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  Director : Odile PUIJALON (omp@pasteur.fr)


  abstract

 

The main research theme of the Unit is the analysis of factors which condition the outcome of a malarial infection, with the goal to develop a vaccine. Our efforts are focused on: the development of a vaccine targeting blood stage surface antigens ; the analysis of parasite polymorphism and of its consequences on immune responses ; the study of parasite pathogenicity factors; the exploration of the parameters which orient the immune system towards either a protective or a pathogenic response.



  report

cale

The Unit's long term objective is to develop vaccines against Plasmodium blood stages. Four research teams work on complementary research programmes, including a vaccine development programme, studies on field parasite diversity and an integrated analysis of the host/parasite interactions contributing to malaria pathology. 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. The vaccine development programme focuses on P. falciparum and P. vivax asexual blood stage antigens identified as targets of immune protective mechanisms.

MSP1p19 (S. Longacre, S. Bonnet): S. Longacre's group develops a program 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 macaque primates (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 basis and justification for undertaking phase I clinical trials with the baculovirus MSP1p19 antigen, currently being planned. 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. The objective now is to determine the crystallographic structure of MSP1p42.

R23 (O.Puijalon, M. Huynh Quan Dat, A. Schneider) : The R23 antigen is a target of antibodies which opsonize P.falciparum-infected erythrocytes. The immunisation of Saimiri monkeys with a minimal dose of this antigen can induce efficient protection. Our research efforts now aim at optimising immunogenicity and analysing the biological function of this molecule.

2. Studies on field parasite diversity and its consequences on immune responses ( O. Puijalon, H. Jouin, N.Noranate, D. Eisen, M.Guillotte )

The analysis of parasite populations under different conditions of transmission and their temporal and spatial evolution is necessary in order to elaborate rational control measures. For several years, we have studied the polymorphism of parasite populations in different endemic regions. This year, we have set up a program to analyse parasite polymorphism by systematic sequencing and have studied the temporal evolution of the msp1 gene, over a period of ten years, in Dielmo, a village in Senegal where longitudinal studies are performed.

The consequences of the polymorphisme of MSP-1 bloc 2 have been studied in two Senegalese villages which differ in transmission conditions. Responses are analysed using synthetic peptides representing the different sequence variants. The response is associated with a decreased risk of clinical episodes during the following transmission season. Such an association appears as a paradox, since fixed reactivity profiles are observed which are independent of past or present parasite genotypes. The distribution of light and heavy Ig chains remains constant throughout the years. In contrast with what is described for the variant antigen PfEMP1, new anti-msp1 bloc 2 specificities are not accumulated following exposure to an increasing number of alleles. The characteristics of this response suggest a peculiar mode of regulation reminiscent of "clonal imprinting". The capacity to induce a fixed response, which is thereby of inappropriate or non optimal specificity against a high number of strains, could constitute a new mode of parasite immune evasion, favouring partial clearance and chronic parasite carriage.

3. Parasite pathogenicity factors (S. Bonnefoy, P. Pendyala, M. Diez Silva, M. Guillotte, P. David)

In the Saimiri sciureus experimental model of infection, strains inducing lethal infections present certain characteristics, such as a deletion of the resa gene and the existence of a particular allele of the hrp1 locus. We have used a reverse-genetics approach to analyse the contribution of these factors to pathogenicity. Disruption of the resa gene in the avirulent FUP/CB strain led to the modification of the profile of infection, increasing mean parasitaemia in all animals infected. The construction of strains in which the hrp1 gene has been manipulated is underway. Several molecular tools have been developed in order to continue this study; among these are tools which will allow the sequential manipulation of the parasite genome towards the construction of double mutants

We are also developing a promoter-trapping strategy. We have demonstrated the feasibility of positive/negative selection in P.falciparum, using DHFR-TS Herpes simplex virus and Thymidine Kinase. Other negative selection markers are under study. This allows us now, among other applications, to seek genes activated in vivo but inactive under in vitro culture conditions, namely genes which one can reasonably suspect to play an important role in host-parasite interactions.

In parallel, we have initiated the systematic exploration of the parasite transcriptome, using DNA chips to study the parasites expression programme under different physiological conditions. Our first objective has been to explore the modulation of parasite transcription by the spleen, an organ which is known to influence red cell surface expression/exposure of parasite antigens. This has allowed the identification of 185 genes the level of transcription of which is modulated by the spleen. Some of these genes code for polypeptides linked to cytoadherence and/or antigenic variation. It is interesting to note that this analysis has pointed out modulation of the expression of "knob" accessory molecules, in particular hrp1. Up to now, we had access to microarrays through a collaboration established with J. DeRisi (UCSF). This year, in collaboration with the Institut Pasteur Genopole (J.Y. Coppée), we have contributed to the set-up of a DNA microarray platform enabling us to explore the transcriptome of P.falciparum on campus. These arrays are made up of over 6200 oligonucleotides of 70 bases, synthesised according to the available genome sequence data. This opens the possibility to study the consequences on the parasite transcriptome of inactivation or allelic replacement of resa and hrp1 genes, as well as the effects of different environmental conditions (immune pressure, splenectomy, culture conditions).

4. Contribution of immune activation to malaria pathology. (C.Behr, S. Loizon, P.Boeuf, F.Remerand , I. Vigan, J.C.Michel).

gdT lymphocytes are highly activated during acute malaria attacks. In order to understand the consequences of such an activation, we have performed a detailed repertoire analysis in healthy children and in children suffering from a malaria attack. The peripheral representation of the various gdT cell subsets in healthy African subjects (children and adults) differs from what is described in Europeans. In children with acute malaria, the Vd1 subset is transiently activated. Vg chain analysis did not show any preferential chain association. Immunoscope analysis did not outline any specific clonal expansion. In children with acute malaria, Vg9 T cells are also activated, but to a lesser extent. Ex-vivo intracellular staining showed that the Vg 9 T cells are high producers of TNF-a, while Vd 1 are high producers of IFN-g. Overall numbers of circulating Tgd cells and the Vd 1/ Vg9 ratio did not significantly differ in the various clinical groups (non complicated, severe anaemia and cerebral malaria). Our results are compatible with the proposed role for the Vd1 population in the regulation of the immune system and the return to homeostasis.

Further analysis of the cytokine profiles associated with distinct forms of clinical malaria showed that cerebral malaria is associated with statistically significantly higher levels of IL-2R than severe anaemia or uncomplicated malaria. Severe anaemia is associated with lower levels of TNFR (I and II). All the work concerning African individuals has been performed in collaboration with Dr.D. Akanmori from " Noguchi Memorial Institute " (Ghana) and Dr. L. Hviid from " Righshospitalet " (Denmark).

Sepsis and severe sepsis present an interesting analogy with mild and severe malaria, with systemic inflammation as a major contributor of pathology. In collaboration with the Intensive Care Unit of the Hopital Lariboisière, Paris, we conducted a longitudinal follow up of patients suffering severe sepsis or local intense inflammation. We measured monocyte surface HLA-DR and co-stimulatory molecules CD80 and CD54 by quantitative flow cytometry either ex-vivo or after in vitro LPS stimulation. This showed that "monocyte deactivation" is a general phenomenon that induces down regulation of HLA-DR, but also of other co-stimulatory molecules that play a major role in T cell activation We are now analysing the consequences of such an "immunodepression" on the dendritic cells.

We have devoted considerable effort to establish the experimental conditions and the immunological and genetic tools necessary for physiopathology studies in the squirrel monkey. Monoclonal antibodies have been screened towards functional identification of mononuclear cell subsets, and primers have been identified for the amplification of the major pro and anti-inflammatory cytokines. This has allowed us to establish the normal range of cellular subset and activation profiles in non infected naive animals, as well as to analyse the kinetics of activation of the various subsets during a primary P.falciparum infection.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

LECUILLER Frédérique, Secretary I.P. (flecuil@pasteur.fr)

BEHR Charlotte, Researcher CR1 CNRS, (charlotte.behr@pasteur.fr)

BONNEFOY Serge, Researcher I.P., (sbf@pasteur.fr)

BONNET Sarah, Postdoc I.P., (sbonnet@pasteur.fr)

DAVID Peter, Researcher DR2 CNRS, (pdavid@paseur.fr)

DELRIEU Isabelle, Postdoc I.P. (idelrieu@pasteur.fr)

LONGACRE Shirley, Researcher DR2 CNRS (longacre@pasteur.fr)

MICHEL Jean-Claude, Head of laboratory IPOM, (jcmichel@pasteur.fr)

PUIJALON Odile, Head of laboratory I.P., Head of Unit, (omp@pasteur.fr)

BŒUF Philippe, PhD student, (pboeuf@pasteur.fr)

DIEZ SILVA Monica, PhD student, (mdiez@pasteur.fr)

NORANATE Nitchakarn, PhD student, (noranate@pasteur.fr)

REMERAND Francis, PhD student, (remerand@pasteur.fr)

SCHNEIDER Achim, postdoctoral trainee, (achimsch@pasteur.fr)

VIGAN Inès, postdoctoral trainee, (ivigan@pasteur.fr)

GUILLOTTE Micheline, Technician I.P., (mguillot@pasteur.fr)

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

JOUIN Hélène, Engineer I.P., (hajouin@pasteur.fr)

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


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