Unit: Parasite Molecular Immunology - CNRS URA 1960
Director: PUIJALON Odile
The main research theme of the Unit is the analysis of factors that condition the outcome of a malarial infection, with the goal to develop a vaccine preventing pathology. Two main axis are explored. The first relates to basic research on Plasmodium falciparum pathology, both in man and in the experimental infection of the Saimiri sciureus monkey. It includes analysing the activity of immuno-competent cells during malarial attacks, exploring the role of the spleen through the use of an isolated/perfused organ experimental approach, identifying novel parasite pathogenicity factors and investigating parasite diversity in the field and its biological consequences. The second axis relates to the development of vaccines aimed at conserved surface molecules identified as potential targets of the protective immune response acquired in endemic regions.
The Unit's long term objective is to develop vaccines aimed at preventing malaria morbidity and mortality. Research is based on complementary programmes, including an integrated analysis of the host/parasite interactions contributing to malaria pathology and a vaccine development program.
Four approaches are used in the coordinated analysis of the elements contributing to malaria pathology: First, studying the activation of the immune response in different clinical forms of P.falciparum malaria, towards a better definition of the T-cell contribution to the balance between pathology and protection, in order to identify the parasite molecules involved. Second, identifying novel parasite virulence factors though reverse genetics and the systematic analysis of the parasite transcriptome under different physiological conditions. Third, analysing the role of the spleen during infection, in particular its implication in parasite-clearance mechanisms and in modulating the expression of infected erythrocyte surface antigens. Four, studying the diversity of parasite populations circulating in endemic regions and its consequences on the occurrence of malaria attacks, their severity and on immune responses. The development of vaccines focuses on conserved antigens of P.falciparum and P.vivax expressed by erythroctic stages, and identified as targets of parasite clearance mechanisms.
One of the characteristics of the Unit is to privilege in vivo studies in humans and in experimental monkey models. This implies resorting to experimental models of infection as well as to field studies: molecular epidemiology studies with the Réseau International des Instituts Pasteur; hospital-based studies on clinical malaria in Ghana; studies on experimental monkey infections in French Guiana and Sri Lanka.
1. Role of immune activation in the patho-physiology of malaria. (C.Behr, S.Loizon, P.Boeuf, F.Remerand , I.Vigan, J.C.Michel).
Severe forms of malaria are characterised by a profound systemic inflammation and a local accumulation of infected erythrocytes in deep organs. Systemic Inflammation is a factor of poor prognosis due to the complications it entails. The cascade of immunological events leading to either simple or severe malaria manifestations remains poorly known, but several lines of evidence suggest different cascades for different clinical forms. Results obtained in man and in rodent models suggest that the innate immune system could play a major role in this cascade, especially cells of the myeloid lineage.
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 suffering from non complicated malaria, severe anaemia or cerebral malaria. Our results show that the main difference between the last two severe forms lies in T lymphocytes. Our further analysis lies on a real time RT-PCR technique we developed, which allows the absolute quantification of cytokine mRNAs. In parallel, we are studying the phenotypic and functional state of monocytes and dendritic cells in these children.
Gamma delta T lymphocytes have been involved in the early regulation of innate immunity. This aspect is studied in the model infection by P.falciparum of an experimental host, the squirrel monkey Saimiri sciureus, using a battery of tools we have developed to study the responses of immuno-competent cells in this animal. Our results indicate that T V gamma 9 cells are involved in the parasite clearance phase. We are currently exploring the functional role of these lymphocytes in vitro and in vivo.
2. Parasite pathogenicity factors (S. Bonnefoy, M. Diez Silva, I. Delrieu, M. Guillotte, P. David, O.Natalang).
We are exploring the role of the var 0 adhesion molecule, exposed on the surface of the infected erythrocyte, which allows binding of non infected (rosetting) or infected (auto-agglutination) erythrocytes, and of the RESA factor inserted in the infected erythrocyte membrane.
The var O molecule is composed of 6 distinct adhesion domains. We have initiated the cloning and expression of these different domains as recombinant proteins, in collaboration with the Unit of Structural Immunology (G. Bentley) and D. Arnot's laboratory (Edinburgh University), in order to identify the domains active in rosetting and auto-agglutination, the cellular receptors involved as well as 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 RESA factor is associated with virulence in the Saimiri monkey model of infection. We are using a reverse genetics approach to explore the contribution of this factor to virulence, in collaboration with the Monash Institute (Australia) and the Pasteur Institute of French Guyana. In order to analyse the contribution of this protein to malaria pathology, we inactivated the resa gene of the avirulent strain FUP/CB. Although this had no notable effect on parasite growth in vitro, the loss of expression of the corresponding protein induced a modification of the infection profile, with an increase in mean parasitaemia in all infected monkeys. Functional and rheological studies of erythrocytes infected with the mutant parasites show that the RESA protein is involved neither in membrane rigidity, nor in cytoadherence properties which contribute to pathology, thus invalidating some hypothesis on the potential functions of this protein.
Due to the difficulties relating to low transfection efficiency, in parallel with the tools for genetic engineering developed in a PRT coordinated by S. Bonnefoy, we have developed an alternate strategy based on exploring the transcriptome. In collaboration with the Genopole (PT2, J.Y.Coppée), a DNA microarray platform has been set up to explore the transcriptome of P.falciparum. The study of parasites isolated from patients infected with P.falciparum, made possible through a clinical research programme with the CMIP and several Paris hospital emergency wards, should allow the identification of genes specifically activated in vivo. These could contribute to virulence and to a better adaptation of the parasites to their host. In parallel with our use of DNA microarrays to study pathogenicity factors, we co-ordinate the use of the platform for the research programs of 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 :
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 be crucially conditioning 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.
4. Studies on field parasite diversity and its consequences on immune responses ( O. Puijalon, H. Jouin, N.Noranate, D. Eisen, M.Guillotte).
Analysis of parasite populations under different transmission conditions and of their temporal and spatial evolution is necessary in order to elaborate rational control measures. We have recently set up a programme aimed at the molecular analysis of parasite polymorphism by systematic sequencing genes. We have studied the temporal variations over ten years of the parasite populations circulating in the village of Dielmo, in Senegal. We have analysed the evolution of allelic diversity of the Pccrt and Pfdhfr genes and of the msp1 locus block 2.
Through a multi-centric collaboration with T. Fandeur (IP of Cambodia), F. Ariey and M.Randriavelojosia (IP of Madagascar), R. Jambou (IP Dakar) and E. Legrand (IP Cayenne), we have studied allelic diversity of parasite populations in 4 different geographic zones. This programme has allowed the description of a large number of new mutations in target-loci of anti-malarials and a high number of alleles of the msp1 bloc 2.
The vaccine development programme focuses on P. falciparum and P. vivax asexual blood stage antigens identified as targets of immune protective mechanisms.
Merozoite Surface Proteins (S. Longacre, S. Bonnet, H. Polson, S. Rosario):
S. Longacre's group develops a research programme exploring the conserved 19 kDa C-terminus of the merozoite surface protein 1 (MSP1p19) from P. falciparum and P. vivax expressed as a recombinant protein produced in the baculovirus-insect cell expression system. The efficacy and/or immunogenicity of different formulations of the MSP1p19 antigen, including several experimental adjuvants, have been evaluated in mice and in macaques (toque, rhesus monkeys). Several vaccination trials have provided solid evidence that the MSP1p19 antigen is very immunogenic, and is a potent protective antigen with a long lasting effect against homologous and heterologous strains. This vaccine research programme has provided the grounds for undertaking phase I clinical trials with the baculovirus MSP1p19 antigen, currently being planned and sponsored by the Pasteur Institute.
R23 (O.Puijalon, M. Huynh Quan Dat, A. Schneider, M.Guillotte) :
The R23 antigen is a target of antibodies that opsonize P.falciparum-infected erythrocytes. The immunisation of Saimiri monkeys with a minimal dose of this antigen in Alum has been shown to induce efficient protection against lethal infection in the squirrel monkey. We are currently exploring how the fine specificity of antibodies induced in mice, rats and monkeys by immunisation with the R23 antigen influences recognition of the infected erythrocyte surface. Continued efforts are dedicated to optimise immunogenicity of this vaccine candidate.
Keywords: Malaria, Pathology, Diversity, Vaccine, Molecular Genetics