A major virulence factor of the human malaria parasite Plasmodium falciparum is Plasmodium falciparum erythrocyte membrane protein 1(PfEMP-1). This protein is inserted into the erythrocyte membrane, giving cytoadherence properties. A family of genes called var, located sub-telomerically and in chromosome central clusters encode this protein. Var genes are expressed in a mutually exclusive manner, how this is controlled is unclear. A non-coding RNA (ncRNA) termed the GC-rich element (GRE) had been identified that is only located at the central clusters and is transcribed throughout the parasite lifecycle. A screen of the P. falciparum genome for novel ncRNAs identified ncRNAs from known classes. Novel transcripts were identified, but none in the proximity of var genes. We have investigated the role of the GRE in var gene regulation. A set of qRT-PCR primers have been designed and tested to follow var gene expression in the HB3 isolate, these are not cross-reactive with a published set for the 3D7 isolate. Alterations were made to the 3D7 set to remove cross-reactivity with HB3. Var gene expression was studied in 31 HB3 clones and progeny of the 3D7xHB3 genetic cross. Following var switching over five months in eleven HB3 clones showed that all of the clones ended up expressing var genes from the same central cluster on chromosome 4. GRE Transcription in these clones is linked to a specific class of var gene. Transcription from a single GRE locus occurs only when a var gene of the central UpsC class is expressed from the same cluster. Expression of other classes of var gene gives multiple transcripts from different GRE loci. Investigations into the in vitro binding properties of the GRE revealed an RNA:protein complex that can be resolved by electrophoresis. Proteomic analysis of the complex revealed predominantly ribosome proteins and translation factors.

Epidemiological observations have linked increased host iron with malaria susceptibility. At the same time, blood-stage malaria infection is associated with potentially life-threatening anemia. To improve our understanding of these relationships, this work presents an examination of the mechanisms controlling the upregulation of the hormone hepcidin, the master regulator of iron metabolism, in malaria infection. Chapter 2 presents data from a mouse model of malaria infection which indicate that hepcidin upregulation in malaria infection is associated with increased activity of the sons of mothers against decapentaplegic (Smad) signaling pathway. Although the canonical Smad pathway activators, bone morphogenetic proteins (Bmp) are not increased at the message level following infection, activin B, which has been recently shown to increase hepcidin through the Smad signaling pathway in conditions of inflammation and infection, is upregulated in the livers of malaria-infected mice. Chapter 3 shows that both activin B and the closely related protein activin A upregulate hepcidin in vitro and in vivo. Chapter 3 also explores the effects of the activin-binding protein follistatin in both systems and in the same malaria-infected mouse model as presented in Chapter 2. The work presented in Chapter 4 extends these studies to human infections by demonstrating that activin A protein co-increases with hepcidin in human serum during malaria infection. Taken together, these findings are consistent with a novel role for activin proteins in controlling hepcidin upregulation in the context of malaria infection. This work may form a basis for the development of novel therapeutics that speed recovery from malarial anemia by inhibiting activins’ actions. Chapter 5 examines the role of infected red blood cell-derived microparticles in the initial recognition of a P. falciparum malaria infection, and subsequent hepcidin upregulation. Microparticles stimulate production of cytokines from peripheral blood mononuclear cells (PBMC), which also upregulate activin A message in response to both microparticles and whole infected red blood cells. These data are consistent with a model in which malaria-derived stimuli such as microparticles trigger the systemic release of activin proteins, which then act on the liver to upregulate hepcidin. Evidence has shown that cytokine levels at birth are related to malaria risk. In Chapter 6, hepcidin is measured in cord blood samples from participants in a large-scale clinical study in a malaria-endemic area, and shown to be elevated in cord blood from neonates with a clinical history of placental malaria. Cord blood hepcidin is also compared to birth levels of iron markers and other cytokines, and future clinical outcomes. Finally, the contributions of DNA methylation levels to cord hepcidin and cytokine levels are assessed by comparison of CpG methylation, at sites in genes encoding hepcidin and cytokines, to the serum concentrations of the genes’ protein products. Several intriguing associations are noted which indicate a possible novel role for DNA methylation in the determination of birth cytokine and hepcidin levels. Chapter 7 synthesizes the data presented in this thesis, interprets the possible significance of the major findings, and offers suggestions for future work.

The apicomplexan parasite Plasmodium falciparum is the causative agent of the most severe and deadly form of human malaria. The production of an efficacious malaria vaccine is seen as one of the key steps towards the eradication of the disease, however to date only one candidate has progressed to application for licensure. Candidate malaria vaccines target the different stages of the P. falciparum lifecycle through induction of a functional immune response. Vaccines targeting the blood-stage parasite require induction of high titre neutralising antibodies. To achieve this, vaccine regimens have been designed specifically to maximise antibody induction and maintenance in humans. The ultimate test of any candidate vaccine is clinical efficacy and controlled human malaria infection (CHMI) is a powerful tool for measuring this. This model can also be used to study how vaccine induced antigen-specific components of the immune system respond to native antigen exposure in the context of parasitic infection In this Thesis I describe the induction and maintenance of B cell responses, including memory B cells (mBC) and antibody secreting cells (ASC) to the candidate blood-stage malaria antigens MSP1 and AMA1 following vaccination with a variety of regimens and CHMI. These B cell populations along with peripheral blood T follicular helper (Tfh) cells correlate strongly with antibody induction. Within these populations I have identified a number of phenotypically distinct subsets which contribute to a functional response to vaccine and/or parasite antigen. From single cell sorting of ASC at day seven post-boost I have managed to produce the first fully human monoclonal antibodies (hmAbs) specific for AMA1, one of which shows significant growth inhibitory activity (GIA). Despite promise the vaccine candidates MSP1 and AMA1 have been disappointing in terms of human efficacy. In this Thesis I have attempted to provide explanations on a cellular level as to why there is such disparity between pre-clinical and human data and ultimately why these candidates may have failed to provide efficacy. Such work will provide a strong basis for analysing future clinical trials of alternative candidate blood-stage vaccines and allow accurate characterisation of immune correlates and clinical efficacy when it is achieved.

The virulence of Plasmodium falciparum is largely attributed to the ability of asexual blood-stage parasites to cytoadhere to the microvascular endothelium of the human host. This pathogenic behavior is mediated by the primary parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), an understanding of which is crucial to develop interventions to ameliorate the morbidity and mortality of P. falciparum malaria. The work presented in this thesis describes the application of a phenotype-to-genotype experimental approach to identify novel parasite proteins involved in the trafficking and display of PfEMP1. Guided by the overall hypothesis that the in vitro culture-adapted parasite line 3D7 harbors 1 or more genetic determinants of impaired PfEMP1 trafficking, surface PfEMP1 levels were first measured in 3D7, the presumably trafficking-competent parasite line HB3, and 16 unique progeny from an HB3 x 3D7 genetic cross (chapter 2). These phenotypes were then combined with genome-wide SNP data in QTL analysis to identify genetic polymorphisms potentially responsible for the impaired trafficking in 3D7 (chapter 3). A near-significant QTL containing a single protein-coding gene, the putative kinesin Pf3D7_1245600, was identified, characterized, and investigated in CRISPR-Cas9-driven allele-exchange parasite transfection experiments to establish a causal link between the gene and PfEMP1 trafficking (chapter 4). The parasite transfections were unsuccessful, but the potential role of Pf3D7_1245600 in PfEMP1 trafficking was indirectly assessed through the disruption of microtubules with colchicine (chapter 4), which significantly impacted the surface PfEMP1 levels of HB3 but not 3D7. The findings of this thesis suggest that kinesins and microtubules may play previously unconsidered roles in the regulation, production, or trafficking of PfEMP1.

This thesis investigates patterns and mechanisms of gene expression in P. falciparum. The rapidly cycling patterns of genes during the asexual stages confounds the analysis of gene expression in culture and in patients. In order to overcome this problem, we develop statistical models to estimate the temporal progression of malaria parasites by using the observed gene expression values and known reference sets. We extend this framework to account for lineage commitment, and show that, similar to asexual development, it is also possible to recover information about parasite sexual differentiation given observed gene expression values. Using datasets from our own lab as well as those available in the literature, we establish that the patterns of expression in patients are similar to those observed in culture but in additive mixtures whose proportions can vary between patients. We then investigate epigenetic and spatial factors that are important in regulating gene expression. Using second-generation DNA sequencing, we generate genomic maps of chromatin state and chromosome interactions. These maps provide the first global view of chromosome folding and locus-specific affinities in malaria. They also highlight the importance of epigenetic modifications in imposing structure on the spatial organization of the genome. After generating an initial set of genomic maps, we apply these tools to study chromatin reconfigurations during var gene switching. We show that when the active var gene changes, reconfigurations can be seen in the local three-dimensional chromatin structure of the activated locus. Overall, our results contribute new methods for analyzing malaria microarray data, highlight the importance of lineage mixtures in patient infections, generate an atlas of spatial interactions in the nucleus at a resolution of approximately 5 kilobases, and establish a link in malaria between the spatial configuration of active loci and the local chromatin environment.

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The Hepatitis B core antigen (HBcAg) has been proposed to be an ideal candidate for use as an adjuvant due to its immunogenicity, and tolerance to manipulations such as insertions of epitopes or covalent attachment of ligands. HBcAg is a complex macromolecule containing protein and nucleic acid. We investigated the effect of the removal and reconstitution of nucleic acids upon its structure. It’s been shown that the RNA content of hepadnavirus core antigens can be reduced significantly, but not be completely removed. Following removal of some of the RNA, antigens retain the ability to bind added nucleic acids, in particular, "immune-enhancing" synthetic oligonucleotides without affecting the structure of antigen or disrupting its ability to spontaneously self-assemble into core particles. The removal and addition of nucleic acids was successfully applied to an altered woodchuck core antigen, with a nucleic acid-based malaria epitope addition, giving rise to a potential vaccine adjuvant platform for malaria.

Les analogues de choline constituent une nouvelle classe d'antipaludiques qui inhibent la biosynthèse de la phosphatidylcholine (PC) de Plasmodium, parasite responsable du paludisme. Les études conduites ont mis en relief des particularités uniques de ces composés. Nous avons élucidé le mécanisme d'action biochimique de l'albitiazolium, actuel candidat clinique, caractérisant chacune des 5 étapes conduisant à la biosynthèse de PC. L'albitiazolium affecte en premier lieu l'entrée de choline dans le parasite intraerythrocytaire, choline et albitiazolium utilisant le même transporteur et affecte de façon différentielle les autres étapes de synthèse. L'activité antipaludique est fortement antagonisée par la choline indiquant que le mécanisme d'action primaire est bien l'inhibition de la synthèse de PC. L'accumulation des analogues de choline dans le parasite intracellulaire leur permet de restreindre leur toxicité aux seuls érythrocytes infectés. Des études comparatives réalisées chez Plasmodium et Babesia montrent une double compartimentation de l'albitiazolium uniquement chez Plasmodium, l'une d'elles correspondant à la vacuole digestive. L'accumulation chez Plasmodium est glucose-dépendante et exige aussi le maintien des gradients ioniques dans la cellule. Bien que les analogues de choline exercent leur effet antiparasitaire dès les premières heures de contact, l'effet dit « cheval de Troie » exige des conditions particulières pour les mesures d'activités pharmacologiques, nous amenant à comparer différents tests d'activité. Seuls les tests isotopiques basés sur l'incorporation d'hypoxanthine ou d'éthanolamine après un cycle parasitaire entier et le test fluorescent au SYBR green appliqué après 72h obtiennent des résultats fiables quel que soit le mécanisme d'action des antipaludiques. Enfin, des études de pharmacocinétique / pharmacodynamie montrent une exposition plasmatique supérieure chez les souris infectées par Plasmodium, due au recyclage de l'albitiazolium après son accumulation dans l'érythrocyte infecté. / Choline analogues form a new class of antimalarial drugs that inhibit the biosynthesis of phosphatidylcholine (PC) in Plasmodium, the malaria-causing parasite. The studies presented here highlighted the unique features of these compounds. We elucidated the biochemical mechanism of action of albitiazolium, the current clinical candidate, characterizing each of the 5 steps leading to the biosynthesis of PC. Albitiazolium primarily affects the entry of choline into the intraerythrocytic parasite and choline and albitiazolium use the same carrier. The other steps of synthesis are differentially affected. Antimalarial activity is strongly antagonized by choline indicating that the primary mechanism of action is the inhibition of PC synthesis Accumulation of choline analogs in the intracellular parasite allows them to restrict their toxicity to infected erythrocytes. Comparative studies in Plasmodium and Babesia show a double compartmentalization of albitiazolium only in Plasmodium, one of them corresponding to the food vacuole. Accumulation in Plasmodium is glucose-dependent and requires maintaining ionic gradients in the cell.Although choline analogues exert their antiparasitic effect in the first hours of contact, the “Trojan horse effect” requires specific conditions for the determination of pharmacological activity, leading us to evaluate various tests of activity. Only the isotopic tests based on hypoxanthine or ethanolamine incorporation after one parasite cycle and the fluorescent SYBR green assay applied after 72 hours give reliable results regardless of the mode of action of the tested antimalarials. Finally, pharmacokinetics/pharmacodynamics studies in Plasmodium-infected mice revealed that albitiazolium is recycled after its accumulation in the infected erythrocyte leading to increased plasma levels.

Entre juin et septembre 2009, nous avons réalisé une étude transversale pour évaluer l'état de la prise en charge du paludisme chez la femme enceinte à Bangui. Les résultats de cette évaluation ont montré que dans les services de consultation prénatale (CPN) de Bangui, 28,8% des femmes enceintes reçoivent à titre curatif au moins une prescription de médicament antipaludique pendant leur grossesse. La quinine et les combinaisons à base d'artémisinine, antipaludiques compatibles avec la grossesse, sont prescrites dans des proportions de 56,7% et 26,8% respectivement. Par contre, la confirmation du paludisme par un examen de laboratoire est réalisée seulement dans 18,9% des cas avant la prescription du traitement. Les deux doses recommandées de traitement préventif intermittent du paludisme par la sulfadoxine-pyrimethamine (TPIsp) sont administrées à 30,5% des femmes pendant leur grossesse. Les moustiquaires imprégnées d'insecticide à longue durée (MIILD) sont utilisées par 42,4% des femmes enceintes. Malgré ce, la prévalence de la parasitémie placentaire à l'accouchement est relativement faible (4%). Ces données nous ont amené à réaliser une étude dont l'objectif était d'évaluer l'intérêt de l'introduction d'un test de diagnostic rapide (TDR) sur la rationalisation du traitement du paludisme chez les femmes enceintes lors des CPN. Entre octobre 2009 et octobre 2011, nous avons réalisé une étude sur une cohorte de 76 femmes enceintes. Le nombre de traitements antipaludiques après confirmation du paludisme par TDR Paracheck-Pf® lors des CPN a été déterminé sur cette cohorte. / From June to September 2009, we designed a cross-sectional study aiming to assess malaria management during pregnancy in antenatal health care in Bangui. Our findings showed that antimalarials are prescribed to 28.8% of pregnant women attending antenatal clinics (ANCs) in Bangui. Quinine and artemisinin combined therapies are widely used (56.7% and 26.8% respectively). However, laboratory diagnosis of malaria infection is performed for solely 18.9% of consultants. The recommended two doses of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTsp) are given to 30.5% of pregnant women, while 42.4% of them use the insecticides treated nets (ITNs). Nonetheless, the prevalence of placental malaria at delivery is relatively low (4%). From those preliminary data of our study we assessed the impact of a systematic rapid diagnosis test (RDT) of malaria during pregnancy on antimalarials prescription, during the period from October 2009 and October 2011. The proportions of antimalarial treatment episodes were compared in two groups of women: a cohort of 76 pregnant women presenting at their ANCs visits, in which a systematic screening of malaria with the RDT Paracheck-Pf® was performed and a control group of women who delivered in the same period. Our findings showed that in the cohort, there was a proportion of 13.8 % of positive RDT, hence requiring antimalarial treatment, while the proportion of antimalarials prescriptions in the control group was 26.3% (P = 0.0001). The avoidable rate of unnecessary antimalarials prescriptions was estimated at 47%.

A eux seuls, le Nigeria, la République Démocratique du Congo, l'Ouganda, l'Ethiopie et la Tanzanie représentent 50% de la mortalité liée au paludisme et 47% du total des cas de paludisme. Cependant, la diminution du nombre de cas et l'augmentation des activités de lutte à l'échelle mondiale sont autant de bonnes nouvelles encourageantes qui font déclarer à l'OMS que le paludisme peut être contrôlé et plus tard éliminé. Les recommandations des experts de la lutte contre le paludisme à l'échelle mondiale recommandent de cibler les zones où l'élimination est possible, les zones de faible transmission, et de là progresser vers les zones plus impaludées. Cela impose une connaissance précise de la situation du paludisme dans la région concernée. Les ensembles géographiques régionaux où la transmission du paludisme connait une diminution sensible, comme la Corne d'Afrique, et ayant des frontières extrêmement poreuses aux mouvements de populations humaines donc aux pathogènes, sont donc à cibler en priorité. Cela nécessite une évaluation du niveau de transmission et du risque de résurgence potentiel, posé par l'importation de nouvelles souches, afin d'aboutir à une élimination durable. Dans ce contexte, la République de Djibouti, ayant montré une prévalence extrêmement faible au cours de ces dernières années, s'est lancée dans une tentative de pré-élimination du paludisme. L'objectif de notre étude a été d'observer l'évolution, sur une durée de onze années (1998-2009), du taux d'incidence de l'infection palustre, du niveau de transmission du paludisme, de la possibilité d'importation depuis les pays voisins et enfin, de la distribution des vecteurs de la maladie. / Nigeria, the Democratic Republic of Congo, Uganda, Ethiopia and Tanzania represent 50% of malaria deaths and 47% of total malaria cases. However, the decrease in the number of cases and worldwide increased control activities were encouraging news which lead the WHO to declare that malaria can be controlled and eliminated later. The recommendations of experts in the worldwide fight against malaria recommend targeting areas where elimination is possible, areas of low transmission, and hence moving towards areas more affected by malaria. This requires a precise knowledge of the malaria situation in the region. Geographical regional assemblies where malaria transmission is experiencing a significant decrease, as the Horn of Africa, and having extremely porous frontiers to the movement of human populations, therefore pathogens, must be the priority target. Thus an assessment of the level of transmission and the risk of potential recurrence, posed by the importation of new strains, was indispensable in order to achieve sustainable elimination. In this context, the Republic of Djibouti, who showed extremely low prevalence in recent years, is embarked on an attempt to pre-eliminate malaria. The aim of our study was to observe changes, over a period of 11 years (1998-2009), in the level of malaria occurrence, malaria transmission, but also the level of its potential importation from neighboring countries and finally the vectors population dynamics. These results could serve as a basis of reflection, for the health authorities of the country or international partners, to a possible adjustment of the current policy of fight against malaria.