. falciparum ubiquitin-proteasome system (UPS) could yield a combination therapy to tackle ART-resistance. DNA-damage inducible protein 1 (DDI1) is a previously uncharacterised essential aspartyl protease in P. falciparum. Recent studies have shown that the catalytic domain of human DDI2 upregulates the UPS in mammalian cells. In other organisms, DDI1 plays a role in shuttling proteins to the proteasome for degradation via its ubiquitin-like domain. We hypothesise PfDDI1 is an active aspartyl protease and plays a role in the parasite’s UPS. To investigate the role of DDI1 in the UPS and parasite survival, we identified a DDI1 orthologue in P. falciparum and characterised this using several strategies. We utilised CRISPR-Cas9 to knock out, tag and inducibly knock down DDI1 across the asexual lifecycle of P. falciparum, and study the effect of this on parasites. Expression of recombinant DDI1 proteins provided insight into the protease activity and substrate repertoire of PfDDI1. Together these studies provide insight into the domain architecture, essentiality and function of PfDDI1 and clues into its potential as an antimalarial target. Development of an antimalarial to block parasite transmission between humans and mosquitos is also a viable strategy to reduce malaria burden. In this study, we also explore a potential transmission-blocking target, Plasmepsin VII (PMVII) and create tools to enable further study of this aspartyl protease in sexually reproductive gametocytes. These tools are vital to determine the function and substrate repertoire of PMVII and elucidate its potential as an antimalarial target.
-
ItemThe molecular epidemiology of Plasmodium spp. in Solomon Islands( 2018)Malaria is a major global disease burden with significant mortality rates amongst young children. The renewed commitment to eradicate malaria along with intensified control efforts has drastically reduced malaria transmission worldwide. In Solomon Islands, the malaria burden had steadily reduced over the last 20 years, achieving 90% reduction in malaria cases, and now the country is aiming to eliminate malaria by 2030. The journey towards malaria elimination requires knowledge of the local malaria epidemiology to identify the population at risk, as well as to understand the underlying risk of infection. In 2013, a longitudinal cohort study was conducted in Ngella. This study was a follow up from the 2012 cross-sectional survey of all ages in Ngella, which previously identified with a relatively higher prevalence of P. vivax infections (13.4% or 468 P. vivax infections) compared to only five P. falciparum infections, with majority of infections asymptomatic and submicroscopic (Waltmann et al., 2015). However, the underlying Plasmodium spp. infection dynamics in Ngella is unclear and it is unknown if P. falciparum infections has disappeared. This follow up study therefore aimed to investigate the local epidemiology of asymptomatic and submicroscopic Plasmodium spp. infections among a cohort of children aged between six months to twelve years old, which will be presented in this thesis. During the 2013 paediatric cohort study, monthly active case detection (ACD) and passive case detection (PCD) were conducted from May 2013 until April 2014. Plasmodium spp. infections were detected by light microscopy, rapid diagnostics test (RDT) and quantitative polymerase chain reaction (qPCR). The qPCR-detectable infection prevalence for P. vivax infections among the 860 children that attended at least six ACD visits was 11.9% (989 episodes), while P. falciparum infections was 0.3% (27 episodes). Most infections detected were submicroscopic (68%) and/or asymptomatic (92.8%). Of children who had at least one P. vivax infection, two thirds had subsequent infections. Genotyping of these P. vivax infections revealed that the subsequent infections have similar genotypes as the initial infections. Since the recorded primaquine (PQ) treatment among the cohort children was uncommon, this indicated that relapses from hypnozoites contributed a majority of P. vivax infections and anti-relapse treatment is important. Heterogeneous transmission and strong individual variation in risk were observed, suggesting that future malaria control program should tailor made accordingly to the varying risk of infections within Ngella and individuals. Drug treatment with PQ among P. vivax infected individuals is nonetheless challenging. It requires the individual to be glucose-6-phosphate dehydrogenase (G6PD) normal to avoid haemolysis during treatment and have a normal cytochrome P450 2D6 (CYP2D6) phenotype for an efficient treatment. All cohort children were screened for G6PD deficiency prior to the start of the study using the BinaxNOW G6PD Test (Alere Inc., USA), a qualitative rapid diagnosis test for G6PD enzyme activity in human whole blood. If any children were identified with P. vivax infections during the study, only G6PD normal children were administered with treatment for both liver (PQ) and blood (artemether-lumefantrine) stages. Meanwhile, P. vivax infected G6PD deficient children were treated with blood stage drug only. Without PQ treatment, these G6PD deficient children were observed to have significantly higher risk of P. vivax infection. This highlights G6PD deficiency is an obstacle for hypnozoite treatment. As such, it is important to screen individuals for G6PD and CYP2D6 prior to PQ drug treatment. This leads to one of the main aims of this thesis, which is to develop a high throughput population genetic screening assay with long read sequencing capability of both G6PD and CYP2D6 genes. A G6PD and CYP2D6 population based genotyping assay can serve to identify populations at risk of unsafe or ineffective treatment with PQ, thus improving the safety and efficacy of PQ treatment among P. vivax infected individuals in future. The occurrence of P. falciparum infections in Ngella was observed to be sporadic yet persisting throughout the year. By employing ten microsatellite markers and 192 SNP barcodes, the genetic diversity, structure and connectivity of the P. falciparum population in Ngella was studied. Comparative examination of the 27 P. falciparum infections from the cohort along with the five P. falciparum infections from previous cross-sectional survey in Ngella, against other villages (Auki and Tetere) of neighbouring islands of P. falciparum infections revealed that the P. falciparum population in Ngella is genetically related and inbred. Evidence of bottleneck event among the Ngella’s P. falciparum population was observed as well. The data presented in this thesis highlighted the risk factors associating with P. vivax infections, as well as the genetic structure of residual P. falciparum in Ngella. The development of large-scale genotyping assay for G6PD and CYP2D6 with long read sequencing allows population level genetic risk assessment for future radical cure of P. vivax infections. Collectively, these findings may support follow up elimination strategies in Solomon Islands or in other low malaria transmission setting with both P. falciparum and P. vivax coexist. For instance, a safe and efficient PQ mass drug administration can be organised among residents after genetic risk evaluation and high P. vivax transmission pockets identification. Evidence from population genetic approaches such as the identified population bottleneck event and low genetic diversity among the P. falciparum population can be useful knowledge that malaria control program has work effectively and further strengthening of the local control program could possibly eliminate P. falciparum.
-
ItemIdentity by descent analysis with applications to epilepsy studies and Plasmodium causing human malaria( 2017)Relatedness mapping is concerned with identifying genomic regions that have been inherited from a common ancestor. Such regions are said to be identical by descent (IBD) and detecting IBD has proven useful in many applications including disease mapping, discovery of familial relatedness and determining loci under selection. Relatedness mapping is typically performed on humans. As such, methodologies are widely available for diploid genomes. This readily allows for analysis of autosomal chromosomes; however, algorithms are generally not applicable to the X chromosome. This is because females have two copies of the X chromosome while males have one copy of the X chromosome, requiring a more complicated model to account for the difference in chromosomal numbers between males and females. As a result, the X chromosome is generally excluded from analysis. This is unfortunate as an abundance of disorders, such as intellectual disability, epilepsy and autism, would greatly benefit from X chromosome IBD analysis. This thesis describes the first probabilistic methodology for identifying pairwise IBD that is applicable to both the X chromosome and autosomes. Genotype data, extracted from either SNP arrays or next generation sequencing platforms, is used to infer IBD and issues surrounding genotyping errors, missing data and linkage disequilibrium are accounted for. Statistical and bioinformatics analyses are carried out to demonstrate the performance of the methodology and an analysis of a small cohort of individuals with a rare form of epilepsy is successfully performed. The lack of methodologies for haploid chromosomes has implications that extend beyond the context of the X chromosome. In particular, microorganisms with haploid genomes, such as the malaria causing parasite, Plasmodium, and bacterium Staphylococcus aureus, are unable to be analysed for relatedness. Such analyses would be invaluable for the study of these, and other, diseases with the recent emergence of antimicrobial drug resistance, whereby a number of microorganisms have become resistant to first-line and/or last-resort antimicrobial treatments. IBD can be used to identify loci under selection that are associated with antimicrobial resistance as well as monitor the genetic diversity in populations to track disease control efforts. One of the main difficulties with analysing genomic data of microorganisms, collected from a human infection, is the occurrence of multiple genetically-distinct strains within an infection. The genomic data can no longer be treated as though it is haploid and requires special treatment. Like the human X chromosome, these samples are commonly excluded from analysis, which can greatly reduce the power of studies in regions where multiple infections are common. As such, this thesis additionally describes the necessary extensions for the X chromosome IBD methodology to be applicable to non-human haploid organisms, where multiple infections may be present. The algorithm successfully identifies antimalarial drug resistance loci under positive selection in a global dataset of the deadliest species of malaria, P. falciparum, which has recently become resistant to the first line treatment, prompting a global health crisis. This thesis also demonstrates the valuable insights that can be gained from IBD analysis of malaria, which are applicable to other infectious diseases.
-
ItemMolecular mechanism of cell traversal by Plasmodium falciparum( 2016)Malaria is an infectious mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Each year, malaria affects over 200 million people, causing considerable morbidity and mortality. A central feature of the virulence of malaria parasites is the ability of the liver-infective form of the parasite, known as sporozoites, to migrate from the mosquito bite site in the skin through host tissues to the target organ, the liver. The ability of sporozoites to traverse through different host cell types is crucial for the establishment and development of parasites within the mammalian host. Over the past decade, our understanding of traversal has become clearer through important studies using rodent models of malaria, such as P. berghei and P. yoelii. However, it remains unclear how these findings apply to malaria parasite species that infect humans, such as P. falciparum and P. vivax. Furthermore, proteins involved in the process, as well as a step-wise molecular model of it, remain unknown. In order to address these questions, the work presented in this thesis utilises molecular genetics and cellular biology to investigate the role of proteins in the traversal mechanism. Overall, this study has identified a novel role for two well-known proteins, Apical Membrane Antigen 1 (AMA1) and Merozoite Apical Erythrocyte Binding Ligand (MAEBL), in the traversal process. Furthermore, this study has validated the role Sporozoite Protein Essential for Cell Traversal (SPECT) and Perforin-Like Protein 1 (PLP1) in P. falciparum sporozoites, which are two proteins that previously have been identified as playing a crucial role in traversal using rodent models of malaria. Using mice engrafted with human hepatocytes, this study also demonstrated the importance of traversal for P. falciparum sporozoites to establish infection of human hepatocytes in vivo. Together, these findings provide the first molecular understanding of cell traversal by P. falciparum and give valuable insights into the complexity of traversal and allowed the formation of a basic molecular model for this process.
-
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.
-
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.
-
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.
-
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.
-
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.