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  Director : Pierre DRUILHE (druilhe@pasteur.fr)


  abstract

 

The Biomedical Parasitology Unit is focused on the understanding of Plasmodium falciparum / human immune interactions, both at the level of the pre-erythrocytic stages (the sporozoite and the liver-stage) and of the merozoite stage of the erythrocytic cycle. The main activities are aimed at facilitating vaccine and, to some extent, drug discovery. We have completed this year the analysis of the first vaccine trial with MSP-3 which has produced results which are beyond our most optimistic expectations. Besides, other phase-I and —II trials with other formulations of MSP-3 and several of LSA-3, a first field trial in Africa will be initiated in 2003 as an outcome of the findings of 2002.



  report

cale

I — Immunity to erythrocytic stages

We have chosen an approach where clinical observations, interactions between P. falciparum and humans and material derived from these studies, are at the origin of the paradigms and the research orientations. By passively transfering protection by African immune IgG into naive infected subjects, we identified a mechanism of defence where antibodies cooperate with monocytes, called ADCI. The latter was used to screen a P. falciparum genomic library to identify an antigen, called MSP-3 and to identify the critical role of cytophilic antibodies, IgG1 and IgG3, able to cooperate with monocytes. Studies conducted this year are inkeeping with this bio-clinical approach of an antimalarial vaccine, they were directed at collecting futher evidence of the role of MSP-3 , conducting a clinical trial with it, identifying in detail the epitopes targeted by protective antibodies, and identifying further targets of the same defence mechanism.

The first clinical trial was conducted in 36 healthy volunteers and was organised as an opened, randomised, dose-escalating phase-I trial with 2 adjuvants, alum and Montanide ISA720. Results showed an excellent safety and immunogenicity, and yielded numerous findings that were not predicted by pre-clinical models ; e.g. : alum was as effective as Montanide, safer, whereas it was totally uneffective in rodents and primates. Results showed that using a simple molecule and a non-toxic adjuvant it was possible to readily induce in naive volunteers a type of immune response that requires over 10 years of daily exposure to billions infected red blood cells under field conditions. Responses were directed to fully conserved epitopes, were very long-lasting and, above all, showed a strong biological activity against P. falciparum, able to block the division or kill the parasite, both under in vitro conditions in the ADCI assay and under in vivo conditions by passive transfer into P. falciparum infected immunocompromised mice. The antiparasitic effect obtained was as strong as, or sometimes stronger than that induced by antibodies from immune African adults which have the strongest type of protection against malaria known to occur in human beings.

Using a series of 6 overlapping peptides covering the conserved C-terminus of MSP-3 and 2 recombinants, we have characterized in detail the T-helper and B-cell epitopes in this region, have identified 3 out of the 6 non cross-reactive B-cell epitopes as being a target for protective antibodies, whereas the remaining 3 are not.

We have identified a family of genes related to MSP-3 by their overall organization and by the presence of either one of the 3 above mentioned B-cell epitopes, which are fully conserved or highly homologous in the remaining members of the gene family. We have expressed as recombinant His-tailed proteins, either the corresponding conserved region of each of the members of the family or, to the opposite, a unique region of the same genes, in order to study in detail a) the cross-reactivity between the members of that family, and b) the proteic expression of each gene on the merozoite surface.

After showing that GLURP and P126-SERP were other targets of the ADCI mechanism, we have recently completed a detailed immuno-epidemiological study aimed at defining the respective role in clinical protection of antibodies directed to each of them. This study confirms the interest of each target but moreover showed that they could act in complementary manner, particularly in medium transmission areas, where antibodies to either 1 or 2 of the 3 could take over, in the potential absence in some individuals of antibodies to the 3rd, and vice versa.

We have identified a new antigen which might be a 4th target of the same mechanism and have re-expressed 3 regions of this molecule to perform in vitro and in vivo studies of the biological activity of the corresponding antibodies and immuno-epidemiological studies of the association of responses to them with protection.

We have also initiated this year a study of the affinity of antibodies to the above 3 targets of ADCI, as compared to controls. The analysis of results is not completed, but indicates major differences in the affinity of antibodies among different groups of people, either prone to malaria attacks or witth acquired immunity to malaria.

We have produced a fully human recombinant antibody to MSP-3-b and have shown this year its biological activity when passively transferred into P.falciparum infected Hu-RBC immunocompromised SCID mice. The role of the heavy chain was analysed by comparing the same Hu Mab expressed either as an IgG1 or an IgG3. We have performed, at the end of 2002, a passive transfer of this antibody in chimpanzees infected by the particular parasite of that host, P. reichenovii, which has 100 % homology in the target epitopes of MSP-3. We are now considering the development of this recombinant human antibody at low cost, expressed in plants.

We have initiated a systematic study of the mechanism of ADCI, both of the afferent arm when cytophilic antibodies bridge a merozoite to the Fc-receptor of monocytes and the efferent arm, the mediators and their effect upon intra-erythrocytic surrounding parasites. This already led to major improvements in the standardization of the assay, the development of high throughput methods better fitted to the screening of new molecules derived from genome data, and to an analysis of the influence of the structure of the Fc fragments from IgG1 and IgG3, the various allotypes in different genetic human backgrounds and the structure of the Fc-g R2 receptors.

II — Pre-erythrocytic stages

We have shown that what has long been considered an " anti-sporozoite immunity " is in fact related to the development of a liver-phase trophozoite and have focused therefore our efforts on the study of immunity to liver stages. The lead molecule LSA-3 has been identified by differential screening of immune responses from protected vs non-protected human volunteers, both immunised by irradiated sporozoites. We have recently completed the characterization of an additional 29 new pre-erythrocytic genes.

We have characterized this year the potential value of a new pre-erythrocytic antigen, LSA-5, which is able to induce protective responses in Aotus monkeys against a challenge by P. falciparum sporozoites.

Simultaneously, we have gathered further evidence in favour of the protective value of the lead molecule, LSA-3, by immunisation of Aotus monkeys with 2 long synthetic peptides preparations adjuvated by ASO2 from GSK. This brings to 5 the number of antigen delivery systems employed in primates with this molecule that have led to a state of protection against a massive dose of infective sporozoites of P. falciparum.

We have introduced recently a number of modifications in the cultivation protocole of hepatic stages in vitro, that resulted in a major breakthrough whereby the yields can be increased by several orders of magnitude, thus opening at once inaccessible avenues of research. This breakthrough has started to be applied to either an improved analysis of the mechanisms of defence taking place at intra-hepatic level and an analysis of the transcriptome of the liver-stages, the least known of the whole malaria cycle.

Recently, mRNA could be obtained from these cultures, a cDNA library prepared for the P.yoelii hepatic stages, 2200 clones successfully sequenced at the Genopole and 400 P.yoelii sequences from those stages characterized. We are currently preparing similar steps to construct a cDNA library from the P. falciparum hepatic stages.

In collaboration with other groups, it was shown that there is a stage-specific expression of the multi-gene family P.yoelii 235, i.e. able to express a given repertoire of genes at erythrocytic stage and different ones at sporozoite and / or liver stage. This is aimed at being exploited to investigate the molecular mechanism underlying the control of gene expression in malaria parasites. Completion of the repertoire of the Py235 gene is in progress. The flanking sequences will be isolated and cloned with a GFP reporter gene. Transfection of P.yoelii with a vector and transient expressoin in liver stage has been successfully achieved.

In a PAL+ funded project, the diversity of T-helper epitopes of the P. falciparum CS gene was analysed and revealed a highly biased frequency distribution. These studies are now completed in 2 locations in Africa to analyse the reason for this biais.

Finally, we have furthered our study of the grafting of human hepatocytes in varous kind of immunocompromised mice, namely BXN, SCID-NOD, UPA-SCID, FAH-Rag 2.

Keywords: Plasmodium falciparum, immunity, vaccines, merozoite, hépatic stages, gene expression



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Laurence GANGLOFF lganglof@pasteur.fr Georges SNOUNOU, DR2 CNRS, (snounou@pasteur.fr)

Christian ROUSSILHON, IP (roussilo@pasteur.fr)

Jean-Louis PERIGNON, Faculte de Medecine Necker (perignon@pasteur.fr)

Daw SOE SOE, IP (soesoe@pasteur.fr)

Jean-Pierre SAUZET, Ingénieur de Recherche, jpsauzet@pasteur.fr

Stéphanie HEZ, doctorant (Université de Rouen) shez@pasteur.fr

Subhash SINGH, doctorant (Université de Rouen)subhash@pasteur.fr

Ali JAFARSHAD, doctorant (Paris VII) jaffarsha@pasteur.fr

Pedro MEJIA, doctorant (Université de Rouen) pmejia@pasteur.fr

Anne-Charlotte GRUNER (post-doc) gac@pasteur.fr

Fouad BENHNINI (DEA, Paris VI) benhnini@pasteur.fr

Sébastien GALLIEN (DEA, Paris I Necker), sgallien@pasteur.fr

Catherine BLANC, Technician, catblanc@pasteur.fr

Sophie CHAOUCHE, Technician, chaouche@pasteur.fr

Nicolas PUCHOT, Technician, npuchot@pasteur.fr

Danielle QUADOUR, Laboratory worker


Activity Reports 2002 - Institut Pasteur
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