Rosetting is an adhesion property of malaria parasites whereby infected erythrocytes bind to two or more uninfected erythrocytes, forming a so-called rosette. Rosetting of Plasmodium falciparum is associated with disease severity and high parasitaemia in sub-Saharan Africa, although currently the function of rosetting remains unknown. An early IFNg response elicited from the innate immune system is associated with resolution of malaria infection in mice. Published data suggests that optimal IFNg production may require contact between peripheral blood mononuclear cells and P. falciparum infected erythrocytes. The first part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy to hide infected erythrocytes from detection by innate immune cells. Across five laboratory strains of P. falciparum rosetting was not associated with differential IFNg production when parasites were grown in group O blood. Reappraisal of the data with respect to blood group for one strain found that rosetting significantly reduced the IFNg response to parasites grown in group A blood (P=0.022, Wilcoxon signed-rank test), where it is known that rosettes are bigger and stronger. This is consistent with the hypothesis that rosetting is an immune evasion strategy and the first study to find evidence for a function of rosetting. Further work is needed in order to generalise this finding. The cytokine response to P. falciparum varies between people and this variation may be indicative of disease progression. In mice infected with malaria it is also apparent that parasite strain can determine the cytokine response of the host. It is unclear whether P. falciparum strains vary in their ability to induce cytokines. The second part of this thesis investigates variation in cytokine induction between P. falciparum strains. Across four laboratory strains of P. falciparum, IFNg production was significantly dependent on parasite strain (F3,178= 48.49, P<0.001). Production of GM-CSF, IL-1b, IL-6, IL-10 and TNFa significantly correlated with production of IFNg (P<0.001, Pearson correlation) and followed the same strain-dependent pattern. The ratio of pro-inflammatory cytokines to IL-10 was also dependent on parasite strain. These data provide strong evidence for P. falciparum strain-dependent cytokine responses which may be an important determinant of disease outcome. Phagocytosis by splenic macrophages is proposed to be the principle mechanism of parasitaemia control in malaria infection. CD36 mediated phagocytosis may by an important mechanism of non-opsonic parasite clearance. The final part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy of P. falciparum in order to evade phagocytic clearance, in particular that mediated by CD36. Overall the data obtained were inconsistent. Phagocytosis was significantly reduced in rosetting versus non-rosetting parasites in some strains (e.g. R29; P=0.048, paired T test), whereas others showed no effect (e.g. Muz12; P=0.228, paired T test) or increased versus non-rosetting parasites (e.g. HB3, P=0.004, paired T test). The relationship between CD36 binding and phagocytosis was also unclear, and anti-CD36 antibody did not effectively block phagocytosis, suggesting the involvement of alternative mechanisms. Further experiments are needed to clarify these observations. Data presented in this thesis are suggestive that rosetting in non-group O blood may be an immune evasion strategy with regard to IFNg production by innate immune cells, mechanistically linking rosetting with enhanced parasitaemia and disease severity. Furthermore, parasite strain significantly affects cytokine production and may be a determinant of disease outcome. This thesis demonstrates the importance of continued research into the effect of parasite virulence on the immune response, with particular emphasis on rosetting.

The human immune-regulatory protein, complement receptor type 1 (CR1, CD35), occurs on erythrocytes where it serves as the immune adherence receptor. It interacts with C3b, C4b, C1q and mannan-binding lectin (MBL). It additionally binds the Plasmodium falciparum protein, Rh4, in the non-sialic acid-dependent erythrocye-invasion pathway, and is also important for rosetting, via an interaction with P. falciparum erythrocyte membrane protein 1 (PfEMP1). A C3b/C4b, and PfEMP1 binding site lies in CCP modules 15-17 (out of 30 in CR1), while polymorphisms that afford advantage to some populations in dealing with severe malaria occur in CCPs 24-25, begging the question central to this thesis – do these polymorphism modulate function, and if so how? We hypothesized that the CR1 architecture apposes CCPs 15-17 and CCPs 24-25 using the exceptionally long linker between CCPs 21 and 22 as a hinge, thus polymorphic variants in CCPs 24-25 modulate functionality in CCPs 15-17. To test this, a panel of recombinant CR1 protein fragments (CCPs 21, 21-22, 20-23, 15-17, 17, 10-11, 17-25, 15-25 and 24-25) were produced in Pichia pastoris along with polymorphic forms of the relevant constructs. After purification, biophysical and biological methods were used to assess whether the linker does indeed act as a hinge, and the comparative abilities of the CCPs 15-25 variants (along with soluble CR1 (sCR1), CCPs 1-3 and the panel of CR1 fragments) to interact with a range of ligands were measured. We found no evidence from NMR for face-to-face contacts between CCPs 21 and 22 that would be consistent with the long linker permitting a 180-degree bend between them. Indeed, based on scattering and analytical ultracentrifugation data, CCPs 20-23 form an extended rather than a bent-back structure. All of the four Knops blood-group variants of the CCPs 15-25 proteins produced similar results according to dynamic light scattering and AUC indicating no structural difference or change in self-association state between variants. In addition, based on the data collected from surface plasmon resonance (SPR), ELISA and fluid-phase cofactor (for factor I) assays, there were no evidence of any difference between the polymorphic forms with respect to their interactions with C3b, C4b, C1q and MBL. Only weak interaction was observed for sCR1, and all CCPs 15-25 variants, with the relevant part of PfEMP1, and there was no measurable difference amongst the variants in disrupting rosettes. The sCR1-Rh4.9 interaction was confirmed by SPR; affinities measured between the binding domain of Rh4 and the panel of CR1 fragments identified CCPs 1-3 (site 1) as the main interaction site. It seemed unlikely therefore that CCPs 24 and 25 could modulate Rh4 binding; indeed none of the four CR1 15-25 variants bound Rh4.9 appreciably. Thus we concluded that allotypic variations in CCPs 24-25 have no measurable effect on the architecture as well as binding of CR1 to its host or parasite ligands The inferred selective pressure acting on these variants likely arise from some other (i.e. besides malaria) geographically localised infectious diseases.

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by Variable major proteins (Vmp), in the outer membrane. Through a non reciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene at a telomeric expression locus downstream from a common expression site. B. hermsii before and after the switch from serotype 7 to serotype 21, was examined in detail. The nucleotide sequence of the vmp7 and vmp21 genes and flanking regions was determined. The vmp7 and vmp21 are 77% identical in their coding sequence, and the deduced translation products are 63% identical. No antigenic cross reactivity is observed between Vmp7 and Vmp21. This suggests a folding of the proteins in which the similar regions are buried, and not exposed when it is presented at the bacterial surface. Vmp7 and Vmp21 have consensus sequences of prokaryotic lipoproteins and are processed as such when expressed in E. coli. The 5' regions of silent and expressed vmp7 and vmp21 were compared. Silent and active vmp7 and vmp21 genes shared a block of homologous sequence at their 5' ends. Sequences upstream of silent vmp7 and vmp21 genes lacked a promoter and differed substantially from each other. In this antigenic switch a vmp gene was activated by a recombination event which placed it downstream of a promoter. The vmp gene promoter is preceded by a poly(dT dA) ran and three imperfectlyrepeated elements of 2 kb. Each of the 2 kb repeats contains inverted repeats of approximately 0.2 kb at their termini. There is no evidence of the presence of similar elements elsewhere in the genome of B. hermsii. One or more of these elements may stimulate vmp gene switch or expression. The African relapsing fever species Borrelia crocidurae and the American species B. hermsii display many similarities. In both species the vmp genes are localised to linear plasmids, and the vmp genes are activated on the transcriptional level. The nucleotide sequence of their expression sites, however, are not related. Still, the possibility that the switch is mechanistically similar in B. crocidurae and B. hermsii, cannot be ruled out. The binding of B. crocidurae causes aggregation of erythrocytes around the spirochete. The aggregation is reminiscent of the erythrocyte rosetting seen in malarial infections. The erythrocytes at the B. crocidurae surface may protect them from clearance by the host. Thus, the rosetting may constitute an additional mechanism in B. crocidurae for the evasion of the immune reaction. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser.</p> / digitalisering@umu

Le paludisme est une infection parasitaire de répartition mondiale notamment en zones intertropicales où l’infection par Plasmodium falciparum est responsable de centaines de milliers de morts par an principalement chez les enfants de moins de cinq ans. Le paludisme constitue également un problème en France par l’importation de cas de paludisme chez le voyageur de retour de zone d’endémie. L’infection à Plasmodium falciparum dans cette population, considérée comme à risque de développer les formes graves de la maladie, peut se présenter sous différentes formes cliniques plus ou moins associées au risque de mortalité. Même si certains facteurs de risque de gravité tels que l’âge et l’immunité ont été identifiés, cette interaction complexe hôte-parasite n’a été largement étudiée que chez l’enfant en zone d’endémie et peu de données sont disponibles pour le paludisme d’importation. L’objectif de ces travaux de thèse repose sur l’analyse des facteurs d’hôte et des facteurs parasitaires intervenant dans le paludisme d’importation. A travers le réseau de surveillance du centre national de référence du paludisme en France métropolitaine, l’ensemble des données démographiques, épidémiologiques, cliniques et biologiques des cas de paludisme d’importation, notifiés entre 2011 et 2015, ont été collectées ainsi que les échantillons ayant servis au diagnostic. Après expertise diagnostique, le plasma obtenu après centrifugation a été utilisé pour les dosages des antipaludéens, pour la quantification d’HRP2 ainsi que pour la sérologie anti-palustre. L’ARN extrait par le TRIZOL® à partir du culot globulaire a été utilisé pour l’étude de l’expression des gènes var et des domaines cassettes par qRT-PCR. Le culot de globules rouges parasités a été mis en culture pour la maturation des formes parasitaires en vue de l’étude du phénotype de cytoadhérence sur les récepteurs solubles CD36, ICAM-1, EPCR et du phénomène de rosetting . L’ensemble de ces études a été réalisé sur une population de patients dans le cadre du paludisme d’importation groupée en migrants de première génération, migrants de deuxième génération et voyageurs/expatriés et dont la présentation clinique du paludisme d’importation a été classée en paludisme « très grave », paludisme « grave » et paludisme « simple ». L’ensemble des données épidémiologiques, cliniques et biologiques recueillies au cours de l’étude a permis d’identifier l’âge élevé, l’origine ethnique, la profondeur de la thrombopénie et l’absence d’antécédents de paludisme comme des facteurs de risque associés à la survenue d’un accès palustre « très grave », entité clinique caractérisée pour une biomasse parasitaire séquestrée élevée. L’effet de la pré-exposition au parasite, reflété par le statut sérologique des patients, semble être à l’origine de la présentation clinique de la maladie en limitant notamment la biomasse parasitaire séquestrée au cours de l’accès palustre. L’étude de l’expression des gènes var et des domaines cassettes réalisée dans cette population, en fonction de la présentation clinique, de l’origine ethnique et du statut sérologique des patients, a révélé une surexpression du groupe de gènes var A et B et des motifs protéiques composant les domaines cassettes DC4, DC8 et DC13 dans le paludisme « grave » et « très grave » d’importation au sein de cette population hétérogène de patients. L’étude du phénotype de cytoadhérence et du rosetting, réalisée dans un autre groupe de patients rencontré dans le cadre du paludisme d’importation, a identifié le rosetting comme le phénotype d’adhérence à l’origine de l’accès palustre « très grave ». Le profil d’expression des gènes var et domaines cassettes correspondants à cette population a confirmé les observations antérieures et corrèle le phénotype de rosetting à l’expression des motifs protéiques DBLß3 et DBLa2 de DC4 et DC8 (...) / Malaria is a worldwide parasitic infection especially in tropical area where Plasmodium falciparum infection is responsible for hundreds of thousands annually mainly among children under five years old. Malaria is also a problem in France by the importation of malaria cases in travelers coming from endemic area. The Plasmodium falciparum infection in this population, considered at risk of developping severe malaria, can present different clinical forms more or less associated with mortality.While some risk factors for severity like age and immunity have been identified, this complex host-parasite interactions have been widely studied in children in endemic areas and few data are available for imported malaria. The aim of the thesis work is based on analysis of host factors and parasite factors in imported malaria.Through the monitoring network of the French National reference center of malaria, all the demographic, epidemiological, clinical and laboratory of imported malaria cases, notified between 2011 and 2015, were collected and also samples of the parasitological diagnosis. After diagnostic expertise, the plasma obtained after centrifugation was used for determinations of antimalarial drugs, for quantification of plasmatic HRP2 and for serological tests. RNA extracted by the Trizol® from red cells pellets was used to study the expression of var genes and domain cassettes by qRT-PCR. The pellet of parasitized red blood cells were cultured for maturation of parasitic forms for the study of phenotype cytoadherence on soluble receptor CD36, ICAM-1 and EPCR and for the study of the rosetting phenomenon. All of these studies was conducted in an imported malaria context,in a population of patients composed by first-generation migrants, second-generation migrants and travelers / expatriates and whose clinical presentation of imported malaria was classified into very severe (VSM), mild severe (MSM) and uncomplicated malaria (UM).All the epidemiological, clinical and biological data collected during the study identified the high age, ethnicity, depth of thrombocytopenia and no history of malaria as factors risk associated with the occurrence of very severe malaria, clinical entity characterized by high sequestered parasite biomass. The effect of pre-exposure to the parasite, reflected by the serological status of patients, seems to be the cause of the clinical presentation of the disease in particular by limiting parasite biomass sequestered during malaria. The study of the expression of var genes and domain cassettes performed in this population, according to clinical presentation, ethnicity and the serological status of patients, revealed an overexpression of the group of var genes A and B and protein patterns of the domain cassette DC4, DC8 and DC13 in mild severe and very severe malaria within this heterogeneous patient population. The study of cytoadherence phenotype and rosetting, made in another group of patients in imported malaria context, identified the rosetting as adhesion phenotype causing very severe malaria. The expression profile of var genes and domain cassettes corresponding to this population confirmed earlier observations and correlates rosetting phenotype to the expression of DBLß3 and DBLa2 of DC4 and DC8 (...)