Unit: Parasite Molecular Immunology - CNRS URA 2581
Director: PUIJALON Odile
The Unit conducts an integrated analysis of the factors that condition the outcome of a P.falciparum infection. Four main axis are explored: analysis of the activation of immuno-competent cells during clinical attacks, identification of parasite pathogenicity factors, exploration of the role of the spleen using an experimental isolated/perfused organ approach and study of parasite field diversity.
The Unit's long term objective is to develop vaccines aimed at preventing malaria morbidity and mortality. Research is based on complementary programmes aiming at a better understanding of the respective role of host and parasite factors in infection outcome and clinical manifestations.
1. Role of immune activation in the patho-physiology of malaria. (C.Behr, S.Loizon, P.Boeuf, F.Remerand , J.C.Michel).
We study the activation of the immune system in different clinical forms of P. falciparum malaria. Severe malaria is accompanied by a profound systemic inflammation and local accumulation of infected red blood cells within the deep vasculature. In collaboration with the Korle-Bu hospital (Dr Goka) and the Noguchi Institute (Dr. Akanmori) in Ghana, and the Righshospitalet in Denmark (Dr. Hviid and Kurtzhals), we performed a detailed analysis of T lymphocyte and monocyte activation profiles in children with uncomplicated malaria, severe anaemia or cerebral malaria. Our results show that the main difference between the two severe forms concerns T lymphocytes. Using CytProQuant, a real time RT-PCR technique that we have developed in order to allow an absolute quantification of cytokine mRNAs, we have demonstrated distinct T lymphocytes activation profiles in severe anaemia and cerebral malaria.
The role of gamma delta T lymphocytes is studied in the experimental model of infection of Saimiri sciureus by P.falciparum. Our results indicate that activation and expansion of T Vgamma 9 cells is associated with parasite clearance. We are currently exploring the functional role of these lymphocytes in vitro and in vivo.
2. Parasite pathogenicity factors (S.Bonnefoy, P.Buffet, P.David, M.Diez Silva, M.Guillotte, O.Natalang, I.Vigan)
Two potential virulence factors are explored. The first one is the var0 adhesion molecule, which is exposed on the surface of the infected erythrocyte where it promotes binding of non infected (rosetting) or infected (auto-agglutination) erythrocytes. We have shown that the varO-mediated cytoadherence represents a novel type of adhesion. The var O molecule is composed of 6 distinct adhesion domains. We are currently cloning and expressing these domains as recombinant proteins in order to explore the feasibility of a vaccine targeting such interactions in the Saimiri monkey model. Analysis of the immunological properties of these domains is underway.
The second factor is RESA, a parasite protein that is discharged to the host cell membrane following erythrocyte invasion, where it establishes specific interactions with spectrin. In order to explore its biological role, mutant parasites with an invalidated resa gene were constructed. Analysis of these mutants indicated that lack of RESA had no notable influence on parasite growth in vitro and only slightly modified the in vivo infection profile in Saimiri monkeys. However, it resulted in a marked susceptibility of the infected red blood cells to thermal shock, which was not observed with parental parasites or isogenic controls (see figure). The biological role of RESA is thus to modify the host cell cytoskeleton to allow the erythrocyte membrane to resist deleterious effects of elevated temperatures during febrile episodes.
In addition, we are pursuing an alternate strategy for identifying novel parasite virulence factors based on exploring the transcriptome. A DNA microarray platform has been established at the Génopole (PT2, J.Y.Coppée) to explore the transcriptome of P.falciparum. The study of parasites isolated from patients with P.falciparum malaria recruited through a clinical research programme (GLOBEX) with the Centre Médical de l'Institut Pasteur and several Paris hospital emergency wards, aims at identifying genes specifically activated in vivo. These may contribute to virulence and/or to fitness of the parasites to their human host. In parallel, we co-ordinate the use of the platform for 15 French laboratories working on different aspects of P.falciparum biology.
3. Exploration of the function of the human spleen during P.falciparum infection using an ex vivo isolated-perfused organ (P.Buffet, P.David , V. Brousse).
The spleen plays a major role in parasite elimination, as well as in modulation of the parasite phenotype. We have undertaken a research program aimed at exploring human splenic functions at the early stages of infection, when they may critically influence the future outcome of infection. The first objective has been to establish an isolated-perfused organ system allowing survival of the spleen ex vivo for several hours. Given the encouraging results of a feasibility study performed with pig spleens, the ex vivo perfusion system is now applied to human taken from patients operated in the context of gastric or pancreatic neoplasia. These spleens are perfused with P.falciparum-infected erythrocytes, allowing different functional and histochemical studies to be performed (in collaboration with : G.Milon, U. d'Immunophysiologie et Parasitisme Intracellulaire. M.Huerre U. de Recherche et d'Expertise Histotechnologie et Pathologie)
4. Studies on field parasite diversity and its consequences on immune responses (O. Puijalon, MT Ekala, H. Jouin, N.Noranate, M.Guillotte).
Parasite polymorphism is a key parameter in the host/parasite relationship. It contributes to clinical attacks in preventing acquired immune responses from controlling novel strains, and also contributes to severity. We have established a program aiming at analysing field parasite diversity using systematic sequencing. We have studied in detail the parasite population in Dielmo, a Senegalese village with holoendemic malaria. We also study several other endemic settings with different transmission conditions. We co-ordinate a multicentric study within the Réseau des Instituts Pasteur [T. Fandeur (I.P. Cambodge), F. Ariey et M. Randrianarivelojosia (I.P. Madagascar), R. Jambou (I.P. Dakar) et E. Legrand (I.P. Guyane)], together with the Génopole (PT1 C. Bouchier). Within this frame, we study geographical and temporal heterogeneity of parasite populations in four distinct geographic areas. This has identified numerous novel mutations in drug target genes as well as a very high number of alleles for the msp1 block2 locus. We currently study the immunological consequence of such a diversity on the acquired humoral response. This has outlined a biased response, with restricted specificity and use of Immunoglobulin light chains.
Red blood cells infected with parasites expressing the RESA (upper panels) no longer vesiculate and undergo the drastic size reduction observed with uninfected red blood cells or with red blood cells infected with the ÆR1C5 RESA-KO parasites (lower panels) upon short exposure to 50°C. The RESA protein was visualised by its positive reaction with specific antibodies detected using an AlexaFluor-488 labeled reagent. The parasite nuclei were stained with Hoechst 33342.
Keywords: Malaria, Pathology, Diversity, Vaccine, Molecular Genetics