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

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 SUITABLE FOR INDUCING IN VIVO ANTIBODIES THEY themselves CAPABLE OF INHIBITING THE INVASION OF RED CELLS BY MEROZOITES OF

P.FALCIPARUM, RELATED PRODUCTS AND THEIR APPLICATION TO THE PRODUCTION OF VACCINANT COMPOSITIONS

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.

 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.

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).

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:

 S V T G N I L V E G

S V T E E V V G E E K

L V S E E I V T E E G

S V A Q E I V E E D A

P A T E E I D E I E

S V T E E V V E E E G

P V D E E I V Q E E G

T V T E E I I Q G E

S K V E E V V E E Q G

S E N E E I F V E E V

S A S Q E I V Q N E

S G T E E I L E K V

S A S Q E I V Q D G

SVTEQIIEELF PVTEEVVNEEE

 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.

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.

 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).

 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).

 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):

 This presents respectively:

- a restriction map of the Pf332 gene, - the respective positions of the selected fragments (C1, G90, G1 and G9),

 - 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.

 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).

 Parts of these fragments and more particularly a sub-fragment of G9 (fragment

BamH1, 441 base pair EcoR1) was subcloned into a plasmid pGEX3 (32). The sequence of

(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.

 The EB200 protein and the corresponding antibodies gave rise to the following observations:

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,

 - anti-EB200 serum reacted with the surface of monkey red blood cells infected with P. falciparum,

- the recombinant antigen PfEB200 is capable of inhibiting the phagocytosis of opsonized parasitized red blood cells (technique described in (ii) by monocytes of monkeys.

 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.

 The EB200 protein or fractions of it still have the following characteristics:

- 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;

 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.

 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.

 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.

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.

 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 .

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 ).

 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.

 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.

 (b) S V T E E V V G E E K.

 (c) L V S E E I V T E E G.

 (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.

(g) P V D E E I V Q E E G.

 (h) T V T E E I I Q G E.

(i) SKVEEVVEEQ G. (j) SENEEIFVEE V.

 (k) S A S Q E I V Q N E.

 (l) S G T E E I L E K V.

 (m) S A S Q E I V Q D G.

 (n) S V T E Q I I E E L F.

 (o) P V T E E V V N E E E.

 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.

 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 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.

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.

 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.

 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.

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.

 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).

 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. 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.

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 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").

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.

 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 ...

 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.

 The literature mentions other types of macromolecular supports which can be used, which generally have a molecular weight greater than 20,000.

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.

 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. Among 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.

 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.

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.

 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. 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.

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. 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.

 As additional experimental data, the following facts are also mentioned.

 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.

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.

 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.

 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.

 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:

 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:

- 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;

- 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.

 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:

1/1 31/11

 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 arg

61/21 91/31

 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 asp

121/41 151/51

 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 thr

181/61 211/71

 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 lys

241/81 271/91

 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 ala

301/101 331/111

 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 gly

361/121 391/131

 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 asn

421/141 451/151

 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 lys

481/161 511/171

 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 phe

541/181 571/191

 TTT TTT TTT TTA CAT TTT TOT AAA TTA ATA TGA ATT

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.

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:

CT

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 Ile

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 Glu

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

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 Val

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 Met

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

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

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

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

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

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

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

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

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

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

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 GAA

Glu 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

 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

Asp 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

Leu 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

His 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

Pro 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

 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

Ser 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

His 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

Asn 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

Phe 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

Ala 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

 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

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

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.

 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

 LysSerAspSerAsnHisLysSerAspHisAsnHisLysSerAspSerAsn

HisMetSerAspHisAsnHisMetSerAspHisAsnHisLysSerAspHisAsnHisLys

SerAspAsnAsnHisLysSerAspSerAsnHisLysSerAspSerAsnHisLysSerAsp

SerAsnHisLysSerAspHisAsn

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.

 It is understood that in associations:

 EB 200 and i72,

 EB 200 and R 23 respectively,

 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.

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.

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Claims

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 E E V V G E E K

L V S E E I V T E E G

S V A Q E I V E E D A

P A T E E I D E I E

S V T E E V V E E E G

P V D E E I V Q E E G

T V T E E I I Q G E

S K V E E V V E E Q G

S E N E E I F V E E V

S A S Q E I V Q N E

S G T E E I L E K V

S 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

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 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 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. Part of a polypeptide according to any one of claims 1 to 3, characterized in that it consists of one of the sequences

(a) to (o) below:

 (a) S V T G N I L V E G.

 (b) S V T E E V V G E E K.

 (c) L V S E E I V T E E G.

 (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.

(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.

 (k) S A S Q E I V Q N E.

(l) S G T E E I L E K V.

 (m) S A S Q E I V Q D G.

 (n) S V T E Q I I E E L F.

(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 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 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 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. Antibodies specific for the polypeptide or part of the polypeptide according to any one of claims 1 to 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. 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. 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. Cell culture transformed by the vector according to claim 10.

 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 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. 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.