WO2004037856A2 - Facteur de virulence du var o du plasmodium falciparum - Google Patents

Facteur de virulence du var o du plasmodium falciparum Download PDF

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WO2004037856A2
WO2004037856A2 PCT/EP2003/013341 EP0313341W WO2004037856A2 WO 2004037856 A2 WO2004037856 A2 WO 2004037856A2 EP 0313341 W EP0313341 W EP 0313341W WO 2004037856 A2 WO2004037856 A2 WO 2004037856A2
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seq
antibody
polynucleotide
isolated
polypeptide
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WO2004037856A3 (fr
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Odile Puijalon
Cécile LE SCANF
Anne Lavergne
Graham Bentley
Cyril Badaut
Sébastien IGONET
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Institut Pasteur
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Publication of WO2004037856A3 publication Critical patent/WO2004037856A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to Plasmodium parasite virulence factors involved in the pathogenesis of malarial infections. More particularly, this invention concerns novel peptide, polypeptide and vaccine compositions for the diagnosis, treatment and prevention of malaria.
  • parasite density is a critical component of clinical malaria. In children living in endemic areas acute malaria is defined as febrile episodes with possibly additional symptoms associated with a parasite density above a certain threshold (Rogier C. et al. (1996). Am. J. Trap. Med. Hyg. 54, 613-619). In severe cases, parasite density is higher than in matched mild cases (Robert F. et al. (1996). Trans. R. Soc. Trap. Med. Hyg. 90, 704-711 ; Pain A. et al. (2001 ) Proc. Natl Acad. Sci. U.S.A. 98 (4), 1805-1810; Heddini A. et al. (2001 ) Infect.
  • Such a sequestration is not restricted to specific organs, since it involves the physical size of the cellular agglutinate rather than local binding of the infected red blood cells (IRBC) to the endothelial lining through specific ligand / receptor interactions.
  • IRBC infected red blood cells
  • targetting (preventing or reverting) rosetting/auto- agglutination should have broader consequences than preventing homing to certain territories by targetting the specific ligand.
  • Adhesive phenotypes can only be evidenced and hence studied after in vitro maturation until pigmented stage. Not all parasites mature to that stage in vitro (Heddini A. et al. (2001 ) Infect. Immun. 69, 5849-5856; Carlson J. et al. 1990. The Lancet 336, 1457-1460; Roberts D.J. et al. (2000). The Lancet 355, 1427-1428. The association of certain adhesive phenotypes with severity is therefore based upon only a fraction of the circulating parasite pool.
  • cytoadherence in malaria parasites have been recently clarified.
  • the cytoadherence phenotype acquired by mature Plasmodium falciparum- ' mfected erythrocytes is mediated by variant PfEMPI adhesins exposed onto the surface of infected RBC from the trophozoite stage on.
  • PfEMPI adhesins are encoded by a repertoire of approximately 50 var genes (Smith et al., (2000) Mol. Biochem. Parasitol., 110, 293-310; Smith et al., (2001 ) Trends Parasitol., 17 (11 ) 538-545).
  • Exons 1 and 2 codes for a relatively well conserved domain implicated in interaction with the erythrocyte cytosqueleton.
  • Exon 1 codes for the variable extra-cellular region of the molecule and has a modular organisation with Duffy Binding Like domains (known as "DBL"), Cysteine Rich Interdomain Regions (known as "CIDR”) and C2 domains. Based on sequence homology, the DBL domains are grouped into five distinct classes (alpha to epsilon) and the CIDR into three classes (alpha to gamma).
  • DBL Duffy Binding Like domains
  • CIDR Cysteine Rich Interdomain Regions
  • the prototypical PfEMPI extracellular region consists in a NTS (a globular N terminal segment) followed by a duplicated arrangement of the DBL-CIDR tandem.
  • the first tandem is almost invariably DBL1alpha-CIDR1 alpha and the second is generally DBL2delta-CIDR2beta.
  • the number of DBL domains varies from 2-7 and the number of CIDR varies from 1-2.
  • DBLbeta is invariably associated with C2.
  • var genes associated with rosetting have been described to date.
  • the var gene 2182041 (Y13402 in Genbank) has been associated with the rosetting phenotype of the R29 clone in the strain It (which de facto is FCR3) (Rowe A. et al. (1997). Nature. 388, 292-295).
  • the gene has 4 DBL domains and 1 CIDR (see figure 2).
  • the first DBL1-CIDR1 association is atypical it consists in DBL1alpha-CIDR1 gamma
  • the rosetting receptor on the erythrocyte surface has been identified. It is Complement Receptor 1 (CR1 ).
  • CR1 Complement Receptor 1
  • DBL1 alpha The domain responsible for rosetting is DBL1 alpha.
  • DBL1 alpha The domain responsible for rosetting is DBL1 alpha.
  • DBL1 alpha the single domain binding erythrocytes.
  • var gene 2961468 also called FCR3S1.2 from the
  • FCR3 strain (Chen Q. et al. (1998). J. Exp. Med. 187, 15-23). This gene has been identified as mediating rosetting by single-cell PCR of micro-manipulated rosette forming cells. It is a very short gene, which contains two DBL domains (1 alpha & 2delta ) and two CIDR (alpha and beta) (see figure 2). This gene codes for a multi- adhesive protein.
  • the DBL1 alpha GST binds heparin-Sepharose, glycosaminoglycans and binds erythrocytes.
  • the var gene 15991381 Flick et al. (2001 ) Science 293, 2098-2100 (AF366567 in Genbank) derived from clone TM284S2 which forms giant rosettes, mixed rosettes and autoagglutinates.
  • the var gene 15991381 codes for a PfEMPI adhesin which binds IgG and through this IgG bridge, binds to the placenta IgG receptor. It has four DBL1 domains and two CIDRs (see figure 2). The six extra-cellular domains were expressed as GST-fusion proteins. This showed that DBL2beta is the IgG binding domain.
  • the present invention concerns the characterization of a var gene, more particularly, the varO gene.
  • the present invention also concerns at least one polypeptide encoded by the varO gene and expressed in Plasmodium species.
  • an object of the present invention is to provide an isolated or purified polynucleotide having a nucleic acid sequence being at least 65% identical to any one of SEQ ID NO 1 , SEQ ID NO.13 to SEQ ID NO.21 and fragments thereof.
  • Another object of the present invention is to provide an isolated or purified polypeptide comprising an amino acid sequence encoded by the polynucleotide sequence as defined above and/or biologically active fragments thereof.
  • a further object of the present invention is to provide an isolated or purified polypeptide having at least 80% sequence identity with amino acid sequence of SEQ ID NO 2.
  • Another object of the invention is to provide an isolated or purified oligonucleotide which can be used as a primer for hybridization with a polynucleotide of the invention.
  • Yet another object of the present invention is to provide an isolated or purified polypeptide comprising an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complement of the polynucleotide of the present invention and having the ability to induce cytoadherence in cells infected Plasmodium related species.
  • Still another object of the present invention is to provide a cloning or expression vector comprising a polynucleotide sequence having SEQ ID NO. 1 or 13 to 21.
  • the present invention is directed to a plasmid comprising at least one var O gene fragment selected from the group consisting of the plasmids deposited under number CNCM I-2929, CNCM I-2930, CNCM I- 3096, CNCM I-3097 and CNCM I-3098.
  • Another object of the present invention is to provide a recombinant Escherichia coli cell selected from the group consisting of the cells deposited under number CNCM 1-2929, CNCM 1-2930, CNCM 1-3096, CNCM 1-3097 and CNCM 1-3098.
  • Yet another object of the present invention is to provide an antibody that specifically binds to the isolated or purified polypeptide of the instant invention.
  • Still another object of the present invention is to provide a composition
  • a composition comprising the isolated or purified polynucleotide and/or the isolated or purified polypeptide of the instant invention; and/or the antibody specific to the polypeptide encoded by the polynucleotide of the invention or biologically active fragments thereof, and an acceptable carrier.
  • Another object of the present invention is to provide a vaccine comprising the isolated or purified polynucleotide and/or the isolated or purified polypeptide and/or the antibody of the instant invention, and an acceptable carrier.
  • Yet another object of the present invention is to provide a method for treating and/or preventing a Plasmodium species related disease, for example malaria, in a mammal, comprising the step of administering to the mammal an effective amount of : - the isolated or purified polynucleotide, polypeptide, antibody, composition and/or vaccine of the instant invention.
  • Still another object of the present invention is to provide an in vitro diagnostic method for the detection of the presence or absence of antibodies indicative of Plasmodium species, which bind with the polypeptide of the present invention to form an immune complex, comprising the steps of a) contacting the polypeptide of the invention with a biological sample for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).
  • the present invention also provides a diagnostic kit for the detection of the presence or absence of antibodies indicative of Plasmodium species, comprising: a polypeptide of the invention; a reagent to detect polypeptide-antibody immune complex; a biological reference sample lacking antibodies that immunologically bind with said polypeptide; and a comparison sample comprising antibodies which can specifically bind to said polypeptide; wherein said polypeptide, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.
  • Still another object of the present invention is to provide an in vitro diagnostic method for the detection of the presence or absence of polypeptides indicative of Plasmodium species, which bind with the antibody of the present invention to form an immune complex, comprising the steps of: a) contacting the antibody of the invention with a biological sample for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).
  • Still another object of the present invention is to provide an in vitro diagnostic method for the detection of the presence or absence of a polynucleotide indicative of Plasmodium species, comprising the steps of: a) contacting at least one oligonucleotide of the invention with a biological sample for a time and under conditions sufficient for said oligonucleotide to hybridize to said polynucleotide; and b) detecting the presence or absence of an hybridization between said oligonucleotide and polynucleotide.
  • Still another object of the present invention is to provide a diagnostic kit for the detection of the presence or absence of polypeptide indicative of Plasmodium species, comprising: - an antibody of the present invention; a reagent to detect polypeptide-antibody immune complex; a biological reference sample lacking polypeptides that immunologically bind with said antibody; and a comparison sample comprising polypeptides which can specifically bind to said antibody; wherein said antibody, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.
  • Yet another object of the present invention is to provide a diagnostic kit for the detection of the presence or absence of polynucleotide indicative of Plasmodium species, comprising: an oligonucleotide of the present invention; a reagent to detect polynucleotide-oligonucleotide hybridization complex; - a biological reference sample lacking polynucleotides that hybridise with said oligonucleotide; and a comparison sample comprising polynucleotides which can specifically hybridise to said oligonucleotide; wherein said oligonucleotide, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.
  • Figure 1 is a schematic representation of the structure of exon 1 of the var O gene and the sub-cloned domains expressed as recombinant proteins.
  • Figure 2 is a schematic representation of the Palo Alto var O deduced protein domains and scores of homology with other var protein domains.
  • Figure 3 is the nucleic acid sequence and the deduced amino acid sequence of the var O gene and identified as SEQ ID NO. 1.
  • Figure 4 is the amino acid sequence of the polypeptide encoded by the var O gene and identified as SEQ ID NO. 2.
  • Figure 5 is the amino acid sequence of the DBL 1 alpha domain encoded by the var O gene and identified as SEQ ID NO. 3.
  • Figure 6 is the amino acid sequence of the DBL 2 beta domain encoded by the var O gene and identified as SEQ ID NO. 4.
  • Figure 7 is the amino acid sequence of the DBL 3 gamma domain encoded by the var O gene and identified as SEQ ID NO. 5.
  • Figure 8 is the amino acid sequence of the DBL 4 epsilon domain encoded by the var O gene and identified as SEQ ID NO. 6.
  • Figure 9 is the amino acid sequence of the DBL 5 epsilon domain encoded by the varO gene and identified as SEQ ID NO.7.
  • Figure 10 is the amino acid sequence of the CIRD gamma domain encoded by the varO gene and identified as SEQ ID NO. 8.
  • Figure 11 is the amino acid sequence of the C2 domain encoded by the var O gene and identified as SEQ ID NO. 9.
  • Figure 12 is the amino acid sequence of the ID 3 domain encoded by the var O gene and identified as SEQ ID NO.10.
  • Figure 13 is the amino acid sequence of the ID 4 domain encoded by the var O gene and identified as SEQ ID NO. 11.
  • Figure 14 is the amino acid sequence of the transmembrane segment encoded by the var O gene and identified as SEQ ID NO. 12.
  • Figure 15 is the nucleic acid sequence of the DBL 1 alpha domain of the var O gene identified as SEQ ID NO. 13.
  • Figure 16 is the nucleic acid sequence of the DBL 2 beta domain of the var O gene identified as SEQ ID NO. 14.
  • Figure 17 is the nucleic acid sequence of the C2 domain of the var O gene identified as SEQ ID NO. 19.
  • Figure 18 is the nucleic acid sequence of the DBL 3 gamma domain of the var O gene identified as SEQ ID NO. 15.
  • Figure 19 is the nucleic acid sequence of the ID 3 inter-domain of the var O gene identified as SEQ ID NO. 20.
  • Figure 20 is the nucleic acid sequence of the DBL 4 epsilon domain of the var O gene identified as SEQ ID NO. 16.
  • Figure 21 is the nucleic acid sequence of the ID 4 inter-domain of the Mar O gene identified as SEQ ID NO. 21.
  • Figure 22 is the nucleic acid sequence of the DBL 5 epsilon domain of the var O gene identified as SEQ ID NO.17.
  • Figure 23 is the nucleic acid sequence of the DBL1 alpha CIRD gamma tandem domain of the var O gene identified as SEQ ID NO. 18.
  • Figure 24 shows the expression of the polypeptide varO DBL1 alpha in E.coli from the pET22-clone G plasmid according to a preferred embodiment of the invention.
  • Figure 25 shows the purification result of the varO DBL1 alpha polypeptide shown in Fig. 24.
  • Figure 26 shows the recognition of the varO C2 domain by agglutinating Saimiri sera.
  • Figures 27 A and B show Saimiri Red Blood cell binding to the surface of COS7L cells transiently transfected with recombinant pDisplay plasmids according to the invention that express the CIDR (Fig.27A), or the DBL1 var O domain (Fig. 27B).
  • Figure 27C shows absence of binding on COS7L cells transiently transfected with the pDisplay vector.
  • Figures 28, 29 and 30 show purification results at different stages of the recombinant soluble polypeptide CIDR1 v according to a preferred embodiment of the invention.
  • the present invention is directed to a polynucleotide encoding a Plasmodium virulence factor and its use in the preparation of compositions and vaccines. More specifically, the present invention is concerned with compositions, vaccines and methods for providing an immune response and/or a protective immunity to mammals against a Plasmodium species as well as oligonucleotides and methods for the diagnosis of Plasmodium infection.
  • compositions, vaccines and methods of the present invention will be useful against disorders caused by P. falciparum.
  • immune response refers to the T cell response or the increased serum levels of antibodies to an antigen, or presence of neutralizing antibodies to an antigen, such as a Plasmodium falciparum virulence factor, for instance, a var peptide.
  • the term "immune response” is to be understood as including a humoral response, a cellular response and an inflammatory response.
  • protection refers herein to the ability of the serum antibodies and cellular response induced during immunization to protect
  • compositions or vaccines of the invention will experience limited growth and spread of an infectious P. falciparum.
  • protection also means cure of an ongoing infection for instance by administration of a component reducing parasite density by disrupting cellular interaction of the parasite with host cells or autoagglutination.
  • the term "mammal” refers to any mammal that is susceptible to be infected by a Plasmodium species causing malaria.
  • mammals which are known to be potentially infected by a P. species, there are humans, apes, birds, and bovines.
  • the present invention concerns an isolated or purified polynucleotide encoding a P. falciparum virulence factor, namely the var O protein. Therefore, the polynucleotide of the invention has a nucleic acid sequence which is at least 65% identical, more particularly 80 % identical and even more particularly 95% identical to any one of SEQ ID NO 1 , 13 to 21 as shown in figures 3, 12 to 23.
  • the terms "Isolated or Purified” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a protein/peptide naturally present in a living organism is neither “isolated” nor purified, the same polynucleotide separated from the coexisting materials of its natural state, obtained by cloning, amplification and/or chemical synthesis is “isolated” as the term is employed herein.
  • a polynucleotide or a protein/peptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism.
  • Amino acid or nucleotide sequence “identity” and “similarity” are determined from an optimal global alignment between the two sequences being compared. An optimal global alignment is achieved using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453). "Identity” means that an amino acid or nucleotide at a particular position in a first polypeptide or polynucleotide is identical to a corresponding amino acid or nucleotide in a second polypeptide or polynucleotide that is in an optimal global alignment with the first polypeptide or polynucleotide . In contrast to identity, "similarity" encompasses amino acids that are conservative substitutions.
  • a “conservative” substitution is any substitution that has a positive score in the bIosum62 substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919).
  • sequence A is n% similar to sequence B
  • sequence B is meant that n% of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides and conservative substitutions.
  • sequence A is n% identical to sequence B is meant that n% of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides.
  • polynucleotide(s) generally refers to any polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • This definition includes, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. "Polynucleotide(s)" embraces short polynucleotides or fragments often referred to as oligonucleotide(s).
  • polynucleotide(s) as it is employed herein thus embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells which exhibits the same biological function as the polypeptide encoded by SEQ ID NO.1.
  • polynucleotide(s) also embraces short nucleotides or fragments, often referred to as “oligonucleotides”, that due to mutagenesis are not 100% identical but nevertheless code for the same amino acid sequence.
  • the present invention concerns an isolated or purified polypeptide comprising an amino acid sequence encoded by a polynucleotide as defined previously.
  • the polypeptide of the present invention preferably has an amino sequence having at least 80% homology, or even preferably 85% homology to part or all of SEQ ID NO:2 as shown in figure 4. Yet, more preferably, the polypeptide comprises an amino acid sequence substantially the same or having 100% identity with SEQ ID NO:2.
  • the polypeptide of the present invention comprises at least one amino acid sequence selected from the group consisting of amino acid sequence having SEQ ID NO. 3 (figure 5), SEQ ID NO.4 (figure 6), SEQ ID NO. 5 (figure 7), SEQ ID NO.6 (figure 8), SEQ ID NO.7 (figure 9), SEQ ID NO.8 (figure 10), SEQ ID NO.9 (figure 11), SEQ ID NO.10 (figure 12), SEQ ID NO.11 (figure 13), SEQ ID NO.12 (figure 14), and biologically active fragments thereof.
  • biological active refers to a polypeptide or fragment(s) thereof that substantially retain the capacity of forming var O-receptor complex.
  • the isolated and purified polypeptide of the present invention comprises an amirio acid sequence encoded by a nucleic acid which hybridizes under stringent conditions to the complement of SEQ ID NO 1 or fragments thereof.
  • a polypeptide has the ability to induce cytoadherence in cells infected with Plasmodium related species.
  • to hybridize under conditions of a specified stringency describes the stability of hybrids formed between two single-stranded DNA fragments and refers to the conditions of ionic strength and temperature at which such hybrids are washed, following annealing under conditions of stringency less than or equal to that of the washing step.
  • high, medium and low stringency encompass the following conditions or equivalent conditions thereto :
  • high stringency 0. 1 x SSPE or SSC, 0. 1 % SDS, 65° C
  • medium stringency 0. 2 x SSPE or SSC, 0. 1 % SDS, 50° C
  • polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins.
  • a peptide according to the invention preferably comprises from 2 to 20 amino acids, more preferably from 2 to 10 amino acids, and most preferably from 2 to 5 amino acids. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post- translational modifications, but also by chemical modification techniques.
  • Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, selenoylation, sulfation and transfer-RNA mediated addition of amino acids to proteins
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • the present invention concerns also the fragments of said polypeptide containing between 2 to 20 amino acids.
  • the fragment may further be a molecule (natural or synthetic) that inhibits the interaction of var O protein with its receptor.
  • the fragment may be an analog, an antibody or a molecule specifically designed to bind the active site of var O protein (site of interaction of var O protein with its receptor).
  • the invention is also directed to a host, such as a genetically modified cell, comprising any of the polynucleotide sequence according to the invention and more preferably, a host capable of expressing the polypeptide encoded by this polynucleotide.
  • the host cell may be any type of cell (a transieritly-transfected mammalian cell line, an isolated primary cell, or insect cell, yeast (Saccharomyces cerevisiae, Ktuyveromyces lactis, Pichia pastoris), plant cell, microorganism, or a bacterium (such as E. coli). More preferably the host is Escherichia coli bacterium.
  • IMP 537, IMP 538, IMP 539, IMP 540 and IMP 541 relating to Escherichia coli comprising respectively an expression vector encoding for DBL1-CIDR1 domains, DBL1-DBL5 domains, DBL1 domain, CIDR1 domain and DBL1- CIDR1 domains were registered at the Collection Nationale des Cultures de Microorganismes (CNCM) under accession numbers I-2929 (on August 30, 2002), I-2930 (on August 30, 2002), 1-3096 (on October 3, 2003), I-3097 (on October 3, 2003) and I-3098 (on October 3, 2003) respectively.
  • the invention is further directed to cloning or expression vector comprising a polynucleotide sequence as defined above, and more particularly directed to a cloning or expression vector which is capable of directing expression of the polypeptide encoded by the polynucleotide sequence in a vector-containing cell.
  • vector refers to a polynucleotide construct designed for transduction/transfection of one or more cell types.
  • Vectors may be, for example, "cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors” which are designed for expression of a nucleotide sequence in a host cell, or a “viral vector” which is designed to result in the production of a recombinant virus or virus-like particle, or "shuttle vectors", which comprise the attributes of more than one type of vector.
  • cloning vectors which are designed for isolation, propagation and replication of inserted nucleotides
  • expression vectors which are designed for expression of a nucleotide sequence in a host cell
  • viral vector which is designed to result in the production of a recombinant virus or virus-like particle
  • shuttle vectors which comprise the attributes of more than one type of vector.
  • a number of vectors suitable for stable transfection of cells and bacteria are available to the public (e.g.
  • plasmids adenoviruses, baculoviruses, yeast baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses), as are methods for constructing such cell lines. It will be understood that the present invention encompasses any type of vector comprising any of the polynucleotide molecule of the invention.
  • the invention features purified antibodies that specifically bind to the isolated or purified polypeptide as defined above or fragments thereof, and more particularly to a protein encoded by the P. falciparum var O gene.
  • the antibodies of the invention may be prepared by a variety of methods using the var O protein or polypeptides described above.
  • the var O polypeptide, or antigenic fragments thereof may be administered to an animal in order to induce the production of polyclonal antibodies.
  • antibodies used as described herein may be monoclonal antibodies, which are prepared using hybridoma technology (see, e.g., Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, 1981 ).
  • the present invention is preferably directed to antibodies that specifically bind P. falciparum var O polypeptides, or fragments thereof.
  • the invention features "neutralizing” antibodies.
  • neutralizing antibodies is meant antibodies that interfere with any of the biological activities of any of the P. falciparum var O polypeptides, particularly the ability of P. falciparum to induce the rosetting/autoagglutination cytoadherence phenotype of infected and non-infected red blood cells. Any standard assay known to one skilled in the art may be used to assess potentially neutralizing antibodies.
  • monoclonal and polyclonal antibodies are preferably tested for specific var O proteins recognition by Western blot, immunoprecipitation analysis or any other suitable method.
  • Antibodies that recognize var O expressing cells and antibodies that specifically recognize varO proteins (or fragments varO), such as those described herein, are considered useful to the invention.
  • Such an antibody may be used in any standard immunodetection method for the detection, quantification, and purification of varO proteins.
  • the antibody may be a monoclonal or a polyclonal antibody and may be modified for diagnostic purposes.
  • the antibodies of the invention may, for example, be used in an immunoassay to monitor varO expression levels, to determine the amount of varO or fragment thereof in a biological sample and evaluate the presence or not of a varO strain of Plasmodium.
  • the antibodies may be coupled to compounds for diagnostic and/or therapeutic uses such as gold particles, alkaline phosphatase, peroxidase for imaging and therapy.
  • the antibodies may also be labeled (e.g. immunofluorescence) for easier detection.
  • the term "specifically binds to” refers to antibodies that bind with a relatively high affinity to one or more epitopes of a protein of interest, but which do not substantially recognize and bind molecules other than the one(s) of interest.
  • the term “relatively high affinity” means a binding affinity between the antibody and the protein of interest of at least 10 6 M “1 , and preferably of at least about 10 7 M “1 and even more preferably 10 8 M “1 to 10 10 M “1 . Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which is common knowledge to one skilled in the art.
  • antibody and “antibodies” include all of the possibilities mentioned hereinafter: antibodies or fragments thereof obtained by purification, proteolytic treatment or by genetic engineering, artificial constructs comprising antibodies or fragments thereof and artificial constructs designed to mimic the binding of antibodies or fragments thereof. Such antibodies are discussed in Colcher et al.
  • F(ab') 2 fragments include complete antibodies, F(ab') 2 fragments, Fab fragments, Fv fragments, scFv fragments, other fragments, CDR peptides and mimetics. These can easily be obtained and prepared by those skilled in the art. For example, enzyme digestion can be used to obtain F(ab') 2 and Fab fragments by subjecting an IgG molecule to pepsin or papain cleavage respectively. Recombinant antibodies are also covered by the present invention.
  • the antibody of the invention may be an antibody derivative.
  • Such an antibody may comprise an antigen-binding region linked or not to a non- immunoglobulin region.
  • the antigen binding region is an antibody light chain variable domain or heavy chain variable domain.
  • the antibody comprises both light and heavy chain variable domains, that can be inserted in constructs such as single chain Fv (scFv) fragments, disulfide-stabilized Fv (dsFv) fragments, multimeric scFv fragments, diabodies, minibodies or other related forms (Colcher et al. Q J Nucl Med 1998; 42: 225-241 ).
  • Such a derivatized antibody may sometimes be preferable since it is devoid of the Fc portion of the natural antibody that can bind to several effectors of the immune system and elicit an immune response when administered to a human or an animal. Indeed, derivatized antibody normally do not lead to immuno-complex disease and complement activation (type III hypersensitivity reaction).
  • a non-immunoglobulin region is fused to the antigen-binding region of the antibody of the invention.
  • the non-immunoglobulin region is typically a non-immunoglobulin moiety and may be an enzyme, a region derived from a protein having known binding specificity, a region derived from a protein toxin or indeed from any protein expressed by a gene, or a chemical entity showing inhibitory or blocking activity(ies) against the Plasmodium virulence- associated polypeptide.
  • the two regions of that modified antibody may be connected via a cleavable or a permanent linker sequence.
  • the antibody of the invention is a human or animal immunoglobulin such as lgG1 , lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD carrying rat or mouse variable regions (chimeric) or CDRs (humanized or "animalized”).
  • the antibody of the invention may also be conjugated to any suitable carrier known to one skilled in the art in order to provide, for instance, a specific delivery and prolonged retention of the antibody, either in a targeted local area or for a systemic application.
  • humanized antibody refers to an antibody derived from a non- human antibody, typically murine, that retains or substantially retains the antigen- binding properties of the parent antibody but which is less immunogenic in humans. This may be achieved by various methods including (a) grafting only the non-human CDRs onto human framework and constant regions with or without retention of critical framework residues, or (b) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Such methods are well known to one skilled in the art.
  • the antibody of the invention is immunologically specific to the polypeptide of the present invention and immunological derivatives thereof.
  • immunological derivative refers to a polypeptide that possesses an immunological activity that is substantially similar to the immunological activity of the whole polypeptide, and such immunological activity refers to the capacity of stimulating the production of antibodies immunologically specific to the Plasmodium virulence-associated protein or derivative thereof.
  • immunological derivative therefore encompass "fragments", “segments", “variants”, or “analogs" of a polypeptide.
  • polypeptides of the present invention may be used in many ways for the diagnosis, the treatment or the prevention of Plasmodium related diseases and in particular malaria.
  • the present invention relates to a composition for eliciting an immune response or a protective immunity against a P. species.
  • the present invention relates to a vaccine for preventing and/or treating a Plasmodium associated malarial disease.
  • the term “treating” refers to a process by which the symptoms of malaria are alleviated or completely eliminated.
  • the term “preventing” refers to a process by which a Plasmodium associated malarial disease is obstructed or delayed.
  • the composition or the vaccine of the invention comprises a polynucleotide, a polypeptide and/or an antibody as defined above and an acceptable carrier.
  • an acceptable carrier means a vehicle for containing the polynucleotide, a polypeptide and/or an antibody that can be injected into a mammalian host without adverse effects.
  • Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions.
  • Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i. e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
  • compositions of the invention may also comprise agents such as drugs, immunostimulants (such as ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator factor (M-CSF), interleukin 2 (IL2), interleukin 12 (IL12), and CpG oligonucleotides), antioxidants, surfactants, flavoring agents, volatile oils, buffering agents, dispersants, propellants, and preservatives.
  • immunostimulants such as ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator factor (M-CSF), interleukin 2 (IL2), interleukin 12 (IL12), and CpG oligonucleotides
  • antioxidants such as antioxidants, surfactants, flavoring agents, volatile oils, buffering agents, dispersants
  • the amount of polynucleotide, a polypeptide and/or an antibody present in the compositions or in the vaccines of the present invention is preferably a therapeutically effective amount.
  • a therapeutically effective amount of polynucleotide, a polypeptide and/or an antibody is that amount necessary to allow the same to perform their immunological role without causing, overly negative effects in the host to which the composition is administered.
  • the exact amount of polynucleotide, a polypeptide and/or an antibody to be used and the composition/vaccine to be administered will vary according to factors such as the type of condition being treated, the mode of administration, as well as the other ingredients in the composition.
  • the present invention relates to methods for treating and/or preventing Plasmodium related diseases, such as malaria in a mammal.
  • the method comprises the step of administering to the mammal an effective amount of the isolated or purified polynucleotide, the isolated or purified polypeptide, the composition as defined above and/or the vaccine as defined above.
  • the vaccine, antibody and composition of the invention may be given to a mammal through various routes of administration.
  • the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally- acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os.
  • the vaccine and the composition of the invention may also be formulated as creams, ointments, lotions, gels, drops, suppositories, sprays, liquids or powders for topical administration. They may also be administered into the airways of a subject by way of a pressurized aerosol dispenser, a nasal sprayer, a nebulizer, a metered dose inhaler, a dry powder inhaler, or a capsule. Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the mammal to be treated. Any other methods well known in the art may be used for administering the vaccine, antibody and the composition of the invention.
  • the present invention is also directed to an in vitro diagnostic method for the detection of the presence or absence of antibodies indicative of Plasmodium species (for instance P. falciparum), which bind with the polypeptide as defined above to form an immune complex.
  • Such method comprises the steps of a) contacting the polypeptide of the present invention with a biological sample for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).
  • a diagnostic kit for the detection of the presence or absence of antibodies indicative of Plasmodium species comprises: a polypeptide as defined above; a reagent to detect polypeptide-antibody immune complex; a biological reference sample lacking antibodies that immunologically bind with the polypeptide; and - a comparison sample comprising antibodies which can specifically bind to the polypeptide; wherein the polypeptide, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform the detection.
  • the present invention also proposes an in vitro diagnostic method for the detection of the presence or absence of polypeptides indicative of Plasmodium species, which bind with the antibody of the present invention to form an immune complex, comprising the steps of: a) contacting the antibody of the invention with a biological sample for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).
  • a diagnostic kit for the detection of the presence or absence of polypeptides indicative of Plasmodium species comprises: an antibody as defined above; a reagent to detect polypeptide-antibody immune complex; a biological reference sample lacking polypeptides that immunologically bind with the antibody; and - a comparison sample comprising polypeptides which can specifically bind to the antibody; wherein said antibody, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform the detection.
  • an in vitro diagnostic method for the detection of the presence or absence of a polynucleotide indicative of Plasmodium species comprises the steps of: a) contacting at least one oligonucleotide as defined above with a biological sample for a time and under conditions sufficient for said oligonucleotide to hybridize to said polynucleotide; and b) detecting the presence or absence of an hybridization between the oligonucleotide and the polynucleotide.
  • a diagnostic kit for the detection of the presence or absence of polynucleotide indicative of Plasmodium species comprises: - an oligonucleotide as defined above; a reagent to detect polynucleotide-oligonucleotide hybridization complex; a biological reference sample lacking polynucleotides that hybridise with the oligonucleotide; and - a comparison sample comprising polynucleotides which can specifically hybridise to the oligonucleotide; wherein said oligonucleotide, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform the detection.
  • the oligonucleotide referred to in the diagnostic methods and kits hybridises to the polynucleotide having SEQ ID NO 18 or fragments thereof.
  • EXAMPLE 1 Cloning and characterization of the P. falciparum var O gene. Characterization of the var O gene as one virulence factor was done using the experimental model of infection with the O and R Palo Alto variants in the splenectomized Saimiri sciureus monkey. The large cellular aggregates which are normally filtered by the spleen in spleen-intact individuals, circulate in splenectomized monkeys (Contamin H. et al. (2000) Microbes & Infection 2. 945-954). Unlike what is observed in human infections, a very large proportion of parasites (80% and over) are rosette-forming or autoagglutinating within the var O population. This provides the opportunity to study the biological properties of that variant with a high degree of confidence.
  • the inventors of the present invention compared the growth rate of variant O and variant R in vivo.
  • This growth rate comparison has variant O to have a higher multiplication rate in vivo than its sibling var R.
  • the calculated multiplication rate (increase in parasite density) is of 2.7 per day for the var O parasites as opposed to 1.7 per day for the var R parasites. Since the O variant is the largely dominant clone within the population, the inventors could conclude with a fair degree of confidence to the contribution of the rosetting phenotype of variant O to increased multiplication rate. This indicates that rosetting is indeed a virulence factor.
  • the rapid multiplication rate of the O variant is further indicated by the observation of re-emerging rosetting/autoagglutinating parasites from two independent variant lines, namely var R and "pic2", that were propagated in the Saimiri monkey most probably due to outgrowth of parasites that had switched back to express the var O type.
  • Consecutive infections with the same variant have shown that the absence of antibodies reacting with the var O surface, as determined by Fluorescence Activated Cell Sorter or by agglutination on the day of infection, is usually associated with sensitivity to subsequent infection by variant O. Inversely, the inventors have observed that the presence of antibodies reacting with the surface is associated with failure of the var O parasites to develop. Both lines of arguments suggest that the rosetting/autoagglutination cytoadherence phenotype associated with var O parasites represents a virulence factor, and that specific immunity against that phenotype protects against further infection by this variant. This is consistent with published associations obtained in human studies.
  • the results show a direct correlation of a highly dominant cytoadherence phenotype (rosetting/autoagglutination) with specific infection outcome.
  • the specific var gene [var O] specifically expressed by rosetting FUP/SP parasites, which has been identified by RT-PCR as being the dominant var gene expressed by var O parasites was characterized by gene walking and RT-PCR.
  • the inventors proceeded by RT-PCR using degenerate primers targeted to the DBL1 domain.
  • O and R parasite RNAs were extracted from highly parasited blood samples (over 20% infected red blood cells) containing a 70 to 90% proportion of mature parasites.
  • RNA from these samples therefore contained transcripts from non mature forms of parasites, abortive transcripts that do not correspond to the majority sequence translated by mature forms.
  • the analogous sequences for the O parasites break down into the following : a group of 28 identical sequences (specifically identified from O cDNAs), a group of 12 identical sequences, a group of 2 identical sequences and 7 unique sequences from 49 analyzed clones.
  • the 20 sequences analyzed break down as follows : 2 groups of 5 identical sequences (one of which is also expressed by the O parasites), 2 groups of 2 identical sequences and 6 unique sequences. parasites
  • Table 1 Distribution of the different sequences of DBL1 clones obtained after RT- PCR using universal primers (alpha AF/alpha BR) for the extracted RNAs of O and R parasites propagated in the splenectomized monkey.
  • chromosome walking which permitted to obtain several fragments on either side of the identified domains
  • RT-PCR which by combining specific primers and conserved primers generated two large fragments, a DBL1-DBL3 fragments and a DBL3-exonll.
  • Exon 1 which codes for the extracellular part of the molecule, features five DBL domains, one CIDR domain adjacent to the DBL1 domain and three interdomains (C2, ID3 and ID4). Alignment of the sequences with the DBL and CIDR domains described in Smith et a/.'s phylogenetic study (2000) MBP 110:293- 310) allowed the inventors to identify and class the var O domains according to the new nomenclature.
  • the DBL 1 domain corresponds to the DBLalpha group
  • the DBL2 belongs to the DBLbeta group
  • the DBL3 to the DBLgamma group
  • the DBL4 and DBL5 to the DBLepsilon group
  • the CIDR corresponds to CIDRgamma.
  • the 2459 amino acid deduced var O protein sequence delimits the different protein domains (with conserved amino acids underlined) which can be seen in figures 5 to 14.
  • the 2459 amino acid deduced var O protein sequence shows five DBL and one CIDR domains, with a particular organization (figure 1).
  • the var O protein is a composite of domains, each of which shows a best identity match with a different PfEMPI protein for a specific domain.
  • Var O differs from the recently described and well conserved var sub-families which share homology all over their sequences (Salanti A. et al. (2002) Mol. Biochem. Parasitol. 122, 111-115; Rowe J.A. et al. (2002) J. Inf. Dis. 185, 1207-1211), the prototype being the var CSA gene (Buffet P. et al. (1999) Proc. Natl Acad.
  • Var O differs substantially from the previously characterized var CSA gene(Buffet P. et al. (1999) Proc. Natl Acad. Sci. U.S.A. 96, 12743-12748). Maximal identity observed was 64%, suggesting unique features to this protein ( Figure 2). The deduced var O protein sequence has several important features.
  • DBL1alpha-CIDR1 gamma association is atypical and has been so far described in only one case, namely the PfEMPI protein coding for the rosette-forming variant R29 of the It line.
  • the R29 DBL1 alpha domain has been implicated in binding to the erythrocyte receptor CR1. Homology with the R29 PfEMPI protein is restricted to this region. Additional domains share substantial identity with domains from other PfEMPI molecules that have been implicated in other binding specificities.
  • the var O PfEMPI protein presents the so far unique and unexpected feature of having no CIDRalpha domain.
  • CIDRalpha is responsible for binding to CD36.
  • the absence of a canonical CD36 binding domain in var O PfEMPI is consistent with the incapacity to demonstrate binding to human CD36. This is particularly interesting because CD36 binding is a common phenotype in Plasmodium falciparum isolates, with quite important consequences in terms of stimulation of the immune system.
  • CIDRalpha has been shown to stimulate naive CD4 T cells to produce interferon (IFN) gamma and interleukin (IL) 10 (Allsop C.E.M. et al. (2002) J. Inf. Dis. 185, 812-819).
  • IFN interferon
  • IL interleukin
  • the various DBL domains, the CIDR domain, the variable inter-domains and the DBL1alpha-CIDR1 gamma head structure have been sub-cloned for expression in a bacterial expression system, the pET system, for expression into the periplasmic space (Novagene).
  • the pET system is a proven expression system for a large number of recombinant molecules, particularly those possessing numerous disulfide bonds.
  • the pET22b vector was chosen in order to increase the chances of obtaining soluble and active proteins.
  • This vector carries the pelB signal sequence that promotes periplasmic orientation of target proteins. This cellular compartment provides a favorable environment for disulfide bond formation and correct protein folding.
  • the pET22b vector also carries a C terminal poly-histidine tail allowing recombinant molecule detection and affinity purification.
  • Cloning step was effected in two stages. Recombinant plasmids were first isolated from the Novablue strain. Two bacterial strains namely, BL21(DE3) and BL21 (DE3)PlysS, possessing the T7 polymerase gene under the control of the IPTG-inducible lacUV ⁇ promoter, were then transformed with these plasmids and used for overexpression.
  • Clone IMP 538 is the name given to the library (mixture) of clones containing the clones IMP 529, IMP 530, IMP 531, IMP 532, IMP 533, IMP 534, IMP 535 and IMP 536 described as follows.
  • SEQ ID NO. 22 sens 5DBLIB : 5TAC AAC GAG GAT CCA AAG CCT TGT TAT GGA AGG (+2aa); SEQ ID NO. 23: anti-sens 3DBLIX : 5'AGT TTT TTT CCA CTC GAG AAT TTC ATA GAA TAT TTA AGG TTT TG.
  • P Z M15 : Tn10 (Tc R )] carries plasmid pET22b-Clone A containing the DBL2 domain coding sequence, e.g. nucleotides 2497 to 3312 of the var O Palo Alto nucleotide sequence as shown in figure 16.
  • the following set of amplification primers were used :
  • SEQ ID NO. 24 sens 5DBL2B : 5'CGT TCT GGT TAG GAT CCT AAA GGA CCA
  • TGT ACA G SEQ ID NO. 25: anti-sens 3DBL2X : 5TGC AAT TTT TGC TTT CTC GAG TAA
  • SEQ ID NO 26 Sens 5ID2B : 5TGG AAA CAA ATG GAT CCA AAA TAC AAG GAT TTA TAC;
  • SEQ ID NO 27 Anti-sens 3ID2X : 5'CCA ATT TAC CTC GAG TTT TAT ATT GCA CCC ATA TAT TGA.
  • P Z M15 : Tn10 (Tc R )] carries plasmid pET22b-Clone C containing the DBL3 domain coding sequence, e.g. nucleotides 3713 to 4461 of the var O Palo Alto nucleotide sequence as shown in figure 18.
  • the following set of amplification primers were used :
  • SEQ ID NO 28 Sens 5DBL3B : 5'AAG GGC GAA ACG GAT CCA ATA TAT GGG
  • Sens 5ID3B AAA ACT CAA TAG GAT CCA CGA AGC CAA AAA TTT ACA AGA;
  • SEQ ID NO 31 Anti-sens 3ID3X : AGA AAC TTC CTC GAG TTT ACA TGT TTC
  • SEQ ID NO 32 Sens 5DBL4B : AAT CAA AAT GGG GAT CCA TGT AAA TTT AAA GAA GTT; SEQ ID NO 33: Anti-sens 3DBL4X : AGC ATT TTT CTC GAG TTC TTT ATA TGC TTT GTT TTG TGT TTC.
  • SEQ ID NO 34 Sens 5ID4B : GCC CAA TTG GAT CCA CAA AAC AAA GCA TAT AAA G;
  • SEQ ID NO 35 Anti-sens 3ID4X : GAA ATT TTT CTC GAG ACA ACT ACC TAT
  • P Z M15 : Tn10 (Tc R )] carries plasmid pET22b-Clone M coding for the DBL5 domain, e.g. nucleotides 6103 to 6813 of the var O Palo Alto nucleotide sequence as shown in figure 22.
  • the following set of amplification primers were used : SEQ ID NO 36: Sens 5DBL5B : TGT AAG AAG TAG GAT CCA GGT AGT TGT
  • SEQ ID NO 37 Anti-sens 3DBL5X : TAG TGT CTT CTC GAG TTC TTT ATC ATA
  • SEQ ID NO 38 Sens 5DBLIB : 5TAC AAC GAG GAT CCA AAG CCT TGT TAT GGA AGG;
  • SEQ ID NO 39 Anti-sens 3CIDRX : 5' TCC TAT TTT AAA CCT CTC GAG ATT TTT ACC TGT ACA TGG.
  • the pET22b recombinant plasmid containing the wild type var O DBL1 alpha domain cloned in frame with the His tag, extracted from IMP538 and retransformed into BL21 (DE3) pLys (Novagen) (lane 1 ) and a control plasmid carrying an insert with an internal stop codon (lane 2) were induced for 1 hour at room temperature.
  • the bacteria were centrifuged, resuspended in Bug Buster as recommended by the supplier, and the soluble fraction loaded on a 12.5% SDS PAGE under reducing conditions, and immunobloted using an anti-His tag monoclonal antibody (Novagen) under the conditions recommended by the manufacturer.
  • the apparent molecular mass of the band visualised in lane 1 corresponds to the predicted DBL1 alpha mass.
  • FIG 25 the results of mass purification of the DBL1 alpha domain by urea extraction is illustrated. More specifically, the pET22b recombinant plasmid containing the wild type var O DBL1 alpha domain cloned in frame with the His tag, extracted from IMP538 and retransformed into BL21 (DE3) pLys (Novagen) was cultivated and induced and protein was extracted as described above. Aliquots were loaded on a 12.5% SDS PAGE under reducing conditions. The proteins were stained with Coomassie Blue.
  • Lane 1 unfractionated Bug Buster lysate of induced bacteria
  • Lane 2 Bug Buster soluble fraction
  • Lane 3 2M urea soluble fraction
  • Lane 4 Molecular mass Markers (indicated on the right of the figure)
  • FIG. 26 shows immunoblot of the induced BL21 (DE3) harbouring plasmid pET22b clone I containing the C2 domain in frame with the poly His tail, probed with Saimiri monkey sera diluted 1/100.
  • Lane 1 serum from monkey 95013 (collected on day 34 of its second infection with Palo Alto parasites (this serum agglutinates var O parasites)
  • Lane 2 serum from monkey 95095 (collected on day 34 of its second infection with Palo Alto parasites (this serum agglutinates var O parasites)
  • Lane 3 pool of malaria naive Saimiri sera
  • a particularly novel aspect of the invention is the inclusion of the DBL1alpha-CIDR1 gamma expression product for further analysis, including of possible immunomodulatory activities.
  • the invention concerns also the collection of individual domains, including the non adhesive domains sub-cloned into an Escherichia coli expression vector. This forms a comprehensive tool for analysis of naturally acquired immune response and a versatile tool for a final vaccine composition including one or multiple individual domains.
  • the DBL1 ⁇ , CIDR1 ⁇ and DBL1 ⁇ -CIDR1 ⁇ varO domains were amplified from Palo Alto var O genomic DNA using i/arO-specific primers containing one artificial ⁇ g/lll and Sail restriction site for the sense and anti-sense primer, respectively, thus permitting unidirectionnal cloning.
  • - DBL1p was amplified using the 5DBL1 ⁇ glll - 3DBLI Sail primer pair
  • the amplification product was examined by agarose gel electrophoresis, cut out from the gel and purified.
  • the PCR product was digested with ⁇ glll and Sail (Phamarcia) under the conditions recommended by the supplier, and purified using the QIAquick Gel Extraction Kit under the conditions recommended by the supplier (Qiagen).
  • the pDisplay plasmid was digested with ⁇ glll and Sail (Pharmacia) and purified using the same procedure.
  • Ligation of 500 ng of insert with 200 ng of vector was done overnight in a final volume 10 ⁇ l at 4°C using T4 DNA ligase (Boerhinger Mannheim), as recommended by the supplier.
  • Competent Escherichia coli TOP10 (Invitrogen ) were transformed with the ligation mixture, as recommended. Transformants were selected on LB plates supplemented with 100 ⁇ g/mL Ampicillin. The plasmid haboured by the transformed, ampicillin-resistance bacteria was purified using the "QIAprep Spin Miniprep kit " (QIAgen). Presence of an insert was verified using Bglll and Sail restriction. The plasmids containing an insert of predicted size were sequenced. Sequences were aligned manually using the undertaken MUST software.
  • coli comprising a pDisplay vector encoding for respectively DBL1 ⁇ , CIDR1 ⁇ and DBL1 ⁇ -CIDR1 ⁇ domains were registered at the CNCM on October 3, 2003 under accession numbers I-3096, I-3097 and I-3098 respectively.
  • Cytoadherence assays of Saimiri red blood cells were carried as follows. Three mL of uninfected Saimiri blood was collected by veinipuncture under heparin, and washed three times with 1X PBS. Hematocrit was adjusted to 5 -10 % either in RPMI medium, 2 % FCS either in 1X PBS. Three mL of this suspension was added onto the transfected COS7L cells obtained according to the protocol described above. After a 2 - 3 hour incubation et 37°C with gentle agitation, unbound cells were washed out in PBS/BSA 0.1% or in RPMI +2 % FCS and observed under a transmission microscope.
  • Figures 27 A, B and C illustrate the results of binding assays between COS7L cells expressing at their surface the var O DBL1 ⁇ or var O CIDR1 ⁇ domains and Saimiri red blood cells.
  • Saimiri Red Blood cell bind to the surface of COS7L cells transiently transfected with recombinant pDisplay plasmids expressing the CIDR1 ⁇ (Fig. 27A), or the DBL1 ⁇ var O domain (Fig. 27B).
  • Figure 27C shows absence of binding on COS7L cells transiently transfected with the pDisplay vector.
  • CIDR1 (gamma) domain pair in homogenous form and in quantity using the baculovirus/insect cell expression system
  • PfEMPI Erythrocyte Membrane Protein 1 from malaria parasite, Plasmodium falciparum, (PfEMPI ) forms a large family of protein variants encoded by the var gene family. (59 var genes have been identified in the sequenced genome of the laboratory clone 3D7). PfEMPI is expressed and exported to the surface of the infected erythrocyte during the trophozoite and schizont stages of parasite development. In association with other Plasmodium proteins, it radically modifies the topology of the erythrocyte membrane by forming protuberances, or knob structures, which are distributed over the entire red blood cell surface.
  • the var genes are divided into two exons.
  • the 3' exon encodes a highly conserved trans-membrane region and a cytoplasmic C-terminal segment that associates with other proteins forming the knob structures, while the 5' exon encodes the highly variable extra-cellular region of PfEMPI .
  • the extra-cellular moiety begins with a semi-conserved N-terminal segment (NTS), followed by a series of Duffy Binding Like (DBL) domains, Cysteine Rich Interdomain Regions (CIDR) and C2 domains. From sequence analysis, the DBL domains have been grouped into 5 classes ( ⁇ to ⁇ ) and the CIDR into 3 classes ( ⁇ to Y).
  • DBL ⁇ -CIDR ⁇ domain pair usually follows immediately after the NTS, and DBL ⁇ is invariably followed by C2.
  • the protein includes from 2 to 7 DBL domains and up to 2 CIDR modules.
  • PfEMPI has been identified as an adhesin, with the different variants displaying specificities for different receptor molecules present on vascular endothelium or on the erythrocyte surface itself. Only one PfEMPI variant is expressed on the erythrocyte surface at a time, thus conferring a defined receptor specificity upon a given parasite.
  • the different receptor specificities are associated with different DBL and CIDR domains; for example, certain members of the CIDR ⁇ class bind to CD36 while certain members of the DBL ⁇ class bind to ICAM-1.
  • PfEMPI as a virulence factor in severe malaria
  • the severity of malaria depends on a number of complex and often poorly understood factors, such as acquired immunity, the nature of the immune response, and parasite virulence. It is widely accepted that the PfEMPI promotes sequestration of infected erythrocytes by cyto-adherence in specific organs. Certain parasites variants cause adhesion of uninfected to an infected erythrocytes (termed rosetting), or the formation of auto-agglutinates of infected erythrocytes. Cyto-adhesion and adhesion of infected erythrocytes are thought to be correlated with parasite virulence. For example, rosetting and auto- agglutination are the only factors that have been associated with disease severity in African children.
  • the receptor specificity and thus the organ(s) susceptible to sequestration, is considered to be determinant in the severity and outcome of the disease.
  • PfEMPI or at least certain of its variants, have be considered as parasite virulence factors, although direct evidence is lacking in most cases. Nonetheless, the absence of particular variant-specific anti-PfEMP1 antibodies in the serum of naturally immune individuals correlates with susceptibility to infection by those variants.
  • the squirrel monkey, or Saimiri sciureus is susceptible to P. falciparum infection and is thus a useful model system for human malaria to study various host/parasite relationships, such as strain-specific immunity and virulence factors.
  • a variant of the Palo Alto FUP/SP line of P. falciparum which has adapted to infect the Saimiri monkey and referred to as var O, causes rosetting and auto- agglutination, whereas a closely related variant, var R, that was derived from var O when placed under immune pressure, shows no such adhesion. Since the spleen of Saimiri is able to clear red blood cells parasitized by P. falciparum, certain studies require the use of splenectomised animals.
  • the present example concerns the expression of different domains of the extra-cellular region of PfEMPI from the var O variant in the baculovirus/insect cell system. Because the inventors intend to validate, or otherwise, these recombinant polypeptides as malaria vaccine candidates, use of this eukaryote expression system ensures that the PfEMPI domains are expressed as correctly folded polypeptides that should mimic the functional properties ' of the native antigen.
  • the present invention covers polypeptides expressed from any gene segment contained within the 5' exon encoding PfEMPI -var O, and covers deletions and mutations that render the recombinant products more amenable to manipulation by conferring, for example, increased solubility and greater stability, and which contribute to its optimisation as a vaccine.
  • Introduced changes can also include N- to-Q mutations in all NXST triplets, which form potential glycosylation sites in the eukaryotic expression system. (P. falciparum has no cellular machinery for posttranslational glycosylation, and thus native proteins do not carry N- or O-glycans; the baculovirus/insect cell expression system could, however, introduce these modifications).
  • the nucleotide sequence of the var O 5' exon has been determined by
  • NST-DBL1 ⁇ -CIDR1 ⁇ -DBL2 ⁇ -C2-DBL3 ⁇ -DBL4 ⁇ -DBL5 ⁇ It has the infrequently occurring DBL1 ⁇ -CIDR1 ⁇ domain association that has been described in only one other case to date: this is the rosette-forming variant R29 from the It parasite line; available evidence suggests that R29 binds to CR1 on erythrocytes.
  • homology between the PfEMPI molecules of the var O and R29 strains is restricted to the DBL1 ⁇ -CIDR1 ⁇ region : sequence identity between the two variants is 64% for DBL1 ⁇ and 29% for CIDR ⁇ .
  • the other var O domains share sequence similarity with domains from other PfEMPI variants that have been implicated in other binding specificities.
  • the various extra-cellular domains of PfEMPI var O have been cloned, and it has been shown that COS cells expressing either DBL1 ⁇ or CIDR1 ⁇ on their surface bind monkey erythrocytes.
  • the inventors have inserted the gene sequence of the DBL1 ⁇ -CIDR1 ⁇ and CIDR1 ⁇ -encoding regions into the baculovirus genome and transfected into insect cells for their expression as recombinant products : the CIDR1 ⁇ domain and the DBL1 ⁇ -CIDR1 ⁇ double domain.
  • Each gene been flanked by a signal sequence on the 5' end to ensure that the recombinant protein is expressed as a secreted product, and by a hexahistidine-encoding segment at the 3' end to facilitate its purification using metallo-affinity techniques.
  • the inventors have isolated a recombinant product expressed in insect cells transfected with the CIDR1 ⁇ var O gene using metallo-affinity and gel filtration chromatography. Analysis by SDS-PAGE in both reducing and non- reducing conditions reveals an intact protein with the molecular mass expected on CIDR1 ⁇ , which is identified too by a Western blot using an anti-histidine tag monoclonal antibody.
  • the novel aspect of this invention is that the inventors have obtained correctly folded recombinant CIDR1 ⁇ in soluble, homogeneous form.
  • the procedure of expression in baculovirus/insect cells has allowed us to obtain product in milligram quantities that will allow further physico-chemical and functional characterisation.
  • the domains DBL1 ⁇ -CIDR1 ⁇ and ClDR1 ⁇ have been amplified from the plasmid pET22b-Clone V using specific oligonucleotides containing sites recognisable by the restriction enzymes BamH1 and Xhol and a polyhistidine coding sequence.
  • the DBL1 ⁇ -CIDR1 ⁇ has been amplified using oligonucleotides A and B
  • the CIDR1 ⁇ has been amplified using oligonucleotides C and B
  • the quality of the PCR amplification product is examined by agarose gel electrophoresis then purified with a QIAquick (QIAgen) purification kit.
  • the fragment is then inserted into the plasmid pBlueBac 4.5/V5-HisTOPO TA according to the manufacturer's protocol (Invitrogen) and the bacteria TOP 10 are transformed with the ligation product.
  • the plasmids are screened by PCR and profiling after enzyme restriction and the positive plasmids are sequenced and purified with the Qiaprep spin (QIAgen) kit.
  • the signal peptide has been amplified by PCR from a plasmid containing the gene MSPI (Unite d'lmmunologie Moleisme des Parasites).
  • the amplified nucleotide sequence is the following: 5'-
  • TCC-3' and its expression will add on the amino-terminal side of the protein the following sequence: MKALLFLFSFIFFVTKCQCETESYKQLVANVDGD which would be cleaved just before ETESY
  • the DNA fragment coding for the signal peptide, generated by PCR, is purified by the QIAgen kit, then cleaved with the enzyme BamH1 (Roche) following the manufacturer's protocol.
  • the plasmid containing the gene is also cleaved by BamH1 then, after heath inactivation of the enzyme, the two restriction products are mixed then Iigated by the kit 'Rapid DNA ligation' (Roche).
  • the bacteria XL10 are transformed then selected on an Ampicilline containing medium. All the insertions and their directions are verified by PCR or by migration profile after restriction with the adequate enzymes.
  • viruses are manufactured then isolated by the method of limiting dilution and amplified in stock I, II then III, following the protocol of the manufacturer (Invitrogen).
  • the column is washed with 100 ml of the same medium and the supernatant is re-deposited. After a second washing with 100 ml of the same buffer, the protein is eluted with 15 ml of the buffer solution to which 500 mM of imidazole is added. One ml fractions are recuperated and deposited on a gel. The fractions containing the protein are reassembled, concentrated on ultrafree-MC (Millipore) then injected in a Superdex S200 16/60 column (Pharmacia). 1 ml fractions which contain the protein are recuperated and they would afterwards be concentrated with the same protocol as above.
  • Figure 28 shows SDS acrylamide 8/25% gels coloured with Coomassie blue, lanes 1 to 10 correspond to the elution of the protein with the buffer containing 500 mM imidazole.
  • Lane 29 it is shown a ponceau red coloured membrane.
  • Lane 1 corresponds to a control protein with a histidine terminal (positive control).
  • Lane 2 corresponds to the exiting sample of the column Talon, before S200 and lane 3 corresponds to fraction n°33 exiting of S200.
  • Lane 4 corresponds to fraction n°34 exiting of S200.
  • Figure 30 shows the immunoblot of Figure 29 with an antibody anti-His tag, the lanes are identical to Figure 29.
  • the control in lane 1 is well recognised.
  • lane 2 a weak amount is recognised at the expected size.
  • lane 3 the protein CIDR ⁇ is recognised.

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Abstract

L'invention porte: sur la découverte: du gène var O spécifique de la souche Palo Alto FUP/SP-O du Plasmodium falciparum, de la protéine correspondante, et des adhésines qui médient la formation de rosettes de globules rouges et leur auto-agglutination dans la pathogénie de la malaria. Elle porte en outre sur de nouveaux outils et procédés biologiques utilisant les précédents, dont des molécules d'acide nucléique, des polypeptides, des anticorps, des vecteurs recombinés, des cellules hôtes recombinées, ainsi que sur des applications diagnostiques et thérapeutiques correspondantes des précédents.
PCT/EP2003/013341 2002-10-25 2003-10-24 Facteur de virulence du var o du plasmodium falciparum WO2004037856A2 (fr)

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WO2011066995A1 (fr) * 2009-12-05 2011-06-09 Universität Heidelberg Vaccins antipaludiques à base de ferlines d'apicomplexa, protéines de type ferline et autres protéines contenant le domaine c2

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WO1996033736A1 (fr) * 1995-04-27 1996-10-31 Affymax Technologies N.V. Peptides du paludisme et vaccins associes

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BARUCH DROR I ET AL: "Immunization of Aotus monkeys with a functional domain of the Plasmodium falciparum variant antigen induces protection against a lethal parasite line" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 99, no. 6, 19 March 2002 (2002-03-19), pages 3860-3865, XP002279798 March 19, 2002 ISSN: 0027-8424 *
CHEN Q ET AL: "IDENTIFICATION OF PLASMODIUM FALCIPARUM ERYTHROCYTE MEMBRANE PROTEIN 1 (PFEMP1) AS THE ROSETTING LIGAND OF THE MALARIA PARASITE P.FALCIPARUM" JOURNAL OF EXPERIMENTAL MEDICINE, TOKYO, JP, vol. 187, no. 1, 5 January 1998 (1998-01-05), pages 15-23, XP000918017 ISSN: 0022-1007 cited in the application *
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LE SCANF CECILE ET AL: "Novel target antigens of the variant-specific immune response to Plasmodium falciparum identified by differential screening of an expression library" INFECTION AND IMMUNITY, vol. 67, no. 1, January 1999 (1999-01), pages 64-73, XP002279712 ISSN: 0019-9567 *
LE SCANF CECILE ET AL: "Plasmodium falciparum: Altered expression of erythrocyte membrane-associated antigens during antigenic variation" EXPERIMENTAL PARASITOLOGY, vol. 85, no. 2, 1997, pages 135-148, XP002279713 ISSN: 0014-4894 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011066995A1 (fr) * 2009-12-05 2011-06-09 Universität Heidelberg Vaccins antipaludiques à base de ferlines d'apicomplexa, protéines de type ferline et autres protéines contenant le domaine c2
JP2013512866A (ja) * 2009-12-05 2013-04-18 ルプレヒト−カールス−ウニヴェルジテート ハイデルベルク アピコンプレクサＦｅｒｌｉｎ、Ｆｅｒｌｉｎ様タンパク質、及び他のＣ２ドメイン含有タンパク質に基づくマラリアワクチン
US8968750B2 (en) 2009-12-05 2015-03-03 Ruprecht-Karls-Universität Heidelberg Malaria vaccines based on apicomplexan ferlins, ferlin-like proteins and other C2-domain containing proteins

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