CA2409897A1 - Plasmodium falciparum virulence factor var o - Google Patents

Abstract

The present invention relates to the discovery of the var O gene specific to the Palo Alto FUP/SP-O strain of Plasmodium falciparum and the corresponding protein, an adhesin that mediates rosetting and auto-agglutination of red blood cells in malaria pathogenesis.
Novel biological tools and methods of use of the foregoing are disclosed including nucleic acid molecules, polypeptides, antibodies, recombinant vectors, recombinant host cells, and corresponding diagnostic and therapeutic applications of the foregoing.

Description

PLASMODIUM FALCIPARUM VIRULENCE FACTOR VAR O
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
Malariotherapy infections in neurosyphilitic patients conducted in the first half of the 20~h century have shown that Plasmodium species, in particular, Plasmodium falciparum strains differ in the clinical manifestations they provoke (James S.P. et al.
(1932) Proc. R.
Soc. Med. 25, 1153-1181). This observation was confirmed in experimental infections of Saimiri sciureus monkeys (Fandeur T. et al. (1996). Exp. Parasitol. 84, 1-15) and suggests the existence of parasite virulence factors. Identification of the parasite factors which critically influence infection outcome is pivotal in devising intervention strategies aiming at preventing or curing malaria morbidity.
To date, little is known about the parasite factors involved at the various steps of the pathogenesis cascade. The current view on P. falciparum pathogenesis is that severe malaria results from an inappropriate immune response to high parasite density, with excessive production of pro-inflammatory cytokines and massive colonisation of specific organs by parasites expressing specific adhesin(s). The relationship between mild and severe malaria is uncertain. The fact that spreading of drug resistance is associated with an increased malaria-attributable mortality (Trape J.F. (2001) Am. J. Trop.
Med. Hyg. 64 S 12-17) suggests a direct continuum, with progression to severe complications if mild 3CI malaria is not rapidly treated.
It is clear that 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. Trop. 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.
Trop. 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. Immun. 68, 5849-5856) and correlates with prognosis (Molyneux M.E. et al. (1989). Quat. J. Med. 71, 446-474). This strongly suggests that the parasite factors that favour an increased multiplication rate/development are contributing to pathogenesis. Indeed, parasites from severe malaria Thai patients have a 3-fold higher in vitro multiplication potential and a lower selectivity for red blood cells than parasites from mild malaria patients (Chotivanich K. et al. (2000) J. Inf. Dis. 181, 1206-1209).
The concept that expression of parasite adhesins promotes massive organ-specific sequestration has only been validated for the placenta (Fried M. & Duffy P.E.
(1996) Science 272, 1502-1504; Beeson J.G. et al. 2001. Trends Parasitol. 17(7), 331-337). So far, the capacity of infected red blood cells with mature parasite stages to bind uninfected red blood cells (called "rosetting") {Carlson J. et al. 1990. The Lancet 336, 1457-1460;
Rowe A. et al. (1995). Infect. Immun. 63, 2323-2336) or to form large auto-agglutinates of infected red blood cells (Pain A. et al. (2001 ) Proc. Natl Acad. Sci. U.S.A.
98 (4), 1805-1810; Roberts D.J. ef al. (2000). The Lancet 355, 1427-1428) associated with the absence of antibodies disrupting rosettes (Carlson J. et al. 1990. The Lancet 336, 1457-1460) are the only factors associated with severity in African children. The large cellular aggregates formed by rosettes and/or auto-agglutinates provoke microvascular obstruction. 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. Thus, targetting (preventing or reverting) rosettinglauto-agglutination should have broader consequences than preventing homing to certain territories by targetting the specific ligand.
So far, identification of parasite factors contributing to pathology in humans has relied on association studies, looking for specific parasite characteristics associated with more or less severe clinical forms (Robert F. et al. {1996). Trans. R. Soc.
Trop. 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. 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; Newbold C.I.
et al.
(1997). Am. J. Trop. Med. Hyg. 57, 389-398; Ariey F. et al. (2001 ). J.
Infect. Dis., 184, 237-241; Kun J.F. et a!. (1998). Trans. R. Soc. Trop. Med. Hyg. 92(1), 110-114).

Interpretation of these studies is difficult because of the very large field Plasmodium falciparum diversity, with numerous strains circulating in any given place.
Investigation of variant phenotypic adhesion specificity is further complicated by the rapid var switching rate, resulting in clonal phenotypic heterogeneity with numerous adhesive phenotypes expressed at the time of blood collection (Newbold C.I. et al. (1997). Am. J.
Trop. Med.
Hyg. 57, 389-398). The number of possible receptors, the list of which is most probably not closed, is so large that an exhaustive analysis of binding properties of patient isolates cannot be conducted.
Isolates with rosette-forming parasites [67% -100%] (Carlson J. et al. 1990.
The Lancet 336, 1457-1460; Rowe A. et al. (1995). Infect. Immun. 63, 2323-2336;
Rogerson S.J. et aL (2000) Infect. Immun., 68 391-393) or autoagglutinating parasites [47-86] (Pain A. et al. (2001 ) Proc. Natl Acad. Sci. U.S.A. 98 (4), 1805-1810; Roberts D.J.
et al. (2000).
The Lancet 355, 1427-1428) are frequent in African children and in adults (Ho M. et aL
(1991 ) Infect. Immun. 59, 2135-2139; Rogerson S.J. ef al. (2000) Infect.
Immun., 68 391-393), but the percentage of all infected erythrocytes forming cellular aggregates within an isolate is low. Rosette-forming infected erythrocytes account for 2.8-9% of the all infected red blood cells (Rogerson S.J. ef al. (2000) Infect. Immun., 68 391-393), 1%
in mild and 5% in severe malaria (Rowe A, et al. (1995). Infect. Immun. 63, 2323-2336) and autoagglutinating erythrocytes for 6.6% of ail IRBC present in severe cases and 2.1 % mild cases (Roberts D.J. et al. (2000). The Lancet 355, 1427-1428). Thus, the association of the capacity of rosetting and autoagglutination with severity was statistically significant in several studies, but figures rather unsatisfactory.
Several factors limit such studies in humans and their interpretation. The inclusion criteria applied differ from one study to the other e.g. high parasite density is >4%
(Heddini A. et al. (2001) Infect. Immun. 69, 5849-5856), 0.2% (Roberts D.J. et al. (2000).
The Lancet 355, 1427-1428) or 0.3% (Roberts D.J. et al. (2000). The Lancet 355, 1427-1428), associated with removal of monocytes and granulocytes (Heddini A. et al. (2001 ) Infect. Immun. 69, 5849-5856), presence or not of platelets in the in vitro assays (Pain A.
et al. (2001) Proc. Natl Acad. Sci. U.S.A. 98 (4), 1805-1810) etc.... 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.
Furthermore, these studies all make the assumption that circulating parasites are fairly representative of the sequestered pool, which is actually causing disease. However, recent data indicate that such is not the case and that in 86% of the cases examined some sequestered genotypes (strains) are not present in the peripheral blood (Schleiermacher D., et al.
2002 Inf. Genet.
Evol. 46, 1-9).
The molecular mechanisms underlying cytoadherence in malaria parasites have been recently clarified. The cytoadherence phenotype acquired by mature Plasmodium falciparum-infected erythrocytes is mediated by variant PfEMP1 adhesins exposed onto the surface of infected RBC from thetrophozoite stage on. PfEMP1 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).
The structural organisation established in 1995 by two independent groups has been clarified by numerous subsequent studies. In brief, var genes have two exons, exons 1 and 2. Exon 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). Within each class, there exist unique consensus motifs that can be used to characterise and classify PfEMPI molecules(Smith et al., (2000) Mol. Biochem. Parasitol., 110, 293-310; Smith et al., (2001 ) Trends Parasitol. 17 (11 ) 538-545). The arrangement and sequence of DBL and CIDR
differ between different PfEMP1 proteins.
The prototypical PfEMP1 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 DBLlalpha-CIDR1alpha 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. Mapping of the PfEMPI adhesive domains has indicated that rosetting is associated with DBL1 alpha , binding to ICAM-1 with the tandem DBLbeta -C2, binding to CD31 with the DBLdelta , binding to CSA is associated with DBLgamma and binding to CD36 with CIDR1 alpha(Smith et al., (2001 ) 5 Trends Parasitol. 17 (11 ) 538-545).
Three var genes associated with rosetting have been described to date.
Firstly, 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 i:lgure 2).
The first DBL1-CIDR1 association is atypical it consists in DBL1alpha-CIDRlgamma .The rosetting receptor on the erythrocyte surface has been identified. It is Complement Receptor 1 (CR1 ). Common CR1 African polymorphism reduced binding of IRBC.
The domain responsible for rosetting is DBL1aIpha. Expression of the different DBL1 - 4 and CIDR1 domains onto the surface of COS cells identified DBL1 alpha as the single domain binding erythrocytes.
Secondly, there is the 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 (1alpha & 2delta ) and two CIDR (alpha and beta) (see figure 2). This gene codes for a multi-adhesive protein. The DBL1alpha GST binds heparin-Sepharose, glycosaminoglycans and binds erythrocytes.
Lastly, there is the var gene 15991381 Flick et al. (2001 ) Science 293, 2098-(AF366567 in Genbank) derived from clone TM284S2 which forms giant rosettes, mixed rosettes and autoagglutinates. The var gene 15991381 codes for a PfEMP1 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 demonstration that specific parasite factors contribute to pathology requires an experimental model of infection, where the size of the inoculum, the time of injection, the parasite strains and possible surface phenotype be well defined and as homogeneous as s possible. Comparison of the course of infection induced by 14 different strains in the splenectomized Saimiri sciureus (a highly susceptible model of infection) has shown substantial differences in the course of infection, some strains inducing afulminant (lethal if untreated) infection, whereas others induce self-curing infections reaching moderate, low or very low peak parasite densities (Fandeur T. et al. (1996). Exp.
Parasitol. 84, 1-15).
Phenotyping and genotyping these strains for more than 20 characters showed that the lethal strains Palo Alto FUP/SP, and 2 other ones collected from fatal human cases, formed rosettes and large autoagglutinates (Fandeur T. et al. (1996). Exp.
Parasitol. 84, 1-15).
To study the possible contribution of rosetting in pathogenesis, two antigenic variants of the Palo Alto FUP/SP line, variants O and R were studied. Variant O forms rosettes and auto-agglutinates while variant R, which is derived from variant O under immune pressure, no longer forms rosettes or autoagglutinates (Fandeur T et al., (1995) J Exp Med 181, I5 283-295). While both variants have the same genetic make-up, they nevertheless present distinct adhesive phenotypes. To minimize sequestration and gain access to circulating pigmented parasites, removal of the spleen is necessary (David P. et al.
(1983) Proc. Natl Acad. Sci. U.S.A. 80, 5075-5079). While there are few experimental models of infection available to identify virulence factors, there exists a 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. ef 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.
Thus, there is a need to identify virulence factors of Plasmodium parasite since it is clear from the above discussion that identification of the parasite factors which critically influence infection outcome is pivotal in devising intervention strategies aiming at preventing or curing malaria morbidity.
The present invention fulfills that need and also other needs, which will be apparent to those skilled in the art upon reading the following specification.

SUMMARY OF THE INVENTION
The present invention concerns the characterization of a var gene, more particularly, the var O gene.
The present invention also concerns at least one polypeptide encoded by the var O gene and expressed in Plasmodium species.
More precisely, 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 N0.13 to SEQ ID N0.21 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 and 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 !D NO. 1 and 13 to 21.
!n a particular embodiment, 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 and CNCM I-2930.

Also another object of the present invention is to provide a transformed or transfected cell containing the polynucleotide sequence of the present invention.
Still a further object of the present invention is to provide a host cell comprising the cloning or expression vector of the instant invention.
Another object of the present invention is to provide a recombinantEscherichia coli cell selected from the group consisting of the cells deposited under number and CNCM I-2930.
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 comprising the isolated or purified polynucleotide and/or the isolated or purified polypeptide of the instant invention; andlor the antibody specific to the polypeptide encoded by the polynucleotide of the invention or biologically active fragments thereof, and an acceptable carrier.
zo Also 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 oligonucieotide 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 antibodies 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 5 - 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.
10 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.
BRIEF DESCRIPTION OF THE DRAWINGS
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 Paio 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 1D NO. 6.
Figure 9 is the amino acid sequence of the DBL 5 epsilon domain encoded by the var O
gene and identified as SEQ ID N0.7.
Figure 10 is the amino acid sequence of the CIRD gamma domain encoded by thevar O
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 thevar0 gene and identified as SEQ ID N0.10.
Figure 13 is the amino acid sequence of the ID 4 domain encoded by thevar O
gene and identified as SEQ ID NO. 11.
Figure 14 is the amino acid sequence of the transmembrane segment encoded by the var 3U 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 var 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 N0.17.
Figure 23 is the nucleic acid sequence of the DBL1 alpha CIRD gamma tandem domain of the var0 gene identified as SEQ ID NO. 18.
DETAILED DESCRIPTION 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.
A non-exhaustive list of P. species against which the methods, compositions and vaccines of the invention may be useful, includes those which affect humans and preferably those that cause malaria, such as P. vivax, P. ovate, P. malariae and P.
falciparum. In a preferred embodiment, the compositions, vaccines and methods of the present invention will be useful against disorders caused by P. falciparum.
As used herein, the term "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.
The term "protection" or "protective immunity" refers herein to the ability of the 1C1 serum antibodies and cellular response induced during immunization to protect (partially or totally) against malaria caused by an infectious agent, such as aP.
falciparum. Thus, a mammal immunized by the compositions or vaccines of the invention will experience limited growth and spread of an infectious P. falciparum.
15 As used herein, the term "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.
As used herein, the term "mammal" refers to any mammal that is susceptible to be 20 infected by a Plasmodium species causing malaria. Among the mammals which are known to be potentially infected by a P. species, there are humans, apes, birds, and bovines.
1. Polynucleotides and polypeptides 2~~
In a first embodiment, 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%
30 identical to any one of SEQ ID NO 1, 13 to 21 as shown in figures 3, 12 to 23.
As used herein, 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. For example, a polynucleotide or a protein/peptide naturally 3~~ 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.
Moreover, 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 palypeptide 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 blosum62 substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad. Sci. USA
89: 10915-10919). By the statement "sequence A is n% similar 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 and conservative substitutions. By the statement "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.
As used herein, the term "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. In addition, "polynucleotide"
as used herein 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. As used herein, 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. Moreover, DNAs or RNAs comprising 5 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). The term 10 "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
N0.1. The term "polynucleotide(s)" also embraces short nucleotides or fragments, often 15 referred to as "oligonucleotides", that due to mutagenesis are not 100%
identical but nevertheless code for the same amino acid sequence.
In a second embodiment, 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
N0:2 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 N0:2.
According to a preferred embodiment, 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 N0.4 (figure 6), SEQ ID
NO. 5 (figure 7), SEQ ID N0.6 (figure 8), SEQ ID N0.7 (figure 9), SEQ ID N0.8 (figure 10), SEQ
ID N0.9 (figure 11 ), SEQ ID N0.10 (figure 12), SEQ ID N0.11 (figure 13), SEQ
ID N0.12 (figure 14), and biologically active fragments thereof.
As used herein, the expression "biological active" refers to a polypeptide or fragments) thereof that substantially retain the capacity of forming var O-receptor complex.

According to another preferred embodiment, the isolated and purified polypeptide of the present invention comprises an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions to the complement of SEQ ID NO 1 or fragments thereof. Such a polypeptide has the ability to induce cytoadherence in cells infected with Plasmodium related species. As used herein, 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. Typically high, medium and low stringency encompass the following conditions or equivalent conditions thereto 1 ) high stringency : 0. 1 x SSPE or SSC, 0. 1 % SDS, 65° C
2) medium stringency : 0. 2 x SSPE or SSC, 0. 1 % SDS, 50° C
3) low stringency : 1. 0 x SSPE or SSC, 0. 1 % SDS, 50° C.
As used herein, the term "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.
Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. 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, GP6 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, such as arginylation, and ubiquitination. See, for instance:
PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci.
663: 48-62(1992). 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 ofvar O protein with its receptor.
Thus, 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).
2. Vectors and Cells In a third embodiment, 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 transiently-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. colt'. More preferably the host is Escherichia coli bacterium. The following biological deposits named IMP 537 and IMP 538 relating to Escherichia coli comprising an expression vector encoding for DBL1-CIDR domains and DBL1-DBLS domains were registered at the Collection Nationale des Cultures de Microorganismes (CNCM) under accession numbers I-2929 and I-2930 on August 30, 2002, respectively.
In a fourth embodiment, 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.
As used herein, the term "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.
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.
3. Antibodies In a fifth embodiment, 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. For example, the var O polypeptide, or antigenic fragments thereof, may be administered to an animal in order to induce the production ofpolyclonal antibodies. Alternatively, 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 ).
As mentioned above, the present invention is preferably directed to antibodies that specifically bind P. falciparum var O polypeptides, or fragments thereof. In particular, the invention features "neutralizing" antibodies. By "neutralizing" antibodies is meant antibodies that interfere with any of the biological activities of any of theP. falciparum var O polypeptides, particularly the ability of P, falciparum to induce the rosettinglautoagglutination 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. Once produced, 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 anitbodies that specifically recognize var0 proteins (or fragments var0), 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 var0 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 var0 expression levels, to determine the amount of var0 or fragment thereof in a biological sample and evaluate the presence or not of a var0 strain of Plasmodium. In addition, 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.
With respect to antibodies of the invention, 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 ones) of interest. As used herein, the term "relatively high affinity" means a binding affinity between the antibody and the protein of interest of at least 106 M-', and preferably of at least about 10' M-' and even more preferably 10a M~' to 10'° M-'.
Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which is common knowledge to one skilled in the art. As used herein, "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. (Q J Nucl Med 1998; 42: 225-241 ).
They 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 5 and Fab fragments by subjecting an IgG molecule to pepsin or papain cleavage respectively. Recombinant antibodies are also covered by the present invention.
Alternatively, 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 10 region. The antigen binding region is an antibody light chain variable domain or heavy chain variable domain. Typically, 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 JNucl Med 1998; 42: 225-241 ). Such a i5 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).
Alternatively, 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.
Preferably, the antibody of the invention is a human or animal immunoglobulin such as IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD carrying rat or mouse variable regions (chimeric) or CDRs (humanized or "animalized"). Furthermore, 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.
The term "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.
As mentioned above, the antibody of the invention is immunologically specific to the polypeptide of the present invention and immunological derivatives thereof. As used herein, the term "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. The term "immunological derivative"
therefore encompass "fragments", "segments", "variants", or "analogs" of a polypeptide.

4. Compositions and vaccines The polypeptides of the present invention, the polynucleotides coding the same, and polyclonal or monoclonal antibodies produced according to the invention, may be used in many ways for the diagnosis, the treatment or the prevention of Plasmodium related diseases and in particular malaria.
In a sixth embodiment, the present invention relates to a composition for eliciting an immune response or a protective immunity against aP. species. According to a related aspect, the present invention relates to a vaccine for preventing and/or treating a Plasmodium associated malarial disease. As used herein, the term "treating"
refers to a process by which the symptoms of malaria are alleviated or completely eliminated. As used herein, 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.
As used herein, the expression "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.
Further agents can be added to the composition and vaccine of the invention.
For instance, the composition of the invention may also comprise agents such as drugs, irnmunostimulants (such as a-interferon, p-interferon, y-interferon, granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator factor (M-CSF), interleukin 2 (/L2), interleukin 12 (/L_12), and CpG oligonucleotides), antioxidants, surfactants, flavoring agents, volatile oils, buffering agents, dispersants, propellants, and preservatives. For preparing such compositions, methods well known in the art may be used.
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 pertorm 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 compositionlvaccine 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.

5. Methods of use In a seventh embodiment, the present invention relates to methods for treating and/or preventing Plasmodium related diseases, such as malaria in a mammal are provided.
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. For instance, 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).
In a further embodiment, a diagnostic kit for the detection of the presence or absence of antibodies indicative of Plasmodium species is provided. Accordingly, the kit 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).
In a further embodiment, a diagnostic kit for the detection of the presence or absence of polypeptides indicative of Plasmodium species is provided.
Accordingly, the kit comprises:
- an antibody as defined above;
- a reagent to detect polypeptide-antibody immune complex;
- a biological reference sample lacking polypeptides that irnmunologically 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.
Yet, in another embodiment, an in vitro diagnostic method for the detection of the presence or absence of a polynucleotide indicative of Plasmodium species is provided, accordingly, the method 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.
Yet, according to a further embodiment, a diagnostic kit for the detection of the presence or absence of polynucleotide indicative of Plasmodium species is provided.
5 accordingly, the kit 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 10 - 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.
15 In a preferred embodiment, the oligonucleotide referred to in the diagnostic methods and kits hybridises to the polypeptide having SEQ ID NO 18 or fragments thereof.
The present invention will be more readily understood by referring to the following example. This example is illustrative of the wide range of applicability of the present 20 invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.

FY~MPI F
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 thesplenectomized 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 (rosettinglautoagglutination) 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.
In order to identify the DBL1 domain of the var gene specifically expressed by the O parasites, 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.
Several sets of primers described in the literature were tested: UNIEBP (Smith J.
D. et al., (1995), Cell 82:101-110), VarA5.1/VarE3.2 (Hernandez-Rivas et al., (1997) Mol. Cell. Biol. 17:604-611 ), and DBL1.1/DBL1.2 (Chen et al., (1998) J. Exp.
Med.
187:15-23), alpha AF/alpha BR (Taylor et al., (2000) MBP 105:13-23).
Following RT-PCR amplification using universal primers (alpha AF alpha BR), 69 DBL1 sequences (49 clones from the O cDNAs and 20 clones from the R cDNAs) were analyzed. In all, 18 different DBL1 sequences were obtained from these samples. This relatively important diversity indicates that the universal primers permit the identification of a large number of sequences. In fact, the parasites propagated in the splenectomized monkey develap asynchronously and the enrichment method, consisting of eliminating trophozoites and schizonts by sorbitol treatment and allowing rings to mature in culture over 24 hours, offered variable rates among samples. The purified 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.
In order to identify the relevant sequences, it was therefore necessary to analyze a great number of clones. Alignment of these 69 sequences evidenced specific majority DBL1 clones for parasites O and R. These specific sequences respectively represent 60% and 25% of clones O and R analyzed.
As illustrated below in Table 1, 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. For the R parasites, 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 DBL1 Sequences var O -varR -2 1.2........................._...._._....__...._2;
_._...._._._.___...__......._....._....__.__..
..~

~___.........._......_............._.___..._._.._...__.___~._....~

4 1..._...__.._........_......__.......__..._._._.~
._......._..
~..._..._..._...._._......_.. ....._.__.._.__._....._....__..___.

1..........,....._...._..._......._.._._..
___._.._.._.._..._..___...__~
L...~......_ . _ __...._...... ... _ _____._._._._..___.._.___.~

Total 49 20 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.
Using a set of UNIEBP degenerated primers, of less restrained specificity, different amplification profiles were again observed with O and R parasite cDNAs. A
450bp band was mainly detected in the O samples and inversely, a 500 by band was mainly detected in samples R confirming that the difference between O and R
parasites is at the phenotypic (gene expression) level. As predicted, amplification of genomic sequences result in identical profiles. Alignment with the var genes described in the literature indicates two different DBL3 domains amplified using the UNIEBP
primers.
This result was reproduced with 8 different parasite RNA preparations (4 Os' and 4 Rs'). The two bands corresponding to specific amplification products were sequenced.
As a result of these analyses, two specific domains of the var O gene : the domain and the DBL3 domain were identified. Comparison of these sequences with those described in the literature showed that the var O DBL1alpha domain identified presents a strong homology with that encoded by the var gene implicated in rosetting decribed by Rowe et a1.((1997) Nature 388:292-295) but little homology with the other DBL1alpha domain implicated in rosetting described by Chen et a1.((1998) J.
Exp. Med.
187:15-23) (Figure 2).
From these specific sequences, the inventors were able to define new primers and proceed with the sequencing of the entire var O gene. Two strategies were developed: (1 ) chromosome walking which permitted to obtain several fragments on either side of the identified domains, and (2) RT-PCR, which by combining specific primers and conserved primers generated two large fragments, a DBL1-DBL3 fragments and a DB3-exonll.
Analysis of these different fragments allowed the inventors to define an open reading frame of 7378 by disclosed in figure 3.
Exon 1 which codes for the extracellular part of the molecule, features five DBL

domains, one CIDR domain adjacent to the DBL1 domain and threeinterdomains (C2, ID3 and ID4). Alignment of the sequences with the DBL and CIDR domains described in Smith et al.'s phylogenetic study (2000) MBP 110:293-310) allowed the inventors to identify and class the var O domains according to the new nomenclature. As illustrated 5 in figure 1, 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, and finally, the CIDR corresponds to CIDRgamma.
The 2459 amino acid deduced var O protein sequence delimits the different 10 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 15 composite of domains, each of which shows a best identity match with a different PfEMP1 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) 20 Proc. Natl Acad. Sci. U.S.A. 96, 12743-12748). Var O differs substantially from the previously chracterized 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).
25 The deduced var O protein sequence has several important features. The DBL1alpha-CIDR1gamma association is atypical and has been so far described in only one case, namely the PfEMP1 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 PfEMP1 protein is restricted to this region.
30 Additional domains share substantial identity with domains from other PfEMP1 molecules that have been implicated in other binding specificities.
The var O PfEMP1 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 PfEMP1 is consistent with the incapacity to demonstrate binding to human CD36. This is particularly interesting because CD36 binding is a comman 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).
Intact infected red blood cells cytoadhere to dendritic cells via a CIDRalpha interaction. Upon binding on dendritic cells, they inhibit dendritic cell maturation and function (Urban B. et al. (1999) Nature 400, 73-77). In this regard, the absence of a CIDRalpha from the var O PfEMP1 protein is particularly interesting as it should present the unexpected advantage no such impairment of T cell function and hence a better immunogenicity of the head domain.
The various DBL domains, the CIDR domain, the variable inter-domains and the DBL1 alpha-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)PIysS, possessing the T7 polymerase gene under the control of the IPTG-inducible IacUVS promoter, were then transformed with these plasmids and used for overexpression.
Use of the pET system required the preparation of new inserts providing adequate cloning sites for unidirectional insertion into the pET22b vector. To date, only the DBL1 domain of the var genes has been implicated in red blood cell adhesion, however, certain associations of tandem domains have been observed (e.g.
DBLalpha CIDRalpha, DBLbeta C2). These associations might have a functional explanation. For these reasons, the DBLalpha-CIDR segment (Figure 1 ) was also expressed. The different constructs were isolated from the Escherichia coli K12 Novablue strain Novagene) endA1 hsdRl7 (rk,2 -- mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ !ac n ZOM15 : : Tn 10 (TcF~)J, then transformed into the expression strains BL21 (DE3) and BL21 (DE3)PIysS. These bacterial clones were registered at the Authorized International depositary known as the Collection Nationale de Cultures de Microorganismes {CNCM) on August 30, 2002 under accession numbers I-2929 (clone IMP 537- clone V) and I-2930 (clone IMP 538).
Clone N°IMP 529 Escherichia coli K12 Novablue strain ( Novagene) endA1 hsdRl7 (rk,2 ' mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ lac i° ZAM15 Tn10 (TcR)J carries plasmid pET22b-Clone G containing the DBL1 domain coding sequence e.g. nucleotides 283 to 1206 of the var O Palo Alto nucleotide sequence as shown in figure 15. The following set of amplification primers were used:
SEQ ID NO. 22: sens SDBLIB : 5'TAC 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.
Clone N°IMP 530 Escherichia coli K12 Novablue strain ( Novagene) endA1 hsdR17 (rk,2 - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+8+ lac ~° ZOM15 TnlO (TcR)J 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-sees 3DBL2X : 5'TGC AAT TTT TGC TTT CTC GAG TAA ATC CTT
GTA
TTT TTG TTC.
Clone N°IMP 531 Escherichia coli K12 Novablue strain ( Novagene) endA1 hsdRl7 (rk,2 - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+8+ lac i° Z~M15 TnlO (TcR)J carries plasmid pET22b-Clone B containing the C2 domain coding sequence, e.g. nucleotides 3301 to 3735 of the var O Palo Alto nucleotide sequence as shown in figure 17. The following set of amplification primers were used:
SEQ ID NO 26: Sens 51D2B : 5'TGG AAA CAA ATG GAT CCA AAA TAC AAG GAT TTA TAC;

SEQ ID NO 27: Anti-sens 31D2X : 5'CCA ATT TAC CTC GAG TTT TAT ATT GCA CCC ATA
TAT
TGA.
Clone N°IMP 532 Escherichia coli K12 Novablue strain ( Novagene) endA1 hsdRl7 (rk,z - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B' lac i° ZOM15 Tn10 (TcR)J 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 TG;
SEQ ID NO 29: Anti-sens 3DBL3X : 5'TTT TCC TTT CTC GAG TGT AAA TTT TTG GCT TCG
TTT
TTC.
Clone N°IMP 533 Escherichia coli K12 Novablue strain ( Novagene) endA1 hsdRl7 (rk,z - mk,z +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ lac ~Q

Tn10 (TcR)J carries plasmid pET22b-Clone D containing the ID3 domain coding sequence, e.g. nucleotides 4444 to 4896 of the var O Palo Alto nucleotide sequence as shown in figure 19. The following set of amplification primers were used SEQ ID NO 30: Sens 51D3B : AAA ACT CAA TAG GAT CCA CGA AGC CAA AAA TTT ACA
AGA;
SEQ ID NO 31: Anti-sens 31D3X : AGA AAC TTC CTC GAG TTT ACA TGT TTC CCC ATT
TTG
ATT.
Clone N°IMP 534 Escherichia coli K12 Novablue strain ( Novagene) endA1 hsdR17 (rk,z - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ !ac i° Z~M15 TnlO (TcR)J carries plasmid pET22b-Clone L containing the DBL4 domain coding sequence, e.g. nucleotides 4891 to 5799 of the var O Palo Alto nucleotide sequence as shown in figure 20. The following set of amplification primers were used 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.
Clone N°IMP 535 Escherichia coli K12 Novablue strain ( Novagene) endA1 hsdRl7 (rk,z - mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac jF' pro A+B+ lac ~° Z~M15 TnlO (TcR)J carries plasmid pET22b-Clone O containing the ID4 domain coding sequence, e.g. nucleotides 5779 to 6111 of the var O de Palo Alto nucleotide sequence as shown in figure 21. The following set of amplification primers were used SEQ ID NO 34: Sens 51D4B : GCC CAA TTG GAT CCA CAA AAC AAA GCA TAT AAA G;
SEQ ID NO 35: Anti-sans 31D4X : GAA ATT TTT CTC GAG ACA ACT ACC TAT ACC ATA
CT'f CTT.
Clone N°IMP 536 Escherichia coli K12 Novablue strain ( Novagene) endA1 hsdRl7 (rk~2 - mk~2 +) supE44 thi-1 recA 1 gyrA96 relA1 lac [F' pro A+B+ lac ~° ZOM15 TnlO (TcR)j carries plasmid pET22b-Clone M coding for the DBLS 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: Sans 5DBL5B : TGT AAG AAG TAG GAT CCA GGT AGT TGT CCA GAA;
SEQ ID NO 37: Anti-sans 3DBL5X : TAG TGT CTT CTC GAG TTC TTT ATC ATA TTT ATC
CTT
TTG AAT TTC.
Clone N°IMP 537 Novablue strain ( Novagene) endA1 hsdR17 (rk,2 -mk,2 +) supE44 thi-1 recA1 gyrA96 relA1 lac [F' pro A''8+ !ac i° 7~M15 : : TnlO
(TcR)J
carries plasmid pET22b-Clone V containing the DBL1-CIDR tandem domain, e.g.
coding sequence, e.g. nucleotides 283 to 2520 of the var O Palo Alto nucleotide sequence as shown in figure 23. The following set of amplification primers were used SEQ ID NO 38: Sans 5DBLIB : 5'TAC AAC GAG GAT CCA AAG CCT TGT TAT GGA AGG;
SEQ ID NO 39: Anti-sans 3CIDRX : 5' TCC TAT TTT AAA CCT CTC GAG ATT TTT ACC
TGT
ACA TGG.
Expression assays of the different domains were also conducted. The bacterial protein extracts, induced and non-induced, were analyzed by SDS-PAGE
(Coomassie blue staining), and western blot (detection with a monoclonal anti-his antibody).
Different expression rates are observed depending on the construct. The C2 domain is recognized by E1 variant var O infected monkey antibodies.
This collection of clones represents a unique tool to investigate by the methods known to the person skilled in the art the binding specificity of the various domains, identify the naturally immunogenic regions, and to devise a rational intervention and diagnosis based on that molecule.
As an example of the usefulness of such a compehensive array of sub-clones is the observation that interdomain 2 called C2 induces antibodies in monkeys infected with var O parasites. A collection of polyclonal and monoclonal antibodies that recognize the different epitopes encoded by the var O gene and its different domains may be produced by the methods known by the person skilled in the art.
Interventions based on this molecule will reduce the incidence of clinical 5 malaria by interfering with in vivo propagation, capillary obstruction and possibly reduce in this way systemic inflammation. A particularly novel aspect of the invention is the inclusion of the DBt-1 alpha-CI DR1 gamma expression product for further analysis, including of possible immunomodulatory activities. The invention concerns also the collection of individual domains, including the non 10 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.

Claims (35)

1. An isolated or purified polynucleotide having a nucleic acid sequence being at least 65% identical to any one of SEQ ID NO 1, 13 to 21 and fragments thereof.

2. An isolated or purified polynucleotide having a nucleic acid sequence being at least 80% identical to any one of SEQ ID NO 1, 13 to 21 and fragments thereof.

3. An isolated or purified polynucleotide having a nucleic acid sequence being at least 95% identical to any one of SEQ ID NO 1, 13 to 21 and fragments thereof.

4. An isolated or purified oligonucleotide which can be used as a primer for hybridization with a polynucleotide as defined in any one of claims 1 to 3.

5. The isolated or purified oligonucleotide of claim 4 comprising a nucleotide sequence selected from the group consisting of SEQ ID No 22 à 39.

6. An isolated or purified polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence of any one of claims 1 to 3 and fragments thereof.

7. An isolated or purified polypeptide having an amino acid sequence being at least 80% identical to SEQ ID NO 2 and fragments thereof.

8. An isolated or purified polypeptide having an amino acid sequence being at least 95% identical to SEQ ID NO 2 and fragments thereof.

9. An isolated or purified polypeptide having an amino acid sequence substantially the same or having 100% identity to SEQ ID NO 2 and fragments thereof.

10. An isolated and purified polypeptide comprising an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complement of the polynucleotide of any one of claims 1 to 3 and having the ability to induce cytoadherence in cells infected Plasmodium related species.

11. The isolated and purified polypeptide of any one of claims 6 to 10, comprising at least one amino acid sequence selected from the group consisting of amino acid sequence having SEQ ID NO. 3, SEQ ID NO.4, SEQ ID NO. 5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ
ID NO.12, and biologically active fragments thereof.

12. A cloning or expression vector comprising a polynucleotide sequence as defined in any one of claims 1 to 3.

13. The cloning or expression vector of claim 12, wherein said vector is capable of directing expression of the polypeptide encoded by said polynucleotide sequence in a vector-containing cell.

14. The cloning or expression vector of claim 13, is selected from the group consisting of pET22b- Clone G, pET22b-Clone A, pET22b-Clone B, pET22b-Clone C, pET22b-Clone D, pET22b-Clone G, pET22b-Clone L, pET22b-Clone M, and pET22b-Clone O.

15. A transformed or transfected cell containing the polynucleotide sequence as defined in any one of claims 1 to 3.

16. A transformed or transfected cell containing a cloning or expression vector of any one of claims 12 to 14.

17. The cell of claim 15 or 16, wherein said cell consists of a Escherichia coli bacterium.

18. The cell of claim 17, wherein the Escherichia coli bacterium is selected from the group consisting of the cells deposited under accession numbers CNCM I-2929 and CNCM I-2930.

19. An antibody that specifically binds to the isolated or purified polypeptide as defined in any one of claims 6 to 11 and/or fragments thereof.

20. The antibody of claim 19, wherein said antibody consists of a monoclonal or polyclonal antibody.

21. A composition comprising the isolated or purified polynucleotide of any one of claims 1 to 3; the isolated or purified polypeptide of any one of claims 6 to 11;
and/or the antibody of claim 19 or 20, and an acceptable carrier.

22. The composition according to claim 21, for preventing and/or treating a Plasmodium species related disease.

23. The composition according to claim 22, wherein the disease is malaria.

24. A vaccine comprising the isolated or purified polynucleotide of any one of claims 6 to 11; the isolated or purified polypeptide of any one of claims 6 to 11;
and/or the antibody of claim 19 or 20, and an acceptable carrier.

25. The vaccine according to claim 24, for preventing and/or treating malaria.

26. A method for treating and/or preventing malaria in a mammal, comprising the step of administering to the mammal an effective amount of:

-the isolated or purified polynucleotide as defined in any one of claims 1 to 3;

-the isolated or purified polypeptide as defined in any one of claims 6 to 11;

-the composition as defined in any one of claims 21 to 22;

-the antibody of claim 19 or 20;
and/or -the vaccine as defined in any one of claims 24 to 25.

27. An in vitro diagnostic method for the detection of the presence or absence of antibodies indicative of Plasmodium species, which bind with the polypeptide according to any one of claims 6 to 11 to form an immune complex, comprising the steps of a) contacting the polypeptide according to any one of claims 6 to 9 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).

28. The method of claim 27, wherein the Plasmodium species consist of P.
falciparum.

29. A diagnostic kit for the detection of the presence or absence of antibodies indicative of Plasmodium species, comprising:

- a polypeptide according to any one of claims 6 to 9;

- a reagent to detect polypeptide-antibody immune complex;

- a biological reference sample lacking antibodies that immunologically bind with said peptide; and - a comparison sample comprising antibodies which can specifically bind to said peptide;

wherein said polypeptide, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.

30. The diagnostic kit according to claim 29, wherein the P. species consists of P.
falciparum.

31. 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 claim 18 or 19 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).

32. 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 according to claim 4 or 5 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.

33. The method of claim 31 and 32, wherein the Plasmodium species consist of P.
falciparum.

34. A diagnostic kit for the detection of the presence or absence of polypeptides indicative of Plasmodium species, comprising:

- an antibody according to any one of claims 19 and 20;

- 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.

35. A diagnostic kit for the detection of the presence or absence of polynucleotide indicative of Plasmodium species, comprising:

- at least one oligonucleotide according to claim 4 or 5;

- 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.