The present invention relates generally to a nucleic acid molecule from Plasmodium species which encodes a product involved in, associated with or which otherwise facilitates cytoadherence of cells parasitised with said Plasmodium species to other cells. The identification of this nucleic acid molecule permits the rational design of and screening for molecules capable of inhibiting or otherwise antagonising interaction between the translation product of the nucleic acid molecule on cells parasitised with the Plasmodium species and a receptor on cells which cytoadhere to said parasitised cells. The present invention extends to a family of nucleic acid molecules encoding cytoadherence molecules wherein the family comprises paralogues either binding to different ligands or at different stages of a parasite's life cycle. The present invention further provides a therapeutic agent useful for the prophylaxis and/or treatment of disease conditions caused or exacerbated by infection with Plasmodium species.

Throughout this specification, unless the context requires otherwise, the word"comprise", or variations such as"comprises"or"comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The subject specification contains nucleotide and amino acid sequence information prepared using the programme PatentIn Version 2.0, presented herein after the bibliography. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <BR> <BR> <BR> <BR> <BR> <210> followed by the sequence identifier (e. g. <210>1, <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> <BR> <BR> <BR> <BR> <BR> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1, <400>2, etc).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C

represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.

The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. This is particularly the case with respect to vaccine development for parasitic infection although the recombinant approach has not been applicable to all parasites. Species of the genus Plasmodium represent one important class of parasite for which hithertofore a recombinant approach to the development of therapeutic and prophylactic agents has proved somewhat elusive.

Malaria is a major disease caused by Plasmodium species. Of the human malaria parasites, Plasmodiumfalciparum is the most significant, being the major cause of morbidity and mortality in malaria-endemic areas. In addition, resistance to anti-malarial drugs is now widespread which is restricting the scope of therapeutic agents available to combat this debilitating disease. There is a need, therefore, for alternative therapeutic strategies in the prophylaxis and treatment of infection by Plasmodium species.

Severe malaria is associated with cytoadherence of infected red blood cells to the endothelial hning of capillaries and vesicles of various tissues including the brain. Ligands on the surface of parasitised red blood cells can bind to a number of endothelial cell receptors including CD36, ICAM1, thombospondin, chrondroitin-4-sulphate, VCAM-1, E selectin and PECAM-1 (1,2).

A thorough understanding of the mechanisms behind cytoadherence is required before therapeutic agents blocking cytoadherence can be rationally designed.

The importance of electron-dense structures ("knobs") on the surface of the parasitised red blood cells to cytoadherence has long been recognised. A major constituent of knobs is the knob- associated histidine rich protein [KAHRP] (3), localised under the red cell membrane. During in vitro culture, some lines of P. falciparum lose the ability to produce knobs (4) and these organisms generally lose the ability to induce cytoadherence in parasitised host cells. This is a consequence of subtelomeric deletions in the region of chromosome 2 bearing the KAHRP gene (5). However, KAHRP is not the only molecule associated with cytoadherence since a clone has been reported to being able to adhere to melanoma cells although it is KAHRP-and knob- (6).

Despite this, a targeted recombinational knockout of the KAHRP gene has been used to demonstrate that KAHRP itself is essential for knobs and for stable cytoadherence under physiological shear-stress levels (7).

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMPI) is a variable molecule of approximately 250 KDa located on the surface of the parasitised red blood cell (8). PFEMP I is now used as a collective term for any product of the multigene var family and it is clear that the parasite can undergo clonal antigenic variation by switching on the expression of different members of this set of about 50 polymorphic genes (9-11). As switching can occur at up to 2% per generation, clonal parasite populations can express a mixture of PFEMP I types even though only one or at most a few are expressed per cell. The PfEMP 1 type expressed has an important role in determining the receptor specificity of the parasitised red blood cell (12).

However, these well-established components of cytoadherence represent only part of the picture.

For example, the results of protease sensitivity studies have been explained by postulating a protease-insensitive common ligand in addition to the protease-sensitive variable PfEMPl (12).

Furthermore, there is considerable evidence that modification of the erythrocyte protein band 3 plays a role. Another candidate molecule with properties of a ligand has been termed "Sequestrin" (13). Currently, therefore, no coherent model can accommodate all these observations.

In accordance with the present invention, the inventors have now identified and cloned a genetic sequence from Plasmodium encoding a product required for cytoadherence. The genetic

sequence represents a member of a family of cytoadherence linked asexual genes (clags). The identification of clag genetic species and the products encoded by these sequences enables a range of therapeutic agents to be rationally designed and/or identified and which are useful for the prophylaxis and treatment of disease conditions caused or exacerbated by infection by Plasmodium species.

A generalized gene encoding a cytoadherence linked asexual molecule is referred to herein as "clag". The translation product is"CLAG". A particular paralogue is identified by the chromosome for which it is resident and number if there is more than one gene. For example, the clag gene described in Australian Patent Application No. PP2580 filed 25 March, 1998 is "clag9". The two clag genes on chromosome 3 are referred herein as clag3.1 and clag3.2. The clag gene on chromosome 2 is referred to herein as"clag2".

In accordance with the present invention, a genetic sequence has been identified which encodes a protein which is involved in, associated with or which otherwise facilitates cytoadherence of red blood cells parasitised with a Plasmodium species to other cells.

Accordingly, one aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein involved in, associated with or which otherwise facilitates host cells parasitised with a Plasmodium species cytoadhering to other cells.

The cells parasitised by Plasmodium species are red blood cells. Generally, the nucleic acid molecules are in an isolated or purified form.

The present invention is particularly directed to and is exemplified by Plasmodium falciparum, the causative agent of malaria as the parasitising Plasmodium species. This is done, however, on the understanding that the subject invention extends to any species of Plasmodium which induce parasitised host cells to cytoadhere. Examples of Plasmodium species contemplated by the present invention include but are not limited to Plasmodium malariae, Plasmodium ovale, Plasmodium cynomolgi, Plasmodium knowlesi, Plasmodium berghei and Plasmodium yoelii.

Conveniently, the genetic sequence encoding the cytoadherence protein is referred to herein as a cytoadherence"gene". The term"gene"is used in its broadest context to refer to a genomic nucleotide sequence having introns and exons and which exons encoded a functional cytoadherence protein. The term"gene"also refers to a cDNA corresponding to the coding regions of the genomic gene and which encodes a functional cytoadherence protein. The cytoadherence gene of the present invention represents a family of cytoadherence genes located on different chromosomes. Each gene is a paralogue of each other. A paralogue is a gene or gene product generally involved in cytoadherence but which functions either at different levels of receptors or ligands or at different stages of the life cycle.

The cytoadherence gene of the present invention is one which encodes a product which: (i) facilitates red blood cells parasitized with Plasmodium species cytoadhering to C32 melanoma cells; (ii) facilitates red blood cells parasitized with Plasmodium species cytoadhering to endothelial cells; (iii) is capable of binding or otherwise interacting with purified CD36; (iv) facilitates red blood cells parasitized with Plasmodium species cytoadhering to endothelial cells at a particular life cycle stage; and/or (v) is capable of interacting with the PfEMPl-KAHRD complex.

The product of the cytoadherence gene is not PfEMP1.

One clag gene is located within an approximately 55 kbp region of chromosome 9 of P. falciparum distal to the break point which commonly occurs in culture in vitro or a homologue of this region on another chromosome of P. falciparum or on a chromosome in another species of Plasmodium or a derivative of this region. This is referred to as clag9. Two other genes are located on chromosome 3 and are referred to as clag3.1 and clag3.2. A gene on chromosome 2 is referred to as clag2.

Preferably, clag9 is located within an approximately 7 kbp region of the 55 kbp portion of the

right hand end of chromosome 9 of P. falciparum or a homologue or derivative thereof. Even more preferably, the approximately 7 kbp region comprises at least about 3, still more preferably at least about 4, even more preferably at least about 5 exons, and still more preferably at least about 6-9 exons such as 9 exons.

Accordingly, another aspect of the present invention is directed to a sequence of nucleotides encoding or complementary to a sequence encoding a protein involved in, associated with or which otherwise facilitates red blood cells parasitised with Plasmodium species cytoadhering to C32 melanoma cells or endothelial cells or purified CD36 wherein said nucleic acid molecule corresponds to a nucleotide sequence located within the 55 kbp region at the right hand end of chromosome 9 and comprises at least about 5 exons within a region of 7 kbp located just distal to a common break point of P. falciparum or a homologue or derivative of said region or a paralogue of this nucleic acid molecule.

The"break point"refers to the point on the chromosome in which cleavage commonly occurs such as when the parasite is cultured.

The cytoadherence gene of the present invention comprises, in a particularly preferred embodiment, a nucleotide sequence which encodes an amino acid sequence substantially as set forth in <400>2 (CLAG9) or <400>11 (CLAG3.1) or <400>12 (CLAG3.2) or <400>13 (CLAG2) or an amino acid sequence having at least 35% similarity to at least about 20 contiguous amino acids of <400>2 or <400>11 or <400>12 or <400>13. Preferably, the 35% similarity is determined with respect to the entire amino acid sequence of <400>2 or <400>11 or <400>12 or <400>13.

The term"similarity"as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level,"similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level,"similarity"includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational

levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity. Any number of programs are available to compare nucleotide and amino acid sequences. Preferred programs have regard to an appropriate alignment. One such program is Gap which considers all possible alignment and gap positions and creates an alignment with the largest number of matched bases and the fewest gaps. Gap uses the alignment method of Needleman and Wunsch (J. Mol. Biol. 48: 443-453,1970). Gap reads a scoring matrix that contains values for every possible GCG symbol match. GAP is available on ANGIS (Australian National Genomic Information Service) at website http://mell. angis. org. au.

Alternative percentage similarities to <400>2 or <400>11 or <400>12 or <400>13 encompassed by the present invention include at least about 40% at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or above.

The amino acid sequence represented by <400>2 corresponds to a protein referred to herein as "CLAG9"and is the product of clag9. <400>11 and <400>12 correspond to CLAG3.1 and CLAG3.2, respectively which are encoded by clag3.1 and clag3.2. <400>13 corresponds to CLAG2 encoded by clag2. Reference herein to"CLAG9"or"CLAG3.1" or"CLAG3. 2" or "CLAG2"includes reference to the protein defined by the amino acid sequence of <400>2 or <400>11 or <400>12 or <400>13, respectively or a derivative or homologue thereof having at least 35% similarity to the amino acid sequence of <400>2 or <400> 11 or <400> 12 or <400> 13.

A"derivative"includes a fragment, portion or part of CLAG as well as any single or multiple amino acid substitutions, additions and/or deletions to the amino acid sequence set forth in <400>2 or <400>11 or <400>12 or <400>13. As stated above, CLAG is preferably from P. falciparum although the present invention extends to any homologue or paralogue having at least 35% similarity to <400>2 encoded by a gene on another chromosome of P. falciparum or by a gene in the genome of another species of Plasmodimn. An example of a paralogue is a similar genetic sequence on chromosome 3 of P. falciparum which encodes an amino acid sequence having approximately 60% similarity to <400>5 or <400>11 or <400>12.

According to these embodiments, there is provided a sequence of nucleotides encoding or complementary to a sequence encoding a protein involved in, associated with or which otherwise facilitates red blood cells parasitised with Plasmodium species cytoadhering to C32 melanoma cells or endothelial cells or to purified CD36 wherein said nucleotide sequence encodes an amino acid sequence substantially as set forth in <400>2 or <400> 11 or <400>12 or <400>13 or an amino acid sequence having at least 35% similarity to at least about 20 contiguous amino acids of <400>2 or <400> 11 or <400> 12 or <400> 13.

In a particularly preferred embodiment, CLAG9 is encoded by a nucleotide sequence substantially as set forth in <400>1 or a nucleotide sequence having at least about 35% similarity to at least about 60 contiguous nucleotides of <400>1. Preferably, however, the 35% similarity is over the entire nucleotide sequence of <400>1. Alternative percentage similarities to the nucleotide sequence of <400>1 include at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or above.

The nucleotide sequence of <400>1 is referred to as the clag9 gene and encodes CLAG9.

Reference herein to a clag gene includes all derivatives, homologues and paralogues thereof. A homologue or a paralogues of clag includes a nucleotide sequence on a different chromosome of P. falciparum or on a chromosome of another species of Plasmodium.

A"derivative"of clag9 includes fragments, portions and parts of the nucleotide sequence set forth in <400>1 as well as single or multiple nucleotide substitutions, additions and/or deletions to the nucleotide sequence of <400>1.

A derivative and homologue is also conveniently defined as any nucleotide sequence capable of hybridizing to <400>1 under low stringency conditions.

Reference herein to a low stringency includes and encompasses from at least about 0% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for

hybridisation, and at least about 1M to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31 % v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions. Generally, low stringency conditions are determined at from about 25 to about 48°C such as 42°C medium stringency in from about 40 to 55°C. High stringency in general from about 50 to about 65°C.

Accordingly, another aspect of the present invention is directed to a sequence of nucleotides encoding or complementary to a sequence encoding a protein involved in, associated with or which otherwise facilitates red blood cells parasitised with Plasmodium species cytoadhering to C32 melanoma cells endothelial cells and/or purified CD36 wherein said nucleotide sequence is as set forth in <400>1 or a nucleotide sequence having at least 35% similarity to 60 contiguous nucleotides of <400> 1.

Another aspect of the present invention provides an isolated nucleic acid molecule having one or more of the following characteristics: (i) encodes a product capable of facilitating cytoadherence of Plasmodium sp parasitized red blood cells to C32 melanoma cells; (ii) encodes a product capable of facilitating cytoadherence of Plasmodium sp parasitized red blood cells to endothelial cells; (iii) encodes a product capable of binding or otherwise interacting with CD36; (iv) encodes a product capable of facilitating cytoadherence of Plasmodium sp parasitized red blood cells to endothelial cells at a particular life cycle stage; (v) encodes a product capable of interacting with PfEMPl-KAHRP complex but is not PfEMP1; (vi) comprises a nucleotide sequence substantially as set forth in <400>1 or a nucleotide sequence having at least 35% similarity to <400>1 and/or is capable of hybridising to <400>1

under low stringency conditions; and/or (vii) encodes a product comprising an amount and sequence substantially as set forth in <400>2 or <400> 11 or <400>12 or <400> 13 or an amino acid sequence having at least 30% similarity thereto.

In a particularly preferred embodiment, the present invention provides an isolated nucleic acid molecule having the following characteristics: (i) is derived from the region of about 55 kbp of the right hand end of chromosome 9 of P. falciparum or an equivalent region on another chromosome of P. falciparum or a chromosome of another Plasmodium species; (ii) encodes a protein enabling cells expressing said protein to adhere to melanoma cells; (iii) encodes a protein having an amino acid sequence substantially as set forth in <400>2 or an amino acid sequence having at least 35% similarity to <400>2 and (iv) comprises a nucleotide sequence which: (a) is as substantially set forth in <400>1 or (b) has at least 35% similarity to <400>1 and/or (c) is capable of hybridizing to <400>1 under low stringency conditions at 42°C.

The present invention further extends to antisense molecules to the clag genetic sequence as well as ribozyme and deoxyribozyme molecules and molecules suitable for use as co-suppression agents.

Accordingly, another aspect of the present invention comprises an oligonucleotide comprising at least about 10 nucleotides, preferably at least about 13-18 nucleotides and more preferably at least about 20 nucleotides capable of hybridizing under low stringency conditions to mRNA transcribed from clag DNA.

More particularly, this aspect of the present invention contemplates an antisense molecule comprising at least about 10 nucleotides, preferably, preferably at least about 13-18 nucleotides and more preferably at least about 20 nucleotides capable of hybridizing or otherwise forming

a duplex with mRNA corresponding to all or part of the nucleotide sequence set forth in <400>1 or to a nucleotide sequence having at least 35% similarity thereto or a sequence capable of hybridizing to <400>1 under low stringency conditions and wherein said antisense molecule is capable of reducing the amount of protein translated from said mRNA.

The reduction in CLAG protein in accordance with the above embodiment of the present invention means a reduction in at least about 10%, more preferably at least about 20%, still more preferably at least about 30%, even still more preferably at least about 40% and yet even more preferably greater than 50% compared to expression of clag in the absence of the antisense molecule.

The antisense molecule is conveniently at least 10 nucleotides in length but is more preferably at least about 13 nucleotides or greater such as at least about 20 nucleotides, 30 nucleotides or 40 nucleotides. Alternatively, the entire clag gene is used in its reverse orientation or a derivative, part, portion or fragment of the clag gene in reverse orientation.

Ribozymes may be constructed by any convenient means such as by referring to in International Patent Application Publication No. WO 98/05852 or the disclosure of Haselhoff and Gerlach (18). Ribozymes are constructed with a hybridizing region which is complimentary to at least part of a target RNA which, in this case, encodes CLAG. The activity of ribozymes is measurable, for example, on Northern blots.

According to this embodiment, there is provided a ribozyme comprising a hybridizing region and a catalytic region wherein the hybridizing region is capable of hybridizing to at least part of a target mRNA sequence transcribed from a genomic gene corresponding to <400>1 or a gene<BR> having at least 35% similarity to a cDNA molecule corresponding to <400>1 or a nucleotide<BR> sequence capable of hybridizing under low stringency conditions to <400>1 and wherein said catalytic domain is capable of cleaving said target mRNA to reduce or inhibit translation of the mRNA molecule.

The present invention further extends to molecules capable of co-suppression of an endogenous

clag gene or its derivatives or homologues. Generally, the entire clag sequence or its derivative or homologue is used or a 3'end portion, a 5'end portion or an internal fragment may also be used.

The present invention further extends to a range of genetic constructs comprising the clag gene or derivatives or homologues thereof. Generally, the genetic construct comprises a clag gene sequence or a derivative or homologue thereof operably linked to a promoter. The clag gene sequence may also be fused to another genetic sequence such as a reporter gene sequence or a sequence encoding an amino acid sequence to facilitate purification of CLAG such as a nucleotide sequence encoding FLAG.

Yet another aspect of the present invention contemplates a purified CLAG protein.

According to this aspect of the present invention, there is provided an isolated protein involved in, associated with or which otherwise facilitates host cells parasitised with Plasmodium species cytoadhering to other cells.

More particularly, the present invention is directed to an isolated protein involved in, associated with or which otherwise facilitates red blood cells parasitised with Plasmodium species cytoadhering to melanoma cells.

In a preferred embodiment, the present invention contemplates an isolated protein involved in, associated with or which otherwise facilitates red blood cells parasitised with Plasmodium species cytoadhering to C32 melanoma cells or endothelial cells or purified CD36.

A further preferred embodiment provides an isolated protein having one or more of the following characteristics: (i) encodes a product capable of facilitating cytoadherence of Plasmodium sp parasitized red blood cells to C32 melanoma cells; (ii) encodes a product capable of facilitating cytoadherence of Plasmodium sp parasitized red blood cells to endothelial cells;

(iii) encodes a product capable of binding or otherwise interacting with CD36; (iv) encodes a product capable of facilitating cytoadherence of Plasmodium sp parasitized red blood cells to endothelial cells at a particular life cycle stage; (v) encodes a product capable of interacting with a PfEMP1-KAHRP complex but is not PfEMP 1; (vi) comprises a nucleotide sequence substantially as set forth in <400>1 or a nucleotide sequence having at least 35% similarity to <400>1 and/or is capable of hybridising to <400>1 under low stringency conditions; and/or (vii) encodes a product comprising an amount and sequence substantially as set forth in <400>2 or <400>11 or <400>12 or <400>13 or an amino acid sequence having at least 30% similarity thereto.

In a particularly preferred embodiment, the present invention is directed to an isolated protein involved in, associated with or which otherwise facilitates red blood cells parasitised with Plasmodium species cytoadhering to C32 melanoma cells or endothelial cells or purified CD36 wherein said protein comprises an amino acid sequence substantially as set forth in <400>2 or <400>11 or <400>12 or <400>13 or an amino acid sequence having at least 35% similarity to at least about 20 contiguous amino acids of <400>2 or <400>11 or <400>12 or <400>13.

The protein of this aspect of the present invention is referred to as CLAG. Preferably, CLAG is in recombinant form. Reference herein to"CLAG"includes all derivatives, homologues and mimetics thereof as well as recombinant, synthetic or isolated naturally occurring forms of CLAG. A derivative as stated above includes mutants, fragments, parts and portions thereof such as single or multiple amino acid substitutions, additions and/or deletions of CLAG. A derivative of CLAG also encompasses chemical analogues of CLAG and peptides and peptide mimetics of certain regions of CLAG such as regions comprising B-cell and T-cell epitopes, a transmembrane domain, an extracellular domain and/or a cytoplasmic domain. The use of chemical analogues may be useful in stabilizing the CLAG molecule for use as a diagnostic agent or in screening for agents capable of interacting with CLAG such as in immunoassays or natural product screening.

Analogues of CLAG contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5- phosphate followed by reduction with NaBH4.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.

Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.

TABLE 1 Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyricacid Abu L-N-methylalanine Nmala a-amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane-Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyricacid Aib L-N-methylcysteine Nmcys aminonorbornyl-Norb L-N-methylglutamine Nmgln<BR> carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys D-asparticacid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr a-methyl-aminoisobutyrate Maib<BR> <BR> <BR> <BR> <BR> <BR> D-valine Dval a-methyl-y-aminobutyrate Mgabu D-a-methylalanine Dmala a-methylcyclohexylalanine Mchexa <BR> <BR> <BR> <BR> D-a-methylarginine Dmarg a-methylcylcopentylalanine Mcpen D-a-methylasparagine Dmasn a-methyl-a-napthylalanine Manap D-a-methylaspartate Dmasp a-methylpenicillamine Mpen <BR> <BR> <BR> <BR> D-a-methylcysteine Dmcys N- (4-aminobutyl) glycine Nglu<BR> <BR> <BR> <BR> <BR> <BR> D-a-methylglutamine Dmgln N-(2-aminoethyl) glycine Naeg<BR> <BR> <BR> <BR> <BR> <BR> D-a-methylhistidine Dmhis N- (3-aminopropyl) glycine Norn D-a-methylisoleucine Dmile N-amino-a-methylbutyrate Nmaabu D-a-methylleucine Dmleu a-napthylalanine Anap <BR> <BR> <BR> <BR> D-a-methyllysine Dmlys N-benzylglycine Nphe<BR> <BR> <BR> <BR> <BR> <BR> D-a-methylmethionine Dmmet N- (2-carbamylethyl) glycine Ngln<BR> <BR> <BR> <BR> <BR> <BR> D-a-methylornithine Dmorn N-(carbamylmethyl) glycine Nasn<BR> <BR> <BR> <BR> <BR> <BR> D-a-methylphenylalanine Dmphe N-(2-carboxyethyl) glycine Nglu<BR> <BR> <BR> <BR> <BR> <BR> D-a-methylproline Dmpro N- (carboxymethyl) glycine Nasp D-a-methylserine Dmser N-cyclobutylglycine Ncbut D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep D-a-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec <BR> <BR> <BR> <BR> D-a-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct <BR> <BR> <BR> <BR> D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methylaspartate Dnmasp N- (2,2-diphenylethyl) glycine Nbhm D-N-methylcysteine Dnmcys N- (3,3-diphenylpropyl) glycine Nbhe <BR> <BR> <BR> <BR> D-N-methylglutamine Dnmgln N- (3-guanidinopropyl) glycine Narg<BR> <BR> <BR> <BR> <BR> <BR> D-N-methylglutamate Dnmglu N- (l-hydroxyethyl) glycine Nthr<BR> <BR> <BR> <BR> <BR> <BR> D-N-methylhistidine Dnmhis N-(hydroxyethyl)) glycine Nser<BR> <BR> <BR> <BR> <BR> <BR> D-N-methylisoleucine Dnmile N-(imidazolylethyl)) glycine Nhis D-N-methylleucine Dnmleu N- (3-indolylyethyl) glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet <BR> <BR> <BR> <BR> D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe <BR> <BR> <BR> <BR> N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro<BR> <BR> <BR> <BR> <BR> <BR> N- (1-methylpropyl) glycine Nile D-N-methylserine Dnmser<BR> <BR> <BR> <BR> <BR> <BR> N- (2-methylpropyl) glycine Nleu D-N-methylthreonine Dnmthr<BR> <BR> <BR> <BR> <BR> <BR> D-N-methyltryptophan Dnmtrp N- (1-methylethyl) glycine Nval<BR> <BR> <BR> <BR> <BR> <BR> D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen y-aminobutyric acid Gabu N- (p-hydroxyphenyl) glycine Nhtyr L-t-butylglycine Tbug N- (thiomethyl) glycine Ncys <BR> <BR> <BR> <BR> L-ethylglycine Etg penicillamine Pen<BR> <BR> <BR> <BR> <BR> <BR> L-homophenylalanine Hphe L-a-methylalanine Mala L-a-methylarginine Marg L-a-methylasparagine Masn <BR> <BR> <BR> <BR> L-a-methylaspartate Masp L-a-methyl-t-butylglycine Mtbug L-a-methylcysteine Mcys L-methylethylglycine Metg <BR> <BR> <BR> <BR> L-a-methylglutamine Mgln L-a-methylglutamate Mglu L-a-methylhistidine Mhis L-a-methylhomophenylalanine Mhphe <BR> <BR> <BR> <BR> L-a-methylisoleucine Mile N-(2-methylthioethyl) glycine Nmet<BR> <BR> <BR> <BR> <BR> <BR> L-a-methylleucine Mleu L-a-methyllysine Mlys L-a-methylmethionine Mmet L-a-methylnorleucine Mnle <BR> <BR> <BR> <BR> L-a-methylnorvaline Mnva L-a-methylomithine Morn L-a-methylphenylalanine Mphe L-a-methylproline Mpro L-a-methylserine Mser L-a-methylthreonine Mthr <BR> <BR> <BR> L-a-methyltryptophan Mtrp L-a-methyltyrosine Mtyr

L-a-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N- (N- (2,2-diphenylethyl) Nnbhm N- (N- (3, 3-diphenylpropyl) Nnbhe carbamylmethyl) glycine carbamylmethyl) glycine 1-carboxy-1-(2, 2-diphenyl-Nmbc ethylamino)cyclopropane These types of modifications may be important to stabilise CLAG if administered to an individual or for use as a diagnostic reagent.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2) n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.

In addition, peptides can be conformationally constrained by, for example, incorporation of C,, and Na-methylamino acids, introduction of double bonds between C ! ; and Cp atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention further contemplates chemical analogues of CLAG capable of acting as antagonists of CLAG or which can act as functional analogues of CLAG. Chemical analogues may not necessarily be derived from CLAG but may share certain conformational similarities.

Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of CLAG. Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening of environments such as the ocean, coral, seabeds, rivers, riverbeds, antarctic regions, plants, bacteria and eukaryotic organisms.

The identification of CLAG permits the generation of a range of molecules capable of

modulating expression of clag or modulating the activity of CLAG. Modulators contemplated by the present invention include antagonists of clag expression. Antagonists of clag expression include antisense molecules, ribozymes and co-suppression molecules. Antagonists of CLAG activity include antibodies and inhibitor peptide fragments.

Another embodiment of the present invention contemplates a method for modulating expression of clag in a mammal, said method comprising contacting the clag gene encoding CLAG with an effective amount of a modulator of clag expression for a time and under conditions sufficient to down-regulate or otherwise modulate expression of clag. For example, an anti-sense nucleic acid molecule to clag or a derivative thereof may be introduced into a cell to reduce the clag expression to reduce cytoadherence of that cell to another cell.

Another aspect of the present invention contemplates a method of modulating activity of CLAG in a mammal, said method comprising administering a modulating effective amount of a molecule for a time and under conditions sufficient to decrease CLAG activity. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of CLAG or its receptor or a chemical analogue or truncation mutant of CLAG or its receptor.

The preferred mammal is a human.

The present invention further contemplates a composition comprising CLAG or a derivative thereof or a modulator of CLAG expression or clag activity and one or more pharmaceutically acceptable carriers and/or diluents. These components are referred to as the"active ingredients".

Composition forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol,

polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active ingredients in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by an appropriate form of sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1 % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 gag and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active ingredients may be incorporated into sustained-release preparations and formulations. The active ingredients may also be administered sequentially or simultaneously with other active compounds such as anti-malaria drugs, anti-biotics or fever reducing compounds. The term"sequentially"includes the administration of two or more compounds within seconds, minutes, hours, days or weeks.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly

dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 pg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ug to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

Alternatively, active ingredients may be administered in amounts ranging from about 0.1 ug/kg body weight to about 10 mg/kg body weight. They may be administered per hour, day, two days, week or months.

The formulation of compositions is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pennsylvania, USA.

A particularly useful form of the composition is a recombinant active ingredient produced, for example, in a vaccine vector, such as but not limited to a vaccinia virus vector or bacterial cell capable of expressing the active ingredient. The active ingredient may be produced with a patient or in cell culture and then administered to a patient.

The present invention clearly extends to recombinant compositions in which the active ingredient at least is contained within killed vaccine vectors prepared, for example, by heat, formalin or other chemical treatment, electric shock or high or low pressure forces. According to this embodiment, the active ingredient of the composition is generally synthesized in a live vaccine vector which is killed prior to administration to an animal.

Furthermore, the vaccine vector expressing the active ingredient may be non-pathogenic or attenuated. Within the scope of this embodiment are non-pathogenic or attenuated viruses and bacteria which express the active ingredient of the composition and non-pathogenic or attenuated viruses which express the active ingredient and are contained within a non- pathogenic or attenuated host cell.

Attenuated or non-pathogenic host cells include those cells which are not harmful to an animal to which the subject composition is administered. As will be known to those skilled in the art, "live vaccines"can comprise an attenuated virus vector expressing the active ingredient or a host cell comprising same, which is capable of replicating in an animal to which it is administered, albeit producing no adverse side-effects therein. Such vaccine vectors may colonise the gut or other organ of the vaccinated patient. Such live vaccine vectors are efficacious by virtue of their ability to continually express the active ingredient in the host animal for a time and at a level sufficient to confer protective immunity against a pathogen which expresses an immunogenic or functional equivalent of said active ingredient. The present invention clearly encompasses the use of such attenuated or non-pathogenic vectors and live vaccine preparations.

The vaccine vector may be a virus, bacterial cell or a eukaryotic cell such as an avian, porcine or other mammalian cell or a yeast cell or a cell line such as COS, VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, baby hamster kidney (BHK) or MDCK cell lines.

Suitable prokaryotic cells include Mycobacterium spp., Corynebacterium spp., Salmonella spp., Escherichia coli, Bacillus spp. and Pseudomonas spp, amongst others. Bacterial strains which are suitable for the present purpose are well-known in the relevant art.

Such cells and cell lines are capable of expression of a genetic sequence encoding a peptide, polypeptide or protein of the present invention which antagonises CLAG function in a manner effective to induce a protective response in the patient. For example, a non-pathogenic bacterium could be prepared containing a recombinant sequence capable of encoding a CLAG antagonist. The recombinant sequence would be in the form of an expression vector under the control of a constitutive or inducible promoter. The bacterium would then be permitted to

colonise suitable locations in a patient's gut and would be permitted to grow and produce the recombinant molecule in amount sufficient to induce a protective response against Plasmodium.

In a further alternative embodiment, the composition may comprise a DNA vaccine comprising a DNA molecule encoding a peptide, polypeptide or protein of the present invention and which is injected into muscular tissue or other suitable tissue in a patient under conditions sufficient to permit transient expression of said DNA to produce an amount of peptide, polypeptide or protein effective to induce a protective response.

Still another aspect of the present invention is directed to antibodies to CLAG and its derivatives. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to CLAG or may be specifically raised to CLAG or derivatives thereof. In the case of the latter, CLAG or its derivatives may first need to be associated with a carrier molecule. Particularly useful derivatives are peptide fragments of CLAG proteins such as but not limited to peptides defined in <400>14, <400>15 and <400>16 or homologues or derivatives thereof. The antibodies and/or recombinant CLAG or its derivatives (including peptide fragments) of the present invention are particularly useful as therapeutic, diagnostic and/or selection agents.

For example, CLAG and its derivatives can be used to screen for naturally occurring antibodies to CLAG. Alternatively, specific antibodies can be used to screen for CLAG. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of CLAG levels may be important for diagnosis of certain disease conditions such as malaria or a predisposition to disease conditions such as malaria or for monitoring certain therapeutic protocols.

Antibodies to CLAG of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A"synthetic antibody"is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy in the

prophylaxis and treatment of Plasmodium infection and may also be used as a diagnostic tool for assessing Plasmlodium infection or monitoring the program of a therapeutic regimin.

For example, specific antibodies can be used to screen for CLAG proteins. The latter would be important, for example, as a means for screening for levels of CLAG in a cell extract or other biological fluid or purifying CLAG made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of CLAG.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of CLAG, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Another aspect of the present invention contemplates a method for detecting CLAG in a

biological sample from a mammal said method comprising contacting said biological sample with an antibody specific for CLAG or its derivatives or homologues for a time and under conditions sufficient for an antibody-CLAG complex to form, and then detecting said complex.

The presence of said complex would then be indicative of the presence of CLAG.

The presence of CLAG may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043,4,424,279 and 4,018,653. These, of course, include both single-site and two-site or"sandwich"assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody specific for CLAG or a molecule fused to CLAG is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody- CLAG complex, a second antibody specific to CLAG, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of another complex of antibody-CLAG-labelled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody.

These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In addition, CLAG receptors may be employed to form a complex or cytoadherence or inhibitor of cytoadherence may be employed in the assay. In accordance with the present invention, the sample is one which might contain CLAG including cell extract, tissue biopsy or whole blood. The sample is, therefore, generally a biological sample comprising

biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody having specificity for the CLAG or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a sufficient period of time (e. g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e. g. from room temperature to 37°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to CLAG.

An alternative method involves immobilizing the target CLAG molecules in a biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.

Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target- first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By"reporter molecule"as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i. e. radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta- galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-CLAG complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-CLAG-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample."Reporter molecule"also extends to use of cell cytoadherence or inhibition of cytoadherence such as red blood cells on latex beads and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-CLAG complex. After washing off the unbound reagent, the remaming tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of CLAG. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The nucleotide sequence corresponding to clag9 has been deposited in GenBank under Serial No. 181394,25 March, 1998. The nucleotide sequence of clag3.1 and clag3.2 is under Serial Number Z97348 and clag2 is under Serial Number AE001428 of GenBank.

The present invention is further described by the following non-limiting Figures and Examples.

In the Figures: Figure 1 is a diagrammatic representation of the relationship of the clag gene to deletions at the right end of chromosome 9. The right end of chromosome 9 from cytoadherent isolate 1776 (19), from non-cytoadherent clone C10 derived from 1776 (19), and from B8, a cytoadherent clone derived from ItG2 (6) are shown. The telomere of each chromosome is indicated (Tel). YAC 773 containing the cytoadherence locus has been described (14). Splicing of clag mRNA is indicated. The four shaded regions of the CLAG protein indicate the transmembrane regions predicted with the highest degree of certainty in Figure 3.

Figure 2 is a photographic representation showing expression of clag9 in blood stages. mRNA prepared using a Pharmacia Quick Prep mRNA purification kit according to the manufacturers instructions was copied with reverse transcriptase using a Pharmacia Read to Go T-primed first strand kit. The resulting cDNA was amplified by PCR using oligonucleotide CAG TGT TTT TAA TAG TGA TC [<400>3] and AAG ATT TGC AGG TGT TTC G [<400>4]. Track 1, 3D7 DNA; Track 2,3D7 cDNA.

Figure 3 is a graphical representation showing hydrophobicity of CLAG9. The clag9 cDNA sequence was analysed using the program TopPredl 1.

Figure 4A is a diagrammatic representation showing the structure of antisense construct HC. ASCl, based on vector HC 1 (16). Arrows show the positions of the PCR primers used.

HSP86,3'Heat shock protein 86 3'untranslated sequence. Cam 5', Calmodulin 5' untranslated region. Tg DHFR-TS, Toxoplasma gondii dihydrofolate reductase-thymidylate synthase. PcDT 5', Plasmodium chabaudi DHFR-TS 5'untranslated region. HRP2 3', Histidine rich protein 2 3"untranslated region.

Figure 4B is a photographic representation of PCR reactions to test for the presence of episomal and integrated forms of HC. ASCI. M, 1/Hind III markers. Lanes 1-4,3D7 DNA.

Lanes 5-8,3D7. HC. ASC1 DNA at 9 weeks at culture. Lanes 9-12,3D7. HC. ASC1 DNA at 15 weeks of culture. Lanes divergent primers Cso 46 and Xho R (test for circular vector). Lanes 2,6,10, primers Xho F plus Xho R (test for vector insert). Lanes 3,7,11, primers 5X'plus 3X' (clag gene test). Lanes 4,8,12, primers S2E5 plus Xho R (integration test). A PCR product could only have been present in lanes 8 and 12 if HC. ASC1 had integrated into chromosome 9 of 3D7 as primer S2E5 lies 5'to the region of clag9 inserted into the HC1 vector.

Figure 5 is a photographic representation showing southern blotting experiments to test for the presence of episomal and integrated forms of HC. ASC1.

A. 3D7 (Lane 1) and 3D7. HC. ASC1 (Lane 2) genomic DNA digested with Bg/TI (which linearises the HC. ASC1 plasmid, see Figure 4) was fractionated by agarose gel electrophoresis, blotted and probed for the clag insert present in the plasmid.

B. Genomic DNA digested with Bg/l was fractionated by pulsed field electrophoresis and probe for the 5'region of clag that is not present in the plasmid. 3D7 (Lane 1) and 3D7. HC. AS (Lane 2) have an approximately 180 kb Bgll fragment whereas a 3D7 clone with a vector integrated into the clag gene (Lane 3) has a 70 kb fragment as the integrated vector introduces an additional Bg/I site.

Figure 6A is a diagrammatic representation showing structure of the chromosomal copy of clag9. Arrows indicate primer locations.

Figure 6B is a diagrammatic representation showing structure of the knockout construct pAC4-CLAG. Large arrows indicate the plasmid backbone, small arrows indicate primer locations.

Figure 6C is a photographic representation showing PCR reactions testing for the presence of vector integrated into the chromosomal copy of clag9. Lanes 1-5 3D7 DNA, lanes 6-10 clag transfectant DNA 19 weeks post transfection. Lanes 1 and 6, primers for the KAHRP gene.

Lanes 2 and 7, primers Csol and S2EB (test for uninterrupted clag 9). Lanes 3 and 8, primers Csol and DCH42 (integration test). Lanes 4 and 9, primers LAV2 and S2EB (integration test).

Lanes 5 and 10, negative control.

Figure 7 is a photographic representation of indirect immunofluorescence staining. The figure shows the staining pattern produced on acetone fixed film of P. falciparum trophozoites by antibodies to CLAG9 peptide 3 (<400>16).

Figure 8 is a photographic representation of the clag gene family. A probe derived from a relatively conserved region and generated by PCR with the corresponding primers was hybridised to chromosomes of 3D7 after separation by pulsed field electrophoresis.

Figure 9 is a representation of amino acid sequences for CLAG3.1 (<400>11), CLAG3.2 (<400>12), CLAG2 (<400>13) and CLAG9 (<400>2).

Figure 10 is a graphical representation of hydrophobicity profiles of three clag gene family members. Predicted hydrophobicity profiles a) clag2, b) clag3.1, and c) clag9 using the TropPred II algorithm.

Figure 11 is a photographic representation showing surface proteins of clone 3D7 and TGD clone 11E were iodinated with 1251. The samples were then divided into two and half of each was subjected to mild trypsinisation. The samples were then extracted sequentially with Factor xlOO followed by SDS. The SDS extracts were fractionated on an SDS-polyacrylamide gel and autoradiographed.

TABLE 2 SUMMARY OF SEQUENCES IN SEQUENCE LISTING

Numeric Indicator Sequence <400> 1 Nucleotide sequence of clag9 <400>2 Amino acid sequence of CLAG9 <400>3 PCR oligonucleotide for clag9 <400>4 PCR oligonucleotide for clag9 <400>5 Nucleotide sequence of paralogue clag9 on chromosome 3 <400>6 Amino acid sequence of paralogue of CLAG9 on chromosome 3 <400>7 Oligonucleotide sequence <400>8 Oligonucleotide sequence <400>9 Oligonucleotide sequence <400> 10 Oligonucleotide sequence <400>11 Amino acid sequence of CLAG3.1 <400>12 Amino acid sequence of CLAG3.2 <400>13 Amino acid sequence of CLAG2 <400> 14 Peptide fragment of CLAG9 <400> 15 Peptide fragment of CLAG9 <400> 16 Peptide fragment of CLAG9

EXAMPLE 1 The Cytoadherence Locus on Chromosome 9 The inventors have identified a gene product which is essential for cytoadherence. During in vitro cultivation, isolates of P. falciparum commonly undergo loss of cytoadherence, as measured by binding to C32 melanoma cells (4). The inventors have associated this loss with subtelomeric deletions of chromosome 9 [19-21] (Figure 1). Independent deletion breakpoints in a number of independent strains were tightly clustered (14). Binding a mixed parasite population (containing deleted and non-deleted forms derived from the same parental line) to melanoma cells resulted in selection of parasites with the undeleted form of chromosome 9 in all 4 lines tested. The inventors proposed that a gene essential for cytoadherence is located in this region.

The product of such a gene may associate with the PfEMPI-KAHRP complex or may be independently expressed at the surface of the red cell. Alternatively it may be necessary for transport, processing or regulation of var expression as PfEMP I was not detectable on the surface of cells bearing chromosome 9 deletions. While it is not clear which endothelial receptors are involved, C32 cells express high levels of CD36 and low levels of ICAM 1.

Melanoma cell binding assays (15) confirmed that a clone from an independent isolate with a chromosome 9 deletion did not bind to melanoma cells although its parental undeleted line did.

While it was possible to increase the level of binding of this clone to ICAM 1 by selection on endothelial cells, selection on melanoma cells did not result in any increase in the level of binding. As PfEMP1 is implicated in binding to ICAM-1, this result clearly indicates that, as is the case with KAHRP which clearly has an important role in cytoadherence to C32 melanoma cells, the cytoadherence molecule may not be essential for all PfEMPI-receptor interactions.

PfEMPI could not be detected on the surface of 3 independent chromosome 9 deletion lines although it was clearly present on the parental lines and this indicates that the chromosome 9 product is in most cases necessary for assembly of PfEMP 1 on the surface, but as with KAHRP, this can be circumvented in some situations. Alternatively, the chromosome 9 product may be required for only a subset of endothelial receptor interactions.

Clone ItG2 differs from many other parasite lines in that cytoadherence is stable over many generations (22). ItG2 is an established model to study cytoadherence. Cytoadherent clones derived from ItG2 (eg. B8) possess a chromosome 9 of intermediate size (Figure 1) between that of cytoadherent isolate 1776 and its non-cytoadherent derivative clone C10 (19). This was due to a deletion of intermediate size at the right end of the chromosome, as well as an internal deletion of about 15kb which deleted an ORF at the site of the most common breakpoints in other isolates (14). The remaining segment of about 55kb is colinear with its counterpart in 3D7 and this segment, therefore, defines the cytoadherence locus on chromosome 9. As no var genes were detectable in this region (14), it must contain a novel cytoadherence gene.

EXAMPLE 2 Identification of clag9 A 55kb region at the right end of chromosome 9 has cloned in YACs and a detailed map generated (Figure 1). Technical problems caused by the high AT content of P. falciparum DNA were eventually overcome by using long PCR (21) with low temperature (60°) extensions (10) to amplify individual segments from this region. Segments of up to 16 kb have been amplified using primers derived from sequence tag sites mapped to this region. These individual fragments cover the entire 55 kb region containing the putative cytoadherence locus. Subclones were generated from Dra I and Rsa I fragments of these long PCR products to facilitate sequencing.

This region is sequenced using a YAC clone referred to as YAC1039.

The sequence revealed a prime candidate gene: it is located just distal to the common breakpoint and at least five exons were intially demonstrated within a region of at least 7 kb. The exons and introns were initially distinguished by the considerably higher AT content of the introns and the open reading frames of the exons. These predictions were then tested by RT-PCR from mRNA prepared from gelatin-purified trophozoite stage parasites of clone 3D7. The PCR products, of which one example is shown (Figure 2), were clearly of the sizes expected for the spliced products rather than the genomic size. The inventors confirmed this by sequencing the RT-PCR products across each splice junction found. The inventors conclude that this gene is expressed in P. falciparum clone 3D7 and further sequencing of genomic DNA showed that there are a

total of 9 exons. This is shown in Fig 1.

The gene was named the cytoadherence-linked asexual gene (clag). As there are a number of clag genes in the genome of P. falciparum, the nomenclature adopted by the inventors is to attach the number of the chromosome of origin and where necessary when more than one gene is present on a chromosome, a dot number. The nucleotide sequence and corresponding amino acid sequence of clag9 is represented in <400>1 and <400>2, respectively. Other clag genes, clag3.1, clag3.2 and clag2, encode amino acid sequences set forth in <400>11,12 and 13, respectively.

A hydrophobicity plot generated by the program TopPred II predicts several potential transmembrane domains in the protein CLAG9 (Figure 3). The prediction indicated with certainty that four of these domains are localized in a membrane, presumably that of the red cell.

Southern hybridization to Hind III fragments of DNA from a number of P. falciparum falciparum and clones revealed two hybridizing fragments (expected because there is an internal Hind III site) of 4kb and 8.5kb present in isolates/clones with an intact chromosome 9 (CSL2 and 3D7) and in ItG2 clone B8 but absent from those clones (D10, E12, C10, NF7,7G8) with large chromosome 9 deletions. Pulsed field electrophoresis confirmed that clag9 was located on chromosome 9. Further, clag9 could be amplified by PCR using YACs 773 and 1039 as templates. As these have been mapped on chromosome 9 in detail (14), there can be no doubt that clag9 is located in the deletable region of chromosome 9. This is confirmed by sequencing of YAC 1039. clag9 is transcribed in ItG2 clone B8.

EXAMPLE 3 Inhibition of C32 melanoma binding by an antisense construct of clag In order to examine whether clag9 function was required for cytoadherence to melanoma cells, the inventors generated an antisense construct HC. ASC1, consisting of two exons of clag9 in vector HC 1 (16) (Figure 4). In this construct, the clag9 exons are inserted in the 3'-5' orientation, 3'to the powerful calmodulin promoter and so it would be expected that anti-clag9

RNA would be expressed at a high level in transfectants. HC. ASC1 was electroporated into stably cytoadherent P. falciparum clone 3D7 under the conditions described (17,23) and the cells were cultured for 10 weeks in 0.1 mM pyrimethamine.

The resulting pyrimethamine-resistant line 3D7. HC. ASC1 was tested for cytoadherence to C32 melanoma cells after 10 weeks of culture. In duplicate experiments it initially showed 3 fold lower binding to melanoma cells than did the parental clone 3D7 (Table 3). Over the next four weeks binding decreased to 15 fold lower, using 3D7 transfected with an unrelated recombinant HC 7 at the same time as HC. ASC1 and cultured in parallel as the control (Table 3). This is consistent with the hypothesis that clag9 is essential for cytoadherence to C32 melanoma cells.

Line 3D7. HC. ASC1 was tested by PCR to examine whether it had integrated into the clag9 gene in order to distinguish between a targeted gene disruption or inhibition due to antisense RNA production. These reactions demonstrated the presence of HC. ASC 1 and the presence of an intact chromosomal clag9 gene (Figure 4). However a test for homologous recombination between a region of clag 9 5'to that included in HC. ASC1 and the HC 1 vector was negative (Figure 4).

As these PCR reactions were carried out on DNA prepared from 3D7. HC. ASC 1 at weeks 9 and 15 weeks of culture, the inventors conclude that it had not integrated during the course of these experiments but continued replicating as an episome.

To confirm that the lowered binding was determined by the presence of the plasmid expressing antisense-clag9 RNA, we cultured 3D7. HC. ASC1 in the absence of pyrimethamine for several weeks after the first experiment shown in Table 3. Under these conditions it has been demonstrated that the plasmid, and, hence, pyrimethamine resistance, is lost, unless it has integrated into the chromosome. After 4 weeks the line so-obtained, 3D7. HC. ASClp-exhibited 2.7 fold greater binding to melanoma cells than 3D7. HC. ASC 1 cultured throughout this time in pyrimethamine (Table 3) while after 7 weeks without pyrimethamine it had regained full binding ability (Table 3). This result strongly supports the conclusion that the presence of plasmid HC. ASC 1 rather than some unrelated change such as var gene switching in the parasite line is responsible for the change in phenotype. It would appear that loss of all copies of the plasmid from all cells took more than 4 weeks.

Southern blotting of the 3D7. HC. ASC 1 with a clag9 sequence present in the construct revealed a very high copy number (at least 100x the chromosomal intensity of clag9 in 3D7) while hybridization with a clag9 sequence not present in the construct revealed that the BglII site of the plasmid had not been introduced into clag9 (Fig5).

One possibility was that 3D7. HC. ASC 1 had undergone a subtelomeric deletion of chromosome 2 and that the loss of ability to bind was due to its conversion to a knobless phenotype by a chromosome 2 deletion (5). This was highly unlikely as a gelatin separation was always employed before binding was measured, and it has been established that this selects for cells with knobs (24). Furthermore, the KAHRP gene was present as determined by PCR. In order to confirm that 3D7. HC. ASC1 continued to produce knobs it was examined by transmission electron microscopy. All 3D7. HC. ASC1 cells examined had characteristic electron-dense knobs whereas these were not present in the knobless control B8.

EXAMPLE 4 Inhibition of Melanoma Binding by a Targeted Gene Disruption of clag in Clone 3D7 The inventors generated a construct of clag suitable for a TGD (Figure 6). It consisted of an incomplete copy of clag9 cloned in plasmid pTgD-TS. C5/H3 (23) and was designated pAC4. CLAG. It was electroporated into P. falciparum isolate 3D7 under the conditions described (17) and cultured for 3 weeks in 0. ImM pyrimethamine. Pyrimethamine was then withdrawn for 3 weeks to allow loss of the plasmid. This cycle was repeated twice more. Cells into which the plasmid had become integrated were then selected by exposure again to pyrimethamine for 3 weeks.

To examine whether homologous recombination had in fact occurred a PCR test was employed.

This relied on sequences in the chromosomal copy of clag9 which were outside, either 3'or 5', to the segment of clag9 included in pAC4. CLAG. Primers located in these regions together with primers located in vector regions could only amplify the predicted regions of DNA if pAC4. CLAG had inserted into chromosome 9 via recombination with clag9. Both the 5'and 3' primer sets generated fragments of sizes consistent with integration (Figure 6), but only after 18

weeks of culture. As this event must have produced two incomplete copies of the gene, a targeted gene disruption has been produced.

Cells in this uncloned mixture were then tested for cytoadherence to melanoma cells. They bound 10-20-fold less than 3D7 when assayed simultaneously (Table 3). Furthermore they showed considerably lower binding to purified CD36 in the one experiment conducted to date (30 cf 120 cells/30 fields). Transmission electron microscopy revealed that all cells examined continued to express knobs.

Clones were generated and one particular clone, 11E, studied in depth. I IE shows only background levels of binding to melanoma cells. Southern blotting of a pulsed field gel after BgIII digestion demonstrated that the BglII site of the plasmid had in fact been introduced into clag9 as shown by the much smaller BglII fragment in Fig5. Attempts to increase the level of binding of clone l lE to melanoma cells by upselection failed. As well, the inventors carried out an independent knockout with a distinguishable insert and obtained the same phenotype (Table 3): it shows a binding level to purified CD36 which is 60 times lower than that of the parental line 3D7.

EXAMPLE 5 Cellular Location of CLAG9 The inventors purified pGEX fused polypeptides corresponding to 6 different regions of clag9. These correspond to sequences on either side of two of the hydrophobic regions and so if CLAG9 does indeed traverse the red cell membrane, at least two of these sequences should be on the outside and be detectable by immunofluorescence (Figure 7) and/or agglutination of the unfixed cells. The affinity purification procedure widely used for pGex fusions resulted in considerable degradation. The purification procedures as modified utilising ion exchange chromatography which avoided the degradation problem and preparations are now used for rabbit antibody preparation.

Antibodies to 3 chemically synthesized peptides <400>14, <400>15 and <400>16 were prepared

and antibodies raised to them in rabbits as described in Example 5.

Results obtained demonstrate that CLAG9 is in the extra-parasite space rather than confined to the parasite.

EXAMPLE 6 Antibodies to CLAG9 Synthetic Peptides Three 16 mer synthetic peptides derived from the translated clag9 nucleotide sequence were synthesised. Each peptide contained 15 CLAG9 amino acid residues plus a single cystine residue at the amino terminal end to facilitate coupling to carrier protein. Peptide 1 corresponds to the CLAG9 amino acid sequence RKYISIYLLEELEKL <400>14 comprising residues 650-664.

Peptide 2 corresponds to the CLAG9 amino acid sequence SIDWQVGYAISHGLS <400>15 comprising residues 1160-1174. Peptide 3 corresponds to the CLAG9 amino acid sequence SHRRNDDVSMNNIFM <400>16 comprising residues 910-924.

For each peptide two rabbits were injected intramuscularly with 300, ug of peptide which had been conjugated to diphtheria toxoid as a carrier protein. The initial dose was given as 1: 1 emulsion with Freund's Complete Adjuvant and the 4 subsequent doses, each of 300ug, given in conjunction with Freund's Incomplete Adjuvant. Doses were spaced at 3 weekly intervals. Pre- bleed bloods were collected from each rabbit before immunization commenced. Final Test Bleed's were collected after the 5'immunization.

ELISA assay's were carried out using pre-bleed and Final Test Bleed rabbit sera from all the rabbits immunized with the peptides. ELISA assays were performed in 96 well immuno-assay plates. Peptides 1-3 were bound separately to individual plates for 16 hours at 4°C in carbonate buffer. The trays were then blocked by 5% w/v skim-milk in Tris/NaCl buffer for 3 hours at room temperature. Rabbit Pre-Bleed and Final Test Bleed sera was diluted in the blocking buffer and added to the appropriate tray. This was incubated for 1 hour at room temperature, then washed in Tris/NaCl buffer. Anti-Rabbit IgG conjugated to Horse-radish peroxidase was added to a concentration of 1 in 1000. The trays were incubated for 1 hour at room temperature. The trays

were again washed in Tris/NaCl buffer and 2'2-azino-di [3-ethylbenzthiazoline sulfonate] and H20, added. The resulting colour reaction was read in a spectrophotometer at 405nm wavelength.

For peptide 1 one rabbit showed no detectable immune response to the peptide. The second rabbit had an ELISA titre of > 1: 2000. For peptide 2 both rabbits showed detectable immune responses to the peptide. The first rabbit had an ELISA titre of between 1: 1000 and 1: 2000. The second rabbit had an ELISA titre of > 1: 2000. For peptide 3 one rabbit showed no detectable immune response to the peptide. The second rabbit had an ELISA titre of > 1: 2000. No rabbit sera showed detectable immune responses to any peptide in the Pre Bleed samples.

The antibodies may be used, for example, in immunoflorescence analysis to determine expression patterns of the clag genes. One example of immunoflorescence is shown in Figure 7. This figure shows the staining pattern produced on acetone fixed film of P. falciparum trophozoites by antibodies to CLAG9 peptide 3 (<400>16).

EXAMPLE 7 Clag9 is a member of a gene family Nucleotide sequences of a 60-70% similarity to clag9 were observed on chromosomes 1,2,3 and 4. After aligning the sequences, a hybridization probe was designed to a relatively conserved region. This hybridized at low stringency to at least 9 of the chromosomes separated by pulsed field electrophoresis (Fig. 8). Hence, the clag gene family has at least 9 members. As the clag9 knockout did not retain the ability to cytoadhere to CD36, it is likely that they are not homologues that carry out the same function. It is proposed, therefore, that they are paralogues with related functions.

The complete sequences of the 2 clag genes on chromosome 3 (clag3.1 and clag3.2) of P. falciparum clone 3D7 have been determined as has the complete sequence of a single clag gene on chromosome 2 (clag2). Fragments of other sequences from chromosomes 1,4,11 and 13 have also been identified. The complete sequences all have the same predicted splicing pattern.

clag9 has the same pattern of 9 exons. The arrangement of hydrophobic regions also appears to be substantially the same (Fig 9).

The degree of homology varies widely. clags3.1 and 3.2 appear to result from a duplication and it is possible that clag3.2 is an inactive pseudogene as it does not appear to be expressed in red blood cells whereas clag3.1 and clag2 are. Alternatively, this may be an example of a clag that is expressed elsewhere in the life cycle. A number of clag3 cDNA and genomic clones were sequenced and this established that only clag3.1 was expressed. An amino acid sequence alignment for all current completed amino acid sequences is shown in Fig 9.

The inventors have established that clag3.1 is expressed in blood stages of 3D7 since they demonstrated the presence of spliced mRNA by RT-PCR. This is also the case for line Dd2 since the inventors found an almost identical sequence in an EST (ie. cDNA) database. Clags 2,3.1 and 3.2 are all located between 100-150kb from one end of the chromosome, a similar location to that of clag9. It is notable that the majority of malaria genes which encode products that are transported out to the red cell or its surface are located in such recombinationally active regions.

Examples are var (PfEMP 1), STEVOR, PfEMPIII, KAHRP, RESA, FIRA, and HRPIII.

On the other hand, the fact that the cDNA from Dd2 is almost identical to clag3, and a fragmentary cDNA sequence from isolate 1916 is identical to that of clag9, suggest that the clag family may have diverged long ago and individual members are now highly conserved. This is supported by the fact that the HindIII fragments of clag9 were identical in the three totally independent isolates.

The nucleotide sequence for clag3.1 and clag3.2 is shown in accession number Z97348 of GenBank. Clag2 is shown under accession number AE001428.

EXAMPLE 8 CLAG and PfEMPI A number of molecules are involved in cytoadherence, either individually or in combination although the way in which these molecules interact is not well understood. PfEMP1 is generally

thought to be of major importance in binding to the cell adhesion molecule CD36. However, red cells containing parasites which do not have a functional clag9 gene and which are not able to bind to CD36 still express PFEMP I on their surface, bringing into question the role of PFEMP I as the omnipotent ligand for CD36.

The inventors carried out a series of experiments in which trophozoite stage parasites from the parental line 3D7 (which has a functional clag9 gene) and from the TGD clone 1 ive (which does not have a functional clag9 gene) were surface labelled with 125 Iodine The samples were then divided into two and half of each was subjected to trypsinisation. PfEMPI is rapidly cleaved from the red cell surface by mild trypsinization. All samples were then extracted sequentially with Triton X-100 followed by SDS. PFEMP I is soluble in sodium dodecyl sulphate (SDS) but not in Triton X-100. I'25-SDS extracts were fractionated on an SDS-PAGE gel and the resulting autoradiograph showed strong bands of the correct size for PfEMPI in lanes containing 3D7 and I lE parasite extracts (Fig. 10). Furthermore the intensity of these bands was reduced dramatically for trypsin treated parasite extracts. This indicates that a molecule of the same size and exhibiting the same characteristics as PfEMPI is expressed on the surface of 3D7 and 3D7 TGD parasitised RBC's.

In a second experiment, samples of trophozoite stage parasites of lines 3D7, clone 1 and unrelated clone C10 (which has a short chromosome 9 and is known not to express PFEMP I) were divided into two and half of each was subjected to trypsinisation followed by sequential extraction with Triton X-100 and SDS. Following SDS fractionation and transfer to nylon membrane, blots were probed with an antibody to the internal domain of PfEMP 1. As expected, no bands were visible in lanes containing C10 parasite extracts; however strong bands were seen with untrypsinised 3D7 and 1 lE parasites and these showed greatly reduced intensity in lanes containing trypsinised parasite extracts. This surprisingly shows that PfEMP1 is still expressed on the surface of clag9 TGD parasites. One possible interpretation is that a truncated CLAG9 is expressed and is sufficient to maintain PfEMPI on the surface. Another is that there is a second relevant gene in the deleted region.

EXAMPLE 9 Stages of the life cycle where clag paralogues might function One inference is that the role of clag genes in cytoadherence may be in determining, together with var, receptor-binding specificity. Alternatively, a clag paralogue could be one or more of the receptor-parasite ligand interactions which occur in the mosquito host, such as adherence of the ookinete to the midgut lumen or invasion of salivary gland cells by sporozoites. It is further possible that a clag paralogue is involved in binding of merozoites to red cells. If this latter model is correct and a chemotherapeutic that inhibited an active site common to all clag paralogues could be found, then simultaneous mutations in a number of paralogues would be required in order for dru resistance to develop.

EXAMPLE 10 Cellular locations of proteins of the CLAG family Agglutination, indirect fluorescent antibody staining and immunoelectron microscopy are used to determine the cellular locations of CLAG paralogues. The patterns of reactivity with human sera are also determined. clag9 has been successfully expressed as a series of fragments fused to glutathione S-transferase using vector pGEX 2T. As hydrophobic domains can be difficult to express, clag cDNA is amplified by PCR, fragmented by sonication and fragments of greater than 300bp are inserted into the SmaI site of pGEX 2T. A large number of colonies are screened for production of abundant fused polypeptides by PAGE and several where isolated. Sequence runs establish which segment is encoded in each and this allowed the selection of representative clones from each region. Fused polypeptides were prepared from clones corresponding to each domain of CLAG by ion exchange chromatography and are being used for production of rabbit antibodies.

The information derived from this should allows the design of constructs of the clag paralogues bounded at points precisely equivalent to those which in clag9 results in stable soluble pGEX fused polypeptides. If clag9 indeed traverses the red cell membrane, antibodies to external

domains should agglutinate the cells and should react with intact infected cells by indirect immunofluorescence.

The generation of antibodies specific for individual clag paralogues permits the use of a variety of approaches to examine questions about the expression of clag paralogues at the single cell level. While it is clear that 3D7 expresses clag 2,3.1 and 9 mRNAs, this does not necessarily mean that every parasite in the culture expresses both. Two-color immunofluorescent studies (25) can be used to establish whether both CLAG9 and CLAG3 are present in the same cells. This approach is extended in pairwise-fashion to other paralogues as antibodies become available. Ex t 1 Expt 1 Expt 2 Expt 3 Expt 4 Expt 5 Expt 6 (6 weeks) (7 weeks) (10 weeks) (16 weeks) (17 weeks) 3D7. HC. ASCI 35 37 19 16 8 11 8 6 3D7. HC. ASCIp. 24 28 142 148 3D7. HC 7 144 138 3D7l l0 105 192 183 189 153 192 183 145 14X 3D7.Pac4 Clag 8 21 Clone 3D7.11E 8 12 TABLE E 3. Inhibition of cytoadherence to melanoma cells aller transfection of 3D7 with antisense construct HC. ASC I and knockout vector Pac4 ('lag.<BR> falciparum clone 3D7 was electroporated with Pac4 Clag, HC. ASC 1 or HC 7 (transfection control) and cultured in 0. I mM pyrimethamine. Once<BR> a targeted gene disruption of clag had been confirmed by Southern blotting the transfected line 3D7. Pac4 Clag was cloned After synchronization<BR> by gelatin flotation, parasites were grown to 5-8% parasitaemia and cytoadherence to melanoma cells was then measured as described [10]. Results<BR> are shown as parasitized cells bound per 100 melanoma cells. Duplicates are shown for each point.

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