EP0772679A1

EP0772679A1 - Schistosoma protein and uses thereof

Info

Publication number: EP0772679A1
Application number: EP95922628A
Authority: EP
Inventors: A. Imperial College of Sci. Tech. & Medic. AGNEW; Volker Gutsmann
Original assignee: Imperial College of Science Technology and Medicine
Current assignee: Imperial College of Science Technology and Medicine
Priority date: 1994-06-22
Filing date: 1995-06-21
Publication date: 1997-05-14
Also published as: AU2745695A; WO1995035370A1

Abstract

The use of a protein obtainable from a Schistosoma as a pharmaceutical is described. In particular, the protein can be used as a blood anti-coagulant or as a vaccine. In particular, the protein is antigen HB14 obtainable from a Schistosoma, preferably S. haematobium.

Description

370

Schistosoma protein and uses thereof

The present invention relates to a protein. In addition, the present invention relates to the use of the protein as a pharmaceutical.

In one embodiment the present invention especially relates to a blood processing agent, in particular an anti-coagulant.

In another embodiment the present invention especially relates to a vaccine, in particular an anti-schistosome vaccine.

Schistosoma are parasites that feed on blood, two examples of which are S. haematobium and S.mansoni. Some background teachings on Schistosoma may be found in US-A-5219566 and EP-A-0554064.

Even though there is limited information available concerning the morphology and physiology of the digestive tract of Schistosoma, some ultrastructural studies have been published by Spence & Silk (1970) and Dike (1971). In this regard, it would appear that the alimentary canal of Schistosoma is divided into the fore-gut, which comprises the oral sucker, the pharynx and the oesophagus. The hind-gut begins at the junction between the oesophagus and the fore-gut. The fore-gut surface is essentially an extension of the outer tegument, a syncytium, bounded by outer and basal membranes and connected to numerous nucleated cell bodies. The cell bodies underlie a muscle layer and are connected to the tegument by narrow cytoplasmic extensions. The oesophageal luminal fringe, from the mouth to the posterior end, becomes dramatically more convoluted. The loops appear to be rich in granular inclusions resembling secretory granules and, at the tips, contain numerous small mitochondria and vesicles. Towards the posterior end of the oesophagus, the number and size of cell bodies increase, and are so closely apposed that they form almost a discrete structure, the oesophageal "gland". These cell bodies are particularly rich in rough endoplasmic reticula, poly-ribosomes and dense granules which indicate extensive protein synthesis. 370

When food is not present in the oesophagus, the number of granules decreases. Thus, there is no direct evidence that the oesophagus is a secretory rather than an adsorptive structure. But, on balance, the oesophagus would appear to be a secretory structure.

The following observations have been made about Schistosoma. First, Morris & Threadgold (1968) observed that intact RBC (red blood cells) were not present within or below the oesophagus. Second, Spence (1970) and Dike (1971) observed that erythrocytes are rapidly lysed on entry into the oesophagus. Third, Spence & Silk ( 1970) observed the absence of clotting of the blood in the pharynx and oesophagus.

Schistosome proteins have been studied. In particular, Davis et al (1988) report on an adult S.mansoni cDNA clone 10.3 which encodes an antigen that is recognised by sera from infected humans. They report the presence of tandemly repeated exons which encode 81 -base repeats in the nucleotide sequence. Davis et al do not suggest a use for the 10.3 protein in S.mansoni, let alone organisms other than S.mansoni, let alone homologues from organisms other than S.mansoni. Moreover, on page 4754 the authors say "The broad array of functions observed for proteins with repeating units precludes functional analogies from being drawn for the clone 10.3 protein."

FR-A-2688008 mentions the use of fragments of a gene coding for glutathione-S- transferase obtained from S.bovis and S. haematobium as a vaccine.

WO-A-91/12327 mentions the use of a 26 kd glutathione-S-transferase obtained from S.mansoni in the prevention of or treatment of infection from Schistosoma.

EP-A-0268501 mentions the use of a glutathione-S-transferase obtained from S.mansoni in the prevention of or treatment of infection from Schistosoma.

FR-A-2689906 mentions the use of fragments of a gene coding for glutathione peroxidase obtained from S.mansoni as a vaccine. WO-A-91/09621 mentions the use of fragments of a 28 kd protein obtained from S.mansoni as a vaccine.

EP-A-0251933 mentions the use of antigenic fragments of a 28 kd protein obtained from S.mansoni as a vaccine.

Each of US-A-5219566 and US-A-5051254 mentions a fusion protein which apparently can stimulate a immune response resulting in protective anti-bodies which recognise surface antigens of Schistosoma. In one embodiment a preparation is provided comprising antibodies which bind to protein epitopes expressed on the surface of live schistosomula of S.mansoni.

WO-A-90/02563 mentions a vaccine against S.mansoni which comprises at least one antigen present in the surface membrane of the adult S.mansoni worm (mb-S) and which apparently is capable of provoking an immune response. The vaccine can be mb-S itself.

EP-A-0554064 mentions an immunogenically and an antigenically active protein that apparently is capable of eliciting immunity against Schistosoma parasites. The protein is the S.mansoni integral membrane protein having in the un-glycosylated form a molecular weight of about 25 kD.

Despite the earlier reported work concerning the preparation of vaccines against infection from Schistosoma, there is still a demand for an effective vaccine against infection from Schistosoma (especially S. haematobium and S.mansoni).

It is also well known in the art that invertebrate blood processing agents - such as the anti-coagulant hirudin - have widespread pharmacological and medical applications (see Clinical Pharmacology of Cardiovascular Drugs Chapter 58 pages 370 etc.). But, likewise, there is still a demand for an effective blood processing agent, in particular an anti-coagulant. In this regard, the present invention provides a new protein obtainable from Schistosoma. The present invention also provides a new use of a protein obtainable from Schistosoma. In particular, the present invention provides a new use of a protein obtainable from S. haematobium and S.mansoni and especially the former.

Thus, according to a first aspect of the present invention there is provided a protein obtainable from a Schistosoma for use as a pharmaceutical, wherein the protein is S. haematobium antigen HB14.

According to a second aspect of the present invention there is provided a protein obtainable from a Schistosoma for use as a pharmaceutical, wherein the protein is any one of:

i. a protein comprising the sequence represented as SEQ. I.D. 1, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity;

ii. a protein comprising at least the sequence represented as SEQ. I.D. 2, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity;

iii. a protein comprising at least the sequence represented as SEQ. I.D. 3, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity;

iv. a protein comprising at least the sequence represented as SEQ. I.D. 4, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity;

v. a protein encoded by the nucleotide sequence represented as SEQ. I.D. 5, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity; vi. a protein encoded by at least the nucleotide sequence represented as SEQ. I.D.

6, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity;

vii. a protein encoded by at least the nucleotide sequence represented as SEQ. I.D.

7, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity;

viii. a protein encoded by at least the nucleotide sequence represented as SEQ. I.D. 8, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity;

ix. a protein comprising a hydrophobic signal peptide region, preferably of about 16 amino acids, a hydrophobic carboxy terminal, preferably showing three peaks on a hydrophobicity plot of the protein, and an intermediate region containing tandem repeating hydrophilic units, preferably wherein each repeating unit has about 27 amino acids and more preferably wherein the repeating unit nearest to the carboxy terminal is truncated, and preferably has about 18 amino acids;

x. a protein obtainable from the oesophagus region of a Schistosoma and having a molecular weight in the range of from about 20 kd to about 26 kd, preferably from about 22 kd to about 25 kd, more preferably from about 22.4 kd to about 24.6 kd;

xi. a protein obtainable from the post acetabular glands of cercariae of Schistosoma and having a molecular weight in the range of from about 20 kd to about 26 kd, preferably from about 22 kd to about 25 kd, more preferably from about 22.4 kd to about 24.6 kd. With this aspect of the present invention, the protein may comprise a plurality of any combination of the features i. to xi. as listed above.

According to a third aspect of the present invention there is provided a protein obtainable from a Schistosoma for use as a pharmaceutical, wherein the protein is coded by at least any one of:

i. the nucleotide sequence represented as SEQ. I.D. 5, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity;

ii. the nucleotide sequence represented as SEQ. I.D. 6, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity;

iii. the nucleotide sequence represented as SEQ. I.D. 7, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity;

iv. the nucleotide sequence represented as SEQ. I.D. 8, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity.

According to a fourth aspect of the present invention there is provided a blood processing composition comprising a protein obtainable from a Schistosoma admixed with or contained within a suitable carrier, diluent or excipient, wherein the protein is as defined above.

According to a fifth aspect of the present invention there is provided a vaccine composition comprising an immuno logically active protein obtainable from a

Schistosoma admixed with or contained within a suitable carrier, diluent or excipient, wherein the protein is as defined above.

According to a sixth aspect of the present invention there is provided the use of a protein obtainable from a Schistosoma in the manufacture of a blood processing agent, wherein the protein is as defined above.

According to a seventh aspect of the present invention there is provided the use of a protein obtainable from a Schistosoma in the manufacture of an anti-coagulant, wherein the protein is as defined above.

According to an eighth aspect of the present invention there is provided the use of an immunologically active protein obtainable from a Schistosoma in the manufacture of a vaccine, wherein the protein is as defined above.

According to a ninth aspect of the present invention there is provided a protein comprising the sequence represented in any one of SEQ I.D. 1-4; or non-critically amino acid substituted variants, homologues or fragments thereof; but not S. mansoni 10.3.

According to a tenth aspect of the present invention there is provided a nucleotide sequence comprising the sequence represented in any one of SEQ I.D. 5-8; or non- critically nucleic acid substituted variants, homologues or fragments thereof; but not that of S. mansoni 10.3.

According to an eleventh aspect of the present invention there is provided a protein according to the present invention or a protein expressed by the nucleotide sequence according to the present invention, wherein the protein exhibits a blood processing effect after administration to or contact with a subject.

According to a twelfth aspect of the present invention there is provided a protein according to the present invention or a protein expressed by the nucleotide sequence according to according to the present invention wherein the protein exhibits a blood anti-coagulant effect after administration to or contact with a subject.

According to a thirteenth aspect of the present invention there is provided a protein according to the present invention or a protein expressed by the nucleotide sequence according to according to the present invention wherein the protein exhibits an immunological effect after administration to or contact with a subject.

According to a fourteenth aspect of the present invention there is provided an article coated with a protein according to the present invention or a protein expressed by the nucleotide sequence according to the present invention.

According to a fifteenth aspect of the present invention there is provided a protein obtainable from a Schistosoma for use as a blood processing agent.

According to a sixteenth aspect of the present invention there is provided the use of a protein obtainable from a Schistosoma in the manufacture of a blood processing agent.

According to a seventeenth aspect of the present invention there is provided the use of a protein obtainable from a Schistosoma in the manufacture of an anti-coagulant.

According to an eighteenth aspect of the present invention there is provided a blood anti-coagulant composition comprising a protein admixed with or contained within a suitable carrier, diluent or excipient; or a protein for use as an anti-coagulant; or the use of a protein in the manufacture of a blood anti-coagulant; wherein the protein has the protein sequence represented in SEQ I.D. 1, or non-critically amino acid substituted variants, homologues or fragments thereof capable of having a blood anti¬ coagulant effect, or a protein expressed by the nucleotide sequence represented in SEQ I.D. 5, or non-critically nucleic acid substituted variants, homologues or fragments thereof and wherein the expressed protein is capable of having an anti-coagulant effect. According to a nineteenth aspect of the present invention there is provided a vaccine composition comprising a protein admixed with or contained within a suitable carrier, diluent or excipient; or a protein for use as a vaccine; or the use of a protein in the manufacture of a vaccine; wherein the protein has the protein sequence represented in SEQ I.D. 1 , or non-critically amino acid substituted variants, homologues or fragments thereof having immunological activity, or a protein expressed by the nucleotide sequence represented in SEQ I.D. 5, or non-critically nucleic acid substituted variants, homologues or fragments thereof encoding a protein having immunological activity.

A further aspect of the present invention includes a method of treating a subject in need of, or likely to be in need of, anti-coagulant treatment wherein the method comprises administering to the subject a protein according to the present invention, or a protein expressed by the nucleotide sequence according to the present invention, or a composition according to the present invention.

A further aspect of the present invention includes a method of treating a subject in need of, or likely to be in need of, immunisation against infection from Schistosoma wherein the method comprises administering to the subject a protein according to the present invention, or a protein expressed by the nucleotide sequence according to the present invention, or a composition according to the present invention.

Preferably the Schistosoma is S. haematobium.

Preferably the protein is for use as a blood processing agent.

Preferably the protein is for use as an anti-coagulant.

__*

Preferably the protein is for use as a vaccine.

Preferably the protein is for use as a vaccine against infection from Schistosoma. Preferably the protein is for use as a vaccine against S. haematobium infection or S.mansoni infection.

Preferably the subject is a vertebrate animal, such as a human.

Preferably the protein according to the present invention comprises the sequence represented in any one of SEQ. I.D. 1-4, preferably that shown in Seq I.D. 1, or it comprises a sequence that has substantial homology therewith, or a fragment, homologue or variant thereof capable of exhibiting a pharmaceutical effect, in particular a blood processing activity, preferably a blood anti-coagulant effect, or an immunological effect capable of immunising a subject.

The present invention therefore provides a protein capable of acting as a pharmaceutical, in particular a blood processing agent, preferably a blood anti- coagulant, or a vaccine.

In a highly preferred embodiment the protein is not expressed by the natural genomic DNA sequence when in its natural environment. Thus, highly preferred embodiments include the protein when prepared by use of recombinant DNA techniques, including the expression of cDNA or a synthetic nucleotide sequence. Preferably the protein is expressed by a cDNA sequence that is the same as that shown in any one of SEQ. I.D. 5-8, preferably that shown in Seq I.D. 5, or it is expressed by a sequence that is complementary to any one of SEQ. I.D. 5-8, or having substantial homology therewith, or containing any suitable codon substitutions for any of those shown in Figure 1 and wherein the resultant protein is capable of acting as a pharmaceutical, in particular a blood processing agent, preferably a blood anti-coagulant, or a vaccine.

The present invention also provides a nucleotide sequence capable of coding for a pharmaceutical, in particular a blood processing agent, preferably a blood anti- coagulant, or a vaccine. In a highly preferred embodiment the nucleotide sequence is not in its natural environment and under the control of the promoter with which it is naturally associated which is also in its natural environment. Thus, highly preferred embodiments include the use of recombinant DNA techniques using for example cDNA or a synthetic nucleotide sequence. Preferably the nucleotide sequence is a cDNA sequence that is the same as that shown in any one of SEQ. I.D. 5-8, preferably that shown in Seq I.D. 5, or it is a sequence that is complementary thereto, or having substantial homology therewith, or containing any suitable codon substitutions for any of those shown in Figure 1 and wherein the resultant protein is capable of acting as a pharmaceutical, in particular a blood processing agent, preferably a blood anti¬ coagulant, or a vaccine.

The term "protein" includes un-modified and modified proteins such as post- translationally modified proteins and glycosylated proteins.

The term "obtainable from" includes directly or indirectly obtaining the protein. Examples of indirectly obtaining the protein include expressing the cDNA by means of a suitable expression system.

The term "blood processing agent" includes any agent that has an effect on blood such as an anti-coagulant, which may or may not work by binding to prothrombin and thereby inhibit the clotting cascade. Preferably, the term means an anti-coagulant.

The term "vaccine" includes a material producing in an animal, both human and non- human, an immune reaction and an acquired immunity to an organism such as a parasite - i.e. providing immunisation against infection from a particular organism. The term also includes the technique of naked DNA immunisation, wherein for the vaccine of the present invention the DNA that is used is the nucleotide sequence according to the present invention. The term also includes the technique of carrier mediated immunisation, such as by using Salmonella transformed with the nucleotide sequence according to the present invention. The term "suitable carrier, diluent or excipient" includes any suitable medium for administration, such as oral administration or administration via injection - such as IM or IV. Such mediums are well known in the art. such as water or saline. The term also includes the protein bound to a solid support. The term also covers the use of a recombinant delivery system as a suitable carrier, such as a transformable Salmonella - i.e. a Salmonella transformed with the nucleotide sequence according to the present invention.

The terms "variant", "homologue" or "fragment" include any substitution of, variation of, modification of, replacement of, deletion of or addition of one or more amino acid(s)/nucleic acid from or to the sequence providing the resultant protein has pharmaceutical activity, such as a blood processing agent, preferably a blood anti¬ coagulant, or a vaccine.

The expression "substantial homology", which can be otherwise expressed as "substantial similarity", includes homology with respect to structure and/or nucleotide components, providing the resultant protein has pharmaceutical activity, such as a blood processing agent, preferably a blood anti-coagulant, or a vaccine. With respect to sequence homology (i.e. similarity), preferably there is at least 50 %, preferably at least 60 %, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% homology.

The term "article" includes any appropriate appliance for transplantation. The term also includes any appropriate device for delivery of the protein of the present invention to a subject.

Other embodiments of the present invention include: a transformed host organism capable of producing the protein according to the present invention as a consequence of the introduction of a nucleotide sequence as herein described, preferably wherein the host organism is a microorganism, such as E. coli.; a method for preparing the protein according to the present invention comprising expressing a nucleotide sequence contained in the host organism and isolating the expressed protein; and a vector incorporating a nucleotide sequence as hereinbefore described, preferably wherein the vector is a transformed pBLUESCRIPT plasmid. For example, the protein HB14 can be expressed in E. Coli, baculovirus, yeast or mammalian cell expression systems.

With regard to expression of the nucleotide sequence according to the present invention in, for example, E. coli, it can be preferable to exclude part of nucleotide sequence coding for the COOH terminal (such as amino acids 131-188) when using, for example, the pMAL system or other similar expression systems. In this regard, primer- linkers can be designed to produce PCR products covering nucleotides 20-427. These PCR products can then allow in-frame, directional cloning into pMAL. Expression of the fragmented HB14 as a fusion protein to maltose-binding protein is increased 10000 fold when the hydrophobic C-terminal (amino acids 131-188) is omitted.

A preferred embodiment of the present invention relates to the non-parasitological application of HB14 as a novel anti-coagulant for animals, in particular humans.

An alternative preferred embodiment of the present invention relates to the use of HB14 as a vaccine for animals, such as humans.

The preferred protein, which is S. haematobium antigen HB14, has the protein sequence represented by SEQ. I.D. 1. This protein has the following features:

1. a hydrophobic signal peptide region of 17 amino acids;

2. a hydrophobic carboxy terminal having three distinct hydrophobic groups, each comprising about 8 amino acids;

3. an intermediate region containing tandem repeating hydrophilic units, wherein each repeating unit has 27 amino acids and the repeating unit nearest to the carboxy terminal is truncated and has 18 amino acids. In addition, there is a possible cleavage site between positions 17 and 18 which suggests that the first 17 amino acids are a signal sequence.

These features are evidenced by the accompanying hydrophobicity plots shown as Figures 5 and 6 (which are discussed later).

An example of a homologue to S. haematobium antigen HB14 is S.mansoni 10.3, as evidenced by the protein alignment characteristics shown in the accompanying figures and the accompanying hydrophobicity plots. Thus, in one aspect of the present invention there is provided the use of S.mansoni 10.3 as a pharmaceutical, such as a blood processing agent, for example an anti-coagulant, or as a vaccine.

The use of a protein obtainable from the oesophagus of a Schistosoma, or the post acetabular glands of cercariae of a Schistosoma, as a pharmaceutical, in particular a blood processing agent, more in particular an anti-coagulant, is surprising, especially as protein has not been previously isolated from, for example, the post acetabular glands of cercariae.

An E. Coli bacterial stock containing the plasmid pBluescript containing a 680 bp fragment of cDNA for HB14 (Sk HB14) was deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on 22 June 1994, and was allocated deposit number NCIMB 40654.

The plasmid was formed by the insertion of the gene into the poly-cloning site using the restriction sites EcoRl and ^Tzol. The nucleotide sequence coding for HB14 may be isolated therefrom by use of the restriction enzymes EcoRl and Xho\ and then inserted in any suitable expression system and subsequently expressed by suitable means. Thus, highly preferred embodiments of the present invention include any one of the aforementioned aspects of the present invention but wherein the protein or the nucleotide sequence is obtainable from deposit NCIMB 40654. For example, highly preferred embodiments include:

1. A protein obtainable from a Schistosoma for use as a pharmaceutical, wherein the protein is S.haematobium antigen HB14, and wherein the protein is obtainable from deposit NCIMB 40654.

2. A blood anti-coagulant composition comprising a protein admixed with or contained within a suitable carrier, diluent or excipient; or a protein for use as an anti¬ coagulant; or the use of a protein in the manufacture of a blood anti-coagulant; wherein the protein has the protein sequence represented in SEQ I.D. 1, or non- critically amino acid substituted variants, homologues or fragments thereof capable of having a blood anti-coagulant effect, or a protein expressed by the nucleotide sequence represented in SEQ I.D. 5, or non-critically nucleic acid substituted variants, homologues or fragments thereof, wherein the expressed protein is capable of having an anti-coagulant effect; and wherein the protein is obtainable from deposit NCIMB 40654.

3. A vaccine composition comprising a protein admixed with or contained within a suitable carrier, diluent or excipient; or a protein for use as a vaccine; or the use of a protein in the manufacture of a vaccine; wherein the protein has the protein sequence represented in SEQ I.D. 1, or non-critically amino acid substituted variants, homologues or fragments thereof having immunological activity, or a protein expressed by the nucleotide sequence represented in SEQ I.D. 5, or non-critically nucleic acid substituted variants, homologues or fragments thereof encoding a protein having immunological activity; and wherein the protein is obtainable from deposit NCIMB 40654. 5370

16

As mentioned above, the protein of the present invention is obtainable from the oesophagus region of a Schistosoma. or the post acetabular glands of cercariae of a Schistosoma, or it is derived or derivable from such a protein - e.g. an active fragment thereof. In this regard, it would appear that wild type HB14 protein is naturally expressed in the oesophageal region in adult S. haematobium. Staining experiments revealed that the protein is usually observed with the oesophagus itself, with faint staining with the oesophageal gland. Further studies with immuno-gold detection and electron microscopy were used to localise HB14 at the sub cellular level. In these experiments, HB14 was less well represented in the nucleated sub-tegumental cells than in the oesophageal luminal fringe and lumen. Since the sub-tegumental cell bodies are the site of protein synthesis for the syncytium, it is believed that the wild type protein is produced here. Furthermore, it would appear that HB14 is then transported through the muscle layer packed in dense secretory granules into the luminal fringe as there gold-labelled secretory granules are clearly visible in the vastly elongated syncytial folds. The strongest immuno-reactivity, however, was observed with extra cellular, amorphous material within the lumen of the posterior oesophagus.

The present invention will now be described only by way of examples, in which reference shall be made to the following Figures, in which:

Figure 1 is the nucleotide sequence of S.haematobium cDNA clone HB14;

Figure 2 is the deduced amino acid sequence of S.haematobium cDNA clone HB14;

Figure 3 is the nucleotide sequence of S.haematobium cDNA clone HB14 and the deduced amino acid sequence of S.haematobium cDNA clone HB14;

Figure 4 shows the alignment of different regions of the open reading frame of S.haematobium cDNA clone HB14 and S.mansoni cDNA clone 10.3. 5370

17

Figure 5 is a hydrophobicity plot of the predicted amino acid sequence of S.haematobium cDNA HB14;

Figure 6 is a hydrophobicity plot of the predicted amino acid sequence oϊ S.mansoni cDNA clone 10.3;

Figure 7 is a map of HB14; and

Figure 8 is a plot of partial thromboplastin time kaolin (PTTK).

In more detail, Figures 1 to 3 show the nucleotide and deduced amino acid sequence of S.haematobium cDNA clone HB14. The cDNA clone is 680 bp long and contains one long open reading frame of 567 bp (in capital letters) which encoded a 188 amino acid protein. The initiation and termination codon are denoted as such. The internal EcoRl site is labelled »«--.

Figure 4 shows the alignment of different regions of the open reading frame of HB14 and S.mansoni 10.3. Nucleotide homology is denoted by a vertical straight line and positions of homology within the amino acid sequence are in bold. Area A) denotes the region 5' up to the beginning of the repeats. Area B) denotes one 81 bp-repeat unit. Area C) denotes the region from the repeats to 3 ' end. Between the clones of the two species there is an overall DNA identity of 66% but only 40% identity on the amino acid level. Despite this low level of protein homology, the hydrophobicity of both proteins is similar. These two molecules are homologues on a morphological/structural basis. It is possible that the number of direct tandem repeats can vary in HB14 and its homologues. Up to date, transcripts with 2, 3 and 4 repeat units have been found to be present in S.haematobium. In this regard, the migratory larval stages of the parasite were found to co-transcribe all three versions, whereas adult worms and the invasive stages predominantly transcribe the smallest, 2-repeat version. 370

18

Figure 5 and Figure 6 show hydrophobicity plots of the predicted amino acid sequence of S.haematobium cDNA HB14 and the predicted amino acid sequence of S.mansoni cDNA clone 10.3. The hydrophobicity plots were conducted in accordance with Kyte and Doolittle. The plots have a window size of 7 amino acids. S.haematobium cDNA HB14 has two repeats; whereas S.mansoni cDNA clone 10.3 has five repeats.

Fig 7 shows in-frame PCR products for cloning into pMALc2, covering different regions of HB14. In the Figure X = hydrophobic signal peptide region, Y = hydrophilic tandem repeats and Z = hydrophobic carboxy terminal. PCRl includes the coding region of the amino terminal and the repeat region. MBP-PCR2: amino terminal excluding the repeat region and MBP-PCRr: the repeat region only. The in- frame inserts were produced by PCR amplification of pBLUESCRIPT-HB14 with the following linker-primers:

PCR 1 VG%-BamHING6-PstI5' end to the end of the repeat region

PCR 2 VG8-BamHIfVG7-PstI5^, end to the beginning of the repeats

PCRr VG4-EcoRI/VG6-PstI repeat region only

The regions of HB14 covered by these MBP constructs are illustrated in Figure 7. The sequence for PCRl is represented as SEQ. I.D. 6, the sequence for PCR2 is represented as SEQ. I.D. 7 and the sequence for PCRr is represented as SEQ. I.D. 8.

Figure 8 is discussed in more detail later.

EXAMPLES

A. SELECTION PROCESS

S.bovis/S.haematobium-speciήc cDNA clones were previously selected by using the following strategy: 370

19

Pooled sera from mice, singly vaccinated with 20krad irradiated cercariae of S. bovis and demonstrably protected against S.haematobium challenge, were used to screen a S.haematobium adult \-gtll expression library. Positive clones were selected and screened with pooled sera from singly S.haematobium vaccinated mice. Clones, recognised in both screening steps, were selected and counter-screened with pooled sera from singly S.mansoni vaccinated mice. This time, positive clones were discarded. The resulting selection of clones represented antigens whose epitopes are shared by S.haematobium and S. bovis, but not by S.mansoni. Serum from multiply S. bovis vaccinated rabbits transferred protection to naive mice in a S.haematobium group-specific manner (Agnew, 1986). The selected clones were screened with an individual S. bovis- vaccinated rabbit serum and positive clones selected. One further selection step was included. The clones should be recognised by S.haematobium -infected humans, but not by patients with schistosomiasis mansoni. 50 sera obtained from S.haematobium infected patients and 50 from S.mansoni patients were used to screen the selected clones. Not all S.haematobium patients recognised all the clones, but no S.mansoni patients had an antibody response to any of the clones. This selection process produced 13 λ-gtl l cDNA clones, which encoded S.haematobium/ S. bovis specific epitopes. Several of these clones encoded the HB14 protein.

B, E.COLI EXPRESSION

Expression of HB14 as fusion protein to MBP in the pMAL system

B.l Preparation of inserts from λ-gtll derived clones for sub-cloning into pMAL

Inserts, originally derived from λ-gtl 1 clones, are in-frame with the malE reading frame, and were subcloned into the pMALc2 vector (New England Biolabs). 370

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B.2 Preparation of open-reading frames of HB14 by PCR

Procedure:

1. HB 14 specific internal primers were designed which contained different restriction sites to allow directional cloning. The design was such that cloning into pMALc2 would be in frame with the malE gene.

2. 1 ng of pBLUESCRIPT-HB14 template was amplified in a 100 microlitre reaction containing 300 ng of each primer.

3. The reaction was carried out for only 20 cycles of 1 min 94°C, 75 seconds 60°C and 1 min 72°C to maintain copy accuracy.

4. The PCR products were subcloned. Briefly, the reaction was extracted with an equal volume of phenohchloroform and the PCR products blunt-ended.

5. The blunt-ended PCR products were ligated into Smal-digested pUC18 and transformed into competent host cells of the strain DH5o_.

6. Recombinant clones were isolated and plasmid prepared by mini-preparation.

7. The insert was cut out by digestion with the two restriction enzymes whose recognition sites had been incorporated in the primer-linkers. The insert was purified and was then ready for cloning into appropriately prepared pMAL vector. 370

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B.3 Cloning of inserts or PCR products into pMALc2

Procedure:

1. After ligation of the DNA fragment of interest into pMALc2, competent E. coli cells of the strain DH5α were transformed.

2. Although selection of recombinant clones by α-complementation was possible, this approach was not used since induction of expression of fusion protein at this stage might prevent the establishment of recombinant clones. Instead, PCR amplification was used to find recombinant clones. To achieve this, individual colonies were picked with a sterile tooth pick, streaked onto a master plate and the remaining cells on the tooth pick transferred into 20 microlitre of complete PCR reaction mixture containing insert-specific primers. After PCR amplification, the products were then analysed by agarose gel electrophoresis.

3. Plasmid DNA was prepared from clones positive in the PCR reaction and digested with appropriate restriction enzymes to cut out the insert.

4. Clones that showed the presence of the insert were grown in 10 ml LB at 37°C and with good aeration, and induced with 0.3 mM IPTG final concentration at an OD₆₀₀=0.5.

5. After 2 hours, cells of a 1 ml aliquot were pelleted, resuspended in 100 microlitre 2x SDS-PAGE loading buffer containing 2-mercaptoethanol and boiled for 5 min.

6. The samples were analysed on a 12.5% SDS-polyacrylamide gel, stained with Coomassie Blue or further analysed by immuno-blotting. 5370

22

B.4 Preparation of Maltose-Binding-Protein (MBP) fusion protein by affinity purification

The affinity of MBP to amylose can be exploited to purify MBP-fusion protein in a one-step-procedure (Guan et al., 1987; Maina et al., 1988).

Reagents:

LB Medium, 50 mg Ampicillin/ml 1 M IPTG, sterile

Amylose resin (New England Biolabs) Column buffer: 20 mM Tris-HCl, pH 7.4 200 mM NaCl 1 mM EDTA Elution buffer: Column buffer, 10 mM Maltose

Procedure:

1. Typically, 11 LB medium was inoculated with 10 ml of an o/n culture of a recombinant clone that expressed the fusion protein in the pilot experiment. Cells were grown at 37°C with good aeration until OD600 = 0.5. IPTG was added to a final concentration of 0.3 mM and incubation was continued for 2.5 hours.

2. The cells were harvested at 5000 x g (20 min, 4°C).

3. The cell pellets were resuspended in 30 ml column buffer and frozen at -20°C o/n.

4. Cells were thawed and sonnicated until the release of protein (as measured by Bradford assay) reached a plateau.

5. Lysate was spun at 9000x g (30 min, 4°C) 70

._.-.

6. The pellet was resonicated if necessary and respun. The supernatants were pooled.

7. The protein concentration of the lysate was measured by the method of Bradford and diluted with column buffer until protein concentration was approximately 2.5 mg/ml.

8. A 5 ml disposable syringe was plugged with glass wool and packed with amylose resin (up to 3 ml bed volume).

9. The column was washed with 10 volumes of column buffer.

10. The lysate was applied at a flow rate of [lOx (diameter of column in cm)2]ml/hr.

1 1. At least 10 bed volumes of column buffer were used to wash the column.

12. The MBP-fusion protein was eluted with elution buffer in 1 ml fractions.

13. The protein concentration of fractions was estimated by spectrometry at 280 nm.

14. The fractions containing protein were pooled and protein concentration measured by the method of Bradford.

C_ BACULOVIRUS EXPRESSION

Expression of HB14 in the Baculovirus system

Most of the following procedures except the cloning of insert into the transfer vector are described in detail in O'Reilly et al. (1992). Cl Cloning of HB14 into the transfer vector pEVmXIN

Reagents:

lOx One-Phor-All restriction enzyme buffer (Pharmacia) 5M ΝaClO₄ isopropanol Phenolxhloroform (1 :1)

Procedure:

1. 6 microgram of plasmid pEVmXIV were digested with 30 U of Kpnl in lx One-Phor-All (OPA) buffer at 37°C for 2 hours.

2. The OPA buffer concentration was adjusted to 2x and the plasmid DNA was further digested with 30 U of EcoRI at 37°C for 2 hours.

3. The digested DNA was extracted with an equal volume of phenol:chloroform and precipitated by adding 0.2 volumes of 5M NaClO₄ and 0.6 volumes of isopropanol for 10 min at RT and centrifugation for 20 min at lOOOOx g. These conditions prevent precipitation of very small fragments (like the poly linker).

4. The pellet was resuspended in 20 microlitre of dH₂O and 2 microlitre were analysed on a agarose gel for quantification.

5. Since HB14 contained an internal EcoRI site, a pilot experiment of a partial EcoRI digest had to be carried out: 5 microgram of pBLUESCRIPT-HB14 in a 50 microlitre reaction volume were linearised with 50 U of Kpnl in 1 x OPA buffer for 4. hours at 37°C. 5 microlitre were set aside as timepoint to. The OPA buffer concentration of the remaining digest was adjusted to 2x and 5 U of EcoRI were added. The digest was incubated at 37°C and 5 microlitre aliquots were snap frozen at 1, 2, 5, 15 and 30 min.

6. The time course was analysed by agarose gel electrophoresis and the timepoint when the largest EcoRI fragment had the highest concentration was determined.

7. 10 microgram of pBLUESCRIPT-HB14 was partially digested under conditions determined by the pilot experiment and the insert isolated from the agarose.

8. The insert was purified by extraction of phenol hloroform, precipitated, resuspended in 20 microlitre of dH₂O and 2 microlitres were analysed on a agarose gel.

9. Ligation was carried out at a molar ratio of vectoπinsert of 1:5.

10. The ligation was transformed into E.coli strain XL-1 BLUE and potential recombinant clones analysed by PCR with insert-specific primers.

11. PCR positive clones were analysed by a EcoRI and Kpnl restriction digest of a plasmid minipreparation.

C.2 Co-transfection of SF-21 insect cells with virus and plasmid DNA

Co-transfections and amplification of the primary virus stock were carried out as described in O'Reilly et al. (1992).

Briefly, 2xl0⁶ cells were co-transfected with 1 microgram of viral BaculoGold-DNA and 2 microgram of recombinant transfer plasmid in transfection buffer. The cells were incubated for five days and the supernatant containing the virus particles removed. This primary virus stock was used to infect insect cells (overlaid by agarose) to form plaques. Plaques were then picked and amplified for two rounds to gain a stable secondary virus stock. 370

26

C.3 Analysis of recombinant baculovirus clones

It is necessary to analyse the viral genomic organisation to confirm the correct insertion of the gene into the virus genome. All methods used are described in O'Reilly et al. (1992, chapter 13).

C.4 Metabolic labelling of baculovirus-infected insect cells

A detailed method is given in O'Reilly et al. (1992, chapter 14). Briefly, 3 batches of 2x106 SF-21 cells were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 15. The same procedure was repeated for wild-type virus (L-l).

After infection the cells were re-fed with 2 ml of complete medium and incubated at

27°C. After 22 h the medium was removed and replaced with 0.5 ml methionine-free culture medium to deplete the endogenous pool of methionine. At 24 h p.i. (post infection) the first batch of cells were washed and re-fed with 0.5 ml methionine-deficient medium supplemented with 25 microCi 35S-methionine. The cells were incubated at RT (room temperature) for 1 hour. The radioactive supernatant was discarded and the cells washed three times. Cells were then lysed with 50 microlitre NP-40 lysis buffer. The lysate was analysed by SDS-PAGE and autoradiography. This procedure was repeated at 36 h and 48 h.

C.5 Detection of recombinant protein in the culture medium

Reagents:

TC100 culture medium supplemented with FCS to 10% (O'Reilly et al., 1992) TCI 00 culture medium

NP 40 lysis buffer: 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% NP-40 PBS, pH 6.2 Procedure:

1. 2 batches of 2x106 SF-21 cells were infected with a selected recombinant baculovirus clone at a MOI of 15 and incubated at 27°C in TCI 00 containing 10% FCS.

2. At 12 hours p.i., one petri dish was rinsed and re-fed with 1 ml TCI 00 without FCS and incubated for a further 24 hours.

3. At 36 hours p.i. both serum-free and serum-containing culture medium was collected. The cells of the serum containing petridish were also harvested and lysed in 50 microlitre of NP-40 lysis buffer.

4. The same experiment was carried out on a different time scale: Re-fed at 24 hours and harvested at 48 hours p.i.

5. All samples were analysed by SDS-PAGE and immunoblotting.

Ω. ANTI-COAGULATION ASSAY

D.l Thrombin Clotting Time

For the anti-coagulation assay, measurements of the thrombin time were used to evaluate the last step in the clotting cascade - i.e. the conversion of fibrinogen to fibrin initiated by the action of thrσmbin. A delay of thrombin time indicates a degree of inhibition of the clotting process.

The thrombin time measures the conversion of fibrinogen to fibrin. It is a measure of both quality and quantity of clottable protein. THROMBIN Fibrinogen — =_- — ====Fibrin

As it is understood in the art. methods vary according to the concentration of thrombin used and the presence or absence of calcium, and these factors will influence the clotting times obtained.

D.2. Equipment Required

KC10, PT100 or PT50

100 ul pipette and disposable tips KC10 Cuvettes Stopwatches KC10 override plug

D.3. Reagents

3.1 Platelet-poor plasma

3.2 Owren's buffered saline 3.3 Thrombin (e.g. Diagen Bovine)

The concentrated frozen stock (100 u/ml) is diluted in normal saline to give a clotting time with control plasma of 14-15 seconds.

D.4. Methods

4.1 Pipette 0.1 ml control plasma into each cuvette.

4.2 Add test reagent in 0.1 ml Owren's buffered saline to each test cuvette and warm to 37°C for two minutes. 4.3 Add 0.1 ml Owren's buffered saline to a control cuvette and warm to 37°C for two minutes.

4.4 Transfer tray with test and control cuvettes to KC10 and insert override plug.

4.5 Add 0.1 ml diluted thrombin to each cuvette in turn, while simultaneously activating each corresponding channel on KC10.

4.6 Record average of clotting times to the nearest second.

D.5. Interpretation

A prolonged thrombin time indicates partial inactivation of the clotting cascade at some point prior to the conversion of fibrinogen to fibrin. The normal range is 13-16 seconds.

D.6. Results

In our experiments, a delay of thrombin time is observed with the protein extracts for male and female schistosome heads. In particular, thrombin time is also significantly delayed by the addition of recombinant HB14 protein prepared by either E. Coli expression (i.e. of PCRl, PCR2 and PCRr - see above) or Baculovirus expression (HB14 - see above). Thus the proteins of the present invention, and the proteins expressed by the nucleotide sequences of the present invention, are blood processing agents. In particular, the proteins act as anti-coagulants.

E, FURTHER ANTI-COAGULATION STUDIES

The fragment of HB14 described above for expression studies using pMAL, was expressed as recombinant protein in the pET system to avoid fusion to a carrier protein. In this regard, the expression vector pET 15b (Novagen) allows IPTG- induced expression of recombinant protein that is not fused to a carrier or a His-tag. An ATG is provided within the 5' primer- linker. The region amplified by PCR comprises nucleotides 74-427 gave rise to a 1 19 AA polypeptide with an approximate molecular weight of 14 kDa. The recombinant vector was transformed into the host cell B 21 pLys.

The purified resultant 14 kDa polypeptide was tested in vitro for delay of coagulation of normal donor plasma. Partial thromboplastin time kaolin (PTTK) was measured with a KC10 automated clot timer.

The results, which are shown in Figure 8, revealed that there is a concentration dependent delay in clot formation for the protein of the present invention.

Also, in some cases, prolongation was also characterised by the apparent need for a 20 min pre- incubation at 37°C.

In more detail, Figure 8 shows the concentration-dependent and time-dependent effect of a recombinant fragment of HB 14 on PTTK. As mentioned, a lysate of E. coli over- expressing a truncated HB14 (AA 18-163, omitting the signal peptide and the hydrophobic carboxy terminal) was added at the indicated dilutions to normal human donor plasma. After 15 or 30 minutes, the clotting reaction was initiated. The delay is calculated with respect to a non-recombinant control lysate.

These further studies confirm that the proteins of the present invention, and the proteins expressed by the nucleotide sequences of the present invention, are blood processing agents. In particular, the proteins act as anti-coagulants. FURTHER ANTI-COAGULATION EVIDENCE

By use of immuno electron microscopy, HB 14 was located in the post acetabular glands of the invasive larval (cercariae) stage. The contents of these glands are released after skin penetration and before intravascularization, which is an ideal time at which to release an anticoagulant as the parasite must break through endothelium entering the blood vessel. This is of particular interest as no molecule has previously been identified from this location.

G. VACCINE STUDIES

The proteins of the present invention, in particular S.haematobium antigen HB14, are useful for use as vaccines - especially against the human parasite S.haematobium.

The use of S.haematobium antigen HB14 as a vaccine is based on the phenomenon that the antigen HB14 was immunologically recognised only by animals that were shown to be protected against specific challenge from Schistosoma. This protection conformed to the specific cross protection of phylogenetically closely related species of Schistosoma (Agnew, 1989; Navarrete, 1994).

It is believed that the use of HB14 as a vaccine will prove to be successful for a number of reasons. First, no homologue has been found in mammalian organisms so induction of auto-immunity is extremely unlikely. Second, any process that restricts feeding of the adult parasite would have a profound effect on the nutritional status of the parasite. If not a lethal effect, at least a reduction in fecundity could be expected. As S.haematobium is a high egg producer, this would greatly reduce pathology and transmission of the parasite. Third, if HB 14 has a function for the invasion, a vaccine could also reduce establishment of re-infection. Without wishing to be bound by theory it is believed that the vaccine works by preventing migration of the parasite into the host (such as via the dermal capillaries in humans) and/or preventing the parasite feeding on the blood of the host in the later stages.

The protein of the present invention could be used as a valuable component of a multistage vaccine as it would be associated with larval entry and adult blood feeding.

In summation, therefore, the present invention relates to a novel protein and, in addition, its use as a pharmaceutical.

In one aspect, the present invention relates to the use of a protein obtainable from a Schistosoma as a blood processing agent. More in particular, the present invention relates to the use of a protein obtainable from a Schistosoma as a blood processing agent. Even more in particular, the present invention relates to the use of S.haematobium protein HB14 as a blood processing agent.

In another aspect, the present invention relates to the use of an antigenic protein obtainable from a Schistosoma as an anti-schistosome vaccine, and more especially the S.haematobium antigenic protein HB14.

In particular, the present invention provides a protein obtainable from a Schistosoma, wherein the protein is S.haematobium antigen HB14, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity, but not S. mansoni 10.3 per se.

The present invention also provides a protein obtainable from a Schistosoma for use as a pharmaceutical, wherein the protein is S.haematobium antigen HB14, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity. In addition, the present invention provides a pharmaceutical method comprising administering to a subject a protein obtainable from a Schistosoma. wherein the protein is S.haematobium antigen HB14, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity.

Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

REFERENCES

Agnew A M (1986). PhD Thesis University of London

Agnew A M, et al. (1989a). Parasite Immunology. 11:341.

Davis R. et al. (1988). Molecular and Cellular Biology. 8:4745.

Dike S (1971). American Journal of Tropical Medicine and Hygiene. 20:552.

Guan (1987). Gene 67 page 21

Kyte and Doolittle (1982). J Mol Biol 157 page 105

Maina (1988). Gene 74 page 365

Morris G, et al. (1968). Journal of Parasitology. 54:15.

Navarrete S, et al. (1994). Parasite Immunology. 16:19.

O'Reilly (1992). Baculovirus Expression Vectors - Laboratory Manual; Publ. W.H. Freemann & Co NY.

Spence I, et al. (1970). South African Journal of Medical Science. 35:93. SEQUENCE LISTINGS

INFORMATION FOR SEQ I.D. 1

i . SEQUENCE CHARACTERISTICS

A. Length: 188

B. Type: Protein

C. Strande ness: Single D. Topology: Linear

ii. SEQUENCE DESCRIPTION

INFORMATION FOR SEQ I.D. 2

i . SEQUENCE CHARACTERISTICS A. Length: 130 B. Type: Protein

C. Strandedness: Single

D. Topology: Linear

ii. SEQUENCE DESCRIPTION

INFORMATION FOR SEQ I.D. 3

i . SEQUENCE CHARACTERISTICS A. Length: 85 B. Type: Protein

C. Strandedness: Single

D. Topology: Linear

ii. SEQUENCE DESCRIPTION

INFORMATION FOR SEQ I.D. 4

i . SEQUENCE CHARACTERISTICS A. Length: 45 B. Type: Protein

C. Strandedness: Single

D. Topology: Linear

ii. SEQUENCE DESCRIPTION

N T 45

INFORMATION FOR SEQ I.D. 5

i . SEQUENCE CHARACTERISTICS

A. Length: 680

B. Type: Nucleic acid

C. Strandedness: Double

D. Topology: Linear

ii. SEQUENCE DESCRIPTION

AAAATTGAAA TTCATTAAGA TGAAATTATT

TTTTCAGTTT GCTCGTTTCA CAAGAATATG GTGAATGATG CAAAACAAAA ACAACATTTA GAATCAATAT TTACAGTCAA GGGGGATTAA AATTCTACAA AATCATATAC GATAGATTAA AAAAGCGATG TCCGCATAAT TGAAAAAGAA AAAGCATGTA GATAATGAGA CATCAGACAG πCAGAAAGA AGTCTCCAAA CGTCCAGTGC ACATCAGACA GATTTCAATC CATATCAGAT GTGGATTCCT TTATGGATTG TAAATCCCCT TCATGCAAAT TATGGCATTT TTACTAGTAG CAAATGCCAG CATACTATTA CGATACATCA CAGTATAGH AAπCAACAC GTAACTAAAA ATMTTTTAG TTTTAACCTT TGCATAAAAT

INFORMATION FOR SEQ I.D. 6

i . SEQUENCE CHARACTERISTICS A. Length: 390 B. Type: Nucleic acid

C. Strandedness: Double

D. Topology: Linear

ii. SEQUENCE DESCRIPTION

ATGAAATTAT TTTTGATTAG TATAATTGGC ATTTTCAGTT 40

TGCTCGTTTC ACAAGAATAT GGTTATACGG TTGATATCAA 80

TGTGAATGAT GCAAAACAAA AACAACATTT AATAACCAAA 120

CTTAATTCAT TGAATCAATA TTTACAGTCA AGGGGGATTA 160 ACAAACAATT CACTGAAGAC GAATTCTACA AAATCATATA 200

CGATAGATTA AACAAGCATA TTGAAGACGA AAAAAGCGAT 240

GTCCGCATAA TTGAAAAAGA AGTCTCCAAA CGTCCAGTGC 280

AAAAGCATGT AGATAATGAG ACATCAGACA GTATGCATGA 320

TAAACAACCT ATTCAGAAAG AAGTCTCCAA ACGTCCAGTG 360 CAAAAGCATG TAGATAATGA GACATCAGAC 390

INFORMATION FOR SEQ I.D. 7

i . SEQUENCE CHARACTERISTICS A. Length: 255 B. Type: Nucleic acid

C. Strandedness: Double

D. Topology: Linear

ii. SEQUENCE DESCRIPTION

ATGAAATTAT TTTTGATTAG TATAATTGGC ATTTTCAGTT 40

TGCTCGTTTC ACAAGAATAT GGTTATACGG TTGATATCAA 80

TGTGAATGAT GCAAAACAAA AACAACATTT AATAACCAAA 120

CGATAGATTA AACAAGCATA πGAAGACGA AAAAAGCGAT 240

GTCCGCATAA TTGAA 255

INFORMATION FOR SEQ I.D. 8

i . SEQUENCE CHARACTERISTICS

A. Length: 135

B. Type: Nucleic acid

C. Strandedness: Double

D. Topology: Linear

ii. SEQUENCE DESCRIPTION

AAAGAAGTCT CCAAACGTCC AGTGCAAAAG CATGTAGATA 40

ATGAGACATC AGACAGTATG CATGATAAAC AACCTATTCA 80

GAAAGAAGTC TCCAAACGTC CAGTGCAAAA GCATGTAGAT 120

AATGAGACAT CAGAC 135

INDICATIONS RELATING TO Λ DCPOSITCD MICROORGANISM

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1^ ft- λM»

B [DE.NTIFIC- TIOIN OF DEPOSIT -.ii- _.ϊ3CSι(3 ιr: _c i o ^3nsϊ! D _r*-.; zι -e o. όr ts(ιt-.(.on

The National Collections ot^" Industrial and Marine Bacteria Limited (NCIMB)

ACCrtSS Ot OeOObiCi. / in_.li.Uuon oositil code JΛ-. -Ouniri)

23 St Machar Drive

Aberdeen

Scotland

AB2 1RY

United Kingdom

Date ot Jeπosit Accession Numoer 22 June 1994 NCIMB 40654

C. ADDITIONAL INDICATIONS (leave otanx </ not aooticaole) inis in.ormacion is continued on an additional sneet | j

In respect of those designations in which a European patent is sougnt, and any other designated state having equivalent legislation, a sample of the deoosited microorganism will be made available until the publication of the mention of the grant of the patent or until the date on which the apolication has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the samole.CcfRule 28(4) EP

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if ike indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave olaπk if not aoplicaola)

Tae indications listed below wnl be submitted to Che International Bureau later (rcwc-ό-'-t-* g er l nature of the indications e j, 'Accessⁱon numoer of Deposit")

For receiving Office use only For International Bureau use only

0T This sheet was received with the international aoplication I I This sheet was received by the International Bureau on

Authorized officer Authorized officer

JS_js

Claims

1. A protein obtainable from a Schistosoma for use as a pharmaceutical, wherein the protein is S.haematobium antigen HB14.

2. A protein obtainable from a Schistosoma for use as a pharmaceutical, wherein the protein is any one of: i. a protein comprising the sequence represented as SEQ. I.D. 1, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity; ii. a protein comprising at least the sequence represented as SEQ. I.D. 2, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity; iii. a protein comprising at least the sequence represented as SEQ. I.D. 3, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity; iv. a protein comprising at least the sequence represented as SEQ. I.D. 4, or a fragment, homologue or variant thereof capable of exhibiting pharmaceutical activity; v. a protein encoded by the nucleotide sequence represented as SEQ. I.D. 5, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity; vi. a protein encoded by at least the nucleotide sequence represented as SEQ. I.D.

6, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity; vii. a protein encoded by at least the nucleotide sequence represented as SEQ. I.D.

7, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity; viii. a protein encoded by at least the nucleotide sequence represented as SEQ. I.D.

8, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity; ix. a protein comprising a hydrophobic signal peptide region, preferably of about 16 amino acids, a hydrophobic carboxy terminal, preferably showing three peaks on a hydrophobicity plot of the protein, and an intermediate region containing tandem repeating hydrophilic units, preferably wherein each repeating unit has about 27 amino acids and more preferably wherein the repeating unit nearest to the carboxy terminal is truncated, and preferably has about 18 amino acids; x. a protein obtainable from the oesophagus region of a Schistosoma and having a molecular weight in the range of from about 20 kd to about 26 kd, preferably from about 22 kd to about 25 kd, more preferably from about 22.4 kd to about 24.6 kd; xi. a protein obtainable from the post acetabular glands of cercariae of a Schistosoma and having a molecular weight in the range of from about 20 kd to about 26 kd, preferably from about 22 kd to about 25 kd, more preferably from about 22.4 kd to about 24.6 kd.

3. A protein obtainable from a Schistosoma for use as a pharmaceutical, wherein the protein is coded by at least any one of: i. the nucleotide sequence represented as SEQ. I.D. 5, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity; ii. the nucleotide sequence represented as SEQ. I.D. 6, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity; iii. the nucleotide sequence represented as SEQ. I.D. 7, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity; iv. the nucleotide sequence represented as SEQ. I.D. 8, or a fragment, homologue or variant thereof and wherein the expressed protein is capable of exhibiting pharmaceutical activity.

4. The protein according to any one of claims 1 to 3 wherein the Schistosoma is S.haematobium.

5. The protein according to any one of the preceding claims wherein the protein is for use as a blood processing agent.

6. The protein according to any one of the preceding claims wherein the protein is for use as an anti-coagulant.

7. The protein according to any one of claims 1 to 4 wherein the protein is for use as a vaccine.

8. The protein according to claim 7 wherein the protein is for use as a vaccine against infection from Schistosoma.

9. The protein according to claim 8 wherein the protein is for use as a vaccine against S.haematobium infection or S.mansoni infection.

10. A blood processing composition comprising a protein obtainable from a Schistosoma admixed with or contained within a suitable carrier, diluent or excipient, wherein the protein is as defined in any one of claims 1 to 4.

1 1. A vaccine composition comprising an immunologically active protein obtainable from a Schistosoma admixed with or contained within a suitable carrier, diluent or excipient, wherein the protein is as defined in any one of claims 1 to 4.

12. The use of a protein obtainable from a Schistosoma in the manufacture of a blood processing agent, wherein the protein is as defined in any one of claims 1 to 4.

13. The use of a protein obtainable from a Schistosoma in the manufacture of an anti-coagulant, wherein the protein is as defined in any one of claims 1 to 4.

14. The use of an immunologically active protein obtainable from a Schistosoma in the manufacture of a vaccine, wherein the protein is as defined in any one of claims 1 to 4.

15. A protein comprising the sequence represented in SEQ I.D. 1 ; or non-critically amino acid substituted variants, homologues or fragments; but not S.mansoni 10.3 itself.

16. A protein comprising the sequence represented in SEQ I.D. 2; or non-critically amino acid substituted variants, homologues or fragments thereof; but not S.mansoni 10.3 itself.

17. A protein comprising the sequence represented in SEQ I.D. 3; or non-critically amino acid substituted variants, homologues or fragments thereof; but not S.mansoni 10.3 itself.

18. A protein comprising the sequence represented in SEQ I.D. 4; or non-critically amino acid substituted variants, homologues or fragments thereof; but not S.mansoni 10.3 itself.

19. A nucleotide sequence comprising the sequence represented in SEQ I.D. 5; or non-critically nucleic acid substituted variants, homologues or fragments; but not the sequence for S.mansoni 10.3.

20. A nucleotide sequence comprising the sequence represented in SEQ I.D. 6; or non-critically nucleic acid substituted variants, homologues or fragments thereof; but not the sequence for S.mansoni 10.3.

21. A nucleotide sequence comprising the sequence represented in SEQ I.D. 7; or non-critically nucleic acid substituted variants, homologues or fragments thereof; but not the sequence for S.mansoni 10.3.

22. A nucleotide sequence comprising the sequence represented in SEQ I.D. 8; or non-critically nucleic acid substituted variants, homologues or fragments thereof; but not the sequence for S.mansoni 10.3.

23. The protein according to any one of claims 1 to 4, or any one of claims 15 to 18, or a protein expressed by the nucleotide sequence according to any one of claims 19 to 22, wherein the protein exhibits a blood processing effect after administration to or contact with a subject.

24. The protein according to any one of claims 1 to 4, or any one of claims 15 to 18, or a protein expressed by the nucleotide sequence according to any one of claims 19 to 22, wherein the protein exhibits a blood anti-coagulant effect after administration to or contact with a subject.

25. The protein according to any one of claims 1 to 4, or any one of claims 15 to 18, or a protein expressed by the nucleotide sequence according to any one of claims 19 to 22, wherein the protein exhibits an immunological effect after administration to or contact with a subject.

26. The protein according to claim 23 or claim 24 wherein the subject is a vertebrate animal.

27. An article coated with a protein as defined in any one of claims 1 to 4, or any one of claims 15 to 18, or a protein expressed by the nucleotide sequence according to any one of claims 19 to 22.

28. A protein obtainable from a Schistosoma for use as a blood processing agent.

29. The use of a protein obtainable from a Schistosoma in the manufacture of a blood processing agent.

30. The use of a protein obtainable from a Schistosoma in the manufacture of an anti -coagulant.

31. A blood anti-coagulant composition comprising a protein admixed with or contained within a suitable carrier, diluent or excipient; or a protein for use as an anti- coagulant; or the use of a protein in the manufacture of a blood anti-coagulant; wherein the protein has the protein sequence represented in SEQ I.D. 1, or non- critically amino acid substituted variants, homologues or fragments thereof capable of having a blood anti-coagulant effect, or a protein expressed by the nucleotide sequence represented in SEQ I.D. 5, or non-critically nucleic acid substituted variants, homologues or fragments thereof and wherein the expressed protein is capable of having an anti-coagulant effect.

32. A vaccine composition comprising a protein admixed with or contained within a suitable carrier, diluent or excipient; or a protein for use as a vaccine; or the use of a protein in the manufacture of a vaccine; wherein the protein has the protein sequence represented in SEQ I.D. 1, or non-critically amino acid substituted variants, homologues or fragments thereof having immunological activity, or a protein expressed by the nucleotide sequence represented in SEQ I.D. 5, or non-critically nucleic acid substituted variants, homologues or fragments thereof encoding a protein having immunological activity.

33. A method of treating a subject in need of, or likely to be in need of, anticoagulant treatment wherein the method comprises administering to the subject a protein as defined in any one of claims 1 to 4, or any one of claims 15 to 18, or a protein expressed by the nucleotide sequence according to any one of claims 19 to 22, or a composition according to claim 31.

34. A method of treating a subject in need of, or likely to be in need of, immunisation against infection from Schistosoma wherein the method comprises administering to the subject a protein as defined in any one of claims 1 to 4, or any one of claims 15 to 18, or a protein expressed by the nucleotide sequence according to any one of claims 19 to 22, or a composition according to claim 32.

35. An anti-coagulant composition substantially as described herein.

36. A vaccine composition substantially as described herein.