Unit: Parasite Molecular Immunology - CNRS URA 2581

Director: PUIJALON Odile

The Unit's central interest is to understand the factors that influence Plasmodium falciparum infection outcome, with the long term objective to prevent malaria morbidity and mortality. Research programmes include investigation of field parasite diversity, analysis of virulence factors, exploration of handling and clearance of infected red blood cells by the spleen using a perfused organ set up and the analysis of immune activation in malaria patients. Our recent findings include the first description of in vitro resistance to artemisinin derivatives, a major concern for malaria control. Pf155/RESA was identified as a novel type of virulence factor that helps the parasite resist exposure to febrile temperatures. VarO rosetting was shown to involve a novel parasite/receptor interaction and was classified amongst the "common" variant antigens. Analysis of the parasite transcriptome identified novel common themes in the early responses to several stresses inflicted to the parasites possibly involved in parasite adaptation to varying physiologic conditions. Exploration of handling of artesunate-treated infected red blood cells by the isolated-perfused human spleen showed rapid clearance in the red pulp by two mechanisms, pitting and phagocytosis. Finally, we have obtained the first demonstration of a protective role for V gamma9 T cells in vivo.

Field parasite diversity Marie-Thérèse Ekala, Nitchkarn Noranate, Odile Puijalon.

During the last years, our longstanding interest in field parasite diversity has focussed on drug resistance, the most pressing issue for malaria control. Within a network of Overseas Institutes Pasteur, we have described a high prevalence of numerous drug resistance mutations, some of which at fixation, both in Cambodia and French Guiana. In Madagascar, where in contrast the parasite population mostly carries susceptibility alleles, we have witnessed the first Pfcrt mutants. The most worrying finding was the observation of isolates from French Guiana with markedly elevated IC50 (and IC90) to artemisinin derivatives associated with a specific S769N mutation in the putative target the SERCA-PfATPase 6. This first description of artemisinin in vitro resistance is a major concern, since artemisinins are the cornerstone of most currently deployed combination therapies.

We have studied the temporal heterogeneity of the parasite population in Dielmo, a Senegalese village with holoendemic malaria. This identified novel mutations in drug target genes and provided new insights on the dynamics of gene flows under accurately recorded drug pressure.

Role of Pf155/RESA in maintaining the infected erythrocyte membrane integrity under conditions of thermal stress Serge Bonnefoy, Monica Diez Silva, Peter David

During invasion of the erythrocyte, the Ring Erythrocyte Surface Antigen (Pf155/RESA) is inserted into the host cell membrane, where it interacts with host's spectrin. Using a resa1 knock in/knock out strategy to create isogenic parasite lines, we could show that loss of RESA expression resulted in an increased physical susceptibility of the infected red blood cells to elevated temperatures. Furthermore their growth rate was impaired after a few hours incubation at a "febrile" temperature of 41°C. This indicates that association of RESA with the cytoskeleton in the ring stages protects it from the deleterious effect of exposure to febrile temperatures. We have therefore identified here a novel type of virulence factor that serves indeed the parasite to resist a very common non-adaptative host response to infection.

VarO-mediated rosetting, virulence and immune responses Inès Vigan, Micheline Guillotte, Solène Fastenackels, Odile Puijalon

The capacity of red blood cells infected with mature parasite stages to bind uninfected red blood cells ("rosetting"), together with the absence of antibodies disrupting rosettes are so far the only factors consistently associated with severe malaria in African children. We have developed a model for this phenotype using the Palo Alto varO antigenic variant. The same cytoadherence phenotype can be studied in vivo in the Saimiri monkey and in vitro in cultures in human red blood cells. In vivo varO parasites have a higher multiplication rate than non-rosetting siblings, indicating that rosetting indeed contributes to virulence. The adhesion domain has been identified (Figure 1). VarO rosetting differs from all interactions described so far and involves a novel parasite/receptor interaction. Studies in two Senegalese settings have highlighted a remarkably elevated seroprevalence in both villages indicating that varO is a "common" serotype, thus representing a potentially interesting vaccine target.

Pathways of response to a variety of physiological stresses Onguma Natalang, Guillaume Deplaine, Peter David

To identify novel parasite virulence factors contributing to parasite adaptation to physiological stresses, we have used a strategy based on exploring the parasite transcriptome. In collaboration with the Génopole PT2 (J.Y. Coppée and E. Bischoff), a DNA micro array platform has been used to explore the modulation of P. falciparum transcription under artesunate drug pressure, in resa-KO and resa wild type parasites and under a variety of physiologic stresses. Analysis of the data including adjustment of the developmental stage of the cultures by crigging is done in collaboration with J.P. Vert (Ecole des Mines). This has allowed identifying common themes in the early responses to most stresses that we have inflicted to the parasites so far.

Exploration of the function of the human spleen during P. falciparum infection using an ex vivo isolated-perfused organ Pierre Buffet, Peter David , Valentine Brousse.

The spleen plays a major role in parasite elimination, as well as in modulation of the parasite phenotype but this remains largely unexplored due to lack of appropriate experimental set up. We have established an isolated-perfused human spleen allowing survival of the spleen ex vivo for several hours. This was used to explore handling of artesunate-treated infected red blood cells by the spleen. Infected erythrocytes were rapidly and efficiently processed in the red pulp, essentially through two mechanisms: pitting of the parasites away from the host cell (Figure 2A) and intense phagocytosis of parasitised cells and parasite remnants (Figure 2B) (in collaboration with G.Milon, U. d'Immunophysiologie et Parasitisme Intracellulaire. M.Huerre U. de Recherche et d'Expertise Histotechnologie et Pathologie)

Immune activation in malaria patients. Charlotte Behr, Séverine Loizon, Philippe Boeuf, Jean Claude Michel.

Analysis of the Vbeta repertoire of peripheral T cells in Ghanaian children with malaria has outlined a remarkably stable profile in all clinical groups investigated, except for an increased frequency of two chains in CD4+ cells of cerebral malaria patients. This points to a specific T cell stimulation by some superantigens in cerebral malaria. Development of CytoQuant, an absolute quantitative RT PCR based on RNA standard curves has allowed us to explore the cytokine profile of children with severe malaria, thus providing novel insights into stimulation pathways in severe anaemia and cerebral malaria. Both approaches indicate that cerebral malaria and severe anaemia are two quite distinct pathophysiological syndromes. This is a collaborative work with Dr. B. Goka (Korle-Bu Hospital) and Prof B. Akanmori (Noguchi Institute) in Ghana.

The role of Vgamma9 T cells on infection outcome has been explored in vivo in the Saimiri sciureus monkeys. Modulating their function by synthetic antagonist or agonist molecules showed a protective role in vivo for Vgamma9 T lymphocytes that correlates with their cytotoxic activity against infected red blood cells in vitro. This first demonstration of a protective role for Vgamma9 T cells in vivo indicates that their pharmacologic modulation has therapeutic potential in malaria.

Figure Legends

Fig 1 a/ Var O rosette : the infected red blood cell, in the center, is surrounded by uninfected red blood cells firmly attached to its surface. b/ surface expression by Trichopulsa ni (High Five) cells of the var O adhesin binding domain, cloned in baculovirus, detected with a monoclonal antibody directed against the poly-His tail added to the construct C-terminus. c/ binding of uninfected erythrocytes to the surface of Trichopulsia ni (High Five) expressing the adhesive domain of varO.

Figure 2 A. Pitting of an infected blood cell as it squeezes to cross the sinus wall, from cord (co) to sinus lumen (sl), in the red pulp of an isolated-perfused human spleen. The parasite is left behind on the cordal side, leaving the " once infected " red blood cell reach the sinus lumen. (Giemsa staining).

Figure 2B. Phagocytosis of a parasitized erythrocyte in the red pulp of a human isolated-perfused spleen. Parasitized erythrocyte, containing pigment, can be seen within a macrophage (Giemsa staining on left ; immunohistochemistry labelling of macrophage with an anti CD68 antibody).  

Keywords: Malaria, Diversity, Virulence, Thermal stress, Spleen, T Lymphocytes, pathology

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