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  Director : Odile PUIJALON (omp@pasteur.fr)


  abstract

 

The main research theme of the Unit is the analysis of the factors that condition the outcome of a malarial infection, with the goal to develop a vaccine preventing pathology. We have undertaken an integrated basic research program on Plasmodium falciparum pathology, both in man and in the experimental infection of the Saimiri sciureus monkey, along with the development of a vaccine targeting asexual blood stage conserved antigens. The integrated study of malaria pathology includes: 1) analysing the activation of immuno-competent cells during malarial attacks, 2) identifying novel parasite pathogenicity factors combining reverse genetics and transcriptome analysis using DNA microarrays, 3) exploring the role of the spleen through the use of an isolated/perfused organ experimental approach, and 4) investigating parasite polymorphism and its consequences on immune responses. Our vaccine development programmes focus on conserved merozoite or infected red blood cell surface antigens.



  report

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The Unit's long term objective is to develop vaccines against Plasmodium blood stages. Four research teams work on complementary research 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. 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 associated with a profound systemic inflammation and a local accumulation of infected erythrocytes resulting from parasite cytoadherence in deep organs. 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 the cytokine profiles in different clinical forms of malaria in African children. Our previous work had shown that severe anaemia and cerebral malaria are two distinct immunopathological syndromes, characterised by different TNF alpha/IL-10 ratios. Using intracellular labelling, we have shown that TCD8high and gdT lymphocytes are the main producers of gIFN, whereas the T CD4+ lymphocytes are an important source of TNF alpha . T CD4+ lymphocytes are the only cells secreting IL-10.

To better understand the differences observed between the various clinical forms of malaria, we developed new sensitive techniques of real time RT-PCR, which allow us to measure the expression of 15 cytokines in the limited blood samples obtained from children suffering from severe malaria. In parallel, we are studying the phenotypic and functional state of monocytes and dendritic cells in these children. The numerous analogies between the immunological disorders occurring during severe malaria and those associated with severe sepsis led us to perform a similar investigation on a cohort of patients hospitalised for severe sepsis. In collaboration with the intensive care units of the Hopital Lariboisière (Pr D. Payen) and the Hopital Piité-Salpétrière (Pr J.J. Rouby) in Paris, we performed a longitudinal follow-up of patients suffering from septic or aseptic choc. We showed that these patients presented a severe monocyte "deactivation", as reflected by the important decrease in monocyte surface expression of HLA-DR, as well as the CD80 and CD54 T cells co-stimulator molecules.

gdT 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 Vg9 cells are implicated upstream from the parasite clearance phase.

2. Parasite pathogenicity factors (S. Bonnefoy, M. Diez Silva, I. Delrieu, M. Guillotte, P. David, O.Natalang)

Genetic engineering of P. falciparum is the strategy we adopted to study parasite virulence in the experimental model infection of the Saimiri sciureus monkey. Parasite strains which result in lethal infections present certain characteristics, among which a deletion of the resa gene and the presence of a particular allele of the hrp1 locus. These two genes code for proteins localised in the membrane of the infected erythrocyte, and interacting with other parasite proteins as well as with the erythrocyte cytoskeletton. In order to analyse the contribution of these factors to malaria pathology, we first 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. The genetic tools necessary for sequential mutagenesis that we have developed are currently being exploited to study the contribution of the hrp1 gene to pathology in this model. Other molecular tools, such a positive/negative selection systems, are currently being developed in the laboratory.

In parallel with the development of tools for genetic engineering, we have established an alternative strategy based on exploring the transcriptome under different physiological conditions. Our goal is to identify genes specifically activated in vivo, which could contribute to virulence and to a better adaptation of the parasites to their host and genes contributing to parasite evasion mechanisms. We have established the conditions for the study the parasite transcriptome in the monkey model as well as in natural human infections. 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 probes, made of 7500 70 base-oligomers synthesised according to the parasite genome data and spotted onto glass slides, allowed us to screen parasite RNA extracted at different stages of parasite development. The differential gene expression profiles obtained indicate that the platform is indeed functional. In parallel with our use of DNA microarrays to study pathogenicity factors, we will 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. In several experimental systems, splenectomy is followed by increased parasitaemia and alteration of the parasite phenotype (infected erythrocyte surface antigens and cytoadherence properties). In human infections, the spleen has only been studied upon autopsy, i.e. at a quite late infection stage. Consequently, virtually nothing is known about implication of the spleen early in infection, when it may be crucially conditioning the future outcome of infection. We therefore have begun a research program aimed at exploring splenic functions at the early stages of infection. The first objective has been to establish an isolated-perfused organ system allowing survival of the spleen ex vivo for several hours. A feasibility study performed with pig spleens has led to most encouraging results. A collaborative network has been set up with several Paris hospitals, providing access to human spleens taken from patients operated in the context of gastric or pancreatic neoplasia. These spleens will be 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. To explore geographic heterogeneity, we recently established a multicentric molecular analysis of parasite polymorphism by systematic sequencing genes coding for drug targets and surface antigens and have developed a new strategy for typing field isolates using a collection of neutral microsatellite markers. To investigate potential temporal variations, we have carried out a retrospective ten year study 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.

The consequence of MSP-1 block 2 polymorphism on immune responses of individuals living in endemic areas was studied in two Senegalese villages. This showed that the presence of antibodies against this domain at the beginning of the transmission season is associated with a markedly decreased risk of clinical attack during the subsequent transmission season. Such an association is paradoxical, in as much as fixed antiu-MSP1 block2 reactivity profiles are observed, independent of past or present parasite genotypes. Novel anti-msp1 bloc 2 specificities are not accumulated following exposure to novel alleles, constrasting with the progressively enlarging antibody repertoire acquired to the variant antigen PfEMP1. Such a response, with an inappropriate or sub-optimal specificity, suggests a new parasite immune evasion mechanism, leading to partial parasite clearance and chronic carriage.

In collaboration with the Institut Pasteur de la Guyane, we studied the immune responses against variant antigens in the model experimental infection of the Saimiri monkey. This enabled us to show that there was no direct relation between response to the variant erythrocyte surface antigen and protection.

5. 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): We have explored 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. In parallel, the analysis of polymorphism in the 42 kDa C-terminal MSP1 (MSP1p42) of P. vivax and P. falciparum has been explored and the immune response to the C-terminal conserved and polymorphic domains of P. vivax MSP1has been studied in man. The crystalline structure of the P. falciparum MSP1p19 has been determined in collaboration with the Unité d'Immunologie Structurale; it supports the notion that similar complex folding of the two EGF domains occurs in MSP1p19 from all Plasmodium species. Recently this group has begun to evaluate other merozoite surface antigens for their potential as malaria vaccine candidates. In addition novel modifications of the antigens and new adjuvant systems are being investigated for general vaccine use.

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 antigen in order to improve sustaiend production of protective antibodies.

Keywords: Malaria, Pathology, Diversity, Vaccine, Molecular Genetics



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  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  LECUILLER Frédérique, (flecuil@pasteur.fr) BEHR Charlotte, C.R.1, CNRS, (charlotte.behr@pasteur.fr)

BONNEFOY Serge, C.R., IP, (sbf@pasteur.fr)

DAVID Peter, D.R.2, CNRS (pdavid@pasteur.fr)

LONGACRE Shirley, D.R.2, CNRS (longacre@pasteur.fr)

MICHEL Jean-Claude, C.L., Réseau IP, (jcmichel@pasteur.fr)

PUIJALON Odile, Chef d’Unité, C.L., IP (omp@pasteur.fr)

BOEUF Philippe, PhD student

BONNET Sarah, Postdoc

DIEZ SILVA Monica, PhD student

DELRIEU Isabelle, Postdoc

NATALANG Onguma, PhD student

REMERAND Francis, PhD student

SCHNEIDER Achim, Postoc

VIGAN Inès, Postdoc

FAGNIERES Plamenna, Technician, IP

GUILLOTTE Micheline, Technician, IP (mguillot@pasteur.fr)

HUYNH QUAN DAT Myoura, Technician, IP (dat@pasteur.fr)

JOUIN Hélène, Engineer, IP (hajouin@pasteur.fr)

LOIZON Séverine, Engineer, CNRS (sloizon@pasteur.fr)


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