Cyclooxygenase Cox

Cs

Claudia Demarta-Gatsi

Leanna Smith

Sabine Thiberge

Roger Peronet

Pierre-Henri Commere

Mariette Matondo

Lionel Apetoh

Pierre Bruhns

Robert Ménard

Salaheddine Mécheri
email: smecheri@pasteur.fr

Blood-brain barrier CD Cluster of differentiation CM Cerebral malaria

Keywords: APC, allo phyco cyanine, DTR, diphtheria toxin receptor, FA, formic acid, GAP, genetically attenuated parasite, gDNA, genomic DNA, hDHFR, human dihydrofolate reductase, HPRT, hypoxanthine phosphoribosyltransferase, HRF, histamine-releasing factor, iRBC, infected RBC, p.i., postinfection, qPCR, quantitative PCR, UTR, untranslated

Circumsporozoite CSP Chemoprophylaxis sont à risque vis-à-vis du paludisme. Cela revient à aborder la prise en charge du paludisme en prenant en compte la susceptibilité des individus aux pathologies allergiques. Ainsi une prise en charge thérapeutique originale pourrait consister à proposer des combinaisons médicamenteuses, incluant des antipaludiques et des anti-histaminiques. D'autre part les mécanismes immunologiques révélés pas l'action de notre mutant HRF conduisent à proposer cet outil comme un nouveau GAP (Genetically Attenuated Parasite) à portée vaccinale visant le développement d'une protection durable.

Almost four years have passed from the start of my thesis and many things have changed.

Not only my scientific knowledge has been improved but I also developed new job skills such as identifying problems and solving them. This thesis represents not only my work at the keyboard, but it is a milestone after more than three years of work at the Institut Pasteur and specifically within the BIHP Unit. My experience has been nothing short of amazing. I have been given unique opportunities and I have taken advantage of them. This includes working with incredible scientists of all levels and participating at many lectures that enabled my achievements as a scientist. The content of this thesis represents the lessons learnt from the study of malaria disease based on the creation of a special tool: Genetically Attenuated Parasites (GAPs). This work is the result of many experiences I have encountered and of different people I wish to thank.

First and foremost I would like to express my sincere gratitude to my advisor, Dr. Salah Mecheri, for the continuous support during my Ph.D training and related research, for his patience, motivation, and immense knowledge. His guidance helped me throughout the time of my research and writing of this thesis (a hard moment!). I remember he used to say something like " we arrive in the morning and you're already here, we go home at night and we leave you behind us, it is as if you had a bed camp here!, this is " to encourage me to go home and to relax". I couldn't have imagined having a better advisor and mentor for my Ph.D training.

Similar, deep gratitude goes to our TONTON Roger who welcomed me to the lab and introduced me to mouse experiments showing me all the secrets of the profession and tried to turn my passion for the dissection of the mosquitoes that was never born! I really need to thank you for coming early at 6 a.m. to start mouse dissections and stay all day long with me spending hours in front of the FACS machine laughing because our eyelids grew heavy closed; we really built a good work team and, even if you didn't like it, a speedy work team.

My gratitude to The Helmut Horten Foundation for their financial support which allowed me to benefit from this experience and to strengthen my scientific knowledge.

3 3 I would also like to thank my committee members, Pr. Dominique Mazier, Pr. Marita Troye Blomberg, Pr. Frédéric Tangy and Dr. Nicolas Blanchard for being part of my thesis committee even at hardship. I also want to thank you for letting my defence be an enjoyable moment, and for your brilliant comments and suggestions, many thanks to you. Special mention goes to Pr. Artur Scherf who gave me the opportunity to be hosted in his laboratory and who always tackled me with puzzling questions.

I am grateful to Dr. Robert Menard and Dr. Shruthi Vembar for the help in developing my writings tools, to Sabine Tiberge who provided me with a fantastic lab training in IFA and sporozoite infections, to Dr. Sylvie Garcia and Dr. Rogerio Amino who were available whenever I needed their advices in various fields, and Pierre-Henri Commere for his kind help with FACS sorting.

To my friends in every corner of the world, starting for the ones who stay at home Noemi, David, Ariella, Paki, Elisabetta, Anna, Flora, Nina, Kim, Giona, Augustin, Tugba, Messina twins, Andrea & Cindy, and the one who was there when I needed him the most, Alessandro. Their unconditional support has been essential all these years. The ones who I left somewhere Bridget, sister Immaculate and sister Carla, Simon, Julius, Meck, Sheila, Colin, Kevin, Blessing, Paul, Karanjia and all the others who always remember me what is really important and give me the strength to fight for them. And all the good people who I have met during these years and who helped me to keep smile on my face. Irina for the blabla between one experiment and the other or in the animal facility, Raquel for the lunch times and the discovery of Paris, Laura for the help in French language and culture, Carmen who was an amazing friend but left so quickly, Shuai for a lot of things starting from blabla and ending with running moments, Doro for the running and walking moments, Shruthi for the picnic and advices, Anne -with you, French Administration was not so hard anymore-, Malika for animating the lab, Sara for the Swiss meals and the photography weekends, Patricia for the scientific advices, Laura-Ines-Memo for the amazing time spending with you during the month-long vaccinology course, the Swiss-Italian and the South American culture are finally not so far, Carolie-Alexander-Gopaul and iGEM students for the evenings at the Institut Pasteur trying to develp the Plasticure project and for the wonderful trip to Boston.

4

Finally, I want to thank Anthony because without you things would be harder, thank you for the cappuccino breaks, for the swimming and running times; at the end you become more fanatic than me but also thanks for always listening to and enduring me, weekends spent in the laboratory were not so long in your company.

A special THANKS to my family. Words cannot express how grateful I am to my mother Rita, and father Marco for all of the sacrifices that you've made on my behalf. Your prayer for me was what sustained me thus far. Non avete mai mollato la presa nonostante qualche volta, a causa di una testa assai dura, ho dato i numeri. Per le domeniche mattina a fare matematica e le gare di francese contro il papi, che alla fine mi hanno servito. Per le ore in colonna tra Agno-Caslano con la mamma dopo le mie lunghe sedute in piscina e per la compagnia durante i nostri viaggi in giro per il mondo. To my sister Valeria who is so different from me but in the end we are not so different. Perché questa tesi ci ha avvicinato un po di più, senza di te starei ancora cercando di dare una forma alla tesi! At the end, I would like to express appreciation to my beloved husband Nico who fought with me for our survival in France, who always pushed me beyond my limits because he believes in me more than myself, and was always my support in the moments when there was no one to answer my queries.

THANKS TO ALL, Claudia

Résumé en Française

Au seuil du troisième millénaire, le paludisme constitue et demeure un problème majeur de santé publique. Avec le sida (VIH) et la tuberculose, le paludisme fait partie des trois maladies infectieuses les plus mortelles au monde. Bien qu'en diminution, il s'agit de la maladie parasitaire la plus dévastatrice car elle concerne 95 pays, avec environ la moitié de la population mondiale exposée au risque d'infection. En 2015, les estimations font état d'environ 500'000 décès en Afrique sub-saharienne dont 65% étant des enfants de moins de 5 ans (WHO, 2015). Outre son impact sanitaire, dans les régions hautement endémiques, le paludisme représente un sérieux obstacle au développement socio-économique avec une diminution de près de 1,3% du produit intérieur brut [START_REF] Sachs | The economic and social burden of malaria[END_REF]. Si la maladie est essentiellement retrouvée dans la plupart des régions tropicales et subtropicales de la planète, avec un risque particulièrement élevé en Afrique, des cas d'importation ont également été recensés en Europe.

Le paludisme est du à une infection par un protozoaire intracellulaire du genre Plasmodium, transmis entre individus par la piqûre de certaines espèces de moustique femelles du genre

Anopheles. Dans la nature il existe plus de 250 espèces de Plasmodium, mais seulement cinq espèces infectent naturellement l'homme (Plasmodium malariae, P. vivax, P. ovale, P. knowlesi et P. falciparum), dont P. falciparum est responsable de la majorité des décès et des formes cliniques sévères du paludisme. D'autres espèces plasmodiales qui infectent les singes (par exemple P. knowlesi, P. cynomolgi, etc.) ou les rongeurs (P. yoelii, P. berghei, P. chabaudi et P. vinckei) sont étudiées comme modèles expérimentaux.

Le cycle de vie de l'agent causal du paludisme est complexe et implique un hôte intermédiaire vertébré et un hôte définitif (vecteur). Chez l'hôte vertébré, l'infection par Plasmodium débute avec la phase pré-érythrocytaire au cours de laquelle la forme sporozoite déposée dans la peau par le moustique rejoint le foie via la circulation sanguine afin de se différencier en une forme infectant les globules rouges. Lors de sa phase érythrocytaire, le parasite s'engage dans un cycle de maturation et multiplication, c'est alors que les symptômes de la maladie vont apparaître. Les premiers symptômes (fièvre, maux de tête, frissons et vomissements) apparaissent au bout de 10 à 15 jours après la piqûre de moustique infectante. Dans des cas plus graves, la maladie peut se développer en neuropaludisme (NP) où les érythrocytes infectés ainsi que des leucocytes sont séquestrés au niveau des veinules post-capillaires du cerveau. Les patients atteints de cette forme sévère de la maladie présentent des taux élevés de cytokines pro-inflammatoires (IFNγ, TNFα, IL-1, IL-6) dans de nombreux tissus [START_REF] Perlmann | IgE and tumor necrosis factor in malaria infection[END_REF][START_REF] Stevenson | Innate immunity to malaria[END_REF].

Le diagnostic peut se faire par évaluation de la parasitémie au microscope ou l'utilisation de tests de diagnostic rapide. Le traitement de choix pour le paludisme réside en une association médicamenteuse contenant de l'artémisinine. Mais au cours des dernières années, l'augmentation des phénomènes de résistance associés à l'utilisation d'antipaludiques de synthèse a conduit à réexaminer les stratégies de lutte pour réduire la mortalité, la morbidité et la transmission vectorielle imputées à la maladie. Dans ce contexte, la mise au point d'un vaccin contre le paludisme est l'approche la plus prometteuse pour parvenir à contrôler de manière significative le paludisme, en particulier en Afrique, où l'habitat écologique est tel que le contrôle des moustiques est difficile, voire impossible à réaliser. En outre, le programme de vaccination lancé par l'Organisation Mondiale de la Santé (OMS) en 1974 a démontré que le contrôle des principales maladies infectieuses est réalisable à l'échelle mondiale grâce aux vaccins. Actuellement, aucun vaccin contre le paludisme n'existe sur le marché. La complexité du parasite qui inclut trois stades de développement (stade pré-érythrocytaire, stade sanguin asexué et stade sanguin sexué) chez l'homme et le choix d'un antigène capable d'induire une réponse immunitaire durable sont des obstacles à la sélection des cibles vaccinales. En effet, plusieurs milliers de protéines (plus des glucides et des lipides) sont synthétisées par les parasites au cours de l'infection. Ces composés peuvent servir de cibles antigéniques pour deux types de réponses immunitaires: humorale avec la sécrétion d'anticorps (IgG) contre les parasites et cellulaire avec l'activation des cellules T qui peuvent attaquer les cellules infectées et stimuler la production d'anticorps. De plus, de nombreuses protéines antigéniques varient entre les différentes souches de Plasmodium pendant l'infection. Pour rendre les choses encore plus complexes, le parasite peut également commuter la sélection de protéines qui apparaissent à la surface des globules rouges infectés et échapper ainsi aux anticorps de l'hôte. Le paludisme, par conséquent, constitue un défi complexe pour le système immunitaire humain et la compréhension des mécanismes régissant la pathogenèse et les réponses immunitaires de l'hôte contre les parasites Plasmodium représente un défi majeur. Dans ce contexte, un vaccin efficace peut sembler utopique, malgré le fait qu'une immunité naturelle est de facto développée au fil des ans chez les personnes constamment exposées au parasite.

Des études récentes suggèrent une forte relation entre susceptibilité au paludisme de forme sévère et réponse de type allergique. En effet, des niveaux plasmatiques et tissulaires élevés d'histamine, un amine vaso-active connue pour ses propriétés pro-inflammatoires et immuno-régulatrices, ont été associés à la sévérité de la maladie chez l'homme infecté par P. falciparum et dans de nombreux modèles animaux (MacDonald et al., 1995). Récemment, notre laboratoire a mis en évidence le rôle crucial de l'histamine dans la pathogénèse de la maladie chez le rongeur (Beghdadi et al., 2008). L'utilisation de souris traitées avec des antihistaminiques ou génétiquement déficientes en histamine ont montré une résistance au NP, suggérant un rôle important de la voie de signalisation de l'histamine pendant l'infection palustre (Beghdadi et al., 2008). Cette résistance est caractérisée par une préservation de l'intégrité de la barrière hémato-encéphalique avec une diminution de l'expression d'ICAM-1 par les cellules endothéliales et une absence de séquestration d'érythrocytes infectés et d'adhésion de leucocytes au niveau des vaisseaux sanguins cérébraux (Beghdadi et al., 2008). Au cours de l'infection, l'histamine peut être libérée par les basophiles et les mastocytes via le complexe IgE-FceRI [START_REF] Perlmann | IgE and tumor necrosis factor in malaria infection[END_REF] ou via l'activation des cellules par le facteur de libération de l'histamine (HRF), une protéine ubiquitaire multifonctionnelle hautement conservée chez les eucaryotes (Bommer and Thiele, 2004a), y compris Plasmodium (MacDonald et al., 2001). Impliquée dans la réversion tumorale et dans l'allergie, HRF a également été décrite comme cible d'antipaludiques tels que l'artémisinine [START_REF] Bhisutthibhan | The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin[END_REF]. En outre, une étude réalisée au Mali a montré qu'une concentration élevée de la protéine parasitaire HRF dans le sérum est liée au développement d'une forme sévère de paludisme, mettant ainsi en évidence l'importance de cette protéine dans le développement de la maladie (MacDonald et al., 2001). D'après les observations précédentes, il est raisonnable de postuler que la libération d'histamine induite par le parasite depuis son entrée dans la peau de l'hôte suivie par son développement dans le foie et, finalement, dans le sang périphérique, peut contribuer au développement des propriétés pathogènes du parasite en modulant la réponse immunitaire de l'hôte.

Pour évaluer l'importance de la protéine parasitaire HRF pendant le développement de la réponse immunitaire et son impact sur la sévérité de la maladie associée à la production d'histamine, il était nécessaire de générer des lignées de parasite déficientes en hrf.

Contrairement à la souris chez qui la délétion du gène est létale au stade embryonnaire, chez P. berghei (Pb), nous avons réussi à générer deux parasites murins déficients pour la protéine HRF (hrfΔ): PbANKA GFP et PbNK65 GFP, une souche mortelle du paludisme murin qui, contrairement à PbANKA, n'engendre pas de NP, mais provoque une forte anémie et nous permet de suivre la réponse immunitaire sur une plus longue période.

Tout d'abord nous avons étudié le rôle de la protéine parasitaire pendant le cycle de vie et surtout dans le développement du parasite chez le moustique en déterminant par qRT-PCR ou par comptage le nombre de parasites (ookinète et sporozoites) au cours du temps. Les résultats obtenus ont démontré que la délétion du gène hrf chez Plasmodium n'affecte pas le développement du parasite en soi, ce qui signifie que la suppression n'est pas létale pour le parasite. En outre, nous avons observé que la mutation ne montre aucune anomalie significative dans la motilité et dans la formation des formes exo-érythrocytaires in vitro et dans la traversée cellulaire in vivo.

Les souris infectées avec des sporozoïtes PbANKA-hrfΔ ont montré une diminution de la fréquence du NP associée à un retard de deux jours entre l'apparition des formes sanguines de PbANKA-hrfΔ par rapport à PbANKA WT. L'absence de Plasmodium HRF induit une diminution de la sévérité de la maladie, ainsi qu'une anomalie au niveau du développement des parasites mutants pendant le stade hépatique qui est associée à une augmentation précoce systémique d'IL-6 (Mathieu et al., 2015), une cytokine qui inhibe fortement le développement des stades hépatiques du Plasmodium (Pied et al., 1991). L'effet inhibiteur de PbHRF sur la production de IL-6 induite par LPS souligne le rôle spécifique et l'effet direct de la protéine parasitaire HRF dans l'inhibition d'une cytokine qui présente une activité antiparasitaire (Mathieu et al., 2015). De plus l'augmentation de la population des cellules B dans le foie des souris infectées par des parasites mutants reflète la possibilité que la protéine Plasmodium HRF puisse interférer avec la mémoire des lymphocytes B et désactiver le développement des cellules B, possiblement via l'induction d'apoptose.

Après cette première observation, les parasites PbNK65-hrfΔ ont été utilisés pour étudier le rôle de HRF dans la pathogenèse. L'infection par les parasites PbNK65-hrfΔ est caractérisée par l'élimination du parasite au 12 ème jour après l'infection qui conduit à une protection de longue durée et au développement d'une mémoire immunitaire. Contrairement à l'infection par le parasite de type sauvage, la souche atténuée induit une immunité contre différentes souches de Plasmodium et aussi contre les différents stades. D'ailleurs, nos données ont montré le rôle protecteur joué par les cellules B et T sachant que les souris dépourvues de ces lymphocytes ont été incapables de contrôler le développement du parasite dans le sang.

En outre, nous avons observé que l'absence de la protéine HRF augmente la survie des cellules T par l'inhibition de l'expression du récepteur "programmed cell death 1 (PD1)" sur des cellules de type CD4 + et CD8 + . La production de cytokines telles que l'IL-12 et IL-6 sont également augmentée chez les souris infectées par PbNK65-hrfΔ et chez les souris déficientes en IL-6, nous avons observé une augmentation de la parasitémie ce qui suggère un rôle important de l'IL-6 dans le contrôle du développent du parasite. Il a été rapporté que l'IL-6 favorise la prolifération des cellules B ainsi que la différenciation et la sécrétion d'anticorps. Avec cette information à l'esprit nous avons testé si des anticorps spécifiques (IgG) pour les antigènes du parasite ont été produits chez les souris protégées. Les analyses sur le sérum de ces souris ont montré qu'en effet la réponse immunitaire contre PbNK65 WT ou d'autres souches de Plasmodium est associée à la production d'anticorps spécifiques IgG2c qui reconnaissent des protéines de Plasmodium telles que MSP-1, MSP-8, SERA-1, et SERA-2 de façon spécifique. De plus, ces protéines de Plasmodium sont connues pour susciter des anticorps associés à la protection chez les personnes vivant dans les zones endémiques [START_REF] Banic | Immune response and lack of immune response to Plasmodium falciparum P126 antigen and its amino-terminal repeat in malariainfected humans[END_REF][START_REF] Okech | Natural human immunoglobulin G subclass responses to Plasmodium falciparum serine repeat antigen in Uganda[END_REF] et ont déjà été présentées comme des possibles candidats vaccins. En outre, l'importance des anticorps dans la protection contre le paludisme a été clairement démontrée par des études cliniques où le transfert passif de sérum hyper-immunisé ou d'Ig purifiées d'adultes asymptomatiques aux enfants malades peut modifier le cours de l'infection en réduisant la parasitémie et les symptômes cliniques [START_REF] Bouharoun-Tayoun | Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes[END_REF][START_REF] Cohen | Gamma-globulin and acquired immunity to human malaria[END_REF]. Enfin, pour déterminer si les anticorps IgG induits par les parasites PbNK65-hrfΔ peuvent être des biomarqueurs à l'exposition aux parasites ou constituent des corrélats de protection stérile, des anticorps purifiés provenant de souris protégées ont été transférés passivement à des souris naïves, infectées par la suite avec des parasites PbNK65 WT. Les souris traitées avec des IgG purifiées à partir d'individus protégés ont développé une parasitémie moindre que les souris non traitées, ce qui suggère une activité anti parasitaire partielle mais significative des anticorps IgG protecteurs. Cela nous a conduit à suggérer que le transfert de cellules CD11b + ayant acquit une fonction phagocytaire plus accrue partir de souris protégées par PbNK65-hrfΔ serait nécessaire et suffisant pour protéger les souris naïves d'une infection par le parasite sauvage. En conséquence, nous avons réalisé des expériences de transfert adoptif de cellules CD11b + , obtenues à partir de souris sauvages ou de souris protégées par PbNK65-hrfΔ, suivi d'une infection avec des parasites WT.

Le transfert de cellules CD11b + à partir de souris protégées par PbNK65-hrfΔ, mais pas de souris naïves, protège les souris receveuses contre des infections avec le parasite sauvage.

Les résultats obtenus suggèrent que les anticorps sécrétés sont importants pour l'immunité protectrice à P. berghei, mais l'absence apparente de protection complète suggère que ces anticorps doivent agir avec des cellules effectrices CD11b + FcγR + "amorcées" telles que les monocytes, les macrophages, les cellules NK et d'autres qui existent chez les souris infectées par des parasites hrfΔ, mais pas chez les souris naïves. A l'appui des résultats obtenus, plusieurs études in vitro ont indiqué la pertinence biologique des sous-classes de FcγRII et IgG dans la résistance au parasite en montrant l'importance du mécanisme "antibodydependent cellular inhibition (ADCI)" lors de la protection contre P. falciparum en phase sanguine [START_REF] Bouharoun-Tayoun | Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes[END_REF][START_REF] Bouharoun-Tayoun | Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages[END_REF][START_REF] Druilhe | Mechanisms of defense against P. falciparum asexual blood stages in humans[END_REF][START_REF] Shi | Differential effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on the growth of asexual stage Plasmodium falciparum parasites[END_REF][START_REF] Zhou | CD14(hi)CD16+ monocytes phagocytose antibody-opsonised Plasmodium falciparum infected erythrocytes more efficiently than other monocyte subsets, and require CD16 and complement to do so[END_REF]. En outre, des études humaines ont également montré l'implication de FcγR à engendrer une puissante immunité antipaludique [START_REF] Mcintosh | The importance of human FcgammaRI in mediating protection to malaria[END_REF].

Dans ce travail nous avons démontré que le ciblage précis d'un gène du parasite responsable de la régulation des processus inflammatoires, dans ce cas précis en modulant la voie de signalisation de l'histamine au niveau de l'hôte vertébré, se révèle être une stratégie clé pour le déverrouillage du système immunitaire protecteur de l'hôte induisant une diminution de la charge parasitaire et de la pathogénicité. Ces travaux ont contribué à décrypter les liens entre l'inflammation en général, et de type allergique en particulier, et le pouvoir pathogène du parasite. Cela nous permet d'émettre l'hypothèse que les individus atopiques/allergiques

General Introduction

1.1 MALARIA Malaria, sometimes called the "King of Diseases", is a terrible disease, one of humankind's oldest and deadliest parasitic diseases in the world. It is caused by protozoan parasites (a group of single-celled microorganisms) belonging to the genus Plasmodium that infects humans, birds, reptiles, and other mammals through the intermediary of an infected female mosquito vector of the genus Anopheles.

Today half of the world's population is at risk. It hits the young, the poor and the vulnerable the hardest. A farmer sick for just some weeks has a huge consequence when his crops go to waste and his family goes hungry. This is happening to families around the world, causing untold misery and hampering development. In the last two decades of the 20th century the number of malaria cases worldwide seems to be increasing due to appearance of resistance phenomena associated with synthetic antimalarial drugs in use. Under this condition, identification and functional characterisation of key parasite and host proteins involved in this multi-system disorder with many similarities with the sepsis syndromes are a major challenge of the post-genomic era of Plasmodium research.

HISTORY OF MALARIA

The origin

Phylogenetic analyses of Apicomplexa genome sequences suggest that the genus Plasmodium evolved around 130 million years ago [START_REF] Escalante | Evolutionary origin of Plasmodium and other Apicomplexa based on rRNA genes[END_REF], before the dinosaurs extension. These ancestral malaria parasites were characterized by the two-host life cycle involving blood-feeding Dipterans and land vertebrates [START_REF] Carter | Evolutionary and historical aspects of the burden of malaria[END_REF].

Mitochondrial genome sequences studies suggest that malaria may have affected human beings ever since the emergence of Homo sapiens 200,000 years ago [START_REF] Jongwutiwes | Mitochondrial genome sequences support ancient population expansion in Plasmodium vivax[END_REF].

The climatic and ecological changes associated with an early and stable development of agriculture in sub-Saharan Africa with the loss of the forest cover beginning around 8,000 to 10,000 years ago contributed to the transmission of malaria in humans [START_REF] Coluzzi | The eradication of malaria. A world-wide challenge[END_REF][START_REF] Joy | Early origin and recent expansion of Plasmodium falciparum[END_REF]. The forest agriculture development with culture of crops, including breaking up the soil and clearing the forest cover created new breeding conditions that favoured the reproduction of Anopheles mosquitoes that could have accelerated the transmission of Plasmodium species among forest-dwelling agriculturalists was pivotal to the subsequent evolution and history of human malaria [START_REF] Carter | Evolutionary and historical aspects of the burden of malaria[END_REF][START_REF] Coluzzi | The eradication of malaria. A world-wide challenge[END_REF].

Malaria in the ancient world

The illness is probably from Africa and followed human migrations toward the Mediterranean coasts, to India and Southeast Asia. In the past, malaria was frequent in the Pontinses swamps, around Rome and its name has derived from a medieval italian term "mal'aria" (born in Venezia, in 1571) or bad air.

The first traces of malaria seem back to ancient Egypt, as evidenced by cases of splenomegaly [START_REF] Ruffer | Studies in palaeopathology in Egypt[END_REF] and presence of specific Plasmodium falciparum (P. falciparum) genes such as apical membrane antigen (ama) 1 and merozoite surface protein (msp) 1 found in 2010 by DNA amplification and sequencing performed on bone samples from the Egyptian Pharaoh Tutankhamon mummified in 1324 B.C at the age of 19 years [START_REF] Hawass | Ancestry and pathology in King Tutankhamun's family[END_REF].

Historical records of ancient civilizations (Sumerians, Assyrians, Babylonians, Chinese, Egyptians and Indians) report testimonials, in medical and religious texts, of the presence of seasonal or intermittent fevers that attest the antiquity of the malaria. But at this time the malarial fevers were attributed to demons or to different gods (Crotti, 2005). The Chinese classical medical book Huang Di Nei Jing (the Canon of Medicine), edited in 2007 B.C by the Emperor Huang Ti, describes enlargement of spleen connected with different types of fever.

In this document three malaria symptoms (headache, chills, and fever) are represented in the form of three demons: one with a hammer, one with a pail of cold water and one with a stone (Crotti, 2005).

At the end of the fifth century B.C in Greece, Hippocrates (460-377 B.C) attributes the origin of the various diseases to natural causes related to climatic and environmental conditions. In the Hippocrates corpus, he reported the occurrence of seasonal fevers characterized by fever peaks recurring every 48 or 72 hours and associated with rigors, cold and hot spells, profuse perspiration and splenomegaly [START_REF] Cunha | Brief history of the clinical diagnosis of malaria: from Hippocrates to Osler[END_REF].

In Italy, during the Roman Empire (circa 50 B.C to 400 A.D), malaria was excluded for several centuries, by drainage, husbandry, and building development. New episodes of malaria appeared with the decline of Empire. The presence and the disappearance of malaria have been associated with corresponding rising and falling agricultural and economic prosperity. This behavior, still observable, reflects the dependence of malaria on prevailing human activity and life-style.

All the medical knowledge acquired during the Roman Empire was lost and the malaria fevers returned to be confused with other common fevers. This situation was the cause of the spread of malaria in the cooler countries, Central and Eastern Europe. Around the sixteenth century, with the first colonization, malaria was widespread in the Americas.

Scientific discoveries

The scientific studies on malaria made their first significant advance the 6 November 1880 when a French military doctor in Algeria, Alphonse Laveran (Nobel prize 1907), discovered the malarial parasites by studying, under a microscope, the blood of patients not treated with quinine (natural alkaloid from the bark of a Peruvian tree, the tree of china). Due to the movement of the observed parasite, Laveran named the parasite Oscillaria malariae.

But the confirmation of an association between malaria and parasites occurred only in the late nineteenth century with the Italians Angelo Celli, Ettore Marchiafava and Camillo Golgi (Nobel Prize 1906). The first two described how malaria parasites, which they called Plasmodium, grow and multiply within the red blood cells before lysing the host cell to infect fresh erythrocytes and Golgi linked the onset of intermittent fever with the destruction of red blood cells and the spread of parasites into the blood [START_REF] Ferroni | Angelo Celli and research on the prevention of malaria in Italy a century ago[END_REF]. At the same time, morphological differences of the various agents responsible for fever were identified and P. falciparum was individualized as the species responsible for fatal cases of malaria [START_REF] Ferroni | Angelo Celli and research on the prevention of malaria in Italy a century ago[END_REF]. These findings allowed the description of malaria parasite life cycle stages in human blood.

While Golgi described the intra-erythrocyte parasite multiplication, Sir Ronald Ross (Nobel prize 1902), a medical officer in the British Colonial Medical Service, identified in the gut of infected mosquitoes Plasmodium sp. oocysts that broke and release sporozoites that migrated to the mosquitoes salivary glands, highlighting the role of mosquitoes in malaria transmission. This discovery allowed Giambattista Grassi in collaboration with Amico Bignami and Giuseppe Bastianelli to identify the specific species of mosquito responsible for malaria transmission to humans [START_REF] Ferroni | Angelo Celli and research on the prevention of malaria in Italy a century ago[END_REF]. They showed that the mosquitoes belonging to the genus Anopheles were the specific carriers of malaria in Italy, and described the development of the Plasmodium in the mosquito [START_REF] Ferroni | Angelo Celli and research on the prevention of malaria in Italy a century ago[END_REF]. To confirm these results Sir Patrick Manson, a Scottish physician working in London (where there was no P. falciparum), exposed his son and one of his colleagues to the infected mosquitoes coming from Italy confirming previous observations. In 1948, Shortt and Garnham showed that the cyst-like bodies (schizonte), filled with thousands of merozoites, are formed in the liver of a rhesus monkey that had been bitten by infected mosquitoes [START_REF] Cox | History of the discovery of the malaria parasites and their vectors[END_REF]. Henceforth, the life cycle of the parasite was fully known.

With the development of the scientific techniques it was possible to initiate the genome sequencing of the P. falciparum and Anopheles gambiae in 1996 and 1998, respectively. The published results (Gardner et al., 2002a;[START_REF] Winzerling | Iron metabolism in insect disease vectors: mining the Anopheles gambiae translated protein database[END_REF] made possible different studies on parasite biology and entomology in order to promote new strategies destined to control the malaria such as diagnoses, medicines and vaccines.

DISTRIBUTION AND IMPACT

Despite the rise in the knowledge gained on Plasmodium parasites and the tremendous efforts to control this disease, malaria remains one of the most deadly infectious diseases in the world with a particularly devastating effect in Africa, where over 90% of deaths due to malaria occur (Fig. 1). The endemic areas are mostly found in tropical and subtropical regions at an altitude below 1800 meters. Malaria infects not only in Sub-Saharan Africa, but also in Asia, Latin America, Middle East and even some areas of Europe are at risk (Fig. 1).

According to the World Health Organisation, estimated 214 million cases leading to 438,000 deaths where reported in 2015, among the poorest populations in the world (WHO, 2015).

The same year, 65% of deaths occur in children under 5 years of age in Africa.

1 21 21

In highly endemic areas, in addition to an enormous amount of human pain, malaria plays a critical role in the socio-economic development (working and education days lost, costs of health care, and loss of investment and tourism), especially in the widespread regions. The morbidity and mortality constitute a serious obstacle to socio-economic development with a decrease of nearly 1.3% in the gross domestic product of countries [START_REF] Sachs | The economic and social burden of malaria[END_REF].

Moreover, in recent years there was a return of malaria in countries where it had been eradicated in the past. This return may be the result of factors such as increasing insecticide-resistance in the vector, increasing multiple drug resistance in the parasite, increasing of large population migrations, increasing of the world proportion climatically suitable for transmission (global warming) [START_REF] Nabi | Is Global Warming likely to cause an increased incidence of Malaria?[END_REF] and increasing of tourism between non-endemic and endemic countries [START_REF] Moorthy | Malaria vaccines[END_REF].

PARASITES AND DISEASES

Malaria parasites

Malaria parasites are members of the genus Plasmodium, belonging to the Apicomplexa family a monophyletic group composed of at least 6000 species [START_REF] Levine | Progress in taxonomy of the Apicomplexan protozoa[END_REF][START_REF] Morrison | Evolution of the Apicomplexa: where are we now?[END_REF], the majority of which have an obligatory intracellular lifestyle. The phylum contains other parasites that are important causative agents of human and animal diseases, such as [START_REF] Shetty | The numbers game[END_REF] Toxoplasma and Cryptosporidium [START_REF] Beck | Molecular approaches to diversity of populations of apicomplexan parasites[END_REF]. In nature there are over 250 species of Plasmodium (Fig. 2), but only five species naturally infect humans: P. falciparum, P. vivax, P. ovale, P. malariae, and sometimes P. knowlesi. The five species differ by clinical criteria, their geographic distribution and their ability to develop resistance to antimalarial drugs. P. vivax is the most prevalent world-wide in most countries outside of sub-Saharan Africa, and P. falciparum is the most dangerous and virulent species, which is associated with severe complications such as cerebral malaria, renal failure and pulmonary affection leading to death. The other human malaria species can cause acute, severe illness but mortality rates are low. The last species, P. knowlesi, has been recently added to the list, originally a malaria parasite of long-tailed macaque monkeys, which has jumped the species barrier and can naturally infect humans [START_REF] Daneshvar | Clinical and laboratory features of human Plasmodium knowlesi infection[END_REF][START_REF] Singh | A large focus of naturally acquired Plasmodium knowlesi infections in human beings[END_REF].

Rodent malaria parasites (animal models in experimental malaria)

Rodent malaria parasites are mostly used to study experimentally the development of the disease to understand how parasites adjust traits during infections in response to changes in the host environment to maximise their fitness and escape the host immune response.

Rodent malaria parasites were discovered in Central Africa (Cameroon, Central African Republic, Congo, Democratic Republic of the Congo andNigeria) between 1948 and1974. During this period, four species -P. berghei (Pb), P. vinckei (Pv), P. chabaudi (Pc) and P. Yoelii (Py) -were described, encompassing 13 sub-species of various strains (e.g. Pb ANKA and Pb NK65) [START_REF] Ramiro | Molecular evolution and phylogenetics of rodent malaria parasites[END_REF]. Like all mammalian malaria parasites, they undergo several rounds of asexual replication in the vertebrate host and sexual reproduction in Anopheline mosquitoes. All four rodent malaria parasites are known to be infective to mice by blood inoculation and by mosquito transmission with some differences in the susceptibility to laboratory animals. Infections caused by parasites such as P. berghei, and some strains of P. vinckei (Pv vinckei), P. yoelii (Py YM, Py XL) and P. chabaudi (Pc CB) cause lethal infections in mice, whereas infections with some strains of P. yoelii (Py 17XNL), P. chabaudi (Pc chabaudi AS, Pc adami) and P. vinckei (Pv petterei) are cleared after the initial acute parasitaemia [START_REF] Wykes | What have we learnt from mouse models for the study of malaria?[END_REF]. Moreover, P. berghei and P. yoelli have a marked predilection to invade reticulocytes with a relatively asynchronous development in laboratory rodents whereas P. vinckei and P. chabaudi invade both immature and mature RBCs populations showing a synchronous development during the blood stage [START_REF] Wykes | What have we learnt from mouse models for the study of malaria?[END_REF]. These differences are due to some changes in morphology, developmental time and size of different stages and iso-enzymes that influence host-parasite interactions.

Consequently, there is no single model that reflects exactly infections in humans, but taken together, different systems could provide valuable informations on the mechanisms of immunity and immunopathogenesis. Lethal infections are better suited for testing putative vaccine candidates and also examining the efficacy and safety of antimalarial drugs. Nonlethal infections have been used as best models to investigate the mechanism of acquired immunity. For example, Pb ANKA and Py 17X, in C57BL/6J mice are used as experimental models to study the development of cerebral malaria (CM) [START_REF] Sanni | Mouse models for erythrocytic-stage malaria[END_REF][START_REF] Wykes | What have we learnt from mouse models for the study of malaria?[END_REF], and Pc chabaudi or Py 17XNL are usually used to explore immune response mechanisms. Moreover, P. yoelii is used to compare two strains (non-lethal 17XNL and lethal YM) with vastly different pathogenicity to deduce which factors may contribute to more serious malaria infections.

Malaria Genome

The recently available sequence of the P. falciparum (clone 3D7) genome provides powerful information for understanding resistance mechanisms and opens exciting new avenues for drug and vaccine development [START_REF] Arav-Boger | Molecular mechanisms of resistance in antimalarial chemotherapy: the unmet challenge[END_REF].

In addition to P. falciparum, other genome sequences of Plasmodium parasites have been published. The rodent malaria Py 17XNL clone [START_REF] Carlton | Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii[END_REF], Pb ANKA clone and Pc AS clone [START_REF] Hall | A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses[END_REF], the human malaria P. vivax Salvador 1 (Sal 1) strain (Carlton et al., 2008) and the human/simian malaria P. knowlesi H strain [START_REF] Pain | The genome of the simian and human malaria parasite Plasmodium knowlesi[END_REF].

Comparative analysis of the available Plasmodium genomes revealed that they are all haploid and composed of 22.8 megabases distributed among 14 chromosomes that vary from approximately 0.643 to 3.29 megabases. A remarkable feature is its base composition that varies among the different species, with the P. falciparum and rodent genomes extremely A + T rich (80.6% on average and close to 90% in introns and intergenic regions in P. falciparum (Gardner et al., 2002a;[START_REF] Winzeler | Malaria research in the post-genomic era[END_REF] in contrast with the more G + C rich P. knowlesi and P. vivax genomes (37.5% and 42.3%, respectively) (Carlton et al., 2008;[START_REF] Pain | The genome of the simian and human malaria parasite Plasmodium knowlesi[END_REF].

Gene research identified around 5000-6000 protein-encoding genes and, about 60% (approximately two-thirds) of the predicted proteins appear to be unique to this organism and another 5% presents significant similarities to proteins of other organisms [START_REF] Plouffe | In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen[END_REF][START_REF] Ward | Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote[END_REF]. The two-thirds of the proteins have poor similarities to proteins of other organisms indicating a great evolutionary distance between Plasmodium and other eukaryotes (Fig. 3) (Gardner et al., 2002a).

The evolutionary distance between the host (human) and the parasite proteins makes the use of inhibitors or genetic modifications, targeting the parasite, more interesting since they do not disturb the human protein and allow the development of new anti-malarial drugs or vaccines that target only the parasite vital functions without any influence on the host.

Life cycle

Malaria parasites have a complex life cycle which involves more than one host, an invertebrate primary host and a vertebrate secondary host (Fig. 4). The cycle can be divided into three main steps: (i) the mosquito phase characterized by sexual and asexual multiplications allowing the formation of sporozoites (12-14 days) (ii) the pre-erythrocytic phase (2-14 days) characterized by an asymptomatic asexual multiplication of the parasite in the liver of the vertebrate host and the (iii) the erythrocytic phase (48-72h) where the parasite undergoes an asexual multiplication within red blood cells leading to the symptoms of the disease. [START_REF] Ward | Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote[END_REF].

Mosquito host

During a blood meal when a female Anopheles mosquito bites an infected host, it ingests mature male and female gametocytes [START_REF] Kappe | That was then but this is now: malaria research in the time of an eradication agenda[END_REF]. As soon as the gametocytes are in the tract of the insect, triggered by the temperature drop and by chemicals [START_REF] Billker | Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito[END_REF], they escape from their surrounding erythrocyte membrane. In the midgut the gametocytes undergo gametogenesis, resulting in the development of female gametocyte into macrogametes while the male gametocyte undergoes exflagellation whereby it develops into eight flagella called microgametes. The male and female gametes fuse together (sexual reproduction) to form a diploid zygote that undergoes meiotic nuclear division and develops into a motile form, called ookinete, which penetrates the midgut epithelium and forms oocysts on the outer surface. Within the oocyst the parasite undergoes several rounds of asexual replication (sporogony) producing thousands sporozoites, elongate slightly curved cells approximately 10 µm long with a central nucleus and apical organelles [START_REF] Schrevel | Vesicle trafficking during sporozoite development in Plasmodium berghei: ultrastructural evidence for a novel trafficking mechanism[END_REF]. Rupture of the mature oocyst releases the sporozoites into the hemocoel (body cavity) of the mosquito. The sporozoites migrate to and [START_REF] Klein | Antimalarial drug resistance: a review of the biology and strategies to delay emergence and spread[END_REF].

invade the salivary glands, thus completing the life cycle [START_REF] Aly | Malaria parasite development in the mosquito and infection of the mammalian host[END_REF] for onward transmission, ready to be delivered to the skin of the host during mosquito bite [START_REF] Zhang | The Plasmodium eukaryotic initiation factor-2alpha kinase IK2 controls the latency of sporozoites in the mosquito salivary glands[END_REF].

Pre-erythrocytic phase

This obligatory hepatic phase is the only classically described life cycle of the parasite where the parasite development requires invasion of nucleated host cells. Upon mosquito injection of motile salivary gland sporozoites into the human dermis, most of them start migrating in the cutaneous tissues and enter into the blood stream or the lymphatic system [START_REF] Amino | Quantitative imaging of Plasmodium transmission from mosquito to mammal[END_REF]. In mice, around 50% of the inoculated parasites remain in the skin where ~10% can initiate development and lead to the production of new infectious parasites inside skin cells [START_REF] Gueirard | Development of the malaria parasite in the skin of the mammalian host[END_REF]. Another 15% enter lymphatic vessels and reach the proximal draining lymph node where the parasites are eventually degraded. Finally, the remaining 35% invade bloodstream by entering capillaries in a process that usually lasts less than a minute [START_REF] Amino | Quantitative imaging of Plasmodium transmission from mosquito to mammal[END_REF] after the sporozoite deposition in the skin, but it can extend to hours. Once in the circulation, sporozoites migrate passively to the liver sinusoids where they migrate along the endothelial cell layer and cross the sinusoidal barrier. The parasite traverses different cells, including Kuppfer cells and several hepatocytes, before settling and establishing a parasitophorous vacuole (PV) derived from the host-cell membrane [START_REF] Frevert | Intravital observation of Plasmodium berghei sporozoite infection of the liver[END_REF][START_REF] Mota | Migration of Plasmodium sporozoites through cells before infection[END_REF] which is rapidly remodelled by parasite proteins [START_REF] Bano | Cellular interactions of Plasmodium liver stage with its host mammalian cell[END_REF]. Once the PV vacuole is established the parasite start to develop [START_REF] Frevert | Intravital observation of Plasmodium berghei sporozoite infection of the liver[END_REF] via a process of asexuate multiplication, called hepatic merogony, which leads to the generation of hundreds or thousands of uninucleated merozoites contained vesicles called merosomes.

After the lysis of the hepatocytes the merosomes are carried away in the circulation and finally reach the pulmonary vasculature where they break releasing hepatic merozoites in the blood.

Infections of P. vivax and P. ovale differ from the other types of malaria in that some sporozoites may develop into a dormant forms termed hypnozoites. Under this form the parasite can remain in the liver and relapses months or even years after the primary infection.

Erythrocytic phase

Red blood cells (RBCs) are the 'central stage' for the asexual development of the malaria parasite. Within RBCs, repeated cycles of parasitic development occur with precise periodicity. Once release in the blood, hepatic merozoites invade healthy erythrocytes by an active and rapid process, accomplished within 30 seconds of initial attachment to the erythrocyte surface minimizing the parasite surface antigens exposure, thereby protecting these parasite forms from the host immune response [START_REF] Cowman | Invasion of red blood cells by malaria parasites[END_REF][START_REF] Greenwood | Malaria: progress, perils, and prospects for eradication[END_REF] (Fig. 5A). This invasion is a process which depends on successive major events: (i) initial weak attachment of merozoite to the erythrocyte surface by random collision mediated by MSP-1 and MSP-9 co-ligand complex that binds erythrocyte membrane transport glycoprotein [START_REF] Gaur | Parasite ligand-host receptor interactions during invasion of erythrocytes by Plasmodium merozoites[END_REF], (ii) reorientation of the merozoite mediated by AMA-1 [START_REF] Mitchell | Apical membrane antigen 1, a major malaria vaccine candidate, mediates the close attachment of invasive merozoites to host red blood cells[END_REF] and creation of a distinctive tight junction between the apical end of the parasite and the erythrocyte followed by protein and lipid material secretion, and (iii) starting of the invagination process that involves direct interactions of parasite ligands at the apical end with erythrocyte receptors [START_REF] Gaur | Parasite ligand-host receptor interactions during invasion of erythrocytes by Plasmodium merozoites[END_REF]. Formation of an invasive pit with the tight junction moving along the surface of merozoite as it penetrates into the erythrocyte, closing of the invasion pit on the erythrocyte membrane and isolation of the parasite within PV in the erythrocyte cytoplasm. At this point the parasite develops (asexual replication) through a change of its morphology (stages) inside the PV (Fig. 5C):

• Ring stage (0-24 hours),

• Trophozoite stage (24-36 hours)

• Schizont stage (40-48 hours) At the end of the cycle the infected RBCs (iRBCs) lyse and release around 16-32 new merozoites that in turn will infect new RBCs. After several rounds of erythrocytic schizogony, some merozoites differentiate into the extracellular sexual stage gametocytes which develop into male and female gametocytes and are taken by mosquitoes during the blood meal.

One of the Plasmodium unique features is the ability to remodelling the host erythrocyte in order to overcome the lack of standard biosynthetic pathways and intracellular organelles in the RBCs that create obstacles for the parasites fast-growing. During the ring stage the major nutrient is the hemoglobin that is ingested into a food vacuole and degraded to obtain amino acids utilized for protein biosynthesis. The remaining toxic heme is converted into harmless hemozoin (malaria pigment) by heme polymerase and sequestred [START_REF] Coronado | Malarial hemozoin: from target to tool[END_REF]. As the parasite grows and multiplies within the cell there is a dramatic expansion of the surface area through the formation of a tubulovesicular network and the parasite exports a variety of proteins to the erythrocyte cytoplasm and cell surface remodeling the RBCs structure (Fig. 5B). These exported proteins have functions in cytoadherence (such as falciparum erythrocyte membrane protein 1 (PfEMP-1)), nutrient acquisition and evasion of the host immune response [START_REF] Charpian | Protein transport across the parasitophorous vacuole of Plasmodium falciparum: into the great wide open[END_REF][START_REF] Winzeler | Malaria research in the post-genomic era[END_REF]. The parasite develops in a parasitophorous vacuole (PV) through the ring (0-24 hours), trophozoite (24-36 hours) and schizont stages (40-48 hours). In mature-stage (>24 h) news membrane-bound structures appear in RBC (Knobs, Maurer's clefts, etc.). After approximately 48 hours there are the infected RBC lyse and 16-32 daughter merozoites are released [START_REF] Maier | Malaria parasite proteins that remodel the host erythrocyte[END_REF].

MALARIA PATHOGENESIS

Malaria pathogenesis is caused by the asexual multiplication of parasites in erythrocytes.

The severity and the incubation period of the symptoms vary depending on the species of Plasmodium that causes the parasitosis. The red blood cells destruction leads to fever and flu-like symptoms, such as chills, headache, muscle aches, nausea, vomiting and diarrhea.

These symptoms usually appear 10-15 days after being bitten. The disease can progress extremely rapidly and if it is not treated quickly with effective drugs, the parasite burden continues to increase and severe malaria may occur. However, symptoms can sometimes appear after weeks or months in patients who have not been appropriately treated. In epidemic region in people who have recently survived an infection, re-infection typically causes milder or no symptoms (uncomplicated malaria) but this partial resistance disappears over months to years if there is no on-going exposure to malaria.

Asymptomatic malaria

In areas of high malaria transmission, P. falciparum infections are characterized by persistence of low levels of chronic parasitaemia in the blood stream without manifestation of any clinical symptoms in adult patients [START_REF] Hamad | Chronic Plasmodium falciparum infections in an area of low intensity malaria transmission in the Sudan[END_REF]. These persistent or repeated "asymptomatic" infections allow the development of a natural acquired partial immunity, which controls but not completely eliminate the infection [START_REF] Doolan | Acquired immunity to malaria[END_REF]. Moreover, microscopic diagnosis is often challenged by very low parasite density and individuals who are not treated with antimalarials remain reservoirs of gametocyte for perpetuating the malaria transmission cycle [START_REF] Bousema | Plasmodium falciparum gametocyte carriage in asymptomatic children in western Kenya[END_REF].

During pregnancy, malaria infection is usually asymptomatic associated with placental sequestration of parasite and significant reduction of maternal haemoglobin level [START_REF] Leke | Detection of the Plasmodium falciparum antigen histidine-rich protein 2 in blood of pregnant women: implications for diagnosing placental malaria[END_REF][START_REF] Matangila | Asymptomatic Plasmodium falciparum infection is associated with anaemia in pregnancy and can be more cost-effectively detected by rapid diagnostic test than by microscopy in Kinshasa, Democratic Republic of the Congo[END_REF] particularly in primigravidae [START_REF] Ndyomugyenyi | Anaemia in pregnancy: Plasmodium falciparum infection is an important cause in primigravidae in Hoima district, western Uganda[END_REF]. It is only during subsequent pregnancies that they acquire a form of pregnancy-associated immunity [START_REF] Rowe | The role of Plasmodium falciparum var genes in malaria in pregnancy[END_REF].

Uncomplicated malaria

Malaria infection usually results in an uncomplicated, mild febrile disease in which intermittent episodes of fever and peaks of parasitemia are controlled by the body's immune defences and eventually eliminated. Nevertheless, clinical symptoms and signs of malaria infection are not specific and shared with other febrile diseases as viral or bacterial diseases and precise analysis, as blood smear or rapid diagnostic test, are necessary to confirm malaria infection. Moreover, the presence of hepatosplenomegaly, thrombocytopenia and anaemia are clearly associated with malaria, particularly in children [START_REF] Grobusch | Uncomplicated malaria[END_REF]White et al., 2014).

The clinical features have been attributed to the release of numerous know and unknown substances, such as red cell membrane products, hemozoin pigment [START_REF] Sherry | Malaria-specific metabolite hemozoin mediates the release of several potent endogenous pyrogens (TNF, MIP-1 alpha, and MIP-1 beta) in vitro, and altered thermoregulation in vivo[END_REF], and other toxin factors including parasite-specific glycosylphosphatidylinositol (GPI) [START_REF] Schofield | Glycosylphosphatidylinositol toxin of Plasmodium up-regulates intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression in vascular endothelial cells and increases leukocyte and parasite cytoadherence via tyrosine kinasedependent signal transduction[END_REF][START_REF] Schumann | Malarial fever: hemozoin is involved but Toll-free[END_REF] into the blood. These products, in particularly the GPI, are potent stimulators of cytokines and inflammatory mediators synthesis by macrophages and endothelial cells [START_REF] Gazzinelli | Innate sensing of malaria parasites[END_REF], such as tumor necrosis factor (TNF), interferon (IFN)-γ, interlukin (IL)-1β, macrophage inflammatory proteins (MIP)-1α and MIP-1β [START_REF] Sherry | Malaria-specific metabolite hemozoin mediates the release of several potent endogenous pyrogens (TNF, MIP-1 alpha, and MIP-1 beta) in vitro, and altered thermoregulation in vivo[END_REF], and nitric oxide (NO) [START_REF] Gazzinelli | Innate sensing of malaria parasites[END_REF]. Additionally, the plasmodial DNA is highly proinflammatory and able to induce cytokinemia and fever.

Hemozoin internalizes the DNA that is recognized by the toll-like receptor (TLR)-9 and activate the synthesis of pro-inflammatory cytokines. Cyclooxygenase (COX)-2 release upregulates the production of prostaglandins and leads to fever [START_REF] Schumann | Malarial fever: hemozoin is involved but Toll-free[END_REF] (Fig. 6).

Severe malaria

The progression of the infection results in progressive and dramatic modification of RBCs that can worsen into severe malaria responsible for a broad spectrum of clinical features accompanying P. falciparum infection. Nevertheless in the recent years, several cases of severe infection and even deaths have been reported in non-falciparum infections such as P.

vivax [START_REF] Naing | Is Plasmodium vivax malaria a severe malaria?: a systematic review and meta-analysis[END_REF] and P. knowlesi [START_REF] Cox-Singh | Severe malaria -a case of fatal Plasmodium knowlesi infection with post-mortem findings: a case report[END_REF][START_REF] Rajahram | Falling Plasmodium knowlesi Malaria Death Rate among Adults despite Rising Incidence, Sabah, Malaysia, 2010-2014[END_REF] infections.

The manifestations of severe falciparum malaria depend on age [START_REF] Dondorp | The relationship between age and the manifestations of and mortality associated with severe malaria[END_REF]. In infants and young children the development of metabolic acidosis (respiratory distress), cerebral malaria (CM) and severe malarial anaemia (SMA) are the major complications causing high mortality [START_REF] Gazzinelli | Innate sensing of malaria parasites[END_REF][START_REF] Schofield | Immunological processes in malaria pathogenesis[END_REF]. In non-immune adults only a minority of infections become severe with the involvement of single-organ, multiple-organ or systemic impairment. Multiple complications could occur in an individual [START_REF] Schumann | Malarial fever: hemozoin is involved but Toll-free[END_REF] at the same time and can develop rapidly and progress to death within hours or days if appropriate treatment is not provided in time.

Severe Malaria Anaemia (SMA)

Anemia is a common manifestation of all types of malaria, especially in children and during pregnancy, and is globally the leading cause of malaria related to mortality and morbidity.

The pathophysiological processes in SMA can include a number of distinct, as well as overlapping features, such as the rupture of infected and uninfected RBCs [START_REF] Dondorp | Red blood cell deformability as a predictor of anemia in severe falciparum malaria[END_REF], splenic sequestration of RBCs [START_REF] Buffet | Retention of erythrocytes in the spleen: a double-edged process in human malaria[END_REF], impaired erythopoiesis and dyserythropoiesis [START_REF] Abdalla | The anaemia of P. falciparum malaria[END_REF][START_REF] Dormer | Ineffective erythropoiesis in acute human P. falciparum malaria[END_REF][START_REF] Kurtzhals | Reversible suppression of bone marrow response to erythropoietin in Plasmodium falciparum malaria[END_REF]Perkins et al., 2011), co-infections with bacteria [START_REF] Were | Bacteremia in Kenyan children presenting with malaria[END_REF], virus (ex. HIV-1) [START_REF] Davenport | Hematological predictors of increased severe anemia in Kenyan children coinfected with Plasmodium falciparum and HIV-1[END_REF] or hookworm [START_REF] Price | Factors contributing to anemia after uncomplicated falciparum malaria[END_REF][START_REF] Stoltzfus | Malaria, hookworms and recent fever are related to anemia and iron status indicators in 0-to 5-y old Zanzibari children and these relationships change with age[END_REF].

The spleen uptake of infected, altered or uninfected RBCs (uRBCs) is responsible for the clearance of those cells by the phagocytic cells such as monocytes, macrophages and neutrophils due to adherence of malarial IgG or complement binding to their surface [START_REF] Haldar | Malaria, erythrocytic infection, and anemia[END_REF]. The elimination of uRBCs may also occur by antibody-dependent cellmediated cytotoxicity (ADCC). In confirmation of this hypothesis, recent studies have detected on the surface of ring stage iRBCs membrane as well as in uRBCs P. falciparum rhoptry protein 2 [START_REF] Sterkers | Members of the low-molecular-mass rhoptry protein complex of Plasmodium falciparum bind to the surface of normal erythrocytes[END_REF]. Moreover, the impaired erythropoiesis due to the involvement of cytokines and other mediators of inflammation (such as hemozoin) causes SMA. The dysregulation of the innate immune response due to the phagocytosis of the malarial pigment hemozoin by effector cells such as neutrophils, macrophages and monocytes down-regulate IL-12 levels, associated with a decreased production of IFN-γ and IFN-α, through an IL-10-dependent mechanism allowing the development of the parasite and SMA [START_REF] Haldar | Malaria, erythrocytic infection, and anemia[END_REF][START_REF] Keller | Acquisition of hemozoin by monocytes down-regulates interleukin-12 p40 (IL-12p40) transcripts and circulating IL-12p70 through an IL-10-dependent mechanism: in vivo and in vitro findings in severe malarial anemia[END_REF]. Increased circulating levels of TNF, IL-6, IL-1b, IL-1RA, MIP-1α, MIP-1β are observed in sick children as well as prostaglandin E, NO, macrophage migration inhibitory factor (MIF), chemokine (C-C motif) ligand 5 (CCL5 or RANTES) and stem cells growth factor suppression. Further, in children with SMA, reduced NO production promotes ineffective parasite killing and may suppress, in the bone marrow, the erythropoiesis [START_REF] Haldar | Malaria, erythrocytic infection, and anemia[END_REF]. Recent studies in mice using P. chabaudi AS have highlighted the important role of the Th2 cytokine IL-4 in erythropoiesis suppression [START_REF] Thawani | STAT6-mediated suppression of erythropoiesis in an experimental model of malarial anemia[END_REF].

Cerebral Malaria (CM)

Cerebral malaria (CM) due to P. falciparum parasites sequestration in the brain is the most striking severe malaria syndrome characterized by gradual neurological disorders [START_REF] Idro | Clinical manifestations of severe malaria in the highlands of southwestern Uganda[END_REF] starting from prostration, loss of consciousness, convulsions that occur in one third of the cases and progressive paralysis leading to coma even with appropriate medical intervention [START_REF] Lovegrove | Parasite burden and CD36-mediated sequestration are determinants of acute lung injury in an experimental malaria model[END_REF]. Disorders develop in 1% of patients infected with P.

falciparum which cause a mortality rate of 20-30% among infected people, while more than 10% of survivors present neurological sequelae such as spasticity, ataxia, hemiplegia and blindness [START_REF] Armah | Cerebrospinal fluid and serum biomarkers of cerebral malaria mortality in Ghanaian children[END_REF]. Furthermore, numerous differences exist between paediatric and adult CM. Epidemiological data show that in endemic areas severe malaria is a complication almost exclusively associated with infancy. However, CM does not occur in children upon first infection, but after one or several infections [START_REF] Artavanis-Tsakonas | The war between the malaria parasite and the immune system: immunity, immunoregulation and immunopathology[END_REF].

The immunopathogenesis of cerebral pathology in both human CM and experimental cerebral malaria (ECM) is a complicated series of inter-related events involving multiple organs, parasite and host immune factors over a period of some 5-10 days. Several mechanisms, which fall under three major hypotheses [START_REF] Polimeni | Host matrix metalloproteinases in cerebral malaria: new kids on the block against blood-brain barrier integrity? Fluids and barriers of the[END_REF] are supported today to explain the CM pathogenesis: 1) the mechanical theory, 2) the permeability theory, and 3) the immunological theory.

The mechanical theory proposes that CM pathology is as a result of mechanical obstruction of cerebral microvasculature by iRBCs due to their impaired deformability [START_REF] Coltel | Cerebral malaria --a neurovascular pathology with many riddles still to be solved[END_REF] or cytoadherence to receptors expressed on endothelial cell surfaces (Fig. 7A) [START_REF] Miller | The pathogenic basis of malaria[END_REF], causing flow perturbations and hypoxia of the surrounding brain parenchyma [START_REF] Turner | Cerebral malaria[END_REF] and increased lactate production due to anaerobic glycolysis [START_REF] Planche | Metabolic complications of severe malaria[END_REF]. The cytoadherent properties increased in mature parasites (trophozoïtes, older schizonts) is associated with the expression on iRBCs surface of parasite proteins, such as

PfEMP-1, that interact with specific host receptors in the microvascular endothelium, including intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), thrombospondin, CD36, and E-elastin [START_REF] Polimeni | Host matrix metalloproteinases in cerebral malaria: new kids on the block against blood-brain barrier integrity? Fluids and barriers of the[END_REF]. Other phenomena such as rosetting [START_REF] Kaul | Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions[END_REF], characterized by iRBCs forming a flower-like cluster around a uRBCs, clumping a cluster of iRBCs and platelets [START_REF] Cox | The role of platelets in the pathogenesis of cerebral malaria[END_REF][START_REF] Pain | Plateletmediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria[END_REF], and decreased deformability of the iRBCs further increase the clogging of the microcirculation (Fig. 7A).

The permeability theory highlights the importance of the blood-brain barrier (BBB) alterations, caused by secondary signalling events activated by sequestered iRBCs, that allow toxic compounds to enter the brain and cause neurological dysfunction [START_REF] Gitau | Review Article: blood-brain barrier in falciparum malaria[END_REF][START_REF] Hunt | Immunopathogenesis of cerebral malaria[END_REF]. Vascular permeability is found to be mildly increased and associated with cerebral edema in several animal models, however, in human no definite evidence of cerebral edema has been found on imaging studies [START_REF] Medana | Human cerebral malaria and the blood-brain barrier[END_REF]. A correlation between malaria disease severity and the role of histamine, a vaso-active amine, has also been demonstrated in P. falciparum infections; with a markedly increased brain uptake of histidine in infected children that would favour an elevated brain level of histamine [START_REF] Enwonwu | Hyperphenylalaninaemia in children with falciparum malaria[END_REF]. Accordingly, increased levels in plasma and tissue histamine have been associated with disease severity in human P. falciparum infections (Enwonwu et al., 2000). Moreover, our laboratory has highlighted the crucial role of histamine in the pathogenesis of the disease in mice. Mice deficient in histamine due to disruption of the histidine decarboxylase gene (HDC -/-) or treated with antihistamines or deficient for the H1 and H2 receptors of histamine are more resistant a to ECM, suggesting a harmful role of histamine pathway signalling through these receptors (Beghdadi et al., 2008).

The involvement of histamine in the severity of the disease was confirmed by the absence of neuropathology observed in histamine-deficient mice. This resistance is characterized by the preservation of the integrity of the BBB with a decrease in the expression of ICAM-1 by endothelial cells and the absence of sequestration of iRBCs or adhesion of leukocytes to the cerebral blood vessels (Beghdadi et al., 2008).

The immunological theory, based on animal testing and poorly established, suggests that host factors such as cytokines and chemokines can enter the brain parenchyma after increased BBB permeability and aggravate pathological consequences. Curiously, in contrast 1 36 36 to other encephalitis-causing pathogens, such as Trypanosoma spp. or Toxoplasma gondii [START_REF] Elsheikha | Protozoa traversal of the blood-brain barrier to invade the central nervous system[END_REF], P. falciparum appears to remain in the vascular space without entering the brain parenchyma. Murine and human studies, during the development of CM, have demonstrated the recruitment of leukocytes and platelets in the cerebral microvasculature, caused by the production of local toxins from the sequestered iRBCs, followed by an exaggerated immune response through excessive release of proinflammatory cytokines (IL-1, IL-6, IL-12, TNF-α, IFN-γ, lympotoxine, and NO) [START_REF] Hunt | Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria[END_REF][START_REF] Jain | Modulation of cerebral malaria by curcumin as an adjunctive therapy. The Brazilian journal of infectious diseases : an official publication of the[END_REF][START_REF] Schofield | Immunological processes in malaria pathogenesis[END_REF] and microparticles [START_REF] Jain | Modulation of cerebral malaria by curcumin as an adjunctive therapy. The Brazilian journal of infectious diseases : an official publication of the[END_REF]. The cellular recruitment is a series of steps, in a first time neutrophils and monocytes via chemotaxis migrate to the brain where they bind to the brain endothelium and stimulate more chemokine and cytokine production [START_REF] Renia | Pathogenic T cells in cerebral malaria[END_REF]. Furthermore, in mice studies, depletion of neutrophils that contribute to the brain lesions and are important source of cytokines and chemokines, prevents the development of CM by downregulation of the expression of T helper (Th)1 cytokines and monocyte sequestration [START_REF] Schofield | Immunological processes in malaria pathogenesis[END_REF]. In contrast to mice, neutrophil contribution to CM is unknown in humans. Several days post-infection (p.i.), after neutrophils and monocytes recruitment in the brain, naïve T cells fully primed by spleen CD11c high CD8 + DCs [START_REF] Piva | Cutting edge: Clec9A+ dendritic cells mediate the development of experimental cerebral malaria[END_REF] upregulates chemokinereceptor CXCR3 on CD8 + T cells [START_REF] Dewalick | Cutting edge: conventional dendritic cells are the critical APC required for the induction of experimental cerebral malaria[END_REF][START_REF] Miyakoda | Malariaspecific and nonspecific activation of CD8+ T cells during blood stage of Plasmodium berghei infection[END_REF], influenced by IFN-γ derived from NK cells, and migrate to the brain in a full assault of the BBB representing the central component of ECM immunopathogenesis also responsible for severe symptoms and mortality. CD8 + T cells accumulate within the cerebral vessels, recognise and kill endothelial cells presenting parasite antigen by MHC I on their surface [START_REF] Monso-Hinard | Expression of major histocompatibility complex antigens on mouse brain microvascular endothelial cells in relation to susceptibility to cerebral malaria[END_REF] and initiate BBB tight junction disruption by promoting the central nervous system vascular permeability under neuroinflammatory conditions (Fig. 7B).

Additionally mice studies shown that depletion of CD8 + T cell at both early and late infection prevents ECM pathology [START_REF] Belnoue | On the pathogenic role of brain-sequestered alphabeta CD8+ T cells in experimental cerebral malaria[END_REF][START_REF] Hermsen | Depletion of CD4+ or CD8+ T-cells prevents Plasmodium berghei induced cerebral malaria in end-stage disease[END_REF][START_REF] Yanez | Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria[END_REF].

A"

B" The iRBCs mature forms have the ability to bind to a range of host cells, such as endothelium, uRBCs (rosetting) and platelets (platelet-mediated clumping) [START_REF] Rowe | Adhesion of Plasmodium falciparuminfected erythrocytes to human cells: molecular mechanisms and therapeutic implications[END_REF]. (B) Schematic representation of severe malarial disease events. iRBCs adherence to receptors expressed by brain microvascular endothelial cells is followed by merozoites releasing. The release of parasite GPI induces an inflammatory response. A local acute-phase response then occurs, which involves activation of the endothelium and local production of cytokines and chemokines resulting in upregulation of expression of cell-adhesion molecules by endothelial cells. GPI can also function as a ligand for CD1d-restricted natural killer T (NKT) cells, leading to their activation. Activated NKT cells can regulate the differentiation and activation of CD4 + T cells into Th1 or Th2 cells. In addition, chemokines recruit monocytes and activate neutrophils. Recruited monocytes can then differentiate into macrophages and become arrested in brain microvessels. Macrophages can also be activated by GPI, a process that is amplified by IFN-γ. Local activated macrophages produce more chemokines amplifying the infiltration of cells, sequestration of iRBCs and release of microparticles. After several more cycles, γδ T cells and CD8 + T cells might become involved, releasing more chemokines and cytokines both systemically and locally and possibly inducing perforin-mediated lesions in the endothelium. Together with locally arrested macrophages, platelets are sequestered and participate in altering endothelial-cell functions. More microparticles of platelet, endothelial-cell and monocyte origin are released, which leads to the dissemination of pro-inflammatory and pro-coagulant effects. Finally, damage to the endothelium, with possible perivascular haemorrhage, axonal injury, and neurotransmitter and metabolic changes, can ensue. [START_REF] Schofield | Immunological processes in malaria pathogenesis[END_REF].

MALARIA PROTECTIVE IMMUNITY

In 1980, Bruce-Chwatt wrote, "malaria immunity may be defined as the state of resistance to the infection brought about by all those processes involved in parasite destroying or by limiting their multiplication" [START_REF] Bruce-Chwatt | Essential malariology[END_REF]. Immunity to malaria is complex, and is essentially both species and stage specific. The multi-stage nature of the Plasmodium life cycle, the differential and specific expression of genes at each stage, and the predominantly intracellular lifestyle of the parasite pose challenges to the ability of the vertebrate host to mount an effective defense [START_REF] Ferreira | Antigenic diversity and immune evasion by malaria parasites[END_REF]. Other factors, including the age and genetic background of the host, pregnancy, nutritional status and co-infection, also influence the development of antimalarial immunity [START_REF] Baird | Host age as a determinant of naturally acquired immunity to Plasmodium falciparum[END_REF][START_REF] Doolan | Acquired immunity to malaria[END_REF]. In humans, various types of acquired or adaptive immunity against plasmodia have been defined (Fig. 8) [START_REF] Doolan | Acquired immunity to malaria[END_REF]):

• Antidisease immunity, rapidly acquired and results in reduced mortality or severe clinical disease.

• Antiparasite immunity, slowly acquired confers protection against parasitemia by affecting the parasites density and the attendant risk of severe disease [START_REF] Marsh | Host-parasite interaction and morbidity in malaria endemic areas[END_REF].

• Premunition, phenomenon that describes the asymptomatic status observed in adults with high degree of immune responsiveness together with the nearly permanent presence of relatively low densities of parasite [START_REF] Sergent | L'immunite, la premunition et la resistance innee[END_REF]. Asymptomatic infection (green), mild disease (febrile episodes caused by malaria; blue) and severe or lifethreatening disease (red) (White et al., 2014).

An#disease)immunity)) An#parasite)immunity)) Premuni#on) Severe)) Mild)) Asymptoma#c)infec#on))
In malaria endemic areas the inhabitants are usually infected repeatedly with malaria parasites and acquired immunity gradually. Sterilizing immunity against infections is never fully achieved. A general trend of increased serological response is seen with age, this is probably due to cumulative exposure over time to various parasite antigens. Acquisition of immunity appears to be sequential, with the ability to limit parasite growth and multiplication, followed by essentially complete protection against severe and then mild clinical disease, and culminating with partial protection against infection. Maintenance of this malaria immunity is dependent on persistent sub-clinical infection and is lost when people leave malaria-endemic areas.

Innate immunity

Animal and human studies have highlighted the importance of innate immune mechanisms to limit the initial phase of parasite replication, controlling the first wave of parasitemia and allowing the host to develop specific adaptive responses that will be enable to clear the infection [START_REF] Molineaux | Malaria therapy reinoculation data suggest individual variation of an innate immune response and independent acquisition of antiparasitic and antitoxic immunities[END_REF][START_REF] Stevenson | Innate immunity to malaria[END_REF]. The initial trigger of the innate immune response is the activation of different cell types including dendritic cells (DCs), monocytes/macrophages, natural killer (NK) cells, natural killer T (NKT) cells, and γδ T cells resulting in the production of different factors among which several inflammatory mediators in particular cytokines and chemokines [START_REF] Stanisic | Escaping the immune system: How the malaria parasite makes vaccine development a challenge[END_REF]. This early inflammatory response is involved in various effector mechanisms such as parasite phagocytic clearance through the production of reactive oxygen and nitrogen species, and secretion of antimicrobial peptides, that all aim at destroying the parasites.

The production of inflammatory cytokines is controlled by the type of interactions between host cells and parasite. The innate immune system recognizes Plasmodium components (such as hemozoin, CpG-containing DNA motifs bound to hemozoin or AT-rich DNA motif), known as pathogen-associated molecular patterns (PAMPs), via a limited number of patternrecognition receptors (PRRs). These receptors include the TLRs family that recognize parasite molecules [START_REF] Coban | Pathological role of Toll-like receptor signaling in cerebral malaria[END_REF] or hemozoin [START_REF] Coban | Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin[END_REF] and cell surface receptors such as CD36 [START_REF] Stevenson | Innate immunity to malaria[END_REF], a co-receptor for TLR2 [START_REF] Langhorne | Immunity to malaria: more questions than answers[END_REF].

The strongest evidence of the importance of these interactions comes from mouse models, specifically P. berghei, P. yoelii and P. chabaudi. In vitro studies show the capacity of Plasmodium GPI-anchored molecules to activate and stimulate the production of proinflammatory cytokines (TNF-α and IL-1) and NO from mice and human macrophages trough TLR2 and TLR4 [START_REF] Krishnegowda | Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity[END_REF][START_REF] Nebl | Stimulation of innate immune responses by malarial glycosylphosphatidylinositol via pattern recognition receptors[END_REF][START_REF] Schofield | Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria parasites[END_REF]. In early infection activation of DCs via TLRs appears to cause inflammatory cytokines, IL-12, release from T cells in mice [START_REF] Perry | Dendritic cells from malariainfected mice are fully functional APC[END_REF] and IL-12 production by NK cells in appropriate dose has been shown to be useful in induction of protective immunity during P.

chabaudi infection [START_REF] Stevenson | IL-12-induced protection against bloodstage Plasmodium chabaudi AS requires IFN-gamma and TNF-alpha and occurs via a nitric oxide-dependent mechanism[END_REF]. However, TLR-MyD88-mediated IL-12 production, during blood stage infection with P. berghei in mice, was shown to be associated with perforin-dependent liver injury [START_REF] Adachi | Plasmodium berghei infection in mice induces liver injury by an IL-12-and toll-like receptor/myeloid differentiation factor 88dependent mechanism[END_REF]. Recently, the role of the CD36

receptor was shown to be involved in opsnonin-independent phagocytosis of P. falciparum iRBCs by monocytes from non-immune individuals [START_REF] Serghides | CD36 and malaria: friends or foes?[END_REF]. Moreover, the in vitro binding of P. falciparum iRBCs to CD36 on the surface of host LPS-activated DCs induces the secretion of IL-10 rather than IL-12 and reduces their ability to activate T cells in an allogeneic mixed lymphocyte reaction or to activate memory CD4 + T cells [START_REF] Urban | Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells[END_REF].

Another important cytokine implicated in the early inflammatory response and essential for controlling the initial wave of parasitaemia in mice is IFN-γ release by NK cells and γδ + T cell that strongly expanded during the early phases of malaria infection (Fig. 9) [START_REF] Hensmann | Cellular basis of early cytokine response to Plasmodium falciparum[END_REF][START_REF] Inoue | Roles of IFN-gamma and gammadelta T Cells in Protective Immunity Against Blood-Stage Malaria[END_REF][START_REF] Stevenson | Innate immunity to malaria[END_REF], and enhances phagocyte activity, resulting in the elimination of extracellular parasites. Indeed, IFN-γ deletion during P. chabaudi infection led to impaired cytokine responses, uncontrolled parasitemia and increased mortality [START_REF] Favre | The course of Plasmodium chabaudi chabaudi infections in interferon-gamma receptor deficient mice[END_REF][START_REF] Su | Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection[END_REF]. Additionally, IL-12p40 subunit deficiency in resistant mice reduced IFN-γ induction and increased mortality [START_REF] Su | IL-12 is required for antibody-mediated protective immunity against blood-stage Plasmodium chabaudi AS malaria infection in mice[END_REF], while administration of recombinant IL-12 to susceptible mice enhanced IFN-γ production, parasite control, and survival [START_REF] Stevenson | IL-12-induced protection against bloodstage Plasmodium chabaudi AS requires IFN-gamma and TNF-alpha and occurs via a nitric oxide-dependent mechanism[END_REF].

A study of P. falciparum infection showed that IFN-γ is produced in major quantities by γδ T cells in contrast to NK cells in response to iRBCs before and after P. falciparum infection [START_REF] Teirlinck | Longevity and composition of cellular immune responses following experimental Plasmodium falciparum malaria infection in humans[END_REF]. Moreover, IFN-γ-producing responses of malaria antigen-specific γδ T cells were shown to be important for the development of a protective immunity against P.

falciparum infection in Papua New Guinea children [START_REF] D'ombrain | Association of early interferon-gamma production with immunity to clinical malaria: a longitudinal study among Papua New Guinean children. Clinical infectious diseases : an official publication of the[END_REF]. In contrast, several in vitro culture studies demostrated the rapid production of IFN-γ by NK cells in response to malaria infection via IL-12 and IL-18 signalling from antigen-presenting cells (APCs) [START_REF] Micallef | Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-gamma production[END_REF][START_REF] Okamoto | Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12[END_REF]. Additionally, different studies showed that IFN-γ is also produced by DCs [START_REF] Suzue | In vivo role of IFN-gamma produced by antigen-presenting cells in early host defense against intracellular pathogens[END_REF] and is important for priming lymphocytes, nevertheless it not clear if IFN-γ production from DCs enhances protective immunity against Plasmodium infection.

Liver stage production of IFN-γ, capable to inhibit parasite growth in vito hepatocyte cultures, seems to be mediated by NKT cells [START_REF] Pied | Liver CD4-CD8-NK1.1+ TCR alpha beta intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro[END_REF]. Furthermore, during P. yoelii low-doses blood infection in nude mice liver NKT cells are required for partial protection, and adoptive transfer of these cells was able to passively transfer resistance to naïve mice [START_REF] Mannoor | Essential role of extrathymic T cells in protection against malaria[END_REF][START_REF] Schmieg | The role of natural killer T cells and other T cell subsets against infection by the pre-erythrocytic stages of malaria parasites[END_REF]. In addition, it has been reported that NKT cells are capable to regulate IgG antibody responses against P. falciparum GPI protein of pre- erythrocytic parasites [START_REF] Hansen | CD1d-restricted NKT cells contribute to malarial splenomegaly and enhance parasitespecific antibody responses[END_REF]. Nevertheless, additional investigations are required because other reports have shown the opposite [START_REF] Schmieg | The role of natural killer T cells and other T cell subsets against infection by the pre-erythrocytic stages of malaria parasites[END_REF].

The role of neutrophil activation in the host response to P. falciparum needs further clarification. Host resistance to malarial infection is not classically attributed to a neutrophil response [START_REF] Stevenson | Innate immunity to malaria[END_REF]. However, examining gene-expression profiles in whole blood from Kenyan children, a cluster associated with neutrophil activity, including absolute neutrophil count and neutrophil-related gene expression was identified [START_REF] Griffiths | Genomewide analysis of the host response to malaria in Kenyan children[END_REF]. A significant increase in parasite gene-expression intensity corresponded to a significant increase in neutrophil count in subjects who had acute malaria as compared to convalescent subjects. Indeed, it has been observed that neutrophils have been linked with acute malaria [START_REF] Ladhani | Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome[END_REF].

Adaptive immunity

The parasite-mediated innate inflammatory immune response is the first step of the induction of the subsequent adaptive response responsible for the effector mechanisms against pathogens and ultimately leading to the memory response (Fig. 10). However, during malaria infection long-term memory (central memory) doesn't exist because continued exposure to malarial antigens is needed for: (i) the generation of effector memory cells and

(ii) the persistence of memory and effector cells. The underlying mechanisms and antigenic specificity of protective immunity against malaria parasite are not well understood. The acquired anti-malaria immunity has been demonstrated to be strain and stage specific, with some cross reactivity

Cellular immunity

Control of asexual stages depends on both humoral and cellular immune response mechanisms. Nevertheless, the acquisition and maintenance of protective immunity is largely both CD4 + and CD8 + T cell dependent [START_REF] Troye-Blomberg | T-cell control of immunity to the asexual blood stages of the malaria parasite[END_REF]. In fact depletion of T cells in animal models does not allow recovery from infections and reconstitution with T cells from immune donors of severe combined immunodeficient mice suppress P. chabaudi adami growth suggesting that T cells contribute to immunity [START_REF] Van Der Heyde | The resolution of acute malaria in a definitive model of B cell deficiency, the JHD mouse[END_REF]. In contrast, animals unable to make antibodies recover normally from some primary infections + T cells, and differentiated CD4 + T cells primes antigen presenting cells to produce cytokines and chemokines, further assists DC maturation, and also activates macrophages, resulting in phagocytosis and killing of parasites via the production of nitric oxide and reactive oxygen intermediates. CD4

+ T cells can also produce IL-10, which can induce apoptosis of DCs. Once activated, macrophages can also participate in antigen presentation. IL-2 produced by activated CD4 + T cells further activates NK cells to produce IFN-γ. (Stanisic et al., with P. chabaudi and when rescued by chemotherapy from other species of malaria parasite develop lasting, nonsterile immunity [START_REF] Langhorne | A role for B cells in the development of T cell helper function in a malaria infection in mice[END_REF][START_REF] Von Der Weid | Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection[END_REF].

The most important cells implicated in regulating the human immune responses to asexual blood stages of P. falciparum are CD4 + T cells. These cells are polarized to make some defined cytokine response and provide help to the humoral component. Activation of malaria-specific αβ TCR + CD4 + T cells can be initiated soon after the antigen presentation by APCs [START_REF] Bruna-Romero | Dendritic cells can initiate protective immune responses against malaria[END_REF][START_REF] Chemtai | T cell-mediated immunity in murine malaria. II. Induction of protective immunity to P. chabaudi by antigenfed macrophages and antigen-educated lymphocytes[END_REF]. The role of CD4 + T cells was observed in in vitro experiments in which CD4 + T cells from malaria exposed individuals respond to a diversity of P. falciparum antigens by proliferation or by production of IFN-γ or IL-4 (Troye-Blomberg et al., 1990). The T cell production of IL-4 instead of IFN-γ was correlated to Abs levels in exposed individuals [START_REF] Troye-Blomberg | Production by activated human T cells of interleukin 4 but not interferon-gamma is associated with elevated levels of serum antibodies to activating malaria antigens[END_REF]. While the production of IL-4 is typically observed at the end of the acute phase characterized by a Th2 response, the production of IFN-γ is predominant in the early acute P. chabaudi infection highlighting a Th1 response involved in the activation of mononuclear and polymorphonuclear leukocytes which phagocytose or lyse iRBCs [START_REF] Kharazmi | Enhanced inhibition of in vitro multiplication of Plasmodium falciparum by stimulated human polymorphonuclear leucocytes[END_REF][START_REF] Orago | Cytotoxicity of human natural killer (NK) cell subsets for Plasmodium falciparum erythrocytic schizonts: stimulation by cytokines and inhibition by neomycin[END_REF]. This high production of IFN-γ declines as the parasitemia decreases, and is replaced by IL-4 and IL-10 production [START_REF] Taylor-Robinson | A model of development of acquired immunity to malaria in humans living under endemic conditions[END_REF][START_REF] Taylor-Robinson | B cells are required for the switch from Th1-to Th2regulated immune responses to Plasmodium chabaudi chabaudi infection[END_REF]. Moreover, activation of Th2 cells in the early acute phase of P. chabaudi infection in susceptible A/J mice results in a severe and lethal course of malaria [START_REF] Stevenson | Differential induction of helper T cell subsets during bloodstage Plasmodium chabaudi AS infection in resistant and susceptible mice[END_REF]. In human infection with P. falciparum, the shift from a Th2 response to a more pronounced Th1 response is associated with the resolution of infection [START_REF] Winkler | Reciprocal regulation of Th1-and Th2-cytokine-producing T cells during clearance of parasitemia in Plasmodium falciparum malaria[END_REF]. These results show that both Th1 and Th2 cells are involved in protective immunity against blood stage malaria, at different times of infection and maybe according to localization.

Infective dose and kinetics also appears to be important and influence Th1/Th2 response in malaria. The increase in inoculum size in susceptible A/J mice leads to fulminating parasitaemia associated with elevated Th2 responses [START_REF] Taylor-Robinson | Infective dose modulates the balance between Th1and Th2-regulated immune responses during blood-stage malaria infection[END_REF]. In murine model infected with P. chabaudi, changes over time of the ability of splenic DCs (CD11c + CD8 + or CD11c + CD8 -) to present antigens to CD4 + T cells was observed. High mortality in CD8 + DCs population followed by an increase in the number of CD8 -DCs responsible to induce IL-4 and IL-10 production from MSP-1-specific T cells was observed in coincidence with the Th1 to Th2 switch during the immune response to the pathogen [START_REF] Sponaas | Malaria infection changes the ability of splenic dendritic cell populations to stimulate antigen-specific T cells[END_REF].

Although CD4 + T cells are critical for protection against malaria, at the same time they could play a role in the development of lethal complications. The depletion of CD4 + T cells before the infection with P. berghei can prevent the convulsions due to ECM [START_REF] Hermsen | Convulsions due to increased permeability of the blood-brain barrier in experimental cerebral malaria can be prevented by splenectomy or anti-T cell treatment[END_REF].

While CD4 + T cells are important during the blood stage, available evidence indicates that MHC class 1-restricted CD8 + T cells, with a cytotoxic potential, seem to have important effector functions in pre-erythrocytic immunity [START_REF] Crompton | Malaria immunity in man and mosquito: insights into unsolved mysteries of a deadly infectious disease[END_REF][START_REF] Nardin | T cell responses to pre-erythrocytic stages of malaria: role in protection and vaccine development against pre-erythrocytic stages[END_REF] and contribute to protection against severe malaria [START_REF] Aidoo | Field studies of cytotoxic T lymphocytes in malaria infections: implications for malaria vaccine development[END_REF][START_REF] Hill | Common west African HLA antigens are associated with protection from severe malaria[END_REF]. In animal model, CD8 + T cell were shown to directly lyse the infected hepatocytes and destroy intracellular parasite via an IFN-γ-dependent mechanism [START_REF] Tse | Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development[END_REF]. Nevertheless, this CD8 + T cells response appears to be dependent on CD4 + T cells and the persistence of parasite antigens [START_REF] Cockburn | Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites[END_REF][START_REF] Overstreet | CD4+ T cells modulate expansion and survival but not functional properties of effector and memory CD8+ T cells induced by malaria sporozoites[END_REF]. Moreover, It has been proposed that CD8 + T cells may regulate immunosuppression in acute malaria and down-modulate inflammatory responses.

Nevertheless, no available evidence for a protective role of CD8 + T cells against P. falciparum blood stage has been reported. This is supported by the fact that RBC do not express classical MHC class I molecules, hence lacking the antigen processing machinery, suggesting that RBC do not represent a target for CD8 + T cells. However, in contrast to the MHCrestricted αβ T cells, the MHC-unrestricted γδ T cells may have a direct cytotoxic potential on the asexual blood stages of malaria parasites, as demonstrated by their inhibitory activity in P. falciparum cultures (Troye-Blomberg et al., 1999b).

Despite the important role of T cells in the development of anti-malaria immunity, some mechanisms are opposed to its effectiveness. In rodent models the development of memory CD4 + T cells during malaria infection occurs but this memory decays over time in the absence of re-infection. Indeed, mice infected with a chronic P. chabaudi are more protected in absence of anti-malaria drug treatment than treated mice suggesting that the persistence of the parasite was necessary for the establishment of the immunity. These non-treated mice presented enhanced Th1 response compared to those treated with drug [START_REF] Stephens | Effector memory Th1 CD4 T cells are maintained in a mouse model of chronic malaria[END_REF][START_REF] Stephens | Homeostatic proliferation and IL-7R alpha expression do not correlate with enhanced T cell proliferation and protection in chronic mouse malaria[END_REF]. In humans Th1 memory responses gradually decline in the absence of infection, whereas central memory IL-10 regulatory responses were maintained [START_REF] Wipasa | Short-lived IFN-gamma effector responses, but long-lived IL-10 memory responses, to malaria in an area of low malaria endemicity[END_REF].

Moreover, in chronic diseases it was shown that repeated antigen stimulation through the T cell antigen receptor drives the sustained expression of T cell inhibitory receptors, including programmed death 1 (PD-1; also known as PDCD1) and lymphocyte-activation gene 3 (LAG3), on virus-specific CD8+ T cells [START_REF] Ishida | Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death[END_REF]. Transcriptional changes that negatively regulate proliferation and the expression of pro-inflammatory cytokines by virusspecific CD8 + T cells are induced [START_REF] Barber | Restoring function in exhausted CD8 T cells during chronic viral infection[END_REF][START_REF] Blackburn | Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection[END_REF]. Recently it was reported that in mice, infection with nonlethal P. yoelii parasites drives specific T cell dysfunction called exhaustion [START_REF] Butler | Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection[END_REF]. Therapeutic blockade of PD-1 ligand (PD-L1) and the inhibitory receptor LAG3 in mice demonstrated a restoration of CD4 + T cells and B cell responses resulting in enhanced parasite control, even in chronically infected mice, suggesting that regulatory pathways may also promote chronicity of infection [START_REF] Butler | Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection[END_REF][START_REF] Horne-Debets | PD-1 dependent exhaustion of CD8+ T cells drives chronic malaria[END_REF][START_REF] Stanisic | Escaping the immune system: How the malaria parasite makes vaccine development a challenge[END_REF]. The combined blocking of PD-L1 ad LAG3 was also associated with increased numbers of CD4 + T FH cells and germinal center B cells resulting in higher Abs titres which contribute to a better control of blood stage malaria [START_REF] Butler | Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection[END_REF]. Recently, studies using a PD1-KO mouse model infected chronically with P. chabaudi confirmed the importance of PD1 and highlighted the important role of CD8 + T cells in the development of a long-term protection against murine malaria. In fact, depletion of CD8 + T cells during a secondary infection in PD1-KO mice results in the development of the parasite in the blood stage, the opposite is observed with CD4 + T cell depletion (Horne- et al., 2016). Furthermore, P. falciparum-infected children present higher expression of the inhibitory receptor PD-1 associated with T cell dysfunction [START_REF] Butler | Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection[END_REF], and the increased frequency of CD4+ T cells expressing PD-1 alone and in combination with LAG-3 appeared to be driven by persistent P. falciparum exposure [START_REF] Illingworth | Chronic exposure to Plasmodium falciparum is associated with phenotypic evidence of B and T cell exhaustion[END_REF].

Debets

Humoral immunity

The role of Abs in malaria protective immunity is not completely understood. However different anti-parasite effector functions have been attributed to the strong Abs responses reported during the infection: (i) the inhibition of the invasion of hepatocytes and erythrocytes, (ii) the inhibition of the cytoadherence, (iii) the complement-mediated opsonization of iRBCs, and (iv) monocyte-dependent antibody-mediated cellular cytotoxicity (ADCC) and monocyte-dependent antibody-mediated cellular inhibition (ADCI) [START_REF] Perlmann | Malaria and the immune system in humans[END_REF].

The importance of antibodies in protection against malaria has been clearly shown by clinical studies where passive transfer of hyper-immune serum or purified Igs from asymptomatic adults to sick children can modify the course of infection by reducing parasitaemias and clinical diseases [START_REF] Bouharoun-Tayoun | Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes[END_REF][START_REF] Cohen | Gamma-globulin and acquired immunity to human malaria[END_REF]. Moreover, until 6 month of age children remain remarkably resistant to P. falciparum and P. vivax infection.

This protection has been associated with maternally derived immunoglobulin G (IgG)

antibodies [START_REF] Logie | Plasma immunoglobulin concentrations in mothers and newborn children with special reference to placental malaria: Studies in the Gambia, Nigeria, and Switzerland[END_REF], acquired by the fetus in utero through the passage of Abs across the placenta by an active receptor-mediated transport system (hFcRn) [START_REF] Ellinger | HFcRn-mediated transplacental immunoglobulin G transport: protection of and threat to the human fetus and newborn[END_REF][START_REF] Malek | Evolution of maternofetal transport of immunoglobulins during human pregnancy[END_REF]. During breastfeeding, infant protection may be associated with parasite growth-inhibitory factors such as lactoferrin and secretory IgA found in maternal milk. As observed in humans in animal models the passive immunization with mAbs [START_REF] Quinn | Mechanisms of action of hyperimmune serum in mediating protective immunity to rodent malaria (Plasmodium berghei)[END_REF][START_REF] Siddiqui | Merozoite surface coat precursor protein completely protects Aotus monkeys against Plasmodium falciparum malaria[END_REF], adoptive transfer of B cells [START_REF] Langhorne | A role for B cells in the development of T cell helper function in a malaria infection in mice[END_REF] and increased susceptibility of B-cell-deficient hosts [START_REF] Von Der Weid | Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection[END_REF] have highlighted the important role of antibodies in malaria immune response.

However, the protection obtained in vivo studies with passive transfer of Igs is lost around 10 months after the Abs treatment [START_REF] Cohen | Gamma-globulin and acquired immunity to human malaria[END_REF]. In vitro, clinically effective Igs obtained from the sera of P. falciparum immune adults had no detectable inhibitory effect on either parasite invasion or growth, but was shown to supress the parasite growth in cooperation with human monocytes in an antibody-dependent cellular inhibition (ADCI) assay [START_REF] Bouharoun-Tayoun | Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes[END_REF]. Additional studies in humans and squirrel monkeys have shown that opsonic or cytophilic antibodies (IgG1 and IgG3 in humans) are associated with the protective effect of the Abs (Bouharoun-Tayoun and [START_REF] Bouharoun-Tayoun | Plasmodium falciparum malaria: evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity[END_REF][START_REF] Groux | Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses[END_REF][START_REF] Shi | Differential effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on the growth of asexual stage Plasmodium falciparum parasites[END_REF]. At this point cooperation between cells of the immune system and Abs appears to be crucial in parasite elimination and acquired protective immunity.

The elimination of the parasite trough an Abs-cell complex is mediated by the capture of Abs on the surface of cells via FcγRs receptors that bind the Fc part of the Ab, while the Fab part of the Ab is bound to antigens on the surface of either merozoites [START_REF] Bouharoun-Tayoun | Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes[END_REF] or late infected erythrocytes [START_REF] Gysin | In vitro phagocytosis inhibition assay for the screening of potential candidate antigens for sub-unit vaccines against the asexual blood stage of Plasmodium falciparum[END_REF] enhancing their phagocytosis by immune cells (neutrophils, monocytes and macrophages) and making parasites available for clearance by the spleen [START_REF] David | Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes[END_REF][START_REF] Del Portillo | The role of the spleen in malaria[END_REF], thus protecting against CM [START_REF] Carlson | Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes[END_REF]. Different Abs with a protective function are produced (IgG, IgM and IgE) but in clinically immune individuals the predominance of the cytophilic IgG1 and

IgG3 subclasses has been associated with either lower parasitaemia or a lower risk of malaria attack [START_REF] Bouharoun-Tayoun | Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages[END_REF]. The non-cytophilic IgG4, present in low doses in immune individuals, may inhibit effector mechanisms by competing with cytophilic Ab and are considered nonprotective [START_REF] Aucan | High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to Plasmodium falciparum malaria[END_REF][START_REF] Garraud | Malaria-specific antibody subclasses in immune individuals: a key source of information for vaccine design[END_REF][START_REF] Leoratti | Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria[END_REF].

In contrast, recent data suggest that, under certain circumstances, the non-cytophilic IgG2 could be correlated with protection in individuals. This protection is due to a rare mutation on the allele encoding an FcγRIIa that can bind IgG2 [START_REF] Aucan | High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to Plasmodium falciparum malaria[END_REF]. Moreover, highest levels of IgG1, IgG2 and IgG3 Abs were observed in individuals with asymptomatic and uncomplicated malaria infection, while high levels of IgG4, IgM and IgE Abs were predominant among individuals with complicated malaria infection in individuals naturally exposed to malaria living in different regions of Brazil [START_REF] Leoratti | Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria[END_REF].

Althoug many functions have been attributed to IgM Abs in infectious diseases, no specific function has been ascribed to this class of Abs during malaria infection. However, it was observed that natural non-specific IgM bind to the surface of some P. falciparum infected erythrocytes via the Fc, and this was shown to correlate with severe malaria in laboratory strains and field isolates [START_REF] Ghumra | Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)[END_REF][START_REF] Rowe | Nonimmune IgM, but not IgG binds to the surface of Plasmodium falciparum-infected erythrocytes and correlates with rosetting and severe malaria[END_REF]. Therefore, the understanding of the role of non-immune IgM and its contribution to the development of a protective immunity needs further investigation.

While the role of IgG subclasses has been characterized in some detail, there is some controversy as to the role of specific IgE Abs and whether they serve to protect against the disease or to make the illness more severe [START_REF] Desowitz | Plasmodium-specific immunoglobulin E in sera from an area of holoendemic malaria[END_REF][START_REF] Perlmann | IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria[END_REF]Troye-Blomberg et al., 1999a). Elevated levels of IgE and anti-plasmodial IgE Abs were observed in human and experimental malaria infections [START_REF] Desowitz | Plasmodium falciparum-specific immunoglobulin G (IgG), IgM, and IgE antibodies in paired maternal-cord sera from east Sepik Province, Papua New Guinea[END_REF][START_REF] Helmby | Immunoglobulin E elevation in Plasmodium chabaudi malaria[END_REF][START_REF] Perlmann | IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria[END_REF]. The induction of IgE Abs reflects a switch in the CD4 + T helper cells, from Th1 to Th2, due to repeated exposures to the parasite [START_REF] Perlmann | IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria[END_REF].

However, the rise in IgE levels is controlled by a variety of environmental and genetic factors [START_REF] Perlmann | IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria[END_REF]. IgE bind to high-affinity receptors (FcεRI) on mast cells and basophils as well as to low-affinity receptors (FcεRII/CD23) expressed on the surface of B cells, activated macrophages and eosinophils amongst other cell types [START_REF] Stone | IgE, mast cells, basophils, and eosinophils[END_REF]. Crosslinking of receptor bound IgE by the antigens triggers the release of pharmacologically active substances from mast cells and basophils, including histamine, leukotrienes and peptides attracting neutrophils and eosinophils.

Some studies conducted in endemic areas suggest that IgE could play a role in the pathogenesis of malaria. Increased levels of IgE where found in individuals suffering from severe malaria in comparison to uncomplicated malaria [START_REF] Perlmann | IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria[END_REF]Perlmann et al., 1997;[START_REF] Perlmann | Contrasting functions of IgG and IgE antimalarial antibodies in uncomplicated and severe Plasmodium falciparum malaria[END_REF][START_REF] Seka-Seka | The role of serum immunoglobulin E in the pathogenesis of Plasmodium falciparum malaria in Ivorian children[END_REF]. Furthermore, immunohistological studies on brain sections revealed the presence of IgE deposits in brain microvessels and on infected erythrocytes from cerebral malaria patients as well as in placentas infected with P.

falciparum [START_REF] Maeno | IgE deposition in brain microvessels and on parasitized erythrocytes from cerebral malaria patients[END_REF][START_REF] Maeno | Immunoglobulin complex deposits in Plasmodium falciparum-infected placentas from Malawi and Papua New Guinea[END_REF]. Additionally, the expression of high levels of IgE during cerebral P. falciparum infection was related to the deepness of the coma [START_REF] Maeno | IgE deposition in brain microvessels and on parasitized erythrocytes from cerebral malaria patients[END_REF]. In vitro studies using malaria patient's sera showed high levels of TNFα, known to contribute to pathogenesis of CM [START_REF] Clark | Proposed link between cytokines, nitric oxide and human cerebral malaria[END_REF], from activated monocytes.

In contrast, P. falciparum specific IgE in the form of immune complexes, with either antigen aggregates or with IgG anti-IgE autoantibodies, crosslink the CD23 receptor expressed on monocytes which results in TNF-α and NO production [START_REF] Elghazali | Elevated plasma levels of IgE in Plasmodium falciparum-primed individuals reflect an increased ratio of IL-4 to interferon-gamma (IFN-gamma)-producing cells[END_REF][START_REF] Perlmann | IgE and tumor necrosis factor in malaria infection[END_REF]. The CD23/NO pathway is believed to play a protective role during the development of malaria pathogenesis by down regulating the expression of ICAM-1 and decreasing iRBCs cytoadherence [START_REF] Pino | Induction of the CD23/nitric oxide pathway in endothelial cells downregulates ICAM-1 expression and decreases cytoadherence of Plasmodium falciparum-infected erythrocytes[END_REF]). However, it has been shown in Thai patients that during severe malaria the release of CD23 from the cell surface giving rise to increased sCD23 levels in plasma was higher compared to uncomplicated and asymptomatic malaria [START_REF] Kumsiri | IgE low affinity receptor (CD23) expression, Plasmodium falciparum specific IgE and tumor necrosis factor-alpha production in Thai uncomplicated and severe falciparum malaria patients[END_REF]. Indeed, the amount of sCD23 correlates with disease severity [START_REF] Rujeni | Soluble CD23 levels are inversely associated with atopy and parasite-specific IgE levels but not with polyclonal IgE levels in people exposed to helminth infection[END_REF][START_REF] Vouldoukis | IgE mediates killing of intracellular Toxoplasma gondii by human macrophages through CD23-dependent, interleukin-10 sensitive pathway[END_REF] and is recognized as an important indicator of disease progression [START_REF] Schwarzmeier | Regulation of CD23 expression by Notch2 in B-cell chronic lymphocytic leukemia[END_REF] in other diseases. In the opposite, several findings have demonstrated that high levels of IgE increase with age in parallel with the acquisition of immunity [START_REF] Desowitz | Plasmodium falciparum-specific immunoglobulin G (IgG), IgM, and IgE antibodies in paired maternal-cord sera from east Sepik Province, Papua New Guinea[END_REF][START_REF] Maeno | Immunoglobulin complex deposits in Plasmodium falciparum-infected placentas from Malawi and Papua New Guinea[END_REF] and in the non-comatose patients compared to comatose patients [START_REF] Calissano | IgE antibodies to Plasmodium falciparum and severity of malaria in children of one ethnic group living in Burkina Faso[END_REF]. In support of these results studies on the Fulani group, known to be less susceptible to malaria infections, showed a correlation between the decrease of infected cells with the increase of anti-malaria IgG and IgE levels [START_REF] Farouk | Different antibody-and cytokine-mediated responses to Plasmodium falciparum parasite in two sympatric ethnic tribes living in Mali[END_REF]. In Tanzania, elevated anti-malaria IgE were associated with the reduction of the subsequent malaria attacks risk [START_REF] Bereczky | Elevated anti-malarial IgE in asymptomatic individuals is associated with reduced risk for subsequent clinical malaria[END_REF].

The impact of malaria parasite on B cells and subsequently on Abs production and in maintenance of B cell memory in individuals chronically re-infected with P. falciparum was highlighted during epidemiological studies in malaria endemic areas where atypical memory B cells were reported to be significantly expanded in Pf-exposed Malian adults and children.

In chronically infected asymptomatic children the number of these atypical MBCs was higher compared with uninfected children, suggesting that the chronic presence of the parasite may drive expansion of these distinct MBCs [START_REF] Weiss | Atypical memory B cells are greatly expanded in individuals living in a malaria-endemic area[END_REF].

Protective immunity against malaria appears to result in a delicate balance, temporally and spatially regulated, between an appropriate innate immunity able to drive a quick Th1 response for the control of the parasite density followed by a Th2 response avoiding Th1mediated deleterious effects, and maintaining the long term protection against parasite rise.

MALARIA TREATMENT

Public health efforts to combat malaria take the three-pronged approach of insecticidetreated bed nets, case management of malaria illness and preventative treatment. Despite the intensive efforts over the years to reduce the malaria burden the situation has gradually worsened in the recent years with the increase in resistance phenomena associated with synthetic antimalarial drugs [START_REF] Ashley | Spread of artemisinin resistance in Plasmodium falciparum malaria[END_REF][START_REF] Packard | The origins of antimalarial-drug resistance[END_REF] and insecticides in use [START_REF] Brown | The insecticide-resistance problem: a review of developments in 1956 and 1957[END_REF] in addition to the economic instability of affected countries and the lack of infrastructure required to instigate these public health measures en masse. For this reason, to further combat the disease, researchers and politicians need to invest in a combination of both traditional and new public health interventions by revising antimalarial drugs and strategy policies. This may take the form of developing new drugs and insecticides, along with novel approaches such as transgenic mosquitoes resistant to malaria parasites and new vaccine design.

Another important aspect, indispensable to prevent the over-medication and avoid the development of resistance, is the development of a reliable diagnostic test. Unfortunately in the past the diagnostic was based on clinical symptoms and sometimes associated to a microscopic examination of a drop of peripheral blood. In recent years new diagnostic techniques have been developed as the rapid diagnostic test based on the identification of three different types of Plasmodium antigens (histidine-rich protein 2, Plasmodium lactate dehydrogenase and aldolase) [START_REF] Moody | Rapid diagnostic tests for malaria parasites[END_REF].

Antimalarial chemotherapy

Several drugs are available to kill the malaria parasite. They can be differentiated into 5 categories: the quinolines, the hydroxynaphthoquinones, the antifolates, the antibiotics not antifolates, and the artemisinin derivatives, depending on their activity on different stages of the parasite life cycle or by their mechanism of action [START_REF] Arav-Boger | Molecular mechanisms of resistance in antimalarial chemotherapy: the unmet challenge[END_REF].

Antimalarials have been known for a long time but their mode of action has still not been completely elucidated. The commonly used were chloroquine, which inhibits haemoglobin digestion, and sulfadoxine or pyrimethamine that inhibit enzymes in the parasite folate synthesis pathway. With the development and spread of drug resistance to chloroquine and the antifolate drugs alkylating agents, such as artemisinin, are becoming more widely used.

Malaria Vaccine

Over the years one of the greatest challenges in the malaria eradication program has been the development of an effective and safe vaccine. Studies on malaria vaccine started in the persistent efforts, the lifelong sacrifice from dedicated physicians and scientists, and the investment of hundreds of millions of dollars, the malaria vaccine has remained elusive.

Development of an effective vaccine seems to be a daunting task, due to the complexity of Plasmodium biology (Gardner et al., 2002a;[START_REF] Scherf | Antigenic variation in Plasmodium falciparum[END_REF] which allows the parasite to escape the host immune system following different mechanisms. Additionally, natural infection with malaria does not induce immune protection in fact only partially effective immunity is acquired after repeated and prolonged exposures to malaria parasite over several years and is short-lived and highly stage-and strain-specific [START_REF] Langhorne | Immunity to malaria: more questions than answers[END_REF][START_REF] Stevenson | Innate immunity to malaria[END_REF].

In spite the evidence that an effective malaria vaccine should be possible, numerous challenges need to be overcome. Essentially two main strategies for malaria vaccine development, targeting the different phases of the parasite's life cycle, are currently being pursued, largely in parallel: (i) the subunit approach, and (ii) the whole organism approach.

Subunit approach

The first peptide-based candidate vaccine tested extensively in endemic areas was the SPf66 developed in Colombia and appeared to be effective in South America [START_REF] Patarroyo | Induction of protective immunity against experimental infection with malaria using synthetic peptides[END_REF]. Unfortunately, field efficacy trials in Africa and Asia failed to demonstrate protection.

In 1982 the identification of the circumsporozoite (CS) protein as the major component of the sporozoite coat has promoted the development of a sporozoite vaccine. The RTS,S/AS, a recombinant protein that fuses a part of the P. falciparum CS protein with the hepatitis B surface antigen [START_REF] Proietti | The case for a rational genome-based vaccine against malaria[END_REF], is the most advanced subunit vaccine in development. RTS,S/AS induces antibodies and T cell function that are believed to diminish the capacity of the malaria parasite to infect, survive, and develop in the human liver.

However, due to the low vaccine efficacy or cover (around 30%) and the development of only a partial protection that is lost about two years after vaccination, alternative strategies need to be considered (Agnandji et al., 2012;Agnandji et al., 2011;[START_REF] Rts | Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial[END_REF]. Moreover, the RTS,S/AS candidate has been tested only in the Africa continent whit no data obtained from outside the continent while it should be effective worldwide. 53

Other vaccine candidates have been developed (Fig. 11) such as MSP-1 or 2 and AMA-1, targeting the asexual blood-stage, both tested in clinical trials still the lack of clear efficacy [START_REF] Mccarthy | A phase 1 trial of MSP2-C1, a blood-stage malaria vaccine containing 2 isoforms of MSP2 formulated with Montanide(R) ISA 720[END_REF][START_REF] Ogutu | Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya[END_REF][START_REF] Sagara | A randomized controlled phase 2 trial of the blood stage AMA1-C1/Alhydrogel malaria vaccine in children in Mali[END_REF]. Different approaches have been used to make the immune response and the protection more efficient, such as the conjugation of Plasmodium antigens to an immunogenic carrier protein [START_REF] Que | Effect of carrier selection on immunogenicity of protein conjugate vaccines against Plasmodium falciparum circumsporozoites[END_REF] or the generation of chimeric proteins comprising domains of two or more vaccine candidates [START_REF] Malkin | A phase 1 trial of PfCP2.9: an AMA1/MSP1 chimeric recombinant protein vaccine for Plasmodium falciparum malaria[END_REF] (ex. PfCP2.9, comprising domain III of the apical membrane antigen AMA1 and the C terminal epidermal growth factor-like domains of the merozoite surface protein MSP1), without any positive result. Although it has been shown that several

Plasmodium antigens can induce some degree of protection in laboratory animal models and induce antibody response able to inhibit P. falciparum development in vitro [START_REF] Richards | The future for blood-stage vaccines against malaria[END_REF], the antigenic complexity of the parasite is an obstacle of greater importance in the development of an efficient subunit vaccine and contributes to some disillusionment with the recombinant protein subunit approach.

Whole organism approach

In recent years the use of live attenuated parasites (LAPs) as vaccines due to their ability to induce sterile long-lasting immunity has become popular. The advantage of whole-parasite vaccines is to induce immune response to a broad range of parasite antigens, and thus potentially more protective. The necessity of using viable parasite for whole parasite immunization has become evident because protection cannot be induced by heat-killed sporozoites, which are unable to invade and develop in the liver [START_REF] Alger | Plasmodium berghei: heat-treated sporozoite vaccination of mice[END_REF].

The first LAP appeared in 1967 when Ruth Nussenzweig and co-workers showed the necessity to use whole parasite for complete immunization using radiation-attenuated sporozoites (RAS) [START_REF] Vanderberg | Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. V. In vitro effects of immune serum on sporozoites[END_REF]. In mice RAS, whose attenuation is based on multiple random DNA breaks, shows an be arrest in the earliest phase of liver-stage, causing no blood infection and conferring sterile, protective immunity against subsequent challenge with wild type sporozoites [START_REF] Suhrbier | Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells[END_REF][START_REF] Vanderberg | Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. V. In vitro effects of immune serum on sporozoites[END_REF] over at least 10 months [START_REF] Hoffman | Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites[END_REF]. Mice studies carried out to understand the mechanism involved during the immune response highlighted the role of CD8 + T cells (Schofield et al., 1987;[START_REF] Weiss | CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites[END_REF] and sporozoite-specific antibodies against the major surface protein CSP [START_REF] Doolan | The complexity of protective immunity against liver-stage malaria[END_REF]. Interestingly, recent studies have shown the possible implication of CD4 + T cells and IFN-γ in protection [START_REF] Oliveira | Class II-restricted protective immunity induced by malaria sporozoites[END_REF]. Moreover, it is known that activation of CD8 + T cells is followed by the production of IFN-γ, which in turn provokes the secretion of IL-12, responsible for stimulating NK cells to produce more IFN-γ in a positive feedback loop [START_REF] Yazdani | Immune responses to asexual blood-stages of malaria parasites[END_REF]. This causes the activation of the NO pathway, which induces the killing of the infected hepatocyte [START_REF] Doolan | IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model[END_REF][START_REF] Doolan | The complexity of protective immunity against liver-stage malaria[END_REF]. Consistently, humans immunized by the bite of irradiated P.

falciparum infected mosquitoes were effectively protected from subsequent challenges with homologous and heterologous infectious P. falciparum sporozoites (efficacy > 90%) [START_REF] Hoffman | Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites[END_REF][START_REF] Vanderberg | Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. V. In vitro effects of immune serum on sporozoites[END_REF]. Given the high levels of protection achieved by irradiated sporozoites in many malaria models the development of P. falciparum RAS vaccine, called PfSPZ, was proposed by Stephen Hoffman (Sanaria) and is actually tested in a

Phase I clinical study with encouraging results (100% protection after challenge) [START_REF] Richie | Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines[END_REF][START_REF] Seder | Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine[END_REF].

Although this vaccine presents some safety problem as the efficacy of the irradiated sporozoites that depend on the number of irradiated sporozoites injected (> 100,000 RAS [START_REF] Luke | Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine[END_REF]) and on the precise irradiation dose; up to 200 Gray can confer complete protection [START_REF] Chattopadhyay | The Effects of radiation on the safety and protective efficacy of an attenuated Plasmodium yoelii sporozoite malaria vaccine[END_REF], less doses allow the parasite to complete the liver stage development and cause blood stage infection, higher doses completely inactivate the sporozoites resulting in no significant protection [START_REF] Silvie | Effects of irradiation on Plasmodium falciparum sporozoite hepatic development: implications for the design of pre-erythrocytic malaria vaccines[END_REF][START_REF] Suhrbier | Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells[END_REF]. In addition to challenges of manufacturing and delivering a viable cryopreserved whole parasite vaccine, it remains to solve the limit of mass-production of vaccines using the sporozoites stage. Besides, animal studies show that several higher doses are needed to protect mice with cryopreserved irradiated sporozoites as compared to fresh ones, demonstrating that the cryopreservation might inactivate the sporozoites [START_REF] Epstein | Live attenuated malaria vaccine designed to protect through hepatic CD8(+) T cell immunity[END_REF].

Another approach has been possible with the availability of the whole Plasmodium genome sequences [START_REF] Carlton | Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii[END_REF]Gardner et al., 2002a;Gardner et al., 2002b) and the advancement of gene manipulation technologies (de Koning- Ward et al., 2000;[START_REF] Menard | Structure-function analysis of malaria proteins by gene targeting[END_REF][START_REF] Thathy | Gene targeting in Plasmodium berghei[END_REF] which has led to the individualization of genes essential for parasite survival at distinct points during its life cycle. Genetically arrested parasites (GAP) generated by targeted gene deletion(s) designed to arrest at specific points of livers-stage development (Fig. 12) (Butler et al., 2011;Mueller et al., 2005a;Mueller et al., 2005b;van Dijk et al., 2005) were able to successfully invade the liver but arrest prior to liver stage maturity, generating complete protective immunity against wild type sporozoite challenge in a manner comparable to radiation attenuated sporozoites (Butler et al., 2011;[START_REF] Khan | Genetic engineering of attenuated malaria parasites for vaccination[END_REF]Mueller et al., 2005b). This immune response is mediated by MHC class I-dependent IFN-γ producing CD8 + T cells [START_REF] Jobe | Genetically attenuated Plasmodium berghei liver stages induce sterile protracted protection that is mediated by major histocompatibility complex Class I-dependent interferon-gamma-producing CD8+ T cells[END_REF][START_REF] Mueller | Genetically attenuated Plasmodium berghei liver stages persist and elicit sterile protection primarily via CD8 T cells[END_REF], which results in sterile protracted protection [START_REF] Jobe | Genetically attenuated Plasmodium berghei liver stages induce sterile protracted protection that is mediated by major histocompatibility complex Class I-dependent interferon-gamma-producing CD8+ T cells[END_REF][START_REF] Wang | Advances and challenges in malaria vaccine development[END_REF] against sporozoite challenge in rodent models. Moreover, a late-blocked liver-stage

GAPs have been shown to induce superior protective immunity against sporozoite challenge in outbred mice compared to both early-blocked liver-stage GAP and RAS vaccine (Butler et al., 2011;[START_REF] Nganou-Makamdop | Liver or blood-stage arrest during malaria sporozoite immunization: the later the better?[END_REF]. Similar transgenic parasites have been produced in P. falciparum and studied in clinical assays. Unfortunately the first clinic study showed considerable but incomplete attenuation [START_REF] Spring | First-in-human evaluation of genetically attenuated Plasmodium falciparum sporozoites administered by bite of Anopheles mosquitoes to adult volunteers[END_REF], indicating that complete attenuation of the parasite in humans remains challenging [START_REF] Bijker | Novel approaches to whole sporozoite vaccination against malaria[END_REF].

More recently, the development of blood-stage GAPs (Table 1) as the P. yoelii lacking purine nucleoside phosphorylase (Ting et al., 2008) or nucleoside transporter 1 (Aly et al., 2010) and P. berghei ANKA lacking plasmepsin-4 (Spaccapelo et al., 2010) where shown to attenuate the parasite development in the blood stage, resulted in long-lasting protection against sporozoite and iRBCs re-infection respectively. Alternative strategies have been developed by combining wild type sporozoite immunizations with drugs acting at various stages of parasite life cycle. Several treatments have been tested as primaquine (liver stages elimination) [START_REF] Putrianti | Vaccine-like immunity against malaria by repeated causal-prophylactic treatment of liver-stage Plasmodium parasites[END_REF], chloroquine (blood stages elimination) [START_REF] Belnoue | Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment[END_REF][START_REF] White | Assessment of the pharmacodynamic properties of antimalarial drugs in vivo[END_REF], clindamycin and azithromycin (targeting the apicoplast resulting in non-infectious merozoites) [START_REF] Friesen | Natural immunization against malaria: causal prophylaxis with antibiotics[END_REF]. David

Pombo and co-workers demonstrated that different challenges of low doses of live bloodstages wild type parasites treated early by appropriate drug treatment could induce a strong cell-mediated immunity to the same strain of malaria parasite in human adults [START_REF] Belnoue | Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment[END_REF]. These results were confirmed in murine models where was highlighted the need of three and more repeated malaria infections followed by drug treatment to obtain semiimmune mice [START_REF] Bao | CD19(+) B cells confer protection against experimental cerebral malaria in semi-immune rodent model[END_REF][START_REF] Bao | Splenic CD11c+ cells derived from semi-immune mice protect naive mice against experimental cerebral malaria[END_REF][START_REF] Evans | Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes[END_REF]. The fact that the infection and the acquisition of immunity are thereby the result of natural wild type infection, as opposed to the attenuated infection induced by GAPs or RAS, the use of this model seems to be difficult in endemic areas where drug resistance is high and the majority of people have already had malaria episodes with the development of an humoral or cellular response.

The pathogenesis of malaria is complex, and development of pathogenesis depends on the parasite species that cause infection and the immune status of the infected host. While current blood stage sub-unit preparations have not yielded any protection in field trials [START_REF] Proietti | The case for a rational genome-based vaccine against malaria[END_REF]White et al., 2014), live attenuated parasites appear to confer significantly broader and more lasting protection. However, the development of an adequate and effective vaccine definitely requires well and detailed understanding of the mechanisms underlying natural immunity to malaria associated with all environmental cultural, society and territorial parameters that can be found among the different populations living in endemic areas. Histamine Releasing Factor

HISTAMINE RELEASING FACTOR (HRF)

Discovered in the 1980s the histamine releasing factor (HRF) protein, also known as translationally controlled tumour protein (TCTP), P23/21, Q23 or fortilin is a housekeeping protein, highly conserved in many organisms, ranging from unicellular, yeast, plants to vertebrates including all Plasmodium parasite species with both intracellular and extracellular functions (Fig. 13).

At first, the synthesis of HRF was shown to be regulated at the translational level, based on the fact that the cDNA cloned from a mouse tumour cell line, and derived from an abundant mRNA found to mainly exist as messenger ribonucleoprotein (mRNP) particles was unable to interact with translational apparatus within the cells (Chitpatima et al., 1988). Later, it was shown that the protein could also be regulated at the level of transcription [START_REF] Bohm | The 5'-untranslated region of p23 mRNA from the Ehrlich ascites tumor is involved in translation control of the growth related protein p23[END_REF]Chung et al., 2000).

Recently HRF has attracted the attention of an increasing number of researchers due to its strong regulation in response to a wide range of extracellular stimuli effecting calcium concentrations both intracellular and within endoplasmic reticulum [START_REF] Xu | Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level[END_REF]. A series of recent reports highlighted the importance of HRF in different intracellular functions including the cell cycle progression (Gachet et al., 1999;[START_REF] Yarm | Plk phosphorylation regulates the microtubule-stabilizing protein TCTP[END_REF], apoptosis inhibition [START_REF] Amson | Reciprocal repression between P53 and TCTP[END_REF][START_REF] Li | Characterization of fortilin, a novel antiapoptotic protein[END_REF][START_REF] Liu | Stabilization and enhancement of the antiapoptotic activity of mcl-1 by TCTP[END_REF]Rho et al., 2011) and malignant transformation of a variety of cell types [START_REF] Li | Characterization of fortilin, a novel antiapoptotic protein[END_REF][START_REF] Tuynder | Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1[END_REF].

Accordingly, multiple studies have implicated the protein in the regulation of growth and proliferation as well as in the control of organ size. Complete gene knockout of HRF in drosophila [START_REF] Hsu | Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase[END_REF] and mice (Chen et al., 2007;[START_REF] Koide | Embryonic lethality of fortilin-null mutant mice by BMPpathway overactivation[END_REF] provided genetic evidence that it plays a critical role in the normal proliferation and survival of embryonic fibroblasts. Moreover, different cellular proteins involved in cell growth have been reported

to interact with HRF, including tubulin (Gachet et al., 1999), elongation factor eEF1A and its guanine nucleotide exchange factor eEF1Bβ [START_REF] Cans | Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A[END_REF][START_REF] Langdon | Identification of the interaction between the human recombinant histamine releasing factor/translationally controlled tumor protein and elongation factor-1 delta (also known as eElongation factor-1B beta)[END_REF], Mcl-1 [START_REF] Li | Characterization of fortilin, a novel antiapoptotic protein[END_REF][START_REF] Liu | Stabilization and enhancement of the antiapoptotic activity of mcl-1 by TCTP[END_REF], TSAP6 (Amzallag et al., 2004) and Na,K-ATPase [START_REF] Jung | Translationally controlled tumor protein interacts with the third cytoplasmic domain of Na,K-ATPase alpha subunit and inhibits the pump activity in HeLa cells[END_REF].

A series of publications have highlighted the role of HRF in anti-apoptotic activities since a decrease at the protein levels leads to apoptosis or reversion of malignant phenotypes [START_REF] Amson | Reciprocal repression between P53 and TCTP[END_REF][START_REF] Li | Characterization of fortilin, a novel antiapoptotic protein[END_REF][START_REF] Liu | Stabilization and enhancement of the antiapoptotic activity of mcl-1 by TCTP[END_REF]Rho et al., 2011). In tumor cells the expression of HRF is upregulated while during reversion of cells from the malignant phenotype the HRF levels are considerably reduced. Consistently, the inhibition of HRF expression, by anti-sense cDNA or small interfering RNA molecules, results in the suppression of the malignant phenotype [START_REF] Tuynder | Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1[END_REF].

The extracellular function of HRF presents a cytokine-like activity leading to the release of histamine from mast cells and basophils via IgE antibody-mediated mechanisms during latephase acute allergy. Moreover, investigations of the actions of HRF on the immune response first indicated that the human recombinant HRF (HrHRF) induces the secretion of histamine, IL-4 and IL-13 from basophils (Schroeder et al., 1997) and IL-8 from eosinophils (Bheekha-Escura et al., 2000) from a subset of allergic donors in a IgE-dependent and IgE-independent manner. Recombinant murine HRF was found to stimulate B cell proliferation in a dosedependent manner (in vitro and in vivo) [START_REF] Kang | Molecular identification of IgE-dependent histamine-releasing factor as a B cell growth factor[END_REF] and the HrHRF was found to modulate cytokine release from peripheral blood T cells (PBTs) by inhibition of IL-2 and IL-13 secretion, but not IFN-γ (Vonakis et al., 2003).

Unfortunately so far no connection has been made between the extracellular functions of HRF with the variety of intracellular processes attributed to the protein family and many intracellular and extracellular functions that have been ascribed to HRF seem to be contradictory. Therefore overall functions of HRF still remained to be elucidated.

HRF structure

The translationally controlled tumor protein consists of 170 amino acids in average and has a mean weight of 18-23 kDa.

Bioinformatic analysis was performed to investigate the sequence and predicted structure of the HRFs from many species [START_REF] Hinojosa-Moya | Phylogenetic and structural analysis of translationally controlled tumor proteins[END_REF]. Extensive sequence analysis revealed that most species, including malaria parasites, have only a single hrf gene.

Mammals appear to harbour more hrf genes (eg. in mouse genome seven hrf genes were identified) [START_REF] Fiucci | Genomic organization and expression of mouse Tpt1 gene[END_REF]. Additionally, no similarity to other proteins or functional domains was discovered, so the protein was listed as a 'family' on its own in the databases. . hrf is a key gene in the process of tumour reversion and a pharmacological target for sertraline, thioridazine and artemisinin. Well conserved through phylogeny as a pro-survival, growth-stimulating and antiapoptotic factor, it is also a transcription factor for OCT4. HRF binds to CHFR and to elongation factor eEF1A and eEF1Bβ, leading to roles during mitosis and in protein synthesis, respectively. GDI, GDP dissociation inhibitor. [START_REF] Telerman | The molecular programme of tumour reversion: the steps beyond malignant transformation[END_REF] Elucidation of the HRF structure, resolved by NMR spectroscopy and X-ray crystallography, demonstrated that the HRF molecule consists of three distinct domains, the core β-sheet domain, a α-helical domain and a flexible loop structure (Fig. 14) [START_REF] Thaw | Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones[END_REF].

Additional analysis confirmed that this principal structure is highly conserved through phylogeny over a long period of evolution supporting the hypothesis that HRF plays and important role in the cellular physiological functions [START_REF] Bini | Two-dimensional gel electrophoresis of Caenorhabditis elegans homogenates and identification of protein spots by microsequencing[END_REF]Bommer and Thiele, 2004b;[START_REF] Thaw | Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones[END_REF]. Moreover, the sequence alignment demonstrates that 9% of HRF amino acids are absolutely conserved (Bommer and Thiele, 2004b). The invariant residues are largely clustered on one side of the β-strain domain indicating that this side is important for interaction. In fact, three conserved amino acids (Glu12, Leu74 and Glu134) which form a binding surface for G protein interaction are localized in this domain suggests a possible role for HRF in proliferation and growth [START_REF] Hinojosa-Moya | Phylogenetic and structural analysis of translationally controlled tumor proteins[END_REF]. The other major domains, the flexible loop and the helical domain, are specific for HRF. The flexible loop is predominantly hydrophilic containing a highly conserved area named HRF1. The α-helical domain is characterized by a tubulin-binging region (Gachet et al., 1999) and a Ca 2+ -binding area [START_REF] Kim | Identification of the calcium binding sites in translationally controlled tumor protein[END_REF]. However, the calcium-binding domain position is controversial.

Additionally detailed studies localized the domain in the residues N131, Q132 and D150, located in the β-stranded core domain close to the connection with the α-helical domain [START_REF] Feng | Solution structure and mapping of a very weak calciumbinding site of human translationally controlled tumor protein by NMR[END_REF] or in the residues E58 and E60 located in the floppy loop (Graidist et al., 2007).

Interesting, 3D structure of P. knowelsi an extra α-helix segment, which in other HRFs correspond to a β-sheet, is present and could potentially interfere with G protein interactions (Fig. 14) [START_REF] Hinojosa-Moya | Phylogenetic and structural analysis of translationally controlled tumor proteins[END_REF].

HRF secretion

It has been reported that HRF protein lacks the NH 2 -terminus signal and no precursor protein has been described suggesting that HRF secretion proceeded via an endoplasmic reticulum/Golgi-independent or nonclassical pathway [START_REF] Muesch | A novel pathway for secretory proteins?[END_REF]. Interestingly, immune fluorescence assay (IFA) analysis shows a partially co-localisation of HRF with tumour suppressor activated pathway-6 (TSAP6, a p53-regulated gene) in vesicular-like structures at the plasma and nuclear membranes (Amzallag et al., 2004). Furthermore, the overexpression of TSAP6 considerably enhanced the secretion of both endogenous and exogenous HRF in epithelial cells as well as in hematopoietic cell lines (Amzallag et al., 2004).

More importantly, these studies show that the TSAP6 gene product could facilitate the secretion of HRF in exosomes (Fig. 15) (Amzallag et al., 2004). Exosomes are nanovesicles (50-150nm in diameter) of cup shape appearance originating from large multivesicular endosomes containing nucleic acids, proteins, lipid rafts and exposed phosphatidylserine [START_REF] Thery | Membrane vesicles as conveyors of immune responses[END_REF] that induce phenotypic changes in the recipient cells [START_REF] Raposo | Extracellular vesicles: exosomes, microvesicles, and friends[END_REF]. Due to their stability, exosomes protect their cargo against degradation and denaturation in the extracellular environment. Moreover, they play an important role in many biological processes including pathogen spread: various pathogens utilize the exosomal pathway to facilitate their survival and propagation, including transfer of virulence factors to host target cells [START_REF] Feng | A pathogenic picornavirus acquires an envelope by hijacking cellular membranes[END_REF][START_REF] Ramakrishnaiah | Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells[END_REF].

Recently, another pathway that proposes the involvement of H, K-ATPase was used to describe HRF secretion (Fig. 15) [START_REF] Kim | Dimerization of TCTP and its clinical implications for allergy[END_REF]. A screening of possible HRF secretion inhibitors show that proton pumps inhibitors modulated the protein export. Indeed, the use of omeprazole and pantoprazole, two peptic ulcer drugs, block HRF secretion from HEK293 and U937 cells in a concentration-dependant fashion, corroborated by the observation that H, K-ATPase inhibitor ameliorated the allergic symptoms.

HRF dimerization

The comparison of basophil histamine releasing abilities of mouse recombinant HRF (MrHRF), human recombinant (HrHRF), HRF from nasal lavages, and HRF from supernatants of peripheral blood mononuclear cells (PBMC) cultures have shown that recombinant HRF were less active than the native HRF purified from the biological fluids. Dimerization of HRF seems to be essential for its cytokine-like activity, as the cytokine-releasing activity of extracellular HRF is generated only when the NH 2 -terminal is truncated allowing the dimerization via the formation of an intermolecular disulfide bond [START_REF] Kim | Dimerization of translationally controlled tumor protein is essential for its cytokine-like activity[END_REF].

Moreover, dimerized form of the protein was found in sera in allergic patients and bronchoalveolar lavage fluids from airway-inflammed mice [START_REF] Kim | Dimerization of TCTP and its clinical implications for allergy[END_REF][START_REF] Kim | Dimerization of translationally controlled tumor protein is essential for its cytokine-like activity[END_REF]. Subsequent studies suggested that allergic status might affect the normal biochemistry composition and physiology of the cellular environment, leading to posttranslational modification of the protein, and the activation of inflammatory cells that promote the late phase reaction. Three possible contributors were described: (i) proteolytic enzymes which catalysed the truncation of the NH 2 -terminus, (ii) oxidants responsible for the formation of intermolecular disulfure bonds, and (iii) HRF-reactive IgE that possibly confer autoantigen activity [START_REF] Kim | Dimerization of TCTP and its clinical implications for allergy[END_REF](Fig. 15).

ROLE OF PLASMODIUM HRF DURING MALARIA INFECTION

Evidence of P. falciparum HRF (PfHRF) secretion was observed in in vitro cultures and in malaria infected individuals. Trophozoite stage culture supernatants of P. falciparum contained 87±21µg of PfHRF per 10 9 iRBCs, while schizont stage culture supernatants contained 44±6µg of PfHRF per 10 9 iRBCs. Therefore around half of the total parasite HRF was released during schizogony (MacDonald et al., 2001). PfHRF has been identified in the serum of mildly (0.6µg/mL) and severely (1.4µg/mL) P. falciparum infected Malawian children (Janse et al., 2006;MacDonald et al., 2001). There was no correlation between percentage parasitemia of infected individuals and serum PfHRF levels, suggesting that the parasite protein might have local rather than systemic effects. Active structure of HRF now can bind to its specific receptor (dHRF-R) to stimulate the mediator release from basophils. In addition to dHRF-R, subset of IgE can serve as a receptor for HRF dimer in FcεRI-expressing cells. Since these modifications might be possible at inflamed site, extracellular HRF will affect around cells only during the late phase or chronic allergic inflammation. [START_REF] Kim | Dimerization of TCTP and its clinical implications for allergy[END_REF] At the amino acid level, PfHRF is highly homologous to mammalian protein by sharing 33% identity and 54% similarity with human HRF. Molecular mass of recombinant Plasmodium HRF molecules is between 20 and 23 kDa for the monomeric form and around 45 kDa for the dimeric form [START_REF] Bhisutthibhan | Immunoprecipitation of [(H]dihydroartemisinin translationally controlled tumor protein (TCTP) adducts from Plasmodium falciparuminfected erythrocytes by using anti-TCTP antibodies[END_REF]. Sequence homology between HRF molecules is very high between different species of Plasmodium parasites. It was fond that P. falciparum share 88% identity and 97% similarity in HRF amino acids sequence with P. yoelli [START_REF] Walker | Mechanisms of artemisinin resistance in the rodent malaria pathogen Plasmodium yoelii[END_REF] and 90% identity with P. chabaudi HRF. Aditionally, comparisons of HRF amino acid sequence of P. chabaudi with that of P. yoelli and P. berghei orthologues show amino acid identities of 97 and 98%, respectively [START_REF] Afonso | Malaria parasites can develop stable resistance to artemisinin but lack mutations in candidate genes atp6 (encoding the sarcoplasmic and endoplasmic reticulum Ca2+ ATPase), tctp, mdr1, and cg10[END_REF]. IFA and immunolelectron microscopy with immunogold labelling have allowed the localization of intracellular PfTCTP within the cytoplasm, the haem-rich food vacuole and the host or parasite-derived membranes surrounding the parasite [START_REF] Bhisutthibhan | Immunoprecipitation of [(H]dihydroartemisinin translationally controlled tumor protein (TCTP) adducts from Plasmodium falciparuminfected erythrocytes by using anti-TCTP antibodies[END_REF]Bhisutthibhan et al., 1999).

Consequences of histamine release

Generation of histamine-releasing activity was observed in cultured peripheral blood mononuclear cells and their supernatants which were able to induce the release of histamine from basophils [START_REF] Thueson | Histamine-releasing activity (HRA). I. Production by mitogen-or antigen-stimulated human mononuclear cells[END_REF]. Several studies have attempted to chemically characterize the new cytokine that induce the release of histamine from basophils. In 1995, HRF was found and purified from the nasal, skin blister, and bronchoalveolar lavage fluids during late-stage allergic reaction, implicating HRF in the latephase allergic reactions [START_REF] Macdonald | Studies of IgE-dependent histamine releasing factors: heterogeneity of IgE[END_REF]MacDonald et al., 1995). Other studies reported the implication of HRF in promoting the allergic inflammation in the skin and lung from mouse models of asthma and allergy. Furthermore, Ig-interacting HRF peptides, shown to block HRF-Ig interactions in vitro, inhibited IgE-HRF-induced mast cell-dependent inflammation in vivo (Kashiwakura et al., 2012). Collectively, these studies indicate that HRF has pro-inflammatory features during allergic reactions. At first this release was thought to be the consequence of physical interaction between HRF and IgE on the surface of the responding basophils [START_REF] Macdonald | Studies of IgE-dependent histamine releasing factors: heterogeneity of IgE[END_REF]. However, some evidences suggested that the interaction with IgE may not be required for cell activation mediated by human recombinant HRF and that the protein exerts its activity independently of IgE through a human recombinant HRF-specific activation pathway other than through FcεRI (Schroeder et al., 1997).

Recent studies suggest a strong relationship between clinical susceptibility to malaria and severe allergic-type responses [START_REF] Demeure | Anopheles mosquito bites activate cutaneous mast cells leading to a local inflammatory response and lymph node hyperplasia[END_REF][START_REF] Griffiths | Genomewide analysis of the host response to malaria in Kenyan children[END_REF][START_REF] Sakuntabhai | Genetic determination and linkage mapping of Plasmodium falciparum malaria related traits in Senegal[END_REF]. As previously demonstrated in the laboratory, this allergic-type response starts immediately after the mosquito bite by the intermediary of mosquito saliva which exerts immunomodulatory effects through induction of mast cell degranulation, in the absence of IgE Abs, affecting the maturation of adjacent DCs, which fail to ultimately elicit fully activated effector T cells [START_REF] Demeure | Anopheles mosquito bites activate cutaneous mast cells leading to a local inflammatory response and lymph node hyperplasia[END_REF]. Additionally, Plasmodium infection in a murine model of malaria, upon mosquito bite and saliva inoculation, potentiates the pathogenesis via dysregulation of immune signalling and reduction in the recruitment of key inflammatory cells into the inoculation site perturbing the early antiparasite immune response and effecting downstream disease development (Fig. 16) [START_REF] Schneider | Anopheles stephensi saliva enhances progression of cerebral malaria in a murine model. Vector borne and zoonotic diseases[END_REF]. High levels of histamine, a major product of allergic responses derived from basophils and mast cells in plasma and tissue, have been associated with the severity of the disease in humans infected with P. falciparum and in many animal models (Enwonwu et al., 2000;MacDonald et al., 1995;Maegraith and Fletcher, 1972;Srichaikul et al., 1976). In addition, during parasitic infection in human and mouse model, higher levels of IgE, which bind to basophils and mast cells and trigger histamine release, are associated with the severity of the disease (MacDonald et al., 2001;[START_REF] Porcherie | Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria[END_REF]Schroeder et al., 1997).

Elevated histamine synthesis (in situ) within the brain mediated by the histidine decarboxylase enzyme was five-fold more elevated in plasma of infected children with P.

falciparum compared with uninfected controls (Enwonwu et al., 2000). Moreover, the proinflammatory activities via histamine signalling pathway was previously demonstrated in the laboratory to be crucial for the development of ECM pathologies and symptoms exhibited in rodent malaria models. Mice treated with antihistamines or deficient for the H1 and H2 receptors were found more resistant to ECM after infection with P. berghei ANKA sporozoites (Beghdadi et al., 2008) than untreated or wild type mice respectively. Similar results were obtained in HDC -/-mice, not able to synthesize histamine, infected either with P. berghei ANKA iRBCs or sporozoites. This indicated that histamine is produced during all stages of infection and is involved in late stage of pathogenesis (Fig. 16). During the later phase of infection, particularly during the blood stage of parasite development, histamine can be elicited by Plasmodium HRF. Parasite HRF has been found in plasma of patients infected with P. falciparum and was shown to trigger histamine release from basophils and IL-8 secretion from eosinophils (MacDonald et al., 2001). Significantly elevated plasma histamine concentrations by Plasmodium HRF could assist the parasite by inducing vasodilation and increased expression of endothelial adhesion factors such as thrombomodulin, to which P. falciparum-infected erythrocytes adhere (MacDonald et al., 2001). These results show that the existence of a parasite protein able to stimulate host histamine secretion can amplify the host inflammatory response, via histamine signalling and highlight a possible strategy developed by the parasite to create conditions advantageous for its own survival and persistence.

Artemisinin chemo-resistance

The artemisinin removes quickly the parasites and eliminates the symptoms associated with malaria by reducing the carriage of the transmission stages. All the artemisinin drugs have activity throughout the asexual intra-erythrocytic stage, also having effect on young gametocytes. However, their mode of action is not completely clear [START_REF] Klonis | Artemisinin activity against Plasmodium falciparum requires hemoglobin uptake and digestion[END_REF]. Some researchers agree that artemisinin might interact with intraparasitic haem as well as other parasite proteins [START_REF] Meshnick | Artemisinin: mechanisms of action, resistance and toxicity[END_REF][START_REF] Robert | Alkylation of heme by the antimalarial drug artemisinin[END_REF]. Consistently, artemisinin and its derivatives are inactive against the RC strain of P. berghei, which doesn't produce hemozoin [START_REF] Meshnick | Artemisinin: mechanisms of action, resistance and toxicity[END_REF]. This hypothesis supposes that artemisinin is activated by intraparasitic heme into a carbon-centred free radicals mediated by the decomposition of the artemisinin endoperoxide bridge. The carbon-centred free radicals then act as alkylating agents for specific parasite proteins [START_REF] Meshnick | Artemisinin: mechanisms of action, resistance and toxicity[END_REF]. The hypothesis that Plasmodium HRF might be a possible drug target for artemisinin comes from the reaction of Plasmodium HRF with radio labelled dihydroartemisinin ([ 3 H]DHA) both in vitro in the presence of hemin, and in intact infected erythrocytes. Scatchard analysis showed that two hemin-binding sites of modest affinity existed on parasite HRF [START_REF] Bhisutthibhan | The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin[END_REF]. Aditionally, [ 3 H]DHA adducts could be immunoprecipitated using anti-HRF antibodies. Overexpression of Plasmodium HRF at the protein level in artemisinin-resistant parasites of P. yoelii is consistent with this hypothesis [START_REF] Walker | Mechanisms of artemisinin resistance in the rodent malaria pathogen Plasmodium yoelii[END_REF].

The three-dimensional structure of PfHRF created using the nuclear magnetic resonance structure of the S. pombe HRF as a template was used to study the interactions between artemisinin, heme and PfHPF. The center of the peroxide bond of artemisinin was docked within a short distance of 2.6 Å from the iron of the heme, in the same way the center of heme-activated artemisinin (C4-radical) was docked on the PfTCTP sulfur of Cys14 within a distance of 2.48 Å suggesting the possibility of alkylation having occurred [START_REF] Chae | Homology modeling and molecular docking study of translationally controlled tumor protein and artemisinin[END_REF]. To confirm this modelling previous study had shown that blocking the single cysteine of PfTCTP reduced artemisinin binding by 67% [START_REF] Bhisutthibhan | The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin[END_REF].

Calcium binding and self-interaction with malaria HRF

High-affinity binding of HRF to calcium was previously demonstrated from both human and trypanosome [START_REF] Haghighat | Purification of novel calcium binding proteins from Trypanosoma brucei: properties of 22-, 24-and 38-kilodalton proteins[END_REF][START_REF] Xu | Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level[END_REF]. Depletion of endoplasmic reticulum levels of calcium up-regulates HRF transcription, while an increase in cytosolic calcium up-regulates HRF translation showing that HRF expression is regulated by calcium at both transcription and post-transcriptional level [START_REF] Xu | Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level[END_REF].

The primary sequence of PfTCTP lacks a calcium-binding motif; but calcium-binding properties have been demonstrated by PfHRF migration and PVDF membrane transfer followed by calcium ( 45 Ca) incubation or by SDS-PAGE gel migration of recombinant PfHRF in presence of calcium (Bhisutthibhan et al., 1999).

B cell inhibition by malaria HRF

Structural analysis of P. knowelsi HRF and human HRF shows some variations, particularly for the presence in P. knowelsi of an extra α-helix near the GTPase binding pocket while in human HRF is a β-strand. This difference causes a more general structural distortion in the putative G-protein binding pocket suggesting that Plasmodium HRF may act as a dominant negative mutant in the host, as it could potentially block the action of B cells due to its proposed inability to bind G proteins [START_REF] Hinojosa-Moya | Phylogenetic and structural analysis of translationally controlled tumor proteins[END_REF]. Recently it was shown that recombinant PfHRF has a lower B cell stimulatory effect compared to recombinant human HRF. In addition, splenic B cells were shown to incorporate more efficiently and faster the parasite protein than the human one [START_REF] Calderon-Perez | The Plasmodium falciparum translationally controlled tumor protein (TCTP) is incorporated more efficiently into B cells than its human homologue[END_REF]. This result indicated that Plasmodium HRF could interfere with the action of the host HRF by inhibiting B cell responses allowing the parasite to escape the immune response. However, additional work is required to confirm these results.

Basophils activation by malaria HRF

Field studies in Senegal had shown an association between high levels of PfHRF in the peripheral blood circulation and basophils activation. At the same time it was observed that a decrease of basophils in the peripheral blood circulation is associated with the severity of symptoms (Pelleau et al., 2012). A significant accumulation of basophils in the spleen during malaria was similarly observed in the mouse model during P. chabaudi AS infection [START_REF] Poorafshar | MMCP-8, the first lineagespecific differentiation marker for mouse basophils. Elevated numbers of potent IL-4producing and MMCP-8-positive cells in spleens of malaria-infected mice[END_REF]. Moreover, activation of basophils leads to IL-4 production that might play a crucial role in the initiation of Th2 responses, that when activated too early during the infection, increases host susceptibility to malaria disease. However, in this case also additional studies are required to understand whether basophils have a protective or deleterious effect during the development of pathogenesis.

Comparative characteristics of mammal HRF and Plasmodium HRF

Many intracellular and extracellular functions have been ascribed to mammal HRF (Table 2) (Bommer and Thiele, 2004b). Many of the functions that have been determined for HRF are unrelated, and some seem to be contradictory. An overall function of HRF has yet to be determined, and much is left to be learned. Moreover, despite this broad knowledge on mammal HFR, little is know about Plasmodium HRF (Table 2) and the structural differences observed suggest that the parasite protein may have different if not opposite biological functions than the human HRF, especially in influencing the biological responses of immune cells. For this reason, during this work we want to study and identify the possible effects of Plasmodium HRF on the host immune response and their implication in the development of disease pathogenesis.

Mammal HRF

Plasmodium HRF Ref.

Biological function

Calcium binding YES YES [START_REF] Xu | Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level[END_REF], (Bhisutthibhan et al., 1999) Growth and development YES (hrf -/-mouse lethal) NO (Chen et al., 2007;[START_REF] Hsu | Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase[END_REF][START_REF] Koide | Embryonic lethality of fortilin-null mutant mice by BMPpathway overactivation[END_REF][START_REF] Tuynder | Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1[END_REF], (Mathieu et al., 2015) Tubulin binding activity YES ND (Gachet et al., 1999) Chaperon activity YES ND [START_REF] Cans | Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A[END_REF] Anti-apoptotic YES ND [START_REF] Amson | Reciprocal repression between P53 and TCTP[END_REF][START_REF] Li | Characterization of fortilin, a novel antiapoptotic protein[END_REF][START_REF] Liu | Stabilization and enhancement of the antiapoptotic activity of mcl-1 by TCTP[END_REF]Rho et al., 2011) Tumour reversion YES ND [START_REF] Li | Characterization of fortilin, a novel antiapoptotic protein[END_REF][START_REF] Tuynder | Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1[END_REF] Pharmacological target YES YES [START_REF] Tuynder | Translationally controlled tumor protein is a target of tumor reversion[END_REF], [START_REF] Bhisutthibhan | The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin[END_REF]

Histamine release

YES (from basophils) YES (from basophils and mast cells?) [START_REF] Macdonald | Studies of IgE-dependent histamine releasing factors: heterogeneity of IgE[END_REF]MacDonald et al., 1995;[START_REF] Thueson | Histamine-releasing activity (HRA). I. Production by mitogen-or antigen-stimulated human mononuclear cells[END_REF], [START_REF] Briquet | Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity[END_REF]MacDonald et al., 2001) Cytokines release YES (IL-4, IL-13 from basophils and IL-8 from eosinophils)

YES (IL-8 from eosinophils) (Bheekha-Escura et al., 2000;Schroeder et al., 1997), (MacDonald et al., 2001) B cell YES (Proliferation-Stimulation) YES (Inhibition of proliferation) (Mathieu et al., 2015), [START_REF] Calderon-Perez | The Plasmodium falciparum translationally controlled tumor protein (TCTP) is incorporated more efficiently into B cells than its human homologue[END_REF] T cell YES (Inhibition of IL2 and IL13 secretion) ND (Vonakis et al., 2003).

Other Characteristics

Copy nb of hrf genes Several copies Only one copy [START_REF] Fiucci | Genomic organization and expression of mouse Tpt1 gene[END_REF] Structure YES YES [START_REF] Hinojosa-Moya | Phylogenetic and structural analysis of translationally controlled tumor proteins[END_REF][START_REF] Thaw | Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones[END_REF], [START_REF] Hinojosa-Moya | Phylogenetic and structural analysis of translationally controlled tumor proteins[END_REF])

Secretion

Exosomes or H,K-ATPase channel ND (Amzallag et al., 2004;[START_REF] Kim | Dimerization of TCTP and its clinical implications for allergy[END_REF] Active form Dimer ND [START_REF] Kim | Dimerization of TCTP and its clinical implications for allergy[END_REF][START_REF] Kim | Dimerization of translationally controlled tumor protein is essential for its cytokine-like activity[END_REF] Table2 -HRF characteristics.

Objective

SCOPE OF THE THESIS

The purpose of this thesis was to assess the importance of Plasmodium HRF in the modulation of immune mechanisms during malaria infection and evaluate whether its expression is associated with the severity of malaria disease.

Specific Aims

Our specific objectives were as follows

• Design and generation of HRF-deficient parasites (hrfΔ) in a P. berghei-GFP ANKA and NK65-GFP strains by homologous recombination

• The proof of concept that the HRF protein plays a role in malaria pathogenesis

• To assess whether mutant parasites show an altered phenotype that may ultimately cause a sterile immunity

• Characterize the cellular and molecular alterations induced by the presence or the absence of HRF and the immune mechanisms by which the hrf gene modulates the immune response

In the present thesis, the methodological part has been omitted since it is described in detail in the enclosed articles. • The prolonged liver-stage development influenced the clinical outcome of infection by decreasing the frequency of ECM and by increasing the survival rate.

• Impairment in the development of PbANKA-hrfΔ parasite in liver stages was associated with an early rise in liver and systemic IL-6 that in case of WT parasite infection was directly inhibited by Plasmodium HRF production.

• Increase in B-cell population in the liver of mice infected with mutant parasites at 40 h p.i.

in association with early rise of IL-6 reflects the possibility that the HRF molecule could interfere with B-cell memory.

Introduction

Histamine-releasing factor (HRF), originally classified as a tumour protein (translationally controlled tumour protein, TCTP) in mouse erythroleukemia, is found in a wide range of eukaryotes including yeast, plants and animals. The name TCTP was coined as a consequence of cDNA cloning from a human mammary carcinoma and based on the fact that TCTP is regulated at the translational level. Histamine-releasing factor/TCTP was first described as P21, Q23 and P23 by different groups and the mouse and human cDNA were cloned in the late eighties (Chitpatima et al., 1988;[START_REF] Gross | cDNA sequence coding for a translationally controlled human tumour protein[END_REF]. HRF plays many different functions and is involved in many physiological processes such as cell proliferation, stress and heat shock responses, and cell death. As an intracellular product, HRF has a calcium-and tubulin-binding properties and has been shown to transiently associate with microtubules during cell cycle (Gachet et al., 1999). As a calcium-binding protein, HRF was found to be upregulated in response to a loss of calcium homeostasis, which could be part of a role of HRF in general stress response [START_REF] Xu | Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level[END_REF]. HRF was shown to be associated with prostate, breast and colon cancers (Chung et al., 2000;[START_REF] Vercoutter-Edouart | Proteomic detection of changes in protein synthesis induced by fibroblast growth factor-2 in MCF-7 human breast cancer cells[END_REF][START_REF] Arcuri | Translationally controlled tumor protein (TCTP) in the human prostate and prostate cancer cells: expression, distribution, and calcium binding activity[END_REF], which is consistent with its anti-apoptotic activity (Zhang et al., 2002). This was supported by the capacity of HRF to interact with the B-cell lymphoma-extra large (Zhang et al., 2002;[START_REF] Liu | Stabilization and enhancement of the antiapoptotic activity of mcl-1 by TCTP[END_REF][START_REF] Fenner | Expanding the substantial interactome of NEMO using protein microarrays[END_REF][START_REF] Rid | H2O2-dependent translocation of TCTP into the nucleus enables its interaction with VDR in human keratinocytes: TCTP as a further module in calcitriol signalling[END_REF] and to antagonize bax dimerization [START_REF] Susini | TCTP protects from apoptotic cell death by antagonizing bax function[END_REF] and to control the stability of the tumour suppressor p53 (Rho et al., 2011). As a secreted product, HRF has immuno-modulatory roles. In humans, HRF induces the release of histamine and modulates cytokine secretion from basophils, eosinophils and T-cells. Histamine-releasing factor stimulates eosinophils to produce IL-8 (Bheekha-Escura et al., 2000), induces secretion of IL-4 and IL-13 from basophils (MacDonald et al., 1995;Schroeder et al., 1997) and inhibits IL-2, IL-4 and IL-13 production from stimulated primary T-cells (Vonakis et al., 2003). Recently, HRF was found to have an inflammatory role in mouse models of asthma and allergy and to exist as a dimer bound to a subset of IgE and IgG antibodies, suggesting the possibility for HRF to cross-link IgE on the surface of basophils and mast cells (Kashiwakura et al., 2012). HRF is also expressed by a number of eukaryotic parasites, including Plasmodium, the causative agent of malaria. Malaria is the most deadly parasitic disease worldwide, causing almost a million deaths each year. The symptoms of the disease are due to the multiplication of the parasite inside host erythrocytes. Infection starts by an asymptomatic phase, during which the highly motile parasite form injected by mosquitoes reaches the liver, invades hepatocytes and multiplies into the erythrocyteinfecting parasite form. The latter, once released into the bloodstream, invades erythrocytes and initiates the clinical phase of infection, consisting in repeated cycles of parasite multiplication inside erythrocytes. A severe complication of the erythrocytic phase of malaria infection is cerebral malaria (CM), a syndrome caused by the cytoadherence of infected erythrocytes to endothelial cells in brain capillaries, the release of inflammatory cytokines and chemokines and the accumulation of activated CD8 + T-cells.

Plasmodium HRF, which has a high homology to human HRF (their amino acid sequences are 33% identical and 54% similar) and similar activity (MacDonald et al., 2001), has been proposed to play an important role during the erythrocytic phase of malarial infection. Plasmodium falciparum HRF was detected in the plasma of malarial patients in Malawi (MacDonald et al., 2001) and in higher concentration in patients with severe malaria in Senegal (Pelleau et al., 2012). Increased levels of plasma histamine have been associated with disease severity during P. falciparum infection as well as in animal models of malaria (Maegraith and Fletcher, 1972;Srichaikul et al., 1976;Bhattacharya et al., 1988;Enwonwu et al., 2000). Accordingly, CM does not develop in histamine-free, histidine decarboxylase-deficient mice (Beghdadi et al., 2008). Therefore, parasite HRF is thought to play a role in malaria pathogenesis, at least in part through its action on histamine.

Although the contribution of cytokines and other inflammatory molecules during malarial erythrocytic infection is extensively studied, still little is known about host factors that control Plasmodium development in the liver. Work in rodents and cultured hepatocytes showed that several host cytokines strongly inhibit parasite development inside hepatocytes, including IFN-γ, TNF-α, IL-1 and IL-6 (Schofield et al., 1987;[START_REF] Maheshwari | The role of cytokines in malaria infection[END_REF][START_REF] Pied | IL-6 induced by IL-1 inhibits malaria pre-erythrocytic stages but its secretion is downregulated by the parasite[END_REF][START_REF] Vreden | Cytokines inhibit the development of liver schizonts of the malaria parasite Plasmodium berghei in vivo[END_REF]. Both TNF-α-induced [START_REF] Vreden | Cytokines inhibit the development of liver schizonts of the malaria parasite Plasmodium berghei in vivo[END_REF] and IL1-induced (Nussler et al., 1991) inhibitions of liver-stage development are mediated by IL-6. In contrast, recombinant IL-6 is sufficient to reduce liverstage development inside cultured hepatocytes in the absence of non-parenchymal cells [START_REF] Pied | IL-6 induced by IL-1 inhibits malaria pre-erythrocytic stages but its secretion is downregulated by the parasite[END_REF] and exerts its action throughout liver-stage maturation (Nussler et al., 1991). One of the mechanisms by which IL-6 might control parasite liver infection is by regulating iron homeostasis through hepcidin [START_REF] Nemeth | IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin[END_REF], which limits Plasmodium development inside hepatocytes [START_REF] Portugal | Superinfection in malaria: plasmodium shows its iron will[END_REF].

In this work, we investigated the role of HRF in P. berghei, a rodent-infecting species of Plasmodium that can be cycled in vivo. We found that HRF has no contribution during the erythrocytic phase of infection, but plays an important role during the pre-erythrocytic phase of infection in the liver by decreasing IL-6.

Results

HRF is important for P. berghei pre-erythrocytic infection

To test the role of HRF in P. berghei, we replaced in erythrocytic stages of a GFP-expressing P. berghei ANKA clone [START_REF] Ishino | A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell[END_REF] the entire coding sequence of PbHRF (PBANKA_111050) by the hDHFR selectable marker (Supporting Information Fig. S1). Three clones were selected from independent transfection experiments, called HRFΔ1-3, which were verified to harbour the expected HRF null-mutant locus by PCR (Supporting Information Fig. S1A-C), and by Southern blot analysis (Supporting Information Fig. S1D). Blood stages of the wild type (WT) and of the three mutant clones exhibited similar growth rates in the blood of C57BL/6 mice, as measured by Fluorescence-activated cell sorter (FACS) analysis during exponential growth (Fig. 1A). When similar numbers of erythrocytes infected with HRFΔ1 or WT parasites were injected in mice, a similar proportion of mice (> 90%) succumbed to CM in the two groups, with similar kinetics (Fig. 1B). Similar results were obtained with HRFΔ2 and HRFΔ3 parasites (not shown). We conclude that HRF is not essential for P. berghei blood stages.

To test pre-erythrocytic stage infectivity, sporozoites were inoculated into mice by mosquito bite (Fig. 1C andD), intradermal (ID, Fig. 1E) or intravenous (IV, Fig. 1F) injection and emergence of blood-stage parasites monitored by FACS. Only 40% to 50% of animals became infected after injection of HRFΔ1 sporozoites when 100% animals were infected after injection of WT parasites (Fig. 6B). Animals became patent with HRFΔ1 bloodstage parasites on average 2 to 3 days after the WT following infection by mosquito bites (Fig. 1C, insert) or inoculation of isolated sporozoites (Fig. 1E andF), corresponding to a 100-to 1000-fold decrease in infectivity of mutant parasites (day of patency 4 and 6 for WT and HRFΔ1-infected mice, respectively, Fig. 1C; day of patency 4 and 6 for WT and HRFΔ1-infected mice, respectively, Fig. 1E; day of patency 3 and 5 for WT and HRFΔ1-infected mice, respectively, Fig. 1F). Similar results were obtained with clones HRFΔ2 and HRFΔ3 (Supporting Information Fig. S2A andB). As expected, the delayed development of the mutant in the liver greatly decreased its ability to induce CM after sporozoite inoculation, as already reported for other mutants with a developmental block in the liver (Butler et al., 2011) and in humans after RTS,S (repeat and T-cell epitope in the circumsporozoite protein (CSP) of Plasmodium falciparum malaria parasite and a viral envelope protein of the hepatitis B virus, HBsAg) immunization [START_REF] Heppner | Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research[END_REF]. Indeed while 100% C57BL/6 mice developed CM on day 7 or 8 after WT sporozoite inoculation by mosquito bite, only 10% of the mice inoculated with HRFΔ1 sporozoites showed signs of neuropathology (Fig. 1D). Similar CM resistant pheno-type was observed in C57BL/6 mice infected by intravenous injection of isolated sporozoites (Supporting Information Fig. S2). We conclude that in P. berghei HRF is crucial for the pre-erythrocytic, but not erythrocytic phase of infection.

HRF is produced by sporozoites and liver stages

To detect HRF in P. berghei pre-erythrocytic stages, specific antibodies were generated by rabbit immunization with recombinant P. berghei HRF protein (PbHRF). Expression of HRF by WT sporozoites was assessed by immunofluorescence assay (IFA), which showed a diffuse staining pattern suggesting a cytoplasmic localization (Fig. 2A). In contrast, no detectable HRF could be seen in HRFΔ1 parasites (Fig. 2A). Expression of HRF in parasitized hepatocytes was assessed by incubating cultured HepG2 hepatoma cells with WT or HRFΔ1 sporozoites. After 48 h, HRF was detected specifically in fixed/ permeabilized cells infected with WT, not HRFΔ1, parasites (Fig. 2B). Staining of WT and mutant sporozoites, as Fig. 1. Marked differences in parasitaemia between WT and HRFΔ1 Pb ANKA-infected mice are observed only at the pre-erythrocytic stage. C57BL/6 mice (12 mice per group) were infected with 10 6 WT or HRFΔ1 Pb ANKA-infected RBCs (A, B) or via bites of 10 mosquitoes (C, D), or inoculated with 10 000 isolated sporozoites ID (E), or IV (F). In C, D, E, and F panels, as 50% of mice did not show parasitaemia, only six mice per group were shown. Kaplan-Meier survival plots (log-rank test) and parasitaemia (Mann-Whitney test; *P < 0.028; **P < 0.007; ***P < 0.002) were recorded over time. Results are from three independent experiments. well as HepG2 cultures harbouring WT or mutant parasites, with only secondary antibodies were negative.

HRF is important for liver-stage development in vivo

In vitro, HRFΔ1 sporozoites did not display any detectable defect in gliding motility (Supporting Information Fig. S3A-C), host cell traversal (Supporting Information Fig. S3F) or host cell invasion of HepG2 cells (Supporting Information Fig. S3D andE). HRFΔ1 and WT developed indistinguishably inside HepG2 cells into exo-erythrocytic forms (EEF, liver stages; Supporting Information Fig. S3G). Therefore, HRF does not appear to be important for a basic parasite developmental step detectable in vitro. After transmission to mosquitoes, WT and HRFΔ1-3 parasites generated similar numbers of sporozoites in the insect salivary glands. Indeed, at day 20 following parasite passage into female mosquitoes, similar prevalence of mosquito infection and similar numbers of HRFΔ1 and WT sporozoites per infected mosquito salivary gland were counted (Supporting Information Fig. S3H), indicating that HRF was dispensable for parasite development inside mosquitoes.

We then tested whether HRF might play a role during sporozoite progression in vivo using a putative vasodilatory effect of histamine. A potential role of HRF in facilitating sporozoite passage across the liver sinusoidal endothelium to reach hepatocytes was addressed by intravital imaging. Mice were injected IV with a 1:1 mixture of GFP + HRFΔ1 and RFP + control P. berghei sporozoites and sporozoites present in the parenchyma were counted after 3 h, when sporozoite motility has ceased. As shown in Fig. 3A andB, sporozoites of each type crossed the sinusoidal barrier with similar efficacy. We next tested whether HRF might help sporozoites penetrate blood capillaries in the skin and thus exit the skin. For this, sporozoites were injected IV or ID and parasite loads in the liver were compared by quantitative real-time polymerase chain reaction (qRT-PCR) at 40 hpi, when individual liver stages have initiated nuclear division (Fig. 3C). The HRFΔ1 parasite load was 3.7-and 3.3-fold smaller than that of the WT after IV and ID injections respectively (Fig. 3C). This indicated that the mutant is not more impaired compared with WT after ID than IV injection, i.e. that the mutant is not impaired in exiting the skin. Clones HRFΔ2 and HRFΔ3 gave similar results (Fig. 3D).

To analyse liver-stage development in vivo, mice were injected with sporozoites IV and liver samples collected at 40, 96 or 144 hpi were subjected to qRT-PCR analysis of parasite LISP1 RNA (Fig. 3E). At 40 hpi, the HRFΔ1 parasite load was ∼ 2.5 times lower than that of the WT. At 96 hpi, WT parasites were undetectable, as expected from the release of merozoites from liver stages starting ∼ 55 hpi, while the amounts of HRFΔ1 parasites had risen to levels similar to those of the WT at 40 hpi. At 144 hpi, both WT and HRFΔ1 parasites were undetectable in the liver. Therefore, RT-PCR analysis in the liver indicated a > 48 h delay in the completion of the pre-erythrocytic phase. In parallel, as expected, blood smears and FACS analysis detected WT parasites 2 days earlier than HRFΔ1 parasites (Fig. 3F).

Lack of HRF increases IL-6 production in the infected liver

Since HRF is important for liver-stage development specifically in vivo, we assessed the levels of various molecules of the host immune system during parasite liver infection (Fig. 4). Liver samples from naïve C57BL/6 mice and mice infected with WT or HRFΔ1 parasites were harvested at various hpi and host gene expression measured by RT-qPCR. Strikingly, at 40 hpi, the transcripts for IL-6 were the sole among those tested that were differentially expressed between infections with WT and HRFΔ1 parasites, being increased fivefold in HRFΔ1-infected liver samples. At 96 and 144 hpi, the levels of transcripts for MIP-1β, MIP-2, ICAM-1, TNF-α and IFN-γ were all higher in response to WT compared with HRFΔ1 parasites (Fig. 4), while the levels of IL-6 transcripts had reverted and reached higher levels in WT-infected livers. Transcripts for IL-4, IL-5 and IL-13 were not detectable both in WT and HRFΔ1 parasites. To verify specificity of the IL-6 increase in HRFΔ1-infected livers, we tested P. berghei ANKA parasites lacking the p36 gene, previously shown to be rapidly blocked in development after hepatocyte invasion (van Dijk et al., 2005;[START_REF] Ishino | Two proteins with 6-cys motifs are required for malarial parasites to commit to infection of the hepatocyte[END_REF][START_REF] Labaied | Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection[END_REF]. After IV injection of 10 000 p36Δ sporozoites into C57BL/6 mice, RT-PCR at 40 h post-infection confirmed the arrested development of the mutant in the liver (Fig. 4H). In addition to the abortive intrahepatocytic development, p36Δparasites did not up-regulate IL-6 (Fig. 4I), confirming that the latter was not a mere consequence of parasite developmental arrest in the liver but indeed the consequence of the absence of HRF.

To characterize the IL-6-producing cells regulated by HRF, C57BL/6 mice were inoculated IV with WT or HRFΔ1 sporozoites and, 40 h later, cells were harvested from the liver and characterized for IL-6 production. For this, liver cells gated on CD45 + IL-6 + cells were subsequently labelled for various leukocyte markers. As shown in Supporting Information Fig. S4A, a higher proportion of liver cells producing IL-6 upon infection with HRFΔ1 parasites compared with WT parasites were confined to Ly6G + , 7/4 + , CD11b + and CD11c + cells, presumably neutrophils and inflammatory monocytes. At early time point of infection (40 h post-infection), gated Ly6G + , 7/4 + double-positive neutrophils (R2) were twice more frequent in mice infected with HRFΔ1 compared with WT parasites (Supporting Information Fig. S4B). Further characterization within this gate of neutrophils expressing IL-6 showed higher mean fluorescence intensity for HRFΔ1 parasites (1200) than those induced by WT parasites (800) (Supporting Information Fig. S4B). Other cells potentially expressing IL-6 including CD4, CD8, B-cells and macrophages were apparently not regulated by HRF (Supporting Information Fig. S4A). These data support the view that HRF modulation of IL-6 at early time points during liver infection may involve neutrophils.

HRF impact on host cell recruitment in the infected liver

We then analysed host cell recruitment in livers infected with HRFΔ1 or WT parasites (Fig. 5). Innate immunityassociated leukocytes such as neutrophils, eosinophils, monocytes, macrophages, NK cells and adaptive immunity-related leukocytes such as CD4 + , CD8 + and B-cells were investigated. Leukocyte populations in the livers of C57BL/6 mice were collected from 40 hpi to 4 days post-injection (dpi) after IV injection of HRFΔ1orWT sporozoites. At 40 hpi, 3.5-to 4-fold more CD19 + /B220 + cells, likely B-cells, were found in mice infected with HRFΔ1 parasites and, conversely, twofold fewer NK1.1 + cells than in mice infected with WT parasites. At 4 dpi, WT infection was associated with higher numbers of CD8 + , CD11b + , Ly6-G + (neutrophils) and CD11c + CD11b + dendritic cells. Other leukocyte populations including CD4 + , eosinophils, basophils and macrophages were similarly affected during WT and HRFΔ1 infections.

Since the 40 h time point appears to be the critical point for events distinctly elicited by the HRFΔ1 parasite, namely increased B-cell number, we examined whether activation markers on leukocytes were modulated as well. To test whether distinct activation phenotypes could serve a cellular marker for HRFΔ1 infection in the liver, leukocytes were analysed for their level of CD62L expression, a key adhesion molecule that is expressed on the surface of most circulating leukocytes, including lymphocytes, The asterisk indicates that differences are significant (Mann-Whitney test, 0.01 < P < 0.05), double asterisk indicates that these values are significantly different (Mann-Whitney test, 0.005 < P < 0.01) and triple asterisk indicates that differences are significant (Mann-Whitney test, 0.001 < P < 0.007). (H) C57BL/6 mice (6/group) were inoculated with 10 000 WT, HRFΔ1o rp 3 6 Δ sporozoites. Forty hours later, livers were isolated and total RNA extracted. Parasite load was determined by using primers specific for the P. berghei 18S rRNA by real-time RT-PCR, which expression was normalized relative to HPRT expression. (I) IL-6 expression was determined in the same liver samples using real-time RT-PCR by measuring IL-6 mRNA. Triple asterisk indicates that differences are significant (Mann-Whitney test, 0.003 < P < 0.01). neutrophils, monocytes, eosinophils and basophils and is down-regulated on activated leukocytes. As shown in Supporting Information Fig. S5, we determined the proportion of various subsets of leukocytes including B-cells (B220 + , CD19 + ), CD4 + , CD8 + , NK cells and Ly6G + (neutrophils) that express low levels of CD62L among CD45 + cells taken 40 h after infection from the liver of naïve mice or mice inoculated with WT or with HRFΔ1 parasites. Regardless of the parasite used, only B-cells (B220 + CD62L low and CD19 + CD62L low ) were found to be slightly activated compared with naïve mice. CD4, CD8, NK cells and neutrophils were not affected by any parasite. MHC class II expression used as an activation marker for dendritic cells (CD11c + MHC class II high ) was also found to be indistinctly up-regulated by both parasites. We infer from this analysis that the slightly increased proportion of activated B-cells and dendritic cells in the liver at early time points is independent from HRF, and that the only HRF-specific effect is the increase in the B-cell number.

To assess whether B-cells contributed to impaired infection by HRFΔ1 parasites, B-cell-deficient mice were inoculated IV with 10 000 WT or HRFΔ1 sporozoites, and the parasite load and IL-6 gene expression were exam-ined at 40 hpi. Data showed, similarly to WT C57BL/6 mice, a ∼ 3-fold reduced parasite load for the mutant parasite compared with the WT in B-cell-deficient mice (Supporting Information Fig. S6A). The IL-6 gene was also up-regulated in B-cell-deficient mice infected with HRFΔ1 sporozoites (Supporting Information Fig. S6B). Mortality rates caused by WT and HRFΔ1 sporozoites were similar in B-cell-deficient and WT mice (Supporting Information Fig. S6C), and the absence of CM induced by HRFΔ1 sporozoites was also observed in B-cell-deficient mice. These data suggested that B-cells were not involved in the HRFΔ1 parasite developmental block in the liver.

The mutant phenotype is rescued by decreasing IL-6 levels

To test whether the impaired HRFΔ1 liver-stage growth in vivo might be caused by increased IL-6, we attempted to rescue the phenotype of the mutant by decreasing IL-6 amounts. Mice received anti-IL-6 antibodies IV, were injected IV 24 h later with HRFΔ1 sporozoites, and parasite loads in the liver were measured at 40 hpi by qRT-PCR. Anti-IL-6 antibodies increased the HRFΔ1 load in the liver ∼ 2.5-fold (Fig. 6A), thus largely rescuing the defective phenotype. Moreover, while only 40% of the mice infected with 10 4 HRFΔ1 sporozoites became bloodstage patent (Fig. 6B, triangles), all mice infected similarly and pretreated with anti-IL-6 antibodies developed bloodstage parasites (Fig. 6B, closed circles). Conversely, when WT sporozoites were injected IV into mice immediately before human recombinant IL-6 (5 μg per mouse), WT parasite loads in the liver decreased ∼ 4-fold at 40 hpi (Fig. 6C). As controls and to see whether IL-6 treatment affects liver parasite load in HRFΔ1-infected mice, mice infected with HRFΔ1 plus/minus IL-6 displayed the same liver parasite load (Supporting Information Fig. S7A). To assess whether treatment with exogenous IL-6 would affect endogenous liver IL-6 mRNA expression, HRFΔ1infected mice displayed similar IL-6 mRNA expression in IL-6-treated and untreated mice (Supporting Information Fig. S7B).

Next, experiments were conducted in IL-6-knockout (IL-6 ko ) C57BL/6 mice. First, sporozoites were injected IV into mice and parasite loads in the liver assessed by RT-PCR. While the liver load of HRFΔ1 parasites was lower than that of the WT in WT C57BL/6 mice, the load of HRFΔ1 parasites in IL-6 ko mice was higher than in WT mice and similar to that of WT parasites in WT mice (Fig. 6D). Second, pre-patent periods of liver-stage development of HRFΔ1 parasites were assessed in IL-6 ko and WT mice. Figure 6E shows that HRFΔ1 parasites developed at a higher rate in IL-6-deficient mice than in WT recipients, reverting to a fully complemented phenotype. The control experiment which consists of inoculation of 10 000 SPZ of WT or HRFΔ1 PbANKA parasites in IL-6 KO mice shows that the phenotype of the mutant parasite in IL-6 KO mice is not completely rescued; however, the delay in the patent period is reduced as compared with the WT parasite (1 day instead of 2 to 3 days in WT mice, day of patency 3, and 4 for WT and HRFΔ1-infected IL-6 KO mice, respectively; Supporting Information Fig. S7C) but the mortality caused by HRFΔ1 parasites is re-established as 5 mice died out of 7 as compared with no death in WT mice (shown in Fig. 6F). Also the death rate is not significantly different than those IL-6 KO mice infected with PbANKA WT parasites (6 mice died out of 7; Supporting Information Fig. S7C). Fig. 6. HRFΔ1-mediated regulation of IL-6 expression in the liver is critical for parasite development. C57BL/6 mice were infected with 10 000 WT GFP-HRFΔ1 sporozoites in the presence of rat anti-mouse IL-6 mAbs or normal rat IgG1 as control (A) or with GFP-WT sporozoites treated or not with 5 μg of rHuIL-6 (C), and 40 h later parasite loads in the liver were assessed by measuring parasite 18S rRNA by real-time RT-PCR, which expression was normalized relative to HPRT expression. Data are presented as the means ± SD from one representative experiment out of two (n = 8). Double asterisk indicates that differences are significant (Mann-Whitney test: 0.01 < P < 0.03). (B) Mice were treated as in A, but were followed for parasitaemia and the proportion of mice developing blood-stage parasites were determined (Mann-Whitney test; ***P = 0.006). (D) C57BL/6 or IL-6 KO mice were inoculated IV with 10 000 GFP HRFΔ1 sporozoites. As control, C57BL/6 mice were inoculated with 10 000 WT sporozoites. Forty hours later, livers were isolated and total RNA extracted. Parasite load was determined by using primers specific for the P. berghei 18S rRNA. mRNA expression measured by real-time PCR was normalized to the endogenous control gene HPRT. Data are presented as the means ± SD from two independent experiments (n = 5, *Mann-Whitney test, P = 0.028). (E) C57BL/6 or IL-6 KO mice were infected IV with 10 000 GFP HRFΔ1 sporozoites and parasitaemia (Mann-Whitney test; *P = 0.032, **P = 0.009) and (F) survival rates (log-rank test) were followed over time.

Last, we compared the ability of sporozoites to induce CM in WT and IL-6 ko mice. HRFΔ1 sporozoites injected IV did not induce CM in WT mice, whereas 70% of IL-6 ko mice developed CM (Fig. 6F), similar to the CM frequency caused by WT parasites in WT mice infected via infectious mosquito bites (Fig. 1D). Similar results were obtained with HRFΔ2 and HRFΔ3 parasites. Together, these data indicate that the phenotype caused by lack of HRF in the parasite is mainly, if not only, due to IL-6 overexpression in the host.

HRF is sufficient to decrease IL-6 production

We then tested whether HRF directly affected the production of IL-6 in the liver and systemically in the mouse. For this, recombinant P. berghei HRF (PbHRF) was expressed in pRSETA plasmid, with an N-terminal his tag and purified/collected from Escherichia coli BL21 strain. Mice were injected IV with various doses of recombinant PbHRF prior to the administration of 0.1 mg of Lipopolysacharaide (LPS), a known IL-6 inducer, and 2 h later IL-6 mRNA expression and plasma levels of IL-6 were determined by RT-qPCR and ELISA respectively. IL-6 expression was down-regulated both at the mRNA (Fig. 7A) and protein levels (Fig. 7B) by HRF, in a dosedependent manner. As a control, we tested the ability of another parasite protein, merozoite surface protein 1 fragment 33 (PbMSP1 33), to modulate IL-6 production in the same conditions. MSP133 had no inhibitory effect on the levels of LPS-induced IL-6 mRNA (Fig. 7C) or IL-6 protein (Fig. 7D). Therefore, the differential regulation of IL-6 in the liver in response to infections with WT and HRFΔ parasites can be directly ascribed to HRF itself. The mRNA transcripts for IL-10, IFN-γ, TNF-α and IL-1 (Fig. 7E-H) were also inhibited by the co-injection of HRF, while those for IL-4, IL-5 and IL-13 remained undetectable in mice treated with LPS alone or LPS combined with HRF.

HRF is detected in the extracellular environment in the liver and in the blood

The phenotype of the mutant predicted that HRF would be secreted by the liver stage of the parasite. To test this, the supernatant of 24 h cultures of HepG2 cells harbouring WT parasites was collected and analysed by immunoblot using anti-HRF antibody (Fig. 8A). The presence of HRF (22 000 Da molecular weight) was detected, as well as other bands at around 15 000 and 7000 molecular weights, likely representing fragments from the native protein. No signal was detected in supernatants from HepG2 cells containing HRFΔ1 parasites or uninfected HepG2 cells (Fig. 8A). To confirm that HRF was produced by liver-stage parasites in vivo, liver extracts obtained 40 h post-infection with WT sporozoites were tested for the presence of HRF. As shown in Fig. 8B, HRF was detected in liver extracts. To assess whether HRF is present in the plasma of infected mice during the blood phase of infection, we measured HRF in the plasma of mice with various levels of parasitaemia. In order to correlate the level of parasitaemia with the amount of circulating HRF, selected sera from two mice with 10% parasitaemia (day 7 post-infection), two mice with 7% parasitaemia (day 5 post-infection) and two mice with 3% parasitaemia (day 3 post-infection) were examined for their content of HRF using a quantitative ELISA assay. As shown in Fig. 8C, HRF was present in the plasma and appeared to correlate with the parasite load in the blood. HRF was not detected in the plasma of mice infected with HRFΔ1 parasites.

Discussion

This work shows that, in rodents, plasmodial HRF is involved in the pre-erythrocytic phase of malarial infection. Given the high (97%) similarity between P. falciparum and P. berghei HRF, it is likely that the same applies in humans. Histamine-releasing factor was not involved in the erythrocytic phase of infection in rodents, including in the development of CM. This, however, does not exclude a role for HRF in severe P. falciparum malaria in humans. It was striking to observe that deletion of hrf/tctp gene was not lethal for Plasmodium parasites in contrast to the loss of hrf expression in mice which results in increased spontaneous apoptosis during embryogenesis [START_REF] Susini | TCTP protects from apoptotic cell death by antagonizing bax function[END_REF]. Therefore, in mice, HRF is regarded as both a key intracellular anti-apoptotic regula-tor beside other functions and as a secreted protein exerting extracellular biological functions. A possible explanation for the lack of lethality of HRFΔ mutants we obtained in our laboratory is that Plasmodium parasites have preferentially selected extracellular functions rather than essential intracellular functions of HRF for a better fitness within infected hosts.

The HRF mutant was specifically inhibited during liverstage development in vivo. In vitro, mutant development was normal in cultured hepatocytes and, in vivo, the mutant was not significantly impaired in leaving the skin and reaching the blood, or in leaving the blood and reaching hepatocytes. In the liver, mutant development was slowed though not blocked, as shown by qRT-PCR analysis of parasite amounts over time. However, the impaired development of HRF mutant liver stages in vivo is likely to induce some parasite death, as suggested by intravital imaging studies. This resulted in a 10 2 -t o1 0 3 -fold reduction in the numbers of merozoites initially released in the blood, and an extension of the time between sporozoite injection and a threshold of parasitaemia. This prolonged liver-stage development influenced the clinical outcome of a parasite infection by decreasing the frequency of CM. A link between expression of ECM in susceptible C57BL/6 mice and a lengthy development period of parasite in the liver was already suggested by other P. berghei parasites deficient in apicoplast protein important for liver merozoite formation (PALM) (palm -), which displayed a strong impairment in liver merozoite formation with a severe delay in blood-stage infection and a reduced incidence of ECM [START_REF] Haussig | Inactivation of a Plasmodium apicoplast protein attenuates formation of liver merozoites[END_REF]. Our data strengthen the wider concept that the efficiency of pre-erythrocytic infection controls CM occurrence during erythrocytic infection.

Like other pathogens, Plasmodium must fine-tune activation/inhibition of host immune effector cells to ensure host survival and parasite dissemination. However, few examples exist in the literature describing particular pathogen genes that manipulate specific host immune response pathways, in particular Plasmodiumassociated genes regulating specific host cytokine responses. Three lines of evidence indicate that the mutant defect is mainly caused by increased IL-6 in the infected host. (i) IL-6 increase in the mutant-infected liver was the earliest detectable difference with WT among the tested factors, while the earliest cellular difference was an increase in B-cell counts in the mutant, in line with the B-cell-activating activity of IL-6. (ii) The mutant defect was complemented by decreasing IL-6 levels in the host, using injection of anti-IL6 antibodies in WT mice or infection in IL-6 ko mice. (iii) Recombinant HRF was sufficient to inhibit LPS-induced IL-6 production in the liver and release in the circulation. Therefore, although HRF might still have other targets than IL-6, the defective phenotype of the mutant appears to be largely caused by unrepressed IL-6.

How the parasite controls IL-6 expression via HRF remains unclear. During the initial phase of the liver development, the main target of IL-6-producing cells appears to be neutrophils, in which IL-6 production was enhanced by the HRFΔ1 parasite. Consistent with this finding, IL-6 was previously found in murine neutrophils as a pre-stored protein, which makes it readily available upon stimulation [START_REF] Terebuh | Biologic and immunohistochemical analysis of interleukin-6 expression in vivo. Constitutive and induced expression in murine polymorphonuclear and mononuclear phagocytes[END_REF]. Evidence that HRF is able to directly prevent or inhibit IL-6 production was provided by the direct inhibitory effet of HRF on LPS-induced cytokine production. At the same time, B-cells increase in number, perhaps as a result of increased Il-6 production. The interplay between neutrophils and B-cells via IL-6 may represent the mechanism by which the delayed development of the mutant parasite occurs. Such an interplay between neutrophils and B-cells has been recently elegantly demonstrated by the discovery of a B-helper neutrophil population whereby splenic neutrophils can function as professional helper cells for marginal zone B-cells, leading to the generation of affinity-matured antibodies (Puga et al., 2012). However, using B-cell-deficient C57BL/6 muMT mice, homozygous for the Ighm tm1Cgn targeted mutation, the phenotype of the mutant was similar in B-cell-deficient and WT mice. This suggests that B-cells are not involved in protection during infection with HRFΔ1 parasites.

Both the sporozoite and the intracellular liver stage produce HRF (Fig. 2). The extracellular sporozoite might secrete HRF during gliding motility and/or the liver stage might export HRF through the PVM and out of the infected cell. Here, we showed that HRF from Plasmodium berghei was secreted by infected HepG2 cells in the culture supernatant and in vivo in the liver of infected mice (Fig. 8). Histamine-releasing factor secretion by liver stages might occur via exosomes. In the human embryonic kidney 293T cells, HRF was found secreted via a non-classical route and exosomes (Amzallag et al., 2004). Immunofluorescence and immunoelectron microscopy studies on P. falciparum show that some of the malarial HRF is present in the food vacuole membranes, where it is in proximity to the heme-rich food vacuole (Bhisutthibhan et al., 1999).

Whether mammalian and malarial HRF, despite their high (54%) similarity, have similar targets and modes of action remains uncertain. Mammalian HRF was reported to act on basophils, eosinophils and B-cells [START_REF] Macdonald | Potential role of histamine releasing factor (HRF) as a therapeutic target for treating asthma and allergy[END_REF]. Interestingly, HRF was reported to increase IL-6 production from B-cells. Examination of immune cells at early time point, 40 h post-infection, indicated a unique association between the absence of HRF expression and an increased accumulation of B-cells. One of the characteristic features in malaria infection is the induction of short-lived B-cell memory. It has been shown that malaria parasites interfere with the immune response by disabling B-cell development, possibly undergoing apoptosis (Wykes et al., 2005). In light of these findings, increased B-cell population in the liver of mice infected with mutant parasites reflects the possibility that the HRF molecule could interfere with B-cell memory. In support of this observation, early rise in IL-6 induction, a B-cell-activating and anti-apoptotic cytokine, at 40 h post-infection was induced by mutant parasites. Plasmodium falciparum HRF stimulates histamine release from basophils and IL-8 secretion from eosinophils in vitro (Bheekha-Escura et al., 2000;MacDonald et al., 2001). We showed here that P. berghei HRF is sufficient to decrease IL-6 production in the liver. Numerous cell types produce IL-6 in the liver, including T-cells, macrophages, endothelial cells and hepatocytes themselves, where it eventually exerts its anti-parasite effect. Clearly, HRF does not affect the levels of IL-6 only, as co-injection of HRF with LPS downregulates other pro-inflammatory cytokines like IFN-γ, TNF-α and IL-1. More work is needed to understand the mode of action of malarial HRF.

Few molecules of malaria parasites have been shown to counteract host innate immunity. In early liver stages, the major sporozoite surface protein called circumsporozoite protein translocates into the hepatocyte cytosol and nucleus, where it outcompetes NF-kB nuclear import and suppresses hundreds of genes involved in the host inflammatory response. Another parasite product, the migration inhibitory factor (MIF) orthologue of mammalian MIF, was shown to increase inflammatory cytokine production during the blood phase of infection in rodents and to induce antigen-experienced CD4 T-cells to develop into shortlived effector cells rather than into memory cells, causing decreased CD4 T-cell recall responses to homologous parasites [START_REF] Sun | A Plasmodium-encoded cytokine suppresses T-cell immunity during malaria[END_REF]. However, parasites lacking MIF were shown to have no growth defect throughout the parasite life cycle in P. berghei [START_REF] Augustijn | Functional characterization of the Plasmodium falciparum and P. berghei homologues of macrophage migration inhibitory factor[END_REF] or a growth defect during liver-stage development in P. yoelii [START_REF] Miller | Plasmodium yoelii macrophage migration inhibitory factor is necessary for efficient liver-stage development[END_REF] although in this latter case, the causes of MIF-deficient liver-stage attenuation were not investigated.

In conclusion, Plasmodium HRF is the first secreted parasite molecule that is found to down-regulate host cytokines, in particular IL-6, which inhibits the development of the Plasmodium liver stage. The importance of the contribution of HRF to Plasmodium liver-stage development makes it an attractive target for intervention strategies, given its secreted nature and diffuse action a target of antibody-mediated immunity.

Experimental procedures

Ethics statement

All animal care and experiments described in the present study involving mice were conducted at the Institut Pasteur, approved by the 'Direction Départementale des Services Vétérinaires' de Paris, France (Permit Number N°75-066 issued on September 14, 2009) and performed in compliance with institutional guidelines and European regulations (http://ec .europa.eu/environment/chemical?s/lab_animals/home_en.htm). A statement of compliance with the French Government's ethical and animal experiment regulations was issued by the Ministère de l'Enseignement Supérieur et de la Recherche under the number 00218.01.

Mice

Seven-to eight-week-old female C57BL/6J Rj mice were purchased from Charles River Laboratories. The IL-6 KO mice have been backcrossed 10 times on C57BL/6 mice from the Jackson Laboratory. To make sure the background of IL-6 KO mice does not alter at least the course of infection and disease in our ECM model, comparable survival rates and parasitaemia are shown between IL-6 KO mice and C57BL/6J Rj used as WT control in this study. C57BL/6 muMT mice, homozygous for the Ighm tm1Cgn targeted mutation and lacking mature B-cells were obtained from The Jackson Laboratory, Bar Harbor, ME, USA.

Parasites

Mice were inoculated with red bloods cells (RBCs) infected with either GFP-transgenic P. berghei ANKA (MRA-867) or HRFΔ1 PbANKA parasites. In a few control experiments, HRFΔ2 and HRFΔ3 PbANKA parasites were used. The P. berghei deficient for the Pbs36 gene (van Dijk et al., 2005;[START_REF] Ishino | Two proteins with 6-cys motifs are required for malarial parasites to commit to infection of the hepatocyte[END_REF][START_REF] Labaied | Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection[END_REF], the disruption of which was previously found to nearly abolish sporozoite infectivity in the mammalian liver host was kindly provided by Dr. T. Ishino (Department of Medical Zoology, Mie University School of Medicine, Edobashi, Tsu, Japan).

Murine model of ECM

Six-to eight-week-old C57BL/6 mice were inoculated with 10 5 RBCs infected with PbANKA or their knockout counterparts. Parasites, before every experiment, were reactivated by previous passage in C57BL/6 mice. Parasitaemia was determined by flow cytometry and the results expressed in percentage of pRBCs.

C57BL/6 mice infected with PbANKA or HRFΔ PbANKA were monitored for clinical symptoms of ECM: hemi-or paraplegia, deviation of the head, tendency to roll over on stimulation, ataxia and convulsions.

Quantification of parasite loads in the liver

C57BL/6J Rj mice were injected IV or ID with 10 000 WT or HRFΔ1 sporozoites. Forty hours later, livers were isolated and total RNA extracted. Parasite load was determined by using primers specific for the P. berghei 18S rRNA (forward: 5′-AAGCATTAAATAAAGCGAATACATCCTTAC-3′ and reverse: 5′-GGAGATTGGTTTTGACGTTTATGTG-3′).

Gene mRNA expression measured by real-time RT-PCR is normalized to the endogenous control gene HPRT.

For kinetic studies spanning 6 days after sporozoite inoculation, groups of C57BL/6J Rj mice were injected IV with 10 000 GFP-WT or GFP-HRFΔ1 sporozoites and livers were collected at indicated time points. To avoid possible contamination of liver stages by blood-stage parasites, we decided to use LISP1 as a specific marker for late stage liver merozoites. Parasite loads in the liver were assessed by measuring parasite LISP1 mRNA by real-time RT-PCR. Gene mRNA expression was normalized to the parasite control gene HSP70.

Detection and quantification of HRF

A plasmid containing the DNA encoding for the PbNK65 HRF was obtained from Kim Taylor (RMIT University, Australia). PbHRF plasmid was in pRSET A (Life Technologies Paisley, United Kingdom), with an N-terminal his tag and E. coli BL21 was used for expression. Bacteria grew in a 10 ml overnight preculture in LB medium with 100 μgml -1 ampicillin, and we then inoculated 500 μl of LB with 1-2% of the preculture. The culture was kept at 37°C for ∼ 3 h until the OD at 600 nm was ∼ 0.5. The expression was then induced for 3 h with 1 mM IPTG. We used freeze/thaw cycles and lysozyme to lyse the bacteria, followed by filtration through a 0.2 μm syringe filter. Purification was performed by using the Profinity resin from Bio-Rad (Marnes-la-Coquette, France). Endotoxin was removed by passing HRF solution on Detoxi-Gel Endotoxin Removing Gel column (Thermo Fisher Scientific, Brebières, France). The recombinant protein (100 μg) was injected intradermally into the back skin of a rabbit in the presence of complete Freund's adjuvant followed by two booster injections in the presence of incomplete Freund's adjuvant. Rabbit IgG antibodies were isolated using a protein A column as an immunoabsorbent. For intracellular detection of HRF, isolated sporozoites were washed in phosphate buffered saline (PBS) buffer and resuspended in fixation/permeabilization solution (BD Pharmingen) in the presence of anti-HRF rabbit antibodies for 20 min at 4°C. Fixed/ permeabilized sporozoites were resuspended in Perm/Wash buffer and incubated with Allophycocyanin (APC)-anti-rabbit antibodies before flow cytometric analysis. A total of 4 × 10 5 living cells were analysed using a four-colour FACSCalibur flow cytometer with ProCellQuest software (BD Biosciences, Mountain View, CA, USA). In vivo production of HRF by hepatic merozoites was demonstrated by IV inoculation of 100 000 sporozoite to C57BL/6 mice, and 40 h post-infection liver extracts were made. Quantitative determination of HRF in the liver and in the plasma was performed by using quantitative ELISA sandwich using anti-HRF rabbit antibody as capture and a range of concentrations of recombinant HRF for the titration curve and the same rabbit antibody labelled with horse radish peroxidase for detection. Liver extract from naïve mice was used as negative control.

Western blot

Supernatant samples (14 μl) of HepG2 cells cultured in the presence of sporozoites were loaded onto 4-12% pre-cast gel gradients (Bolt Bis-Tris Plus; Life Technologies, Carlsbad, CA, USA). Gels were placed in the Novex Bolt Mini Gel system (Life Technologies). At the completion of SDS-PAGE, the gel was washed in distilled water and assembled with the iBlot Gel Transfer Stacks (Life Technologies). At the completion of the Western transfer, the membrane was removed from the sandwich and was incubated in blocking solution for 1 h, shaking (5% milk solution in PBS tween 0.01%), then incubated with the rabbit anti-HRF antibody in diluent solution overnight shaking at 4°C.

The membrane was washed three times with PBS tween 0.01% shaking for 10 min, and then incubated in biotinylatedgoat anti-rabbit antibody (Life Technologies) for 1 h, shaking at room temperature. The membrane was washed three times in PBS tween for 10 min shaking, and then was incubated with HRPO-streptavidine for 1 h at room temperature. After several washings, the membrane was incubated in a solution of Supersignal West Pico Chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA) in the dark. The blot was recorded either by scanning the image or by photography.

Sporozoite development in HepG2 cells and intracellular detection of HRF

HepG2 cells (3-4 × 10 4 per well) were plated in eight-well chamber slides (Lab-Tek ® Chamber Slide ™ ) and cultured overnight in DMEM + GlutaMAX-I media (Gibco) supplemented with 10% heat inactivated FBS (Fetal Bovine Serum, Gibco) at 37°C in the presence of 5% CO2. Wild type and/or mutant purified P. berghei salivary gland sporozoites were used for HepG2 infection at a ratio of 1:1 (parasite/cells) for 48 h, at 37°C, 5% CO2 in the presence of PSN (penicillin-streptomycin-neomycin solution, Sigma). The PbHRF was detected by immunofluorescence staining as described above.

HepG2 cells fixation and permeabilization was done with 4% paraformaldehyde + 0.1% triton or acetone 1:1 mix, and blocked with 1% gelatin from porcine skin (Sigma) for 30 min at RT. Thereafter, cells were incubated with anti-HRF rabbit antibodies (diluted 1:500) at 4°C overnight, and then incubated with Alexa Fluor ® 568 donkey anti-rabbit IgG (Life Technologies; diluted 1:500) and 0.02 mg/ml 4′, 6-diamidino-2-phenylindole (DAPI) for nuclear staining for 30 min at RT. The presence of PbHRF was detected under the fluorescence microscope. Similar IFA was performed to detect HRF in sporozoites.

Flow cytometric analysis of liver leukocytes

Liver cells obtained after various periods of time after infection were stained for FACS analysis according to standard protocols in cold PBS containing 2% Fetal calf serum (FCS) and 0.01% sodium azide (FACS buffer) with the following Abs: APClabelled CD4, phycoerythrin (PE)-labelled anti-CD8, Fluorescein isothiocyanate (FITC)-labelled anti-F4/80 antibody, and PE-labelled ant-Ly6G antibody, Alexa Fluor 647-labelled anti-B220, APC-anti-NK1.1 antibodies. Red blood cells were eliminated using cell lysis buffer, and cells were washed in FACS buffer. A total of 4 × 10 5 living cells were analysed using a fourcolour FACSCalibur flow cytometer with ProCellQuest software (BD Biosciences, Mountain View, California).

Cytokine and chemokine quantification in the liver

Cytokine and chemokine expression in the liver taken at different periods of time after infection was analysed by the real-time RT-qPCR. RNA utilized for these assays was isolated by means of a two-step extraction process. First, livers were surgically removed from mice as previously described and placed immediately in RNAlater at 4°C overnight. After RNAlater infused the samples, it was removed and samples were maintained at -80°C until processing. Livers were thawed in 1 ml of Trizol and subjected to bead disruption in a polytron three times from 2 min at a setting of 30 cycles per second. Samples were spun at high speed (10 000× g) for 3 min to remove debris and lipids. Half of the sample was transferred to a new tube and mixed with 500 μl of Trizol reagent by vortexing. Following this step, RNA extraction proceeded according to manufacturer's protocol. Precipitated RNA was resuspended in 100 μl of RNase-free water. The second step of this extraction was followed by Qiagen's protocol for RNA clean-up including steps for removal of protein and DNA (Qiagen RNeasy Kit). Samples were eluted with 50 μlo f RNase-free water and quality and quantity assured by photospectroscopy. Real-time RT-qPCR utilized various primerprobe sets and standard Taqman protocols (Applied Biosystems).

Assessment of cell traversal

To determine the ability of sporozoites to traverse host cells, a standard cell traversal assay was used. GFP-labelled WT and HRFΔ1 sporozoites, isolated from Anopheles stephensi mosquito salivary glands, were incubated with human HepG2 cells, grown in 24-well plates in the presence of rhodamine dextran. After 3 h, the cells were washed with PBS/1% FCS, fixed and analysed by FACS using a BD Bioscience FACSCalibur flow cytometer. Percentage of traversed HepG2 cells was labelled with rhodamine dextran.

Intravital microscopy and development of EEF

Freshly isolated WT or HRFΔ1 sporozoites were resuspended in Ringer's solution containing 1% FBS, and allowed to glide on glass-bottom dishes (MatTek Corp., Ashland, MA, USA) precoated with 2% FCS. Video microscopy was conducted using a Zeiss Axiovert equipped with phase-contrast and epifluorescence microscopy. Time-lapse images and videos were collected under low-light illumination with a Hamamatsu ORCA ER camera (Hamamatsu Photonics KK, Hamamatsu, Japan). Videos were recorded digitally at approximately eight frames per second. Colours represent different frames from a time-lapse movie.

Development of EEF of HRFΔ1 as compared with WT Pb ANKA sporozoites was assessed by plating HepG2 cells (10 5 in 1 ml) in 8-well Labtek chamber slides for 24 or 48 h, respectively, in the presence of 2.5 × 10 4 genetically GFP tagged WT or HRFΔ1 sporozoites. Cells were examined with a fluorescence microscope. Exo-erythrocytic forms in each well were counted and expressed as EEFs ± SD of duplicate wells. Exo-erythrocytic forms were counted randomly and expressed as number of EEF per field.

In vivo treatment with IL-6 and anti-IL-6 antibodies C57BL/6 mice were injected IV with 5 μg of human recombinant IL-6 (kindly provided by Barbara Willi, Novartis Pharma AG, Basel, Switzerland) in 200 μl of sterile PBS 15 min before sporozoite inoculation. To neutralize endogenous IL-6, recipient mice were injected IV with 1 mg per mouse of anti-IL-6 (rat anti-mIL-6 IgG1 MP5-32C11 kindly provided by Dr Paola Minoprio, Institut Pasteur, Paris), or with 1 mg per mouse of isotype control (rat IgG1), 1 day prior to sporozoite inoculation. The rat anti-mouse IL-6 neutralizing mAb is a very potent and specific antagonist of mouse IL-6 bioactivity.

Determination of IL-6 in the plasma of infected mice was performed using ELISA kit assays according to the manufacturer's instructions (BD Biosciences, San Diego, CA, USA).

Statistical analysis

Significant differences in survival were evaluated by generation of Kaplan-Meier plots and log-rank analysis. P < 0.05 was considered statistically significant. For parasitaemia, after verification using a Jarque-Bera test, our data did not follow normal distribution, and thus, when differences between groups of mice were to be compared at a given time point, the Mann-Whitney test was performed with significance set at P < 0.05. [START_REF] Xu | Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level[END_REF] Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and translational level. Biochem J 342: 683-689. Yang, Y., Yang, F., Xiong, Z., Yan, Y., Wang, X., Nishino, M., et al. (2005) An N-terminal region of translationally controlled tumor protein is required for its antiapoptotic activity.

Oncogene 24: 4778-4788. Zhang, D., Li, F., Weidner, D., Mnjoyan, Z.H., and Fujise, K. (2002) Physical and functional interaction between myeloid cell leukemia 1 protein (MCL1) and Fortilin. The potential role of MCL1 as a fortilin chaperone. J Biol Chem 277: 37430-37438.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site: )

! WT! HRFΔ1! WT! HRFΔ1! H! PbANKA!WT!! PbANKA!HRFΔ1!! 3%! 6%! 85%! PbANKA!WT!! PbANKA!HRFΔ1!! 97%! 8%! 13%! LY6G5PE! An95IL565Alexa!647! LY6G5PE! 7/45FITC! mean=800! mean=1200! Figure"S4" 9%! 30%! 12%! 9%! 45%! 15%! 22%! 8%! 8%! 10%! 17%! 1.6%! 17.5%! 10%! 1%! 3.8%! CD11c5PE! LY6G5PE! CD85PE! F4/805PECy5! CD11b5FITC! 7/45FITC! CD45FITC! B2205FITC! CD11c5PE! LY6G5PE! CD85PE! F4/805PECy5! CD11b5FITC! 7/45FITC! CD45FITC! B2205FITC! An95IL565Alexa!647! A" B" % B220 + CD62L low % CD4 +

ARTICLE II

Protection against malaria in mice is induced by blood stage-arresting histamine-releasing factor (HRF)-deficient parasites.

In this work the attention was focused on the role and the possible immune mechanisms modulated by Plasmodium HRF during P. berghei NK65 infections using a mutant parasite where hrf gene was deleted (PbNK65-hrfΔ). Instead of using PbANKA which causes a rapid death of infected mice around day 7-8 p.i., we introduced the same mutation in a distinct lethal stain of parasite, PbNK65, which causes severe anemia and death around day 20-25 p.i., allowing the tracking of anti-blood stage immune responses over a relatively extended period of time.

Highlights

• Identification of hrf as a gene which deletion confers to the lethal parasite strain PbNK65

an attenuated virulence and the ability to induce a long lasting protection and immune memory against homologous and heterologous strains of parasites.

• Elevated IL-6 production in the absence of HRF expression is key in establishing parasite clearance and acquisition of sterile immunity.

• In an effort to identify the cellular source of IL-6 that was negatively regulated by HRF, examination by intracellular cell staining showed that Ly6G + and CD11c + cells are the main targets.

• Furthermore, our data showed the immunological role played by B and T cell as mice lacking these lymphocytes were unable to control the infection.

• Absence of the HRF protein increased the survival of T cells by the down-regulation of PD1 expression in both CD4 + and CD8 + T cells and the production of specific anti-parasite antibodies that recognized multiple Pb antigens.

• Parasite antigen-specific antibodies and FcγR + CD11b + cells play an important part in mutant-induced protection.

Brief Definitive Report

The Rockefeller University Press $30.00 J. Exp. Med. 2016 www.jem.org/cgi/doi/10.1084/jem.20151976

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Live attenuated parasites, in particular genetically attenuated parasites (GAPs), are increasingly being considered as vaccines against malaria. Preerythrocytic GAPs fail to develop in the liver, whereas blood stage GAPs cause abortive infections in the blood. In both cases, GAP infection induces solid protection against challenge.

The notion that attenuated blood stage parasites can confer protection originated in early studies using irradiated parasites (Waki et al., 1982;Miyagami et al., 1987). More recently, it was found that infecting individuals with low doses of Plasmodium falciparum-infected RBCs (iRBCs) followed by rapid curative treatment induced strong cell-mediated immunity and durable protection against challenge (Pombo et al., 2002). Subsequently, blood stage GAPs were generated in rodent parasites, targeting genes involved in the purine salvage pathway in Plasmodium yoelii (Ting et al., 2008;Aly et al., 2010) or genes encoding a protease involved in hemoglobin degradation (Spaccapelo et al., 2010) and a merozoite surface protein involved in adhesion to RBCs (Spaccapelo et al., 2011) in Plasmodium berghei. These GAPs multiply suboptimally in the blood and cause infections that eventually self-resolve. Notably, abortive GAP-induced infections confer lasting protection against challenge with blood stages or mosquito transmission stages and depend on both cellular and humoral immunity (Ting et al., 2008;Aly et al., 2010).

Recently, we described the crucial role of histaminereleasing factor (HRF), also known as translationally controlled tumor protein, during development of P. berghei ANKA (PbANKA) in the host liver (Mathieu et al., 2015). In vivo development of HRF-deicient PbANKA parasites is severely impaired in the liver, caused by elevated levels of IL-6 (Pied et al., 1991). To test whether HRF might also modulate blood stage multiplication, we deleted the HRF-encoding gene in P. berghei NK65 (PbNK65). PbANKA induces cerebral malaria in susceptible mice, with lesions starting at day 5 postinfection (p.i.) and mice dying from day 7-8 p.i. (Beghdadi et al., 2008), which precludes studies on adaptive immunity. In contrast, PbNK65 does not cause cerebral malaria but hyperparasitemia, leading to mouse death by severe anemia around day 25 p.i. It thus provides an opportunity to track immune responses against blood stage parasites over a longer period of time.

RES ULTS AND DIS CUS SION Deletion of the hrf gene in PbNK65 parasites causes abortive blood stage development

We generated hrf knockout PbNK65 parasites (PbNK65-hrfΔ) by replacing the coding sequence of pbhrf (PBA Although most vaccines against blood stage malaria in development today use subunit preparations, live attenuated parasites confer signiicantly broader and more lasting protection. In recent years, Plasmodium genetically attenuated parasites (GAPs) have been generated in rodent models that cause self-resolving blood stage infections and induce strong protection. All such GAPs generated so far bear mutations in housekeeping genes important for parasite development in red blood cells. In this study, using a Plasmodium berghei model compatible with tracking anti-blood stage immune responses over time, we report a novel blood stage GAP that lacks a secreted factor related to histamine-releasing factor (HRF). Lack of HRF causes an IL-6 increase, which boosts T and B cell responses to resolve infection and leave a cross-stage, cross-species, and lasting immunity. Mutant-induced protection involves a combination of antiparasite IgG2c antibodies and FcγR + CD11b + cell phagocytes, especially neutrophils, which are suficient to confer protection. This immune-boosting GAP highlights an important role of opsonized parasite-mediated phagocytosis, which may be central to protection induced by all self-resolving blood stage GAP infections. NKA_111050; UniProt accession no. A0A077XCV2) with the human dihydrofolate reductase (hDHFR)-selectable marker in a GFP-expressing PbNK65 strain (Fig. S1 A). Two clones, PbNK65-hrfΔ1 and 2, were selected from independent transfection experiments and veriied to harbor the mutant locus by PCR (Fig. S1, B-D) and Southern blot analysis (Fig. S1 E). Using speciic rabbit antibodies against recombinant PbHRF (Mathieu et al., 2015), the protein was found to be expressed at all Plasmodium stages tested and to localize to the cytoplasm (Fig. 1, A-D), consistent with previous studies in human cells and PbANKA parasites (Bhisutthibhan et al., 1999;Mathieu et al., 2015). HRF was not detected in PbNK65-hrfΔ1 parasites (Fig. 1, A-D), conirming both antibody speciicity and successful gene knockout.

To assess the efect of pbhrf deletion on parasite blood stage development, C57BL/6 mice were infected i.p. with 10 5 , 1 0 4 , or 10 3 WT or PbNK65-hrfΔ1-infected iRBCs, and parasite growth was monitored by low cytometry. In mice infected with WT parasites, parasitemia increased steadily, resulting in severe malaria and death at approximately day 20 p.i. When using 10 5 or 10 4 infectious doses, PbNK65-hrfΔ1 parasites multiplied like WT until day 10 p.i., reaching parasitemia of ∼20%, and were cleared from mice at day 13 or 14 p.i., respectively. Mouse survival rate after injection of 10 4 or 10 5 PbNK65-hrfΔ1 parasites was ∼90% and ∼70%, respectively. Notably, injection of 10 3 PbNK65-hrfΔ1 parasites did not lead to parasite clearance or mouse survival. Infection with PbNK65-hrfΔ2, a second clone, gave similar results (Fig. S1, F andG). We concluded that infection with parasites lacking HRF can self-resolve and that parasite clearance depends on the initial parasite load and/or time to threshold parasitemia. Further experiments were performed after injection of 10 5 PbNK65-hrfΔ1 parasites. To determine whether clearance of mutant parasites from the circulation could be caused by parasite retention in the spleen, the parasite load in this organ was measured by RT-quantitative PCR (RT-qPCR). After a dramatic retention of PbNK65-hrfΔ1 parasites at day 6, parasites were no longer detected in the spleen at day 14 (Fig. 1 F), indicating that parasite clearance was not caused by retention in the spleen (Fig. 1 G). Moreover, macroscopic examination showed a more important splenomegaly at day 6 in PbNK65-hrfΔ1-infected mice than in WT-infected mice, suggesting leukocyte iniltration in the mutant-infected spleen (Fig. 1, H-J).

IL-6, B cells, and T cells are critical for inhibition of PbNK65-hrfΔ blood stage growth

Because HRF-deicient PbANKA sporozoites induce IL-6 production in the liver during preerythrocytic infection (Mathieu et al., 2015), we compared IL-6 levels in mouse spleens 6 d p.i with WT or PbNK65-hrfΔ1 iRBCs. Levels of IL-6 mRNA and plasmatic IL-6 protein were higher in PbNK65-hrfΔ1-infected mice than in WT-infected mice (Fig. 2, A andB). Given that recombinant PbHRF protein is suicient to down-regulate IL-6 expression in vivo (Mathieu et al., 2015), we tested whether clearance of mutant parasites was the consequence of elevated IL-6 by infecting IL-6 KO mice with mutant parasites. Mutant parasites developed normally in and eventually killed IL-6 KO mice (Fig. 2 C), phenocopying WT parasite behavior in WT mice (Fig. 1 E). We concluded that increased IL-6 accounts for mutant parasite clearance.

Immunostaining with leukocyte surface markers and anti-IL-6 antibodies of cells collected from the spleen at days 6 and 20 p.i. identiied IL-6-producing cells as Ly6G + neutrophils at day 6 and both Ly6G + neutrophils and CD11c + DCs at day 20 (Fig. 2 D). Depletion of neutrophils (Fig. S2, A andB) or DCs (Fig. S2, C andD) in mutant-infected mice reduced splenomegaly (Fig. S2, E, F, H, andI) and cell counts (Fig. S2, G andJ) compared with nondepleted mice, conirming the contribution of neutrophils and DCs in the splenomegaly caused by mutant infection.

IL-6 is known to regulate the acute phase of the immune response and major B and T cell functions [START_REF] Kishimoto | Interleukin-6 and its receptor: a paradigm for cytokines[END_REF][START_REF] Barton | IL-6: insights into novel biological activities[END_REF]. To test whether B or T cells were involved in self-resolution of mutant infection, we infected mice lacking B cells (μS KO ) or T cells (CD3 KO ) with WT or PbNK65-hrfΔ1 parasites and monitored parasite development. B cell-or T cell-deicient mice were unable to control PbNK65-hrfΔ1 blood stage multiplication and died with kinetics similar to WT mice infected with WT parasites (Fig. 2, E andF). The importance of T cells was conirmed by the normal multiplication of the mutant parasite upon mouse treatment of previously protected mice with anti-CD3 antibody (Fig. 2 G and Fig. S2 K). This indicated that B and T lymphocytes contributed to the clearance of mutant parasites.

PbNK65-hrfΔ1 infection confers lasting protection in a species-and stage-transcendent manner

To determine whether resolved PbNK65-hrfΔ1 parasite infection might confer protection against challenge, mutantinfected mice were challenged with 10 5 WT PbNK65 iRBCs at days 20, 35, 68, 168, and 396 p.i. Mice were protected in all cases, displaying no detectable parasitemia at any time point, and survived for more than a year (Fig. 3 A). Mice challenged at days 20 and 23 p.i. with 10 5 RBCs infected with virulent P. berghei ANKA (Fig. 3 B) or P. yoelii YM (Fig. 3 C), respectively, were also protected and did not develop parasitemia. Next, we asked whether mutant-infected mice were also protected against a challenge with WT PbNK65 sporozoites, the mosquito-transmitted parasite stage. Sporozoite challenge did not result in detectable blood stage infection (Fig. 3 D), and parasite genes were not detected in the blood by PCR (not depicted). To ascertain that protection indeed targeted preerythrocytic stages and not just emerging blood stage development, the livers of mice challenged with sporozoites were analyzed by RT-qPCR 40 h after sporozoite inoculation. As shown in Fig. 3 E, the parasite load was signiicantly lower in the liver of PbNK65-hrfΔ1-protected mice compared with control mice. A similar protection was observed against heterologous P. berghei ANKA (Fig. 3 F) and P. yoelii YM (Fig. 3 G) sporozoite challenge. Therefore, infection with HRF-deicient blood stage PbNK65 parasites induces long-lasting protection against malaria in a species-and stage-transcendent manner.

Mutant-induced immunity involves Plasmodiumspeciic IgG2c antibodies

To assess whether antibodies were involved in the anti-PbNK65-hrfΔ1 response, anti-parasite-speciic antibodies of various isotypes were quantiied by ELI SA in sera of mice 15 d p.i. with WT or PbNK65-hrfΔ1 parasites. As shown in Fig. 4 A, mice infected with PbNK65-hrfΔ1 produced higher levels of IgG antibodies than mice infected with WT parasites, essentially belonging to the IgG2c subclass and to a lesser extent to the IgG3 subclass (Fig. 4 A). Next, as shown by Western blot analysis of extracts from WT blood stages (Fig. 4 B), these antibodies recognized multiple P. berghei antigens in contrast to sera from WT PbNK65-infected mice or from naive mice (Fig. 4 B). Interestingly, the IgG2c isotype, expressed in C57BL/6 mice in which the IgG2a heavy chain is deleted, is known to be the predominant isotype generated in antiviral antibody responses [START_REF] Coutelier | IgG2a restriction of murine antibodies elicited by viral infections[END_REF] and the most eicient IgG subclass for antipathogen FcR-mediated efector functions [START_REF] Nimmerjahn | Divergent immunoglobulin g subclass activity through selective Fc receptor binding[END_REF]. In most experimental mouse malaria models, parasite-speciic antibodies have been shown to be predominantly skewed toward the IgG2c isotype [START_REF] Ndungu | Functional memory B cells and long-lived plasma cells are generated after a single Plasmodium chabaudi infection in mice[END_REF].

Immunoprecipitation of P. berghei blood stage proteins with the IgG antibodies from mutant-infected mice and mass spectrometry of the immunoprecipitate revealed ive P. berghei proteins targeted by the protective IgG response (Fig. S3, A andB). These included the vaccine candidates merozoite surface protein 1 on July 21, 2016 jem.rupress.org

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Published July 18, 2016 (MSP1), serine repeat antigen 1 (SERA1), and SERA2 [START_REF] Bodescot | Transcription status of vaccine candidate genes of Plasmodium falciparum during the hepatic phase of its life cycle[END_REF]Putrianti et al., 2010;[START_REF] Alaro | A chimeric Plasmodium falciparum merozoite surface protein vaccine induces high titers of parasite growth inhibitory antibodies[END_REF]. As shown by immunoblots (Fig. S3 C) and ELI SA (Fig. S3 D), only sera from protected mice recognized the recombinant MSP1-33 antigen.

Next, to test whether IgG antibodies may mediate parasite clearance via FcRγ-expressing cells, WT or FcRγ KO C57BL/6 mice were infected with PbNK65-hrfΔ1 parasites. As shown in Fig. 4 C, in contrast to WT mice, FcRγ KO mice were unable to eliminate PbNK65-hrfΔ1 parasites and phenocopied WT mice infected with WT parasites.

Antibodies and CD11b + cells from mutant-infected mice mediate protective immunity

Finally, to assess what immune effectors are important for protection, we performed passive transfer experi-ments. First, antibodies purified from protected mice were transferred to naive mice before challenge with WT PbNK65 parasites. As shown in Fig. 4 D, parasitemia was lower in mice treated with IgG from protected mice than in normal IgG-treated mice, suggesting that antibodies alone provide partial but significant protective activity (Fig. 4 D).

Last, we asked whether CD11b + cells such as phagocytic leukocytes from PbNK65-hrfΔ1-protected mice might be suficient to protect naive mice from WT infection. WT C57BL/6 mice with adoptively transferred CD11b + cells from naive or PbNK65-hrfΔ1-infected mice at day 15 p.i. were challenged with 10 5 WT parasites. As shown in Fig. 4 E, transfer of CD11b + cells from mutant-infected, but not naive, mice eiciently protected against infection. Collectively, these data suggest that This study shows that abortive blood stage infection leading to lasting protection can be achieved not just by impairing parasite intracellular growth, but also by enhancing protective immune responses. Indeed, lack of HRF and an increase in IL-6 do not afect parasite growth per se because PbNK65-hrfΔ blood stages multiply normally in mice until day 10. Rather, IL-6, which is involved in B and T cell diferentiation, boosts antiparasite adaptive responses that clear parasites. Like with previously reported blood stage GAPs that induce abortive infections, the protective response to PbNK65-hrfΔ parasites is both solid, conferring cross-stage and cross-species immunity, and durable. We found that the protective response relies on the combination of antiparasite IgG2c antibodies and FcγR + CD11b + phagocytic cells, in particular neutrophils, which are suicient for solid protection. Interestingly, the discovery of a B helper neutrophil population in the spleen that can act as professional helper cells for marginal zone B cells (Puga et al., 2012) highlights a neutrophil-B cell interplay that may be critical for B cell diferentiation into antibodyproducing plasma cells and may also contribute to inhibition of the well-known Plasmodium capacity to induce short-lived B cell memory (Wykes et al., 2005). Opsonic phagocytosis was also described as a protective mechanism induced by the plasmepsin-4-deicient mutant (Spaccapelo et al., 2010). Whether this represents the essential protective mechanism common to all self-resolving infections remains to be determined. Finally, although not formally demonstrated in our work, the contribution of parasite-speciic CD8 + cells to self-resolution cannot be precluded and awaits further investigation.

MAT ERI ALS AND MET HODS Ethics statements.

All experiments involving mice were conducted at Institut Pasteur, approved by the Direction Départementale des Services Vétérinaires' de Paris, France (permit number N° 75-066 issued on September 14, 2009), and performed in compliance with institutional guidelines and European regulations. A statement of compliance with the French government's ethical and animal experiment regulations was issued by the Ministère de l'Enseignement Supérieur et de la Recherche under the number 00218.01.

Rodents. 5-8-wk-old WT female C57BL/6J Rj and Swiss Webster (SW) mice were purchased from the Janvier laboratory. Transgenic T cell-deicient (CD3 KO ), B cell-deicient (secretory μ chain [μs KO ]), Fcγ receptor-deicient (FcγR KO ) , and IL-6 KO mice strains were provided by B. Ryfel (Institut Pasteur, Paris, France), J.M. Cavaillon (Institut Pasteur), P. Bruhns (Institut Pasteur), and L. Apetoh (Institut National de la Santé et de la Recherche Médicale U866, Dijon, France), respectively. CD11c-DTR (diphtheria toxin receptor)-GFP mice [START_REF] Jung | In vivo depletion of CD11c + dendritic cells abrogates priming of CD8 + T cells by exogenous cellassociated antigens[END_REF] have been used to explore the role of DCs in controlling parasite development. Transgenic mice have all been backcrossed 10 times on C57BL/6 mice from The Jackson Laboratory.

Parasites. Mice were inoculated with RBCs infected with either P. berghei (Pb) NK65 WT or mutant (hrfΔ) GFP-transgenic parasites. In a few control experiments, mice were infected with P. yoelii YM or Pb ANKA-GFP iRBCs, or with PbNK65 or PbANKA GFP-transgenic sporozoites collected from salivary glands of infected Anopheles stephensi. Mosquitoes were provided by the CEP IA (Centre d'élevage, de production et d'infection des anopheles, Institut Pasteur).

Mouse infections and immunization with blood stages. Cryopreserved P. berghei parasites were passaged once through SW mice before being used to infect experimental animals. Mice were infected with blood stages of either GFP-transgenic PbNK65, PbNK65-hrfΔ1, or PbNK65-hrfΔ2 parasites by injecting 10 5 , 10 4 , or 10 3 iRBCs i.p. After injection, blood samples were taken daily from the tail, and parasitemia was assessed by low cytometry. If mice did not develop parasites after challenge, they were recorded as completely protected.

Splenic index. Spleens from uninfected and infected mice were harvested at day 6 p.i. with WT or PbNK65-hrfΔ1 parasites. The splenic index for each individual mouse was calculated as follows: spleen weight (mg)/body weight (mg) × 100.

Sporozoite development in HepG2 cells. HepG2 cells (2-3 × 10 4 /well) were plated in 8-well chamber slides (Lab-Tek) and cultured overnight in DMEM + GlutaMAX-I media (Gibco) supplemented with 10% heat-inactivated FBS (Gibco) at 37°C in the presence of 5% CO 2 . WT or mutant puriied P. berghei salivary gland sporozoites were used for HepG2 in- fection at a ratio of 1:1 (parasite/cells) for 36 h at 37°C with 5% CO 2 in the presence of penicillin-streptomycin-neomycin solution (Sigma-Aldrich). PbHRF was detected by immunoluorescence staining as described in the next paragraph.

Immunoluorescence assays for the intracellular detection of HRF. Fixation and permeabilization of sporozoites, infected HepG2 cells, iRBCs, and puriied gametocytes were performed using 4% paraformaldehyde and 0.1% Triton X-100 and blocked with 1-3% gelatin from porcine skin (Sigma-Aldrich). Thereafter, cells were incubated with speciic rabbit anti-HRF antibodies (diluted 1:500; Mathieu et al., 2015) and then incubated with Alexa Fluor 568-conjugated secondary antibodies (diluted 1:500; Thermo Fisher Scientiic) and 0.02 mg/ml DAPI for nuclear staining. The expression of PbHRF was detected using a luorescence microscope (AxioVert 200; ZEI SS).

Preparation of total RNA and RT-qPCR analysis of mRNA.

The spleens and livers of C57BL/6J mice infected with WT or PbNK65-hrfΔ1 parasites were surgically removed 40 h p.i. or at day 2, 4, 6, 8, 10, 12, 14, and 20 p.i., respectively. Total RNAs were extracted from the spleen as well as from liver samples using the guanidinium-thiocyanate-phenol-chloroform method (all from Invitrogen). RNA was thereafter reverse transcribed by PCR (temperature proile: 65°C for 5 min, 42°C for 50 min, and 70°C for 15 min) using 100 U of SuperScript II reverse transcriptase (Invitrogen), 40 U RNase inhibitor, and 2 µM oligo(dT) 18S rRNA primer (Euroins MWG Operon) per sample. The expression levels of diverse transcripts were analyzed by real-time RT-qPCR using Power SYBR green PCR master mix (Applied Biosystems) and various primer sets (Table S1). All reactions were performed in a real-time PCR machine (temperature proile: 50°C for 2 min, 95°C for 10 min, 40 cycles of 15 s at 95°C, and 60°C for 1 min; ABI PRI SM 7000 Sequence Detection System; Ap-plied Biosystems). The relative abundance of parasite and cytokine rRNA in the spleen was calculated using the ΔC t method and expressed as 2 -∆ C t . The mouse hypoxanthine phosphoribosyltransferase (HPRT) gene was used as an internal control for the variation in input RNA amounts. A notemplate control was included to ensure that there was no cross-contamination during sample preparation.

Flow cytometry analysis of spleen leukocytes. Spleens were mechanically disrupted in 2 ml PBS, and cells were iltered through a 70-mm strainer (BD). Erythrocytes were lysed using Gey's solution for 5 min on ice and washed twice in PBS. Single-cell suspensions were stained for FACS analysis according to standard protocols in cold PBS containing 2% FCS and 0.01% sodium azide (FACS bufer) with the following antibodies: PE-labeled anti-CD4, PE-Cy5-labeled anti-CD45, allo phyco cyanine (APC)-labeled anti-CD8, FITC-labeled anti-CD11b, APC-labeled anti-CD11c, APC-labeled anti-Ly6G, PE-Cy5-labeled anti-F4/80, and PE-labeled anti-IL-6 antibodies (all antibodies from BD). A total of 5 × 10 5 living cells were analyzed using a four-color low cytometer (FAC SCalibur; BD) and ProCellQuest software (BD).

In vivo cell depletion. For neutrophil depletion, C57BL/6 mice were injected with 500 µg of a rat anti-mouse neutrophil (clone NIMP-R14) provided by G. Milon (Institut Pasteur) at day 2 and day 4 p.i. with PbNK65-hrfΔ1. For systemic DC depletion, CD11c-DTR-GFP transgenic mice were injected i.p. with 5.2 ng/g body weight diphtheria toxin (Sigma-Aldrich) in PBS at days 2 and 4 p.i. with PbNK65-hrfΔ1. To determine whether CD3 plays a role in the antiparasitic memory response developed by protected mice, cell-speciic depletion experiments were performed. C57BL/6J Rj-protected mice were injected i.p. with 20 µg anti-CD3 (clone 145-2C11) Armenian hamster IgG (eBioscience) 24 h before the infection with PbNK65 WT and 48 h p.i. The cell depletion was followed and conirmed by low cytometry. Before the infection and every day p.i., 10 µl of blood was collected from the tip of the mouse tail and analyzed to conirm neutrophil, DC, and CD3 cell depletion by FACS analysis.

Detection of speciic IgG antibodies and IL-6 cytokine in the serum of infected mice.

To detect parasite-speciic antibodies, protein extracts from blood stages obtained by saponin lysis (0.1%) of parasite pellets were sonicated in lysis bufer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN 3 , 20 mM MgCl 2 , 1 % Triton X-100, and complex protease inhibitors) and centrifuged (10 ,000 g for 30 min at 4°C). The total amount of proteins in the supernatant was measured using a Bio-Rad Laboratories protein assay. 96-well plates (Nunc-immuno plate; Thermo Fisher Scientiic) were coated with 2 µg/ml PbNK65 WT protein extracts in carbonate bufer, pH 9.6, for using HRP-conjugated goat anti-mouse secondary antibody (diluted 1:2,000; Cell Signaling Technology) followed by the addition of o-phenylenediamine dihydrochloride substrate (Sigma-Aldrich). HCl 1N was used to block the reaction. The optical density was read at 490-630 nm. Each sample was tested against nonimmune serum and PBS as background controls. Amounts of IL-6 in the serum were analyzed following the instructions provided by the ELI SA kit supplier (BD).

Western blotting. 20 µg PbNK65 WT protein extract from asexual blood stages were separated by SDS-PAGE (4-12% Bis-Tris gels; BOLT mini gel system; Thermo Fisher Scientiic) and transferred onto a polyvinylidene luoride membrane (iBLOT system; Thermo Fisher Scientiic). Sera from uninfected, WT, or PbNK65-hrfΔ1 mice were added (1:1,000 dilution) and incubated overnight at 4°C. After washing the membrane with PBS + Tween 20 (Sigma-Aldrich), polyclonal anti-mouse IgGs (1:20,000; P0260; Dako) were added, and speciic bands were visualized with the SuperSignal West Pico kit (Thermo Fisher Scientiic) according to the manufacturer's instructions.

Adoptive transfer of IgG-speciic antibodies and CD11bpositive cells. C57BL/6J mice were infected with either WT or PbNK65-hrfΔ1 parasites as described in the previous paragraph. Speciic IgGs and CD11b + cells were obtained from challenged protected mice at day 15 p.i. Immune sera were collected, and the IgG fraction was puriied on an immunoabsorbent protein G-Sepharose column (BioVision). 100 µg IgGs was transferred i.p. into naive mice 24 h before WT PbNK65 iRBC infection. Mice were then given 100 µg of antibody on days 3 and 6 p.i. Single-cell suspension of CD11b + cells was obtained from naive or PbNK65-hrfΔ1infected mice by FACs sorting of spleen and bone marrow cells stained with FITC-labeled anti-CD11b. Each mouse received intravenous injections of 2.5 × 10 6 CD11b + cells 1 h after WT PbNK65 iRBC infection.

Generation and cloning of PbNK65-hrfΔ parasites. For construction of the targeting vector for pbhrf disruption (PbHRF; plasmid provided by P. Smooker and K. Taylor, RMIT University, Bundoora, Australia), DNA fragments corresponding to the 5′ untranslated (UTR) and 3′UTR regions of the pbhrf gene were ampliied by PCR using P. berghei NK65 genomic DNA (gDNA) as a template. These primers (Table S2) were tailed with restriction sites for ApaI, PstI, KpnI, and EcoRI to facilitate cloning into either side of the hDHFR cassette (de Koning- Ward et al., 2000) in pUC18 backbone. The targeting construct was integrated into the pbhrf gene locus by double crossover recombination, resulting in the disruption of pbhrf and conferring WR99210 or pyrimethamine resistance. Transfection into a GFP-PbNK65 parasite strain and selection of recombinant parasite clones were performed as previously described (Janse et al., 2006). In brief, after overnight culture (37°C with 10% O 2 and 5% CO 2 at 90 rpm) of the blood of infected animals, mature schizonts were puriied using a Nycodenz gradient and collected at room temperature. The electroporation mix was composed of 10 7 -10 8 merozoites resuspended in 50 µl and 100 µl of human T cell Nucleofector solution (Amaxa) and 5 µl DNA (containing 5 µg of digested DNA in water). Parasites were electroporated using the U33 program of the Nucleofector electroporator (Amaxa) and immediately resuspended in PBS and injected intravenously into 3-wk-old female SW mice. Recipient mice were treated with 0.07 mg/ml pyrimethamine in drinking water or with 6 mg/kg WR99210 by subcutaneous injections, starting 24 h after electroporation. At day 6 after electroporation, the emerging parasite population was collected, gDNA was extracted, and genomic integration of the hDHFR cassette (within the pbhrf locus of GFP-PbNK65 transfectants) was conirmed using speciic PCR primers: (a) Apa-5′UTR PbHRF-F and EcoRI-3′UTR PbHRF-R, (b) HRF5′-F and hDHFR5′-R, and (c) HRF3′-R and hDH-FR3′-F. The irst pair of primers (Apa-5′UTR PbHRF-F and EcoRI-3′UTR PbHRF-R) ampliied gDNA encompassed by the ApaI and EcoRI restriction sites and inclusive of the pbhrf 5′ and 3′UTRs. A PCR product of 2,681 bp was indicative of hrfΔ, whereas a PCR product of 1,760 bp denoted a WT genotype. The second pair of primers (HRF5′-F and hDHFR5′-R) ampliied DNA ouside the hDHFR insert (inclusive of the 5′UTR PbHRF) and within the 5′ region of the hDHFR cassette, respectively. A PCR product of 700 bp indicated a hrfΔ clone, whereas absence of a band denoted a WT genotype. The third pair of primers (hDHFR3′-F and HRF3′-R) ampliied gDNA within the 3′ region of the hDHFR cassette and outside the hDHFR insert (inclusive of the 3′UTR pbhrf), respectively. A PCR product of 1,100 bp indicated a hrfΔ clone, whereas absence of a band indicated a WT genotype.

Statistical analysis. All data were analyzed using Prism 5.0 software (GraphPad Software). Unpaired data between two groups at a speciic time point were analyzed by a Mann-Whitney test for nonparametric analysis when data did not it a Gaussian distribution. A p-value <0.05 was considered to be statistically signiicant. All experiments were replicated several times as indicated in the igure legends.

Southern blotting. gDNA was obtained as follows: parasite pellets obtained by saponin lysis of iRBCs were resuspended in PBS and treated with 150 μg/ml proteinase K and 2% SDS at 55°C for 20 min. The DNA was isolated from the parasite pellet using the DNeasy blood and tissue kit (QIA GEN). The DNA was digested with EcoRV and probed with an hrf probe. 9 JEM nano LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) using a chromatograph (EASY-nLC 1000; Thermo Fisher Scientiic) coupled to a mass spectrometer (Q Exactive Orbitrap). About 1 µg of each sample (dissolved in 0.1% formic acid [FA]) was loaded at 250 nl/min on a homemade C18 50-cm capillary column picotip silica emitter tip (75-μm diameter illed with 1.9 μm Reprosil-Pur Basic C18-HD resin [Dr. Maisch GmbH]) equilibrated in solvent A (0.1% FA). The peptides were eluted using a twoslopes gradient of solvent B (0.1% FA in acetonitrile) from 2-30% in 90 min and from 30-80% in 60 min at a 250-nl/ min low rate (total length of the chromatographic run was 180 min). The mass spectrometer (Q Exactive Orbitrap; Thermo Fisher Scientiic) was operated in data-dependent acquisition mode with XCalibur software (version 2.2; Thermo Fisher Scientiic). Survey scan MSs were acquired in the Orbitrap on the 300-1,800-m/z range with the resolution set to a value of 70,000 at m/z = 400 in proile mode (automatic gain control target at 1E6). The 20 most intense ions per survey scan were selected for higher energy collisional dissociation fragmentation (NCE 28), and the resulting fragments were analyzed in the mass spectrometer at 17,500 resolution (m/z of 400). Isolation of parent ion was ixed at 2.5 m/z, and the underill ratio was ixed at 0.1%. Dynamic exclusion was used within 20 s. Each sample was prepared in triplicate.

Data were searched using MaxQuant (version 1.4.1.2; with the Andromeda search engine) against the Plasmo-dium_berghei database (22,006 entries). The following search parameters were applied. Carbamidomethylation of cysteines was set as a ixed modiication. Oxidation of methionine and protein N-terminal acetylation were set as variable modiications. The mass tolerances in MS and MS/MS were set to 10 parts per million for each, respectively. Two peptides were required for protein identiication and quantitation. Peptides and proteins identiied with a false discovery rate <0.01% were considered as valid identiication. Statistical analysis of the data was performed using Perseus, R package, MSstat, and internal tools. Two sample Student's t tests were used to identify signiicantly regulated proteins between two groups. The results were visualized on volcano plots.

Immunoprecipitation. Immunoprecipitation of Pb proteins from parasite extracts was performed using the Pierc Direct immunoprecipitation kit (Thermo Fisher Scientiic). Before immunoprecipitation, 10 µg of puriied IgG antibodies from the serum of protected, infected, and naive mice was directly immobilized onto an agarose support (AminoLink Plus Resin) using a short coupling protocol. 500-700 µg of parasite extracts was incubated with the immobilized antibody to form the immune complex with gentle end-over-end mixing overnight at 4°C. To remove nonbound material, beads were washed three times with wash bufer, and a low pH elution bufer was used to dissociate the bound antigen from the an-tibody. Immunoprecipitated proteins were then used for mass spectrometry analysis.

Online supplemental material. Fig. S1 shows disruption of the pbhrf gene in PbNK65 parasites. Fig. S2 shows assessment of leukocyte depletion and the role of neutrophils and DCs in the occurrence of splenomegaly. Fig. S3 shows identiication of immune sera-derived immunoprecipitated proteins. Table S1 contains a list of oligonucleotides used for RT-qPCR analyses. Table S2 contains a list of oligonucleotides used for PCR of WT and recombinant parasites. Online supplemental material is available at http ://www .jem .org /cgi /content / full /jem .20151976 /DC1. TGC TTT GAG GGG TGA GCA TTT AAA GC PbHRF-5′orf-F CCA TTT GGA AAT GCG GAAT PbHRF-3′orf-R TTT TTC TTC AAA TAA ACC ATC TGA Bold letters refer to the oligonucleotide's position in Fig. S1 (A andB).

Supplementary Results

HRF is important for P. berghei pre-erythrocytic and erythrocytic infection

Similar to previous studies performed on PbANKA strain, we examined the contribution of Plasmodium HRF to the phenotype of PbNK65 both at the level of the pre-erythrocytic phase and at the blood stage. As depicted in Fig. 17A, mice inoculated with PbNK65-hrfΔ sporozoites showed an initial delayed development of the parasite followed by a complete clearance during blood stage at around day 17 p.i., in a similar way as the parasite was inoculated with iRBCs (Fig. 17B). Since similar phenotypes were obtained with both sporozoites and iRBCs, the followup investigation was carried out using iRBCs to explore the mechanisms by which HRF shapes the immune response.

Pattern of cytokines associated with PbNK65-hrfΔ induced protection

It is known from previous studies that both CD4 + and CD8 + T cells play a central role in the elimination of blood-stage malaria parasites through the release of cytokines that activate other effector cells such as NK cells, macrophages and dendritic cells. In addition, Th1 normalization of the weight a few days later (day 10 p.i.). The loss of weight from day 4 to day 8 represented 10% of the weight of control mice at the same age. In contrast, mice infected with WT parasites did not show any loss of weight until day 8 where this was continuously decreasing until the death of the mice (Fig. 17B). Mice that were infected with WT parasites displayed a delayed peak of TNF-α, IL-10 and IL-6 at day 12-14 post infection which in addition to high parasitemia may be associated to a drastic loss of body weight in these mice starting from day 14 (Fig. 18B).

Augmented CD11b + cells during PbNK65-hrfΔ infection

Detailed analyses of two organs (spleen and liver) mainly characterized by high parasitemia following malaria infection show differences in the innate immune cell activation (Fig. 20). As compared to the PbNK65 WT infected spleen, we observed an increase in the total number of F4/80 + macrophages and CD11c + CD11b + dendritic cells in the PbNK65-hrfΔ infected spleen (Fig. 20B), whereas we found the opposite in the infected liver (Fig. 20A). In addition, we observed no differences in the percentage and total number of Ly6G + neutrophils between WT and hrfΔ PbNK65-infected spleens but increased number of neutrophils in the PbNK65

IL-12p70 ! IFN γ ! ! ! 10! 8! 6! 4! 2! 0! ! WT hrfΔ1 CTL! ! ! 3 ! ! 2 ! ! 1 ! ! 0! ! WT hrfΔ1 CTL! C$ A$ WT hrfΔ1 CTL! ! ! 40! 30! 20! 10! 0! ! p = 0.0286 ! IFN γ ! ! ! 300 ! 200! 100! 0! ! WT hrfΔ1 CTL! WT hrfΔ1 CTL! WT hrfΔ1 CTL! IL-6 ! IL-10 ! ! ! 5! 4! 3! 2! 1! 0! ! ! ! 20! 15! 10! 5! 0! ! B$ WT hrfΔ1 CTL! ! ! 120
WT infected liver as compared to the PbNK65-hrfΔ infected liver. Taken together, these data suggest organ-specific innate cell activation after malaria infection.

Protection conferred by mutant parasites is dependent on effector CD4 + T cells.

In order to show that the protection induced by PbNK65-hrfΔ parasites is dependent on effector CD8 + or CD4 + T cells, protected mice were treated with normal mouse IgG, anti-CD8

or with anti-CD4 depleting antibody. The dose of each antibody used was set to 20 mg for anti-CD8 (clone 53-6.7, Armenian hamster IgG, eBiosceince) and 100 mg for anti-CD4 (clone )!

! ! 5! 4! 3! ! 2! 1! ! 0! ! Positive cells (%)! Positive cells (%)! ! ! 2.5! ! 2! ! 1.5! ! 1! ! 0.5! ! 0! ! p = 0.0013 ! 10! 8! 6! 4! 2! 0! ! A! p = 0.0303 ! WT hrfΔ1 CTL ! ! ! 1.5! ! 1.0! ! 0.5! ! 0! ! Positive cells (%)! p = 0.0015 ! WT hrfΔ1 CTL CD11b +! CD11b +! WT hrfΔ1 CTL ! WT hrfΔ1 CTL WT hrfΔ1 CTL ! WT hrfΔ1 CTL B"
Liver" Spleen"

CD11c +! Ly6G +! F4/80 +!
Total nb of cells (10 5

)! ! ! ! 4 ! 3! ! 2! ! 1 ! 0! ! 15! 10! 5! ! 0! !
Total nb of cells (10 5

)!

Total nb of cells (10 5 in animals injected with anti-CD8 and anti-CD4 respectively. Interestingly, the measurement of parasitemia indicated a loss of parasite control upon treatment of protected mice with anti-CD4 antibody but not with anti-CD8 antibody. WT parasite-challenged mice treated with control IgG remained parasite free. This suggests that the activation of effector CD4 + T lymphocytes but not CD8 + are indispensable for clearance of malaria infection in long term immune protection. These results are further supported by previous studies in animal models where depletion or adoptive transfer of the different T cell populations highlighted the important role of CD4 + T cells in the development of protective immunity against blood stages [START_REF] Taylor-Robinson | A model of development of acquired immunity to malaria in humans living under endemic conditions[END_REF][START_REF] Van Der Heyde | The time course of selected malarial infections in cytokine-deficient mice[END_REF].

)! p = 0.0350 ! ! ! 20! 15! ! 10! 5! ! 0! ! ! ! 10! 8! ! 6! ! 4! 2! ! 0! ! Positive cells (%)! Positive cells (%)! p = 0.0007 ! p = 0.0078 ! 2.5! ! 2! ! 1.5! ! 1! ! 0.5! ! 0! ! p = 0.0418 ! ! ! 1.5! ! 1.0! ! 0.5! ! 0! ! Positive cells (%)! p = 0.0012 ! WT hrfΔ1 CTL ! WT hrfΔ1 CTL CD11b +! CD11b +! WT hrfΔ1 CTL ! WT hrfΔ1 CTL WT hrfΔ1 CTL ! WT hrfΔ1 CTL A"

Protection induced by PbNK65-hrfΔ parasites is associated with down-regulation of PD-1 on T cells

As previously observed, mice lacking T cells were unable to control the parasitemia of PbNK65-hrfΔ infected mice, suggesting that the activation of T lymphocytes may be indispensable for immune memory against malaria infection. In order to better characterize the molecular signatures of the T cell response and given that chronic malaria infection results in an increased frequency of T cells expressing surface markers of exhaustion such as programmed cell death-1 (PD-1) [START_REF] Butler | Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection[END_REF], we asked whether the self-resolving nature of PbNK65-hrfΔ infection could be correlated to a change in PD-1 expression on the surface of effector T cells. When we analyzed PD-1 expression by flow cytometry at day 6 and day 20 p.i. (Fig. 22A,B), we observed that WT parasites induced a high proportion of PD1 + CD4 + and PD1 + CD8 + T cells in the spleen hinting at T cell exhaustion. In contrast, PbNK65-hrfΔ parasites induced two-fold fewer PD1 + CD4 + and PD1 + CD8 + T cells at day 6 p.i. as compared to WT parasites, with this number decreasing to basal levels found in uninfected mice at day 20 p.i. (Fig. 22A). We further correlated PD-1 expression to the activation status of T cells using the cell surface marker CD62L: low or undetectable levels of surface CD62L are indicative of T cell activation and effector function [START_REF] Oehen | Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division[END_REF]. At day 6 p.i. we observed in WT parasite-infected mice, 65% and 70% of activated CD4 + CD62L -and CD8 + CD62L -T cells, respectively, were PD-1 + whereas only 40% of activated CD4 + CD62L -and CD8 + CD62L -T cells were positive for PD-1 + in mice infected with PbNK65-hrfΔ parasites (Fig. 22B). The number of PD-1 + activated T cells further decreased at day 20 p.i. in PbNK65-hrfΔ-infected mice, resetting to the basal levels found in uninfected mice (Fig. 22A,B). To investigate whether rPbHRF can directly modulate the expression of PD-1 on the surface of T cells, naïve mice were injected with 100 µg of rPbHRF or BSA as a negative control and 48 h later, PD-1 surface expression examined. We observed an upregulation of PD-1 on CD4 + and CD8 + T cells surface (Fig. 22C) and on activated CD4 + CD62L -and CD8 + CD62L -T cells (Fig. 22D) induced by the rPbHRF. Taken together, these data demonstrate that infection with PbNK65 parasites expressing PbHRF is associated with an increased proportion of PD-1 + T cells in mice, indicative of T cell exhaustion.

! p = 0.0159 ! WT! CTL ! hrfΔ1! WT! CTL ! hrfΔ1! WT! CTL ! hrfΔ1! WT! CTL ! hrfΔ1! 2.5! 2! 1.5! 1! 0! 0.5! 2.5! 2! 1.5! 1! 0! 0.5! 4! 3! 2! 1! 0! 4! 3! 2! 1! 0! 8! 6! 4! 2! 0! 4! 3! 2! 1! 0! 6! 4! 2! 0! 6! 4! 2! 0! 6! 4! 2! 0! 80! 40! 20 ! 0! 60! 80! 40! 20 ! 0! 60! 5! 4! 3! 2! 1! 0! 40! 20! 10 ! 0! 30! 2.0! 1! 0.5 ! 0! 1.5! p = 0.0006 ! p = 0.0041 ! p = 0.0043 ! p = 0.0429 ! p = 0.0012 ! p = 0.0175! p = 0.0006 ! p = 0.0262 ! 4! 3! 2! 1! 0! 5! 4! 3! 2! 1! 0! B Total

Prior exposure to WT parasite followed by drug treatment does not hamper PbNK65-hrfΔ-induced parasite clearance and immune protection

Results obtained from mice infected firstly with PbNK65-hrfΔ parasites and, upon the complete clearance of PbNK65-hrfΔ parasites, with either PbNK65, PbANKA or PyYM WT parasites demonstrated that the sterile protection conferred by this GAP is long-lasting in a species-and stage-transcendent manner. In real field conditions, such GAP delivery would only be given to individuals who had been previously infected with WT parasites and drug treated. In endemic areas pre-exposure to WT parasites followed by antimalarial chemotherapy could induce epigenetic marks that may negatively influence anti-parasite immunity. In this situation the protective effect induced by the PbNK65-hrfΔ mutant will not be effective. The question is to assess whether these individuals who had been exposed to WT parasites and drug cured will be equally efficiently protected following PbNK65-hrfΔ infection as in naïve mice. To address this issue, mice were first infected with 10 5 PbNK65

WT iRBCs, and when parasitemia reached around 2%, were treated for three consecutive days with 6 mg/kg WR99210 by subcutaneous injections. Once the parasites were completely eliminated from the blood stream, at day 19 p.i., mice were infected either with 10 5 WT or with PbNK65-hrfΔ parasites iRBCs (Fig. 23). We observed that in the group of mice which have received WT parasites, parasites develop normally and the mice died around day 20 p.i.. In contrast mice, which received PbNK65-hrfΔ parasites, show a slow development of parasites which were ultimately eliminated around day 15 p.i. from peripheral blood circulation. To assess whether one single infection with the mutant parasite was enough to develop a long-lasting immune response, as we previously observed without the use of drugs, mice which received WT parasites followed by PbNK65-hrfΔ parasites were infected with 10 5 WT iRBCs 4 weeks after the elimination of the mutant parasite from the blood circulation (Fig. 23). A control group, which has been neither infected nor treated by antimalarial drugs, was infected at the same time with 10 5 WT iRBCs. As obtained with WT parasite challenges in protected mice without drug treatment, infection with PbNK65-hrfΔ parasites allows the development of a long-lasting immune protection despite a prior drug treatment. This result shows that a primary infection with WT parasites followed by a drug cure does not hamper the efficacy of NK65-hrfΔ-induced protection. or IgM + IgG -) in the hrfΔ PbNK65-infected spleen. These preliminary results reinforce the hypothesis that the B cells are important in the immune response developed in C57BL/6 mice against the PbNK65-hrfΔ parasites. We can assume that the effector memory cells developed during the first infection with the PbNK65-hrfΔ parasites are important for the maintenance of the immune response during a long period of time, in fact challenges of protected mice with WT parasites show that the parasite is eliminated by the immune system immediately without allowing him to develop. 86

!WT! CTL!!!! hrfΔ1! !WT! CTL!!!! hrfΔ1! Memory!B!cells!(AID/YFP + CD19 + ) ! Total!nb!of!cells!(10 6 )! PosiIve!cells!(%)! 8! 6! 4! 2! 0! 4! 3! 2! 1! 0! 5! Effector!memory!B!cells!(IgM @ IgG + ) ! !WT! CTL!!!! hrfΔ1! !WT! CTL!!!! hrfΔ1! Total!nb!of!cells!(10 6 )! PosiIve!cells!(%)! 4! 3! 2! 1! 0! 2! 1.5! 1! 0.5! 0! 2.5! Central!memory!B!cells!(IgM + IgG @ ) ! !WT! CTL!!!! hrfΔ1! !WT! CTL!!!! hrfΔ1! PosiIve!cells!(%)! Total!nb!of!cells!( 10

Discussion

In spite of continued efforts to control the disease, malaria remains a major health problem in many regions of the world, especially sub-Saharan Africa, and new ways to control or eradicate the disease are urgently needed. Development of an effective malaria vaccine, that could greatly improve global public health by enhancing a rapid and protective immune response that completely eliminates the infection, would provide a better way of alleviating malaria. But scientists have different point of view on the best lines of research to pursue.

Despite the large number of successful vaccines in use today for different diseases (such as polio, tetanus, measles, smallpox or pertussis) the development of malaria vaccine still remains a challenge for scientists. Malaria vaccine research involves the combination of vaccinology, immunology and parasitology (malariology) and there are few established theoretical principles in this field to serve as guidelines. Moreover, the differences between Plasmodium strains, the persistence of infection for months and the capacity of the parasite to both evade and disable the immune system by a variety of mechanisms are the major problems encountered for the development of a vaccine.

Understanding malaria pathogenesis may find its way in the investigation of possible common pathways with allergic inflammation.

Since the beginning of the humanity, the parasite has learned to live and co-evolve with the human immune system. This co-evolution has been performed through a myriad of complex mechanisms that ultimately result in the inability of the immune system to eliminate the parasite and to mediate resistance to subsequent infections. An important part in the development of an effective malaria vaccine is the understanding of the mechanisms developed by the parasite to escape the immune response or the natural immunity to malaria developed by the host. A way to address the mechanisms involved in the pathogenesis of a disease is to find out if the disease in question does not share signalling pathways associated with other pathologies. Following this line of thought we hypothesised that the Plasmodium parasite turns to its advantage the mechanisms involved in the allergic inflammatory response (Fig. 25).

Several studies conducted in endemic areas in humans suggest a strong relationship between clinical susceptibility to malaria and severe allergic-type responses [START_REF] Demeure | Anopheles mosquito bites activate cutaneous mast cells leading to a local inflammatory response and lymph node hyperplasia[END_REF][START_REF] Griffiths | Genomewide analysis of the host response to malaria in Kenyan children[END_REF][START_REF] Sakuntabhai | Genetic determination and linkage mapping of Plasmodium falciparum malaria related traits in Senegal[END_REF]. Indeed, the contribution of host genetic factors to the risk of severe outcome was highlighted in different studies during P.

falciparum infection in African populations. Large scale genetic studies in Senegal revealed that several chromosome regions were linked to clinical susceptibility to malaria. The four genome regions; 5p15-p13 and 13q13-q22 associated with clinical malaria, and 12q21-q23 and 5q31-33 associated with parasite density, evoked have been previously found to be associated to atopic disease or related phenotypes [START_REF] Flori | Linkage and association between Plasmodium falciparum blood infection levels and chromosome 5q31-q33[END_REF][START_REF] Iyengar | Improved evidence for linkage on 6p and 5p with retrospective pooling of data from three asthma genome screens[END_REF][START_REF] Rihet | Malaria in humans: Plasmodium falciparum blood infection levels are linked to chromosome 5q31-q33[END_REF][START_REF] Sakuntabhai | Genetic determination and linkage mapping of Plasmodium falciparum malaria related traits in Senegal[END_REF]. Link between parasite density and the region on chromosome 5q31, previously found to be significantly linked to malaria parasite density and involved in the control of immunity to P. falciparum blood stage, have been shown to be associated to asthma/atopy related traits and to elevated IgE levels [START_REF] Bouzigon | Meta-analysis of 20 genome-wide linkage studies evidenced new regions linked to asthma and atopy[END_REF]; autoimmune diseases, infections diseases, and allergic responses. Indirect evidence from immunoepidemiological studies support that an association exist between malaria pathogenesis and mechanisms involving IgE-mediated inflammatory responses. This latter consist of classical pathway which starts with the binding of antigen (Ag)-specific IgE Abs to FcεRI expressed on various leukocytes (mast cells, basophils, and neutrophils), and upon aggregation, cells will release histamine and other inflammatory mediators. Beside this classical pathway, alternative pathway independent from IgE/ FcεRI complexes, are also able to trigger mediator release. One of the prototypes IgE-independent triggers is the histamine releasing factor (HRF). [START_REF] Flori | Linkage and association between Plasmodium falciparum blood infection levels and chromosome 5q31-q33[END_REF][START_REF] Iyengar | Improved evidence for linkage on 6p and 5p with retrospective pooling of data from three asthma genome screens[END_REF][START_REF] Rihet | Malaria in humans: Plasmodium falciparum blood infection levels are linked to chromosome 5q31-q33[END_REF][START_REF] Sakuntabhai | Genetic determination and linkage mapping of Plasmodium falciparum malaria related traits in Senegal[END_REF]. Previous studies highlighted the importance of this region in immune regulation by its linkage to plasma IgE levels [START_REF] Marsh | Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations[END_REF][START_REF] Meyers | Evidence for a locus regulating total serum IgE levels mapping to chromosome 5[END_REF], bronchial hyper responsiveness, and to Schistosoma mansoni infection intensity [START_REF] Postma | Genetic susceptibility to asthma--bronchial hyperresponsiveness coinherited with a major gene for atopy[END_REF] in humans and with resistance to P. chabaudi in mice [START_REF] Hernandez-Valladares | Mapping of a new quantitative trait locus for resistance to malaria in mice by a comparative mapping approach with human Chromosome 5q31-q33[END_REF]. The 5q31-33 region encodes for several genes mostly Th2 cytokines (IL-3, IL-4, IL-5, IL-9, IL-13) which are responsible for the differentiation of B cells that generate IgE, but also the Th1 cytokine IL-12B and other immunologically active genes such as interferon regulatory factor-1 which are strong candidates for controlling the outcome of malaria infection. The 13q13-q22 and 12q21-q23 regions contain genes, namely PHF1153 and STAT6, known to increase total serum IgE levels [START_REF] Chen | Signal transducer and activator of transcription signals in allergic disease[END_REF]. Moreover, the IL-4, IL-13 and STAT6 pathways have been reported to be associated with IgE and asthma. The role of IL-4 in regulating the antibody response induced by Plasmodium parasite was studied using different approaches. Genetic studies on IL4 T/C single nucleotide polymorphism (SNP) in Mali [START_REF] Vafa | Relationship between immunoglobulin isotype response to Plasmodium falciparum blood stage antigens and parasitological indexes as well as splenomegaly in sympatric ethnic groups living in Mali[END_REF][START_REF] Vafa | Associations between the IL-4 -590 T allele and Plasmodium falciparum infection prevalence in asymptomatic Fulani of Mali[END_REF] and in Burkina Faso [START_REF] Verra | IL4-589C/T polymorphism and IgE levels in severe malaria[END_REF] have highlighted the importance of the IgE antibodies induced by IL-4 pathway. Higher parasite prevalence in asymptomatic Fulani population, known to be naturally less susceptible to malarial infections than their sympatric neighbours, was correlated with the T allele, suggesting that CT genotype carriers are less successful in clearing the infection as compared to the CC genotype carriers [START_REF] Vafa | Associations between the IL-4 -590 T allele and Plasmodium falciparum infection prevalence in asymptomatic Fulani of Mali[END_REF]. Additionally, an increased level of IgE in the T allele group was associated to the presence of malaria parasites [START_REF] Vafa | Relationship between immunoglobulin isotype response to Plasmodium falciparum blood stage antigens and parasitological indexes as well as splenomegaly in sympatric ethnic groups living in Mali[END_REF]. Burkina Faso studies in non-Fulani children correlated CT genotype carriers with higher total IgE levels with severe malaria compared to uncomplicated malaria [START_REF] Verra | IL4-589C/T polymorphism and IgE levels in severe malaria[END_REF].

Elevated plasma levels of IgE and anti-plasmodial IgE Abs which bind to basophils and mast cells have been associated with severity to P. falciparum infections in adults and children living in malaria endemic areas in different African and Asian countries [START_REF] Perlmann | IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria[END_REF][START_REF] Perlmann | IgE and tumor necrosis factor in malaria infection[END_REF]Troye-Blomberg et al., 1999a). Indeed, IgE levels were reduced amongst patients with uncomplicated malaria in comparison to those suffering from severe malaria [START_REF] Seka-Seka | The role of serum immunoglobulin E in the pathogenesis of Plasmodium falciparum malaria in Ivorian children[END_REF] and high IgE levels were found during cerebral P. falciparum malaria related to the deepness of the coma compared to uncomplicated malaria [START_REF] Luty | Immunologic responses to soluble exoantigens of Plasmodium falciparum in Gabonese children exposed to continuous intense infection[END_REF][START_REF] Maeno | IgE deposition in brain microvessels and on parasitized erythrocytes from cerebral malaria patients[END_REF]. In a more recent study, total IgE levels tented to be higher in uncomplicated malaria than in severe cases, while the levels of P. falciparumspecific IgE in severe forms were higher than those found in the uncomplicated cases of the disease [START_REF] Kumsiri | IgE low affinity receptor (CD23) expression, Plasmodium falciparum specific IgE and tumor necrosis factor-alpha production in Thai uncomplicated and severe falciparum malaria patients[END_REF], suggesting that parasite-specific IgE Abs may contribute to the severity of the disease.

During the allergic inflammatory response, the IgE Abs bind to the high-affinity receptor FcεRI and the formation of the antigen-IgE-FcεRI complex activates the cells and stimulates the release of inflammatory mediators such as histamine (Fig. 25). Release of proinflammatory mediators involved in allergic inflammation in the host is one of the events occurring during malaria infection [START_REF] Mecheri | Contribution of allergic inflammatory response to the pathogenesis of malaria disease[END_REF]. Significantly elevated plasma histamine concentrations, derived from basophils and mast cells (MacDonald et al., 2001), in comparison to uninfected controls (P< 0.01) correlate with systemic P. falciparum disease complications within infected children (Bhattacharya et al., 1988;Enwonwu et al., 2000;Srichaikul et al., 1976). A study conducted in a hyperendemic area of malaria in Southwest Nigeria, where chloroquine and amodiaquine treatment failed, demonstrated that treatment of children with a combination of amodiaquine or chloroquine or pyrimethamine/sulfadoxine plus chlorpheniramine, a histamine receptor type 1 antagonist reversed chloroquine resistance in vitro [START_REF] Basco | In vitro reversal of chloroquine resistance with chlorpheniramine against African isolates of Plasmodium falciparum[END_REF], and enhanced the drug efficacy in vivo [START_REF] Sowunmi | Randomized comparison of chloroquine and amodiaquine in the treatment of acute, uncomplicated, Plasmodium falciparum malaria in children[END_REF][START_REF] Sowunmi | Enhancement of the antimalarial efficacy of amodiaquine by chlorpheniramine in vivo[END_REF][START_REF] Sowunmi | Enhanced efficacy of chloroquine-chlorpheniramine combination in acute uncomplicated falciparum malaria in children[END_REF]Sowunmi et al., 1998a;Sowunmi et al., 1998b). In addition a clinical drug library screen using existing drugs for previously unknown activities identifies the nonsedating anti-histamine astemizole as an antimalarial agent able to inhibits the proliferation of three P. falciparum strains (3D7, Dd2 and ItG) [START_REF] Chong | A clinical drug library screen identifies astemizole as an antimalarial agent[END_REF]. These findings strongly suggest that histamine, a major product of the allergic inflammatory cascade, plays a prominent role in malaria pathogenesis.

Moreover, a mouse model for human atopic disease (NC/Jic) was found to be susceptible to P. yoelii infections compared to WT mice [START_REF] Ohno | Chromosomal mapping of the host resistance locus to rodent malaria (Plasmodium yoelii) infection in mice[END_REF]. The Pymr (P. yoelii malaria resistance) locus, able to control host survival and parasitemia after infection, was mapped on chromosome 9. In P. chadaudi infection, the char1 locus (P. chabaudi resistance locus 1) responsible to control host survival and peak parasitemia was mapped to the same region.

Interestingly, the loci of atopic disease susceptibility (quantitative trait locus of derm1) on chromosome 9 are mapped exceptionally close to genes (char1 and pymr) mediating parasitemia control [START_REF] Kohara | A major determinant quantitative-trait locus responsible for atopic dermatitis-like skin lesions in NC/Nga mice is located on Chromosome 9[END_REF][START_REF] Sakuntabhai | Genetic determination and linkage mapping of Plasmodium falciparum malaria related traits in Senegal[END_REF]. This concept has been further supported by several in vivo studies carried out in human and mice. Thus, atopic dermatitis in children is linked with a higher prevalence of clinical malaria attacks in Ethiopia [START_REF] Haileamlak | Early life risk factors for atopic dermatitis in Ethiopian children[END_REF]. In our laboratory, using a mouse model, we highlighted the crucial role of histamine in the pathogenesis of malaria disease. Firstly, by demonstrated that the allergic-type response, by the intermediary of mosquito saliva, starts immediately after the mosquito bite perturbing the early anti-parasite immune response and affecting downstream disease development [START_REF] Demeure | Anopheles mosquito bites activate cutaneous mast cells leading to a local inflammatory response and lymph node hyperplasia[END_REF][START_REF] Schneider | Anopheles stephensi saliva enhances progression of cerebral malaria in a murine model. Vector borne and zoonotic diseases[END_REF]. Secondly, by showing the importance of histamine release during malaria infection and its consequences. Indeed, mice treated with antihistamines or deficient for the H1 and H2 receptors for histamine were more resistant to ECM (Beghdadi et al., 2008). Moreover, the disruption of IgE or the α chain of FcεRI led to the resistance to the development of ECM after infection with PbANKA indicating a pathological role of IgE that acts via FcεRI to promote disease development. This study identified, in mice, neutrophils and eosinophils as alternative FcεRI-expressing cell types having a disease enhancing capacity. Although FcεRIα is essentially absent in naive mouse neutrophils, the receptor was shown to be induced during Plasmodium infection [START_REF] Porcherie | Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria[END_REF]. The transfer of FcεRI + , but not FcεRI -, neutrophils to the otherwise resistant FcεRI-α-KO mice conferred ECM susceptibility highlighting the implication of FcεRI + neutrophils in malaria pathogenesis [START_REF] Porcherie | Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria[END_REF]. Altogether, these findings demonstrate that components of the allergic cascade including IgE, FcεRI, histamine and the newly identified FcεRI-positive neutrophil population, are intimately associated with severe forms of malaria disease. This may have practical consequences on the management of malaria disease by introducing novel anti-allergic therapeutic approaches used individually or in combination with classical anti-malarial chemotherapy.

Recent studies have documented the importance of an alternative mechanism, independent of the classical pathway implicating the IgE-FcεRI complex during histamine release, where cells are directly activated by a Plasmodium-derived protein called Histamine Releasing Factor (HRF) (MacDonald et al., 2001)(Fig. 25). Additionally, high levels of PfHRF have been detected in the serum of mildly and severely P. falciparum infected Malawian children (Janse et al., 2006;MacDonald et al., 2001).

Considering these observations, we chose to provide a comprehensive description of the role of Plasmodium HRF during the parasite development in the host liver and during blood stages infection. To do so, we investigated (1) the influence of the Plasmodium HRF to modulate the immune mechanisms during malaria infection and (2) whether its expression was associated with the severity of malaria disease. For this, we generated a genetically HRFdeficient parasites (hrf∆) firstly in P. berghei ANKA-GFP parasites and later in P. berghei NK65-GFP parasites, and we assessed the impact of this mutation both on the host immune response and on the severity of the resulting malaria disease. Knowing that the loss of hrf expression in mice results in increased spontaneous apoptosis and death during embryogenesis, we analysed the development of mutant parasites during the whole life cycle by determining by qRT-PCR or by counting the number of parasites over time. We found that the deletion of the hrf gene did not affect the parasite development per se, meaning that the deletion was not lethal for the parasite.

Inoculation of PbANKA WT or hrf∆ sporozoites was implemented either via intravenous or intradermic injection, ensuring that a consistent number of sporozoites enters mouse circulatory system, or via mosquito bite. The later involves saliva effects, that include antiplatelet, anti-coagulant, vasodilatory and anti-platelet mediators and was shown to induce more likely a development into blood stage parasitemias than their intravenous inoculated counterparts. We also studied the influence of HRF in the development of the parasite during direct injection of RBCs previously infected with WT or HRF mutant parasites in mice.

Our results showed that infection with sporozoites was characterized by a delay in blood stage development of mutant parasites as compared to PbANKA WT parasites. This delayed development resulted in clinical immunity but without sterile immunity.

Sporozoite infectious capacities are dependent on three characteristics: host cell traversal, host cell invasion and gliding motility, all critical for parasite proceeding development within the liver. Therefore, cell traversal assay was done to deduce a potential role of Plasmodium HRF in mediating sporozoite locomotive capacities. Incubation of hepatic cells (HepG2) with PbANKA WT or hrf∆ sporozoites in vitro did not display any detectable defect in gliding motility or host cell traversal and invasion. A comparative qualitative analysis between PbANKA WT and hrf∆ parasite gliding properties was also observed via intravital (in vivo) microscopy. Injection of PbANKA WT and hrf∆ sporozoites in the tail vein was performed to compare the parasite ability to cross the sinusoidal barrier. The number of moving sporozoites and respective percentage within the liver parenchyma did not show any reduced gliding motility in mutant parasites compared to WT parasites, confirming the previous in vitro results and demonstrated that HRF is not important during the entry into hepatocytes. However, the impaired development of HRF mutant liver stages in vivo is likely to induce some parasite death, as suggested by intravital imaging studies. This resulted in a 10 2-to 10 3-fold reduction in the numbers of merozoites initially released in the blood, and an extension of the time between sporozoite injection and detectable parasitaemia. This prolonged liver-stage development influenced the clinical outcome of infection by decreasing the frequency of ECM as was observed in mice infection by mosquito bite sporozoite inoculation. Indeed, in these conditions, 100% of the mice that received PbANKA WT parasites develop ECM and died on day 7 or 8 p.i., while only 10% of mice infected with PbANKA-hrfΔ parasites showed signs of cerebral complications.

These first results, using PbANKA strain, demonstrated the importance of the Plasmodium HRF during malaria infection and the development of the pathogenesis of cerebral malaria.

In order to confirm and consolidate our results on the role of Plasmodium HRF in parasite and pathogenesis development we decided to extend these observations to a murine parasite strain PbNK65, which causes a lethal infection in C57BL/6 mice that is less acute than PbANKA, and anemia and mortality occur within 20-25 days p. pathology, the infection course with the PbNK65-hrfΔ iRBCs gave rise to a particular phenotype characterized by an initial growth similar to that of the WT parasite, followed by a clearance of the parasite at day 12 p.i.. This phenotype was reminiscent of an infection with the WT P. chabaudi chabaudi parasite strain, which mimics a similar pattern of an initial growth followed (around day 12) by a tight control of the parasite. This phenotype was well documented in terms of the mechanisms involved in the anti-parasite immune response. Indeed, parasite elimination was preceded by a Th1 response with a marked IFN-γ production during acute infection followed by a switch to a Th2 response crucial for the total clearance of the parasite [START_REF] Muxel | The spleen CD4+ T cell response to blood-stage Plasmodium chabaudi malaria develops in two phases characterized by different properties[END_REF].

HRF, a key regulator of IL-6 production: a prototype example of how a parasite gene product directly modulates host cytokine responses (Fig. 26).

A more detailed analysis showed that the impairment in the development of PbANKA-hrfΔ parasites in liver stages was associated with an early rise in systemic IL-6 at 40 hours post infection from neutrophils and inflammatory monocytes. The importance of IL-6 was confirmed by injection of anti-IL-6 antibodies or infection in IL-6-deficient mice during PbANKA-hrfΔ infection that rescue WT phenotype. In parallel, injection of human recombinant IL-6 in mice infected with PbANKA WT parasite shows a decrease in parasite load in the liver. Additionally, we demonstrated the inhibitory effect of HRF on LPS-induced IL-6 cytokine production confirming a direct role of parasite HRF in the down-regulation of anti-parasite cytokines. Previous studies have shown that in addition to the cellular mechanisms involving both CD8 + and CD4 + T cells [START_REF] Crispe | APC licensing and CD4+T cell help in liver-stage malaria[END_REF][START_REF] Van Braeckel-Budimir | CD8 T-cell-mediated protection against liver-stage malaria: lessons from a mouse model[END_REF], the production of type I IFN responses [START_REF] Haque | Hepatocytes break the silence during liver-stage malaria[END_REF][START_REF] Liehl | Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection[END_REF] has an important role in parasite killing within hepatocytes. Additionally, IL-6 with IL-1 and TNF-α have been involved in induction of acute phase proteins in the liver [START_REF] Gabay | Acute-phase proteins and other systemic responses to inflammation[END_REF][START_REF] Gauldie | Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells[END_REF][START_REF] Gruys | Acute phase reaction and acute phase proteins[END_REF][START_REF] Heinrich | Interleukin-6 and the acute phase response[END_REF][START_REF] Ramadori | Interleukin 6, the third mediator of acute-phase reaction, modulates hepatic protein synthesis in human and mouse. Comparison with interleukin 1 beta and tumor necrosis factor-alpha[END_REF], some of which being known to interfere with malaria parasite development or the outcome of disease [START_REF] Aggrey | Platelet induction of the acute-phase response is protective in murine experimental cerebral malaria[END_REF][START_REF] Pied | C-reactive protein protects against preerythrocytic stages of malaria[END_REF]. Consistently, addition of recombinant IL-6 during P. yoelii infection in in vitro hepatic cultures resulted in a specific dose-dependent inhibition of parasite development exerting its action during both the early phase of infection and during the subsequent maturation of the schizonts. These observations support the hypothesis that production of IL-6 during liver stage could be important for the outcome of the disease (Nussler et al., 1991;Pied et al., 1991). Consistent with these reports, our data also highlight the link between the early rise of IL-6 induction and the B-cell-activation at 40 h p.i. induced by mutant parasites. One of the features in malaria infection is the induction of short-lived B-cell memory resulting in the short-lived nature of many anti-malarial antibody responses [START_REF] Dorfman | B cell memory to 3 Plasmodium falciparum blood-stage antigens in a malaria-endemic area[END_REF] and the increase of the frequency and absolute number of apoptotic cells (T cells, B cells, and macrophages) in spleen [START_REF] Helmby | Cellular changes and apoptosis in the spleens and peripheral blood of mice infected with blood-stage Plasmodium chabaudi chabaudi AS[END_REF]. Accordingly, studies using lethal P. yoelii YM infection have reported that malaria parasites interfere with the immune response by disabling B-cell differentiation, possibly by inducing apoptosis (Wykes et al., 2005). In our murine malaria model, the increase in B-cell population in the liver of mice infected with mutant parasites reflects the possibility that the HRF molecule could interfere with B-cell function and memory possibly via the down-regulation of IL-6 production. Despite the observation that recombinant murine HRF stimulates proliferation and increased serum Abs production in splenic B cells both in vivo and in vitro [START_REF] Kang | Molecular identification of IgE-dependent histamine-releasing factor as a B cell growth factor[END_REF], recent studies have shown that Plasmodium HRF could inhibit B cell functions. Indeed murine splenic B cells were shown to incorporate more efficiently and faster the parasite protein than the human ones, indicating that Plasmodium HRF could be responsible for the altered immune response, thus allowing the parasite to escape the immune response [START_REF] Calderon-Perez | The Plasmodium falciparum translationally controlled tumor protein (TCTP) is incorporated more efficiently into B cells than its human homologue[END_REF]. This result is supported by structural studies on Plasmodium HRF, highlighting some differences between parasite and mammalian protein structure, which could impact the functionality of the protein [START_REF] Hinojosa-Moya | Phylogenetic and structural analysis of translationally controlled tumor proteins[END_REF].

Interestingly, the production of IL-6 observed in mice infected with the hrfΔ PbNK65 parasite confirms what was previously observed with PbANKA-hrfΔ where IL-6 strongly suppressed the liver-stage development. More importantly, in the PbNK65 model, the elevated IL-6 production during blood stage in the absence of HRF expression seems to be important in establishing parasite clearance and acquisition of sterile immunity during PbNK65-hrfΔ infection. Based on two main observations: first, a marked upregulation of IL-6 expression induced by PbNK65-hrfΔ parasites found in spleen and plasma at day 6 p.i. as compared to WT parasites, and second, a normal development of hrfΔ parasites and acquisition of the WT phenotype when the mutant parasites were inoculated to the IL6-KO mice allow us to conclude that the IL-6 pathway, a component of innate immunity, plays a central role in controlling PbNK65-hrfΔ blood stage parasitemia. In support of our findings, Prakash and colleagues, by studying the clusters of cytokines that determine the severity of the disease during P. falciparum infection in endemic areas of central India showed that the induction of serum IL-6 was inversely correlated with the disease severity [START_REF] Prakash | Clusters of cytokines determine malaria severity in Plasmodium falciparum-infected patients from endemic areas of Central India[END_REF]. In addition, co-infection with Schistosoma mansoni followed by P. knowlesi in baboons showed that animals with chronic schistosomiasis were protected from severe malaria by an increase of IL-6 levels compared to animals not infected with [START_REF] Nyakundi | Protective Effect of Chronic Schistosomiasis in Baboons Coinfected with Schistosoma mansoni and Plasmodium knowlesi[END_REF]. In an effort to identify the cellular source of IL-6 during PbNK65-hrfΔ infection that was negatively regulated by HRF, examination by intracellular cell staining flow cytometry analysis showed that neutrophils are the main cell targets. Nonetheless, how IL-6 affects parasite growth inside erythrocytes, and the contribution of other cytokines such as IL-12 or TNF-α to the deficient growth of the PbNK65-hrfΔ mutant parasite, remain open questions. IL-6 is a cytokine with well-defined pro-and anti-inflammatory properties. Among the biological activities, IL-6 plays a role in T cell stimulation [START_REF] Tormo | IL-6 activates STAT5 in T cells[END_REF], B cell proliferation and differentiation [START_REF] Hirano | Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin[END_REF], monocytes/macrophages differentiation [START_REF] Chomarat | IL-6 switches the differentiation of monocytes from dendritic cells to macrophages[END_REF] and neutrophils activation [START_REF] Borish | Activation of neutrophils by recombinant interleukin 6[END_REF]. Additionally, IL-6 deficiency can lead to dysfunctional innate and adaptive immunity against many infections [START_REF] Hoge | IL-6 controls the innate immune response against Listeria monocytogenes via classical IL-6 signaling[END_REF][START_REF] Van Der Poll | Interleukin-6 gene-deficient mice show impaired defense against pneumococcal pneumonia[END_REF]. For example, IL-6 was shown to play a protective role in many bacterial, viral and fungal infections [START_REF] Imanishi | Expression of cytokines in bacterial and viral infections and their biochemical aspects[END_REF]. Consistently, IL-6-deficient mice are significantly more susceptible to some fungal infections like candida [START_REF] Basu | Mice lacking both G-CSF and IL-6 are more susceptible to Candida albicans infection: critical role of neutrophils in defense against Candida albicans[END_REF] and herpes simplex virus type 1 infections. In this later infection, IL-6 ensures survival of infected mice, but does not substantively affect herpes simplex virus replication [START_REF] Leblanc | Lack of interleukin-6 (IL-6) enhances susceptibility to infection but does not alter latency or reactivation of herpes simplex virus type 1 in IL-6 knockout mice[END_REF].

HRF mutant parasite infection is associated with potent effector immune mechanisms (Fig. 26).

The reasons for the abortive infection and subsequent protection by PbNK65-hrfΔ parasites are likely multifactorial. Analysis of cytokine levels at early time points (day 6 p.i) indicated an increase of several pro-inflammatory cytokines, such as IL-12 cytokine family including IL-12p35, IL-12p40, IL-23, and Ebi3, IFN-γ and IL-6, in the spleen, liver and plasma of PbNK65-hrfΔ-infected mice indicating the development of a Th1 response at early time points following infection. In support of our results, administration of IL-12 antibody to mice infected with P. chabaudi chabaudi AS resulted in the elimination of the parasite with an increased cell numbers of NK and CD4 + T cells that enhance IFN-γ production [START_REF] Mohan | Natural killer cell cytokine production, not cytotoxicity, contributes to resistance against blood-stage Plasmodium chabaudi AS infection[END_REF]. A similar mechanism operates in a distinct experimental model where administration of recombinant IL-12 before inoculation of rhesus monkeys with P. cynomolgi provides 100% protection through an IFN-γ-dependent antiplasmodial mechanism [START_REF] Hoffman | Sterile protection of monkeys against malaria after administration of interleukin-12[END_REF]. A parallel can be made in the human situation since during P. falciparum infection, children with mild malaria infection have higher levels of plasma IL-12 than children with severe malaria infection, and the levels of IL-12 are inversely correlated with parasitemia and numbers of malaria pigment-containing neutrophils [START_REF] Luty | Low interleukin-12 activity in severe Plasmodium falciparum malaria[END_REF][START_REF] Perkins | Reduced interleukin-12 and transforming growth factor-beta1 in severe childhood malaria: relationship of cytokine balance with disease severity[END_REF][START_REF] Prakash | Clusters of cytokines determine malaria severity in Plasmodium falciparum-infected patients from endemic areas of Central India[END_REF]. Additionally, an increase in IL-12 levels during the acute phase of uncomplicated P. falciparum malaria is thought to reflect an early and effective immune response regulated by proinflammatory Th1 cytokines [START_REF] Prakash | Clusters of cytokines determine malaria severity in Plasmodium falciparum-infected patients from endemic areas of Central India[END_REF]. Furthermore, IL-12 production by macrophages in response to infection mediates resistance to Trypanosoma cruzi [START_REF] Aliberti | Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes[END_REF]. Studies regarding strategies toward cancer immunotherapy showed that optimal target-cell clearance required DC support and IL-12 production allowing the development of CD4 + cytotoxic T lymphocytes [START_REF] Nelles | Murine splenic CD4(+) T cells, induced by innate immune cell interactions and secreted factors, develop antileukemia cytotoxicity[END_REF].

Knowing that in cancer cells the IL-12 production is almost non-existant and that the HRF is implicated in cancer development [START_REF] Li | Characterization of fortilin, a novel antiapoptotic protein[END_REF][START_REF] Tuynder | Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1[END_REF], assumption can be made that HRF maybe responsible for the inhibition of IL-12 production, function that during malaria infection allows the parasite to escape the initial immune response. Some evidence suggests that IFN-γ have a protective role during malaria infections. Indeed, enhanced IFN-γ protective immunity was observed during P. chabaudi and P. yoelii 17XL infection in IFN-γ-deficient mice where absence of cytokine production increases the severity of the infection resulting in death or require additional time for the resolution of the infection in comparison to WT mice [START_REF] Van Der Heyde | The time course of selected malarial infections in cytokine-deficient mice[END_REF]. Mice infected with P. chabaudi and treated with neutralizing anti-IFN-γ Ab failed to suppress acute infection developing high levels of parasitemia and administration of IFN-γ to intact C57BL/6 mice slightly decreased and partially delayed parasitemia [START_REF] Meding | Role of gamma interferon during infection with Plasmodium chabaudi chabaudi[END_REF]. In humans, the protective capacity of IFN-γ during malaria infections appears to be related to the timing of IFN-γ production. Early appearance of IFN-γ after infection is correlated in humans and animal models with protection against the development of clinical symptoms of malaria as CM [START_REF] D'ombrain | Association of early interferon-gamma production with immunity to clinical malaria: a longitudinal study among Papua New Guinean children. Clinical infectious diseases : an official publication of the[END_REF][START_REF] Villegas-Mendez | IFN-gammaproducing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain[END_REF]. The Fulani ethnic group in Mali present elevated levels of IFN-γ suggesting a protective role for IFN-γ against malaria [START_REF] Mccall | Early interferon-gamma response against Plasmodium falciparum correlates with interethnic differences in 124 susceptibility to parasitemia between sympatric Fulani and Dogon in Mali[END_REF][START_REF] Prakash | Clusters of cytokines determine malaria severity in Plasmodium falciparum-infected patients from endemic areas of Central India[END_REF]. Moreover, IFN-γ is a key mediator of inflammatory immune responses induced primarily by IL-12 and its secretion is essential for control of intracellular pathogens (such as Mycobacteriul tuberculosis or Listeria) and tumours. Indeed, it was demonstrated that during Ebola infection, a disease that at the beginning of the infection present the same symptoms of malaria, the administration of IFN-γ in mice profoundly inhibits Ebola virus infection of macrophages reducing morbidity and serum viral titres [START_REF] Rhein | Interferon-gamma Inhibits Ebola Virus Infection[END_REF]. Once adaptive immunity is initiated IFN-γ secreted by CD4 + T cells is critical for optimal activation of cytotoxic CD8 + T cells [START_REF] Janssen | CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes[END_REF], induction of B cell class-switching to the cytophilic IgG2a antibodies [START_REF] Su | Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection[END_REF][START_REF] Xu | Stat1-dependent synergistic activation of T-bet for IgG2a production during early stage of B cell activation[END_REF] and induction of inflammatory monocytes in bone marrow that migrate into spleen [START_REF] Sponaas | Migrating monocytes recruited to the spleen play an important role in control of blood stage malaria[END_REF] or expression of a number of proteins such as FcγRI on macrophage surface enhancing their phagocytic abilities [START_REF] Waddell | Dissecting interferon-induced transcriptional programs in human peripheral blood cells[END_REF], all of which perform vital roles in the control of Plasmodium infection. At day 6 p.i. analysis also show that the parasite load in the spleen was higher in mice infected with PbNK65-hrfΔ parasite than the ones infected with WT parasite. Moreover, macroscopic examination of spleens showed a splenomegaly in mice infected with PbNK65-hrfΔ confirmed by quantitative cell analysis. The importance of the spleen in elimination of parasites and in the development of the immune response during malaria infection was demonstrated in different studies both in humans and in experimental murine models.

Several studies in the African population showed that an enlarged spleen was more frequently seen among the Fulani compared to the Dogon [START_REF] Bereczky | Spleen enlargement and genetic diversity of Plasmodium falciparum infection in two ethnic groups with different malaria susceptibility in Mali, West Africa[END_REF][START_REF] Greenwood | Ethnic differences in the prevalence of splenomegaly and malaria in The Gambia[END_REF][START_REF] Oomen | Differences in blood status of three ethnic groups inhabiting the same locality in Northern Nigeria. Anaemia, splenomegaly and associated causes[END_REF]. Indeed, during acute P. falciparum infection patients with splenomegaly accelerate the clearance of iRBCs compared to patients with normal spleen sizes [START_REF] Looareesuwan | Dynamic alteration in splenic function during acute falciparum malaria[END_REF] and splenectomised patients show an increase in parasitemia [START_REF] Bachmann | Absence of erythrocyte sequestration and lack of multicopy gene family expression in Plasmodium falciparum from a splenectomized malaria patient[END_REF], demonstrating the crucial role of spleen in parasite clearance. It is known that the first lines of defence against blood stage in spleen are the red pulp macrophages and the splenic marginal zones DCs able to capture malaria antigens and activate T cells. During P. chabaudi infection two population of DCs responsible for the activation of CD4 + T cells in spleen with different kinetics. At the beginning of the infection splenic CD8 + DCs cells or macrophages through the production of IL-12 induce the proliferation of CD4 + T cells and their production of IFN-γ (Th1 response) [START_REF] Ing | Dendritic cell and NK cell reciprocal cross talk promotes gamma interferon-dependent immunity to blood-stage Plasmodium chabaudi AS infection in mice[END_REF]Sam and Stevenson, 1999a;Sam and Stevenson, 1999b). The switch of the immune response from Th1 to Th2 starts at the peak of infection when CD8 + DCs cells undergo apoptosis in the spleen and CD8 -DCs cells activated CD4 + T cells to produce IL-4 and IL-10 (Helmby et al., 2000;[START_REF] Sponaas | Migrating monocytes recruited to the spleen play an important role in control of blood stage malaria[END_REF]. These mechanisms allow the generation of a robust splenic immune response to the parasite, including prominent germinal centre formation and generation of a long-lived memory B and CD4 + T cell responses as well as long-lived plasma cells and protective antibodies [START_REF] Del Portillo | The role of the spleen in malaria[END_REF].

Additionally, we show that immunological effectors are required to clear PbNK65-hrfΔ parasites and induce protection. In concordance with findings on previous protective blood stage mutants (Aly et al., 2010;Spaccapelo et al., 2011), PbNK65-hrfΔ-induced protection was T cell-and B cell-dependent, with the involvement of two types of effectors. Indeed, this mutant parasite developed normally in T-and B-cell deficient mice. By reflexion, these findings raise the question why WT parasites develop normally within the host environment without being cleared. Two key findings were presented in the present work: first, no trace of anti-parasite antibodies could be detected at any time of the infection course with the WT parasite and the B cell compartment seems to be completely non functional, and second, a significant proportion of CD4 + CD62L -and CD8 + CD62L -cells, which are memory effector T cells, have their surface PD-1 expression upregulated during infection with WT parasites in contrast to hrfΔ parasites. These two combined mechanisms may explain why the WT parasite remains unaffected by the host immune system.

During PbNK65-hrfΔ infection antibodies of the IgG2c subclass were dramatically increased in PbNK65-hrfΔ-infected mice. A clear evidence of the association of parasite clearance and the subsequent sterile immunity was provided by the strong antigen-specific antibody response demonstrated by immunoblot and by ELISA assays. Among these antigens, MSP-1, MSP-8, SERA-1, and SERA-2 which are known to elicit antibodies associated with protection in people living in malaria-endemic areas [START_REF] Banic | Immune response and lack of immune response to Plasmodium falciparum P126 antigen and its amino-terminal repeat in malariainfected humans[END_REF][START_REF] Okech | Natural human immunoglobulin G subclass responses to Plasmodium falciparum serine repeat antigen in Uganda[END_REF] were identified by mass spectrometry. Moreover, BALB/c mice infected with P. Yoelii YM-sera2Δ but not SERA-1 mutant parasites, showed a clearance of the parasite at day 28-30 p.i. compared to the lethal WT parasite which killed mice within 6-8 days indicating that SERA2 during malaria infection is an important antigen and that its inhibition or blocking could help parasite elimination [START_REF] Huang | The role of serine-type serine repeat antigen in Plasmodium yoelii blood stage development[END_REF]. In contrast, in a distinct murine model of malaria using NMRI mice (originally from Swiss strain but maintained as an inbred strain), namely during infection with P. berghei, SERA1 and SERA2 seem not to play a vital role in vivo, since loss-of-function parasite lines progressed normally through the parasite life cycle (Putrianti et al., 2010). These murine models highlight inconsistencies regarding the GAP phenotypes depending both on the parasite and the mouse strain. In order to gain more insight into the role of genes of potential interest, novel studies must be carried out to design GAPs using the same parasite line tested in the same mouse strain. We found that secreted antibodies from PbNK65-hrfΔ protected mice were able to reduce parasitemia following a passive transfer to naïve mice but were not able to completely clear the infection. In addition FcγR -/- mice were unable to clear infection by mutant parasites and CD11b + cells, most likely macrophages and dendritic cells, were necessary and sufficient for adoptive transfer of immunity. These results suggest that secreted antibodies are important for the protective immunity to P. berghei but the apparent lack of complete protection suggests that these antibodies need to act in concert with "primed" FcγR + effector cells such as monocytes, macrophages, NK cells, and others that are existing in hrfΔ parasites-infected but not in naive mice. This picture of antibody-dependent cellular cytotoxicity involved in protection was earlier reported in the case of the plasmepsin-4-deficient mutant (Spaccapelo et al., 2010) and in host resistance to blood stage P. berghei XAT infection [START_REF] Yoneto | A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection[END_REF]. In vivo experiments using rodent malaria parasites transgenic for P. falciparum MSP1 19 in mice also transgenic for human Fc-receptors (FcRs) after three i.p. inoculations of functional humans anti-MSP1 19 IgG1 Abs effectively suppressed a lethal blood stage challenge with P. berghei. In contrast in non transgenic mice, human anti-MSP1 19 IgG1 Abs where not protective suggesting that FcγRI recruitment is crucial for parasite elimination [START_REF] Mcintosh | The importance of human FcgammaRI in mediating protection to malaria[END_REF]. Several in vitro studies have indicated the biologic relevance of the FcγRII and IgG subclasses in resistance to high-density parasitemia by showing the importance of ADCI mechanism during protection against blood-stage P. falciparum parasitemia [START_REF] Bouharoun-Tayoun | Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes[END_REF][START_REF] Bouharoun-Tayoun | Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages[END_REF][START_REF] Druilhe | Mechanisms of defense against P. falciparum asexual blood stages in humans[END_REF][START_REF] Shi | Differential effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on the growth of asexual stage Plasmodium falciparum parasites[END_REF][START_REF] Zhou | CD14(hi)CD16+ monocytes phagocytose antibody-opsonised Plasmodium falciparum infected erythrocytes more efficiently than other monocyte subsets, and require CD16 and complement to do so[END_REF]. The ADCI function is triggered by the binding of the IgG-merozoite complex to monocytes via the FcγRII receptor [START_REF] Bouharoun-Tayoun | Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages[END_REF], and correlates with malaria-specific IgG [START_REF] Bouharoun-Tayoun | Plasmodium falciparum malaria: evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity[END_REF][START_REF] Shi | Differential effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on the growth of asexual stage Plasmodium falciparum parasites[END_REF].

Human studies also showed the capacity of FcγR to mediate potent antimalarial immunity [START_REF] Mcintosh | The importance of human FcgammaRI in mediating protection to malaria[END_REF]. Studies on the Fulani population demonstrate a relation between FcγRs polymorphisms and susceptibility to malaria, which is associated with higher antibody levels [START_REF] Cherif | Distribution of FcgammaR gene polymorphisms among two sympatric populations in Mali: differing allele frequencies, associations with malariometric indices and implications for genetic susceptibility to malaria[END_REF][START_REF] Modiano | Different response to Plasmodium falciparum malaria in west African sympatric ethnic groups[END_REF]. A recent study on the allelic frequencies of rs396991 (FCGR3A) and rs3933769 (FCGR2C) between the Fulani and Dogon populatations in Mali revealed differences in these allelic frequencies suggesting that the genes may contribute to the interethnic variability in malaria susceptibility seen among the Fulani and their sympatric neighbours. For example, the rs396991 mutant allele occurs at a higher frequency in the Dogon, who are more susceptible to malaria relative to Fulani, suggesting that this SNP may play a role in malaria pathogenesis [START_REF] Cherif | Distribution of FcgammaR gene polymorphisms among two sympatric populations in Mali: differing allele frequencies, associations with malariometric indices and implications for genetic susceptibility to malaria[END_REF]. However, further studies will be needed to determine the possible roles of these SNPs in malaria pathogenesis. Additionally, a study performed in Kenyan infants, residing in an area of high malaria transmission, provides evidence that genetic polymorphism of FcγRIIa is associated with protection against high-density P. falciparum. Indeed, infants with FcγRIIa-Arg/Arg131 genotype, which binds IgG1 or IgG3 Abs, were less likely to have repeated high-density P. falciparum infections during the first year of life suggesting a protective effect of this FcγRIIa genotype. Moreover, the study showed that infants with FcγRIIa-His/Arg131 genotype were at greater risk for high-density P. falciparum infection compared to infants who were at low risk [START_REF] Shi | Fcgamma receptor IIa (CD32) polymorphism is associated with protection of infants against high-density Plasmodium falciparum infection. VII. Asembo Bay Cohort Project[END_REF]. The clinical importance of FcγRIIa polymorphism has been evaluated during encapsulated bacterial infections where FcγRIIa-His/His131 genotype was associated to protection, whereas the poorly IgG2-binding allotype FcγRIIa-Arg/Arg131 was associated with increased susceptibility to these pathogens [START_REF] Musser | Global genetic structure and molecular epidemiology of encapsulated Haemophilus influenzae[END_REF][START_REF] Platonov | Association of human Fc gamma RIIa (CD32) polymorphism with susceptibility to and severity of meningococcal disease. Clinical infectious diseases : an official publication of the[END_REF][START_REF] Yee | Association between FcgammaRIIa-R131 allotype and bacteremic pneumococcal pneumonia. Clinical infectious diseases : an official publication of the[END_REF].

One of the characteristic features of malaria infection is the inability to generate an acquired protective immunity suggesting that memory T cells develop inadequately or their maintenance is not assured. Using our mutant strain of parasite, we observed a dramatic difference with the WT parasites in modulating the expression of PD-1 on the surface of both CD4 + and CD8 + T cells. PD1 and its ligand PD-L1/PD-L2 belong to the family of immune checkpoint proteins that act as co-inhibitory factors transmitting an inhibitory signal into the T cell, which reduces cytokine production and suppresses T-cell proliferation (Day et al., 2006;[START_REF] Hofmeyer | The PD-1/PD-L1 (B7-H1) pathway in chronic infectioninduced cytotoxic T lymphocyte exhaustion[END_REF][START_REF] Wherry | T cell exhaustion[END_REF]. The PD1/PD-L1 pathway represents an adaptive immune resistance mechanism that is exerted by tumour cells in response to endogenous anti-tumour activity. Moreover, T cells exhaustion by upregluation of PD1 expression of cell surfaces was described in humans in the context of chronic viral infection such ad HIV and hepatitis (B and C) [START_REF] El-Far | T-cell exhaustion in HIV infection[END_REF][START_REF] Kim | Features of responding T cells in cancer and chronic infection[END_REF][START_REF] Wherry | T cell exhaustion[END_REF]. Interruption of PD1/PD-L1 binding, with monoclonal antibodies against PD1 and PD-L1, has shown to rescue tumours and HIV-infected cells from this state of anergy or 'exhaustion' [START_REF] Porichis | Role of PD-1 in HIV pathogenesis and as target for therapy[END_REF][START_REF] Swaika | Current state of anti-PD-L1 and anti-PD-1 agents in cancer therapy[END_REF]. Examining the expression of PD-1, an inhibitory receptor expressed on T cells, we found that throughout the infection period starting from day 6 p.i., the absence of HRF was associated with a significantly reduced expression of this receptor. This suggests that this marker, found early during infection (day 6 p.i.) and lasted throughout the infection period (day 20 p.i.), may have an influence on both clearance of primary infection and induction of lasting protection. Moreover using the P. berghei recombinant HRF protein we demonstrated the direct involvement of HRF in the upregulation of PD-1 receptor. Interestingly, an involvement of PD-1 in malaria blood stage control has already been reported: in P. falciparum infections, higher expression of PD-1 was associated with T cell dysfunction and the blockade of PD-1 ligand in a murine model of infection rapidly cleared blood-stage malaria in a B-and T-cell dependent manner resulting in enhanced parasite control [START_REF] Butler | Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection[END_REF]. In the P. chabaudii rodent model of chronic blood stage infection [START_REF] Good | Crossspecies malaria immunity induced by chemically attenuated parasites. The Journal of clinical investigation Graidist[END_REF], parasite-specific protective CD8 + T cells undergo significant PD-1-dependent exhaustion (up to 95% reduction), which exacerbates acute blood stage infection and drives chronic disease. A parallel that can be made with our results is that our mutant parasite allows a rapid clearance of the parasite, a down-regulation of PD-1 expression and the establishment of a long lasting memory which are all dependent on both B and T cells.

After confirming the importance of T cell responses during parasite clearance such as helper function for B cells to produce Abs that are essential for parasite clearance or production of cytokines able to amplify the phagocytosis and the parasitocidal response of the innate immune system, we wanted to determine the importance of T cells for the maintaining of the immune response and to identify which population of T cells are implicated in the memory. Depletion of CD4 or CD8 T cells in protected mice followed by PbNK65 WT reinfection showed no differences in parasitemia in the control group and in CD8 depleted mice. In contrast, all of CD4 depleted mice had detectable parasitemia (until 30%) indicating the contribution of CD4 + T cells to long term immunity. Studies performed in both mice and humans have shown the importance of CD4 + T cells in parasite-specific memory [START_REF] Brake | Adoptive protection against Plasmodium chabaudi adami malaria in athymic nude mice by a cloned T cell line[END_REF][START_REF] Vinetz | Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria[END_REF]. Depletion of CD4 + or CD8 + T cells in mice during P. yoelii 17X infection and adoptive transfer into a naïve mice showed that mice which received CD8 + T cells failed to control the infection in contrast to mice which received CD4 + T cells which were able to control the infection, suggesting that CD8 + T cells are not responsible for blood-stage immunity [START_REF] Vinetz | Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria[END_REF]. Additionally, parasite-specific memory CD4 + T cells were found in the blood of human volunteers deliberately exposed to very low doses of malaria parasites with very few or no parasite-specific antibodies (Pombo et al., 2002;[START_REF] Roestenberg | Protection against a malaria challenge by sporozoite inoculation[END_REF]. and the maintaining of the efficacy after storage. More recent work has demonstrated that in humans, repeated infections that were initiated by ultra-low doses of P. falciparum and rapidly cured by appropriate drug treatment, induced protective immunity. The resulting protection was associated with CD4 + and CD8 + T cell proliferative responses [START_REF] Fell | Inhibition of Plasmodium falciparum growth in vitro by CD4+ and CD8+ T cells from non-exposed donors[END_REF], nitric oxide synthase activity and IFN-γ production, but did not appear to involve parasite-specific antibodies (Pombo et al., 2002). Likewise, low doses of killed P. falciparuminfected red blood cells in the adjuvant CpG-ODN demonstrated strong protective efficacy, mainly dependent on CD4 + T cells, IFN-γ and NO (Pinzon-Charry et al., 2010). Nonetheless, human studies are highly regimented and require the use of rodent and ape models for initial validation.

Given the advances in genetic manipulation techniques for rodent malaria parasites, the identification of GAP vaccines against infections by pre-erythrocytic and blood stage malaria infections has become more feasible. Some of the pre-erythrocytic GAP studies have been extended to human malaria parasites: for example, in 2013, P. falciparum Δp52Δp36 GAPs, that arrest in pre-erythrocytic stages, were tested in human adult volunteers [START_REF] Spring | First-in-human evaluation of genetically attenuated Plasmodium falciparum sporozoites administered by bite of Anopheles mosquitoes to adult volunteers[END_REF]; and they conferred partial protection, similar to the equivalent murine model P. berghei GAP PbΔp52Δp36 [START_REF] Annoura | Two Plasmodium 6-Cys familyrelated proteins have distinct and critical roles in liver-stage development[END_REF]. However, few candidates for blood stage GAP vaccines have been explored. For example, P. yoelii lacking genes involved in the purine salvage pathway (Aly et al., 2010;Ting et al., 2008) or P. berghei lacking an aspartic protease, plasmepsin-4, involved in hemoglobin degradation (Spaccapelo et al., 2010) and a merozoite surface protein 7 (Spaccapelo et al., 2011) displayed various degrees of attenuation, from limited peak parasitemia to delayed blood stage growth. Crucially, all these mutants generated self-resolving infections that induced strong and lasting immunity. Protection was effective against both erythrocytic and pre-erythrocytic stages of the parasite as well as across Plasmodium species (Aly et al., 2010;Spaccapelo et al., 2011;Spaccapelo et al., 2010), and lasted up to at least one year (Spaccapelo et al., 2010). Moreover, both antibodies and T cells (Aly et al., 2010), as well as antibody-dependent cellular cytotoxicity in which antibodies function by enhancing clearance of infected erythrocytes by splenic macrophages (Spaccapelo et al., 2011;Spaccapelo et al., 2010), were shown to be involved in protection.

Interestingly, P. berghei plasmespin-4 KO parasites were shown to be more rapidly cleared and induced stronger protective immunity than WT parasites treated by pyrimethamine (Spaccapelo et al., 2010). Taken together with our observations, we conclude that regardless of the specific effect of the genetic mutation, self-resolving, protective blood stage GAPs induce similar immune responses that are B-and T-cell dependent (Aly et al., 2010;this work) and rely on opsonized phagocytosis (Spacapello et al., 2010;this work). Importantly, this protection is stage-transcendent against homologous and heterologous challenges. In contrast there are no evidence of heterologous protection using liver stage GAPs.

Furthermore, it was shown that Plasmodium blood stage infection can suppress, through modulation of DCs maturation and their capacity to activate CD8 + T cell responses against the liver stage, inhibiting the establishment of a protective response during the liver stage leaving the host susceptible for the next infection [START_REF] Ocana-Morgner | Malaria blood stage suppression of liver stage immunity by dendritic cells[END_REF].

Overall, we speculate that early during blood stage infection, protective mechanisms are induced by low amounts of parasites, which might be progressively overwhelmed, and possibly replaced by inhibitory immune mechanisms caused by further parasite multiplication. The balance between protective and inhibitory mechanisms might depend on peak parasitemia (i.e., parasite load), or more importantly, on the parasite growth rate after the initial antigen stimulation in the host. One can then assume that any genetic modification that causes a growth defect would tip the balance towards protective mechanisms, resulting in parasite clearance before inhibitory mechanisms can be fully engaged. We further propose that PD-1 expression might constitute a crucial marker of protective vs. inhibitory responses and needs to be further exploited for both antimalarial drug and vaccine design. Future work should continue to dissect the different protective mechanisms induced by self-resolving blood stage GAP infections, with an emphasis to develop an effective multi-stage malaria vaccine. In order to achieve this aim it would be interesting to compare, using the same murine Plasmodium strain, the phenotypes of all the blood stage GAPs developed until now. This first step could help for the choice of a second mutation in order to develop a double mutant parasite maybe more performing in the development of the immune response. The last step in the vaccine development will be the construction of a P. falciparum mutant parasite, for example Pf3D7-hrfΔ and the assessment of mutation on the parasite viability.

Beside the whole parasite vaccine approach, we could also propose to use Plasmodium HRF as a subunit vaccine since anti-Plasmodium HRF Abs may mimic Plasmodium-hrfΔ induced protection. Nevertheless, the evidence does not support this hypothesis. First, we cannot hide the high sequence homology that exists between the human and the parasite HRF protein, even if structural differences have been highlighted. It cannot be excluded that this homology might generate cross-reactive Abs against human HRF, thus causing autoimmune reactions. Second, a recent study showed that immunization of mice with Plasmodium HRF resulted in a weak protective capacity is against malaria infections [START_REF] Taylor | Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection[END_REF].

Taken together these observations strongly support the fact that, at least in the case of HRF, whole organism approach should be used to generate sterile immunity.

Bibliography Résumé:

De études récentes suggèrent une forte relation entre susceptibilité au paludisme et réponse allergique. En effet, des niveaux élevés d'histamine plasmatique et tissulaire ont été associés à la sévérité de la maladie chez l'homme infecté par P. falciparum et dans de nombreux modèles animaux. Histamine releasing factor (HRF), une protéine proinflammatoire libérant l'histamine au cours des processus allergiques, est produite par le parasite au cours des infections palustres modérées et sévères, suggérant l'implication du HRF dans l'altération des réponses immunitaires et dans la pathogenèse. Les objectifs de ce travail consistaient à évaluer le rôle de la protéine parasitaire HRF dans le développement de la réponse immunitaire et à déterminer si son expression est associée à la sévérité de la maladie en étudiant deux parasites murins, PbANKA et PbNK65, déficients pour la protéine HRF (hrfΔ). Les souris infectées avec des sporozoïtes PbANKA-hrfΔ ont montré une diminution de la fréquence du neuropaludisme associée à un déficit du développement des parasites mutants au cours du stade hépatique et à une augmentation précoce systémique d'IL-6. En outre, l'infection par des parasites PbNK65-hrfΔ a confirmé l'importance de la protéine HRF dans la virulence du parasite. En effet, l'infection par les parasites PbNK65-hrfΔ est caractérisée par l'élimination du parasite qui conduit à une protection durable et au développement d'une mémoire immunitaire caractérisée par une augmentation d' IL-6, une diminution de l'expression de PD-1 sur les cellules T et une amélioration de la phagocytose dépendante des anticorps. La protéine HRF est le premier gène de parasite Plasmodium dont l'effet direct sur la réponse immunitaire de l'hôte est démontré.

Mots clés: Plasmodium, Histamine releasing factor, GAP, IL-6, immunité protectrice, vaccin

Abstract:

Recent findings have raised the hypothesis that clinical susceptibility to malaria may be related to allergy-type response. In human infection with P. falciparum, as well as in murine models of malaria, increased levels of histamine have been shown to be associated with disease severity. Histamine releasing factor (HRF), shown to be implicated in the release of pro-inflammatory histamine during late-phase allergy, was demonstrated to be produced by the parasite during mild and severe malaria infections suggesting that Plasmodium HRF may affect host immune responses and contributes to the pathogenesis. The objectives of this work were to evaluate the role of Plasmodium HRF in the development of the immune response and to determine whether its expression is associated with the severity of malaria disease by studying two HRF-deficient (hrfΔ) murine parasites (PbANKA and PbNK65). Infection with PbANKA-hrfΔ sporozoites showed a decrease in the frequency of ECM due to the impairment of the development of the mutant parasites in liver stages as a consequence of the up-regulation of IL-6. Infection with PbNK65-hrfΔ parasites confirmed the importance of HRF in enhancing the virulence of the parasite. Indeed, PbNK65-hrfΔ infection results in parasite clearance leading to a long-lasting protection and immune memory as reflected by an up-regulation of IL-6, a down-regulation of PD-1 expression on T cells and in the enhancement of Ab-mediated phagocytosis. HRF is the first parasite gene which directly modulates the host immune response. Keywords: Plasmodium, Histamine releasing factor, GAP, IL-6, protective immunity, vaccine

Figure 1 :

 1 Figure 1: Global distribution of Malaria.[START_REF] Shetty | The numbers game[END_REF]

Figure 2 :

 2 Figure 2: Phylogenetic relationships among Plasmodium species (Prugnolle et al., 2010).

Figure 3 :

 3 Figure 3: Phylogenetic tree demonstrating the putative relationships between the major types of eukaryotic organisms[START_REF] Ward | Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote[END_REF].

Figure 4 :

 4 Figure 4: Schematic representation of the Plasmodium life cycle. The cycle consists of asexual development phase in the vertebrate host (liver and erythrocytic stages) and one sexual development phase inside the mosquito hosts (Klein, 2013).

Figure 5 :

 5 Figure 5: Schematic representation of the different stages of P. falciparum development in erythrocyte. (A) Major morphological changes occur during the merozoite invasion of erythrocytes. Initial weak attachment followed by reorientation, formation of tight junction and secretion of apical organelle contents and invagination (Wright and Rayner, 2014). (B) Cellular modifications occurring during parasite growth in the host erythrocyte and confirmed locations of exported proteins within the export. Protein names in dark blue indicate resident proteins of that organelle, names in green indicate transient location, and names in light blue indicate proteins, the of which location has not been confirmed. Abbreviations: ER, endoplasmatic reticulum; MC, Maurer's clefts; PVM, parasitophorous vacuolar membrane; TVN, tubular-vesicular network (Mundwiler-Pachlatko and Beck, 2013). (C) Parasite development process. Merozoite attach and RBCs invasion (0-5min).The parasite develops in a parasitophorous vacuole (PV) through the ring (0-24 hours), trophozoite (24-36 hours) and schizont stages (40-48 hours). In mature-stage (>24 h) news membrane-bound structures appear in RBC (Knobs, Maurer's clefts, etc.). After approximately 48 hours there are the infected RBC lyse and 16-32 daughter merozoites are released[START_REF] Maier | Malaria parasite proteins that remodel the host erythrocyte[END_REF].

Figure 6 :

 6 Figure 6: Potential mechanism of malaria-induced fever. (1) Hemozoin presents or internalizes plasmodial DNA. (2) TLR9 recognizes plasmodial DNA and initiates the innate immune response. (3) NF-kappaB activated pro-inflammatory cytokines induce COX-2. (4) COX-2 upregulates prostaglandins leading to a change in the setpoint of the thermoregulatory center (fever).[START_REF] Schumann | Malarial fever: hemozoin is involved but Toll-free[END_REF]

Figure 7 :

 7 Figure 7: Schematic representation of mechanisms involved in the development of CM. (A) Schematic representation of the adhesion properties of P. falciparum iRBCs to different host cells. The iRBCs mature forms have the ability to bind to a range of host cells, such as endothelium, uRBCs (rosetting) and platelets (platelet-mediated clumping)[START_REF] Rowe | Adhesion of Plasmodium falciparuminfected erythrocytes to human cells: molecular mechanisms and therapeutic implications[END_REF]. (B) Schematic representation of severe malarial disease events. iRBCs adherence to receptors expressed by brain microvascular endothelial cells is followed by merozoites releasing. The release of parasite GPI induces an inflammatory response. A local acute-phase response then occurs, which involves activation of the endothelium and local production of cytokines and chemokines resulting in upregulation of expression of cell-adhesion molecules by endothelial cells. GPI can also function as a ligand for CD1d-restricted natural killer T (NKT) cells, leading to their activation. Activated NKT cells can regulate the differentiation and activation of CD4 + T cells into Th1 or Th2 cells. In addition, chemokines recruit monocytes and activate neutrophils. Recruited monocytes can then differentiate into macrophages and become arrested in brain microvessels. Macrophages can also be activated by GPI, a process that is amplified by IFN-γ. Local activated macrophages produce more chemokines amplifying the infiltration of cells, sequestration of iRBCs and release of microparticles. After several more cycles, γδ T cells and CD8 + T cells might become involved, releasing more chemokines and cytokines both systemically and locally and possibly inducing perforin-mediated lesions in the endothelium. Together with locally arrested macrophages, platelets are sequestered and participate in altering endothelial-cell functions. More microparticles of platelet, endothelial-cell and monocyte origin are released, which leads to the dissemination of pro-inflammatory and pro-coagulant effects. Finally, damage to the endothelium, with possible perivascular haemorrhage, axonal injury, and neurotransmitter and metabolic changes, can ensue.[START_REF] Schofield | Immunological processes in malaria pathogenesis[END_REF].

Figure 8 :

 8 Figure 8: Pattern of P. falciparum infection outcome with age in a malaria endemic population.Asymptomatic infection (green), mild disease (febrile episodes caused by malaria; blue) and severe or lifethreatening disease (red)(White et al., 2014).

Figure 9 :

 9 Figure 9: IFN-γ producers and their activation. Plasmodium antigens are recognized by innate immune cells (γβ T cells, DCs and NK cells) via PPRs (TLRs and TCR) expressed on cells surface and activate the cells to produce IFN-γ. Moreover, IL-12 production and the antigen presentation from activated DCs strong activate the ab T cells leading to high IFN-γ production. Furthermore, activated ab T cells produce IL-2, resulting in the proliferation of γβ T cells and NK cells and ab T cells themselves. IL-12 can also activate NK cells.[START_REF] Inoue | Roles of IFN-gamma and gammadelta T Cells in Protective Immunity Against Blood-Stage Malaria[END_REF]

Figure 10 :

 10 Figure 10: Linking innate and adaptive immunity to blood-stage malaria. In response to parasite ligands recognized by pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and CD36, or inflammatory cytokines, such as interferon-γ (IFN-γ), dendritic cells (DCs) mature and migrate to the spleen. Maturation of DCs is associated with the up regulation of IL-12 secretion, and antigen presentation to CD4 + T cells. DCs can also cross-present antigen to CD8 + T cells. IL-12 activates natural killer (NK) cells to produce IFN-γ and promote adaptive and humoral immune responses via antibody subclass switching. IFN-γ production by NK cells, CD8+ T cells, and differentiated CD4 + T cells primes antigen presenting cells to produce cytokines and chemokines, further assists DC maturation, and also activates macrophages, resulting in phagocytosis and killing of parasites via the production of nitric oxide and reactive oxygen intermediates. CD4+ T cells can also produce IL-10, which can induce apoptosis of DCs. Once activated, macrophages can also participate in antigen presentation. IL-2 produced by activated CD4 + T cells further activates NK cells to produce IFN-γ.(Stanisic et al.,

Figure 11 :

 11 Figure 11: Malaria vaccine candidate antigens. List of P. falciparum and P. vivax candidate antigens indicated the category of malaria vaccine being developed and the life cycle stage targeted. Antigens evaluated in preclinical trials or have entered at least Phase I clinical trails, according to WHO malaria vaccine rainbow tables, are indicated in bold.[START_REF] Barry | Strategies for designing and monitoring malaria vaccines targeting diverse antigens[END_REF]

Figure 12 :

 12 Figure 12: Liver stages of arrest for radiation-attenuated sporozoites (RAS), genetically attenuated parasites (GAP), and wild type sporozoites with chemoprophylaxis (CPS). Shortly after entry, sporozoites form a parasitophorous vacuole (PV) for further development into liver trophozoites, schizonts, and eventually merozoites. Release of merozoites marks the end of the liver stage and the start of blood-stage infection. GAPS are produced by deletion of selected parasite genes (Δ). Both RAS and the GAP ΔP36p/P36, ΔSAP1, ΔSLARP arrest early in the liver before completion or remodeling of the PV. Development into intermediate stages is observed for the GAP (ΔUIS3/UIS4, ΔE1α, ΔE3, ΔFABI, ΔFABB/F, ΔFABZ, and ΔPKG), and the CPS-PYR (pyrimethamine), -CN (centanamycin) and-PQ (primaquine). Later liver-stage arrest is observed for CPS-AZ (azithromycin) and CD (clindamycin). Blood-stage exposure occurs only for CPS-CQ (chloroquine) where the latest parasite arrest is observed (Nganou-Makamdop and Sauerwein, 2013).

Figure 13 :

 13 Figure13: Histamine Releasing Factor (HRF), is secreted through exosomes that are regulated by TSAP6 and p53. hrf is a key gene in the process of tumour reversion and a pharmacological target for sertraline, thioridazine and artemisinin. Well conserved through phylogeny as a pro-survival, growth-stimulating and antiapoptotic factor, it is also a transcription factor for OCT4. HRF binds to CHFR and to elongation factor eEF1A and eEF1Bβ, leading to roles during mitosis and in protein synthesis, respectively. GDI, GDP dissociation inhibitor.[START_REF] Telerman | The molecular programme of tumour reversion: the steps beyond malignant transformation[END_REF]

  Figure 14: HRF structure of S. Pombe and P. knowlesi. (A) Two-and (B) three dimensional structure. The arrow indicated the additional a-helix from residue 22 to 30 in P. knowlesi, which replaces a b-sheet in the oerthologous S. Pombe. Note that in the 3D structure the torsion induced by the α-helix instigates the resulting protrusion exhibited by the two β-sheets. (Hinojosa-Moya et al., 2008)

Figure 15 :

 15 Figure 15: Mechanism of secretion and hypothetical modification of HRF. Secretion of HRF is enhanced by hydrogen peroxide, M-CSF, dioxin, and so forth during late phase allergic inflammation. HRF is secreted outside of cells through H,K-ATPase-or TSAP6-mediated non-classical pathway and undergoes posttranslational modification. (a) Processing of secreted full-length HRF by proteolytic enzyme releases truncated HRF (N11TCTP) and short-length N-terminal fragments (PTD). (b) Oxidants can form an intermolecular disulfide linkage between two of HRF that produces the dimerized HRF (dTCTP). (c) Alternatively, HRF can cross-react with IgE which dimerize the intact HRF (dHRF-IgE) by connecting a HRF monomer with an other HRF monomer.Active structure of HRF now can bind to its specific receptor (dHRF-R) to stimulate the mediator release from basophils. In addition to dHRF-R, subset of IgE can serve as a receptor for HRF dimer in FcεRI-expressing cells. Since these modifications might be possible at inflamed site, extracellular HRF will affect around cells only during the late phase or chronic allergic inflammation.[START_REF] Kim | Dimerization of TCTP and its clinical implications for allergy[END_REF]

Figure 16 :

 16 Figure 16: IgE/FcεRI complex and parasite HRF represent two mechanisms by which histamine release is triggered from its cellular stores during malaria infection. Histamine is continuously released into host body starting from the beginning of the infection which allows the development of ECM.

Highlights•

  In this work we were interested in the role of the P. berghei HRF protein during malaria infection since it could affect the immune responses of the host in vivo, at least through its potential to induce secretion of histamine and to contribute to the pathogenesis of cerebral malaria. To characterize the role of Plasmodium HRF in parasite transmission, development and disease pathogenesis, HRF-deficient P. berghei ANKA (PbANKA-hrfΔ) parasite were generated. The parasite ability to induce ECM was studied comparing mice infected with either WT or mutant PbANKA-hrfΔ parasites. Parasitemia, survival rates, and immunological monitoring were all used to establish the mutant phenotype. Identification of hrf as a gene playing a role during parasite development in preerythrocytic stages. The development of PbANKA-hrfΔ parasite was specifically inhibited during liver stage development in vivo, resulting in reduction of merozoites numbers initially released in the blood, and an extension of the time between sporozoite injection and blood stage development.

3

  Institut Pasteur, Unité de Biologie et Génétique du Paludisme, Paris F-75015, France.SummaryPlasmodium spp., which causes malaria, produces a histamine-releasing factor (HRF), an orthologue of mammalian HRF. Histamine-releasing factor produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre-erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito-injected parasite form into the erythrocyte-infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL-6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti-IL-

Fig. 2 .

 2 Fig. 2. Detection of HRF protein in pre-erythrocytic stages. A. Immunofluorescent detection of HRF in fixed WT and HRFΔ1 GFP-tagged sporozoites. Representative images are shown for anti-HRF antibody together with bright-field images and DAPI stained nuclei. Scale bar 10 μm. B. Intracellular staining for detection of HRF within hepatocytes was performed by incubating HepG2 cells with WT or HRFΔ1 sporozoites at MOI of 1:1 for 48 h. Cells were fixed and stained with anti-HRF rabbit IgG antibodies followed by Alexafluor 568-labelled donkey anti-rabbit IgG antibodies. Experiments were replicated twice with similar results. Scale bar, 10 μm.

Fig. 3 .

 3 Fig. 3. Comparison of the liver infection by the WT and HRFΔ1 parasites. A, B. 5 × 10 5 WT and HRFΔ1 sporozoites were injected together in the tail vein of C57BL/6 mice to compare the parasite ability to cross the sinusoidal barrier. WT and mutant parasites were imaged in the liver of the same infected mice at 3 h post-inoculation. A. Representative intravital confocal images of WT (red) and HRFΔ1 sporozoites (green) that invaded the liver parenchyma at 3 h post-inoculation. The liver parenchyma (black) is delineated by the liver sinusoids (white), which are labelled by the IV injection of Alexa Fluor 647 BSA. Scale bar, 20 μm. B. Percentage of sporozoites that invaded the liver parenchyma (black bar) or not (white bar) after 3 h post-inoculation. Similar numbers of parasites were detected in the imaged area (n = 18 WT sporozoites, n = 22 HRFΔ1 sporozoites). C. Role of HRF in infectivity of pre-erythrocytic stages of the parasite. Forty hours after inoculation of C57BL/6J Rj mice IV or ID with 10 000 WT or HRFΔ1 sporozoites, livers were isolated and total RNA extracted. Parasite loads were determined using real-time RT-PCR by measuring P. berghei 18S rRNA. Gene mRNA expression was normalized to the endogenous control gene HPRT. Data are presented as the means ± SD (n = 5) from two independent experiments. ***0.02 < P < 0.004 (Kruskal-Wallis multiple comparison test). D. Comparative development of independent HRF mutant clones. Analysis of the three mutant clones was performed as in E. Data are presented as the means ± SD from two independent experiments. For statistics, n = 5, **P < 0.01, *** P < 0.001 according to the Kruskal-Wallis test. E, F. Groups of C57BL/6 mice were injected IV with 10 000 GFP-WT or GFP-HRFΔ1 sporozoites and livers and blood were collected at indicated time points. Parasite loads in the liver were assessed by measuring parasite LISP1 mRNA by real-time RT-PCR (E). Gene mRNA expression was normalized to the parasite control gene HSP70. Data are presented as the means ± SD from six individual values. *P < 0.01. In parallel, parasitaemia (F) was determined and means ± SD from six individuals values are shown. Significant differences were obtained using The Kruskal-Wallis multiple comparison test (*P = 0.02, ***P < 0.002). Shown are data from two distinct experiments (E, F).

Fig. 4 .

 4 Fig. 4. Immune response genes are differentially regulated by WT and HRFΔ1 parasites. Transcription of the immune response genes including MIP-1β (A), MIP-2 (B), VCAM-1 (C), ICAM-1 (D), TNF-α (E), IL-6 (F) and IFN-γ (G) in the liver (n = 6 per group) at different time points post-infection as evaluated by real-time RT-PCR. mRNA expression was normalized relative to HPRT expression for each mouse strain.The asterisk indicates that differences are significant (Mann-Whitney test, 0.01 < P < 0.05), double asterisk indicates that these values are significantly different (Mann-Whitney test, 0.005 < P < 0.01) and triple asterisk indicates that differences are significant (Mann-Whitney test, 0.001 < P < 0.007). (H) C57BL/6 mice (6/group) were inoculated with 10 000 WT, HRFΔ1o rp 3 6 Δ sporozoites. Forty hours later, livers were isolated and total RNA extracted. Parasite load was determined by using primers specific for the P. berghei 18S rRNA by real-time RT-PCR, which expression was normalized relative to HPRT expression. (I) IL-6 expression was determined in the same liver samples using real-time RT-PCR by measuring IL-6 mRNA. Triple asterisk indicates that differences are significant (Mann-Whitney test, 0.003 < P < 0.01).

Fig. 5 .

 5 Fig. 5. Leukocyte patterns are differentially regulated by WT and HRFΔ1 parasites. At indicated time points post-infection, livers from C57BL/6 mice infected with 10 000 GFP-WT or GFP-HRFΔ1 sporozoites of Pb ANKA strain (n = 6 per group) were taken and leukocytes associated with liver tissue were analysed by using the following leukocyte markers: CD4 (A), CD8 (B), NK1.1 (C), CD11b (D), CD19 (E), B220 (F), MHCII (G), Ly6G (H), DX5/MAR-1 (I), Siglec-F (J), CD11c/CD11b (K) and F4/80 (L). Data are expressed as number of cells. The asterisk indicates that differences are significant (Mann-Whitney test, 0.018 < P < 0.05), double asterisk indicates that these values are significantly different (Mann-Whitney test, 0.008 < P < 0.025). Values represent the mean ± SD of two experiments.

Fig. 7 .

 7 Fig. 7. Capacity of the recombinant HRF to down-modulate LPS-induced IL6-and other cytokine-mRNA expression and protein synthesis. C57BL/6 mice were injected i.v with the indicated doses of recombinant Pb ANKA HRF (A and B) or with recombinant PbMSP133 (panels C and D) 15 min prior to the administration of 0.1 mg of LPS. Two hours later, livers were harvested and processed for IL-6 mRNA transcript analysis by RT-PCR (A, C) and in parallel plasma were tested for IL-6 content by ELISA (B, D). As control, HRF was injected alone at 0.1 mg per mouse (far left bar). In the same experimental setting, mRNA transcripts for IL-6, IL-10, IFN-γ, TNF-α and IL-1 (E-H) were measured in mice injected with LPS alone, HRF alone, or in combination. One representative analysis out of 2 is shown and the means ± SD of values obtained in three mice. For statistics in all graphs, **P < 0.05 < 0.01, ***P < 0.001 according to the Kruskal-Wallis test.

Fig. 8 .

 8 Fig. 8. Detection and quantification of HRF protein in various biological fluids. A. Supernatants from HepG2 cells cultured in the presence of WT, HRFΔ1 sporozoites, or in the absence of parasite were immunolabelled with anti-HRF antibody. B. Presence of HRF was measured in the liver extract of mice infected with 10 5 WT sporozoite at 40 h post-infection by using ELISA. C. The level of HRF in plasma of infected mice with WT and HRFΔ1 (not shown) was measured along with parasitaemia. One representative experiment among three are shown.

Fig. S1 .

 S1 Fig. S1. Disruption of the pbHRF gene. Schematic representation of the strategy utilized to knock out the PbHRF gene via double-crossover homologous recombination. Red lines represent regions of homology for recombination. Integration of the targeting sequence disrupt the HRF-coding sequence and replace it by drug resistance (hDHFR) marker. Specific PCR primers indicated genomic integration of the hDHFR targeting vector (within the hrf/tctp locus) within the PbANKA clone. A, B and C primers were used for PCR analysis. The amplicons are diagnostic for lane 1, wild-type locus; lane 2, integrated plasmid; lane 3, H2O. (A) Band sizes corresponding to 2681 bp in lane 2 indicated the integration of the plasmid (hDHFR cassette (1600 bp) and 5′ (487 bp) and 3′ (594 bp) PbHRF UTRs). A PCR product of 1760 bp in lane 1 indicated wild-type locus. (B) In lane 2 a PCR product of 700 bp indicated the HRFΔ1 mutant. (C) PCR product of 1100 bp indicated the HRFΔ1 mutant. (D) Southern blot analysis of the PbWT and PbHRFΔ1 mutant locus in Pb ANKA parasites. Genomic DNA was cleaved with EcoRV, transferred and probed with a HRF probe. The sizes of the bands are indicated in blue in locus representation. Line 1 represents the WT locus (1840 bp), whereas line 2 represents the genomic integration of the hDHFR targeting vector (2780 bp). Fig. S2. Assessment of the phenotype of additional HRFdeficient clones. C57BL/6 mice were inoculated with 10 000 isolated WT or HRFΔ2o rH R F Δ3 Pb ANKA. (A) Parasitaemia (Mann-Whitney test; *P < 0.02; ***P < 0.001) and (B) Kaplan-Meier survival plots (log-rank test) were recorded over time. Results are from two independent experiments. Fig. S3. Comparable gliding locomotion capacity, EEF formation, cell traversal ability and in vivo cycling of WT and HRFΔ1 Pb ANKA sporozoites. (A, B) Circular sporozoite motility of WT and HRFΔ1 Pb ANKA parasites expressing GFP fluorescence images of WT and HRFΔ1 sporozoites was tracked on glass cover slides. Individual frames are 2 s apart. The rings indicate the maximum intensity projection over 30 frames showing sporozoite gliding motility (arrows) or passive floating parasites (stars). (C) Percentage of gliding parasites expressed as a ratio between ring forms and total parasites, WT (54% ± 8), HRFΔ1 (48.8% ± 12). (D, E) Comparison of exoerythrocytic forms between cultured GFP-WT and GFP-HRFΔ1 Pb ANKA sporozoites on HepG2 cells for 24 h. EEFs in each well were counted and expressed as EEFs plus SD of duplicate wells.

  Figure S1!

  Figure"S5"

Figure 1 .

 1 Figure 1. PbHRF protein expression and phenotype of mutant parasites. (A-D) Anti-HRF-based immunoluorescence (red) was used to detect HRF in GFP-expressing WT or PbNK65-hrfΔ1 sporozoites (A), liver stages obtained 48 h p.i. of HepG2 cells with sporozoites at a multiplicity of infection of 1:1 (B), infected erythrocytes (C), and gametocytes (D). Nuclear DNA stained with DAPI and phase-contrast images are shown. (A-D) Bars, 6 µm. (E) Blood stage parasitemia and survival of C57BL/6 mice (Kaplan-Meier survival plots) after i.p. injection of 10 5 , 10 4 , and 10 3 WT or PbNK65-hrfΔ1-infected iRBCs measured over several days. (F and G) The kinetics of parasite load in the spleen of WT or PbNK65-hrfΔ1-infected mice was determined by RT-qPCR analysis of P. berghei 18S rRNA expression relative to mouse HPRT mRNA levels (F) or low cytometric analysis of parasitemia (G). (H) Spleen size of WT or PbNK65-hrfΔ1-infected mice at day 6 p.i. (I and J) Splenic index (I) and total cell number (J) were compared at day 6 p.i. between mice infected with 10 5 WT and PbNK65-hrfΔ1 iRBCs. Control: splenic index and cell number from naive mice. Error bars, SEM. Data are representative of three (A-D and H-J), six (E), and two (F and G) independent experiments with ive to six mice per group. *, P = 0.029; **, P = 0.003; Mann-Whitney test. CTL, control; nb, number.

Figure 2 .

 2 Figure 2. IL-6 expression by neutrophils and DCs and T and B cells are essential for the clearance of PbNK65-hrfΔ parasites. (A and B) mRNA levels (RT-qPCR) normalized to HPRT of IL-6 in the spleen (A) and in the serum (ELI SA; B) measured at day 6 p.i. from WT or PbNK65-hrfΔ1-infected mice. Control (CTL): mRNA and sera from naive mice. (C) WT or IL-6 KO C57BL/6 mice were infected i.p. with 10 5 PbNK65-hrfΔ1 iRBCs. Parasitemia and mouse survival (Kaplan-Meier survival plots: log-rank test; P = 0.0046) were followed over time. (D) Frequency and absolute numbers (nb) of IL-6-expressing splenic macrophages, DCs, and neutrophils at day 6 and day 20 p.i. from mice (ive per group) infected with 10 5 WT or PbNK65-hrfΔ1 iRBCs or from naive mice (CTL). (E and F) WT or T cell-deicient (E) or B cell-deicient (F) C57BL/6 mice were infected i.p. with 10 5 PbNK65-hrfΔ1 iRBCs, and parasitemia was followed over time. (G) Protected mice were treated with IgG or with anti-CD3-depleting antibody 1 d prior to a challenge with WT parasites followed by two booster injections of anti-CD3 at days 1 and 3 after challenge. Error bars, SEM. Data are representative of four (A and B), three (C and E-G), and two (D) independent experiments with ive to seven mice per group. *, 0.0028 < P < 0.046; **, P = 0.019; ***, P = 0.0097; Mann-Whitney test.

Figure 3 .

 3 Figure 3. Infection with HRF-deicient blood stage parasites ensures long-lasting cross-species and cross-stage protection. (A) PbNK65-hrfΔ1protected mice were challenged with 10 5 WT PbNK65 iRBCs at the indicated time points where control naive mice were also infected with 10 5 WT PbNK65 iRBCs. (B-D) Parasitemia and survival were measured over time. Parasitemia and Kaplan-Meier survival plots of PbNK65-hrfΔ1-protected mice challenged with 10 5 PbANKA (log-rank test; P = 0.0027; B) or P. yoelii YM (log-rank test; P = 0.0047; C) iRBCs at day 20 and day 23 p.i., respectively, or with 10 4 GFP-expressing WT PbNK65 sporozoites (log-rank test; P = 0.0047) at day 36 p.i. (D). (E) Intrahepatic parasite development in experimental and control mice from D was assessed by RT-qPCR analysis of the liver stage-speciic LSP-2 marker at 40 h p.i. of sporozoites. (F and G) PbNK65-hrfΔ1-protected mice were challenged with 10 4 Pb ANKA (F) or P. yoelii YM (G) sporozoites at day 25 p.i., and parasitemia and survival (log-rank test; P = 0.0082) were determined over time. Naive mice infected on the same day with PbANKA (F) or with P. yoelii YM (G) sporozoites were used as controls. Error bars, SEM. Data are representative of two (A) and three (B-G) independent experiments with four to eight mice per group. **, P = 0.015; Mann-Whitney test. Py, P. yoelii; SPZ, sporozoites.

Figure 4 .

 4 Figure 4. PbNK65-hrfΔ-induced immunity is T and B cell dependent and involves the secretion of Plasmodium-speciic IgG2c antibodies. (A) ELI SA detection and quantiication of antiparasite-speciic antibodies of various isotypes in mouse sera at day 20 p.i. with WT or PbNK65-hrfΔ1 parasites. Control (CTL): sera from naive mice. (B) Separated total protein extracts from WT PbNK65 iRBCs were incubated with the IgG fraction from three independent mice infected with either PbNK65-hrfΔ1 or WT parasites at day 20 p.i. or with normal mouse IgG. (C) C57BL/6 or FcRγ KO mice were infected i.p. with 10 5 PbNK65-hrfΔ1 iRBCs, and parasitemia was followed over time. (D) Puriied IgG antibodies from PbNK65-hrfΔ1-protected mice or from naive mice were injected i.p. 1 d before and 1 d after infection with 10 5 WT PbNK65 iRBCs, and parasitemia was recorded over time. Ab, antibody; Ctl, control. (E) Sorted CD11b + cells from naive or PbNK65-hrfΔ1-protected mice were transferred into WT C57BL/6 mice and immediately infected with 10 5 WT PbNK65 iRBCs. Parasitemia was recorded over time. Error bars, SEM. Data are representative of three (A-D) and two (E) independent experiments with ive to seven mice per group. *, 0.019 < P < 0.03; **, 0.0079 < P < 0.01; ***, P = 0.0002; Mann-Whitney test.

Figure S2 .

 S2 Figure S2. Assessment of leukocyte depletion and role of neutrophils and DCs in the occurrence of splenomegaly. (A and B) In vivo depletion of neutrophils using antineutrophil antibody (B) or normal IgG (A) was assessed by measuring at day 6 p.i. the percentage of residual Ly6G + neutrophils in spleens by FACS analysis. (C and D) Depletion of DCs was performed by injection of diphtheria toxin (DTX; D) or saline (C) into CD11c-DTR-GFP mice, and at day 6 p.i., the percentage of CD11c + cells was determined in spleens by FACS analysis. (E-J) Comparison at day 6 p.i. of splenic indexes and total number of leukocytes in WT and PbNK65-hrfΔ1-infected mice untreated or depleted of neutrophils (E-G) or DCs (H-J). (K) Control of T cell depletion (Fig. 2 G): protected mice received anti-CD3-depleting antibody 1 d before a challenge with WT parasites followed by two booster injections of anti-CD3 at days 1 and 3 after challenge with PbNK65 WT parasites. T cell depletion eficiency was assessed by FACS analysis using anti-CD4-PE or anti-CD8-APC in blood samples from protected mice that were challenged at day 15 p.i. with PbNK65 WT parasites. Analysis was performed 10 d after challenge. Error bars, SEM. Data are representative of two independent experiments with ive to six mice per group. *, P = 0.028; **, P = 0.015; ***, P = 0.009; Mann-Whitney test. CTL, control; FSC, forward side scatter; nb, number.

  -PbHRF-F (a) CGC GGG CCC GCG CAT TAT TAC CGT TGT CA PstI-5′UTR-PbHRF-R CGC CTG CAG GGC TTA TGC AAG TAT CGA ACAA KpnI-3′UTR-PbHRF-F CGC GGT ACC TTG CTA CAT GAC GCA TAA ACC EcoRI-3′UTR-PbHRF-R (a') CGC GAA TTC TGT GAA ATC GAC AAT GTT TTGG HRF5′-F (b) GCG ATA CAA ACA AAT TTA TTC AGC HRF3′-R (c') CGC AAG ATA TCA GAG CTT TTCA hDHFR3′-F (c) TGT TGT CTC TTC AAT GAT TCA TAA ATA GTT GG hDHFR5′-R (b')

Figure 17 :Figure 18 :

 1718 Figure 17: Marked differences in parasitaemia between WT and hrfΔ PbNK65-infected mice in blood stage development. Blood stage parasitemia and survival (Kaplan-Meier survival plots) of C57BL/6 mice infected WT or hrfΔ PbNK65 parasites (A) i.v. with 1000 isolated sporozoites or (B) i.p. with 10 5 iRBCs. Error bars, SEM. Data are representative of two independent experiments with 5 mice per group. (Following methods already described in the previous article (Demarta-Gatsi et al., 2016)).

Figure 19 :

 19 Figure 19: Cytokine production by blood-stage WT and hrfΔ PbNK65 infection. A, B and C) mRNA levels (RT-qPCR) normalized to HPRT of cytokine production in liver cells (A), in spleen cells (B) and in the serum (ELISA) (C) measured at day 6 p.i. from WT or PbNK65-hrfΔ infected mice. CTL: mRNA and sera from naive mice. Error bars, SEM. Data are representative of four (A,B and C) independent experiments with 5 mice per group. (Following methods already described in the previous article (Demarta-Gatsi et al., 2016)).

  GK1.5). The different blood cell populations were monitored by flow cytometry 24h after intravenous injection. Mice were challenged with 10 5 iRBCs of WT PbNK65 parasites and both parasite growth and cells depletion efficacy were monitored daily by flow cytometry in blood samples (Fig.21). The results confirmed the specific reduction of CD8 + or CD4 + T cells

Figure 20 :

 20 Figure 20: Innate immune cells are activated in the spleen and liver after malaria infection. (A) Liver analysis show increased levels of innate immune cells in mice infected with WT PbNK65 parasites. (B) Spleen analysis show increased levels of innate immune cells in mice infected with PbNK65-hrfΔ parasites. CTL: spleen from naive mice. Error bars, SEM. Data are representative of three independent experiments with 9 mice per group. (Following methods already described in the previous article (Demarta-Gatsi et al., 2016)).

  Figure 21: Influence of CD4 + or CD8 + T cells depletion on parasite development in protected mice. Protected mice were treated either with IgG or with anti-CD4-depletion Abs or anti-CD8-depletion Abs one day prior to a challenge with WT parasites followed by two booster injections of anti-CD4 and anti-CD8 at days 1 and 3 after challenge. Error bars, SEM. Data are representative of two independent experiments with 5 mice per group. (Following methods already described in the previous article (Demarta-Gatsi et al., 2016)).

  Figure 22: Protection induced by hrfΔ PbNK65 parasites is associated with reduced induction of PD1 + CD8 + CD62L -and PD1 + CD4 + CD62L -cells in spleen. (A) Representative frequency and absolute number of

Figure 23 :

 23 Figure 23: Drug treatment does not affect the development of long-lasting protection after infection with HRF-deficient blood stage parasites. (A) Protocol used to infect and to treat mice. (B) Blood stage parasitemia of C57BL/6 mice after i.p. injection of 10 5 WT iRBCs and treated at day 4 p.i. with 6 mg/kg WR99210 by subcutaneous injections. At day 19 p.i. infection of mice with either with 10 5 iRBCs WT or PbNK65-hrfΔ iRBCs.Mice group infected with PbNK65-hrfΔ parasites were subsequently, at day 52 p.i., infected with 10 5 WT iRBCs.Error bars, SEM. Data are representative of three independent experiments with 5 mice per group. (Following methods already described in a previous article[START_REF] Demarta-Gatsi | Protection against malaria in mice is induced by blood stage-arresting histamine-releasing factor (HRF)-deficient parasites[END_REF]).

  PbNK65-hrfΔ parasite-induced protection is associated with an increased number of central and effector memory B cellsThe observation that hrfΔ PbNK65 infected mice eliminate the parasite while they acquire a long lasting immune memory and high anti-parasite IgG Abs titers prompted us to analyse the generation of central and memory B cells. The germinal centers are the main sites where somatic hypermutations and class switch recombination occur. During the immune response, B cells express the activation-induced cytidine deaminase (AID) enzyme which acts on Ig gene loci to enhance antibody production and B cell functions. To visualize activated AID transcription in B cells, AID/YFP Tg mice were used, in which memory B cells containing populations can be divided into "central" memory (AID/YFP + CD19 + IgM + IgG) and "effector" memory (AID/YFP + CD19 + IgM -IgG + ) B cells. In order to investigate the generation of the immunological memory during Plasmodium infections AID/YFP Tg mice were infected i.p with 10 5 iRBCs of either WT or hrfΔ PbNK65 parasites. At day 15 p.i., mice were killed and spleen B cell populations analysed by flow cytometry (Fig.24). From our preliminary data we observe a significant difference in the memory B cells between mice infected with either WT or hrfΔ PbNK65 parasites. Additionally, characterization of memory B cells populations show also significant difference in both effector and central memory B cells between the two populations of mice. As compared to the WT PbNK65-infected spleen, we observed an increase in the total number of CD19 + AID/YFP + memory B cells and its population (IgM -IgG +

Figure 24 :

 24 Figure 24: Frequency of memory B cells in PbNK65-hrfΔ protected mice. Representative frequency and absolute number of AID/YFP + CD19 + memory B cells splenic leukocytes at day 15 p.i. with either 10 5 GFP-WT or hrfΔ iRBCs. (A) Representative frequency and absolute number of AID/YFP + CD19 + memory B cells splenic leukocytes expressing IgM -IgG + at day 15 p.i. (B) Representative frequency and absolute number of AID/YFP + CD19 + memory B cells splenic leukocytes expressing IgM + IgG -at day 15 p.i. Error bars, SEM. Data are representative of two independent experiments with 4 mice per group. (Following methods already described in a previous article (Demarta-Gatsi et al., 2016)).

Figure 25 :

 25 Figure 25: Hypotesis: understanding malaria pathogenesis associated with Plasmodium infection and allergic inflammation share common signalling pathways. The figure displays three major known pathologies:autoimmune diseases, infections diseases, and allergic responses. Indirect evidence from immunoepidemiological studies support that an association exist between malaria pathogenesis and mechanisms involving IgE-mediated inflammatory responses. This latter consist of classical pathway which starts with the binding of antigen (Ag)-specific IgE Abs to FcεRI expressed on various leukocytes (mast cells, basophils, and neutrophils), and upon aggregation, cells will release histamine and other inflammatory mediators. Beside this classical pathway, alternative pathway independent from IgE/ FcεRI complexes, are also able to trigger mediator release. One of the prototypes IgE-independent triggers is the histamine releasing factor (HRF).

  i. allowing us to explore the immune mechanisms in more detail over a longer period of time. Mice infection with PbNK65 WT or hrfΔ sporozoites shown, as previously observed with PbANKA, a delay in blood stage parasite development between mutant and WT parasite confirming a prolonged liver-stage development of Plasmodium hrfΔ. Moreover, infection with PbNK65-hrfΔ sporozoites results at day 17 p.i. in complete blood stage clearance of the parasite, in contrast to PbANKA-hrfΔ sporozoite infection that despite the development of a clinical immunity (no ECM) the parasite develops normally without being eliminated at the blood stage. More interestingly, differently from what was observed during PbANKA-hrfΔ iRBCs infection where there was no difference between WT and mutant parasite development and

Table 1 -Blood stage GAPs and their characteristics.

 1

	1 58 58

Plasmodium berghei histamine-releasing factor favours liver-stage development via inhibition of IL-6 production and associates with a severe outcome of disease Cédric Mathieu, 1,2 † Claudia Demarta-Gatsi, 1,2 † Adeline Porcherie, 1,2 Sara Brega, 3 Sabine Thiberge, 3 Karine Ronce, 1,2 Leanna Smith, 1,2 Roger Peronet, 1,2 Rogerio Amino, 3 Robert Ménard 3 and Salaheddine Mécheri 1,2 * 1

  Institut Pasteur, Unité de Biologie des Interactions Hôte Parasites, Paris F-75015, France.

2 Centre National de la Recherche Scientifique, Unité de Recherche Associée 2581, CEDEX 15, Paris F-75724, France.

Table S1 .

 S1 C57BL/6 mice were inoculated IV with 10 000 sporozoites from WT or HRFΔ1 Pb ANKA parasites. Livers were taken 40 h after infection and total RNA extracted. Parasite loads and IL-6 expression were determined using real-time RT-PCR by measuring P. berghei 18S rRNA and IL-6 mRNA respectively. Gene expression was normalized to the endogenous control gene HPRT. Values are expressed as means ± SD obtained in five mice. (C) Rescue of the phenotype of HRFΔ1 Pb ANKA parasite in IL-6 KO mice. IL-6 KO mice (seven mice per group) were infected IV with 10 000 GFP Pb ANKA WT or Pb ANKA HRFΔ1 sporozoites and parasitaemia (Mann-Whitney test; *P = 0.02, **P = 0.007) and survival rates (log-rank test) were followed over time. List of the oligonucleotides used for PCR of wild-type and recombinant parasites. Appendix S1. Supporting experimental procedures.

Table S1 . List of oligonucleotides used for RT-qPCR analyses

 S1

	Primer	Forward /	Sequence (5′-3′)
		Reverse
	Pb 18S	Forward	ATT AAT CTT GAA CGA GGA ATG GCT
		Reverse	TCA ATC GGT AGG AGC GACG
	Pb LSP2	Forward	GCC AAA TGC TAA ACC TAA TG
		Reverse	TGG GTT TGT ATT GTA TGC AC
	Pb HSP70	Forward	TGC AGC TAA TCA AAC TC
		Reverse	ACT TCA ATT TGT GGA ACA CC
	mu IL-23	Forward	CCA CCA GGA CTC AAG GAC AACA
		Reverse	GCA GGC TCC CCT TTG AAGA
	mu EBI3	Forward	CAG AGT GCA ATG CCA TGC TCC
		Reverse	GCC ACA CCG AGC CTG TAA GT
	mu IL-12p35	Forward	TAC TAG AGA GAC TTC TTC CAC AAC AAG AG
		Reverse	GAT TCT GAA GTG CTG CGT TGAT
	mu IL-12p40	Forward	GGA AGC ACG GCA GCA GAA TA
		Reverse	AAC TTG AGG GAG AAG TAG GAA TGG
	mu IFN-γ	Forward	AAA GGA TGC ATT CAT GAG TAT TGC
		Reverse	CGC TTC CTG AGG CTG GATT
	mu IL-6	Forward	AAA GAA ATG ATG GAT GCT ACC AAAC
		Reverse	CTT GTT ATC TTT TAA GTT GTT CTT CAT
			GTA CTC
	mu IL-10	Forward	GGC GCT GTC ATC GAT TTC TC
		Reverse	GAC ACC TTG GTC TTG GAG CTT ATT AA
	mu HPRT	Forward	CTG GTG AAA AGG ACC TCT CG
		Reverse	TGA AGT ACT CAT TAT AGT CAA GGG CA

Table S2 . List of oligonucleotides used for PCR of WT and recombinant parasites

 S2

20

© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 17, 542-558

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PbNK65-hrfΔ1 iRBCs. Control: splenic index and cell number from naive mice. Error bars, SEM. Data are representative of three (A-D and H-J), six (E), and two (F and G) independent experiments with ive to six mice per group. *, P = 0.029; **, P = 0.003; Mann-Whitney test. CTL, control; nb, number. on July 21, 2016 jem.rupress.org Downloaded from Published July 18, 2016

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37°C and then saturated with 1% (wt/vol) BSA (Sigma-Aldrich). Serum samples were assayed using serial dilutions and incubated for 2 h at 37°C. Speciic binding was detected on July 21, 2016 jem.rupress.org Downloaded from Published July 18, 2016 Malaria HRF abrogates antiparasite immunity | Demarta-Gatsi et al.

Mass spectrometry analysis, database search, protein identiication, and statistical analysis. After the immunoprecipitation, proteins were solubilized in denaturation bufer (10 mM Tris, pH 8, and 8 M urea). Proteins were reduced, alkylated, and digested with trypsin. Tryptic peptides were analyzed by on July 21, 2016 jem.rupress.org Downloaded from Published July 18, 2016

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Acknowledgements

Acknowledgements

We thank Peter Smooker and Kim Taylor (Department of Biotechnology and Environmental Biology, RMIT University, Bundoora, Australia) for providing P. berghei HRF plasmid. We are grateful to Barbara Willi, Novartis Pharma AG, Basel, Switzerland, and Lionel Apetoh, Dr Lionel Apetoh (INSERM U866, Dijon, France) for providing human recombinant IL-6 and IL-6 KO mice respectively. We thank CEPIA (Centre d'élevage, de production et d'infection des anopheles, Institut Pasteur) for providing anopheles mosquitoes. We also thank the Helmut Horten Foundation for the financial support provided to CDG.

ACK NOW LED GME NTS

We thank Peter Smooker and Kim Taylor for providing P. berghei HRF plasmid the CEP IA for providing A. stephensi, Shruthi Vembar for her thorough revision of the manuscript, and Veronique Hourdel for her technical assistance for mass spectrometry.

This work was funded by an Institut Pasteur grant to S. Mécheri. C. Demarta-Gatsi is supported by a predoctoral fellowship from the Helmut Horten Foundation.

The authors declare no competing inancial interests.

Submitted: 18 December 2015

Accepted: 26 May 2016

 S2) were used to assess genomic integration of hDHFR in PbNK65-hrfΔ clones. The primers used for PCR analysis include a + a' (B), b + b' (C), and c + c' (D), with gDNA from the following: lane 1, WT parasites; lane 2, hrfΔ clone 1; lane 3, hrfΔ clone 2; and lane 4, H 2 O. (E) Southern blot analysis of the pbhrf locus in PbNK65 WT, PbNK65 hrfΔ1, and PbNK65 hrfΔ2 mutant locus in PbNK65 parasites. WT locus = 1,840 bp, whereas hDHFR insertion = 2,780 bp. (F and G) C57BL/6 mice were inoculated with either 10 5 GFP-expressing WT, PbNK65-hrfΔ1, or PbNK65-hrfΔ2 iRBCs, and parasitemia (F) or survival (Kaplan-Meier survival plots: log-rank test; n = 11; P = 0.007; G) was followed over time. Error bars, SEM. Experiments were replicated three times.

HRF mutant parasites as vaccine candidates

The use of whole parasites to induce protection against malaria blood stage infection has a long history of inventive approaches. Early studies in the 1940s using P. knowlesi or P. falciparum infection in monkeys showed that high doses of killed parasites along with complete Freund's adjuvant provided protection against blood stage challenge (Pombo et al., 2002). In the 1980s, radiation-attenuated derivatives of P. berghei or P. yoelii blood stage parasites which initiate self-resolving infections in immuno-competent hosts, were found to be protective (Miyagami et al., 1987;Waki et al., 1982). Nevertheless, production and use of this live attenuated parasite presents different problems such as the large scale production