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  Pharmaceutical Patents  

 

Title:  Compounds useful in the diagnosis and treatment of pregnancy-associated malaria
United States Patent: 
7,745,580
Issued: 
June 29, 2010

Inventors:
 Theander; Thor Grundtvig (Ishoj, DK), Salanti; Ali (Copenhagen, DK), Hviid; Lars (Copenhagen S, DK), Staalso; Trine (Copenhagen NV, DK), Jensen; Anja Tatiana Ramstedt (Bronshoj, DK), Lavstsen; Thomas (Copenhagen O, DK), Dahlback; Madelaine (Malmo, SE)
Assignee:
  Kobenhavns Universitet (Copenhagen, DK)
Appl. No.:
 10/543,312
Filed:
 December 30, 2003
PCT Filed:
 December 30, 2003
PCT No.:
 PCT/DK03/00938
371(c)(1),(2),(4) Date:
 August 18, 2006
PCT Pub. No.:
 WO2004/067559
PCT Pub. Date:
 August 12, 2004


 

Pharm Bus Intell & Healthcare Studies


Abstract

The present invention relates to nucleic acid molecules related to the var2csa gene family as well as amino acid sequences encoded by such nucleic acid molecules with respect to their role in mediating adhesion of infected red blood cells to chondroitin sulphate A (CSA) in the placenta which is characteristic for the pathogenesis of pregnancy associated malaria (PAM). Accordingly, The invention provides the use compounds that are related to VAR2CSA polypeptides var2csa nucleic acid molecules as medicaments, as well as it provides pharmaceutical compositions, in particular immunological compositions and vaccines, hereunder nucleotide-based vaccines comprising these compounds. In addition, the invention provides the use of the compounds mentioned for the manufacture of compositions, such as immunogenic compositions. Other aspects of the invention relates to methods of treatment and prevention of pregnancy associated malaria wherein these methods are based on the nucleic acid molecules and polypeptides the invention. As these compounds can also be used as biotechnological tools the invention provides in vitro diagnostic methods and kits comprising reagents and IgGs/antibodies designated to the use in such methods. The invention also relates to methods of identifying agents capable of modulating the VAR2CSA dependent adhesion to CSA and agent capable of interacting with VAR2CSA. Finally, a method for identifying polypeptides, which will induce a specific IgG/antibody response upon administration to a subject is provided by the invention.

Description of the Invention

The inventive concept disclosed in the present application is based on the unexpected observation that the mRNA and protein expression of a specific Plasmodium falciparum var gene, var2csa, a member of an unusual class of PfEMP-1 types is up-regulated in all parasite lines and clones selected for CSA adhesion and expressed at high levels by placental parasites. This gene product is gender specifically and parity dependently recognised by immune serum from malaria-endemic areas. These observations indicate that proteins of the VAR2CSA family encoded by var2csa-type var genes are responsible for adhesion of iRBC to CSA. It also follows from these findings that such proteins are useful as therapeutic and prophylactic agents as well as biological tools and diagnostic agents for the study, treatment and prevention of PAM malaria.

Polypeptide Molecules of the invention

In its broadest aspect, the present invention relates to a polypeptide comprising at least one amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 6 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell epitope; with the proviso that the amino acid sequence of SEQ ID NO.: 2 is excluded.

The amino acid sequence SEQ ID NO.: 2 comprises sequences encoded by exon I and exon II, 6 DBL domains, a transmembrane domain and the conserved ATS domain. For the present and any of the following aspects of the invention it applies that the ATS domain could be excluded from the scope of any embodiments of the present invention. The ATS domain consists of amino acids No. 2667 to 3056 of SEQ ID NO.: 2.

Said sub-sequences may be at least 100 amino acids in length and at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 2.

For the present and any of the following aspects of the invention it applies that the preferred polypeptides of the invention have the ability to bind to CSA and may further be subject to gender-specific and parity dependent recognition by antibodies in sera isolated from subjects exposed to Plasmodium falciparum.

The predicted amino acid sequence of VAR2CSA in the parasite line NF54 is provided in the sequence listing as SEQ ID NO.: 2. For all the aspects of the invention, it is apparent that the polypeptides of the invention, which form the basis of the described embodiments of the invention may be less or equal to any length between 9-1250 amino acids, such as but not limited to less than or equal to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250 amino acids in length.

With respect to all aspects of the invention it may be preferred that the polypeptides of the invention may have a length of 6-10, 6-20, 6-30, 6-40, 6-50, 6-60, 6-70, 6-80, 6-90, 6-100, 6-110, 6-120, 6-130, 6-140, 6-150, 6-160, 6-170, 6-180, 6-190, 6-200, 6-225, 6-250, 6-275, 6-300, 6-350, 6-400, 6-450, 6-500, 6-5 600, 6-700, 6-800, 6-900, 6-1000 or 6-1250 amino acids.

In addition to these fragments or sub-sequences of the polypeptide of the invention larger proteins comprising such sub-sequences as part of their sequence, are also embodiments of the present invention.

Preferred embodiments of the present invention include specific sub-sequences of the polypeptide of the invention having a minimum length of 6 amino acids such as sub-sequences that are at least 100 amino acids long. In even more preferred embodiments of the invention, these sub-sequences can be shown by known molecular biological techniques to be involved in the interaction with endothelial receptors, hereunder CSA. It is anticipated that relatively short sequences within the VAR2CSA protein are responsible for mediating adhesion to CSA. In particular, it is possible that certain DBL domains or parts hereof are responsible for the adhesion. In other preferred embodiments of the invention, the sub-sequences of the polypeptide of the invention can be shown to possess one or more antigen epitopes. In particular, such epitopes may be B-cell epitopes. Optionally, the sub-sequences may also comprise one or more T-cell epitopes alone or in combination with the B-cell epitopes. Finally, also larger polypeptides comprising the polypeptide of the invention or sub-sequences hereof with antigen epitopes and/or sequences involved in interaction with CSA are embodiments of the present invention.

It is also apparent that the polypeptide sequences of the invention can be present in the form of fusion proteins. In a further preferred embodiment, this fusion protein will comprise polypeptide sequences, which will facilitate the purification or detection of the protein. These polypeptide sequences may be but are not limited to tags that will facilitate purification and detection using commercially available systems such as the HA- ,-c-myc, His or GST tags.

The polypeptide embodiments of the present invention can therefore exhibit a vast degree of sequence identity to the full-length VAR2CSA sequence. It can for instance be appreciated that a fusion protein carrying within its sequence one or more B-cell epitopes and or regions of the polypeptide of the invention that are involved in adhesion to CSA will have a relatively low overall degree of sequence identity to full-length VAR2CSA. For all the aspects of the invention, it is thus apparent that the polypeptides of the invention may include sequences, which show anywhere between 1-100% sequence identity, such as at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or preferably 100% sequence identity to VAR2CSA or a fragment or sub-sequence thereof.

Preferred embodiments of the invention comprise a fragment of the polypeptide of the invention that is involved in interaction with endothelial receptors such as CSA and thus exhibits adhesion to CSA. Preferably, the sequence has at least 70% sequence identity to a region of comparable length within the sequence of SEQ ID NO.: 2.

A more preferred embodiment pertains to an amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 20 amino acids with the proviso that SEQ ID NO.: 2 and sub-sequences of a) and b), which, when aligned to the best possible fit with SEQ ID NO.: 2, comprise a region which align with less than 90% sequence identity to amino acids No. 2602-2622 of SEQ ID NO.: 2, be excluded.

It is further preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite. It is equally preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

In particularly preferred embodiments the sub-sequence comprises at least one B-cell epitope and/or at least one T-cell epitope, and in other particularly preferred embodiments it comprises one or more GAG-binding motifs.

It is further preferred that the amino acid sequence does not comprise a CIDR domain or DBL-.gamma. domain and that the amino acid sequence is gender specifically recognised. Finally, It is preferred that the amino acid sequence is recognised in a parity dependent manner. In one embodiment of the invention, the sub-sequences of a) and b) are at least 100 amino acids in length and at least 80% identical to a region of comparable length within the sequence of SEQ ID NO.: 2.

It is understood that the polypeptide fragments of the invention may possess one or more types of post-translational modifications when expressed on the cell surface. These modifications may comprise, but are not limited to, glycosylation, phosphorylation, acylation, cross-linking, proteolytic cleavage, linkage to an antibody molecule, a membrane molecule, or another ligand.

The embodiments of the present invention thus relate to polypeptides of the PfEMP1 class or sub-sequences hereof as well as nucleic acid molecules encoding such polypeptides or sub-sequences, wherein said polypeptides and sub-sequences comprise structures that are involved directly or indirectly in the binding to CSA. The var2csa gene is a member of an unusual class of var genes and, in their widest perspective, the embodiments of the invention thus relate to nucleic acid molecules, which are characteristic in that they do not belong to the var1 gene subfamily as defined in Salanti et al. 2002. Furthermore, nucleic acid molecules, which are complementary to the nucleic acid molecules of the invention as described above as well as polypeptides encoded by these nucleic acid molecules are within the scope of the invention.

Nucleic Acid Molecules

One embodiment of the present invention relates to a nucleic acid molecule comprising at least one nucleotide sequence selected from the group consisting of a) SEQ ID NO.: 1 or a sequence complementary thereof; and b) a nucleotide sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 18 nucleic acids; and d) sub-sequences of a) or b) which comprises at least one sequence encoding a B-cell epitope; with the proviso that the nucleotide sequence of SEQ ID NO.: 1 is excluded.

The nucleic acid sequence SEQ ID NO.: 1 comprises exon I and exon II. For the present and any of the following aspects of the invention it applies that the exon II could be excluded from the scope of any embodiments of the present invention. The exon II domain consists of amino acids No. 8001 to 9171 of SEQ ID NO.: 1

It further applies that the nucleic acid sequence having the EMBL database accession number BQ739499; PfESToab46 g01.yl Plasmodium falciparum 3D7 asexual cDNA Plasmodium DE falciparum cDNA 5' similar to TR:Q26030 Q26030 VARIANT SURFACE PROTEIN, deposited by Tang, K. et al. could be excluded from the scope of any embodiment of the present invention.

In particular, the nucleic acid molecule may comprise sub-sequences, which are at least 300 nucleotides in length and at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 1.

The CDNA sequence encoding VAR2CSA in the parasite line NF54 is provided in the sequence listing as SEQ ID NO.: 1. Again, it is apparent for all the aspects of the invention that the nucleic acid molecules of the invention may be less than or equal to any length between 9-4500 nucleotides, such as less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000, 4500 nucleotides in length.

Still with respect to all aspects of the invention it may be preferred that the nucleic acid molecules of the invention may have a length of 6-10, 6-20, 6-30, 6-40, 6-50, 6-60, 6-70, 6-80, 6-90, 6-100, 6-110, 6-120, 6-130, 6-140, 6-150, 6-160, 6-170, 6-180, 6-190, 6-200, 6-225, 6-250, 6-275, 6-300, 6-350, 6-400, 6-450, 6-500, 6-600, 6-700, 6-800, 6-900, 6-1000, 6-1250, 6-1500, 6-1750, 6-2000, 6-2500, 6-3000, 6-3500, 6-4000 or 6-4500 nucleotides.

In some embodiments of the invention, sub-sequences of the nucleic acid molecules of the invention have a minimum length of 18 nucleic acids and in other embodiments these sub-sequences are at least 300 nucleotides long. Preferred nucleic acid embodiments further Include nucleic acids encoding fragments of the polypeptide of the invention that are involved in interaction with endothelial receptors such as CSA and thus exhibit adhesion to CSA. In addition, it is an object of preferred embodiments that sub-sequences of the nucleic acid molecule of the invention comprise nucleic acids encoding one or more B-cell epitopes and/or one or more T-cell epitopes.

Some characteristic structures lie within the peptide sequence of VAR2CSA and therefore also within the nucleotide sequence encoding this peptide sequence. Such structures comprise, but are not necessarily limited to, a string of at least 2 consecutive DBL domains as the N-terminal domains. On the other hand, some common features have been identified for proteins encoded by the var1 gene subfamily including the CIDR domains and the DBL-.gamma. domains. These features are not found within the amino acid sequence of VAR2CSA.

Further embodiments comprise nucleic acid molecules that complement full-length var2csa or sequences identical in part hereto as well as nucleic acid sequences that complement fragments of full-length var2csa or sequences identical in part hereto. Preferred complementary nucleic acid molecules of the invention comprise nucleic acid molecules that are complementary to fragments of var2csa, which have a nucleotide sequence that encodes a polypeptide or parts of a polypeptide that are involved in interaction with CSA. Additionally, preferred complementary nucleic acid molecules of the invention are complementary to sequences encoding one or more B-cell epitopes and/or one or more T-cell epitopes.

As discussed for the polypeptide-based compounds of the invention it is also apparent that the nucleotide based embodiments may represent only part of the full-length sequence. In addition these nucleotide sequences may be present in combination with exogenous sequences. For all the aspects of the invention, it is thus apparent that the nucleic acids molecules of the invention may include sequences that have anywhere between 1-100% sequence identity to the full-length sequence of var2csa, such as at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or, preferably, 100% sequence identity to var2csa or a fragment or sub-sequence thereof.

Preferred embodiments of the invention comprise a nucleotide sequence that encodes a polypeptide, which is involved in interaction with endothelial receptors such as CSA and thus exhibits adhesion to CSA. The nucleotide sequence may have at least 70% sequence identity to a region of comparable length within the sequence of SEQ ID NO.: 1.

More preferred embodiment of the present invention pertains to a nucleotide sequence selected from the group consisting of a) SEQ ID NO.: 1; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 30 nucleic acids with the proviso that SEQ ID NO.: 1 and sequences and sub-sequences of a) or b), which, when aligned to give the best possible fit with SEQ ID NO.: 1, comprise a region of 70 nucleic acid residues or less which align with less than 90% sequence identity to nucleic acids No. 7800-8001 of SEQ ID NO.: 1, and or comprise a region of 40 nucleic acid residues or less which align with less than 90% sequence identity to nucleic acids No. 600-660 of SEQ ID NO.: 1, and/or comprise a region of 30 base pairs which align with less than 90 % sequence identity to nucleic acids No.: 1495-1540 of SEQ ID NO.:1, be excluded.

Especially preferred is a nucleic acid sequence which is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

In particularly preferred embodiments the sub-sequence encodes at least one B-cell epitope and/or at least one T-cell epitope, and in other particularly preferred embodiments it encodes one or more GAG-binding motifs.

It is further preferred that the nucleic acid sequence does not encode a sequence comprising a CIDR domain or DBL-.gamma. domain and that the nucleic acid sequence encodes an amino acid sequence that is gender specifically recognised. Finally, it is preferred that nucleic acid sequence encodes an amino acid sequence, which is recognised in a parity dependent manner.

In one embodiment of the invention, the sub-sequences of a) and b) are at least 300 nucleic acids in length and at least 80% identical to a region of comparable length within the sequence of SEQ ID NO.: 1.

It is to be understood that the nucleotide sequence of SEQ ID NO.: 1 when present within the genome of the intact Plasmodium falciparum parasites as well as the polypeptide sequence of SEQ ID NO.: 2 when present in or on the surface of intact red blood cells infected with P. falciparum are excluded from the scope of the present invention. This applies to all embodiments of the invention described in the present application. Compounds of the invention may however comprise sub-sequences of SEQ ID NO.: 1 and sub-sequences of SEQ ID NO.: 2 isolated and/or purified from the Plasmodium parasites or infected RBC. In addition, recombinant polypeptides comprising sub-sequences of the amino acid sequence of SEQ ID NO.: 2 may be generated by use of the above-mentioned nucleic acid embodiments. These can be cloned into vectors by the use of cloning techniques known in the art. The sequence encoding the polypeptide of interest is thereby linked to a heterologous promoter sequence. It may be preferred to optimise the codon context and codon pairing for the particular expression system. With respect to the polypeptide embodiments of the invention the incorporation of a secretory leader sequence may also be of use. The vector can be an expression vector in any of the mammalian, yeast, amphibian, insect, parasite, plant, or bacterial expression systems known in the art. It is therefore apparent that, with the exception of Plasmodium infected RBC, prokaryotic and eukaryotic cells hereunder mammalian cells and transformed cell lines as well as cells in animals possessing nucleotide and/or amino acid embodiments described herein, are within the scope of the present invention.

Var2csa or homologues hereof can be expressed in different expression systems eukaryotic or prokaryotic. In some instances it could be an advantage to recodonize the var2csa sequence or homologues hereof for expression in other hosts than P falciparum. in one example the sequence can be optimised for expression in different yeast systems, human cell in vitro systems, insect cell systems, in these systems in could be an advantage to purify the protein before using it as an vaccine or therapeutically. In another example the sequence could be optimised for expression in plant derived systems--from these transgenic plants the whole plant organism might be ingested to activate the immune system against PAM parasites, or the proteins could be purified. Plant expression systems could for example be transgenic potatoes, soya been, tobacco, banana, crops used for animal feeding, or other plants that can be made transgenic with known methods. Var2csa or homologues hereof can be delivered to the plant by different means, in one case the DNA can be transferred by Agrobacterium T-DNA vectors or by shooting the DNA inside the nucleus of the plant cell. Transient expression can be obtained with different virus vectors transfection the plant cell.

In a further preferred embodiment nucleic acid sequence is a re-codonised sequence. Particularly preferred are sequences that are recodonised in order to enhance or optimise expression of the resulting protein or polypeptide in a given expression system. Accordingly, in an even more preferred embodiment of the present invention the nucleic acid sequence has been recodonised in order to enhance expression in an expression system selected from the group consisting of: Yeast systems, human cell in vitro systems, insect cell systems and plant expression systems.

An example of such a recodonised nucleic acid is provided in the form of SEQ ID NO.: 3. This sequence represents the entire exon 1 of VAR2CSA including nucleic acids 1 to 8000 subjected to full recodonisation facilitating the expression of VAR2CSA in eucaryotic organisms. Accordingly, a currently most preferred embodiment of the invention is the recodonised sequence of SEQ ID NO.: 3.

Propagation of the cells or cell lines described above may be performed with the intention of providing recombinant forms of one or more of the nucleic acid or polypeptide embodiments of the invention in amounts that are sufficient for further processing or purification. It is therefore within the scope of the present invention to provide preparations of compounds, which comprise polypeptides of the invention as well as nucleic acid molecules encoding these polypeptides. Preparations of such compounds may have a desired degree of purity referring to the relative amounts of the desired polypeptide and for instance whole cell proteins and unwanted variants of the desired polypeptide as defined above. The existence of a wide range of protein purification and concentration techniques is known to the skilled artisan. These techniques include gel electrophoresis, ion-exchange chromatography, affinity and immunoaffinity chromatography, ceramic hydroxyapatite chromatography, differential precipitation, molecular sieve chromatography, isoelectric focusing, gel filtration, and diafiltration.

For the various types of chromatography, the desired molecules are suspended in a buffer, which promotes adhesion of the molecules to the active surface of the resin and are then applied to the chromatography column. Removal of contaminants is performed by washing the resin in a buffer of intermediate ionic strength or pH. Elution of the desired molecules is performed by changing the ionic strength or pH of the buffer to values that will promote the dissociation of the molecules from the active surface of the resin used. In the case of immunoaffinity chromatography, the polypeptide may be purified by passage through a column containing a resin to which is bound antibodies which are specific for at least a portion of the polypeptide. Furthermore, His- or GST tags may be added to the polypeptides of the invention. Subsequently, the resulting fusion proteins can be purified by affinity chromatography on for instance glutathione sepharose 4B and HIS tag Metal Chelate Affinity Chromatography.

It is readily apparent that a person skilled in the art can create a nucleic acid molecules of virtually any length by ligating a nucleic acid molecule encoding VAR2CSA or any part thereof to an exogenous nucleotide sequence. Recombinant nucleic acid molecules generated by this approach are embodiments of the invention. A recombinant construct can be capable of replicating autonomously within a host cell or, alternatively, it can become integrated into the chromosomal DNA. Such a recombinant nucleic acid molecule can comprise a sequence of genomic DNA, cDNA, synthetic or semi-synthetic origin. Again, It is preferred that such nucleic acid molecules are encoding one or more B-cell epitopes and/or one or more T-cell epitopes. The nucleic acid embodiments of the present invention can be altered by genetic engineering so as to introduce substitutions, deletions and/or additions. In preferred embodiments of the invention, these alterations will provide for sequences encoding functionally equivalent molecules or molecules with the same or improved properties. Such changes of the polypeptide embodiments can be generated using techniques that are known to a person skilled in the art, including random mutagenesis and site-directed mutagenesis.

The use of recombinant polypeptides of the invention may be preferred when it is required that the preparations of these polypeptides are essentially free of any other antigen with which they are natively associated, i.e. free of any other antigen from Plasmodium parasites. As an alternative this may also be accomplished by synthesizing the polypeptide fragments by the well-known methods of solid or liquid phase peptide synthesis.

In some aspects, the present invention can be used to both inhibit the adhesion of iRBC to CSA and to generate an immune response directed at var2csa. It is therefore within the scope of the invention to provide uses of any of the polypeptides of the present invention as medicaments that are therapeutically or prophylactically useful or both.

Medicaments

An embodiment of the present invention thus relates to at least one amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 6 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell and/or T-cell epitope for use as a medicament.

It is preferred, that these sub-sequences have a minimum length of 6 amino acids and that they are at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 2. it is even more preferred that sub-sequences are at least 100 amino acids in length.

A more preferred embodiment pertains to an amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 10 amino acids for use as a medicament.

It may be preferred that sub-sequence of a) or b) have a minimum length of 20 amino acids.

It is further preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite. It is equally preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

In particularly preferred embodiments the sub-sequence comprises at least one B-cell epitope and/or at least one T-cell epitope, and in other particularly preferred embodiments It comprises one or more GAG-binding motifs.

It is further preferred that the amino acid sequence does not comprise a CIDR domain or DBL-.gamma. domain or is derived from a gene or a protein which does not comprise a CIDR domain or DBL-.gamma. domain, and that the amino acid sequence is gender specifically recognised. Finally, it is preferred that the amino acid sequence is recognised in a parity dependent manner.

It readily appears that any feature and characteristic that is described for such an amino acid sequence for use as a medicament will also apply by analogy to a method for prevention or treatment of a disease or disorder. A method for prevention or treatment of a disease or disorder constitutes an additional aspect of the present invention. A method for prevention or treatment of pregnancy associated malaria is a preferred embodiment of the present invention.

Alternatively, therapeutic and prophylactic effects can be obtained as a result of the expression of polypeptides of the invention within a diseased subject or a subject at risk of contracting malaria. Therefore, it is also within the scope of the invention to provide uses of any of the nucleic acid molecules of the present invention as medicaments that are therapeutically or prophylactically useful or both.

A preferred embodiment of the present invention thus relates to a nucleic acid molecule comprising at least one nucleotide sequence selected from the group consisting of a) SEQ ID NO.: 1 or a sequence complementary thereof; and b) a nucleotide sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 18 nucleic acids; and d) sub-sequences of a) and b) which comprise at least one sequence encoding a B-cell epitope for use as a medicament.

These sub-sequences have a minimum length of 18 nucleic acids and they may be at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 1. it is even more preferred that sub-sequences are at least 300 nucleotides in length.

An equally preferred embodiment of the present invention pertains to a nucleotide sequence selected from the group consisting of a) SEQ ID NO.: 1; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 30 nucleic acids for use as a medicament.

Especially preferred is a nucleic acid sequence which is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

In particularly preferred embodiments the sub-sequence encodes at least one B-cell epitope and/or at least one T-cell epitope, and in other particularly preferred embodiments it encodes one or more GAG-binding motifs.

It is further preferred that the nucleic acid sequence does not encode a sequence comprising a CIDR domain or DBL-.gamma. domain and that the nucleic acid sequence encodes an amino acid sequence that is gender specifically recognised. Finally, it is preferred that nucleic acid sequence encodes an amino acid sequence, which is recognised in a parity dependent manner.

In a further preferred embodiment the nucleic acid sequence is a re-codonised sequence. Particularly preferred are sequences that are recodonised in order to enhance or optimise expression of the resulting protein or polypeptide in a given expression system. Accordingly, in an even more preferred embodiment of the present invention the nucleic acid sequence has been recodonised in order to enhance expression in an expression system selected from the group consisting of: Yeast systems, human cell in vitro systems, insect cell systems and plant expression systems

An example of such a recodonised nucleic acid is provided in the form of SEQ ID NO.: 3. This sequence represents the entire exon 1 of VAR2CSA including nucleic acids 1 to 8000 subjected to full recodonisation facilitating the expression of VAR2CSA in eukaryotic organisms. Accordingly, a currently most preferred embodiment of the invention is the recodonised sequence of SEQ ID NO.: 3.

Pharmaceutical Compositions

Additional aspects of the present invention relate to pharmaceutical compositions based on any of the polypeptide embodiments of the invention. Preferably, such a composition comprises at least one amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 6 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell epitope.

It is preferred, however, that the sub-sequences have a minimum length of 6 amino acids and that they are at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 2. it is even more preferred that sub-sequences are at least 100 amino acids in length.

Alternatively, the pharmaceutical composition according to the present invention may be based on any of the nucleotide embodiments of the invention. In a preferred embodiment, the pharmaceutical composition comprises a vector containing at least one nucleotide sequence selected from the group consisting of a) SEQ ID NO.: 1 or a sequence complementary thereof; and b) a nucleotide sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 18 nucleic acids; and d) sub-sequences of a) and b) which comprise at least one sequence encoding a B-cell epitope.

It is preferred that these sub-sequences have a minimum length of 18 nucleic acids and that they are at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 1. it is even more preferred that sub-sequences are at least 300 nucleotides in length.

In a particularly preferred embodiment the pharmaceutical composition as described above is an immunogenic composition. It is further preferred that the immunogenic composition comprises an amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 10 amino acids

It is even more preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite. It is equally preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a parasite that has been selected for its ability to mediate adhesion to CSA.

In particularly preferred embodiments the sub-sequence comprises at least one B-cell epitope, and in other particularly preferred embodiments it comprises one or more GAG-binding motifs.

It is further preferred that the amino acid sequence does not comprise a CIDR domain or DBL-.gamma. domain and that the amino acid sequence is gender specifically recognised. Finally, it is preferred that the amino acid sequence is recognised in a parity dependent manner.

Alternatively the pharmaceutical composition may comprise a nucleic acid sequence selected from the group consisting of a) SEQ ID NO.: 1; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 30 nucleic acids

Especially preferred is a nucleic acid sequence which is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placenta parasite.

In particularly preferred embodiments the sub-sequence encodes at least one B-cell epitope and/or at least one T-cell epitope, and in other particularly preferred embodiments it encodes one or more GAG-binding motifs.

It is further preferred that the nucleic acid sequence does not encode a sequence comprising a CIDR domain or DBL-.gamma. domain and that the nucleic acid sequence encodes an amino acid sequence that is gender specifically recognised. Finally, it is preferred that nucleic acid sequence encodes an amino acid sequence which is recognised in a parity dependent manner.

It is preferred that the immunogenic composition described above is characterised in that it induces an IgG/IgM antibody response.

In a further preferred embodiment the nucleic acid sequence is a re-codonised sequence and in a most preferred embodiment of the invention the nucleic acid sequence is the recodonised sequence of SEQ ID NO.: 3.

In a specially preferred embodiment, any of the pharmaceutical compositions described in the present application may further comprise a pharmaceutically acceptable carrier and/or an adjuvant.

Pharmaceutical compositions comprising the nucleotide and polypeptide embodiments of the invention can be produced by conventional techniques so that the said sequences are present as monomeric, multimeric or multimerised agents. Furthermore, antibodies generated from the polypeptide embodiments of the invention may constitute part of such pharmaceutical compositions. In addition to the active ingredients, pharmaceutical compositions may further comprise one or more physiologically acceptable carriers, proteins, supports, adjuvants as well as components that may facilitate the delivery of the active components of the compositions. As described above, a large number of adjuvants are available including but not limited to Freund's adjuvant, mineral gels such as aluminium hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. As a pharmaceutical composition, the nucleic add and peptide embodiments of the invention will be purified and processed through one or more formulation steps. A large variety of formulation buffers will be physiologically acceptable, such as phosphate, citrate, and other organic acids.

It is further understood that a pharmaceutical composition must be clinically safe. More specifically, it must be free of virus and bacteria that can cause infection upon administration of the composition to a subject. It will therefore be necessary to process the composition through on or more steps of virus filtration and/or inactivation. The removal of virus by filtration can be obtained by passing the composition through a nanofilter whereas virus inactivation can be accomplished by the addition of various detergents and/or solvents or other antiviral compounds to the composition.

The polypeptide embodiments of the invention may be used in their purified form to generate various types of antibodies, and it is understood that such antibodies will also be considered as compounds of the invention. These antibodies may include, but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. A person skilled in the art knows that antibodies can be produced by immunisation of various hosts including goats, rabbits, rats, and mice.

Alternatively, antibodies, such as chimeric antibodies, and anybody fragments corresponding to antibodies generated in response to immunisation with the nucleic acid sequences or amino acid sequences of the invention or parts of such antibodies can be produced by recombinant processes well known in the art. Preferred antibody fragments do not contain the Fc region of the antibody molecule. The Fc region is responsible for effector functions of the immunoglobulin (Ig) molecule such as complement fixation, allergic responses and killer T cell activation. The smaller size of the antibody fragment may help improve tissue bloavailability, which may be critical for better dose accumulation in acute disease indications. Furthermore, they have reduced immunogenicity, they do not induce precipitation (Fab only) and they can be used for a variety of in vivo applications and immunoassays.

Antibody fragments can be produced via recombinant methods creating single chain antibodies ("ScFv"), in which the heavy and light chain Fv regions are connected, or by enzymatic digestion of whole antibody.

In particular, Fabs can be converted to whole Ig molecules. The light-chain gene and variable gene fragment of the heavy-chain sequence of each clone can be inserted into a eukaryotic expression vector containing a Ig constant region gene, for instance of human origin.

Such chimeric antibodies, which are of partially human origin are less immunogenic than wholly murine MAbs, and the fragments and single chain antibodies are also less immunogenic. All these types of antibodies are therefore less likely to evoke an immune or allergic response. Consequently, they are better suited for in vivo administration in humans than wholly animal antibodies, especially when repeated or long-term administration is necessary.

Humanized antibodies have a greater degree of human peptide sequences than do chimeric antibodies. In a humanized antibody, only the complementarity determining regions (CDRS) which are responsible for antigen binding and specificity are animal derived and have an amino acid sequence corresponding to the animal antibody, and substantially all of the remaining portions of the molecule (except, in some cases, small portions of the framework regions within the variable region) are human derived and correspond in amino acid sequence to a human antibody. In addition to chimeric antibodies, such humanised antibodies may be preferred for therapeutic applications according to the present invention.

The term `immunisation` refers to the injection of a polypeptide with immunogenic properties. Depending on the host species various types of adjuvants can be used in order to increase the immunological response including but not limited to Freund's adjuvant, mineral gels such as aluminium hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.

An important aspect of the present invention pertains to an antibody or antiserum induced in response to one or more amino acid sequences selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 10 amino acids and/or one or more nucleic acid sequences nucleic acid sequence selected from the group consisting of a) SEQ ID NO.: 1; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 30 nucleic acids

A preferred embodiment of this aspect of the invention pertains to an antibody, which is capable of binding to a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

Another preferred embodiment pertains to an antibody, which is capable of binding specifically to a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite. In this context, the term `specific binding` indicates that the antibody recognises a panel of placental parasites expressing VSA-PAM to a significantly higher level than a panel of non-placental parasites as determined by flow cytometry (Staalsoe, et al. 2001).

Additionally, preferred embodiments of this aspect of the invention pertains to antibodies or parts of antibodies which are capable of preventing or reducing the binding of erythrocytes to CSA. It is conceivable that antibodies generated in response to immunisation with one or more of the nucleic acid sequences or amino acid sequences of the present invention when present in a sufficiently high concentration will provide a hindrance of the VAR2CSA dependent adhesion to CSA. In particular, antibodies of the IgG class may be used for this purpose. Furthermore, based on the molecular structures of the variable regions of the antibodies according to the present invention, a skilled person will be able use molecular modelling and rational molecular design to generate and screen small molecules which mimic the molecular structures of the binding region of the antibodies and prevent or inhibit the adhesion of infected erythrocytes to CSA.

In some embodiments of the present invention, it is preferred to use shorter sequences of the polypeptide of the invention fused to a powerful immunogenic molecule such as keyhole limpet hemocyanin resulting in the production of antibodies against this chimeric molecule. Accordingly, antibodies capable of recognising VAR2CSA can be produced by injection of synthetic peptides consisting of 14 to 150 amino acids corresponding to a particular sequence of the VAR2CSA polypeptide. As an alternative, a more diverse set of antibodies can be generated by injection of a purified polypeptide embodiment of the invention.

As suggested above, monoclonal antibodies directed against a fragment of VAR2CSA, such as a purified polypeptide embodiment of the invention, can be produced using any of the conventional techniques that provide for the production of antibodies from cell lines in continuous culture. These techniques include the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.

It will be readily appreciated that polypeptides of the invention can be incorporated into vaccines capable of inducing protective immunity against a specific subtype of malaria. In relation to the present invention it is preferred that the vaccine is directed specifically against the infectious activity of Plasmodium falciparum in the placenta, which is characteristic of PAM.

One important aspect of the present invention therefore relates to a vaccine comprising one or more B-cell epitopes from a polypeptide encoded by a member of the var2 gene family as defined by Salanti et al. 2002. This vaccine is characterised in that it induces an IgG/antibody response wherein said IgG/antibody specifically recognises a molecule expressed on the surface of an intact erythrocyte infected by placental parasites or parasites that have been selected for their ability to mediate adhesion to CSA. Generally, this molecule is recognised by the antibodies in a gender-specific and parity-dependent manner.

In a preferred embodiment of the present invention, such a polypeptide-based vaccine comprises at least one amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 6 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell epitope.

Sub-sequences of the polypeptide of the invention used in a vaccine may be any of the above mentioned amino acid lenghts and in addition to these fragments or sub-sequences of the polypeptide of the invention, larger polypeptides comprising sub-sequences of the invention as part of their sequence, are also embodiments of the present invention. It is preferred, however, that these sub-sequences have a minimum length of 6 amino acids and that they are at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 2. it is even more preferred that sub-sequences are at least 100 amino acids in length.

In recent years there has been increased focus on nucleotide based vaccines. Other aspects of the present invention therefore concern nucleotide based vaccines such as vaccines based on DNA molecules or on RNA molecules, which result in the expression of one or more B-cell epitopes from a polypeptide encoded by a member of the var2 gene family. As for the polypeptide based vaccine this vaccine is characterised in that it induces an IgG/antibody response wherein said IgG/antibody specifically recognises a molecule expressed on the surface of an intact erythrocyte infected by placental parasites or parasites that have been selected for their ability to mediate adhesion to CSA. It is further desired that this molecule is recognised by the antibodies in a gender-specific and parity-dependent manner.

One embodiment of the present invention relates to a nucleotide based vaccine, which results in the expression of an amino acid sequence comprising one or more B-cell epitopes from a polypeptide encoded by a member of the var2csa gene family, said vaccine characterised in that it is capable of inducing an IgG/antibody response wherein said IgG/antibody specifically recognises a molecule expressed on the surface of an intact erythrocyte infected by placenta parasites or parasites that have been CSA-selected in vitro, and wherein said molecule is recognised by antibodies in a gender-specific and parity dependent manner.

In a preferred embodiment, the present invention relates to a nucleotide-based vaccine, which may be a DNA or RNA vaccine, comprising a vector comprising at least one nucleotide sequence selected from the group consisting of a) SEQ ID NO.: 1 or a sequence complementary thereof; and b) a nucleotide sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 18 nucleic acids; and d) sub-sequences of a) and b) which comprise at least one sequence encoding a B-cell epitope.

The vaccine may thus comprise any of the sub-sequences of the nucleotide sequence of the invention, which may have any of the sequence identities described above. It is preferred, however, that these sub-sequences have a minimum length of 18 nucleic acids and that they are at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 1. it is even more preferred that sub-sequences are at least 300 nucleotides in length.

According to this aspect of the invention one approach is to incorporate the DNA encoding a polypeptide of the invention or parts hereof into a viral or bacterial vector. The following organisms, among numerous others, may be employed for this purpose: Coxsackie virus, vaccinia virus, Salmonella typhi or Salmonella typhimurium (for oral administration). In each case the carrier organism must be acquired by the host cell and the relevant DNA sequences used for production of the polypeptide of the invention or parts hereof. These in turn are recognised as abnormal by the host or recipient and an immune response ensues.

Alternatively, the parasite nucleic acid sequence may be incorporated into an RNA virus or used to prepare viral replicons. This approach allows for the delivery of coding sequences, such as mRNA, to the host cell without risking a replicative, infectious process.

In order to obtain expression of immunogenic polypeptides it is required that elements of a nucleotide-based vaccine are capable of entering into the relevant target cells of the subject receiving such a vaccine. Therefore, it is preferred that the vaccine further comprise one or more agents and/or vectors to facilitate such entry.

It is further preferred that the vector component of a nucleotide-based vaccine comprises a promoter for driving the expression in a mammalian cell line, a nucleotide sequence encoding a leader peptide for facilitating secretion/release of a polypeptide sequence from a mammalian cell, and a terminator.

The simple concept of a nucleotide-based vaccine is the inoculation of a recipient using the relevant DNA sequence alone. This `naked DNA` approach avoids the administration of polypeptide directly, but its effectiveness depends on the ability of the host cell to utilise the injected DNA as a template for RNA and subsequent protein synthesis.

It is anticipated that the principal value of providing PAM-specific protective immunity to sporozoite-induced infection will be for those individuals who have had previous exposure to malaria. In the case of PAM such individuals will be primigravid women who live in endemic areas. However, it is also anticipated that multi-gravid women who have not yet acquired immunity towards placental infections with P. falciparum and previously unexposed pregnant women travelling into endemic areas and women likely to become pregnant during such travelling will benefit from receiving said vaccine.

While not being limited by way of theory it is believed that the protection against malaria obtained by the use of a vaccine is most likely a result of IgGs blocking the interaction between the iRBC and cells in the placenta. It is also possible, however, that opsonized erythrocytes are killed by macrophages or T-cells, either by fagocytosis or by other means.

In a preferred embodiment, the vaccine is therefore capable of inducing an immunoglobulin response and, accordingly, it comprises a polypeptide comprising one or more B-cell epitopes. It is desirable, however, that polypeptides comprising one or more T-cell epitopes are also part of the vaccine since assistance from T-cells may be required in order to obtain a good antibody response.

In another preferred embodiment of the invention, the vaccine is therefore based on the use of polypeptides of the invention wherein said polypeptides comprises one or more B-cell epitopes in combination with one or more T-cell epitopes. In a less preferred embodiment of the invention, the vaccine comprises B-cell epitopes in combination with T-cell epitopes originating from an exogenous molecule, and in an even less preferred embodiment, the peptides of the vaccine comprises only B-cell epitopes. In equally preferred embodiments of the invention, the vaccine is based on nucleotide sequences encoding polypeptides, which have the characteristics with respect to antigen epitopes described above.

Techniques exist for enhancing the antigenicity of immunogenic peptides including incorporation of these into a multimeric structure, binding to a highly immunogenic protein carrier, for example, keyhole limpet hemocyanin, or diptheria toxoid, and administration in combination with adjuvants or any other enhancers of immune response. Furthermore, it will be understood that polypeptides specific for a plurality of Plasmodium stages and species may be incorporated in the same vaccine composition to provide a multivalent vaccine. In addition, the vaccine composition may comprise antigens to provide immunity against other diseases in addition to malaria.

Immunogenic polypeptides of the invention as well as nucleic acid molecules encoding such polypeptides may be injected as is, or for convenience of administration, it can be added to pharmaceutically acceptable carriers or diluents. Suitable pharmaceutically acceptable carriers will be apparent to those skilled in the art, and include water and other polar substances, including lower molecular weight alkanols, polyalkanols such as ethylene glycol, polyethylene glycol, and propylene glycol as well as non-polar carriers.

Routes of administration, antigen dose, number and frequency of injections are all matters of optimisation within the scope of ordinary skill in the art, particularly in view of the fact that there is already experience in the art of providing protective immunity by the injection of irradiated sporozoites. Protective antibodies are usually best elicited by a series of 2 to 3 doses given about 2 to 3 weeks apart. The series can be repeated when concentrations of circulating antibodies in the vaccinee drops. Further, the vaccine can be used to immunise a human against other forms of malaria, that is, heterologous immunisation. The polypeptide is present in the vaccine in an amount sufficient to induce an immune response against the antigenic polypeptide and thus to protect against Plasmodium infection thereby protecting the human against malaria.

Vaccination protocols can include the identification of a subject in need of a vaccine, for instance adolescent females and/or pregnant women living in regions populated with P. falciparum or pregnant women travelling through such regions, and administration of one or more effective doses of the vaccine to this subject.

Pharmaceuticals and Compositions

Another aspect of the present invention is the production of pharmaceuticals based on polypeptides of the invention or sub-sequences hereof or nucleic acid sequences encoding such molecules, as described above. Such pharmaceuticals may also include agents such as but not limited to other polypeptides and in particular antibodies, which are capable of modulating the adhesion of VAR2CSA to CSA.

Accordingly, it is within the scope of the invention to provide the use of at least one amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 6 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell epitope for the manufacture of a composition, such as an immunogenic composition, which is to be administered in order to prophylactically or therapeutically reduce the incidence, prevalence or severity of PAM in a female subject.

These sub-sequences may have a minimum length of 6 amino acids and that they are at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 2. it is even more preferred that sub-sequences are at least 100 amino acids in length.

A preferred embodiment of this aspect of the invention pertains to the use of a polypeptide sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 10 amino acids for the manufacture of a composition which is to be administered in order to prophylactically or therapeutically reduce the incidence, prevalence or severity of pregnancy-associated malaria in a female subject.

Also in this context it is further preferred that the amino acid sequence is capable of Inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite. It is equally preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a parasite that has been selected for its ability to mediate adhesion to CSA.

In particularly preferred embodiments the sub-sequence comprises at least one B-cell epitope, and in other particularly preferred embodiments it comprises one or more GAG-binding motifs.

It is further preferred that the amino acid sequence does not comprise a CIDR domain or DBL-.gamma. domain and that the amino acid sequence is gender specifically recognised. Finally, it is preferred that the amino acid sequence is recognised in a parity dependent manner. In addition, the invention also relates to the use of a nucleic acid molecule comprising at least one nucleotide sequence selected from the group consisting of a) SEQ ID NO.: 1 or a sequence complementary thereof; and b) a nucleotide sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 18 nucleic acids; and d) sub-sequences of a) and b) which comprise at least one sequence encoding a B-cell epitope for the manufacture of a composition, such as an immunogenic composition, which is to be administered in order to prophylactically or therapeutically reduce the incidence, prevalence or severity of PAM in a female subject.

It is preferred that these sub-sequences have a minimum length of 18 nucleic acids and that they are at least 70% identical to a region of comparable length within the sequence of SEQ ID NO.: 1. it is even more preferred that sub-sequences are at least 300 nucleotides in length.

Another preferred embodiment pertains to the use of a nucleotide sequence selected from the group consisting of a) SEQ ID NO.: 1; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 30 nucleic acids for the manufacture of an composition which is to be administered in order to prophylactically or therapeutically reduce the incidence, prevalence or severity of pregnancy-associated malaria in a female subject.

Especially preferred is a nucleic acid sequence which is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placenta parasite.

In particularly preferred embodiments the sub-sequence encodes at least one B-cell epitope and/or at least one T-cell epitope, and in other particularly preferred embodiments it encodes one or more GAG-binding motifs.

It is further preferred that the nucleic acid sequence does not encode a sequence comprising a CIDR domain or DBL-.gamma. domain and that the nucleic acid sequence encodes an amino acid sequence that is gender specifically recognised. Finally, it is preferred that nucleic acid sequence encodes an amino acid sequence which is recognised in a parity dependent manner.

Again the nucleic acid sequence may be a re-codonised sequence and may, in particular be recodonised in order to enhance expression in an expression system selected from the group of expression systems previously mentioned.

A currently most preferred embodiment of the invention pertains to use of the recodonised sequence of SEQ ID NO.: 3.

It should be understood that any feature and/or aspect discussed above in connection with the use of the nucleic acid sequences and amino acid sequences according to the invention apply by analogy to methods of treatment or prevention of PAM according to the invention.

Delivery of these pharmaceuticals can be performed by any conventional route including, but not limited to, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar administration.

In preferred embodiments, antibodies directed against the polypeptides of the invention can be administered to a subject in order to provide protection against the retention and sequestration of iRBC in the placenta which is characteristic of PAM. Effective amounts of an agent that will promote an immune response against a compound of the present invention can be administered to subjects living in endemic areas so as to prevent the contraction of malaria. In another embodiment, a subject believed to be at risk for contracting malaria may be identified either by conventional methods or by one of the in vitro diagnostic techniques, which constitute other embodiments of the present invention. An effective amount of an agent that inhibits VAR2CSA mediated sequestration or elicits an immune response in a subject can then be administered to this subject.

Biotechnological Tools

The use of the nucleic acid and polypeptide-based embodiments of the present invention can also extend to their use as biotechnological tools and as components of diagnostic assays.

Additional embodiments of the invention therefore include an in vitro diagnostic method, which comprises contacting a sample such as a tissue or biological fluid with a polypeptide comprising a sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 6 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell epitope under conditions allowing an in vitro immunological reaction to occur between said polypeptide composition and the antibodies possibly present in the biological sample, and the in vitro detection of the antigen-antibody complexes possibly formed. In one preferred embodiment the polypeptide is immobilised on a solid support.

Other embodiments include an in vitro diagnostic method, which comprises contacting a sample such as a tissue or biological fluid with a nucleotide composition comprising a sequence selected from the group consisting of a) SEQ ID NO.: 1 or a sequence complementary thereof; and b) a nucleotide sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 18 nucleic acids; and d) sub-sequences of a) and b) which comprise at least one sequence encoding a B-cell epitope under conditions allowing an in vitro reaction to occur between said nucleotide composition and the antibodies possibly present in the biological sample, and the in vitro detection of the antigen-antibody complexes possibly formed.

In a preferred embodiment the in vitro diagnostic method comprises contacting a sample with a polypeptide sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 10 amino acids, under conditions allowing an in vitro immunological reaction to occur between the said polypeptide and the antibodies possibly present in said sample, and the in vitro detection of the antigen-antibody complexes possibly formed.

Also for the in vitro diagnostic method it may be preferred that the amino acid used is a sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite. It is equally preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a parasite that has been selected for its ability to mediate adhesion to CSA.

In particularly preferred embodiments the sub-sequence comprises at least one B-cell and/or T-cell epitope, and in other particularly preferred embodiments it comprises one or more GAG-binding motifs.

It is further preferred that the amino acid sequence does not comprise a CIDR domain or DBL-.gamma. domain and that the amino acid sequence is gender specifically recognised. Finally, it is preferred that the amino acid sequence is recognised in a parity dependent manner.

In another preferred embodiment the in vitro diagnostic method comprises contacting a sample with a nucleic acid sequence selected from the group consisting of a) SEQ ID NO.: 1; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 30 nucleic acids, under conditions allowing an in vitro biochemical reaction to occur between the said nucleic acid sequence and nucleic acid sequences possibly present in said sample.

Especially preferred is a nucleic acid sequence which is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

In particularly preferred embodiments the sub-sequence encodes at least one B-cell epitope and/or at least one T-cell epitope, and in other particularly preferred embodiments it encodes one or more GAG-binding motifs.

It is further preferred that the nucleic acid sequence does not encode a sequence comprising a CIDR domain or DBL-.gamma. domain and that the nucleic acid sequence encodes an amino acid sequence that is gender specifically recognised. Finally, it is preferred that nucleic acid sequence encodes an amino acid sequence which is recognised in a parity dependent manner.

In some aspects, the nucleic acid embodiments are employed as nucleic acid probes in hybridisation assays, in cloning, or as primers for polymerase chain reaction (PCR). Similarly, the polypeptide-based embodiments can be used as components of immunological reactions such as ELISA, radio-immunoassays (RIA) and adhesion-blocking assays. The scope of such work can be, for example, to characterise VAR2CSA, or regions of VAR2CSA involved in interaction with CSA as well as other molecules including other VARCSA species that are involved in such interactions.

Diagnostic embodiments of the present invention provides methods and kits for the diagnosis of malaria and pregnancy associated malaria in particular. Malaria, hereunder pregnancy associated malaria can be diagnosed by detecting P falciparum derived compounds related to VAR2CSA in a bodily fluid. These VAR2CSA related compounds can for example be mRNA, DNA, protein-antigen, peptide-antigen or antibody being of any subclass. The methods for in vitro diagnosis of pregnancy associated malaria could be PCR, RT-PCR, ELISA, RIA, Dip stick test or any hybridisation assay as defined:

In some diagnostic embodiments, nucleic acids complementary to the nucleic acid molecules of the invention or fragments hereof are used to identify var2csa nucleic acids (e.g. mRNA) present in a biological sample, for instance a tissue sample or a sample of body fluid such as blood or serum. In a preferred diagnostic embodiment, nucleic acid molecules complementary to fragments of var2csa comprising sequences, which are not found in nucleic acids encoding other VARCSA proteins, are used to identify var2csa nucleic acids (e.g. mRNA) present in a biological sample.

The concentration or expression level in the infected subject of var2csa nucleic acids or other nucleic acids, which encode proteins that can mediate adhesion to CSA will differ depending on the type of Plasmodium infection. Thus, some Plasmodium parasites will only cause the expression of low amounts of VAR2CSA or no expression at all. Likewise it will not be possible to detect any expression of VAR2CSA in subjects that are not carrying a Plasmodium infection. Accordingly, malaria and, more specifically, PAM can be diagnosed by determining the concentration of var2csa gene transcripts in an individual at risk of contracting this disease. In the case of PAM such individuals may be e.g. pregnant women who live in endemic and sub-endemic areas, and previously unexposed pregnant women travelling into endemic areas.

One embodiment of the present invention is therefore an in vitro diagnostic method whereby infection with Plasmodium and more specifically infection with P. falciparum can be detected. In a preferred embodiment, a disease state profile can be created by collecting data on the expression level of var2csa in a large number of infected subjects and subsequent using these sets of data as reference. The concentration or expression level of var2csa transcripts detected in a tested subject can then be compared to this reference material so as to predict or follow the disease-state of that particular individual. Thus, in some embodiments the term "var2csa disease-state profile" refers to the concentration or expression level or concentration range or expression level range of a nucleic acid sequence encoding VAR2CSA or a part hereof that is detected in a biological sample. Arrays comprising nucleic acid probes comprised by the nucleotide sequence of the invention or fragments hereof can be used to create such disease-state profiles.

Accordingly, a particular embodiment of this aspect of the invention is an in vitro diagnostic procedure, wherein a disease-state profile for a tested subject is generated by determining the concentration or expression level in a sample of sequences as defined above.

In a similar fashion to that discussed above, a VAR2CSA disease-state profile comprising concentration levels or concentration range levels of VAR2CSA amino acid sequences in healthy and diseased subjects can be created and used to follow the disease-state of an individual. Accordingly, in some embodiments the term "VAR2CSA disease-state profile" refers to the concentration or concentration range or the expression level or expression level range of a polypeptide corresponding to VAR2CSA or a part hereof in a biological sample. Preferred methods for detecting such proteins or polypeptides include radioactive or non-radioactive immune-based approaches such as ELISA or radio-immunoassays as well as standard membrane-blotting techniques.

The invention also relates to a method for the in vitro detection of antibodies, which correlate with malaria originating from the infection of an individual P. falciparum in a tissue or biological fluid likely to contain such antibodies. This procedure comprises contacting a biological fluid or tissue sample as defined above with a preparation of antigens comprising the polypeptide of the invention or any part hereof under conditions, which allow an in vitro immunological reaction to occur between these antigens and the antibodies possibly present in the tissue or fluid. It further comprises the in vitro detection of the antigen-antibody complexes possibly formed by the use of conventional techniques. As an example, a preferred method involves the use of techniques such as ELISA, as well as immuno-fluorescent or radio-immunological assays (RIA) or equivalent procedures.

Again, such techniques can be used for collecting data on the concentration of antibodies against the polypeptide of the invention or parts hereof in subjects infected with Plasmodium parasites. These data can serve as reference when compared to the concentration of antibodies against the polypeptide of the invention detected in a given subject and a disease-state profile can be generated on the basis hereof. Thus, in some embodiments the term "VAR2CSA disease-state profile" refers to the concentration or concentration range of VAR2CSA antibodies, which are detected in a biological sample.

With respect to the above embodiments, the invention further relates to host cells comprising the above-described nucleic acid molecules. The nucleic acid molecules may be transformed, stably transfected or transiently transfected into the host cell or infected into the host cell by a live attenuated virus. The preferred host cells may include, but are not limited to, prokaryotic cells, such as Escherichia coli, Staphylococcus aureus, and eukaryotic cells, such as Sacchromyces cerevisiae, CHO and COS cells as well as Bachulo virus infected hi-fi insect cells. Transformation with the recombinant molecules can be effected using methods well known in the art.

In other aspects of the invention, kits are provided which will simplify the use of the polypeptide and nucleotide embodiments of the invention for in vitro diagnostic purposes. Such an in vitro diagnostic kit may comprise a sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 6 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell and/or T-cell epitope.

In addition to this component, the kit may comprise reagents for preparing a suitable medium for carrying out an immunological reaction between an IgG/antibody present in a sample of body fluid and said sequence; and reagents allowing the detection of the antigen-antibody complexes formed, wherein said reagents may bear a radioactive or non-radioactive label.

A specific embodiment pertains to an in vitro diagnostic kit comprising a) a nucleic acid sequence and/or an amino acid sequence as defined above for the in vitro diagnostic method b) reagents for preparing a suitable medium for carrying out an immunological reaction between an IgG/antibody present in a sample of body fluid or tissue and said sequence; and c) reagents allowing the detection of the antigen-antibody complexes formed, wherein said reagents may bear a radioactive or non-radioactive label.

It is further preferred that the in vitro diagnostic kit comprises a polypeptide sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 10 amino acids,

Again, it may be preferred that the amino acid used is a sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite. It is equally preferred that the amino acid sequence is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

Additionally, it may be preferred that an in vitro diagnostic kit comprises a nucleic acid sequence selected from the group consisting of a) SEQ ID NO.: 1; and b) a sequence having at least 80% sequence identity to a); and c) a sub-sequence of a) or b) with a minimum length of 30 nucleic acids

It may further be preferred that the nucleic acid sequence is a sequence which is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

In particularly preferred embodiments the sub-sequence encodes at least one B-cell epitope and/or at least one T-cell epitope, and in other particularly preferred embodiments it encodes one or more GAG-binding motifs.

It is further preferred that the nucleic acid sequence does not encode a sequence comprising a CIDR domain or DBL-.gamma. domain and that the nucleic acid sequence encodes an amino acid sequence that is gender specifically recognised. Finally, it is preferred that nucleic acid sequence encodes an amino acid sequence, which is recognised in a parity dependent manner.

Again the nucleic acid sequence may be a re-codonised sequence and may, in particular be recodonised in order to enhance expression in an expression system selected from the group of expression systems previously mentioned.

A currently most preferred embodiment of the invention pertains to use of the recodonised sequence of SEQ ID NO.: 3.

Alternatively, the in vitro diagnostic kit may comprise IgGs/antibodies or antibody fragments as described above, which specifically recognise a sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 20 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell epitope as well as reagents for preparing a suitable medium for carrying out an immunological reaction between said IgG/antibody and a sequence possibly present in a sample of body fluid or tissue and reagents allowing the detection of the antigen-antibody complexes formed. Said agents or said antibodies may optionally bear a radioactive or non-radioactive label.

In a preferred embodiment, the kit comprises a solid support to which the IgGs/antibodies of the kit are coupled. Such a support may for instance comprise an organic polymer.

In an additional embodiment, the kit comprises one or more doses of a vaccine in addition to the diagnostic components as described above. It is contemplated that such a kit may simplify the process of identifying and treating subjects in need of one of the therapeutic or prophylactic embodiments of the invention. Furthermore, the diagnostic components of a kit may be used to determine the presence of IgGs/antibodies and thereby the efficiency of the vaccine in each individual subject.

In certain embodiments a kit comprises preparations of the polypeptide and/or nucleotide embodiments of the invention filled in a number of separate containers. The containers can be entirely separate or can be constituted by separate chambers of the same applicator device. Where the containers are separate, they could be provided in the form of a kit comprising separate dispensers or syringes. Where the containers form part of the same applicator, they could for example, be defined by separate barrels of a multi-barrel syringe. A kit may thus comprise containers and/or barrels, where one container or barrel contains an immunogenic substance and another container or barrel contains a diluent and/or a carrier and/or an adjuvant. Other containers or barrels may contain diagnostic components.

Novel Agents

Within the scope of the present invention are also methods for identifying and/or designing novel agents useful in the prevention or treatment of malaria. Embodied in the invention is therefore a method for identifying an agent, which is capable of disrupting the Plasmodium life cycle, and an agent, which specifically modulates VAR2CSA dependent adhesion to CSA, the method comprising providing a cell expressing an amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 80% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 20 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell and/or T-cell epitope and contacting said cell with the agent and detecting adhesion of said cell to chondroitin sulphate A.

By this approach, an agent, which inhibits adhesion of a polypeptide of the invention to CSA can be identified by contacting CSA or a representative fragment thereof with polypeptides of the invention or sub-sequences thereof in the presence of the agent. Detection is accomplished and successful agents identified--according to their ability to induce a desired modulation of the formation of complexes of CSA and polypeptides of the invention.

In a preferred embodiment, this method is based on the detection of cells, which adhere to CSA immobilised on a solid support. Again, such a support may for instance comprise a resin, a membrane, an organic polymer, a lipid or a cell or part thereof. According to another aspect of the invention a support comprising a polypeptide of the invention or a fragment thereof coupled to it can be used to capture CSA or fragments of CSA and thereby identify substances that are capable of modulating the interaction of CSA and a polypeptide of the invention. The method may be based on directly or indirectly labelled CSA or a labelled polypeptide of the invention as well as the labelling of whole cells using radioactive as well as non-radioactive techniques. Another possibility of using the polypeptide embodiments of the present invention is the development of a method for identifying an agent, which interacts with an amino acid sequence selected from the group consisting of a) SEQ ID NO.: 2; and b) a sequence having at least 70% sequence identity to a); and c) sub-sequences of a) or b) with a minimum length of 6 amino acids; and d) sub-sequences of a) or b) comprising at least one B-cell epitope; said method comprising providing a cell expressing one or more of said polypeptides; contacting said cell with the agent; and detecting the interaction of the agent with one or more of the said polypeptides.

A specific embodiment pertains to a method for testing whether a molecule inhibits binding of an amino acid sequence as disclosed above to a receptor expressed on syncytiotrophoblast cells comprising a) isolating and culturing syncytiotrophoblast cells, b) contacting said syncytiotrophoblast cells with a potential inhibiting-molecule, c) contacting said endothelial cells with RBC infected with parasites which express any of the amino acid sequences disclosed above on their surface, d) measuring the binding of the iRBC with said syncytiotrophoblast cells.

The agents identified by the use of these methods may be monoclonal or polyclonal antibodies.

In addition, the methods described above can be used to identify compounds that will induce a desired immune response in a subject or patient and thereby serve as valuable tools in the development of novel pharmaceutical compositions as for instance vaccines. Therefore, in a preferred embodiment of the invention, the methods described above are used for identifying polypeptides, which will induce a specific IgG/antibody response upon administration to a subject in need hereof, or nucleotide sequences encoding such amino acid sequences. Use of the methods for this purpose comprises injecting into a living organism one or more of the polypeptides defined above, contacting a tissue or a biological fluid sample from said organism with said polypeptides; allowing an in vitro reaction to occur between the polypeptides and antibodies possibly present in the biological tissue; and the in vitro detection of complexes possibly formed.

An additional preferred embodiment is a method as described above wherein said tissue or said biological fluid sample is contacted with polypeptides expressed on the surface of a cell.

An equally preferred embodiment is a method as described above wherein said tissue or said biological fluid sample is contacted with polypeptides expressed on the surface of erythrocytes selected for adhesion to CSA.

Finally, another preferred embodiment of the invention is a method as described above wherein said tissue or biological fluid sample is contacted with polypeptides immobilised on a solid support.

In other embodiments, protein models of the polypeptides of the invention are constructed by the use of conventional techniques within molecular biology. Agents that interact with polypeptides of the invention are constructed and approaches in combinatorial chemistry are employed in the development of agents that modulate VAR2CSA mediated interaction with CSA or are able to induce an immune response. Accordingly, novel agents that interact with VAR2CSA are developed, screened in a VAR2CSA characterisation assay, for Instance a VAR2CSA anti-adhesion assay as described above. The identity of each agent and its performance in the VAR2CSA characterisation assay, its effect on the modulation of VAR2CSA-mediated adhesion to CSA or its ability to induce an immune response is recorded on electronic or non-electronic media. These recorded data can serve as the basis for a library of VAR2CSA modulating agents. Such a library can again be employed to further identify agents that modulate VAR2CSA-mediated adhesion to CSA and can be valuable tools for selecting an appropriate pharmaceutical to treat a particular type of Infection with Plasmodium. It is further expected that the high throughput screening techniques currently in use within the biotech and pharmaceutical industries can readily be applied to the procedures outlined above.

Finally, an additional aspect of the invention provides a method of generating a vaccine against malaria. A specific embodiment of this aspect the invention is a method for generating a vaccine against pregnancy associated malaria comprising a) injecting a nucleic acid sequence or an amino acid sequence according to the invention in a subject under conditions allowing said sequences to induce the generation of antibodies b) purifying said antibodies c) determining whether said antibodies display binding to any of the amino acid sequences according to the invention, when expressed on the surface of a iRBC infected with a parasite.


Claim 1 of 7 Claims

1. An isolated polypeptide comprising an amino acid sequence that is at least 90% identical to a sub-sequence of-SEQ ID NO:2 with a minimum length of 100 amino acids, wherein said amino acid sequence (i) does not comprise a cysteine-rich inter-domain region (CIDR) domain or Duffy-binding like-.gamma. (DBL-.gamma.) domain, and (ii) comprises (a) at least one B-cell epitope, (b) at least one T-cell epitope, or (c) at least one B-cell epitope and at least one T-cell epitope, and (iii) is capable of inducing an immune response against a molecule expressed on the surface of an intact erythrocyte infected by a placental parasite.

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