WO1993003057A1 - Polypeptides capable of in vivo induction of antibodies themselves capable of inhibiting the invasion of red blood corpuscles by merozoites of p.falciparum, related products and their use in producing vaccine compositions - Google Patents

Polypeptides capable of in vivo induction of antibodies themselves capable of inhibiting the invasion of red blood corpuscles by merozoites of p.falciparum, related products and their use in producing vaccine compositions Download PDF

Info

Publication number
WO1993003057A1
WO1993003057A1 PCT/FR1992/000763 FR9200763W WO9303057A1 WO 1993003057 A1 WO1993003057 A1 WO 1993003057A1 FR 9200763 W FR9200763 W FR 9200763W WO 9303057 A1 WO9303057 A1 WO 9303057A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
antibodies
sequence
lys
asn
Prior art date
Application number
PCT/FR1992/000763
Other languages
French (fr)
Inventor
Denise Mattei
Arthur Scherf
Luiz Pereira Da Silva
Odile Mercereau-Puijalon
Serge Bonnefoy
Jurg Gy Sin
Original Assignee
Institut Pasteur
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR9109771A external-priority patent/FR2679909B1/en
Priority claimed from FR9109772A external-priority patent/FR2679776A1/en
Application filed by Institut Pasteur filed Critical Institut Pasteur
Priority to JP5503341A priority Critical patent/JPH06510527A/en
Priority to EP92917738A priority patent/EP0602079A1/en
Publication of WO1993003057A1 publication Critical patent/WO1993003057A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the subject of the invention is new antigens of Plasmodium falciparum and their application in particular with a view to protection against malaria.
  • the parasites responsible for malaria in humans including Plasmodium falciparum or Plasmodium vivax to name only the main ones, present in the human host different morphologies and express different antigens, depending on the stage of their development and their location in the organism of the infected host.
  • the morphological and antigenic differences of these parasites during their life cycle in humans make it possible to define at least four distinct stages of development.
  • the very first stage of development of the parasite in humans corresponds to the sporozoite form introduced into the host's blood, by bites from insects carrying the parasite.
  • the second stage corresponds to the passage of the parasite in the liver and to the infection of the hepatic cells in which the parasites develop to form the hepatic schizonts which, when they are mature (for example for P. falciparum on the 6th day after penetration of the sporozoites), release by bursting hepatic merozoites.
  • the third stage is characterized by the infection of blood erythrocytes by the asexual forms (merozoites) of the parasite; this erythrocyte stage of development corresponds to the pathogenic phase of the disease.
  • the fourth stage corresponds to the formation of forms with sexual potential (or gametocytes) which will become sexual forms or extra-cellular gametes in the mosquito.
  • antigens 11.1 Pf11-1) and (Pf155 / RESA) which themselves contained immunogenic determinants leading to cross-reactions as was observed using a human monoclonal antibody ( 20). More recently Udomsangtepch et al (36) described a human monoclonal antibody mAb 33G2 capable of inhibiting the cytoadherence of parasitized red blood cells, whether or not having protuberances with cells of the C32 melanoma cell line cells. It has also been found that this monoclonal antibody effectively inhibits the invasion of erythrocytes by merozoites in an in vitro test (38).
  • this monoclonal antibody initially selected because of its reactivity with the molecule Pf155 / RESA (38), lacks specificity. It also reacts with other antigens that appear in the bloodstream stage of infection with the parasite, including the antigens Ag11-1 and Ag332 (20,37).
  • the Ag 332 antigen presents undoubtedly a significant potential interest with regard to its ability to induce protective immunity.
  • a recombinant polypeptide including the expression product of a fragment of 303 base pairs derived from Pf332 genes has been found to constitute a particularly effective inhibitor of the monoclonal antibody mAb33G2, as was demonstrated in a test of inhibition of the invasion of red blood cells by merozoites (37).
  • the characterization of Ag332 has so far been inhibited by the cross-reactions to which antibodies which recognize it give rise, with various other antigens of the parasite (20).
  • the present invention results from the identification and characterization of a new gene sequence within the Pf332 gene, of the antigen encoded by this gene sequence and of antibodies inducible in vivo by this antigen and specific for the antigen.
  • the gene sequence in question has been located in the subtelomeric region of chromosome 11.
  • the antigen expressed by this gene sequence has been shown to have a monospecific character allowing it to specifically detect a mega-colortonic protein of the parasite or asexual parasites in the blood stage by immunoblotting and immunoprecipitation reactions, unlike antibodies induced by other antigens, either derived from Ag332 or related to sequences thereof, such as the synthetic dimer Y (SVTEEIAEEDK) 2 (24) or a fusion protein with beta-galactosidase (20). These antibodies also react with various other molecules of the parasite. Antibodies formed against the EB200 antigen also lead to surface reactions with red blood cells infected with Plasmodium falciparum with an efficiency having the same order of magnitude as that of the monoclonal antibody mAb33G2.
  • the peptide sequence of the EB200 antigen, one of the antigens according to the present invention has a characteristic peptide sequence corresponding to that which is framed in the sequence of the clone G9 (FIG. 1 (c)), this sequence having been deduced from the clone G9 resulting from the amplification of one of the fragments of the Pf332 gene under the conditions which will be recalled later.
  • the EB200 antigen more generally belongs to the family of polypeptides having a common amino acid motif or sequence containing all or part of the polypeptide sequence below defined by its amino acid sequence:
  • a peptide which contains only part of this sequence is part of the invention when this peptide is capable, if necessary when it is incorporated into a fusion protein, oligomerizes or conjugated to a carrier protein, in vivo a protective immune response in the squirrel monkey Saimiri sciureus.
  • the distribution of the successive motifs of the amino acid sequence of a preferred polypeptide in accordance with the invention, as presented above, has no other objective than that of revealing the existence of motifs successive repetitions, within the protein, this repetitiveness is not however devoid of degenerations.
  • the blanks (actually direct links) that appear at the end of some of the lines or within some of the lines have no particular meaning. Their only aim is to visually highlight the homologs of partial sequences of these successive repeating patterns. We can notice the frequency of the VTEEV motif within these partially repetitive or "degenerate" motifs.
  • FIG. 2 The same amino acid sequence (using the three-letter representation by amino acids, instead of the one-letter representation in the above definition) is shown in Figure 2.
  • Figure 2 shows the coding sequence of the recombinant clone , named PfEB200, containing an insert of 411 base pairs isolated from the Pf332 gene. It is recalled that this clone had already been isolated from a bank of expression sequences constructed in an ⁇ Amp3 vector with a batch (pool) of immune human sera (20).
  • the present invention derives from the demonstration of the existence within the Ag332 antigen of amino acid sequences which are both specific for this antigen and exposed on the surface of the merozoites of P. falciparum and / or of the globules infected reds.
  • the initial P. falciparum genomic library in the vector ⁇ gtWES had itself been constructed by digestion using DNasel and addition of adapters (linkers) containing EcoRI sites as previously described in (21).
  • the sequenced parts are represented in FIG. 1 by thickening of the line.
  • the enzymes used for the production of the restriction map were: BamH1 (B), Cla1 (C), Dra1 (D), EcoR1 (E) and Hind3 (H).
  • EB200 soluble fusion protein expressed in the form of a soluble fusion protein PfEB200 with a glutathione-S-transferase
  • the expression of the soluble polypeptide was carried out in E.coli Dh5 ⁇ (F end A1 hsdr 17 (rkmk-) sup E44 thi-1, ⁇ -, rec A1 gyr A96 rel A1 f80d lac Z ⁇ M15).
  • the protein was purified by affinity chromatography on agarose beads carrying glutathione groups (32).
  • the degree of purification of the fusion protein was analyzed by electrophoresis on polyacrylamide gel containing 10% SDS and by staining with Coomassie blue. Any conventional technique can be used to produce antibodies specific for the EB200 protein (in the form of a fusion or not). However, in this case, the antibodies tested in the tests referred to below were produced under the following conditions: Balb / c mice were immunized by subcutaneous injection of the recombinant protein EB200 (20 ⁇ g) in presence of Freund's complete adjuvant. Two booster injections, each of 15 ⁇ g of antigen, in incomplete Freund's adjuvant were given 3 weeks and 5 weeks respectively after the first injection.
  • Antibody titers were determined by immunofluorescence on parasitized red blood cells spread in thin layers. It is the antibodies of such a serum that have been found to be monospecific. Tested in immunoprecipitation and by Westerm Blot on parasitic extracts, the anti-PfEB200 antibodies have been shown to be able to react with a protein of apparent molecular weight of approximately 1000 kDa belonging to the parasite Plasmodium falciparum.
  • the antibodies directed against the recombinant protein expressed by the clone EB200 were tested according to their capacity to inhibit, in vitro, the reinvasion of red blood cells by merozoites, in particular in the test described by R. Udomsangpetch et al (37) which itself refers to a publication by Wâhlin, B. et al., entitled "Human antibodies to a M r 155,000 Plasmodium falciparum efficiently inhibit merozo ⁇ te invasion" (human antibodies against a M r 155,000 of Plasmodium falciparum effectively an invasion to merozoites) (1984) Proc. Nat. Acad. Sci. USA 81: 7912.
  • the recombinant antigen PfEB200 is capable of inhibiting the phagocytosis of opsonized parasitized red blood cells (technique described in (ii) by monocytes of monkeys.
  • the EB200 protein or fractions of it still have the following characteristics:
  • polypeptide EB200 In the above, reference was made above all to the polypeptide EB200. It goes without saying that the invention also relates to any part of this polypeptide which is able, if necessary after incorporation into a fusion protein, oligomerization or conjugation to a carrier protein, to induce in vivo a protective immune response on the squirrel monkey Saimiri sciureus.
  • the invention extends to all equivalent peptides resulting from the expression of the corresponding sequences of DNAs originating from infectious Plasmodiae for humans, other than P. falciparum, for example strains 7G8 (Brazil), T9- 96 (Thailand), FcBR (South America), 37D (derived from NF54, Netherlands) and Banjul (Gambia) which are all recognized in immunoprecipitation tests with the serum ⁇ EB200. It also recognizes erythrocytes infected with these parasites.
  • all peptides are structurally differentiating from the previous ones by substitutions, deletions or additions of one or more amino acids is part of the invention since it is recognized by antibodies previously formed against EB200 and, preferably, if necessary after oligomerization, incorporation into a fusion protein, conjugation to a carrier molecule, since it induces in the squirrel monkey Saimiri sciureus antibodies active against parasites of human malaria, in particular P. falciparum.
  • any polypeptide or fragment of polypeptide comprising in common a part of amino acid sequence with EB200 and which is capable of inducing in vivo antibodies capable of inhibiting the invasion of globules red in a proportion at least equal to 50% compared to those observable in preparations of control erythrocytes placed under the same conditions in the presence of the same concentrations of merozoites, but in the absence of the polypeptides or part of polypeptides of the genus in question .
  • the invention naturally relates to any polypeptide containing a part of a sequence extending beyond the ends of the EB200 sequence, since the polypeptide concerned would exhibit protective immunogenic properties. of the same nature.
  • Part beyond the ends of EB200 is meant, for example, the amino acid sequences which adjoin the amino acid structure of EB200 in the larger peptide sequence deduced from the nucleotide sequence of the G9 fragment, from which EB200 is derived (FIG. 1C ).
  • the invention extends its effects to all peptides derived from the preceding ones, except that certain aminoacyl residues of their amino acid sequences would be substituted by others, even deleted or on the contrary intercalated in the represented sequences , without the modified peptides obtained losing the biological properties characteristic of the EB200 polypeptide.
  • the invention relates more particularly to the monomer sequences corresponding to the degenerate repeating units present in EB200, in particular the peptides whose sequences correspond to the sequences (a to o) which follow:
  • the peptides of the genus in question are capable of inducing in vivo a protective immune response in the squirrel monkey Saimiri sciureus or else of inhibiting the invasion of erythrocytes by merozoites.
  • part of the invention is also any peptide equivalent containing motifs which can be represented by the formula:
  • each of the groups X represents, according to the index which follows the signs of parenthesis which surrounds the group considered, a dipeptide or tripeptide each containing , at least one hydrophobic amino-acyl residue, this peptide equivalent being capable of inducing a protective immune response in the squirrel monkey Saimiri sciureus or of inhibiting an in vitro invasion of erythrocytes by P. falciparum.
  • the invention relates to any fusion protein, oligomer or conjugate involving the peptides according to the invention.
  • These peptides can be produced by chemical synthesis. It can also be the same for oligomers of these peptides or peptides conjugated to carrier molecules. Techniques allowing these syntheses to be carried out are recalled in the following, of course without limitation.
  • This synthetic method consists in successively condensing, in the required order, the amino acids or peptides previously formed with other amino acids or peptides previously formed chosen so as to allow the production of a polypeptide having the final sequence chosen as amino acids, being it is understood that care will have been taken, if necessary, to protect beforehand all the reactive functions carried by these amino acids or peptides, with the exception of those of their amine and carboxyl functions which must intervene in the formation of peptide bonds, in particular after activation of said carboxyl functions.
  • aminoacyl used has an additional acid function (in particular in the case of glutamic acid), these functions are protected, for example by t-butylester groups.
  • the synthesis preferably begins with the condensation of the C-terminal amino acid with the amino acid which corresponds to the neighboring amino acid in the desired sequence and, closely in close proximity, with the other amino acids chosen in an appropriate manner, the synthesis ends with the fixing of the N-terminal amino acid.
  • the first C-terminal amino acid in the chain to be produced is attached to a porous polymer resin, acting as an insoluble support through its carboxylic group.
  • the amino function of this first amino acid will normally have been protected beforehand with an appropriate protective group, for example by a t-butyloxycarbonyl group.
  • the oligomerization of the monomer blocks obtained can be carried out in any case as is known. These oligomers contain, for example from 2 to 20 monomer units. There is moreover no limit to the maximum number of monomer units capable of being included in the oligomer, except for the capacity of the oligomer finally obtained to be water-soluble or easily soluble in l 'water.
  • a first method of oligomerization or polymerization of the monomer consists in the reaction of the latter with a crosslinking agent, such as glutaraldehyde.
  • a crosslinking agent such as glutaraldehyde.
  • Another preferred oligomerization process uses the techniques described by Richman and Reese, RT. (1988) Proc. Nat. Acad. a5: 1662-1666 or Posnett, DN. (1988) The Journal of Biological Chemistry Vol. 283, No. 4. These methods involve a small "central peptide matrix" (peptidyl core matrix) formed from a limited number of trifunctional amino acids, in particular lysines, interconnected so as to contain a large number of free functions vis with respect to the molecular weight of this central matrix and forming as many arms or dendrites which can then be conjugated with distinct amino acids or with C-terminal ends.
  • central peptide matrix peptidyl core matrix
  • central matrix based on heptalysins comprising sites for the attachment of 9-16 peptide chains.
  • Cys - SS - C— Peptide Cys - SS - C— Peptide
  • oligomer also includes the conjugates obtained by covalent coupling of several monomer units to a carrier molecule (natural or synthetic), physiologically acceptable and non-toxic, by means of complementary reactive groups respectively carried by the carrier molecule and / or the monomer units. Examples of suitable groupings are illustrated in the following.
  • polylysines or poly (D-L-alanine) -poly (L-lysine) or immunologically inert proteins.
  • Any coupling process can be used which involves, on the one hand, one or more reactive functions of the peptide and, on the other hand, one or more reactive functions of support molecules.
  • these are the carboxyl and amino functions, which can give rise to a coupling reaction in the presence of a coupling agent of the type of those used.
  • a coupling agent of the type of those used.
  • proteins for example, 1- ethyl-3- (3-dimethylaminopropyl) - carbodiimide, N-hydroxybenzotriazole, etc.
  • glutaraldehyde especially when it comes to link together amino groups respectively carried by the peptide and the support molecule.
  • the invention also relates to a variant of the process for producing the oligomers according to the invention, this variant consisting in the transformation of cells of a culture of competent cells with a vector containing a nucleotide sequence coding for an oligomeric polypeptide containing the above-mentioned units. monomers under the control of a promoter recognized by said competent cells, under conditions allowing the expression in these cells of said oligomeric nucleotide sequence, and the recovery of the expression product of said nucleotide sequence.
  • This is also part of the invention. There is no need to insist on the techniques that can be used, except that, preferably, the elements of the vector are preferably such that the expression product is transported outside the cells. competent, even excreted in their culture medium.
  • the invention naturally relates to the corresponding acid fragments, more particularly to all or part of those which follow from FIG. 1 (c) with regard to the sequence G9.
  • This DNA fragment can either be in the state individualized, or recombined within a larger recombinant DNA.
  • these DNAs of larger sizes it is understood that the invention relates to:
  • DNAs coding for fusion proteins involving a DNA sequence coding for a peptide according to the invention with DNA sequences coding themselves for heterologous peptide sequences which do not interfere with the induction properties by the polypeptide sequence according to the invention of a protective immunity against P. falciparum;
  • Any vector containing this polypeptide sequence placed under the control of a promoter authorizing its expression in a competent cellular host and whose polymerases are capable of recognizing said promoter.
  • the invention extends its effects to cell cultures thus transformed by such recombinant vectors or DNA which allow the expression of the polypeptide or fragment of the polypeptide according to the invention.
  • part of the invention forms the specific antibodies of the sequences contained in EB200 or of peptides or fragments of peptides having the same biological properties as defined above.
  • This protection extends its effects to all monoclonal antibodies specific for the same polypeptide sequences, as well as to secretory hybridomas of these monoclonal antibodies. It will be understood that the skilled person is able to produce these monoclonal antibodies and to select them in sorting operations involving the specific recognition by the monoclonal antibodies to be selected, amino acid sequences of peptides characteristic of the invention.
  • the invention likewise relates to any pharmaceutical composition using these polypeptides, if appropriate associated with pharmaceutically acceptable vehicles facilitating their use in vaccination operations in order to induce the production in vivo, in particular in humans, of a protective immune response against infectious malarial parasites.
  • the invention also relates to the antibody preparations, induced by EB200 or the equivalent peptides of the invention, whether these antibodies are polyclonal (sera containing them or immunoglobulin fraction purified from these sera) or monoclonal antibodies obtained as indicated below. -above.
  • the invention relates to pharmaceutical compositions containing such antibodies for the purpose of entering into competition in vivo with the human parasite, in particular in individuals infected or threatened with infection by this parasite, in particular of the P. falciparum type, in order to protect them from the pathogenic effects due to this infection.
  • the antibodies according to the invention are also more particularly characterized by the fact that they do not give rise to crossed immune reactions with the expression products of the Pf11-1 gene (27) and of the Pf155 / RESA gene (38).
  • the clone containing the nucleotide sequence coding for EB200, contained in Escherichia coli DH5 was deposited on July 25 under number I-1128 at the National Collection of Cultures of Microorganisms of the Institut Pasteur de Paris (CNCM). The bibliography to which reference has been made in this description by references to publication numbers supplements this description.
  • the invention also relates to the other applications of these antibodies, in particular their use for the in vitro diagnosis, for example carried out on a serum sample, of an infection by malarial parasites, in particular at the stage of infection of blood erythrocytes, for example by means of a conventional antigen-antibody reaction.
  • the invention finally relates to the application of the peptides themselves to the in vitro detection of the presence of antibodies characteristic of infection by malarial parasites, in particular of the P. falciparum type or the like, and more particularly still at the erythrocyte stage of these parasites, in the form of merozoites.
  • Anti-PfEB200 antibodies have also been used to locate the Pf332 antigen in the parasite.
  • Indirect immunofluorescence on parasites fixed in air and analyzed by confocal microscopy, shows an image formed by structures similar to vesicles of about 0.5-1 ⁇ m. These vesicles were located in the cytoplasm of the parasitized red blood cell, dissociated from the parasite, and transported to the membrane of the erythrocyte.
  • the Pf332 antigen appears to be associated with the membrane of red blood cells. Indeed, immunoelectromicroscopy shows that the Pf332 antigen is transported towards the membrane of the erythrocyte in association with "Maurer's clefts".
  • the anti-PfEB200 serum reacts with the surface of the parasitized monkey red blood cells.
  • the presence of epitopes of Pf332 on the surface of red blood cells confirms that this antigen may be the target of host immunological reactions and therefore be of interest for the development of a vaccine.
  • the recombinant polypeptide PfEB200 inhibits phagocytosis, by monocytes, erythrocytes infected in the presence of hyperimmune monkey serum.
  • the invention also relates to compositions using the peptides previously defined in association with other peptide constituents having vaccinating properties against malaria, in particular due to Plasmodium falciparum, more particularly the ability to induce a protective immune response in vivo, including the production of immunoprotective antibodies capable of destroying the parasites of the blood stage, responsible for the disease in humans, in a primate model represented by the squirrel monkey SAIMIRI SCIUREUS.
  • the "second polypeptide” consists of a polypeptide derived from a major P falciparum antigen, having a molecular weight of the order of 72 kDA, hence the designation "72 kDA antigen” used in the following for the denote, in particular of a polypeptide derived from this 72 kDA antigen having the capacity, like this:
  • This "second polypeptide" is itself often recognized by sera from human patients living in regions where malaria is endemic.
  • the nucleotide sequence coding for the 72 kDA antigen peptide sequence has been identified by LS MATTEI et al European Journal of Immunology (1989) 19: 1823 - 1828.
  • the "second polypeptide” advantageously consists of a polypeptide hereinafter designated “i72" comprising the 153 amino acids of the C - terminal region of the 72 kDA antigen.
  • the amino acid sequence, as well as a nucleic acid sequence encoding the i72 polypeptide, are shown below:
  • second polypeptide can also be constituted by a recombinant polypeptide containing all or part of the sequence of i72, for example a fusion molecule with the glutatione transferase of Schistosoma japonicum, according to the technique described by DB Smith and KS Johnson (1988) Gene 67, 41-48, by transformation of E. Coli with the plasmid pGEX previously modified with the appropriate insertion nucleotide sequence.
  • Coli Escherichia coli DH5 ⁇ transformed by the above plasmid modified by a nucleotide sequence coding for the peptide i72, was deposited on July 24, 1991 with the National Collection of Cultures of Microorganisms of the Institut Pasteur de Paris (CNCM) under number I - 1129.
  • third polypeptide (peptide or oligopeptide) as well as a nucleic acid fragment which codes for this third polypeptide, are characterized in that they contain the following respective sequence elements:
  • GAA CAA TTA AAT AAA AAT AAG GAT GGA TAT GTT GTT TTG GTT ACT GAA
  • TTT TTA AAG TTA TTA ATA AAA TTA CAT AAT GCA GGA TTG GTA CAT CTT
  • a strain of E. Coli, transformed by such a nucleic acid was deposited at the CNCM under the number I-987 on July 27, 1990.
  • a preferred "third peptide” is the recombinant peptide containing the sequence of the peptide "R 23" - of formula
  • a strain of E. Coli containing the corresponding DNA sequence was deposited on July 27, 1990 under number I - 986 at the CNCM.
  • the R23 peptide was likewise produced in the form of a recombinant fusion protein with the glutatione transferase from Schistosoma japonicum, according to the technique already mentioned by D.B. Smith and K.S. Johnson.
  • these different peptides can be replaced by fragments or fusion proteins meeting the conditions which have also been defined above.
  • the invention naturally also relates to the mixtures of specific antibodies which can be obtained from the respective constituents of the above-mentioned mixtures of polypeptides.
  • These mixtures antibodies can be administered in vivo, in particular for the purpose of interfering with parasitaemia due to infection by a human parasite, in particular of the P. falciparum type.
  • the invention likewise extends its effects to mixtures of the nucleic sequences in which the different corresponding nucleic sequences have been recombined with one another, under conditions permitting their expression in the form of a fusion protein comprising peptide sequences corresponding to the sequences of the three constituents (whole or partial) previously taken into consideration.
  • telomere-rclated sequences 39. Veroick, K. D., D. Walliker, and T. F. McCutchan. 1988. Genetic hypervariability of telomere-rclated sequences is associated with meiosis in Plasmodiumfalciparum.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Communicable Diseases (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a new antigen of Plasmodium falciparum and its application to the production of compounds for vaccines against the human parasites responsible for paludism. Said antigen is characterized by the amino acid sequence (I) or by a portion of this sequence.

Description

OLYPEPTIDES APTES A INDUIRE IN VIVO DES ANTICORPS EUX-MEMES CAPABLES D'INHIBER L'INVASION DE GLOBULES ROUGES PAR DES MEROZOITES DE  OLYPEPTIDES SUITABLE FOR INDUCING IN VIVO ANTIBODIES THEY themselves CAPABLE OF INHIBITING THE INVASION OF RED CELLS BY MEROZOITES OF
P.FALCIPARUM, PRODUITS APPARENTES ET LEUR APPLICATION A LA PRODUCTION DE COMPOSITIONS VACCINANTES P.FALCIPARUM, RELATED PRODUCTS AND THEIR APPLICATION TO THE PRODUCTION OF VACCINANT COMPOSITIONS
L'invention a pour objet de nouveaux antigènes de Plasmodium falciparum et leur application notamment en vue de la protection contre le paludisme. The subject of the invention is new antigens of Plasmodium falciparum and their application in particular with a view to protection against malaria.
Les parasites responsables du paludisme chez l'homme, dont notamment Plasmodium falciparum ou Plasmodium vivax pour ne citer que les principaux d'entre eux, présentent chez l'hôte humain des morphologies différentes et expriment des antigènes différents, fonction du stade de leurs développpement et de leur localisation dans l'organisme de l'hôte infecté. Les différences morphologiques et antigeniques de ces parasites au cours de leur cycle de vie chez l'homme, permettent de définir au moins quatre stades de développement distincts.  The parasites responsible for malaria in humans, including Plasmodium falciparum or Plasmodium vivax to name only the main ones, present in the human host different morphologies and express different antigens, depending on the stage of their development and their location in the organism of the infected host. The morphological and antigenic differences of these parasites during their life cycle in humans make it possible to define at least four distinct stages of development.
Le tout premier stade de développement du parasite chez l'homme correspond à la forme sporozoïte introduite dans le sang de l'hôte, par piqûres d'insectes porteurs du parasite. Le second stade correspond au passage du parasite dans le foie et à l'infection des cellules hépatiques dans lesquelles les parasites se développent pour former les schizontes hépatiques qui, lorsqu'ils sont matures (par exemple pour P. falciparum le 6è jour après pénétration des sporozoïtes), libèrent par éclatement des mérozoïtes hépatiques. Le troisième stade est caractérisé par l'infection des érythrocytes sanguins par les formes asexuées (mérozoïtes) du parasite; ce stade érythrocytaire de développement correspond à la phase pathogène de la maladie. Le quatrième stade correspond à la formation des formes à potentiel sexué (ou gamétocytes) qui deviendront des formes sexuées ou gamètes extra-cellulaires chez le moustique. The very first stage of development of the parasite in humans corresponds to the sporozoite form introduced into the host's blood, by bites from insects carrying the parasite. The second stage corresponds to the passage of the parasite in the liver and to the infection of the hepatic cells in which the parasites develop to form the hepatic schizonts which, when they are mature (for example for P. falciparum on the 6th day after penetration of the sporozoites), release by bursting hepatic merozoites. The third stage is characterized by the infection of blood erythrocytes by the asexual forms (merozoites) of the parasite; this erythrocyte stage of development corresponds to the pathogenic phase of the disease. The fourth stage corresponds to the formation of forms with sexual potential (or gametocytes) which will become sexual forms or extra-cellular gametes in the mosquito.
Des études nombreuses ont été consacrées au cours de ces dernières années à l'identification de molécules de parasites exposées à la surface de globules rouges sanguins ou érythrocytes infectés avec le parasite humain pathogène, notamment du type Plasmodium falciparum. En effet, la virulence des parasites au stade sanguin a souvent été associée à l'existence de structures peptidiques ainsi exposées. Elles pourraient être responsables de deux phénomènes souvent observés, à savoir l'obstruction de capillaires vasculaires mettant en jeu une fixation de globules rouges parasités (PRBC, abréviation de l'expression "Parasitized Red Blood Cells) aux cellules endothéliales ou séquestration (2), et la fixation d'érythrocytes non infectés aux PRBCs avec formation de "rosettes" (40). Ces phénomènes paraissent jouer un rôle médiateur important dans la physiopathologie des cas de paludisme sévère, notamment de ceux induits par Plasmodium falciparum.  Numerous studies have been devoted in recent years to the identification of parasite molecules exposed on the surface of red blood cells or erythrocytes infected with the pathogenic human parasite, in particular of the Plasmodium falciparum type. Indeed, the virulence of parasites in the blood stage has often been associated with the existence of peptide structures thus exposed. They could be responsible for two often observed phenomena, namely the obstruction of vascular capillaries involving a fixation of parasitized red blood cells (PRBC, abbreviation of the expression "Parasitized Red Blood Cells) to endothelial cells or sequestration (2), and the attachment of uninfected erythrocytes to PRBCs with the formation of "rosettes" (40). These phenomena appear to play an important mediating role in the pathophysiology of severe malaria cases, in particular those induced by Plasmodium falciparum.
Ces molécules de surface pourraient aussi représenter une cible pour les mécanismes de défense immunitaire de l'hôte dont l'une des actions conduit à la phagocytose des PRBCs (10,11). Il y a quelque temps un gène, exprimé dans les stades tardifs du cycle intra-érythrocytique, à été isolé (20). Le produit d'expression (antigène 332) s'est avéré contenir une série de séquences d'acides aminés, répétitives dégénérées. Il a été montré que des anticorps humains purifiés par affinité au contact d'un polypeptide recombinant contenant la séquence de l'antigène 332, réagissait avec une famille de protéines du parasite qui consistaient en des produits d'expression de gènes différents (20). Parmi les antigènes identifiés au sein de cette famille, on mentionnera les antigènes 11.1 (Pf11-1) et (Pf155/RESA) qui contenaient eux-mêmes des déterminants immunogéniques conduisant à des réactions croisées comme cela fut observé en utilisant un anticorps monoclonal humain (20). Plus récemment Udomsangtepch et al (36) décrivirent un anticorps monoclonal humain mAb 33G2 capable d'inhiber la cytoadhérence de globules rouges parasités comportant ou non des protubérances avec des cellules de la lignée cellulaire de mélanomes C32. Il a de plus été constaté que cet anticorps monoclonal inhibe, de façon efficace, l'invasion d'érythrocytes par les mérozoïtes dans un test réalisé in vitro (38). Néanmoins, cet anticorps monoclonal initialement sélectionné en raison de sa réactivité avec la molécule Pf155/RESA (38), manque de spécificité. Il réagit également avec d'autres antigènes apparaissant au stade sanguin de l'infection par le parasite, notamment les antigènes Ag11-1 et Ag332 (20,37). L'antigène Ag 332 présente sans aucun doute un intérêt potentiel important eu égard à sa capacité à induire une immunité protectrice. En effet, un polypeptide recombinant incluant le produit d'expression d'un fragment de 303 paires de bases issues de gènes Pf332 s'est avéré constituer un inhibiteur particulièrement efficace de l'anticorps monoclonal mAb33G2, comme cela fut démontré dans un essai d'inhibition de l'invasion des globules rouges par les mérozoïtes (37). Néanmoins, la caractérisation de Ag332 a jusqu'à ce jour été inhibée par les réactions croisées auxquelles les anticorps qui le reconnaissent donnent lieu, avec différents autres antigènes du parasite (20). These surface molecules could also represent a target for defense mechanisms host immune system, one of whose actions leads to phagocytosis of PRBCs (10,11). A gene, expressed in the late stages of the intraerythrocytic cycle, has been isolated for some time (20). The expression product (antigen 332) was found to contain a series of degenerate, repetitive amino acid sequences. Affinity-purified human antibodies on contact with a recombinant polypeptide containing the 332 antigen sequence have been shown to react with a family of parasite proteins that consist of different gene expression products (20). Among the antigens identified within this family, mention will be made of antigens 11.1 (Pf11-1) and (Pf155 / RESA) which themselves contained immunogenic determinants leading to cross-reactions as was observed using a human monoclonal antibody ( 20). More recently Udomsangtepch et al (36) described a human monoclonal antibody mAb 33G2 capable of inhibiting the cytoadherence of parasitized red blood cells, whether or not having protuberances with cells of the C32 melanoma cell line cells. It has also been found that this monoclonal antibody effectively inhibits the invasion of erythrocytes by merozoites in an in vitro test (38). However, this monoclonal antibody initially selected because of its reactivity with the molecule Pf155 / RESA (38), lacks specificity. It also reacts with other antigens that appear in the bloodstream stage of infection with the parasite, including the antigens Ag11-1 and Ag332 (20,37). The Ag 332 antigen presents undoubtedly a significant potential interest with regard to its ability to induce protective immunity. Indeed, a recombinant polypeptide including the expression product of a fragment of 303 base pairs derived from Pf332 genes has been found to constitute a particularly effective inhibitor of the monoclonal antibody mAb33G2, as was demonstrated in a test of inhibition of the invasion of red blood cells by merozoites (37). However, the characterization of Ag332 has so far been inhibited by the cross-reactions to which antibodies which recognize it give rise, with various other antigens of the parasite (20).
La présente invention résulte de l'identification et de la caractérisation d'une nouvelle séquence génique au sein du gène Pf332, de l'antigène codé par cette séquence génique et d'anticorps inductibles in vivo par cet antigène et spécifique de l'antigène. La séquence génique en question a été localisée dans la région subtélomérique du chromosome 11. L'antigène exprimé par cette séquence génique s'est avéré avoir un caractère monospécifique lui permettant de détecter spécifiquement une protéine mégadaltonique du parasite ou des parasites asexués au stade sanguin par des réactions d'immuno-buvardage (immunoblot) et d'immunoprécipitation, contrairement aux anticorps induits par d'autres antigènes, soit issus de Ag332, soit apparentés à des séquences de celui-ci, tels que le dimère synthétique Y(SVTEEIAEEDK)2 (24) ou une protéine de fusion avec la béta-galactosidase (20). Ces anticorps réagissent également avec diverses autres molécules du parasite. Des anticorps formés contre l'antigène EB200 conduisent également à des réactions de surface avec des globules rouges infectés par Plasmodium falciparum avec une efficacité ayant le même ordre de grandeur que celle de l'anticorps monoclonal mAb33G2. The present invention results from the identification and characterization of a new gene sequence within the Pf332 gene, of the antigen encoded by this gene sequence and of antibodies inducible in vivo by this antigen and specific for the antigen. The gene sequence in question has been located in the subtelomeric region of chromosome 11. The antigen expressed by this gene sequence has been shown to have a monospecific character allowing it to specifically detect a mega-colortonic protein of the parasite or asexual parasites in the blood stage by immunoblotting and immunoprecipitation reactions, unlike antibodies induced by other antigens, either derived from Ag332 or related to sequences thereof, such as the synthetic dimer Y (SVTEEIAEEDK) 2 (24) or a fusion protein with beta-galactosidase (20). These antibodies also react with various other molecules of the parasite. Antibodies formed against the EB200 antigen also lead to surface reactions with red blood cells infected with Plasmodium falciparum with an efficiency having the same order of magnitude as that of the monoclonal antibody mAb33G2.
La séquence peptidique de l'antigène EB200, l'un des antigène conforme à la présente invention a une séquence peptidique caractéristique correspondant à celle qui est encadrée dans la séquence du clone G9 (figure l(c) ), cette séquence ayant été déduite du clone G9 résultant de l'amplification de l'un des fragments du gène Pf332 dans les conditions qui seront rappelées plus loin.  The peptide sequence of the EB200 antigen, one of the antigens according to the present invention has a characteristic peptide sequence corresponding to that which is framed in the sequence of the clone G9 (FIG. 1 (c)), this sequence having been deduced from the clone G9 resulting from the amplification of one of the fragments of the Pf332 gene under the conditions which will be recalled later.
L'antigène EB200 appartient de façon plus générale à la famille de polypeptides ayant un motif ou une séquence en acides aminés en commun contenant tout ou partie de la séquence polypeptidique ci- dessous définie par sa séquence en acides aminés :  The EB200 antigen more generally belongs to the family of polypeptides having a common amino acid motif or sequence containing all or part of the polypeptide sequence below defined by its amino acid sequence:
S V T G N I L V E G S V T G N I L V E G
S V T E E V V G E E KS V T E E V V G E E K
L V S E E I V T E E GL V S E E I V T E E G
S V A Q E I V E E D AS V A Q E I V E E D A
P A T E E I D E I EP A T E E I D E I E
S V T E E V V E E E GS V T E E V V E E E G
P V D E E I V Q E E GP V D E E I V Q E E G
T V T E E I I Q G ET V T E E I I Q G E
S K V E E V V E E Q GS K V E E V V E E Q G
S E N E E I F V E E VS E N E E I F V E E V
S A S Q E I V Q N ES A S Q E I V Q N E
S G T E E I L E K VS G T E E I L E K V
S A S Q E I V Q D GS A S Q E I V Q D G
S V T E Q I I E E L F P V T E E V V N E E E SVTEQIIEELF PVTEEVVNEEE
Un peptide qui ne contient qu'une partie de cette séquence fait partie de l'invention dès lors que ce peptide est apte, le cas échéant lorsqu'il est incorporée à une protéine de fusion, oligomérise ou conjugué à une protéine porteuse, à induire in vivo une réponse immunitaire protectrice chez le singe écureuil Saimiri sciureus.  A peptide which contains only part of this sequence is part of the invention when this peptide is capable, if necessary when it is incorporated into a fusion protein, oligomerizes or conjugated to a carrier protein, in vivo a protective immune response in the squirrel monkey Saimiri sciureus.
La répartition des motifs successifs de la séquence en acides aminés d'un polypeptide préféré conforme à l'invention, telle qu'elle a été présentée ci-dessus, n'a d'autre objectif que celui de faire apparaître l'existence de motifs répétitifs successifs, au sein de la protéine, cette répétitivité n'étant cependant pas dépourvue de dégénérescences. Les blancs (en fait des liaisons directes) qui apparaissent à la fin de certaines des lignes ou au sein de certaines des lignes n'ont aucune signification particulière. Leur seul but est la mise en évidence visuelle des homologues de séquences partielles de ces motifs répétitifs successifs. On peut remarquer la fréquence du motif VTEEV au sein de ces motifs partiellement répétitifs ou "dégénérés".  The distribution of the successive motifs of the amino acid sequence of a preferred polypeptide in accordance with the invention, as presented above, has no other objective than that of revealing the existence of motifs successive repetitions, within the protein, this repetitiveness is not however devoid of degenerations. The blanks (actually direct links) that appear at the end of some of the lines or within some of the lines have no particular meaning. Their only aim is to visually highlight the homologs of partial sequences of these successive repeating patterns. We can notice the frequency of the VTEEV motif within these partially repetitive or "degenerate" motifs.
La même séquence en acides aminés (utilisant la représentation à trois lettres par acides aminés, au lieu de la représentation à une lettre dans la définition qui précède) est présentée dans la figure 2. Celle-ci fait également apparaître la séquence codante du clone recombinant, nommé PfEB200, contenant un insert de 411 paires de bases isolées à partir du gène Pf332. Il est rappelé que ce clone avait déjà été isolé à partir d'une banque de séquences d'expression construites dans un vecteur λAmp3 avec un lot (pool) de sérums humains immuns (20) . La présente invention découle de la mise en évidence de l'existence au sein de l'antigène Ag332 de séquences en acides aminés qui sont à la fois spécifiques de cet antigène et exposés à la surface des mérozoïtes de P. falciparum et/ou des globules rouges infectés. The same amino acid sequence (using the three-letter representation by amino acids, instead of the one-letter representation in the above definition) is shown in Figure 2. This also shows the coding sequence of the recombinant clone , named PfEB200, containing an insert of 411 base pairs isolated from the Pf332 gene. It is recalled that this clone had already been isolated from a bank of expression sequences constructed in an λAmp3 vector with a batch (pool) of immune human sera (20). The present invention derives from the demonstration of the existence within the Ag332 antigen of amino acid sequences which are both specific for this antigen and exposed on the surface of the merozoites of P. falciparum and / or of the globules infected reds.
La banque génomique initiale de P. falciparum dans le vecteur λgtWES avait elle-même été construite par digestion à l'aide de DNasel et addition d'adaptateurs (linkers) contenant des sites EcoRI comme antérieurement décrits dans (21).  The initial P. falciparum genomic library in the vector λgtWES had itself been constructed by digestion using DNasel and addition of adapters (linkers) containing EcoRI sites as previously described in (21).
Le criblage de la banque génomique initiale d'ADN de P. falciparum (souche Palo Alto à protubérances positives) par digestion à l'aide d'une nucléase et clonage dans un vecteur λgtWES, et à l'aide d'une sonde d'hybridation issue du clone Pf332 a conduit les inventeurs à porter leur attention sur une série de quatre clones C1, G1, G9 et G90 et à les séquencer partiellement. Les séquences nucléotidiques de G1, G9 et G90, chevauchantes, avaient des tailles respectivement de 2,9 Kb; 4,8 Kb et 1,9 Kb. Le criblage d'une banque de cADNs davait conduire à un clone contenant un insérât de 466 paries de bases (clones C1) .  Screening of the initial genomic DNA library of P. falciparum (Palo Alto strain with positive protuberances) by digestion using a nuclease and cloning into a λgtWES vector, and using a probe hybridization from the clone Pf332 led the inventors to focus their attention on a series of four clones C1, G1, G9 and G90 and to partially sequence them. The overlapping nucleotide sequences of G1, G9 and G90 had sizes of 2.9 Kb, respectively; 4.8 Kb and 1.9 Kb. The screening of a library of cDNAs led to a clone containing an insert of 466 base parts (clones C1).
Les positions relatives des séquences de C1, G1, G9 et G90, vis-à-vis du clone initial dont elles sont issues sont schématiquement représentés dans la figure 1(a) :  The relative positions of the sequences of C1, G1, G9 and G90, with respect to the initial clone from which they are derived are schematically represented in FIG. 1 (a):
Celle-ci présente respectivement :  This presents respectively:
- une carte de restriction du gène Pf332, - les positions respectives des fragments retenus (C1, G90, G1 et G9), - a restriction map of the Pf332 gene, - the respective positions of the selected fragments (C1, G90, G1 and G9),
- les séquences en acides aminés déduites de parties des séquences de C1, G90, G1 et G9 (dont la séquence s'étend approximativement dans la seconde moitié de la première colonne puis dans la première moitié de la seconde colonne des séquences représentées dans la figure 1) qui ont plus particulièrement été séquencées.  - the amino acid sequences deduced from parts of the sequences of C1, G90, G1 and G9 (whose sequence extends approximately in the second half of the first column and then in the first half of the second column of the sequences shown in the figure 1) which have more particularly been sequenced.
Les parties séquencées sont représentées dans la figure 1 par des épaississement du trait. Les enzymes utilisés pour la production de la carte de restriction étaient les suivants : BamH1(B), Cla1(C), Dra1(D), EcoR1(E) et Hind3 (H) .  The sequenced parts are represented in FIG. 1 by thickening of the line. The enzymes used for the production of the restriction map were: BamH1 (B), Cla1 (C), Dra1 (D), EcoR1 (E) and Hind3 (H).
Des parties de ces fragments et plus particulièrement un sous-fragment de G9 (fragment Parts of these fragments and more particularly a sub-fragment of G9 (fragment
BamH1, EcoR1 de 441 paires de bases) a été souscloné dans un plasmide pGEX3(32). La séquence deBamH1, 441 base pair EcoR1) was subcloned into a plasmid pGEX3 (32). The sequence of
(ci-après dénommée EB200) protéine exprimée sous forme d'une protéine de fusion soluble PfEB200 avec une glutathione-S-transférase est l'objet d'un encadrement au sein de la partie de G9 qui avait été séquencée. L'expression du polypeptide soluble a été effectuée dans E.coli Dh5 α (F end A1 hsdr 17 (rkmk-) sup E44 thi-1, λ-, rec A1 gyr A96 rel A1 f80d lac ZΔ M15). La purification de la protéine a été faite par chromatographie d'affinité sur des billes d'agarose portant des groupes glutathion (32). Le degré de purification de la protéine de fusion a été analysée par électrophorèse sur gel de polyacrylamide contenant 10 % de SDS et par coloration au bleu de Coomassie. Toute technique classique peut être mise en oeuvre pour produire des anticorps spécifiques de la protéine EB200 (sous forme de fusion ou non). Mais, en l'occurrence, les anticorps testés dans les essais dont il est question plus loin ont été produits dans les conditions suivantes : des souris Balb/c ont été immunisées par injection sous-cutanée de la protéine recombinante EB200 (20 μg) en présence de l'adjuvant complet de Freund. Deux injections de rappel, chaque fois de 15 μg d'antigène, dans de l'adjuvant incomplet de Freund ont été effectuées 3 semaines et 5 semaines respectivement après la première injection. Des sérums sanguins contenant les anticorps produits ont été prélevés sur les souris immunisées 7 jours après la dernière injection. Les titres en anticorps ont été déterminés par immunofluorescence sur des globules rouges parasités étalés en couches minces. Ce sont les anticorps d'un tel sérum qui se sont avérés être monospécifiques. Testés en immunoprécipitation et par Westerm Blot sur des extraits parasitaires, les anticorps anti-PfEB200 se sont avérés capables de réagir avec une protéine de poids moléculaire apparent d'environ 1000 kDa appartenant au parasite Plasmodium falciparum. (hereinafter referred to as EB200) protein expressed in the form of a soluble fusion protein PfEB200 with a glutathione-S-transferase is the subject of a frame within the part of G9 which had been sequenced. The expression of the soluble polypeptide was carried out in E.coli Dh5 α (F end A1 hsdr 17 (rkmk-) sup E44 thi-1, λ-, rec A1 gyr A96 rel A1 f80d lac ZΔ M15). The protein was purified by affinity chromatography on agarose beads carrying glutathione groups (32). The degree of purification of the fusion protein was analyzed by electrophoresis on polyacrylamide gel containing 10% SDS and by staining with Coomassie blue. Any conventional technique can be used to produce antibodies specific for the EB200 protein (in the form of a fusion or not). However, in this case, the antibodies tested in the tests referred to below were produced under the following conditions: Balb / c mice were immunized by subcutaneous injection of the recombinant protein EB200 (20 μg) in presence of Freund's complete adjuvant. Two booster injections, each of 15 μg of antigen, in incomplete Freund's adjuvant were given 3 weeks and 5 weeks respectively after the first injection. Blood sera containing the antibodies produced were collected from the immunized mice 7 days after the last injection. Antibody titers were determined by immunofluorescence on parasitized red blood cells spread in thin layers. It is the antibodies of such a serum that have been found to be monospecific. Tested in immunoprecipitation and by Westerm Blot on parasitic extracts, the anti-PfEB200 antibodies have been shown to be able to react with a protein of apparent molecular weight of approximately 1000 kDa belonging to the parasite Plasmodium falciparum.
La protéine EB200 et les anticorps correspondants ont encore donné lieu aux observations suivantes :  The EB200 protein and the corresponding antibodies gave rise to the following observations:
les anticorps dirigés contre la protéine recombinante exprimé par le clone EB200 ont été testés selon leur capacité à inhiber, in vitro, la réinvasion des globules rouges par les mérozoïtes, notamment dans le test décrit par R. Udomsangpetch et al (37) qui lui même renvoie à une publication de Wâhlin, B. et al., intitulée "Human antibodies to a Mr 155,000 Plasmodium falciparum efficiently inhibit merozoïte invasion" (des anticorps humains contre une Mr 155.000 de Plasmodium falciparum inhibent efficacement une invasion aux mérozoïtes) (1984) Proc. Nat. Acad. Sci. USA 81 : 7912.Dans ce test, les parasites cultivés en présence du sérum antiEB200 ont été inhibés à 84,4 % par rapport aux contrôles ou témoins cultivés en présence de sérum normal. L'anticorps monoclonal 33G2 dans le même test a inhibé 86,7 % de la parasitémie. Les activités de sérums anti-EB200 (anticorps polyclonal) et de l'anticorps monoclonal 33G2 étaient donc du même ordre de grandeur, the antibodies directed against the recombinant protein expressed by the clone EB200 were tested according to their capacity to inhibit, in vitro, the reinvasion of red blood cells by merozoites, in particular in the test described by R. Udomsangpetch et al (37) which itself refers to a publication by Wâhlin, B. et al., entitled "Human antibodies to a M r 155,000 Plasmodium falciparum efficiently inhibit merozoïte invasion" (human antibodies against a M r 155,000 of Plasmodium falciparum effectively an invasion to merozoites) (1984) Proc. Nat. Acad. Sci. USA 81: 7912. In this test, the parasites grown in the presence of antiEB200 serum were 84.4% inhibited compared to controls or controls grown in the presence of normal serum. The monoclonal antibody 33G2 in the same test inhibited 86.7% of the parasitaemia. The activities of anti-EB200 sera (polyclonal antibody) and of the monoclonal antibody 33G2 were therefore of the same order of magnitude,
- le sérum anti-EB200 réagi avec la surface des globules rouges de singe infectés par P. falciparum, - anti-EB200 serum reacted with the surface of monkey red blood cells infected with P. falciparum,
- l'antigène recombinant PfEB200 est capable d'inhiber la phagocytose des globules rouges parasités opsonisés (technique décrite dans (ii) par les monocytes des singes. - the recombinant antigen PfEB200 is capable of inhibiting the phagocytosis of opsonized parasitized red blood cells (technique described in (ii) by monocytes of monkeys.
Ces résultats témoignent donc de l'exposition de la partie de l'antigène Pf332 qui contient une séquence peptidique correspondante à celle de la protéine EB200 à la surface des globules rouges infectés par le parasite ou à la surface des mérozoïtes.  These results therefore testify to the exposure of the part of the Pf332 antigen which contains a peptide sequence corresponding to that of the EB200 protein on the surface of the red blood cells infected by the parasite or on the surface of merozoites.
La protéine EB200 ou des fractions de celle-ci présentent encore les caractéristiques suivantes :  The EB200 protein or fractions of it still have the following characteristics:
- elles sont reconnues par des anticorps protecteurs contenus dans des sérums ou des fractions immunoglobuliniques provenant d'animaux, notamment de singes Saimiri sciureus, résistants aux parasites, lesdits sérums ou fractions étant susceptibles de rendre résistants des singes initialement sensibles à Plasmodium falciparum; - they are recognized by protective antibodies contained in sera or immunoglobulin fractions originating from animals, in particular from Saimiri sciureus monkeys, resistant to parasites, said sera or fractions being capable of rendering resistant monkeys initially sensitive to Plasmodium falciparum;
elles sont inductrices notamment chez le singe, et plus particulièrement chez le singe Saimiri sciureus, d'anticorps actifs contre des parasites du paludisme, plus particulièrement de Plasmodium falciparum.  they are inducing in particular in monkeys, and more particularly in Saimiri sciureus monkeys, of antibodies active against malaria parasites, more particularly of Plasmodium falciparum.
Les conditions dans lesquelles de tels essais peuvent être réalisés ont été décrites, par exemple par Dubois et al, 1984 Proc. Nat. Acad. Sci. USA 81: 229-232; Perrin et al, 1984, J. of Exp. Med. 160: 440-451 et Siddiqui et al, 1987, Proc. Nat. Acad. Sci. USA 84: 3014-3018.  The conditions under which such tests can be carried out have been described, for example by Dubois et al, 1984 Proc. Nat. Acad. Sci. USA 81: 229-232; Perrin et al, 1984, J. of Exp. Med. 160: 440-451 and Siddiqui et al, 1987, Proc. Nat. Acad. Sci. USA 84: 3014-3018.
Dans ce qui précède il a surtout été fait référence au polypeptide EB200. Il va de soi que l'invention se rapporte également à toute partie de ce polypeptide qui est apte, le cas échéant après incorporation à une protéine de fusion, oligomérisation ou conjugaison à une protéine porteuse, à induire in vivo une réponse immunitaire protectrice sur le singe-écureuil Saimiri sciureus.  In the above, reference was made above all to the polypeptide EB200. It goes without saying that the invention also relates to any part of this polypeptide which is able, if necessary after incorporation into a fusion protein, oligomerization or conjugation to a carrier protein, to induce in vivo a protective immune response on the squirrel monkey Saimiri sciureus.
De même l'invention s'étend à tous les peptides équivalents résultant de l'expression des séquences correspondantes d'ADNs issus de Plasmodiae infectieux pour l'homme, autres que P. falciparum, par exemple des souches 7G8 (Brésil), T9-96 (Thaïlande), FcBR (Amérique du sud), 37D (dérivée de NF54, Pays Bas) et Banjul (Gambie) lesquelles sont toutes reconnues dans des tests d'immunoprécipitation avec le sérum αEB200. Il reconnaît de même des érythrocytes infectés par ces parasites. D'une façon générale tout peptide se différenciant structurellement des précédents par substitutions, délétions ou additions d'un ou de plusieurs aminoacides fait partie de l'invention dès lors qu'il est reconnu par des anticorps préalablement formés contre EB200 et, de préférence, le cas échéant après oligomérisation, incorporation dans une protéine de fusion, conjugaison à une molécule porteuse, dès lors qu'il induit chez le singe écureuil Saimiri sciureus des anticorps actifs contre des parasites du paludisme humain, notamment P. falciparum. Likewise, the invention extends to all equivalent peptides resulting from the expression of the corresponding sequences of DNAs originating from infectious Plasmodiae for humans, other than P. falciparum, for example strains 7G8 (Brazil), T9- 96 (Thailand), FcBR (South America), 37D (derived from NF54, Netherlands) and Banjul (Gambia) which are all recognized in immunoprecipitation tests with the serum αEB200. It also recognizes erythrocytes infected with these parasites. Generally, all peptides are structurally differentiating from the previous ones by substitutions, deletions or additions of one or more amino acids is part of the invention since it is recognized by antibodies previously formed against EB200 and, preferably, if necessary after oligomerization, incorporation into a fusion protein, conjugation to a carrier molecule, since it induces in the squirrel monkey Saimiri sciureus antibodies active against parasites of human malaria, in particular P. falciparum.
On peut également avoir recours pour identifier les polypeptides ou peptides entrant dans le cadre de l'invention, au test de d'inhibition de l'invasion de globules rouges par des mérozoïtes, notamment issus de P. falciparum. En particulier doit être considéré comme faisant partie de l'invention tout polypeptide ou fragment de polypeptide comportant en commun une partie de séquence en acides aminés avec des EB200 et qui est apte à induire in vivo des anticorps capables d'inhiber l'invasion des globules rouges dans une proportion au moins égale à 50 % par rapport à celles observables dans des préparations d'érythrocytes témoins placés dans les mêmes conditions en présence des mêmes concentrations de mérozoïtes, mais en l'absence des polypeptides ou partie de polypeptides du genre en question.  It is also possible to use, in order to identify the polypeptides or peptides coming within the scope of the invention, the test of inhibition of the invasion of red blood cells by merozoites, in particular from P. falciparum. In particular should be considered as part of the invention any polypeptide or fragment of polypeptide comprising in common a part of amino acid sequence with EB200 and which is capable of inducing in vivo antibodies capable of inhibiting the invasion of globules red in a proportion at least equal to 50% compared to those observable in preparations of control erythrocytes placed under the same conditions in the presence of the same concentrations of merozoites, but in the absence of the polypeptides or part of polypeptides of the genus in question .
L'invention concerne naturellement tout polypeptide contenant une partie de séquence s'étendant au delà des extrémités de la séquence EB200, dès lors que le polypeptide concerné présenterait des propriétés immunogènes protectrices de la même nature. Par "Partie au delà des extrémités de EB200" on entend par exemple les séquences en acides aminés qui jouxtent la structure en acides aminés de EB200 dans la séquence peptidique plus importante déduite de la séquence nucléotidique du fragment G9, dont EB200 est issu (figure 1C). The invention naturally relates to any polypeptide containing a part of a sequence extending beyond the ends of the EB200 sequence, since the polypeptide concerned would exhibit protective immunogenic properties. of the same nature. By "Part beyond the ends of EB200" is meant, for example, the amino acid sequences which adjoin the amino acid structure of EB200 in the larger peptide sequence deduced from the nucleotide sequence of the G9 fragment, from which EB200 is derived (FIG. 1C ).
De la même façon, l'invention étend ses effets à tous peptides dérivés des précédents, si ce n'est que certains résidus aminoacyle de leurs séquences en acides aminés seraient substitués par d'autres, voire délétés ou au contraire intercalés dans les séquences réprésentées, sans que les peptides modifiés obtenus perdent les propriétés biologiques caractéristiques du polypeptide EB200.  In the same way, the invention extends its effects to all peptides derived from the preceding ones, except that certain aminoacyl residues of their amino acid sequences would be substituted by others, even deleted or on the contrary intercalated in the represented sequences , without the modified peptides obtained losing the biological properties characteristic of the EB200 polypeptide.
En particulier l'invention concerne plus particulièrement les séquences monomères correspondantes aux motifs répétitifs dégénérés présents dans EB200, notamment les peptides dont les séquences correspondent aux séquences (a à o) qui suivent :  In particular, the invention relates more particularly to the monomer sequences corresponding to the degenerate repeating units present in EB200, in particular the peptides whose sequences correspond to the sequences (a to o) which follow:
(a) S V T G N I L V E G.  (a) S V T G N I L V E G.
(b) S V T E E V V G E E K.  (b) S V T E E V V G E E K.
(c) L V S E E I V T E E G.  (c) L V S E E I V T E E G.
(d) S V A Q E I V E E D A.  (d) S V A Q E I V E E D A.
(e) P A T E E I D E I E.  (e) P A T E E I D E I E.
(f) S V T E E V V E E E G.  (f) S V T E E V V E E E G.
(g) P V D E E I V Q E E G. (g) P V D E E I V Q E E G.
(h) T V T E E I I Q G E.  (h) T V T E E I I Q G E.
(i) S K V E E V V E E Q G. (j) S E N E E I F V E E V. (i) SKVEEVVEEQ G. (j) SENEEIFVEE V.
(k) S A S Q E I V Q N E.  (k) S A S Q E I V Q N E.
(l) S G T E E I L E K V.  (l) S G T E E I L E K V.
(m) S A S Q E I V Q D G.  (m) S A S Q E I V Q D G.
(n) S V T E Q I I E E L F.  (n) S V T E Q I I E E L F.
(o) P V T E E V V N E E E.  (o) P V T E E V V N E E E.
dès lors que incorporées à une protéine de fusion, oligomérisés ou conjugués à une protéine porteuse, les peptides du genre en question sont aptes à induire in vivo une réponse immunitaire protectrice chez le singe écureuil Saimiri sciureus ou encore à inhiber l'invasion d'érythrocytes par les mérozoïtes.  as soon as incorporated into a fusion protein, oligomerized or conjugated to a carrier protein, the peptides of the genus in question are capable of inducing in vivo a protective immune response in the squirrel monkey Saimiri sciureus or else of inhibiting the invasion of erythrocytes by merozoites.
Au même titre fait encore partie de l'invention tout équivalent peptidique contenant des motifs représentables par la formules :  In the same way, part of the invention is also any peptide equivalent containing motifs which can be represented by the formula:
(X)3 - EE - (X)2 - EE - (X)2-3 dans lequel chacun des groupes X représente, selon l'indice qui suit les signes de parenthèse qui entoure le groupe considéré, un dipeptide ou tripeptide contenant chacun, au moins un résidu amino-acyle hydrophobe, cet équivalent peptidique étant susceptible d'induire une réponse immunitaire protectrice chez le singe-écureuil Saimiri sciureus ou d'inhiber une invasion in vitro d'érythrocytes par P. falciparum. (X) 3 - EE - (X) 2 - EE - (X) 2-3 in which each of the groups X represents, according to the index which follows the signs of parenthesis which surrounds the group considered, a dipeptide or tripeptide each containing , at least one hydrophobic amino-acyl residue, this peptide equivalent being capable of inducing a protective immune response in the squirrel monkey Saimiri sciureus or of inhibiting an in vitro invasion of erythrocytes by P. falciparum.
Comme cela a déjà été indiqué plus haut, l'invention concerne toute protéine de fusion, oligomère ou conjuguée faisant intervenir les peptides conformes à l'invention. Ces peptides peuvent être produits par synthèse chimique. Il peut encore en être de même des oligomères de ces peptides ou des peptides conjugués à des molécules porteuses. Des techniques permettant d'effectuer ces synthèses sont rappelées dans ce qui suit, bien entendu de façon non limitative. As already indicated above, the invention relates to any fusion protein, oligomer or conjugate involving the peptides according to the invention. These peptides can be produced by chemical synthesis. It can also be the same for oligomers of these peptides or peptides conjugated to carrier molecules. Techniques allowing these syntheses to be carried out are recalled in the following, of course without limitation.
Par exemple, on aura recours à la technique de synthèse en solution homogène décrite par HOUBENWEYL dans l'ouvrage intitulé "Méthode der Organischen Chemie" (Méthodes de la Chimie Organique) édité par E. Wunsch, vol.15-1 et II., THIEME, Stuggart 1974.  For example, we will use the synthesis technique in homogeneous solution described by HOUBENWEYL in the work entitled "Method der Organischen Chemie" (Methods of Organic Chemistry) published by E. Wunsch, vol.15-1 and II., THIEME, Stuggart 1974.
Cette méthode de synthèse consiste à condenser successivement, dans l'ordre requis, les aminoacides ou peptides préalablement formés avec d'autres aminoacides ou peptides préalablement formés choisis de façon à permettre la production d'un polypeptide ayant la séquence finale choisie en aminoacides, étant entendu que l'on aura eu soin, si besoin, de protéger au préalable toutes les fonctions réactives portées par ces aminoacides ou peptides, à l'exception de celles de leurs fonctions aminé et carboxyle devant intervenir dans la formation des liaisons peptidiques, notamment après activation desdites fonctions carboxyle. En variante, on pourra avoir recours à des réactions de couplage mettant en jeu des réactifs de couplage classique, du type carbodiimide, tels que par exemple la 1-éthyl-3-(3- diméthyl-amino-propyl)-carbodiimide.  This synthetic method consists in successively condensing, in the required order, the amino acids or peptides previously formed with other amino acids or peptides previously formed chosen so as to allow the production of a polypeptide having the final sequence chosen as amino acids, being it is understood that care will have been taken, if necessary, to protect beforehand all the reactive functions carried by these amino acids or peptides, with the exception of those of their amine and carboxyl functions which must intervene in the formation of peptide bonds, in particular after activation of said carboxyl functions. As a variant, it is possible to have recourse to coupling reactions involving conventional coupling reagents, of the carbodiimide type, such as for example 1-ethyl-3- (3-dimethyl-amino-propyl) -carbodiimide.
Lorsque l'aminoacyle mis en oeuvre possède une fonction acide supplémentaire (notamment dans le cas de l'acide glutamique), ces fonctions sont protégées, par exemple par des groupes t-butylester. Dans le cas de la synthèse progressive, amino- acide par amino-acide, la synthèse débute de préférence par la condensation de l'aminoacide C- terminal avec l'aminoacide qui correspond à l'aminoacyle voisin dans la séquence désirée et, de proche en proche, avec les autres acides aminés choisis de façon appropriée, la synthèse s'achevant avec la fixation de l'acide aminé N-terminal. When the aminoacyl used has an additional acid function (in particular in the case of glutamic acid), these functions are protected, for example by t-butylester groups. In the case of progressive synthesis, amino acid by amino acid, the synthesis preferably begins with the condensation of the C-terminal amino acid with the amino acid which corresponds to the neighboring amino acid in the desired sequence and, closely in close proximity, with the other amino acids chosen in an appropriate manner, the synthesis ends with the fixing of the N-terminal amino acid.
Il est souvent avantageux d'avoir recours à la technique décrite par R.D. MERRIFIELD dans l'article intitulé "Solid phase peptide synthesis" (J. Am. Soc, 45: 2149-2154).  It is often advantageous to use the technique described by R.D. MERRIFIELD in the article entitled "Solid phase peptide synthesis" (J. Am. Soc, 45: 2149-2154).
Le premier acide aminé C-terminal de la chaîne à produire est fixé sur une résine poreuse de polymère, jouant le rôle de support insoluble par l'intermédiaire de son groupe carboxylique. La fonction aminé de ce premier aminoacide aura normalement été protégée au préalable avec un groupe protecteur approprié, par exemple par un groupe t- butyloxycarbonyle.  The first C-terminal amino acid in the chain to be produced is attached to a porous polymer resin, acting as an insoluble support through its carboxylic group. The amino function of this first amino acid will normally have been protected beforehand with an appropriate protective group, for example by a t-butyloxycarbonyl group.
L'oligomérisation des séquences monomères obtenues peut être conduite de toute façon en soit connue. Ces oligomères contiennent, par exemple de 2 à 20 unités monomères. Il n'y a d'ailleurs pas de limite au nombre maximum d'unités monomères suceptibles d'être inclues dans l'oligomere, si ce n'est la capacité de l'oligomere finalement obtenu d'être hydrosoluble ou facilement solubilisable dans l'eau.  The oligomerization of the monomer blocks obtained can be carried out in any case as is known. These oligomers contain, for example from 2 to 20 monomer units. There is moreover no limit to the maximum number of monomer units capable of being included in the oligomer, except for the capacity of the oligomer finally obtained to be water-soluble or easily soluble in l 'water.
Une première méthode d'oligomérisation ou de polymérisation du monomère consiste dans la réaction de celui-ci avec un agent de réticulation, tel que le glutaraldéhyde. On peut également avoir recours à d'autres méthodes d'oligomérisation ou de couplage, par exemple à celle mettant en jeu des couplages successifs d'unités monomères, par l'intermédiaire de leurs fonctions terminales carboxyle et aminé, en présence d'agents de couplage homo- ou hétéro- bifonctionnels, les autres groupes fonctionnels portés par ces unités monomères ayant, le cas échéant, été protégés au préalable. A first method of oligomerization or polymerization of the monomer consists in the reaction of the latter with a crosslinking agent, such as glutaraldehyde. You can also use other methods of oligomerization or coupling, for example that involving successive couplings of monomer units, via their terminal carboxyl and amine functions, in the presence of homo- or hetero-coupling agents bifunctional, the other functional groups carried by these monomer units having, where appropriate, been protected beforehand.
Un autre procédé d'oligomérisation préféré fait appel aux techniques décrites par Richman et Reese, RT. (1988) Proc. Nat. Acad. a5: 1662-1666 ou encore Posnett, DN. (1988) The Journal of Biological Chemistry Vol. 283, N°4. Ces procédés mettent en jeu une petite "matrice centrale peptidique" (peptidyl core matrix) formée à partir d'un nombre restreint d'acides aminés trifonctionnels, en particulier des lysines, interconnectés entre eux de façon à comporter un nombre de fonctions libres important vis à vis du poids moléculaire de cette matrice centrale et formant autant de bras ou dendrites qui peuvent alors être conjuguées à des aminoacides distincts ou aux extrémités C-terminales. On se reportera plus particulièrement à l'article de Tam, J.P. publié en 1988 dans Proc. Nat. Acad. USA, Vol. 85, 5409-5413, dans lequel les structures de telles matrices centrales produites à partir de lysine ont été représentés. En particulier on peut avoir recours à de telles "matrices centrales" à base d'heptalysines comportant des sites pour la fixation de 9-16 chaînes peptidiques. Une autre façon de combiner des résidus lysine résulte du schéma suivant ("matrice centrale" dite sous la forme d' "oσtopussy") Cys - SS - C— Peptide Cys - SS - C— Peptide Another preferred oligomerization process uses the techniques described by Richman and Reese, RT. (1988) Proc. Nat. Acad. a5: 1662-1666 or Posnett, DN. (1988) The Journal of Biological Chemistry Vol. 283, No. 4. These methods involve a small "central peptide matrix" (peptidyl core matrix) formed from a limited number of trifunctional amino acids, in particular lysines, interconnected so as to contain a large number of free functions vis with respect to the molecular weight of this central matrix and forming as many arms or dendrites which can then be conjugated with distinct amino acids or with C-terminal ends. We refer more particularly to the article by Tam, JP published in 1988 in Proc. Nat. Acad. USA, Vol. 85, 5409-5413, in which the structures of such central matrices produced from lysine have been shown. In particular, use may be made of such "central matrices" based on heptalysins comprising sites for the attachment of 9-16 peptide chains. Another way of combining lysine residues results from the following scheme ("central matrix" called in the form of "oσtopussy") Cys - SS - C— Peptide Cys - SS - C— Peptide
Cys - SS - C— Peptide Cys - SS - C— Peptide  Cys - SS - C— Peptide Cys - SS - C— Peptide
Cys - SS - C— Peptide Cys - SS - C— Peptide Cys - SS - C— Peptide Cys - SS - C— Peptide
Cys - SS - C— Peptide
Figure imgf000020_0001
Cys - SS - C— Peptide dans laquelle K représente la lysine et "Peptide" a la signification sus-indiquée; d'où il résulte un système dénommé MAP (abréviation partielle de l'expression anglaise "Multiple Antigen Peptide System" ou "Système d'Antigène Peptidique Multiple"). Cys - SS - C— Peptide
Figure imgf000020_0001
Cys - SS - C - Peptide in which K represents lysine and "Peptide" has the meaning indicated above; whence results a system called MAP (partial abbreviation of the English expression "Multiple Antigen Peptide System" or "System of Antigen Peptide Multiple").
Partant par exemple d'une "matrice centrale" comportant huit fonctions aminé susceptibles d'être mises en réaction avec les peptides qui doivent y être rattachés, on procède au couplage des peptides à fixer, en particulier ceux conformés à l'invention, à cette matrice centrale par l'intermédiaire des fonctions réactives de celle-ci, et ce par la mise en oeuvre des techniques applicables à la synthèse peptidique. On peut se reporter aux articles précédemment identifiés pour ce qui concerne les techniques préférées susceptibles d'être mises en oeuvre. L'expression "oligomère" inclut également les conjugués obtenus par couplage covalent de plusieurs unités monomères à une molécule porteuse (naturelle ou synthétique), physiologiquement acceptable et non toxique, par l'intermédiaire de groupements réactifs complémentaires respectivement portés par la molécule porteuse et/ou les unités monomères. Des exemples de groupements appropriés sont illustrés dans ce qui suit. Starting for example from a "central matrix" comprising eight amino functions capable of being reacted with the peptides which must be attached thereto, the peptides to be attached, in particular those conforming to the invention, are coupled to this central matrix through the reactive functions thereof, and this by the implementation of techniques applicable to peptide synthesis. Reference may be made to the articles previously identified with regard to the preferred techniques which may be used. The expression "oligomer" also includes the conjugates obtained by covalent coupling of several monomer units to a carrier molecule (natural or synthetic), physiologically acceptable and non-toxic, by means of complementary reactive groups respectively carried by the carrier molecule and / or the monomer units. Examples of suitable groupings are illustrated in the following.
A titre d'exemple de molécules porteuses ou supports macromoléculaires entrant dans la constitution des conjugués selon l'invention, on mentionnera des protéines naturelles, telles que l'anatoxine tétanique, l'ovalbumine, des sérumalbumines, des hémocyanines, une protéine purifiée de la tuberculine ("Purified Protein Derivative" = PPD) etc ...  By way of example of carrier molecules or macromolecular carriers used in the constitution of the conjugates according to the invention, mention will be made of natural proteins, such as tetanus toxoid, ovalbumin, serum albumin, hemocyanins, a protein purified from tuberculin ("Purified Protein Derivative" = PPD) etc ...
A titre de supports macromoléculaires synthétiques, on mentionnera par exemple des polylysines ou des poly(D-L-alanine)-poly(L-lysine) ou des protéines immunologiquement inertes.  By way of synthetic macromolecular supports, mention may, for example, be made of polylysines or poly (D-L-alanine) -poly (L-lysine) or immunologically inert proteins.
La littérature mentionne d'autres types de supports macromoléculaires susceptibles d'être utilisés, lesquels présentent en général un poids moléculaire supérieur à 20 000.  The literature mentions other types of macromolecular supports which can be used, which generally have a molecular weight greater than 20,000.
On peut avoir recours à tout procédé de couplage faisant intervenir, d'une part, une ou plusieurs fonctions réactives du peptide et, d'autre part, une ou plusieurs fonctions réactives de molécules supports. Avantageusement, il s'agit des fonctions carboxyle et aminé, lesquelles peuvent donner lieu à une réaction de couplage en présence d'un agent de couplage du genre de ceux utilisés dans la synthèse des protéines, par exemple, le 1- éthyl-3-(3-diméthylaminopropyl)- carbodiimide, le N-hydroxybenzotriazole, etc... On peut encore avoir recours à la glutaraldéhyde, notamment lorsqu'il s'agit de relier entre eux des groupes aminés respectivement portés par le peptide et la molécule support. Any coupling process can be used which involves, on the one hand, one or more reactive functions of the peptide and, on the other hand, one or more reactive functions of support molecules. Advantageously, these are the carboxyl and amino functions, which can give rise to a coupling reaction in the presence of a coupling agent of the type of those used. in the synthesis of proteins, for example, 1- ethyl-3- (3-dimethylaminopropyl) - carbodiimide, N-hydroxybenzotriazole, etc. We can still use glutaraldehyde, especially when it comes to link together amino groups respectively carried by the peptide and the support molecule.
L'invention concerne encore une variante de procédé de production des oligomères selon l'invention, cette variante consistant dans la transformation des cellules d'une culture de cellules compétentes par un vecteur contenant une séquence de nucléotides codant pour un polypeptide oligomère contenant les susdites unités monomères sous le contrôle d'un promoteur reconnu par lesdites cellules compétentes, et ce dans des conditions autorisant l'expression dans ces cellules de ladite séquence oligomérique de nucléotides, et la récupération du produit d'expression de ladite séquence de nucléotides. Celle-ci fait également partie de l'invention. Il n'est point nécessaire d'insister sur les techniques susceptibles d'être utilisées, si ce n'est que, de préférence, les éléments du vecteur sont de préférence tels que le produit d'expression soit transporté à l'extérieur des cellules compétentes, voire même excrété dans leur milieu de culture.  The invention also relates to a variant of the process for producing the oligomers according to the invention, this variant consisting in the transformation of cells of a culture of competent cells with a vector containing a nucleotide sequence coding for an oligomeric polypeptide containing the above-mentioned units. monomers under the control of a promoter recognized by said competent cells, under conditions allowing the expression in these cells of said oligomeric nucleotide sequence, and the recovery of the expression product of said nucleotide sequence. This is also part of the invention. There is no need to insist on the techniques that can be used, except that, preferably, the elements of the vector are preferably such that the expression product is transported outside the cells. competent, even excreted in their culture medium.
L'invention concerne naturellement les fragments d'acides correspondants, plus particulièrement de tout ou partie de ceux qui découlent de la figure 1(c) à propos de la séquence G9. Ce fragment d'ADN peut être, soit à l'état individualisé, soit recombiné au sein d'un ADN recombiné de taille plus importante. Parmi ces ADNs de tailles plus importantes il est entendu que l'invention concerne : The invention naturally relates to the corresponding acid fragments, more particularly to all or part of those which follow from FIG. 1 (c) with regard to the sequence G9. This DNA fragment can either be in the state individualized, or recombined within a larger recombinant DNA. Among these DNAs of larger sizes, it is understood that the invention relates to:
- des ADNs codant pour des protéines de fusion faisant intervenir une séquence d'ADN codant pour un peptide conforme à l'invention, avec des séquences d'ADN codant elles-mêmes pour des séquences peptidiques hétérologues n'interférant pas les propriétés d'induction par la séquence polypeptidique selon l'invention d'une immunité protectrice contre P. falciparum ;  DNAs coding for fusion proteins involving a DNA sequence coding for a peptide according to the invention, with DNA sequences coding themselves for heterologous peptide sequences which do not interfere with the induction properties by the polypeptide sequence according to the invention of a protective immunity against P. falciparum;
- tout vecteur contenant cette séquence polypeptidique placé sous le contrôle d'un promoteur autorisant son expression dans un hôte cellulaire compétent et dont les polymérases sont aptes à reconnaître ledit promoteur.  - Any vector containing this polypeptide sequence placed under the control of a promoter authorizing its expression in a competent cellular host and whose polymerases are capable of recognizing said promoter.
Il va également de soi que l'invention étend ses effets aux cultures cellulaires ainsi transformées par de tels vecteurs ou ADN recombinants qui permettent l'expression du polypeptide ou du fragment de polypeptide selon l'invention.  It also goes without saying that the invention extends its effects to cell cultures thus transformed by such recombinant vectors or DNA which allow the expression of the polypeptide or fragment of the polypeptide according to the invention.
De même font partie de l'invention les anticorps spécifiques des séquences contenues dans EB200 ou des peptides ou fragments de peptides présentant les mêmes propriétés biologiques telles qu'elles ont été définies plus haut. Cette protection étend ses effets à tous anticorps monoclonaux spécifiques des mêmes séquences polypeptidiques, ainsi qu'aux hybridomes sécréteurs de ces anticorps monoclonaux. Il sera compris que l'homme du métier est à même de produire ces anticorps monoclonaux et de les sélectionner dans des opérations de tri faisant intervenir la reconnaissance spécifique par les anticorps monoclonaux à sélectionner, des séquences en acides aminés de peptides caractéristiques de l'invention. Likewise, part of the invention forms the specific antibodies of the sequences contained in EB200 or of peptides or fragments of peptides having the same biological properties as defined above. This protection extends its effects to all monoclonal antibodies specific for the same polypeptide sequences, as well as to secretory hybridomas of these monoclonal antibodies. It will be understood that the skilled person is able to produce these monoclonal antibodies and to select them in sorting operations involving the specific recognition by the monoclonal antibodies to be selected, amino acid sequences of peptides characteristic of the invention.
L'invention concerne de même toute composition pharmaceutique mettant en oeuvre ces polypeptides, le cas échéant associés à des véhicules pharmaceutiquement acceptables facilitant leur utilisation dans des opérations de vaccination afin d'induire la production in vivo, notamment chez l'homme, d'une réponse immunitaire protectrice contre des parasites malariques infectueux.  The invention likewise relates to any pharmaceutical composition using these polypeptides, if appropriate associated with pharmaceutically acceptable vehicles facilitating their use in vaccination operations in order to induce the production in vivo, in particular in humans, of a protective immune response against infectious malarial parasites.
L'invention est également relative aux préparations d'anticorps, induits par EB200 ou les peptides équivalents de l'invention, que ces anticorps soient polyclonaux (sérums les contenant ou fraction immunoglobulinique purifiée à partir de ces sérums) ou anticorps monoclonaux obtenus comme indiqués ci-dessus. En particulier l'invention concerne des compositions pharmaceutiques contenant de tels anticorps aux fins d'entrer en compétition in vivo avec le parasite humain, notamment chez des individus infectés ou menacés d'infection par ce parasite, notamment du type P. falciparum, afin de les protéger contre les effets pathogènes dus à cette infection.  The invention also relates to the antibody preparations, induced by EB200 or the equivalent peptides of the invention, whether these antibodies are polyclonal (sera containing them or immunoglobulin fraction purified from these sera) or monoclonal antibodies obtained as indicated below. -above. In particular, the invention relates to pharmaceutical compositions containing such antibodies for the purpose of entering into competition in vivo with the human parasite, in particular in individuals infected or threatened with infection by this parasite, in particular of the P. falciparum type, in order to protect them from the pathogenic effects due to this infection.
Les anticorps selon l'invention sont aussi plus particulièrement caractérisés par le fait qu'ils ne donnent pas lieu à des réactions immunitaires croisées avec les produits d'expression du gène Pf11-1 (27) et du gène Pf155/RESA (38). Le clone contenant la séquence nucléotidique codant pour EB200, contenu dans Escherichia coli DH5 a été déposé le 25 juillet sous le nº I-1128 à la Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur de Paris (CNCM). La bibliographie à laquelle il a été fait référence dans la présente description par des renvois à des numéros de publication complète la présente description. The antibodies according to the invention are also more particularly characterized by the fact that they do not give rise to crossed immune reactions with the expression products of the Pf11-1 gene (27) and of the Pf155 / RESA gene (38). The clone containing the nucleotide sequence coding for EB200, contained in Escherichia coli DH5 was deposited on July 25 under number I-1128 at the National Collection of Cultures of Microorganisms of the Institut Pasteur de Paris (CNCM). The bibliography to which reference has been made in this description by references to publication numbers supplements this description.
L'invention concerne également les autres applications de ces anticorps, notamment leur utilisation pour le diagnostic in vitro, par exemple effectué sur un échantillon de sérum, d'une infection par des parasites paludéens, notamment au stade de l'infection des érythrocytes sanguins, par exemple au moyen d'une réaction antigène-anticorps classique. A l'inverse, l'invention concerne enfin l'application des peptides eux-mêmes à la détection in vitro de la présence d'anticorps caractéristiques de l'infection par des parasites paludéens, notamment du type P. falciparum ou analogue, et plus particulièrement encore au stade érythrocytaire de ces parasites, sous forme de mérozoïtes.  The invention also relates to the other applications of these antibodies, in particular their use for the in vitro diagnosis, for example carried out on a serum sample, of an infection by malarial parasites, in particular at the stage of infection of blood erythrocytes, for example by means of a conventional antigen-antibody reaction. Conversely, the invention finally relates to the application of the peptides themselves to the in vitro detection of the presence of antibodies characteristic of infection by malarial parasites, in particular of the P. falciparum type or the like, and more particularly still at the erythrocyte stage of these parasites, in the form of merozoites.
Au titre des données expérimentales supplémentaires, on mentionne encore les faits suivants.  As additional experimental data, the following facts are also mentioned.
La solubilisation de Pf332, comme celle de Pf211, par différents détergents montre que l'antigène est associé avec la membrane des globules rouges parasités sans être associé au cytosquelette.  The solubilization of Pf332, like that of Pf211, by various detergents shows that the antigen is associated with the membrane of the parasitized red blood cells without being associated with the cytoskeleton.
Les anticorps anti-PfEB200 ont aussi été utilisés pour localiser l'antigène Pf332 dans le parasite. L'immunofluorescence indirecte, sur des parasites fixés à l'air et analysée par microscopie confocale, montre une image formée par des structures similaires à des vésicules d'environ 0.5-1μm. Ces vésicules ont été localisées dans le cytoplasme du globule rouge parasité, dissociées du parasite, et transportées vers la membrane de l'érythrocyte. De plus, chez les formes évolutives tardives, l'antigène Pf332 semble être associé à la membrane des globules rouges. En effet, l'immunoélectromicroscopie montre que l'antigène Pf332 est transporté vers la membrane de l'érythrocyte en association avec les "Maurer's clefts". Plus important, par la technique d'immunofluorescence en suspension, le sérum anti-PfEB200 réagit avec la surface des globules rouges parasités de singe. La présence d'épitopes de Pf332 à la surface des globules rouges confirme que cet antigène peut être la cible de réactions immunologiques de l'hôte et, donc, être d'intérêt pour le développement d'un vaccin. Anti-PfEB200 antibodies have also been used to locate the Pf332 antigen in the parasite. Indirect immunofluorescence, on parasites fixed in air and analyzed by confocal microscopy, shows an image formed by structures similar to vesicles of about 0.5-1μm. These vesicles were located in the cytoplasm of the parasitized red blood cell, dissociated from the parasite, and transported to the membrane of the erythrocyte. In addition, in late-onset forms, the Pf332 antigen appears to be associated with the membrane of red blood cells. Indeed, immunoelectromicroscopy shows that the Pf332 antigen is transported towards the membrane of the erythrocyte in association with "Maurer's clefts". More importantly, by the immunofluorescence suspension technique, the anti-PfEB200 serum reacts with the surface of the parasitized monkey red blood cells. The presence of epitopes of Pf332 on the surface of red blood cells confirms that this antigen may be the target of host immunological reactions and therefore be of interest for the development of a vaccine.
Des expériences préliminaires montrent que les anticorps anti-PfEB200 (immunoglobulines totales purifiées ou immunoglobulines purifiées sur une colonne d'affinité couplée avec le recombinant PfEB200) sont capables d'inhiber le développement des parasites en 60-80%, par rapport aux contrôles.  Preliminary experiments show that the anti-PfEB200 antibodies (purified total immunoglobulins or purified immunoglobulins on an affinity column coupled with the recombinant PfEB200) are capable of inhibiting the development of the parasites by 60-80%, compared to the controls.
Le polypeptide recombinant PfEB200 inhibe la phagocytose, par des monocytes, des érythrocytes infectés en présence de sérum hyperimmun de singe.  The recombinant polypeptide PfEB200 inhibits phagocytosis, by monocytes, erythrocytes infected in the presence of hyperimmune monkey serum.
L'invention concerne également des compositions mettant en oeuvre les peptides précédemment définis en association avec d'autres constituants peptidiques ayant des propriétés vaccinantes contre le paludisme, notamment dû à Plasmodium falciparum, plus particulièrement la capacité d'induire in vivo une réponse immunitaire protectrice, y compris la production d'anticorps immunoprotecteurs aptes à détruire les parasites du stade sanguin, responsables de la maladie chez l'homme, dans un modèle de primate représenté par le singe écureuil SAIMIRI SCIUREUS. The invention also relates to compositions using the peptides previously defined in association with other peptide constituents having vaccinating properties against malaria, in particular due to Plasmodium falciparum, more particularly the ability to induce a protective immune response in vivo, including the production of immunoprotective antibodies capable of destroying the parasites of the blood stage, responsible for the disease in humans, in a primate model represented by the squirrel monkey SAIMIRI SCIUREUS.
Parmi les polypeptides avantageusement utilisés en association avec les peptides EB200 ou analogues précédemment décrits, on mentionnera plus particulièrement, soit le "deuxième polypeptide", soit le "troisième polypeptide" respectivement identifiés ci-après :  Among the polypeptides advantageously used in combination with the peptides EB200 or analogues previously described, mention will be made more particularly of either the "second polypeptide" or the "third polypeptide" respectively identified below:
Le "deuxième polypeptide" est constitué par un polypeptide dérivé d'un antigène majeur de P falciparum, ayant un poids moléculaire de l'ordre de 72 kDA, d'où l'appellation "Antigène 72 kDA" utilisée dans ce qui suit pour le désigner, particulièrement d'un polypeptide issu de cet antigène 72 kDA ayant la capacité, comme celui-ci : The "second polypeptide" consists of a polypeptide derived from a major P falciparum antigen, having a molecular weight of the order of 72 kDA, hence the designation "72 kDA antigen" used in the following for the denote, in particular of a polypeptide derived from this 72 kDA antigen having the capacity, like this:
- d'être reconnu par des anticorps protecteurs contenus dans des sérums ou des fractions immunoglobulines provenant d'animaux, notamment de singes SAIMIRI SCIUREUS, devenus résistants aux parasites après les infections répétées contrôlées par chimiothérapie, lesdits sérums ou fractions étant susceptibles de rendre résistants des singes initialement sensibles ; - to be recognized by protective antibodies contained in sera or immunoglobulin fractions originating from animals, in particular SAIMIRI SCIUREUS monkeys, which have become resistant to parasites after repeated infections controlled by chemotherapy, said sera or fractions being capable of rendering resistant initially sensitive monkeys;
- d'être lui-même inducteur, notamment chez le singe, et plus particulièrement chez le singe SAIMIRI SCIUREUS, d'anticorps actifs contre des parasites du paludisme, plus particulièrement P falciparum ou des parasites qui présentent les mêmes caractéristiques biologiques essentielles. - to be itself an inducer, in particular in the monkey, and more particularly in the SAIMIRI SCIUREUS monkey, of antibodies active against malaria parasites, more particularly P falciparum or parasites that have the same essential biological characteristics.
Ce "deuxième polypeptide" est lui-même souvent reconnu par des sérums de patients humains habitant des régions où le paludisme sévit de façon endémique.  This "second polypeptide" is itself often recognized by sera from human patients living in regions where malaria is endemic.
La séquence nucléotidique codant pour la séquence peptidique antigène 72 kDA a été identifiée par L.S. MATTEI et al European Journal of Immunology (1989) 19 : 1823 - 1828. Le "deuxième polypeptide" est avantageusement constitué par un polypeptide ci-après désigné "i72" comprenant les 153 acides aminés de la région C - terminale de l'antigène 72 kDA. La séquence en acides aminés, de même qu'une séquence d'acide nucléique codant pour le polypeptide i72, sont présentées ci-après : The nucleotide sequence coding for the 72 kDA antigen peptide sequence has been identified by LS MATTEI et al European Journal of Immunology (1989) 19: 1823 - 1828. The "second polypeptide" advantageously consists of a polypeptide hereinafter designated "i72" comprising the 153 amino acids of the C - terminal region of the 72 kDA antigen. The amino acid sequence, as well as a nucleic acid sequence encoding the i72 polypeptide, are shown below:
1/1 31/11 1/1 31/11
GAT CGT ATG GTT AAT GAT GCT GAA AAA TAC AAA GCA GAA GAT GAA GAA AAC AGA AAA AGA  GAT CGT ATG GTT AAT GAT GCT GAA AAA TAC AAA GCA GAA GAT GAA GAA AAC AGA AAA AGA
asp arg met val asn asp ala glu lys tyr lys ala glu asp glu glu asn arg lys argasp arg met val asn asp ala glu lys tyr lys ala glu asp glu glu asn arg lys arg
61/21 91/31 61/21 91/31
ATC GAA GCA AGA AAC AOC CTT GAA AAT TAC TOC TAT OOA GTT AAA AGC TCA TTA GAA GAC  ATC GAA GCA AGA AAC AOC CTT GAA AAT TAC TOC TAT OOA GTT AAA AGC TCA TTA GAA GAC
ile glu ala arg asn ser leu glu asn tyr cys tyr gly val lys ser ser leu glu aspile glu ala arg asn ser leu glu asn tyr cys tyr gly val lys ser ser leu glu asp
121/41 151/51 121/41 151/51
CAA AAA ATT AAA GAA AAA TTA CAA CCA GCT GAA ATT GAA ACA TGT ATG AAA ACT ATT ACA  CAA AAA ATT AAA GAA AAA TTA CAA CCA GCT GAA ATT GAA ACA TGT ATG AAA ACT ATT ACA
gln lys ile lys glu lys leu gln pro ala glu ile glu thr cys met lys thr ile thrgln lys ile lys glu lys leu gln pro ala glu ile glu thr cys met lys thr ile thr
181/61 211/71 181/61 211/71
ACC ATA CTT GAA TGG TTA GAA AAA AAC CAA CTT GCT GGA AAA GAT GAA TAT GAA GCC AAA  ACC ATA CTT GAA TGG TTA GAA AAA AAC CAA CTT GCT GGA AAA GAT GAA TAT GAA GCC AAA
thr ile leu glu trp leu glu lys asn gln leu ala gly lys asp glu tyr glu ala lysthr ile leu glu trp leu glu lys asn gln leu ala gly lys asp glu tyr glu ala lys
241/81 271/91 241/81 271/91
CAA AAA GAA GCA GAA TCG GTT TGT GCT CCA ATT ATG TCT AAA ATC TAT CAA GAT GCT GCT  CAA AAA GAA GCA GAA TCG GTT TGT GCT CCA ATT ATG TCT AAA ATC TAT CAA GAT GCT GCT
gln lys glu ala glu ser val cys ala pro ile met serlys ile tyr gln asp ala alagln lys glu ala glu ser val cys ala pro ile met serlys ile tyr gln asp ala ala
301/101 331/111 301/101 331/111
GGT GCA GCC GGT GGT ATG CCA GGA GGT ATG CCC GGT GGA ATG CCA OGT GGA ATG CCA GGT  GGT GCA GCC GGT GGT ATG CCA GGA GGT ATG CCC GGT GGA ATG CCA OGT GGA ATG CCA GGT
gly ala ala gly gly met pro gly gly met pro gly gly met pro gly gly met pro glygly ala ala gly gly met pro gly gly met pro gly gly met pro gly gly met pro gly
361/121 391/131 361/121 391/131
GGT ATG CCA GGT GGT ATG AAT TTC CCA GGA GGT ATG CCC GCA GCA GGA ATG CCA GGA AAT  GGT ATG CCA GGT GGT ATG AAT TTC CCA GGA GGT ATG CCC GCA GCA GGA ATG CCA GGA AAT
gly met pro gly gly met asn phe pro gly gly net pro gly ala gly met pro gly asngly met pro gly gly met asn phe pro gly gly net pro gly ala gly met pro gly asn
421/141 451/151 421/141 451/151
GCC CCA GCT GGA AGT GGA CCA ACA GTT GAA GAA GTT GAT TAA ACT AAT AAA AAA AAA AAA  GCC CCA GCT GGA AGT GGA CCA ACA GTT GAA GAA GTT GAT TAA ACT AAT AAA AAA AAA AAA
ala pro ala gly ser gly pro thr val glu glu val asp OCH thr asn lys lys lys lysala pro ala gly ser gly pro thr val glu glu val asp OCH thr asn lys lys lys lys
481/161 511/171 481/161 511/171
CAT TAA ACA GGA CAA ATA TAA AAA TAT ATA TAT TAT AAA AAT ATA TAT ATA TAT ATT TTT  CAT TAA ACA GGA CAA ATA TAA AAA TAT ATA TAT TAT AAA AAT ATA TAT ATA TAT ATT TTT
his OCH thr gly gln Ile OCH lys tyr Ile tyr tyr lys asn Iletyr Ile tyr ile phehis OCH thr gly gln Ile OCH lys tyr Ile tyr tyr lys asn Iletyr Ile tyr ile phe
541/181 571/191 541/181 571/191
TTT TTT TTT TTA CAT TTT TOT AAA TTA ATA TGA ATT  TTT TTT TTT TTA CAT TTT TOT AAA TTA ATA TGA ATT
phe phe phe leu his phe cys lys leu Ile OPA Ile Ce "deuxième polypeptide" peut encore être constitué par un polypeptide recombinant contenant tout ou partie de la séquence de i72, par exemple une molécule de fusion avec la glutatione transférase de Schistosoma japonicum, selon la technique décrite par D.B. Smith and K.S. Johnson (1988) Gene 67, 41-48, par transformation de E. Coli avec le plasmide pGEX préalablement modifié par la séquence nucléotidique d'insertion appropriée. Une culture de E. Coli (Escherichia coli DH5α) transformé par le susdit plasmide modifié par une séquence nucléotidique codant pour le peptide i72, a été déposée le 24 juillet 1991 auprès de la Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur de Paris (CNCM) sous le numéro I - 1129. phe phe phe leu his phe cys lys leu Ile OPA Ile This "second polypeptide" can also be constituted by a recombinant polypeptide containing all or part of the sequence of i72, for example a fusion molecule with the glutatione transferase of Schistosoma japonicum, according to the technique described by DB Smith and KS Johnson (1988) Gene 67, 41-48, by transformation of E. Coli with the plasmid pGEX previously modified with the appropriate insertion nucleotide sequence. A culture of E. Coli (Escherichia coli DH5α) transformed by the above plasmid modified by a nucleotide sequence coding for the peptide i72, was deposited on July 24, 1991 with the National Collection of Cultures of Microorganisms of the Institut Pasteur de Paris (CNCM) under number I - 1129.
Le "troisième polypeptide" (peptide ou oligopeptide) ainsi qu'un fragment d'acide nucléique qui code pour ce troisième polypeptide, sont caractérisés en ce qu'ils contiennent les éléments de séquence respectifs suivants : The "third polypeptide" (peptide or oligopeptide) as well as a nucleic acid fragment which codes for this third polypeptide, are characterized in that they contain the following respective sequence elements:
CTCT
GAG GAA TGT CAA GGG GAA GTT TAT TTA TTT GTT AAA AAG ATA CCT ATA GAG GAA TGT CAA GGG GAA GTT TAT TTA TTT GTT AAA AAG ATA CCT ATA
Glu Gln Cys Gln Gly Glu Val Tyr Leu Phe Val Lys Lys Ile Pro IleGlu Gln Cys Gln Gly Glu Val Tyr Leu Phe Val Lys Lys Ile Pro Ile
GAG GTA TGG ATA AGA CAG TAC AAC TTG ATG AAT GAT AAT GAT GGA GAA GAG GTA TGG ATA AGA CAG TAC AAC TTG ATG AAT GAT AAT GAT GGA GAA
Glu Val Trp Ile Arg Gln Tyr Asn Leu Met Asn Asp Asn Asp Gly GluGlu Val Trp Ile Arg Gln Tyr Asn Leu Met Asn Asp Asn Asp Gly Glu
TAT TTA TTA GAT GGG GAA AAT TTT ATT ATG GAA GCA GTA GCC TGC GCT TAT TTA TTA GAT GGG GAA AAT TTT ATT ATG GAA GCA GTA GCC TGC GCT
Tyr Leu Leu Asp Gly Glu Asn Phe Ile Met Glu Ala Val Ala Cys Ala Tyr Leu Leu Asp Gly Glu Asn Phe Ile Met Glu Ala Val Ala Cys Ala
TAT TTA AGC GAG CAT TAT CCT GGA CTC ACA CCT AAA TTG TAC AAG GTA TAT TTA AGC GAG CAT TAT CCT GGA CTC ACA CCT AAA TTG TAC AAG GTA
Tyr Leu Ser Glu His Tyr Pro Gly Leu Thr Pro Lys Leu Tyr Lys ValTyr Leu Ser Glu His Tyr Pro Gly Leu Thr Pro Lys Leu Tyr Lys Val
TTA TAT GAG CCT GAA TGT GCT AAC TGT AAT GAA GAG GAT AAG AAT ATG TTA TAT GAG CCT GAA TGT GCT AAC TGT AAT GAA GAG GAT AAG AAT ATG
Leu Tyr Glu Pro Glu Cys Ala Asn Cys Asn Glu Glu Asp Lys Asn MetLeu Tyr Glu Pro Glu Cys Ala Asn Cys Asn Glu Glu Asp Lys Asn Met
AGT GAA GAT AAT CAT AAG AAG GAT AGT AAA CAT AAG GGG GAT AGT AAT AGT GAA GAT AAT CAT AAG AAG GAT AGT AAA CAT AAG GGG GAT AGT AAT
Ser Glu Asp Asn His Lys Lys Asp Ser Lys His Lys Gly Asp Ser Asn Ser Glu Asp Asn His Lys Lys Asp Ser Lys His Lys Gly Asp Ser Asn
CAC AAA AGT GAT AGT AAT CAC AAA AGT GAT AGT AAT CAC AAA AGT GAT CAC AAA AGT GAT AGT AAT CAC AAA AGT GAT AGT AAT CAC AAA AGT GAT
His Lys Ser Asp Ser Asn His Lys Ser Asp Ser Asn His Lys Ser Asp His Lys Ser Asp Ser Asn His Lys Ser Asp Ser Asn His Lys Ser Asp
AGT AAT CAT AAA AGT GAT AGT AAT CAT AAA AGT GGT AGT AAT CAC AAA AGT AAT CAT AAA AGT GAT AGT AAT CAT AAA AGT GGT AGT AAT CAC AAA
Ser Asn His Lys Ser Asp Ser Asn His Lys Ser Gly Ser Asn His Lys Ser Asn His Lys Ser Asp Ser Asn His Lys Ser Gly Ser Asn His Lys
AGT GAT TGT AAT CAC AAG AGT GGT AGT AAT CAC AAG AGT GGT AGT AAT AGT GAT TGT AAT CAC AAG AGT GGT AGT AAT CAC AAG AGT GGT AGT AAT
Ser Asp Cys Asn His Lys Ser Gly Ser Asn His Lys Ser Gly Ser Asn Ser Asp Cys Asn His Lys Ser Gly Ser Asn His Lys Ser Gly Ser Asn
CAC AAG AGT GAT AGT AAT CAT CAA AGT GAT TGT AAT ... ... ... ... His Lys Ser Asp Ser Asn His Gln Ser Asp Cys Asn ... ... ... ... CAC AAG AGT GAT AGT AAT CAT CAA AGT GAT TGT AAT ... ... ... ... His Lys Ser Asp Ser Asn His Gln Ser Asp Cys Asn ... ... ... ...
... ... ... ... ... ... ... ... ... GAT CAT AAT CAC AAA AGC GAT ... ... ... ... ... ... ... ... ... Asp His Asn His Lys Ser Asp... ... ... ... ... ... ... ... ... ... GAT CAT AAT CAC AAA AGC GAT ... ... ... ... .... .. ... ... ... Asp His Asn His Lys Ser Asp
CAT AAC CAC AAG AGC GAT AAT AAC CAC AAG AGT GAT CAT AAT CAC AAA CAT AAC CAC AAG AGC GAT AAT AAC CAC AAG AGT GAT CAT AAT CAC AAA
His Asn His Lys Ser Asp Asn Asn His Lys Ser Asp His Asn His Lys His Asn His Lys Ser Asp Asn Asn His Lys Ser Asp His Asn His Lys
AGT GAT CAT AAA AAA AAT AAT AAC AAT AAT AAG GAT AAT AAA AAT GAC AGT GAT CAT AAA AAA AAT AAT AAC AAT AAT AAG GAT AAT AAA AAT GAC
Ser Asp His Lys Lys Asn Asn Asn Asn Asn Lys Asp Asn Lys Asn Asp Ser Asp His Lys Lys Asn Asn Asn Asn Asn Lys Asp Asn Lys Asn Asp
GAT AAT GAT GAC AGT GAT GCA AGC GAT GCA GTT GAT GAA GAT ATT GAG GAT AAT GAT GAC AGT GAT GCA AGC GAT GCA GTT GAT GAA GAT ATT GAG
Asp Asn Asp Asp Ser Asp Ala Ser Asp Ala Val Asp Glu Asp Ile Glu Asp Asn Asp Asp Ser Asp Ala Ser Asp Ala Val Asp Glu Asp Ile Glu
ATA CTT GAG TCT TAT AGT GAT TTG AAT AAA TTT AAT GAG ATG TTA ACA ATA CTT GAG TCT TAT AGT GAT TTG AAT AAA TTT AAT GAG ATG TTA ACA
Ile Leu Glu Ser Tyr Ser Asp Leu Asn Lys Phe Asn Glu Met Leu Thr Ile Leu Glu Ser Tyr Ser Asp Leu Asn Lys Phe Asn Glu Met Leu Thr
GAA CAA TTA AAT AAA AAT AAG GAT GGA TAT GTT GTT TTG GTT ACT GAA GAA CAA TTA AAT AAA AAT AAG GAT GGA TAT GTT GTT TTG GTT ACT GAA
Glu Gln Leu Asn Lyβ Asn Lys Asp Gly Tyr Val Val Leu Val Thr Glu Glu Gln Leu Asn Lyβ Asn Lys Asp Gly Tyr Val Val Leu Val Thr Glu
TTA TTT GGT GAA GAT CTT TTT CAA TAT ATT AAT AAA AGG AAT GAA AAT TTA TTT GGT GAA GAT CTT TTT CAA TAT ATT AAT AAA AGG AAT GAA AAT
Leu Phe Gly Glu Asp Leu Phe Gln Tyr Ile Asn Lys Arg Asn Glu Asn GAA GAT ACG CGT GTA AGA GAT GAA GAT AAA AAA ATA ATT ATG TTT GAALeu Phe Gly Glu Asp Leu Phe Gln Tyr Ile Asn Lys Arg Asn Glu Asn GAA GAT ACG CGT GTA AGA GAT GAA GAT AAA AAA ATA ATT ATG TTT GAA
Glu Asp Thr Arg Val Arg Asp Glu Asp Lys Lys Ile Ile Met. Phe GluGlu Asp Thr Arg Val Arg Asp Glu Asp Lys Lys Ile Ile Met. Phe Glu
TTT TTA AAG TTA TTA ATA AAA TTA CAT AAT GCA GGA TTG GTA CAT CTT TTT TTA AAG TTA TTA ATA AAA TTA CAT AAT GCA GGA TTG GTA CAT CTT
Phe Leu Lys Leu Leu Ile Lys Leu His Asn Ala Gly Leu Val His Leu Phe Leu Lys Leu Leu Ile Lys Leu His Asn Ala Gly Leu Val His Leu
GAT ATA TCT CCT GAA AAT ATA TTG ATA GAAAAT AAT GGT GAG TTA CGC GAT ATA TCT CCT GAA AAT ATA TTG ATA GAAAAT AAT GGT GAG TTA CGC
Asp Ile Ser Pro Glu Asn Ile Leu Ile Glu Asn Asn Gly Glu Leu ArgAsp Ile Ser Pro Glu Asn Ile Leu Ile Glu Asn Asn Gly Glu Leu Arg
TTA TGT GAT CTA GCT AAA TGT GCT CCA ATG TAT ACA CAC AAT TTA AGA TTA TGT GAT CTA GCT AAA TGT GCT CCA ATG TAT ACA CAC AAT TTA AGA
Leu Cys Asp Leu Ala Lys Cys Ala Pro Met Tyr Thr His Asn Leu ArgLeu Cys Asp Leu Ala Lys Cys Ala Pro Met Tyr Thr His Asn Leu Arg
CAT ATT AAA GGA AAT GGA AAC GAT TTG TAT TCA TTT CAA TCT TGT CAA CAT ATT AAA GGA AAT GGA AAC GAT TTG TAT TCA TTT CAA TCT TGT CAA
His Ile Lys Gly Asn Gly Asn Asp Leu Tyr Ser Phe Gln Ser Cys GlnHis Ile Lys Gly Asn Gly Asn Asp Leu Tyr Ser Phe Gln Ser Cys Gln
CCT TGT GTT GGA AAA ATC CCA TGT ATT CCA AAA GAG TGT TGG GAT ATT CCT TGT GTT GGA AAA ATC CCA TGT ATT CCA AAA GAG TGT TGG GAT ATT
Pro Cys Val Gly Lys Ile Pro Cys Ile Pro Lys Glu Cys Trp Asp IlePro Cys Val Gly Lys Ile Pro Cys Ile Pro Lys Glu Cys Trp Asp Ile
ATT AGA GAA CAT ATA AAA TTA AAA ATA GAT AAT CCT TTT GAA CAT TTA ATT AGA GAA CAT ATA AAA TTA AAA ATA GAT AAT CCT TTT GAA CAT TTA
Ile Arg Glu His Ile Lys Leu Lys Ile Asp Asn Pro Phe Glu His Leu Ile Arg Glu His Ile Lys Leu Lys Ile Asp Asn Pro Phe Glu His Leu
TCA ACT ATT ACT GAT CAA GAA GAA AGA AAA AAA TAT TAT TTT GAT GTA TCA ACT ATT ACT GAT CAA GAA GAA AGA AAA AAA TAT TAT TTT GAT GTA
Ser Thr Ile Thr Asp Gln Glu Glu Arg Lys Lys Tyr Tyr Phe Asp ValSer Thr Ile Thr Asp Gln Glu Glu Arg Lys Lys Tyr Tyr Phe Asp Val
CAT TGT GTA GAT AAA TAT ATG CTT GGT ATA TTT TAT ATA TGG ATA TGG CAT TGT GTA GAT AAA TAT ATG CTT GGT ATA TTT TAT ATA TGG ATA TGG
His Cys Val Asp Lys Tyr Met Leu Gly Ile Phe Tyr Ile Trp Ile TrpHis Cys Val Asp Lys Tyr Met Leu Gly Ile Phe Tyr Ile Trp Ile Trp
AAT TTA AAT TAT ATA TGG AAA CGA GCT GAC CCA CCA AAT GAT AGA ACA AAT TTA AAT TAT ATA TGG AAA CGA GCT GAC CCA CCA AAT GAT AGA ACA
Asn Leu Asn Tyr Ile Trp Lys Arg Ala Asp Pro Pro Asn Asp Arg ThrAsn Leu Asn Tyr Ile Trp Lys Arg Ala Asp Pro Pro Asn Asp Arg Thr
TTT AAT AAT TTC TTA AAA TAT AAT TTA AAT ATA AAT GTA TTT CAG TTA TTT AAT AAT TTC TTA AAA TAT AAT TTA AAT ATA AAT GTA TTT CAG TTA
Phe Asn Asn Phe Leu Lys Tyr Asn Leu Asn Ile Asn Val Phe Gln LeuPhe Asn Asn Phe Leu Lys Tyr Asn Leu Asn Ile Asn Val Phe Gln Leu
GCC CAA CAA TGG CCA AAA GGT CTC AAA GAT ATA ATT AAC GTA AGA TAT GCC CAA CAA TGG CCA AAA GGT CTC AAA GAT ATA ATT AAC GTA AGA TAT
Ala Gln Gln Trp Pro Lys Gly Leu Lys Asp Ile Ile Asn Val Arc TyrAla Gln Gln Trp Pro Lys Gly Leu Lys Asp Ile Ile Asn Val Arc Tyr
ATA AGA AAA AAA AAA AAA AAA AAA AAA ATA ATA ATA ATA ATA ATG ATG ATA AGA AAA AAA AAA AAA AAA AAA AAA ATA ATA ATA ATA ATA ATG ATG
Ile Arg Lys Lys Lys Lys Lys Lys Lys Ile Ile Ile Ile Ile Met Met Ile Arg Lys Lys Lys Lys Lys Lys Lys Lys Ile Ile Ile Ile Met Met
TTT TAT ATA GTA TGT GCA TAG TGAATGTTTT TTTTTTATAT TTTAATACAT TTT TAT ATA GTA TGT GCA TAG TGAATGTTTT TTTTTTATAT TTTAATACAT
Phe Tyr Ile Val Cys Ala *** Phe Tyr Ile Val Cys Ala ***
GAAATGATAT ATATATATAT ATATATATAT ATATTTTCTC TTTATAGAAA TTATTAAGCC TAGAAAGTAG AATGAAGACA GACTTAAATG AATTAACTGA ACATCCATGG TGGATTAATG AAGATTAATT TTAATTTTTT AAGTATTATA TGAATATTTA TTATTAGTAG ACTTTATATA TAACAATAAA TATTGACACG TGCAATATTA AAAAAAAAAA AAAAAAAAAA AAAAAAAAGA ATTAAAAAGG AATGTTTTAT TGTTTAGGAC TATATGGAAA AATAATAATA TATATATGTT TCCACATTAA AAA  GAAATGATAT ATATATATAT ATATATATAT ATATTTTCTC TTTATAGAAA TTATTAAGCC TAGAAAGTAG AATGAAGACA GACTTAAATG AATTAACTGA ACATCCATGG TGGATTAATG AAGATTAATT TTAATTTTTT AAGTATTATA TGAATATTTA TTATTAGTAG ACTTTATATA TAACAATAAA TATTGACACG TGCAATATTA AAAAAAAAAA AAAAAAAAAA AAAAAAAAGA ATTAAAAAGG AATGTTTTAT TGTTTAGGAC TATATGGAAA AATAATAATA TATATATGTT TCCACATTAA AAA
ce "troisième polypeptide" a été décrit dans la demande de brevet français n°90 10039 déposée le 6 août 1990. Ce "troisième polypeptide" est reconnu par des anticorps contre P. falciparum. this "third polypeptide" was described in French patent application no. 90 10039 filed on August 6, 1990. This "third polypeptide" is recognized by antibodies against P. falciparum.
Une souche de E. Coli, transformée par un tel acide nucléique a été déposée à la CNCM sous le n°I-987 le 27 juillet 1990.  A strain of E. Coli, transformed by such a nucleic acid was deposited at the CNCM under the number I-987 on July 27, 1990.
Un "troisième peptide" préféré est le peptide recombinant contenant la séquence du peptide "R 23" - de formule  A preferred "third peptide" is the recombinant peptide containing the sequence of the peptide "R 23" - of formula
LysSerAspSerAsnHisLysSerAspHisAsnHisLysSerAspSerAsn  LysSerAspSerAsnHisLysSerAspHisAsnHisLysSerAspSerAsn
HisMetSerAspHisAsnHisMetSerAspHisAsnHisLysSerAspHisAsnHisLys HisMetSerAspHisAsnHisMetSerAspHisAsnHisLysSerAspHisAsnHisLys
SerAspAsnAsnHisLysSerAspSerAsnHisLysSerAspSerAsnHisLysSerAsp SerAspAsnAsnHisLysSerAspSerAsnHisLysSerAspSerAsnHisLysSerAsp
SerAsnHisLysSerAspHisAsn SerAsnHisLysSerAspHisAsn
Une souche d'E. Coli contenant la séquence d'ADN correspondante a été déposée le 27 juillet année 1990 sous le nº I - 986 à la CNCM. A strain of E. Coli containing the corresponding DNA sequence was deposited on July 27, 1990 under number I - 986 at the CNCM.
Le peptide R23 a pareillement été produit sous forme d'une protéine recombinante du fusion avec la glutatione transférase de Schistosoma japonicum, selon la technique déjà mentionnée de D.B. Smith et K.S. Johnson.  The R23 peptide was likewise produced in the form of a recombinant fusion protein with the glutatione transferase from Schistosoma japonicum, according to the technique already mentioned by D.B. Smith and K.S. Johnson.
Il est entendu que dans les associations :  It is understood that in associations:
EB 200 et i72,  EB 200 and i72,
EB 200 et R 23 respectivement,  EB 200 and R 23 respectively,
ces différents peptides peuvent être remplacés par des fragments ou protéines de fusion répondant aux conditions qui ont également été définies plus haut.  these different peptides can be replaced by fragments or fusion proteins meeting the conditions which have also been defined above.
L'invention concerne naturellement aussi les mélanges d'anticorps spécifiques qui peuvent être obtenus à partir des constituants respectifs des mélanges susdits de polypeptides. Ces mélanges d'anticorps peuvent être administrés in vivo, notamment dans le but d'interférer avec une parasitémie due à une infection par un parasite humain, notamment du type P. falciparum. The invention naturally also relates to the mixtures of specific antibodies which can be obtained from the respective constituents of the above-mentioned mixtures of polypeptides. These mixtures antibodies can be administered in vivo, in particular for the purpose of interfering with parasitaemia due to infection by a human parasite, in particular of the P. falciparum type.
Enfin, l'invention étend de même ses effets à des mélanges des séquences nucléiques dans lesquelles les différentes séquences nucléiques correspondantes auront été recombinées entre elles, dans des conditions autorisant leur expression sous la forme d'une protéine de fusion comprenant des séquences peptidiques correspondant aux séquences des trois constituants (entiers ou partiels) précédemment pris en considération. Finally, the invention likewise extends its effects to mixtures of the nucleic sequences in which the different corresponding nucleic sequences have been recombined with one another, under conditions permitting their expression in the form of a fusion protein comprising peptide sequences corresponding to the sequences of the three constituents (whole or partial) previously taken into consideration.
1. Ahlborg, N., K. Berzins, and P. Perimann. 1991. Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2. Mol. Biochem. Parasitol.46:89-96. 1. Ahlborg, N., K. Berzins, and P. Perimann. 1991. Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2. Mol. Biochem. Parasitol. 46: 89-96.
2. Berendt, A. R., D. J. P. Ferguson and C. I. Newbold. 1990. Sequestration in Plasmodium falciparum malaria: Sricky cells and sticky problems. Parasitol. Today. 6:247-254. 2. Berendt, A. R., D. J. P. Ferguson and C. I. Newbold. 1990. Sequestration in Plasmodium falciparum malaria: Sricky cells and sticky problems. Parasitol. Today. 6: 247-254.
3. Chen, E. J., and P. H. Seeburg. 1985. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA.4:165-170. 3. Chen, E. J., and P. H. Seeburg. 1985. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA.4: 165-170.
4. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochcm. 162:156-161. 4. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochcm. 162: 156-161.
5. Chu, G., D. Vollrath, and R. J. Davis. 1986. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 234:1582-1585. 5. Chu, G., D. Vollrath, and R. J. Davis. 1986. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 234: 1582-1585.
6. Coppel, R. L., J. G. Culvenor, A. E. Bianco, P. E. Crewther, H. D. Stahl, G. V. Brown, R. F. Anders, and D. J. Kemp. 1986. Variable antigen associated with the surface of erythrocytes of Plasmodium falciparum. Mol. Biochem. Parasitol.20:265-277. 6. Coppel, R. L., J. G. Culvenor, A. E. Bianco, P. E. Crewther, H. D. Stahl, G. V. Brown, R. F. Anders, and D. J. Kemp. 1986. Variable antigen associated with the surface of erythrocytes of Plasmodium falciparum. Mol. Biochem. Parasitol. 20: 265-277.
7. Corcoran, L. M., J. K. Thompson, D. Walliker, and D. J. Kemp. 1988. Homologous recombination within subtelomeric rcpeat sequences generates chromosome size polymorphisms in Plasmodium. falciparum. Cell.53:807-813 7. Corcoran, LM, JK Thompson, D. Walliker, and DJ Kemp. 1988. Homologous recombination within subtelomeric rcpeat sequences generates chromosome size polymorphisms in Plasmodium. falciparum. Cell.53: 807-813
8. Gassner, D., Z. Shraideh, and K. Wohlfarth-Bottermann. 1985. A giant titin-like protein in Physarum pofycephalum: evidence for its candidacy as a major component of an elastic cytoskeletal superthin filament lattice. Eur. J. Cell Biol.37:44-62. 8. Gassner, D., Z. Shraideh, and K. Wohlfarth-Bottermann. 1985. A giant titin-like protein in Physarum pofycephalum: evidence for its candidacy as a major component of an elastic cytoskeletal superthin filament lattice. Eur. J. Cell Biol. 37: 44-62.
9. Goding, J. W., and E. Handman. 1984. Electrophoretic analysis of protein antigens. In: Genes and antigens of parasites.383-415. 9. Goding, J. W., and E. Handman. 1984. Electrophoretic analysis of protein antigens. In: Genes and antigens of parasites. 383-415.
10. Groux, H., and J. Gysin. 1990. Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses. Res. Immunol. 141:529-542. 10. Groux, H., and J. Gysin. 1990. Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses. Res. Immunol. 141: 529-542.
11. Groux, H., R. Perraut, O. Garraud, J. P. Poingt, and J. Gysin. 1990. Functional characterization of the antibody-mediated protection against blood stages of Plasmodiumfalciparum in the monkey Saimiri sciureus. Eur. J. Immunol.20:2317-2323. 11. Groux, H., R. Perraut, O. Garraud, J. P. Poingt, and J. Gysin. 1990. Functional characterization of the antibody-mediated protection against blood stages of Plasmodiumfalciparum in the monkey Saimiri sciureus. Eur. J. Immunol. 20: 2317-2323.
12. Kemp, DJ., L.M. Corcoran, R.L. Coppel, H.D. Stahl, A.E. Bianco, G.V. Brown, and R.F. Anders. 1985. Size variation in chromosomes from independent cultured isolates of Plasmodiumfalciparum. Nature (London). 315347-350. 12. Kemp, DJ., L.M. Corcoran, R.L. Coppel, H.D. Stahl, A.E. Bianco, G.V. Brown, and R.F. Anders. 1985. Size variation in chromosomes from independent cultured isolates of Plasmodiumfalciparum. Nature (London). 315 347-350.
13. Koenen, M., A. Scherf, O. Mercereau, G. Langsley, L. Sibilli, P. Dubois, L. Pereira da Silva, and B. MuIler-Hill. 1984. Human antisera detect a Plasmodium falciparum genomic clone encoding a nonapeptide repeat. Nature (London).311:382-384 14. Labeit, S., D. P. Barlow, M. Gautel, T. Gibson, J. Holt, C. L. Hsieh, U. Franke, K. Leonard, J. Wardale, A. Whiting, and J. Trinick.1990. A regular panern of two types of 100-residues motif in the sequence of titin. Nature (London).231.507-509 13. Koenen, M., A. Scherf, O. Mercereau, G. Langsley, L. Sibilli, P. Dubois, L. Pereira da Silva, and B. MuIler-Hill. 1984. Human antisera detect a Plasmodium falciparum genomic clone encoding a nonapeptide repeat. Nature (London). 311: 382-384 14. Labeit, S., DP Barlow, M. Gautel, T. Gibson, J. Holt, CL Hsieh, U. Franke, K. Leonard, J. Wardale, A. Whiting, and J. Trinick. 1990. A regular panern of two types of 100-residues motif in the sequence of titin. Nature (London). 231.507-509
15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London).227:680-685. 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London). 227: 680-685.
16. Langsley, G., J. E. Hyde, M. Goman, and J. G. Scaife. 1983. Cloning and 16. Langsley, G., J. E. Hyde, M. Goman, and J. G. Scaife. 1983. Cloning and
characterisation of the rRNA genes from the human parasite Plasmodium falciparum.  characterization of the rRNA genes from the human parasite Plasmodium falciparum.
Nucl. Acids Res.24:8703-8717.  Nucl. Acids Res. 24: 8703-8717.
17. Leech, J. H., J. W. Bamwell, L. H. Miller, and R. J. Howard. 1984. Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium 17. Leech, J. H., J. W. Bamwell, L. H. Miller, and R. J. Howard. 1984. Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium
falciparum-infected erythrocytes. J. Exp. Med. 159:1567-1575.  falciparum-infected erythrocytes. J. Exp. Med. 159: 1567-1575.
18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
19. Maruyama, K. 1986. Connectin, an elastic filamentous protein of striated muscle. Int 19. Maruyama, K. 1986. Connectin, an elastic filamentous protein of striated muscle. Int
Rev. Cytol. 104:81-114.  Rev. Cytol. 104: 81-114.
20. Mattei, D., K. Berzins, M. Wahlgren, R. Udomsangpetch, P. Perlman, H. W. Griesser, A. Scherf, B. Müller-Hill, S. Bonnefoy, M. Guillotte, G. Langsley, L. Pereira da Silva, and O. Mercereau-Puijalon. 1989. Cross-rcactive antigenic deter minants present on different Plasmodium faIciparum blood-stage antigens.Parasite Immunol. 11:15-30. 20. Mattei, D., K. Berzins, M. Wahlgren, R. Udomsangpetch, P. Perlman, HW Griesser, A. Scherf, B. Müller-Hill, S. Bonnefoy, M. Guillotte, G. Langsley, L. Pereira da Silva, and O. Mercereau-Puijalon. 1989. Cross-reactive antigenic deter minants present on different Plasmodium faIciparum blood-stage antigens.Parasite Immunol. 11: 15-30.
21. Mattei, D., G. Langsley, G. Braun-Breton, M. Guillotte, J. F. Dubremetz, and O. Mercereau-Puijalon. 1988. The S-antigen of Plasmodium falciparum Palo Alto represents a new S-antigen serotype. Mol. Biochem. Parasitol.27:171-180. 21. Mattei, D., G. Langsley, G. Braun-Breton, M. Guillotte, J. F. Dubremetz, and O. Mercereau-Puijalon. 1988. The S-antigen of Plasmodium falciparum Palo Alto represents a new S-antigen serotype. Mol. Biochem. Parasitol. 27: 171-180.
22. Manei, D., A. Scherf, O. Bensaude, and L. Pereira da Silva. 1989. A heat shock-like protein from the human malaria parasite Plasmodium falciparum induces autoantibodies. Eur J Immunol 19:1823-1828. 22. Manei, D., A. Scherf, O. Bensaude, and L. Pereira da Silva. 1989. A heat shock-like protein from the human malaria parasite Plasmodium falciparum induces autoantibodies. Eur J Immunol 19: 1823-1828.
23. McCutchan, T.F., J.L.Hansen, J.B. Dame, and J.A. Mullins, 1984. Mung bean nuclease cleaves Plasmodium genomic DNA at sites before and after genes. Science 225: 625-628. 23. McCutchan, T.F., J.L. Hansen, J.B. Dame, and J.A. Mullins, 1984. Mung bean nuclease cleaves Plasmodium genomic DNA at sites before and after genes. Science 225: 625-628.
24. Mercereau-Puijalon, O., G. Langsley, D. Manei, M. Guillotte, T. Blisnick, K. Berzins, H. W. Griesser, A. Scherf, B. Mü ller-Hill, and L. Pereira da Silva. 1987. Presence of cross-reacting determinants on several blood-stage antigens of Plasmodium falciparum. UCLA Symp.42:343-354. 24. Mercereau-Puijalon, O., G. Langsley, D. Manei, M. Guillotte, T. Blisnick, K. Berzins, H. W. Griesser, A. Scherf, B. Mü ller-Hill, and L. Pereira da Silva. 1987. Presence of cross-reacting determinants on several blood-stage antigens of Plasmodium falciparum. UCLA Symp. 42: 343-354.
25. Pasvol, G., R. J. M. Wilson, M. E. Smalley, and J. Brown. 1978. Separation of viable schizont-infected red blood cells of Plasmodium falciparum from human blood. Ann. Trop. Med. Parasitol.72:87-88. 25. Pasvol, G., RJM Wilson, ME Smalley, and J. Brown. 1978. Separation of viable schizont-infected red blood cells of Plasmodium falciparum from human blood. Ann. Too much. Med. Parasitol. 72: 87-88.
26. Patarapotikul, J., and G. Langsley. 1988. Chromosome size polymorphism in Plasmodium falciparum can involve deletions of the subtelomeric pPFrep20 sequence. Nucl. Acids 26. Patarapotikul, J., and G. Langsley. 1988. Chromosome size polymorphism in Plasmodium falciparum can involve deletions of the subtelomeric pPFrep20 sequence. Nucl. Acids
Res. 16:4331-4340. Res. 16: 4331-4340.
27. Petersen, C., R. Nelson, J. Leech. J. Jensen, W. Wollish, and A. Scherf. 1990. The gene product of the Plasmodiumfalciparum 11.1 locus is a protein larger than one megadalton. Mol. Biochem. Parasitol.42:189-196. 27. Petersen, C., R. Nelson, J. Leech. J. Jensen, W. Wollish, and A. Scherf. 1990. The gene product of the Plasmodiumfalciparum 11.1 locus is a protein larger than one megadalton. Mol. Biochem. Parasitol. 42: 189-196.
28 . Pologe, LG., and J.V. Ravetch. 1988. Large deletions resuit from breakage and healing of P. falciparum chromosomes. Cell.55: 869-874. 28. Pologe, LG., And J.V. Ravetch. 1988. Large deletions resuit from breakage and healing of P. falciparum chromosomes. Cell. 55: 869-874.
29. Ponzi, M., T. Pace, E. Dore, and C. Frontali. 1985. Identification of a telomeric DNA sequence in Plasmodium. berghei. EMBO J. 4:2991-2995. 29. Ponzi, M., T. Pace, E. Dore, and C. Frontali. 1985. Identification of a telomeric DNA sequence in Plasmodium. berghei. EMBO J. 4: 2991-2995.
30. Pudles, J., M. Moudjou, S. Hisanaga, K. Maruyama, and H. Sakai. 1990. Isolation, characterisation and immunochemical properties of a giant protein from sea urchin egg cytomatrix. Exp. Ccll Res. 189:253-260. 30. Pudles, J., M. Moudjou, S. Hisanaga, K. Maruyama, and H. Sakai. 1990. Isolation, characterization and immunochemical properties of a giant protein from sea urchin egg cytomatrix. Exp. Ccll Res. 189: 253-260.
31. Schwartz, D. C, and C. R. Cantor. 1984. Separation of yeast chromosome-sized DNAs by pulse field gradient electrophoresis. Cell.37:67-75. 31. Schwartz, D. C, and C. R. Cantor. 1984. Separation of yeast chromosome-sized DNAs by pulse field gradient electrophoresis. Cell. 37: 67-75.
32. Smith, D. B., and K. S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusion porteins with glutathione S-transferase. 32. Smith, DB, and KS Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusion porteins with glutathione S-transferase.
Gene.29:31-40. Gene. 29: 31-40.
33. Smith, G.E. and, M.D.Summers. 1980. The bidirectional transfer of DNA and RNA to 33. Smith, G.E. and, M.D. Summers. 1980. The bidirectional transfer of DNA and RNA to
nirrocellulose or diazobenzylomethyl-paper. Anal. Biochem. 109, 123-129.  nirrocellulose or diazobenzylomethyl-paper. Anal. Biochem. 109, 123-129.
34. Taylor, D.W., M. Parra, G.B. Chapman, M.E. Stearns, J. Rener, M. Aikawa, S. Uni, S.B. 34. Taylor, D.W., M. Parra, G.B. Chapman, M.E. Stearns, J. Rener, M. Aikawa, S. Uni, S.B.
Aley, L.J. Panton, and R.J. Howard. 1987. Localization of Plasmodium falciparum histidine rich protein I in the erythrocyte skeleton under knobs . Mol. Biochem. Parasitol.25: 165- 174.  Aley, L.J. Panton, and R.J. Howard. 1987. Localization of Plasmodium falciparum histidine rich protein I in the erythrocyte skeleton under knobs. Mol. Biochem. Parasitol. 25: 165-174.
35. Trager, W. T. and J. B. Jensen. 1976. Human malaria parasite in continuous culture. Science. 193:673-675. 35. Trager, W. T. and J. B. Jensen. 1976. Human malaria parasite in continuous culture. Science. 193: 673-675.
36. Udomsangpetch, R., M. Aikawa, K. Berzins, M. Wahlgren, and P. Perlmann. 1989. Cytoadhercnce of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by human monoclonal antibody. Nature (London).338:763-765. 36. Udomsangpetch, R., M. Aikawa, K. Berzins, M. Wahlgren, and P. Perlmann. 1989. Cytoadhercnce of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by human monoclonal antibody. Nature (London) .338: 763-765.
37. Udomsangpetch, R., J. Carlsson, B. Wahlin, G. Holmquist, L. S. Ozaki, A. Scherf, D. Manei, O. Mercereau-Puijalon, S. Uni, M. Aikawa, K. Berzins, and P. Perlmann. 1989. Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens. J. Immunol. 1423620-3626. 37. Udomsangpetch, R., J. Carlsson, B. Wahlin, G. Holmquist, LS Ozaki, A. Scherf, D. Manei, O. Mercereau-Puijalon, S. Uni, M. Aikawa, K. Berzins, and P Perlmann. 1989. Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens. J. Immunol. 1423620-3626.
38. Udomsangpetch, R., K. Lundgren, K. Berzins, B. Wahlin, H. Perlmann, M. Troye- Blomberg, J. Carlsson, M. Wahlgren, P. Perlmann, and A. Björkman. 1986. Human monoclonal antibodies to Pf155, a major antigen of malaria parasite Plasmodium 38. Udomsangpetch, R., K. Lundgren, K. Berzins, B. Wahlin, H. Perlmann, M. Troye- Blomberg, J. Carlsson, M. Wahlgren, P. Perlmann, and A. Björkman. 1986. Human monoclonal antibodies to Pf155, a major antigen of malaria parasite Plasmodium
falciparum. Science.231:57-59.  falciparum. Science 231: 57-59.
39. Veroick, K. D., D. Walliker, and T. F. McCutchan. 1988. Genetic hypervariability of telomere-rclated sequences is associated with meiosis in Plasmodiumfalciparum. 39. Veroick, K. D., D. Walliker, and T. F. McCutchan. 1988. Genetic hypervariability of telomere-rclated sequences is associated with meiosis in Plasmodiumfalciparum.
Nucl. Acid. Res. 16:6973-6985.  Nucl. Acid. Res. 16: 6973-6985.
40. Wahlgren, M. 1986. Antigens and antibodies involved in humoral immunity to Plasmodium falciparum. Thesis, Stockholm, Sweden, Karolinska Institutet 40. Wahlgren, M. 1986. Antigens and antibodies involved in humoral immunity to Plasmodium falciparum. Thesis, Stockholm, Sweden, Karolinska Institutet
41. Walliker, D., I. A. Quakyi, T. E Wellems, T. F. McCutchan, A. Szarfman, W. T. London. 41. Walliker, D., I. A. Quakyi, T. E Wellems, T. F. McCutchan, A. Szarfman, W. T. London.
L. M. Corcoran, T. R. Burkot, and R. Carter. 1987. Genetic analysis of the human malaria parasite Plasmodiumfalciparum. Science.236:1661-1666.  L. M. Corcoran, T. R. Burkot, and R. Carter. 1987. Genetic analysis of the human malaria parasite Plasmodiumfalciparum. Science. 236: 1661-1666.
42. Wang, K., and A. McClure. 1979. Tu:Titin : Major myofibriïlar components of striated muscle. Proc. Natl. Acad. Sci. USA.76:3698-3702. 42. Wang, K., and A. McClure. 1979. Tu: Titin: Major myofibriïlar components of striated muscle. Proc. Natl. Acad. Sci. USA 76: 3698-3702.
43. Wellems, T. E., D. Walliker, C. L. Smith, V. E. Rosario, W. L. Maloy, R. J. Howard, R. Carter, and T. F. McCutchan. 1987. A histidinc-rich pπotein genc marks a linkage group favored strongly in a genetic cross of Plasmodium falciparum. Cell.49:633-642. 43. Wellems, T. E., D. Walliker, C. L. Smith, V. E. Rosario, W. L. Maloy, R. J. Howard, R. Carter, and T. F. McCutchan. 1987. A histidinc-rich pπotein genc marks a linkage group favored strongly in a genetic cross of Plasmodium falciparum. Cell. 49: 633-642.

Claims

REVENDICATIONS
1. Polypeptide contenant la séquence polypeptidique ci-dessous définie par sa séquence en acides aminés : 1. Polypeptide containing the polypeptide sequence below defined by its amino acid sequence:
S V T G N I L V E G S V T G N I L V E G
S V T E E V V G E E KS V T E E V V G E E K
L V S E E I V T E E GL V S E E I V T E E G
S V A Q E I V E E D AS V A Q E I V E E D A
P A T E E I D E I EP A T E E I D E I E
S V T E E V V E E E GS V T E E V V E E E G
P V D E E I V Q E E GP V D E E I V Q E E G
T V T E E I I Q G ET V T E E I I Q G E
S K V E E V V E E Q GS K V E E V V E E Q G
S E N E E I F V E E VS E N E E I F V E E V
S A S Q E I V Q N ES A S Q E I V Q N E
S G T E E I L E K VS G T E E I L E K V
S A S Q E I V Q D GS A S Q E I V Q D G
S V T E Q I I E E L FS V T E Q I I E E L F
P V T E E V V N E E E P V T E E V V N E E E
ou une partie de cette séquence dès lors que celleci est apte, le cas échéant lorsqu'elle est incorporée à une protéine de fusion, oligomérisée ou conjuguée à une protéine porteuse, à induire in vivo une réponse immunitaire protectrice chez le singe écureuil Saimiri sciureus. or part of this sequence when this is capable, if appropriate when it is incorporated into a fusion protein, oligomerized or conjugated to a carrier protein, to induce in vivo a protective immune response in the squirrel monkey Saimiri sciureus.
2. Polypeptide selon la revendication 1, caractérisé en ce qu'il est capable d'induire in vivo des anticorps eux-mêmes capables d'inhiber l'invasion de globules rouges par des mérozoïtes.  2. Polypeptide according to claim 1, characterized in that it is capable of inducing in vivo antibodies themselves capable of inhibiting the invasion of red blood cells by merozoites.
3. Polypeptide selon la revendication 2, caractérisé en ce que ledit polypeptide ou ladite partie de séquence polypeptidique est apte à induire in vivo des anticorps capables d'inhiber l'invasion des globules rouges dans une proportion au moins égale à 50 % par rapport à celles observables dans des préparations d'érythrocytes témoins placés dans les mêmes conditions en présence des mêmes concentrations de mérozoïtes, mais en l'absence des polypeptides ou partie de polypeptides du genre en question. 3. Polypeptide according to claim 2, characterized in that said polypeptide or said part of the polypeptide sequence is capable of inducing in vivo antibodies capable of inhibiting the invasion of red blood cells in a proportion at least equal to 50% compared to those observable in preparations of control erythrocytes placed under the same conditions in the presence of the same concentrations of merozoites, but in the absence of the polypeptides or part of polypeptides of the genus in question.
4. Partie de polypeptide selon l'une quelconque des revendications 1 à 3, caractérisée en ce qu'elle est constituée par l'une des séquences 4. Part of a polypeptide according to any one of claims 1 to 3, characterized in that it consists of one of the sequences
(a) à (o) suivantes : (a) to (o) below:
(a) S V T G N I L V E G.  (a) S V T G N I L V E G.
(b) S V T E E V V G E E K.  (b) S V T E E V V G E E K.
(c) L V S E E I V T E E G.  (c) L V S E E I V T E E G.
(d) S V A Q E I V E E D A.  (d) S V A Q E I V E E D A.
(e) P A T E E I D E I E. (f) S V T E E V V E E E G.  (e) P A T E E I D E I E. (f) S V T E E V V E E E G.
(g) P V D E E I V Q E E G. (g) P V D E E I V Q E E G.
(h) T V T E E I I Q G E. (i) S K V E E V V E E Q G. (j) S E N E E I F V E E V.  (h) T V T E E I I Q G E. (i) S K V E E V V E E Q G. (j) S E N E E I F V E E V.
(k) S A S Q E I V Q N E.  (k) S A S Q E I V Q N E.
(l) S G T E E I L E K V. (l) S G T E E I L E K V.
(m) S A S Q E I V Q D G.  (m) S A S Q E I V Q D G.
(n) S V T E Q I I E E L F.  (n) S V T E Q I I E E L F.
(o) P V T E E V V N E E E. ou par un équivalent peptidique ayant un motif représentable par : (o) PVTEEVVNEE E. or by a peptide equivalent having a motif which can be represented by:
(X)3 - EE - (X)2 - EE - (X)2-3 dans lequel chacun des groupes X représente, selon l'indice qui suit les signes de parenthèse qui entoure le groupe considéré, un dipeptide ou tripeptide contenant chacun, au moins un résidu amino-acyle hydrophobe, cet équivalent peptidique étant susceptible d'induire une réponse immunitaire protectrice chez le singe-écureuil Saimiri sciureus. (X) 3 - EE - (X) 2 - EE - (X) 2-3 in which each of the groups X represents, according to the index which follows the signs of parenthesis which surrounds the group considered, a dipeptide or tripeptide each containing , at least one hydrophobic amino-acyl residue, this peptide equivalent being capable of inducing a protective immune response in the squirrel monkey Saimiri sciureus.
5. Polypeptide selon l'une quelconque des revendications 1 à 4, caractérisé en qu'il est incorporé à une protéine de fusion, oligomérise ou conjugué de façon covalente à une molécule, notamment protéine porteuse.  5. Polypeptide according to any one of claims 1 to 4, characterized in that it is incorporated into a fusion protein, oligomerizes or covalently conjugated to a molecule, in particular carrier protein.
6. Composition capable d'induire in vivo des anticorps aptes à détruire les parasites responsables du paludisme chez l'homme, notamment à leur stade sanguin, caractérisée en ce qu'elle contient un polypeptide ou partie de polypeptide selon l'une quelconque des revendications 1 à 5, en association avec un véhicule pharmaceutiquement acceptable.  6. Composition capable of inducing in vivo antibodies capable of destroying the parasites responsible for malaria in humans, in particular at their blood stage, characterized in that it contains a polypeptide or part of a polypeptide according to any one of claims 1 to 5, in combination with a pharmaceutically acceptable vehicle.
7. Anticorps spécifiques du polypeptide ou de la partie de polypeptide selon l'une quelconque des revendications 1 à 4.  7. Antibodies specific for the polypeptide or part of the polypeptide according to any one of claims 1 to 4.
8. Fragment d'ADN, caractérisé en ce qu'il code pour le polypeptide ou partie de polypeptide selon l'une quelconque des revendications 1 à 4.  8. DNA fragment, characterized in that it codes for the polypeptide or part of a polypeptide according to any one of claims 1 to 4.
9. Fragment d'ADN selon la revendication 8, caractérisé en ce qu'il se trouve à l'état recombiné au sein d'un ADN codant pour une protéine de fusion, dont les éléments d'ADN extérieurs au susdit fragment sont hétérologues vis-à-vis de celui-ci. 9. DNA fragment according to claim 8, characterized in that it is in the recombined state within a DNA coding for a fusion protein, whose DNA elements external to the above fragment are heterologous vis-à-vis the latter.
10. Vecteur modifié par le fragment d'ADN selon la revendication 8 ou la revendication 9, dans lequel ledit fragment se trouve placé sous le contrôle d'un promoteur autorisant son expression dans un hôte cellulaire dont les polymérases reconnaissent ledit promoteur.  10. The vector modified by the DNA fragment according to claim 8 or claim 9, wherein said fragment is placed under the control of a promoter allowing its expression in a cellular host whose polymerases recognize said promoter.
11. Culture cellulaire transformée par le vecteur selon la revendication 10.  11. Cell culture transformed by the vector according to claim 10.
12. Application des polypeptides ou peptides selon l'une quelconque des revendications 1 à 5 au diagnostic in vitro de la présence d'anticorps anti-paludéens dans un échantillon biologique, notamment sérum sanguin, prélevé chez la personne soumise au diagnostic.  12. Application of the polypeptides or peptides according to any one of claims 1 to 5 to the in vitro diagnosis of the presence of anti-malarial antibodies in a biological sample, in particular blood serum, taken from the person subjected to the diagnosis.
13. Application des anticorps selon la revendication 7 au diagnostic in vitro d'une infection par un parasite paludéen dans un échantillon biologique, notamment sérum sanguin chez la personne soumise au diagnostic.  13. Application of the antibodies according to claim 7 to the in vitro diagnosis of an infection by a malarial parasite in a biological sample, in particular blood serum in the person subjected to the diagnosis.
14. Composition selon la revendication 6 qui contient un autre antigène vaccinant, de préférence, soit un antigène contenant la séquence en aminoacides du polypeptide i72, soit un antigène contenant la séquence en amino-acides du polypeptide R 23.  14. The composition as claimed in claim 6, which contains another vaccinating antigen, preferably either an antigen containing the amino acid sequence of the polypeptide i72, or an antigen containing the amino acid sequence of the polypeptide R 23.
PCT/FR1992/000763 1991-07-31 1992-07-31 Polypeptides capable of in vivo induction of antibodies themselves capable of inhibiting the invasion of red blood corpuscles by merozoites of p.falciparum, related products and their use in producing vaccine compositions WO1993003057A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP5503341A JPH06510527A (en) 1991-07-31 1992-07-31 P. Polypeptides capable of inducing in vivo antibodies capable of inhibiting erythrocyte invasion by merozoites of C. falciparum, related products and their application for the production of vaccine compositions.
EP92917738A EP0602079A1 (en) 1991-07-31 1992-07-31 POLYPEPTIDES CAPABLE OF $i(IN VIVO) INDUCTION OF ANTIBODIES THEMSELVES CAPABLE OF INHIBITING THE INVASION OF RED BLOOD CORPUSCLES BY MEROZOITES OF $i(P.FALCIPARUM), RELATED PRODUCTS AND THEIR USE IN PRODUCING VACCINE COMPOSITIONS

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
FR91/09772 1991-07-31
FR91/09771 1991-07-31
FR9109771A FR2679909B1 (en) 1991-07-31 1991-07-31 POLYPEPTIDES SUITABLE FOR INDUCING IN VIVO ANTIBODIES INHIBITING THE INVASION OF RED CELLS BY MEROZOUITES OF P. FALCIPARUM, RELATED PRODUCTS AND THEIR APPLICATION AS VACCINES.
FR9109772A FR2679776A1 (en) 1991-07-31 1991-07-31 Mixture of peptide constituents having vaccinating properties against malaria, in particular caused by Plasmodium falciparum

Publications (1)

Publication Number Publication Date
WO1993003057A1 true WO1993003057A1 (en) 1993-02-18

Family

ID=26228876

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FR1992/000763 WO1993003057A1 (en) 1991-07-31 1992-07-31 Polypeptides capable of in vivo induction of antibodies themselves capable of inhibiting the invasion of red blood corpuscles by merozoites of p.falciparum, related products and their use in producing vaccine compositions

Country Status (6)

Country Link
EP (1) EP0602079A1 (en)
JP (1) JPH06510527A (en)
AU (1) AU2442792A (en)
CA (1) CA2114223A1 (en)
PT (1) PT100757A (en)
WO (1) WO1993003057A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0730466A1 (en) * 1993-09-10 1996-09-11 New York University Compositions and methods for inhibiting hepatocyte invasion by malarial sporozoites

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986000620A1 (en) * 1984-07-02 1986-01-30 Institut Pasteur Molecule comprising at least one peptidic sequence carrying an epitope
WO1987005607A1 (en) * 1986-03-14 1987-09-24 Saramane Pty. Ltd.; Polypeptides providing protective immunity against malaria
EP0276014A2 (en) * 1987-01-23 1988-07-27 Merrell Dow Pharmaceuticals Inc. Anticoagulant peptides
WO1992002549A1 (en) * 1990-08-06 1992-02-20 Institut Pasteur Plasmodium falciparum antigen for eliciting protective antibodies for use in vaccines

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986000620A1 (en) * 1984-07-02 1986-01-30 Institut Pasteur Molecule comprising at least one peptidic sequence carrying an epitope
WO1987005607A1 (en) * 1986-03-14 1987-09-24 Saramane Pty. Ltd.; Polypeptides providing protective immunity against malaria
EP0276014A2 (en) * 1987-01-23 1988-07-27 Merrell Dow Pharmaceuticals Inc. Anticoagulant peptides
WO1992002549A1 (en) * 1990-08-06 1992-02-20 Institut Pasteur Plasmodium falciparum antigen for eliciting protective antibodies for use in vaccines

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GENE. vol. 110, no. 1, 2 Janvier 1992, AMSTERDAM NL pages 71 - 79 D.MATTEI E.A. 'THE PF332 GENE OF P.FALCIPARUM CODES FOR A GIANT PROTEIN THAT IS TRANSLOCATED FROM THE PARASITE TO THE MEMBRANE OF INFECTED ERYTHROCYTES' *
JOURNAL OF IMMUNOLOGY. vol. 142, no. 10, 15 Mai 1989, BALTIMORE US pages 3620 - 3626 R.UDOMSANGPETCH C.S. 'REACTIVITY OF THE HUMAN MONOCLONAL ANTIBODY 33G2 WITH REPEATED SEQUENCES OF THREE DISTINCT PLASM.F. ANTIGENS.' cité dans la demande *
JOURNAL OF IMMUNOLOGY. vol. 143, no. 1, 1 Juillet 1989, BALTIMORE US pages 285 - 292 S.J.RICHMAN 'PEPTIDE MAPPING OF CONFORMATIONAL EPITOPES IN A HUMAN MALARIAL PARASITE HEAT SHOCK PROTEIN' *
O.MERCEREAU-PUIJALON C.S. 'MOLECULAR STRATEGIES OF PARASITIC INVASION' 1987 , A.R.LISS, INC. *
PARASITE IMMUNOLOGY vol. 11, no. 1, Janvier 1989, pages 15 - 30 D.MATTEI C.S. 'CROSS-REACTIVE ANTIGENIC DETERMINANTS PRESENT ON DIFFERENT PLASM.F. BLOOD-STAGE ANTIGENS.' cité dans la demande *
PRESENCE OF CROSS-REACTING DETERMINANTS ON SEVERAL BLOOD-STAGE ANTIGENS OF PLASM.F. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0730466A1 (en) * 1993-09-10 1996-09-11 New York University Compositions and methods for inhibiting hepatocyte invasion by malarial sporozoites
EP0730466A4 (en) * 1993-09-10 1999-04-14 Univ New York Compositions and methods for inhibiting hepatocyte invasion by malarial sporozoites

Also Published As

Publication number Publication date
JPH06510527A (en) 1994-11-24
EP0602079A1 (en) 1994-06-22
CA2114223A1 (en) 1993-02-18
PT100757A (en) 1993-10-29
AU2442792A (en) 1993-03-02

Similar Documents

Publication Publication Date Title
EP0666916B1 (en) Plasmodium falciparum antigens inducing protective antibodies
US7438917B2 (en) Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
Coppel et al. A blood stage antigen of Plasmodium falciparum shares determinants with the sporozoite coat protein.
US20050287166A1 (en) Malarial pre-erythrocytic stage polypeptide molecules
EP0343186B1 (en) MOLECULES COMPRISING AT LEAST ONE PEPTIDIC SEQUENCE CARRYING ONE OR A PLURALITY OF EPITOPES CHARACTERIZING A PROTEIN PRODUCED BY $i(P. FALCIPARUM) IN HEPATOCYTES AND COMPOSITIONS CONTAINING THEM
EP0275196B1 (en) Protein copolymer malaria vaccine
EP0407230B1 (en) Sporozoite and liver-stage protein antigen of P. falciparum
WO1993003057A1 (en) Polypeptides capable of in vivo induction of antibodies themselves capable of inhibiting the invasion of red blood corpuscles by merozoites of p.falciparum, related products and their use in producing vaccine compositions
FR2679909A1 (en) Polypeptides capable of inducing in vivo antibodies which inhibit the invasion of red blood cells by P. falciparum merozoites, related products and their use as vaccine
US6270771B1 (en) Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
GB2193215A (en) Peptides for antimalarial vaccines and diagnostic kits
CA2382977A1 (en) Plasmodium falciparum antigens and their vaccinal and diagnostic applications

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1992917738

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2114223

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 1994 182120

Country of ref document: US

Date of ref document: 19940609

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 1992917738

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1992917738

Country of ref document: EP