Introduction

The Unit's activities are centred along two main themes i) the analysis of parasite and host factors that shape the parasite/host interface in human infections; ii) drug resistance and drug discovery.

P. vivax malaria in Duffy-negative people in Madagascar

The Duffy blood group antigen is the portal of entry of Plasmodiumvivaxinto human red blood cells, and Duffy-negative people are considered resistant to vivaxmalaria. An assault to this paradigm was provided by our observation that P. vivax commonlyinfects and causes disease in Duffy-negative people in Madagascar, where human admixture has created a full range of Duffy erythrocyte expression phenotypes. Multilocus genotyping showed that multiple P. vivax strains have the capacity to infect Duffy-negative people.P. vivax prevalence in Duffy-negatives seems related to the proportion of Duffy-positive and -negative people in the area, suggesting local parasite evolution endowing P. vivax parasites the capacity to bypass the Duffy-negative invasion hurdle. These findings challenge current vaccination strategies targeting the parasite ligand, PvDBP, and raise concern about potential spreading of such strains away from Madagascar.

Parasite factors remodelling the red blood cell membrane of ring stages

The parasite remodels the RBC membrane very early after invasion. We explore the physiologic and mechanistic serologic consequences of early remodelling by the Ring Erythrocyte Surface Antigen (PfRESA), a parasite protein delivered to the red cell cytoskeleton. PfRESA. Association of PfRESA with the red cell cytoskeleton allows the young infected red cell to withstand exposure to febrile temperatures, and decreases membrane deformability.

Rosetting

Rosetting, the binding of uninfected red cells onto the surface of the infected red cell, is a parasite cytoadherence phenotype associated with severe malaria in African children. Rosetting is mediated by a subgroup of PfEMP1 variant adhesins. We have developed monovariant cultures of rosetting parasites, in which all parasites express the same adhesin; individual PfEMP1-derived adhesion domains were produced in their native conformation as recombinant proteins. Seroepidemiological studies in Senegal and Benin showed high seroprevalence of antibodies reacting with Palo Alto/VarO parasites. The response is associated with acquisition of protection against clinical malaria. The serological consequences of allelic polymorphism were investigated using three monovariant parasite lines expressing each an allele of an NTS-DBL1a1 domain implicated in rosetting. Both antibodies elicited in mice to the recombinant adhesion domain and antibodies acquired by humans living in malaria-endemic area display variant-specific surface seroreactivity. Thus, surface-exposed epitopes are allele-specific. Production of a vaccine targeting rosetting PfEMP1 adhesins will require either engineering of the immunogen to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity.

Retention and clearance of infected red blood cells by the spleen

One important role of the human spleen is the clearance of altered red cells. To explore the tissue mechanisms of the spleen clearance function, we established an isolated perfused human spleen model system. Unexpectedly, numerous young ring stages were retained and accumulated upstream from inter-endothelial slits of the red pulp where considerable RBC deformation is needed for the cell to re-enter a blood vessel. The moderately reduced elongation index of rings suggested an innate, mechanical retention process.The geometry of narrow and short inter-endothelial splenic slits was mimicked by filtration through a mixture of 5-25 micron metallic micro-beads. The retention rate of P.falciparum infected red cells inthe micro beads was similar to retention by the isolated-perfused human spleen at various stages of intraerythrocytic development. These in vitro results confirm the importance of the mechanical sensing of infected red cells by the human spleen.

Parasite drug resistance

Resistance of Plasmodium falciparum to multiple drugs is a major concernworldwide. Understanding the evolution of the target genes under changing drug policy regimens is crucial for optimal drug efficacy monitoring and for implementation of novel drug combinations. Collaborative studies with field-based teams and the National Reference Centre for antimalarial resistance allow us to analyse changes in drug target genes in Madagascar, Senegal, French Guiana and Cambodia. We observed local emergence of mutations, dissemination of resistance haplotypes and recently, discordant haplotypes/drug susceptibility associations, indicating that additional, yet unknown loci modulate drug susceptibility in several parasite populations.

Emerging evidence for the reduced efficacy of artemisinin derivatives in Cambodia is of considerable concern. Little is known on the mode of action and on the parasite response to artemisinins. Analysis of the dynamic changes of the parasite transcriptome after exposure to lethal doses of artesunate in vitroshed interesting new light on the mobilization of processes shaping the interface between the parasite and its environment and interference with metabolic pathways [5]. The contribution of some of these genes to the responses tending to overcome drug toxicity has been recently obtained. Genetic manipulation of a laboratory line resulting in over expression of a single gene rendered the parasites tolerant to artemisinin.

Sub proteases and rationale development of antimalarials

Parasite encoded subtilisin proteases implicated in the invasion process are promising novel drug targets. Current efforts concern elucidation of their biological function, characterisation of the catalytic properties of the parasite and recombinant enzymes, development of specific inhibitors using a combination of strategies and their validation using in vitro culture of P. falciparum and in vivo rodent malaria infection.

Keywords: red cell membrane, virulence, fever, rosetting, clearance, Duffy, antimalarials, spleen, malaria, pathogenesis

- Natalang O, Bischoff E, Deplaine G, Proux C, Dillies MA, Sismeiro O, Guigon G, Bonnefoy S, Patarapotikul J, Mercereau-Puijalon O, Coppée JY, David PH. (2008) Dynamic RNA profiling in Plasmodium falciparum synchronized blood stages exposed to lethal doses of artesunate. BMC Genomics. 9:388. PMID: 18706115

- Safeukui I, Correas JM, Brousse V, Hirt D, Deplaine G, Mulé S, Lesurtel M, Goasguen N, Sauvanet A, Couvelard A, Kerneis S, Khun H, Vigan-Womas I, Ottone C, Molina TJ, Tréluyer JM, Mercereau-Puijalon O, Milon G, David PH, Buffet PA. (2008) Retention of Plasmodium falciparumring-infected erythrocytes in the slow, open microcirculation of the human spleen. Blood. 112(6):2520-8. PMID: 18579796

- Ménard D, Barnadas C, Bouchier C, Henry-Halldin C, Gray R, Ratsimbasoa A, Thonier V, Carod J-F, Domarle O, Colin Y, Bertrand O, Picot J, King CL, Grimberg BT, Mercereau-Puijalon O, Zimmerman PA. Plasmodium vivax Clinical Malaria is Commonly Observed in Duffy-negative Malagasy People. Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):5967-71. PMID: 20231434.

- Deplaine G, Safeukui I, Jeddi F, Lacoste F, Brousse V, Perrot S, Biligui S, Guillotte M, Guitton C, Dokmak S, Aussilhou B, Sauvanet A, Cazals Hatem D, Paye F, Thellier M, Mazier D, Milon G, Mohandas N, Mercereau-Puijalon O, David PH, Buffet PA. The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro. Blood. 2010 Dec 20. PMID: 21163923