HK1084403B - Conjugates of malarial pre-erythrocytic stage polypeptide molecules and lipids - Google Patents

Abstract

Polypeptide molecules containing at least 10 consecutive amino acids of the amino acid sequence representing antigen LSA-3 shown in figure 2, with the exception of the polypeptides (1).

Description

The parasites responsible for malaria in humans have different morphologies in the human host and express different antigens depending on their location in the organism. The morphological and antigenic differences of these parasites during their life cycles in humans, allow to define different stages of development in the liver and in the blood : the sporozoite, an infectious form injected by the vector mosquito, quickly transforms into a schizophrenic in the hepatocytes of the host to then infect the erythrocytes.This phase, which is clinically silent, is currently the only one against which a very strong, sterilizing immunity can be experimentally induced in humans by the injection of irradiated sporozoites, which are able to enter the hepatocyte and develop there, but cannot lead to the blood stage of the disease. That is why the inventors have focused most of their efforts on these two pre-erythrocytic stages.The Commission has already taken a number of steps to ensure that the information provided by the Member States is not used in a way that is costly and limited.

To access pre-erythrocyte antigens, the researchers used serums from individuals who had been living in endemic areas for 25 years but were on permanent prophylaxis with chloroquine. These individuals were regularly subjected to bites from infected mosquitoes but did not develop any complete blood infection. Their serum therefore contained antibodies primarily directed against pre-erythrocyte stages, which was verified by Immuno-Fluorescence (IF) and Western Blot on all 3 stages of the parasite.

The use of these serums for screening a genomic DNA bank of the parasitic P.falciparum clone, constructed from vectors of expression in a lambda phage gt11 (see Rosario, Science 212, 1981, p.1037-1038; and Thaithong et al, Transactions of Royal Society of Tropical Medicine and Hygien, 1984, 78 242-245) has led to the identification of pre-erythrocyte polypeptides, including the SALSA polypeptides described in EP A-0407230 and the LSA 1 (for Liver Stage Antigen) described in W 92/013884. The present invention relates to specific polypeptide antibodies of the pre-erythrocyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cyte-cytecyte-cytecytecy-cytecy-cytecy-cy-cytecy-cy-cy-cytecy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-cy-

The invention is the result of the inventors' highlighting of the special properties of a particular antigen called LSA-3 and its fragments, which appear to be high potential candidates for an antimalarial vaccine for the following reasons: (a) when a fraction of LSA-3 was used in combination with another antigen of the same stage of development of the parasite, such as LSA-1, to immunize chimpanzees, the animal responding to both molecules or to LSA-3 alone has the characteristic of having no parasites in the blood, a significant decrease in parasites in the liver, and a significant recruitment of mononuclear cells indicating a cellular immunity response;(c) in eight human volunteers immunized by irradiated sporozoite injection, antibodies directed against LSA-3 were found in each of the four individuals resistant to a sporozoite infection and in none of the other four volunteers who developed a blood infection; (d) antibodies obtained against the peptide; DG729 in WO 92/13884 already describe a cross-reaction with the sporozoite and hepatic stages of the mouse Pyoelii parasite, which allows for significant exploitation of the mouse model.In vitro, human immunoantibodies purified on DG729 are able, even at very low concentrations, to block the penetration of P. yoelii sporozoites into mouse hepatocytes. In vivo, mice immunized by DG729 are fully or partially protected against infection by P. yoelii sporozoites. e) Finally, certain epitopes, particularly in the non-repeated parts of the molecule, stimulate the secretion of interferon γ by monocytes, a mediator which inhibits the intrahepatic development of the parasite (S. Mello et al., The Journal of Immunology, 139, 4192-4195.1987f).(g) information on the structure of the antigen, and in particular of an ER peptide, and in particular on the central repeat region from which the ER peptide was designed and which contains one or more B major epitopes, were obtained from the Hydrophobic Cluster Plot of the sequence available in the T9/96 clone (630 amino acids),The repeated region has an extraordinary regularity in the spacing of valine and isoleucine residues, alternating with acid or proline residues. The arrangement of hydrophobic clusters on the surface of this helix suggests a hydrophobic rim gradually shifting from one side of the helix to the other, following a constant general orientation along the molecule, and probably in relation to a structure or packaging coiled-coil CP which appears in Figure 4b (H) as the hydrophobic cluster of the seventh clot of DG729.(h) After demonstrating a very wide range of immune responses to the LSA-3 antigen, we analysed the ability of the responding cells to locate themselves around the parasites in the liver. In mice immunized by recombinant antigens, intra-portal injection of each of the absorbed peptides onto 10μm polystyrene balls enables an influx of lymphocytes around the antigen (mimicking the parasite) to be visualized after 48 hours, followed by a significant recruitment of cells from the macrophage lineage on day 5.

All these properties, some of which will be demonstrated in detail in the experiments described below, show that the LSA-3 antigen has both good antigenicity and good immunogenicity.

The researchers were able to confirm and specify the specificity of the expression stages of the molecule; in the sporozoite, this expression was confirmed by surface immunofluorescence of several strains and isolates. In Western Blot analysis, the LSA-3 molecule appears as a protein with a molecular weight of 200,000 daltons. While the sporozoite messenger RNA could not be obtained in sufficient quantity for analysis in northern blot, reverse PCR experiments confirmed LSA-3 expression at this stage. In the infected hepatocytes, LSA-3 is observed in the parasite's proteophore using repeated fluorescence and electron microscopy against parasite-infected regions and the immune system.

A LSA-3 fragment called 729S and three peptides called NRI and NRII included in the non-repeated part, and 729 R included in the repeated part were described in application WO 92/13884. However, this document does not address the specific properties mentioned above or other LSA-3 fragments that could be either longer or shorter, included or combined with these fragments that would have particular properties of interest for use in vaccines.

This application describes polypeptide molecules containing at least 10 amino acids following the amino acid sequence shown in Figure 2 and designated as SEQ ID No 2, representing LSA-3, excluding the following polypeptides: - the Commission has decided to authorise the use of the new technology in the production of the new products.

Other molecules of interest contain at least 20 consecutive amino acids or at least 50.

All of these polypeptides and the LSA-3 molecule are in all of the following polypeptides of interest as requested .

The invention relates to a conjugate where a C16 or C18 lipid residue is coupled by a lysine bridge to the NRI, NRII, 729RE or CT1 peptides. It also relates to an immunogenic conjugate whose polypeptide sequence is derived from a P.falciparum isolate and has at least 70% homology to that of a conjugate.

Experimental results and non-repeated sequence comparisons between different isolates of P.falciparum indicate at least 70% homology between equivalent antigens of the hepatic stage of the parasite. Therefore, any peptide molecule showing at least 70% homology with any of the above-defined molecules is included in this description and any peptide molecule showing 70% homology with the following sequence: The following are the main objectives of the programme: between amino acids 140-159 of K1 or 23-42 of T9/96.

In the same way, polypeptide molecules with at least 70% homology to the sequence shown in Figure 3 representing part of LSA-3 in T9/96 are described here: the DNA of this P.falciparum isolate was digested by restriction enzymes and then cloned into lambda gt11 and thus made it possible to constitute the genome of this isolate already described above.

Conjugates consisting of a polypeptide derived from LSA-3 covalently bound by a lysine bridge to saturated or unsaturated lipid residues are also part of the invention, particularly when the lipid residue is a palmitoyl or a palmityl or an oleyl. Residues in C16 or C18 have thus been coupled by a lysine bridge to the NRI peptides NRII, 729 RE and CT1 already represented above. The synthesis method used for these conjugates is described in Bourgault, Journal of Immunology, 149, 3416 (1992) and Rouaix, Vaccine, 12, 1209 (1994).

Other immunogenic epitopes including LSA-1, SALSA, STARP have been described in EP A-0407230 and WO 92/13884 Vaccine compositions according to the invention may advantageously contain a mixture of immunogenic peptides derived from LSA-3 and peptides or SALPs derived from LSA-1, SALSA or STARP; a more interesting mixture may be one consisting of whole NRI, NRII or LSA-3, coupled or not with a lipid residue, and one containing lipid-1SASA2 or antigen or SALSA lipid residue.

All polypeptide molecules meeting the above definition and showing at least 70% homology to LSA-3, CT1, NRI, NRII or 729RE polypeptides may be homologously or heterologously combined with other peptide sequences or derived from another antigen of the different stages of P.falciparum.

The central repeating region of LSA-3 (block 2 of Figure 4) has a variable number of repeating sequences responsible for variability from one isolate to another, as shown by the representation in Figure 4 in which the difference in length between the repeated parts of block 2 of the T9/96 and K1 isolates is strikingly clear, although the t-peptides that make up this repeating region (VEES, VEES, VEEE, VAAP, VAAP, etc.) are very well preserved.

Polyclonal or monoclonal antibodies specifically recognizing the polypeptide molecules of the invention are also described.

These molecules of interest may be used for the implementation of diagnostic methods and the manufacture of kits for detecting the presence of P.falciparum infection: this method may be either a specific antibody assay circulating by the implementation of conventional serological methods by contacting one of the above antigens with a biological fluid of the individual in question, or antigen assay methods using polyclonal or monoclonal antibodies obtained by conventional methods of obtaining antibodies by the corresponding antigens. In the kits or diagnostic kits of the invention, the reagents carrying the antigen/antigen complexes, which may be marked or marked with antigen products, may also be detected by a serological test, depending on whether the antigen/antigen complexes are carried or not.

This application also describes all nucleotide sequences coding for a polypeptide of the invention as well as any recombinant nucleic acid containing at least one nucleotide sequence of the invention, inserted into a nucleic acid that is heterologous to that nucleotide sequence.

The sequences of nucleic acids encoding LSA-3 or its immunogenic fragments and meeting one of the following definitions are also described here: (a) the nucleotide sequence as shown in SQ ID No 1 in Figure 1. or (b) the nucleotide sequence as shown in SQ ID No 2 in Figure 2. (c) a sequence having at least 70% homology with that in Figure 1 or Figure 2. or (d) a sequence of nucleotides complementary to those shown in (a), (b) or (c).

The LSA-3 coding gene is both the gene represented in SEQ ID No 1 in Figure 1 and the cDNA represented in SEQ ID No 2 in Figure 2.

In particular, a recombinant nucleic acid is described, in which the nucleotide sequence of interest is preceded by a promoter (including an inducible promoter) under whose control the transcription of the said sequence is likely to be performed and, if applicable, followed by a sequence encoding for termination signals of the transcription.

The coding sequence from the T9/96 clone, represented in Figure 3 by ID QSES No 3, is also described.

In this sequence, the CT1 fragment is between nucleotide 67 and 126, and the 679 fragment is between nucleotide 206 and the 729 ER fragment is between nucleotide 547 and 630.

Finally, any recombinant vector, used in particular for the cloning of a nucleotide sequence of the invention, and/or for the expression of the polypeptide encoded by that sequence, and characterized by the presence of a recombinant nucleic acid, as defined above, at one of its sites not essential for replication, is described.

As an example of the above vector, plasmids, cosmids, phages or viruses are mentioned.

The specific description is pK plasmid 1.2. registered with the CNCM under No 1573.

A process for preparing a polypeptide described in the application by transforming a cell host with a recombinant vector of the above type, followed by culture of the transformed cell host and recovery of the polypeptide from the culture medium is also described.

Thus, any cell host transformed by a recombinant vector as defined above, including the regulatory elements allowing the expression of the nucleotide sequence coding for a polypeptide described in the application, is described.

The application also describes DNA (or RNA) precursors that can be used in the synthesis of nucleotide and/or polypeptide sequences of interest by the Polymerase Chain Reaction (PCR) technique or any other currently known method for amplifying nucleic acids such as CCR, CPR, ERA, SPA, NASBA, etc.

The application describes a DNA or RNA strand, characterised by being composed of approximately 10 to 25 nucleotides, identical or complementary to the first 10 to 25 nucleotides of the nucleotide sequence coding for a peptide sequence of interest according to the application or identical to the 10 to 25 nucleotide units of that sequence.

Thus, this application also describes a process for preparing a polypeptide of interest according to the application including the following steps: where appropriate, the prior amplification by conventional techniques of the quantity of nucleotide sequences coding for that polypeptide using two appropriately selected DNA strands,the culture in an appropriate culture medium of a cell host previously transformed by a vector containing a nucleic acid according to the invention including the nucleotide sequence coding for that polypeptide, and the recovery from the culture medium of the polypeptide product by that transformed cell host.

As an example of the initiators of DNA or RNA according to the invention, the following sequence pairs are given: The following information is provided for the purpose of the calculation of the amount of the allowance: whose respective positions on the LSA-3/K1 gene represented in Figure 1 are 695 to 722 and 829 to 799 (reverse reading), or the pair: The following is a list of the Member States which have adopted the measures referred to in Article 1 of Regulation (EC) No 882/2004 and the measures taken to implement it: The following positions are given in the table below:

Information on ID No 4, ID No 5, ID No 6 and ID No 7 sequences is detailed at the end of the description.

The peptide of interest may also be prepared on demand by the conventional techniques of peptide synthesis, which may be carried out in homogeneous solution or in solid phase. For example, the homogeneous solution synthesis technique described by HOUBENWEYL in the book Meethode der Organischen Chemie (Method of Organic Chemistry edited by E. Wunsch, vol. 15-I and II. THIEME, Stuttgart 1974) or that described by R..D. MERRIFIELD in the article Solid peptide synthesis (J. Am. Chem. Soc., phase 45, 2149-2154) will be used.

The application also describes the water-soluble oligomers of the above monomeric peptides.

Oligomerization can increase the immunogenicity of monomeric peptides as required, although this numerical indication cannot be considered limiting, it should be noted that these oligomers may contain, for example, 2 to 10 monomer units.

Any polymerization technique commonly used in the field of peptides can be used to achieve oligomerisation, which is carried out until an oligomer or polymer containing the number of monomeric motifs required to achieve the desired immunogenicity is obtained.

One method of oligomerisation or polymerisation of the monomer is by reaction of the monomer with a cross-linking agent such as glutaraldehyde.

Other methods of oligomerisation or coupling may also be used, e.g. one involving successive couplings of monomer units via their carboxyl and amine terminal functions in the presence of homo- or hetero-bifunctional coupling agents.

The invention also concerns conjugates obtained by covalent coupling of peptides according to the invention (or oligomeric suffixes) to carrier molecules which allow in particular to argue for immunogenicity (natural or synthetic), physiologically acceptable and non-toxic, by means of complementary reactive groups carried by the carrier molecule and the peptide respectively.

For example, I will mention polylysines or poly ((D-L-alanine) -poly ((L-lysine) as synthetic macromolecular media.

Hydrocarbon or lipid media shall be saturated or unsaturated fatty acids, preferably C16 or C18 oleyl or palmitoleyl.

Finally, but not limited to, antigens or peptides of interest may be coupled to conventional media or adsorbed on such media, including latex or polystyrene microspheres or balls, or incorporated into Ty1 particles as required.

To synthesize the conjugates of the invention, known processes may be used, such as that described by Frantz and Robertson in Infect. and Immunity, 33, 193-198 (1981), or that described in Applied and Environmental Microbiology, (October 1981), vol. 42, no. 4, 611-614 by P.E. Kauffman using the appropriate peptide and carrier molecule.

The nucleic acids described herein may be prepared either by a chemical process or by other processes.

An appropriate method of preparation of the nucleic acids of the invention containing up to 200 nucleotides (or 200 pb in the case of bicatenous nucleic acids) includes the following steps: DNA synthesis using the automated method of β-cyanethylphosphoramidite described in Bioorganic Chemistry 4; 274-325 (1986), cloning of the nucleic acids thus obtained in a suitable vector and recovery of the nucleic acid by hybridization with an appropriate probe.

A method for the chemical preparation of nucleic acids with a length of more than 200 nucleotides has already been described in WO 92/13884.

The application also describes diagnostic kits that contain one or more specific LSA-3 gene amplification initiators and that can detect the presence of the gene or mRNA in an individual likely to be infected with P.falciparum.

The invention also relates to pharmaceutical or vaccine formulations in which at least one of the products of the invention is combined with pharmaceutically acceptable solid or liquid excipients, suitable for the construction of oral, ocular or nasal forms, or excipients suitable for the construction of rectal forms of administration, or with gelatinous excipients for vaginal administration.

The vaccine formulations of the invention contain an additional vehicle, such as polyvinylpyrrolidone, which facilitates the administration of the vaccine.Instead of polyvinylpyrrolidone, any other type of adjuvant in the classical sense of the term can be used, i.e. a substance which facilitates the absorption of a drug or facilitates its action in the body.Examples of other adjuvants of the latter type include carboxymethylcellulose, aluminium hydroxides and phosphates, saboxine or any other adjuvants of this type, which are well known in the art.They contain an immunological adjuvant, in particular if necessary, wallpaper.

The application also describes pharmaceutical formulations containing as active substance at least one of the previously defined polyclonal or monoclonal antibodies in combination with a pharmaceutically acceptable vehicle.

Finally, the application describes a method of immunisation of an individual likely to be infected by P. falciparum by injection of a peptide molecule or oligomer as described above, alone or in combination with other types of molecules which may protect the individual against further infection, the polypeptide or antigenic molecule or the natural or recombinant lipopeptides are either used alone or adsorbed or coupled to latex or polystyrene microspheres or balls.

Further features of the invention will be shown in the illustrated examples of the following figures, which show the particular characteristics of the molecules of interest in the application in relation to other antigens of the pre-erythrocyte stage of the parasite. Figure 1 represents the 6152-base pair genomic DNA ID sequence 1 of the LSA-3 gene, which is derived from the K1.2 clone itself from a Thai isolate.Figure 2 represents the cDNA ID sequence 2 and the LSA-3 antigen polypeptide sequence.The DNA sequence represents 5361 base pairs.Figure 3 represents the ID sequence 3 of the part sequenced in the parasitic clone T9/96 (1890 base pairs),the upper line being the nucleotide sequence and the lower line being the peptide sequence. In this clone, the CT1 sequence is between nucleotides 67 and 126, the DG 679 fragment proper starting at nucleotide 207. The 729 RE fragment is between nucleotides 547 and 629.Figure 4a schematically represents the relative positions of the repeated and non-repeated sequences, with introns and exons in strains K1 and T9/96, the 679 and 729 clones being derived from the latter.Figure 4b represents the HCP (Hydrophobic Cluster Plot) of the peptide sequence of the DG729 clone.Figure 5 shows the amounts of immunoglobulins produced in the Nuria chimp serum before and after immunization with different LSA-3 peptides.Figure 6 shows the specific antibody titer of different immune mouse species with either a peptide or a corresponding lipopeptide.Figure 7 shows the inhibition of the invasion of liver cells by sporozoites by hyperimmune serums obtained after immunization with different immuno-purified peptides of the entire LSA-3.Figure 8 represents the comparison of an LSA-3 antigen with two other antigens on T-type immunity.Figure 9 represents the induction of interferon-interference in Gerdapan and Dirk issus peptides by the LSA-3 molecule.Figure 10 shows the results of lymphoproliferation of PBMCs from an individual protected by an injection of sporosomes irradiated against LSA-1 and LSA-3 antigen peptides.

Example 1: Cloning and sequencing of the LSA-3 gene 1) sequencing

Initial screening of the genome from the T9/96 parasite clone with the serum of a missionary treated continuously for prophylaxis allowed us to isolate 120 clones corresponding to molecules expressed at the sporozoic and/or hepatic stage of the P.falciparum cycle. The 729S clone was used as a probe to screen a genomic bank of the Thai K1 strain already mentioned above which contains large Eco R1 fragments cloned in the lambda phage gt10. A 6.85 kilobase insert containing the entire gene was purified from this serum and recloned into a generated and characterized p18UC plasmid. In P.falciparum, the plasma is often very rare (80%), and when this is the case, the cloning of certain fragments is impossible and the same approach is used to generate and sequence.

The structure of the gene is shown in Figure 4 and has the following characteristics: (a) a mini exon 1 coding at its 3' end for a hydrophobic signal peptide; (b) a short intron (168 base pairs) included between donor and acceptor splicing sites; (c) a second exon of five kilobases coding for an organized 1.8 kilobase region composed of a 7 block arrangement of 4 amino acids, and a hydrophobic region at 3' which could correspond to a glycosyl-phosphatidyl-inositol (GPI) type ink.

A detailed investigation of LSA-3 polymorphism was carried out by sequencing clone 679 which contains the bulk of the repeated sequences of the LSA-3 gene and a 1 kilobase portion of the unrepeated 3' fraction, the sequence of this fragment being represented in Figure 3 between nucleotides 207 and 1890.

The repetitions of the K1 strain are as follows: The following is a list of the species of plants which are to be classified in Annex I to Regulation (EC) No 1829/2003: (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (aa) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (b) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (EC) No 1830/2004; (EC) the species of plants which are to be classified in Annex I to Regulation (EC) No 1830/2004; (EC) are to Regulation (EC) No 1830/2004; (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC) (EC (EC) (EC) (EC) (EC (EC) (EC) (EC) (EC) (EC) (EC (EC) (EC) (EC) (EC) (EC) (EC) (EC (EC) (EC) (EC (EC) (EC) (EC) (EC

The repetitions in clone T9/96 as determined in patent application No FR 9101286 of 5 February 1991 are as follows: The following shall be added to the list of products which are to be considered as originating in the Union:

Exon 2 of the LSA3 gene has 2 repeating regions that can be broken down into 3 blocks as shown in Figure 4: block 1, coding for a sequence of 14 tetrapeptides. This block is 100% conserved in amino acids and nucleic acids between T9/96 and K1. Only the VEES and VEEN tetrapeptides are found in block 2.block 2, in K1, codes for 127 tetrapeptides corresponding to the sequence of different octapeptides, themselves formed by a combination of 2 of the 7 tetrapeptides or basic motifs (VEES, WES, VEEN, VEEI, VAEN, VAPS, VAPT). The number of repetitions and arrangement of these octapeptides vary according to the motifs and appear to be specific to the K1 clone.2.In clone T9/96, block 2 (53 tetrapeptides) also corresponds to the octapeptide sequence formed from the same 7 basic tetrapeptides (and an 8th VVPS pattern not present in K1), but with a different number and arrangement of these repeats.Block 3 consists of the sequence of 10 degenerate tetrapeptides and is different from blocks 1 and 2.This block was sequenced only in the K1 strain.

The non-repeated regions of exon 2 are particularly well preserved, as sequence comparison between T9/96 and K1 was made on 315 bp in 5' of block 1 and 763 bp in 3' of block 2.

Comparison of the sequences of the P.falciparum T9/96 clone 679 with the corresponding LSA-3 sequence from the K1 isolate shows that the gene is well conserved, with the most notable differences being observed in the repeat region where the 4-amino acid blocks are well conserved but vary in number and organization.

On the contrary, the unrepeated 5' and 3' parts appear to be particularly well preserved showing up to 100% homology in the 5' region where B and T epitopes have already been identified.

DNA amplifications, including PCR of different strains of P.falciparum with 8 pairs of amores spread over the entire LSA-3 gene, showed that, except for those around repeated regions, the entire genome yields similar sized PCR products suggesting that the LSA-3 antigen is well conserved.

Several LSA-3 probes, selected from repeated and non-repeated regions, were low-stringence hybridized with DNA from different Plasmodium species and did not identify any homologous genes to LSA-3, except in the chimpanzee parasite P.reichenowi, confirming the close relationship of this species to P.falciparum.

Surprisingly, the LSA-3 analogue antigen found in P. voelii, which clearly gives immunological cross-reactions on the surface of the sporozoite with antibodies against the 729S fragment, does not appear to be conserved at the nucleotide sequence level. Finally, comparison of LSA-3 sequences with databases revealed no homology with known molecules, except for the repeat region which has some patterns that show strong analogy with repeats of a Staphylococcus xylosis gene, but also with two P. falciparum antigens, RESA and Pf11.1, both expressed during the parasite's blood. This homology is mainly due to the richness of both glucose antigen sequences and LSA-3 repeats.

2) Cloning:

The DG729 insert and other regions of the K1 strain exon 2 were cloned into a pGEX prokaryotic expression vector marketed by In Vitrogene Corp (San Diego USA). This vector produces a fusion protein with Schistosoma mansoni glutathione-S-transferase (GST) and allows easy purification of recombinant proteins by glutathione-agarose ball affinity. for the entire LSA-3 protein: REC protein, or for the 729S fragment: 729PGEX.

Attempts to clone other fragments including 3NSREP, 3NFREP, 5NR, and 5SNREP fragments 1-5 have presented difficulties with either cloning or producing and purifying proteins in sufficient quantities for immunization experiments.

Only fragments 729, NN and 3PC produced and purified corresponding recombinant polypeptides in sufficient quantity for the analysis of the antigenicity of the molecule.

Example 2: Protection of immunised chimps against low or high dose test injections:

2.1 A Dirk chimpanzee previously immunized by a fraction of LSA-3 in combination with another antigen of the same stage of parasite development, and exhibiting the effects described above in (a), was re-immunized a few years later with recombinant peptides and proteins corresponding to the same combination of antigens. Again, this chimpanzee was shown to be protected from a low-dose test infection (2,104 sporozoites) and then a high-dose test infection (5106 sporozoites). As with the first challenge, there was a significant reduction in the number of schizons detected in the liver after the high-dose challenge as well as lympho-monocytic infiltrate around detectable local schizons (indication of a strong defence).

2.2. Partial protection of Gerda: another chimp was immunized only by the LSA-3 antigen (animal described in examples 7 and 8 below), namely lipopeptide NR2 and then by the recombinant proteins (GST-729, GST-NN, GST-3PC) which all cover 95% of the LSA-3 molecule and are adsorbed on latex microspheres.

2.3 Partial protection of the Nuria chimp: a chimp immunised by a fraction of the LSA-3 antigen alone, i.e. a combination of peptides, lipopeptides and then recombinant proteins corresponding to 95% of the LSA-3 molecule and emulsified in the ISA-51 Montanide (SEPPIC, 75 Quai d'Orsay, France), was shown to be partially protected against a test infection at medium dose (1,105 sporozoites). This animal showed a significant delay in the appearance of parasites in the blood compared to 4 controls (chimps predicted by the LSA-1, SALSA or STARP antigen-thyrocyte antigen, and 1 unimmunised), achieving a higher maximum blood test result with a lower blood test dose and a faster drop in parasite infection (24 days), which in this case was achieved in 3 animals, with a stronger blood test resulting in a reduction in the number of forms of parasite infection induced by the virus, and in this case in a reduction in the number of parasite infections caused by the virus.

2.4 Immunogenicity B. T in Demi, Karlien and Iris chimps: Three chimps immunised by LSA-3-NRI and -RE peptides and lipopeptides -NRII and -CT1, as well as by peptides corresponding to a different pre-erythrocyte antigen (LSA-1, SALSA or STARP) in each animal, all showed 3: The antibodies recognise not only the peptides and recombinant but are also highly positive on the native molecules of the parasite, as measured by immunofluorescence on the sporozoites and hepatic stages of Plasmosdium falciparum (but negative on the erythrocyte stages); high and specific lymphoproliferative responses against all 4 LSA-3 peptides and the T-epitopes on the surface of the native Plasmodium sporozoites and Plasmodium falciparum yoelii, in which LSA-3 has a homologue (not yet characterised).

B and T responses to native antigens are important because: (b) they mean that at the time of infection there is a good chance of an anamnesic secondary response; this was indeed observed in the chimpanzee Nuria at the time of the challenge.

Immunogenicity in Aotus: a monkey hippopotamus (Aotus trivirgatus) immunised by the 2 LSA-3-NRI and -RE peptides and the 2 lipopeptides -NRII and -CT1 and then restimulated by the recombinant proteins corresponding to 95% of the LSA-3 molecule and adsorbed on microspheres as described above, has high and specific lymphoproliferative responses against the T-epitopes present on these same peptides.

In terms of in vivo response in the various chimpanzees thus pre-immunized, the results highlighted the excellent immunogenicity (B and T) of LSA-3 in peptide, lipopeptide and recombinant forms in all animal models tested so far, i.e. 6/6 outbred, 1/1 aotus, and all immune mice (>20).

Example 3: Identification of the CTL epitope

The method used to identify CTLs is that described in Fidock et al, (1994), J. Immunol. 153: 190 or in Bottius et al, (1996), J. Immunol 156:2874-2884.

CTL (cytotoxic T cell) epitopes have been identified in NRII, RE and CT1 peptides from cytotoxicity tests performed from the PBMCs of the chimpanzees Dirk, Gerda, Nuria, Demi, Karlien and Iris described above.

In humans, 8 additional CTL epitopes, including 7 in the 3' non-repeating region, were identified from the PBMCs of individuals belonging to 3 different haplotypes (MHC class 1-A2, -B8 and -B53) living in endemic region (Gambia) (results unpublished).

In total, we identified 11 CTL epitopes in the LSA-3 molecule, which is considerable. In addition, 5 chimpanzees/6 developed CTL responses to NRII peptide after immunization with NRII lipopeptide without adjuvant, which is a remarkable result for non-in-conjugate animals. On the other hand, since the antibodies developed by Nuria had no inhibitory activity against the invasion of Plasmodium falciparum sporozoites, it can be assumed that the observed protection was dependent on cellular responses, particularly CTLs.

Example 4: Comparison of antibody titres before and after immunisation with different peptides

4.1 Comparison of antibody responses induced by different peptides in different immune animals.

The method used is that described in Behr et al, (1992), J. Immunol 149: 3321.

The reactivity is expressed in the Elisa ratio, i.e. the optical density measured at 496 nanometres of the serum after immunisation compared to the optical density of the same serum before immunisation. The first column indicates the animal immunised, the second column the immunogen received by the animal, the third column indicates the number of injections given and the medium accompanying the injected peptide: RP means recombinant protein, RP/B means recombinant protein adsorbed on latex balls, P means peptide and LP means lipopeptide. tableau I : REACTIVITE ANTICORPS DES DIFFERENTS PEPTIDES EXPRIMEE EN RATIO ELISA

			LSA1				SALSA		STARP		LSA3
Chimpanzés	Immunogène	nbre et type d'injection	LSA-REP	LSA-J	LSA -NR	LSA-TER	SALSA -1	SALSA -2	STARP-M	STARP-R	LSA 3 -CT1	LSA 3 NR1	LSA 3-NR II	LSA 3-REP	R32T et 32
Animaux immunisés
DIRK	LSA3 et	3RP(d)	7.4	9.0	0.9	0.8	nd	nd	0.5	0.7	1.7	1.0	1.1	8.8	0.7
LSA1	3RP + 3 (P + LP)	20.0	10.0	0.1	0.4	0.2	1.1	1.0	0.6	1.0	1.1	3.1	17.0	0.8
GERDA	LSA3	3LP	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	3.9	nd	0.6
3LP + 3 RP/B	nd	nd	nd	nd	nd	nd	nd	nd	0.7	1.1	3.0	12.3	0.9
DEMI	LSA3 et	2 (P+LP)	8.0	14.1	0.7	16.4	0.6	1.1	nd	nd	0.7	1.5	11.7	19.1	0.7
LSA1	3 (P+LP)	8.4	14.5	1.8	21.5	0.8	0.2	nd	nd	0.8	5.1	14.2	20.7	1.2
3 (P+LP)+ 3RP/B
KAR-	LSA3 et	2 (P+LP)	0.5	1.2	1.0	1.0	1.1	2.1	nd	nd	1.0	3.6	3.1	10.3	0.9
LIEN	SALSA	3 (P+LP)	1.1	0.2	0.5	0.2	1.8	2.5	nd	nd	1.4	4.7	6.8	14.1	0.6
3 (P+LP) + 3 RP/B
IRIS	LSA3 et	2 (P+LP)	nd	nd	nd	nd	nd	nd	10.1	15.9	0.7	2.4	6.7	12.5	0.6
STARP	3 (P+LP)	nd	nd	nd	nd	nd	nd	10.5	16.4	1.3	3.1	6.8	15.3	0.5
3 (P+LP) + 3 RP/B

tableau I : REACTIVITE ANTICORPS DES DIFFERENTS PEPTIDES EXPRIMEE EN RATIO ELISA

COR	β.GAL	3 RP	0.6	0.7	0.8	0.9	0.5	1.0	1.2	0.8	1.1	1.0	0.6	1.1	1.2
PEER	β-GAL	8 RP	1.1	0.8	0.7	0.9	0.8	1.2	1.0	0.9	1.1	0.6	0.9	0.9	0.3
BRAM	GST	2 RP	1.1	0.6	0.5	1.1	0.3	0.8	0.9	1.2	1.1	0.3	0.4	0.7	1.0
3 RP	0.8	0.3	0.8	1.3	0.7	1.2	1.1	1.2	1.6	0.2	1.3	0.6	0.4
FOUAD	PBS	0.9	0.5	1.0	0.6	0.8	1.3	1.0	0.3	1.9	1.3	0.3	0.2	0.9

4.2 Title of the antibodies obtained: - What? Table 2 shows serum antibody titres obtained in chimpanzees by immunofluorescence on the surface native antigens of the different stages (sporosoid, hepatic and blood) of P. falciparum, P. yoelii and P. berghei. tableau II : TITRE EN ANTICORPS IMMUNOFLUORESCENTS

			P. falciparum		P. yoelii	(17XL et 17XNL)
CHIMPANZE	Antigène	SS (NF54)	LS (NF54 et 73OXI)	BS (150)	SS	LS	BS
DIRK	LSA3 et LSA1	800	200	-(<100)	200	200	-(<100)
GERDA	LSA3	400	200	-(<100)	400	200	-(<100)
DEMI	LSA3 et LSA1	100	400	-(<100)
KARLIEN	LSA3 et SALSA	100	200	-(<100)
IRIS	LSA3 et STARP	400	100	-(<100)

tableau II : TITRE EN ANTICORPS IMMUNOFLUORESCENTS

COR	β-GAL	-(<100)	-(<100)	-(<100)	-(<100)	-(<100)	-(<100)
BRAM	GST	-(<100)	-(<100)	-(<100)	-(<100)	-(<100)	-(<100)
FOUAD	PBS	-(<100)	-(<100)	-(<100)	-(<100)	-(<100)	-(<100)

4.3 Lymphoproliferative response of the PBMCs of the different chimpanzees after stimulation in vitro by either the different peptides or by the native antigens present on the surface of the sporozoites. tableau III : INCORPORATION DE THYMIDINE TRITIEE DANS DES PBMC APRES STIMULATION PAR LES PEPTIDES LSA-3

Chimpanzé	Immunogène	LSA3-CT1	LSA3-NRI	LSA3-NRII	LSA3-REP	MSP3-C (a)	PPPD(b)
DIRK	LSA3 et LSA1	94 256	27 125	32 455	69 321	796	89 338
(4.0)	(8.5)	(10.7)	(32.3)	(1.0)	(50.3)
GERDA	LSA3	13 359	1 429	13 236	14 883	485	29 355
(25.1)	(2.8)	(25.6)	(28.6)	(0.9)	(132.3)
DEMI	LSA3 et LSA1	30 036	17 221	4178	52 301	689	167 277
(46.8)	(27.4)	(7.3)	(81.2)	(1.1)	(113.3)
KARLIEN	LSA3 et SALSA	30 025	10 039	18 365	31 312	575	96212
(36.4)	(12.8)	(23.1)	(38.0)	(0.7)	(82.3)
IRIS	LSA3 et STARP	53 312	25 223	6 458	35 078	799	196 223
(62.6)	(34.8)	(9.7)	(47.5)	(0.9)	(62.3)

tableau III : INCORPORATION DE THYMIDINE TRITIEE DANS DES PBMC APRES STIMULATION PAR LES PEPTIDES LSA-3

COR	β-GAL	1399	2599	3625	788	2600	19 395
(0.6)	(1.0)	(1.3)	(0.3)	(1.1)	(22.3)
PEER	β-GAL	1225	1 389	3251	2960	3 962	59 399
(0.2)	(0.3)	(1.2)	(0.9)	(1.5)	(22.3)
BRAM	GST	1201	509	2501	2659	2 745	39 399
(0.4)	(0.2)	(0.7)	(0.6)	(0.7)	(22.3)
FOUAD	PBS	1211	1.310	956	688	655	136258
(1.2)	(1.3)	(0.9)	(0.6)	(0.5)	(82.3)

tableau III : INCORPORATION DE THYMIDINE TRITIEE DANS DES PBMC APRES STIMULATION PAR LES PEPTIDES LSA-3

a) Peptide de contrôle à partir de l'antigène MSP3 dans le sang b) PPD = Dérivé de protéine purifié à partir de Mycobacterium turberculosis.

tableau IV: INCORPORATION DE THYMIDINE TRITIEE DANS DES PBMC APRES STIMULATION IN VITRO AVEC DES SPOROZOITES.

		P. falciparum sporozoïtes	P. yoellii sporozoïtes	P. berghei sporozoïtes
Chimpanzé	Antigène

tableau IV: INCORPORATION DE THYMIDINE TRITIEE DANS DES PBMC APRES STIMULATION IN VITRO AVEC DES SPOROZOITES.

DIRK	LSA3 et LSA1	10 402 (12.1)	5 552 (5.6)	2110 (2.0)
GERDA	LSA3	24 021 (20.5)	18 228 (18.6)	2 430 (0.7)
DEMI	LSA3 et LSA1	2111 (3.2)	935 (1.4)	214 (0.1)
KARLIEN	LSA3etSALSA	4 402 (8,5)	2 228 (3.8)	914 (2.1)
IRIS	LSA3etSTARP	9 816 (14.2)	5 304 (8.1)	614 (2.0)

tableau IV: INCORPORATION DE THYMIDINE TRITIEE DANS DES PBMC APRES STIMULATION IN VITRO AVEC DES SPOROZOITES.

BRAM	GST	245 (0.4)	1 295 (1.8)	514 (1.2)
FOUAD	PBS	997 (1.5)	828 (1.8)	714 (1.1)

Lymphoproliferative responses are expressed as difference in beat count per minute (Δ CPM) between the number of beats obtained in the presence of antigen less the number of beats obtained in the absence of antigen.

The results are considered positive when Δ CPM is greater than 1000 and when the stimulation index is greater than 3.

Comparison of antibody responses in chimpanzees Nuria before and after immunization with different peptides.

Figure 5 shows the amounts of immunoglobulins present in the serum of Nuria chimps before and after immunisation with peptides 729 NRI and 729 ER, and lipopeptides 729 NRII, and CT1.

This experiment shows the superiority of the R antigen in terms of B immunity, especially when it is combined with a lipid residue.

Figure 6 shows that the specific antibody level measured by Elisa against peptide 729 NRII in mice immunized with either peptide 729 NRII or lipopeptide 729 NRII is significantly higher when the lipopeptide is used regardless of the mouse species.

Example 5: Lymphoproliferation of PBMCs from a protected individual by injection of sporosomes irradiated against peptides from LSA-1 and LSA-3 antigens.

In eight human volunteers immunized by irradiated sporozoite injection, antibodies against LSA-3 were found in each of the four individuals resistant to a sporozoite infection; none of the other four volunteers developed a blood infection.

In addition, for the only one of these four protected individuals whose cells were accessible, PBMCs were taken six months after the test infection and incubated in the presence of peptides from LSA-1 and LSA-3 antigens.

Figure 10 shows the results of lymphoproliferation of PBMCs from an individual protected by injection of sporosomes irradiated against LSA-1 and LSA-3 antigen peptides.

Significant lymphoproliferations were observed with each of the three LSA-3 peptides (NRI, NRII and RE) but with none of the LSA-1 peptides. There was a particularly high level of IFN-γ secretion (100 Ul/ ml) after stimulation by the NRI peptide and to a lesser extent by the NR2 peptide (IFN-γ: the cytokine with the strongest blocking effect on hepatic schizophrenia).

Example 6: Effects of antibodies against LSA-3 peptides on the inhibition of sporozoite penetration in mice.

The techniques used to prepare primary cultures of hepatocytes, sporozoites, antibodies and the indirect fluorescence test are described in detail by S.MELLOUK et al, Bulletin of the World Health Organization, 68: 52-59, 1990. Table V below compares the results of immunofluorescence obtained either by antibodies against fragment 679 or by antibodies against fragments from other peptides. The left column shows the number of schizophrenia detected after 48h of culture in P.yoelii-infected Balb c mouse hepatocytes and the right column shows the same parameters after P.Berghei infection. Tableau V:

Clones d'anticorps	P.yoelii			P.berghei
	IFA	Nbre de LS à 48 h		IFA	Nbre de LSà48h
		a)	b)
Contrôle	88	110	119	108
679	++	0	-		47
++	0	-		ND
679	++	1	-	105
679b	++	1	-	133
679c	++	1	-	30
32	++	8	±	103
222	+	5	±		26
667	++	276	143	ND	502
362	+	3
493	++	55		ND	508
α P.b. CSP Mab	82	+++		30
α P.y. CSP Mab	+++	171	138

It is clear that the antibody against peptide 679 has a nearly total inhibition effect on the number of those observed at 48h in liver cells. Similarly, Figure 7 shows the inhibition of the invasion of liver cells by sporozoites by hyperhuman serums obtained after immunization with various peptides and immuno purified against whole LSA-3.

As regards mouse protection, the best results were obtained by immunisation with recombinant antigenes, or antigen preparations on demand, adsorbed on latex or polystyrene microspheres of 0.5 μm diameter: 3/3 of the mice were protected from administration of 10 times the minimum infectious dose3/3 of the mice were protected from the second challenge2/3 of the mice were protected from the third challenge.

The microspheres used are Polybead® Polystyrene Microspheres (Polysciences, Inc.) of 0.50 μm diameter (refer.07307) on which the recombinant or peptides are passively adsorbed. In practice, in mice, 50 μg of antigen is in contact with 50 μl of microbubbles for 1 injection; the exact amount of antigen adsorbed is not determined. In the chimpanzee, the same procedure is performed with 200 μg of antigen and 200 μl of beads.

In addition, recently, the immunisation of mice with the recombinant GST-3PC (corresponding to the 3' unrepeated region of amino acid 869 at the stop codon at 3') has resulted in serums that react very strongly with immunofluorescence on the sporozoites of Plasmodium falciparum.

Example 7: cytotoxicity test against peptide 729 NRII in the Gerda chimp.

The chimp Gerda was immunized intravenously with lipopeptide 729NRII from the LSA-3 antigen. Blood was collected 9 days after the 4th injection. The PBMCs were incubated in vitro with 5μg/ml of the 729NRII peptide (10 U/ml of recombinant IL2 added on day 3). On day 15, cytotoxicity was studied against autologous PHA-generated blasts at 0.5μg/ml. The blasts were pre-incubated at night with 5g/ml of the 729NRII peptide, and with a RESA-control peptide, or μ peptide. The peptides were not added during the test (8 hours). The number of targets per 5000 wells is:

Gerda PBMCs incubated over the same period with 5μg/ml of a control peptide or 729NRI peptide (from the same antigen) do not result in the lysis of autologous blasts pre-incubated or not with the above peptides.

Figure 8 shows the results obtained for an E/C ratio (effector to target) ranging from 12 to 0.03. It is shown that target cells presensitized by the 729NRII peptide are lysed in the presence of effector cells, indicating a specific cytotoxic T-type immune response of this antigen.

The lipopeptide NRII, when injected intravenously, is capable of inducing a specific cytotoxic response without adjuvant.

Example 8: effect of the NRI peptide on interferon γ production.

Interferons have been shown to have inhibitory activity in the development of P.falciparum in cultured human hepatocytes (Sylvie Mellouk et al, The Journal of Immunology, vol. 139 No. 12:41-92, 41-95, 1987). The results obtained with the peptides of the invention are as follows:

Chimpanzee Gerda, immunized by NRII polypeptide and boosted by recombinant DG729, carries PBMCs capable of secreting high levels of IFN-γ in the presence of LSA-3 peptides, particularly peptide 729 NR1. The result was confirmed in chimpanzee Dirk, immunized by the same protein.

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Claims (11)

1. Conjugate, characterised in that a C16 or C18 lipid residue is coupled by a lysine bridge to a peptide of Plasmodium falciparum selected from among NRI, NRII, 729RE or CT1.
2. Conjugate according to claim 1, characterised in that the lipid residue is saturated or unsaturated.
3. Conjugate according to claim 1 or 2, characterised in that the lipid residue is a palmitoyl or a palmityl or an oleyl.
4. Immunogenic conjugate the polypeptide sequence of which originates from an isolate of P. falciparum and has at least 70% homology with that of a conjugate according to one of claims 1 to 3.
5. Immunogenic composition, characterised in that it contains at least one conjugate according to any one of claims 1 to 4 and at least one pharmaceutical vehicle.
6. Immunogenic composition according to claim 5, characterised in that the pharmaceutical vehicle is polyvinylpyrrolidone.
7. Immunogenic composition according to claim 5, characterised in that the composition contains an adjuvant.
8. Immunogenic composition according to claim 5, characterised in that the adjuvant is carboxymethylcellulose, a hydroxide or a phosphate of aluminium, saponine or a muramylpeptide.
9. Anti-malarial vaccine composition, containing among other immunogenic principles a conjugate according to one of claims 1 to 4.
10. Method of in vitro diagnosis of malaria in an individual likely to be infected with P. falciparum, which comprises bringing a tissue or biological fluid taken from an individual into contact with a conjugate according to one of claims 1 to 4, under conditions enabling an immunological reaction between said conjugate and any antibodies present in the tissue or the biological fluid, and detecting in vitro any antigen/antibody complexes that may be formed.
11. Use of a conjugate according to one of claims 1 to 4 in the preparation of an anti-malarial vaccine.