This thesis investigates antigenic variation and its evolution in Plasmodium falciparum, the cause of the most deadly form of human malaria. Antigenic variation is a strategy for evading immunity by switching between antigenic variants during infection. In P. falciparum, such variable antigens confer different binding phenotypes that may affect parasite survival and have also been linked to pathology. Here, a new statistical method is described for determining the switching patterns that underlie antigenic variation. This method is then applied to experimental data to yield a full description of an antigenic switching network in P. falciparum. In light of the findings, theoretical modelling is used to show how immune selection and binding phenotypes may have contributed to the evolution of antigenic repertoire structure, expression order and virulence. Related models are also used to investigate parasite population diversity, providing possible explanations for observations reported here and elsewhere, with implications for vaccine design. Together, these chapters advance understanding of P. falciparum immune evasion and how it relates to pathology. This work further reinforces the role of host immunity in shaping pathogen population diversity at multiple levels.

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.

Os genes var de Plasmodium falciparum codificam a proteína PfEMP1 expressa na superfície de eritrócitos infectados e que medeia os fenômenos de citoaderência e \"rosetting\". Ambos os fenômenos estão diretamente associados à malária grave, e seu domínio mais N-terminal, DBL1alfa, media especificamente \"rosetting\". Análise de seqüências DBL1alfa de isolados brasileiros e de outros países revelou que a similaridade entre elas não pode predizer origem geográfica. Com o objetivo de determinar se existem seqüências DBL1alfa associadas à malária grave, analisamos as seqüências DBL1alfa expressas em parasitas obtidos de pacientes brasileiros com esta manifestação clínica e encontramos que as seqüências predominantemente expressas apresentavam uma ou duas deleções de cisteínas. Significativamente, apesar de freqüentes no genoma de parasitas de pacientes com malária não grave, essas seqüências foram raramente expressas. Esses dados demonstram a primeira associação de seqüências PfEMP1 expressas e malária grave em pacientes da Amazônia Brasileira. / Plasmodium falciparum var genes code for PfEMP1, a protein expressed on the surface of infected erythrocytes, and which mediates cytoadherence and rosetting. Both phenomena are directly associated with severe malaria and the most N-terminal domain, DBL1alfa, specifically mediates rosetting. DBL1alfa sequence analysis from Brazilian and worldwide isolates revealed that sequence similarities cannot predict geographical origin. To determine whether there are DBL1alfa sequences associated with severe malaria, we examined expressed var DBL1alfa sequences in patients with severe malaria from the Brazilian Amazon and found that the predominantly expressed DBL1alfa sequences from these parasites lacked 1-2 cysteine residues. Significantly, these sequences were amply found on the genomic repertoire of parasites from patients with mild malaria and yet they were rarely expressed. These data demonstrate the first association of particular PfEMP1 expressed sequences and severe malaria in patients from the Brazilian Amazon.

Os genes var de Plasmodium falciparum codificam a proteína PfEMP1 expressa na superfície de eritrócitos infectados e que medeia os fenômenos de citoaderência e \"rosetting\". Ambos os fenômenos estão diretamente associados à malária grave, e seu domínio mais N-terminal, DBL1alfa, media especificamente \"rosetting\". Análise de seqüências DBL1alfa de isolados brasileiros e de outros países revelou que a similaridade entre elas não pode predizer origem geográfica. Com o objetivo de determinar se existem seqüências DBL1alfa associadas à malária grave, analisamos as seqüências DBL1alfa expressas em parasitas obtidos de pacientes brasileiros com esta manifestação clínica e encontramos que as seqüências predominantemente expressas apresentavam uma ou duas deleções de cisteínas. Significativamente, apesar de freqüentes no genoma de parasitas de pacientes com malária não grave, essas seqüências foram raramente expressas. Esses dados demonstram a primeira associação de seqüências PfEMP1 expressas e malária grave em pacientes da Amazônia Brasileira. / Plasmodium falciparum var genes code for PfEMP1, a protein expressed on the surface of infected erythrocytes, and which mediates cytoadherence and rosetting. Both phenomena are directly associated with severe malaria and the most N-terminal domain, DBL1alfa, specifically mediates rosetting. DBL1alfa sequence analysis from Brazilian and worldwide isolates revealed that sequence similarities cannot predict geographical origin. To determine whether there are DBL1alfa sequences associated with severe malaria, we examined expressed var DBL1alfa sequences in patients with severe malaria from the Brazilian Amazon and found that the predominantly expressed DBL1alfa sequences from these parasites lacked 1-2 cysteine residues. Significantly, these sequences were amply found on the genomic repertoire of parasites from patients with mild malaria and yet they were rarely expressed. These data demonstrate the first association of particular PfEMP1 expressed sequences and severe malaria in patients from the Brazilian Amazon.

Le paludisme à P. falciparum est l’une des causes majeures de mortalité et de morbidité dans le monde. Ce parasite est responsable de plusieurs manifestations cliniques allant du portage asymptomatique et infections non compliquées aigüe au paludisme grave et compliqué, tel que le neuropaludisme. Nous avons émis l’hypothèse que l’expression différentielle des gènes contribue à la variation phénotypique de parasites, entraînant des interactions spécifiques avec l’hôte, qui à son tour déterminent le type de manifestations cliniques du paludisme. L’objectif principal de cette étude était d’identifier les facteurs génétiques de P. falciparum impliqués dans la pathogenèse du neuropaludisme. Ceci a été réalisé par l’analyse complète du transcriptome d’isolats provenant d’enfants camerounais porteurs asymptomatiques (PA) ou atteints d’accès simple (AS) ou de neuropaludisme (NP). Le transcriptome du clone non sélectionnée (3D7) et la lignée sélectionnée (3D7-Lib) a été également analysé. Les résultats ont montré la surexpression de plusieurs gènes chez des isolats provenant d’enfants atteints de neuropaludisme et chez la lignée 3D7-Lib, par rapport à ceux provenant d’enfants asymptomatiques et 3D7, respectivement. L’analyse de l’ontologie de gène indique que les gènes potentiellement impliqués dans la pathogenèse, la cytoadhérence et l’agrégation des érythrocytes sont surreprésentés parmi les gènes surexprimés chez les isolats de CM et 3D7-Lib. Les résultats les plus marquants étaient la surexpression des gènes var (groups A et B) portant les domaines cassettes DC4, DC5, DC8 et DC13 et les gènes avoisinants rif chez les isolats de NP et la lignée 3D7-Lib, par rapport aux isolats de PA et au clone non sélectionné 3D7, respectivement. Le rôle joué par ces gènes dans la virulence parasitaire est lié à la cytoadhérence, c’est-à-dire la capacité de leurs protéines exprimées à interagir entre les érythrocytes parasités et les récepteurs endothéliaux post capillaires. Parmi ces récepteurs, le CD36 et inter cellular adhesion molecule 1 (ICAM-1) ont été les plus couramment utilisés par les isolats. L’étude sur l’implication de ces deux récepteurs, ainsi que celle des ligands PfEMP-1, dans la pathogenèse du neuropaludisme devrait être approfondie poursuivie. Nous avons analysé le phénotype de cytoadhérence et les profils de transcription des variantes de Pfemp-1 des isolats frais provenant des enfants béninois atteints de NP ou AS à l’aide du test d’adhérence statique aux récepteurs CD36, ICAM-1 et CSPG et au moyen de RT-PCR quantitative pour les groupes A, B, var2, var3, DC8 et DC13. Nos résultats montrent que le niveau de cytoadhérence des parasites associés au neuropaludisme au CD36 est significativement plus important que celui des parasites associés à l’accès simple. En outre, nous n’avons pas trouvé de différence significative entre la cytoadhérence des isolats de deux groupes cliniques à ICAM-1 et au CSPG. En outre, les niveaux d’expression des groupes var A, B, var2, var3 et du DC8 et DC13 sont plus élevés chez les isolats associés au neuropaludisme que chez les isolats associés à l’accès simple. Nos résultats montrent également que, chez les parasites provenant de NP le haut niveau de cytoadhérence des parasites au CD36 est corrélé au niveau de l’expression de groupe B de gènes var. En revanche, les profils d’expression des groupes spécifiques du gène var et le phénotype de cytoadhérence aux récepteurs ICAM-1 et CSPG n’étaient pas corrélés. Nos résultats suggèrent un rôle important du récepteur CD36 et des protéines codées par les variantes de PfEMP-1 codées par le groupe B dans la pathogenèse du neuropaludisme. / Plasmodium falciparum infection is a major cause of mortality and morbidity worldwide. This parasite is involved in several clinical manifestations, ranging from asymptomatic carriage and acute uncomplicated to severe and complicated malaria, including cerebral malaria. We hypothesized that differential gene expression contributes to phenotypic variation of parasites leading to specific interaction with the host which induces several clinical categories of malaria. The principal aim of this study was to identify parasite genetic factors implicated in the pathogenesis of cerebral malaria. We investigated the whole transcriptome of parasites isolated from Cameroonian children with asymptomatic (AM), uncomplicated (UM) and cerebral malaria (CM). We also investigated the transcriptome of 3D7 clone and the selected 3D7-Lib line. Our results revealed the up-regulation of several genes in CM isolates and 3D7-Lib line compared to AM isolates and 3D7 clone respectively. Gene ontology analysis indicates an over-representation of genes implicated in pathogenesis, cytoadherence, and erythrocyte aggregation among up-regulated genes in CM and 3D7-Lib. The most remarkable outcomes were the up-regulation of UPS A and B var genes containing architectural Domains Cassettes DC4, DC5, DC8, and DC13 and their neighboring rif genes in isolates from CM and 3D7-Lib line, compared with isolates from AM and the unselected 3D7 line, respectively. The involvement of these genes in parasite virulence rises from the ability of their encoded proteins to mediate cytoadherence of infected erythrocytes to post-capillary endothelial receptors. Of these receptors, CD36 and Inter Cellular Adhesion Molecule-1 (ICAM-1) were found as the most commonly used by the isolates. The implication of these two receptors, as well as that of PfEMP-1 ligands in the pathogenesis of CM needs to be more elucidated. We examined the adhesive phenotype and the transcription patterns of Pfemp-1 variants of fresh isolates from Beninese children with CM or UM malaria by static binding assay to CD36, ICAM-1 and CSPG and RT-qPCR for groups A, B, var2, var3, DC8, and DC13. Our findings showed that isolates from CM patients bind more to CD36 than those from UM cases. No differences were observed in binding levels to ICAM-1 or CSPG between these two groups. Furthermore, CM isolates transcribed groups A, B, var2, var3, DC8 and DC13 of var genes at higher levels than UM isolates. Interestingly, the high transcription levels of group B in CM parasites correlated with their higher level of binding to CD36. In contrary, the expression profiles of a specific var group and the binding phenotype of isolates to ICAM-1 and to CSPG were not correlated. Our findings support the implication of CD36 along with PfEMP-1 variants encoded by group B in cerebral malaria pathogenesis.