WO2019166832A1

WO2019166832A1 - Disease detection

Info

Publication number: WO2019166832A1
Application number: PCT/GB2019/050590
Authority: WO
Inventors: Richard William Titball; Nicholas Peter LEWIS; Sariqa WAGLEY; Helen MORCRETTE; Monika BOKORI-BROWN
Original assignee: One Health Ventures Ltd
Current assignee: One Health Ventures Ltd
Priority date: 2018-03-02
Filing date: 2019-03-01
Publication date: 2019-09-06
Anticipated expiration: 2020-09-02
Also published as: GB201803387D0

Abstract

There is provideda composition comprising one or more of a first polypeptide consisting of 10-200 amino acids and comprising the sequence YLKKVNVKGN; a second polypeptide of 11-200 amino acids and comprising the sequence GRYNTKYNYLKR; a third polypeptide consisting of 6-200 amino acids and comprising the sequence IVKYRS; a fourth polypeptide consisting of 14-200 amino acids and comprising the sequence QEQKLKSQSFTCKN; a fifth polypeptide consisting of 10-200amino acids and comprising at least 6 contiguous amino acids from the sequence TGVSLTTSYSFANTN; and/or a sixth polypeptide consisting of 13-200 amino acids and comprising the sequence TVGTSIQATAKFT. There is also provided methods of identifying an individual at risk of a condition associated with demyelination comprising use of the composition.

Description

DISEASE DETECTION

FIELD OF THE INVENTION

The invention relates to compositions and methods for identification of a demyelinating condition .

BACKGROUND

Clostridium perfringens is found in the gastrointestinal tracts of many animals. The ability of different strains to cause a range of diseases in human a nd in animals is ascribed largely to the differential production of toxins (1). Epsilon toxin, produced by C. perfringens types B and D, is associated with dysentery and enterotoxaemia in ovines following the growth of bacteria in the intestine and the production of epsilon toxin (2). The toxin crosses the gut wall, accumulating in the kidneys and brain (3). In the brain toxin binds to the synaptosomal membranes (4), myelinated structures (5, 6), glial cells (7) and oligodendrocytes (8) and causes demyelination (6). Peracute enterotoxaemia in ovines appears without clinical signs and results in sudden death, while acute disease leads to convulsions and coma (9). There are only three reports of the isolation from humans of C. perfringens producing epsilon toxin (10, 11), one from a stool of a patient who had presented with multiple sclerosis (MS) three months previously (12) .

Multiple sclerosis (MS) is a demyelinating disease in which the insulating covers of nerve cells in the brain and spinal cord are damaged . This damage disrupts the ability of parts of the nervous system to communicate, resulting in a range of symptoms, which can include double vision, blindness in one eye, muscle weakness, trouble with sensation, or trouble with coordination. The condition often begins as a clinically isolated syndrome (CIS) over a period of time, before being confirmed as Clinically Definite MS (CDMS).

Optic neuritis (ON) is a demyelinating inflammation of the optic nerve. It is frequently associated with multiple sclerosis. The autoimmune disease neuromyelitis optica (NMO) is a heterogeneous condition consisting of the simultaneous inflammation and demyelination of the optic nerve (optic neuritis) and the spinal cord (myelitis).

Antibodies to epsilon toxin are reported to occur in 10% of patients with MS and in 1% of healthy individuals in the USA (12) . Cases et al. (13) recently showed that epsilon toxin affects the propagation of action potentials in isolated optic nerves. As a result of these studies, and the similarities between the symptoms of ovine enterotoxaemia and humans suffering from MS, it has been suggested that epsilon toxin may contribute to the development of MS. SUMMARY OF THE INVENTION

SEQ ID NO : l referred to herein is the amino acid sequence of epsilon toxin as translated, comprising a 32 amino acid export signal at the N-terminal end. This export signa l is removed to provide a toxin precursor having amino acid sequence SEQ ID NO : 18; further amino acids are removed from the N-terminal and C-terminal ends of SEQ ID NO : 18 to form SEQ ID NO: 19, 20 or 21, the activated epsilon toxin. All three forms occur in C. perfringens, SEQ ID NO: 19 being the consequence of processing of SEQ ID NO : 18 by trypsin, SEQ ID NO : 20 being the consequence of processing by chymotrypsin and SEQ ID NO:21 being the consequence of processing by l-protease; each protease has a slightly d ifferent cleavage site. The term "epsilon toxin polypeptide", as referred to throughout this specification, indicates a protein having any of amino acid sequences SEQ ID NO : 19-21, or a protein having such a sequence with one or more amino acid substitutions but which is capable of binding to one or more antibodies raised against SEQ ID NO: 19, 20 or 21. Examples of such proteins and mutation positions are described in WO2013/144636; the tyrosine residues forming the mutation positions mentioned therein (Y29, Y33, Y42, Y43, Y49, Y209) are found at positions 61, 65, 74,

75, 81 and 241 of SEQ ID NO : l herein .

A first aspect of the invention provides a composition comprising one or more of:

(a) a first polypeptide consisting of 10-200 amino acids and comprising the sequence YLKKVNVKGN (SEQ ID NO : 2) ;

(b) a second polypeptide of 11-200 amino acids and comprising the sequence

RYNTKYNYLKR (SEQ ID NO: 5);

(c) a third polypeptide consisting of 6-200 amino acids and comprising the sequence IVKYRS (SEQ ID NO: 7);

(d) a fourth polypeptide consisting of 14-200 amino acids and comprising the

sequence QEQKLKSQSFTCKN (SEQ ID NO : 10);

(e) a fifth polypeptide consisting of 6-200 amino acids and comprising at least 6

contiguous amino acids from within the sequence TGVSLTTSYSFANTN (SEQ ID NO: 11) ;

(f) a sixth polypeptide consisting of 13-200 amino acids and comprising the

sequence TVGTSIQATAKFT (SEQ ID NO: 17) .

The invention does not encompass a composition comprising any of SEQ ID NOs : 1 or 18-21, or any of these sequences comprising one or more amino acid substitutions at any position. Therefore, the invention does not encompass a composition in which the first, second, third, fourth, fifth and sixth polypeptide is contained within a naturally occurring epsilon toxin polypeptide selected from any of SEQ ID NOs : l or 18-21, or any of these sequences comprising one or more amino acid substitutions at any position .

That is, each of the first, second, third, fourth, fifth and sixth polypeptides may be "isolated" in that it is not contained within a polypeptide having amino acid sequence SEQ ID NO : 1, 18, 19, 20 or 21, or within any of these sequence comprising one or more amino acid substitutions at any position.

The first, second, third, fourth, fifth and sixth polypeptides each consist of an amino acid sequence which is not contiguously linked to a further sequence of contiguous amino acids from within SEQ ID NO : l which is not one of SEQ ID NOs: 2-17, i.e., it does not form part of a longer portion of any of SEQ ID NOs : 1 or 18-21. However, any one of the first, second, third, fourth, fifth and sixth polypeptides may be formed as a fusion protein with any further one or more of the first, second, third, fourth, fifth and sixth

polypeptides. That is, the composition may comprise a primary polypeptide formed as a fusion protein with a secondary polypeptide, each of the primary and secondary polypeptides being independently selected from the first, second, third, fourth, fifth and sixth polypeptide. The term "fusion protein" may indicate that the primary and secondary polypeptides are contiguous with one another, or that they may be linked by an intervening linker sequence of one or more a mino acids. The fusion protein may also comprise one or more further polypeptides which may be any amino acid sequence required for successful translation of a fusion protein, and/or one or more further polypeptides each independently selected from the first, second, third, fourth, fifth and sixth polypeptide. In some embodiments, the first, second, third, fourth, fifth and sixth polypeptide is not directly joined at its N-terminal end to an amino acid (i.e., to a carboxylic group) and/or is not directly joined at its C-terminal end to an a mino acid (i.e. , to an amine group).

The composition according to the invention is useful, for example, when included in a diagnostic reagent for use to identify an individual at risk from a condition associated with demyelination and/or to identify an individual suffering from a condition associated with demyelination, as described in further detail below.

The composition may comprise at least the first and the second polypeptide or at least the first and the third polypeptide or at least the first, second and third polypeptide. The composition may optionally additionally comprise the fourth polypeptide.

The first polypeptide may consist of 10-19 or 10-25 amino acids. The first polypeptide may consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence YLKKVNVKGN (SEQ ID NO : 2) or comprise or consist of the sequence AYLKKVNVKGN (SEQ ID NO: 3) or comprise or consist of the sequence YLKKVNVKGNVK (SEQ ID NO:4) . It may comprise SEQ ID NO : 2 and/or 3 and/or 4 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO : 1.

The second polypeptide may consist of 11-19 or 11-25 amino acids. The second polypeptide may consist of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence RYNTKYNYLKR (SEQ ID NO : 5) or comprise or consist of the sequence

IEKGRYNTKYNYLKR (SEQ ID NO: 6). It may comprise SEQ ID NO: 5 and/or 6 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO : 1.

The third polypeptide may consist of 6- 19 or 6-25 amino acids. The third polypeptide may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence IVKYRS (SEQ ID NO : 7) or comprise or consist of the sequence KEKSN DSNIVKYRS (SEQ ID NO : 8) or comprise or consist of the sequence IVKYRS LYIKAPG IK (SEQ ID NO:9) . It may comprise one or more of SEQ ID NOs : 7-9 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO: 1.

The fourth polypeptide may consist of 14-19 or 14-25 amino acids. The fourth polypeptide may consist of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence

QEQKLKSQSFTCKN (SEQ ID NO : 10). It may comprise SEQ ID NO: 10 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO: l .

The fifth polypeptide may consist of 6- 19 or 6-25 amino acids. The fifth polypeptide may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence TGVSLTTSY (SEQ ID NO : 12), or may comprise or consist of the sequence SYSFANTNTN (SEQ ID NO : 13), may comprise or consist of the sequence TTSYSFANTNT (SEQ ID NO : 14), may comprise or consist of the sequence VPFNETGVSLTTS (SEQ ID NO: 15), may comprise or consist of the sequence SFANTNTNTNSKEI (SEQ ID NO : 16) . These sequences overlap with SEQ ID NO: 11 as indicated by the underlined amino acids. It may comprise one or more of SEQ ID NOs : 11-16 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO : l .

The sixth polypeptide may consist of 13-19 or 13-25 amino acids. The sixth polypeptide may consist of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence TVGTSIQATAKFT (SEQ ID NO : 17). It may comprise SEQ ID NO: 17 directly joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO : l .

The composition may be a liquid composition or a lyophilised form of a liquid

composition ; alternatively, the composition may be a surface having one or more of the first, second, third, fourth, fifth and sixth polypeptide adhered thereto.

When the composition is a liquid, it may consist of the first polypeptide in a diluent, carrier or vehicle. It may alternatively consist of the first polypeptide and second polypeptide in a diluent, carrier or vehicle, or may consist of the first polypeptide and the third polypeptide in a diluent, carrier or vehicle, or may consist of the second polypeptide and the third polypeptide in a diluent, carrier or vehicle. In an embodiment, the composition may be a liquid composition which consists of the first and second and third polypeptides in a diluent, ca rrier or vehicle. It may consist of AYLKKVNVKGN (SEQ ID NO : 3) and RYNTKYNYLKR (SEQ ID NO : 5) and KEKSNDSNIVKYRS (SEQ ID NO: 8) in a diluent, carrier or vehicle. It may consist of AYLKKVNVKGN (SEQ ID NO :3) and

RYNTKYNYLKR (SEQ ID NO : 5) and KEKSNDSNIVKYRS (SEQ ID NO: 8) and

QEQKLKSQSFTCKN (SEQ ID NO : 10) in a diluent, carrier or vehicle.

The diluent, carrier or vehicle may be any liquid suitable for carrying the polypeptide components of the composition, such as, for example, an aqueous solvent, non-aqueous solvent, non-toxic excipient, such as a salt, preservative, buffer and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous solvents include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Preservatives include antimicrobial, a nti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the composition are adjustable according to the routine ability of the skilled person . When the composition is a lyophilised form of a liquid composition, it may consist of the first polypeptide in a lyophilised diluent, carrier or vehicle. It may alternatively consist of the first polypeptide and second polypeptide in a lyophilised diluent, carrier or vehicle, or may consist of the first polypeptide and the third polypeptide in a diluent, carrier or vehicle, or may consist of the second polypeptide and the third polypeptide in a lyophilised diluent, carrier or vehicle. In an embodiment, the composition may be a lyophilised form of a liquid composition which consists of the first and second and third polypeptides in a lyophilised diluent, carrier or vehicle. It may consist of AYLKKVNVKGN (SEQ ID NO: 3) and RYNTKYNYLKR (SEQ ID NO: 5) and KEKSNDSNIVKYRS (SEQ ID NO:8) in a lyophilised diluent, carrier or vehicle. It may consist of AYLKKVNVKGN (SEQ ID NO: 3) and RYNTKYNYLKR (SEQ ID NO: 5) and KEKSNDSNIVKYRS (SEQ ID NO: 8) and QEQKLKSQSFTCKN (SEQ ID NO: 10) in a lyophilised diluent, carrier or vehicle. When the composition is a lyophilised form of a liquid composition, it may be prepared by forming a composition according to the first aspect of the invention in liquid form as described herein, and submitting it to a lyophilisation process in order to obtain the lyophilised form.

When the composition is a surface having one or more of the first, second, third, fourth, fifth and sixth polypeptide adhered thereto, it may consist of the first polypeptide adhered to the surface. It may alternatively consist of the first polypeptide and the second polypeptide adhered to the surface, or it may consist of the first polypeptide and the third polypeptide adhered to the surface, or it may consist of the second polypeptide and the third polypeptide adhered to the surface. It may consist of the first and second and third polypeptides adhered to the surface. It may consist of AYLKKVNVKGN (SEQ ID NO:3) and RYNTKYNYLKR (SEQ ID NO: 5) and KEKSNDSNIVKYRS (SEQ ID NO: 8) adhered to the surface. It may consist of AYLKKVNVKGN (SEQ ID NO: 3) and

RYNTKYNYLKR (SEQ ID NO: 5) and KEKSNDSNIVKYRS (SEQ ID NO:8) and

QEQKLKSQSFTCKN (SEQ ID NO: 10) adhered to the surface.

The surface may be a plate, well or bead, or any other solid support such as a testing strip or "dip stick". The polypeptide may be adhered to the surface directly, for example by adsorption onto a surface such as a polystyrene surface, or indirectly, for example by binding to an anti-polypeptide antibody attached to the surface. The polypeptide may be adhered to the surface by direct chemical conjugation thereto. For example, the composition may be prepared by chemical synthesis of the polypeptide on a solid support. The polypeptide may be adhered to an absorbent surface by application of a liquid composition according to the first aspect of the invention to the surface and allowing the liquid components of the composition to evaporate. The composition may be a "diagnostic composition", indicating that further components of the composition, which are not the one or more polypeptide referred to in (a)-(f) above, are components suitable to provide a composition which may be used in a diagnostic test, such as an immunoassay.

The polypeptides as included in the composition according to the first aspect of the invention have been found to be useful in being recognisable in an immunoassay by a sample obtained from an individual with a condition associated with demyelination, such as enterotoxemia (ET), multiple sclerosis (MS), clinically definite MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica spectrum disorder (NMOSD), optic neuritis (ON), neuromyelitis optica (NMO), myelitis, transverse myelitis (TM), a disease or condition characterised by the increase or presence of antibodies against aquaporin-4 (AQP4) and /or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

An assay utilising the polypeptides is capable of distinguishing between :

1. a sample from an individual who does not have such a condition associated with demyelination, the sample containing anti-epsilon toxin antibodies (i.e., antibodies detected using an epsilon toxin polypeptide, as defined above, as the detection antigen) ; and

2. a sample which is from an individual who does have a condition associated with demyelination .

Furthermore, an assay utilising the polypeptides is capable of identifying a sample from an individual who has a demyelinating condition, even if a sample obtained from the individual does not contain anti-epsilon toxin antibodies, according to a detection method which relies on an epsilon toxin polypeptide as a detection antigen.

As outlined above, exposure to epsilon toxin is thought to be associated with the development of some conditions associated with demyelination, such as multiple sclerosis. However, anti-epsilon toxin antibodies are also present in many individuals who do not have such a condition, so that simple detection of antibodies, using an epsilon toxin polypeptide as the detection antigen, does not provide any reliable predictor or indicator of the presence of a demyelinating condition . The present invention provides a method for positive identification of individuals who have such a condition (whether presenting with symptoms or at a pre-symptomatic stage), distinguishing them from individuals who have antibodies against the toxin but who do not have a demyelinating condition. A second aspect of the invention, therefore, provides a method of identifying an individual at risk of a condition associated with demyelination, comprising the steps of:

(a) contacting a biological sample obtained from the individual with a composition according to the first aspect of the invention; and

(b) determining whether a response is observed in an immunoassay.

An "immunoassay" as described herein is any which is capable of detecting an element of the immune system of the individual recognising/interacting with the one or more polypeptides included in the composition. For example, the immunoassay may be an antibody binding assay or a cell-mediated immunity (CMI) assay. When the

immunoassay is a CMI assay, it may be one which detects interferon-g (IFN-y).

The term "at risk of a condition associated with demyelination", as used herein in relation to any aspect of the invention, indicates that the individual has a greater risk of developing a condition associated with demyelination compared to the average risk for a comparable individual. The term encompasses the individual having a condition associated with demyelination, so a method according to aspects of the invention may be a method of identifying an individual having a condition associated with

demyelination. A condition associated with demyelination may be enterotoxemia (ET), multiple sclerosis (MS), clinically definite MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica spectrum disorder (NMOSD), optic neuritis (ON), neuromyelitis optica (NMO), myelitis, transverse myelitis (TM), a disease or condition characterised by the increase or presence of antibodies against aquaporin-4 (AQP4) and /or astrocyte damage, and acute disseminated encephalomyelitis (ADEM) and/or detectable demyelination of nerve cells in the individual. The term "at risk of a condition associated with demyelination" may encompass an individual who does, in fact, have such a condition. The individual may be asymptomatic at the time that a sample is obtained for use in a method according to the invention. Identifying an individual at risk of a condition associated with demyelination may involve assessment of, for example, an individual who has a parent or a sibling who has previously developed MS. The compositions and methods described herein may be useful to assess the relative likelihood, compared to control individuals lacking a family background of MS, of the individual also developing a condition associated with demyelination.

The term "comparable individual" referred to above and throughout this specification indicates an individual (who may also be referred to as "a control individual") having matched characteristics to the individual who is the subject referred to in aspects of the invention, for example, age, body mass index, race, country of domicile, smoking habits, drinking and eating habits, and other common population characteristics which may be readily selected by the skilled person.

The method may comprise the step of observing a response that indicates that an element of the immune system of the individual has recognised/interacted with the one or more polypeptides included in the composition and a step of concluding that the individual is at risk of a condition associated with demyelination or has such a condition. The method may further comprise the step of assigning the individual to a program for follow-up by medical practitioners, for example by way of additional tests to detect whether demyelination of the individual's nerve cells is taking place.

The method may comprise a preceding step of obtaining a biological sample from the individual and determining whether the sample comprises an antibody to a C.

perfringens epsilon toxin polypeptide (e.g., SEQ ID NO: 19, 20 or 21). For example, the sample may have been identified as not comprising such an antibody. It has surprisingly been found that the method according to the invention is capable of eliciting a positive result when conducted on a sample obtained from an individual with a negative result from a Western blot test for the presence of anti-epsilon toxin polypeptide antibodies.

In some embodiments, therefore, the method according to the second aspect of the invention (and referred to in the third and fourth aspects outlined below) may be a useful second-line test for a demyelinating condition, to be used in conjunction with routine tests for the presence or absence of anti-epsilon toxin antibodies. For example, the method according to the second aspect of the invention may comprise the steps of: a) contacting a biological sample obtained from the individual with an epsilon toxin polypeptide, as defined above, and determining whether a response is observed in a first immunoassay; and b) contacting a biological sample obtained from the individual with a composition according to the first aspect of the invention and determining whether a response is observed in a second immunoassay; wherein detection of a response in the first and/or the second immunoassay is indicative of the individual being at risk of a condition associated with demyelination.

Steps (a) and (b) may be conducted in either order.

A related third aspect of the invention provides a method of identifying an individual at risk of a condition associated with de-myelination, comprising the steps of: (a) obtaining a biological sample from an individual suspected to be at risk of having the condition;

(b) contacting the biological sample with the composition according to the first aspect of the invention;

(c) determining whether a response is observed in an immunoassay;

wherein, if a response is observed in step (c), the individual is identified as being at risk of a condition associated with de-myelination .

A fourth aspect of the invention provides the composition according to the first aspect of the invention for use in a method of identifying a n individual at risk of a condition associated with de-myelination, the method comprising the steps of:

(a) obtaining a biological sample from an individual suspected to be at risk of having the condition;

(c) determining whether a response is observed in an immunoassay;

In either the third or the fourth aspect of the invention, the method may be used in conj unction with a second method comprising contacting a biological sample obtained from the individual with an epsilon toxin polypeptide, as defined above, and determining whether a response is observed in a further immunoassay, as described above in conjunction with the second aspect of the invention.

For example, the third aspect of the invention may comprise the steps of: a) obtaining a biological sample from an individual suspected to be at risk of having the condition; b) contacting a first portion of the biological sample with an epsilon toxin

polypeptide, as defined above, and determining whether a response is observed in a first immunoassay; and c) contacting a second portion of the biological sample with a composition according to the first aspect of the invention and determining whether a response is observed in a second immunoassay; wherein detection of a response in the first and/or the second immunoassay is indicative of the individual being at risk of a condition associated with demyelination. Therefore, in the fourth aspect of the invention, the composition may be for use in such a method.

A fifth aspect of the invention provides an affinity reagent capable of binding to (for example, an antibody raised against) a polypeptide selected from :

(b) a second polypeptide of 11-200 amino acids and comprising the sequence

RYNTKYNYLKR (SEQ ID NO: 5);

(d) a fourth polypeptide consisting of 14-200 amino acids and comprising the

sequence QEQKLKSQSFTCKN (SEQ ID NO: 10);

(e) a fifth polypeptide consisting of 6-200 amino acids and comprising at least 6 contiguous amino acids from within the sequence TGVSLTTSYSFANTN (SEQ ID NO: 11) ;

(f) a sixth polypeptide consisting of 13-200 amino acids and comprising the

sequence TVGTSIQATAKFT (SEQ ID NO: 17) .

The term "affinity reagent" as used throughout this specification, in relation to any aspect of the invention, indicates a moiety which is capable of binding to one of the polypeptides and facilitating an immune response in the body of an individual to which the moiety is administered. For example, the moiety may be an antibody which may be a monoclona l antibody or a synthetic antibody, an Affibody^® or other antibody mimetic, an aptamer, a protein scaffold or a major histocompatibility complex (MHC) protein or portion thereof.

The invention does not encompass an antibody raised against a polypeptide comprising SEQ ID NO : 1, 18 or 19, or any of these sequences comprising one or more amino acid substitutions at any position. Therefore, the invention does not encompass an antibody raised against a polypeptide comprising the first, second, third, fourth, fifth or sixth polypeptide contained within a naturally occurring epsilon toxin polypeptide selected from any of SEQ ID NO : 1 or 18-21, or contained within any of these sequence comprising one or more amino acid substitutions at any position. That is, each of the first, second, third, fourth, fifth and sixth polypeptides may be "isolated" in that it is not contained within a polypeptide having amino acid sequence SEQ ID NO : l, 18, 19, 20 or 21, or within any of these sequence comprising one or more amino acid substitutions at any position.

The first polypeptide may consist of 10-19 or 10-25 amino acids. The first polypeptide may consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence YLKKVNVKGN (SEQ ID NO : 2) or comprise or consist of the sequence AYLKKVNVKGN (SEQ ID NO: 3) or comprise or consist of the sequence YLKKVNVKGNVK (SEQ ID NO:4). It may comprise SEQ ID NO: 2 and/or 3 and/or 4 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO: 1. The affinity reagent may be capable of binding to any one of SEQ ID NOs: 2, 3 or 4.

IEKGRYNTKYNYLKR (SEQ ID NO: 6). It may comprise SEQ ID NO: 5 and/or 6 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO : l . The affinity reagent may be capable of binding to SEQ ID NOs : 5 or 6.

The third polypeptide may consist of 6- 19 or 6-25 amino acids. The third polypeptide may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or a bout 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence IVKYRS (SEQ ID NO : 7) or comprise or consist of the sequence KEKSNDSNIVKYRS (SEQ ID NO : 8) or comprise or consist of the sequence IVKYRSLYIKAPGIK (SEQ ID NO :9) . It may comprise one or more of SEQ ID NOs: 7-9 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous a mino acids which are not found in SEQ ID NO : l . The affinity reagent may be capable of binding to any one of SEQ ID NOs: 7, 8 or 9.

QEQKLKSQSFTCKN (SEQ ID NO : 10). It may comprise SEQ ID NO: 10 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO: l. The affinity reagent may be capable of binding to SEQ ID NO: 10.

The fifth polypeptide may consist of 6- 19 or 6-25 amino acids. The fifth polypeptide may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence TGVSLTTSY (SEQ ID NO: 12), or may comprise or consist of the sequence SYSFANTNTN (SEQ ID NO : 13), may comprise or consist of the sequence TTSYSFANTNT (SEQ ID NO : 14), may comprise or consist of the sequence VPFNETGVSLTTS (SEQ ID NO: 15), may comprise or consist of the sequence SFANTNTNTNSKEI (SEQ ID NO : 16) . These sequences overlap with SEQ ID NO: 11 as indicated by the underlined amino acids. It may comprise one or more of SEQ ID NOs : 11-16 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO : l . The affinity reagent may be capable of binding to any one of SEQ ID NOs: 11- 16

The sixth polypeptide may consist of 13-19 or 13-25 amino acids. The sixth polypeptide may consist of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids. It may comprise or consist of the sequence TVGTSIQATAKFT (SEQ ID NO : 17). It may comprise SEQ ID NO: 17 directly joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO : l . The affinity reagent may be capable of binding to SEQ ID NO : 17.

A sixth aspect of the invention provides an immunotherapy composition comprising a composition according to the first aspect of the invention, or comprising an affinity reagent according to the fifth aspect of the invention, or a polynucleotide according to the sixteenth or seventeenth aspects of the invention, in a pharmaceutically acceptable formulation. For example, the immunotherapy composition may be a vaccine

composition comprising a composition according to the first aspect of the invention, or a polynucleotide according to the sixteenth or seventeenth aspects of the invention, and an adjuvant. An adjuvant may be included in any type of immunotherapy composition described herein. Suitable adjuvants may include all acceptable immunostimulatory compounds such as, for example, a cytokine, toxin, or synthetic composition . Commonly used adjuvants include aluminium hydroxide, aluminium phosphate, calcium phosphate, Freund's adjuvants and Quil-A saponin. An adjuvant provided by SEPPIC Inc (New Jersey, USA) as a Montanide™ adjuvant, for example Montanide™ ISA 61VG, may also be a suitable adjuvant. In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM) with a vaccine conjugate to down regulate suppressor T cell activity.

Optionally, an immunotherapy composition, such as a vaccine composition, described herein may include a vaccine carrier. Commonly used vaccine carrier molecules are bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin, mouse serum albumin, rabbit serum albumin and the like. Synthetic vaccine carriers may be used and are readily available. Means for conjugating peptides to vaccine carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N- hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

Possible vehicles for administration of the immunotherapy composition include but are not limited to liposomes, micelles and/or nanoparticles. Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments. Liposomes are similar in composition to cellular membranes and, as a result, liposomes generally can be administered safely and are biodegradable. Techniques for preparation of liposomes and the formulation (e.g., encapsulation) of various molecules with liposomes are well known.

Depending on the method of preparation, liposomes may be unilamellar or multilamellar and can vary in size with diameters ranging from about 0.02pm to greater than about 10pm. Liposomes can also adsorb to virtually any type of cell and then release the encapsulated agent. Alternatively, the liposome fuses with the target cell, whereby the contents of the liposome empty into the target cell. Alternatively, an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by

intralysosomal degradation of liposomal lipids and release of the encapsulated agents. In the present context, the vaccine composition according to the invention can comprise a molecule according to the first aspect of the invention localized on the surface of the liposome, to facilitate antigen presentation without disruption of the liposome or endocytosis. Irrespective of the mechanism or delivery, however, the result is the intracellular disposition of the associated vaccine composition and/or molecule.

Liposomal vectors may be anionic or cationic. Anionic liposomal vectors include pH sensitive liposomes which disrupt or fuse with the endosomal membrane following endocytosis and endosome acidification.

Other suitable liposomes that may be used in the compositions and methods of the invention include multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium-sized unilamellar vesicles (MIN), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MVV), single or oligolamellar vesicles made by reverse-phase evaporation method (REV), multilamellar vesicles made by the reverse-phase evaporation method (MLV- REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV), vesicles prepared by fusion (FUV), dehydration-rehydration vesicles (DRV), a nd bubblesomes (BSV) . Techniques for preparing these liposomes are well known in the art.

Other forms of delivery particle, for example, microspheres and the like, also are contemplated for delivery of the immunotherapy composition .

Alternatively, polynucleotide-based vaccines may be produced that comprise nucleic acid, such as, for example, DNA or RNA, encoding the immunologically active peptide epitope or polyepitope and cloned into a suitable vector (e.g ., vaccinia, canarypox, adenovirus, or other eukaryotic virus vector).

Alternatively, the polypeptide may be administered in the form of a cellular vaccine via the administration of autologous or allogeneic APCs or dendritic cells that have been treated in vitro so as to present the peptide on their surface. Salmonella enterica or Escherichia coli strains harbouring mutations which reduce their virulence and allow them to colonise a host a nimal without causing disease might be used to deliver vaccine antigens, especially for administration to non-human animals. The bacteria used might include strains which are already used as vaccine in livestock where the attenuating lesion is not fully characterised . In addition, strains in which mutations have been deliberately introduced into the bacterium to rationally attenuate virulence could be used to deliver the polypeptide, as described in WO2013/144636. The antigen might also be delivered as a naked DNA vaccine where the gene encoding the epsilon toxoid is cloned into a mammalian expression vector and expressed from a eukaryotic promoter.

In one embodiment, the immunotherapy composition may be included in a foodstuff (i.e., a food material suitable for consumption by a human or an animal) comprising a polypeptide and/or a polynucleotide and/or a vector and/or a cell and/or a subunit vaccine and/or vaccine composition according to preceding aspects of the invention. This may, in non-limiting examples, be in the form of pellets, crumbs or a mash which may further comprise, aga in for example only, grain, grass and/or protein components. The composition may also be included in drinking liquids and/or administered via a spray into the atmosphere surrounding the animal which is, consequently, inhaled by the animal .

The term "pharmaceutically acceptable formulation" as used throughout this

specification, in relation to any aspect of the invention, may indicate a formulation comprising a composition according to any aspect of the invention formulated to be suitable for use with (e.g ., for administration to) a human or animal individual . The formulation may be in a form suitable for administration orally (e.g . in a dietary supplement) and/or parenterally, for example, by injection, inhalation, or by transderma l administration via a patch, lotion or gel. The formulation may comprise a diluent, carrier or vehicle such as, for example, an aqueous solvent, non-aqueous solvent, non-toxic excipient, such as a salt, preservative, buffer and the like. Examples of non-aqueous solvents a re propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous solvents include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases.

A seventh aspect of the invention provides a method for protecting an individual at risk of developing, or treating an individual suffering from, a condition associated with demyelination comprising administering to the individual an immunotherapy composition according to the sixth aspect of the invention . For example, the seventh aspect may provide a method for protecting an individual at risk of developing a condition associated with demyelination comprising administering to the individual a vaccine composition according to the sixth aspect of the invention . The seventh aspect may provide a method of treating an individual suffering from a condition associated with demyelination comprising administering to the individual an immunotherapy composition comprising an affinity reagent accord ing to the fifth aspect of the invention .

An eighth aspect of the invention provides a liquid composition according the first aspect of the invention, or an affinity reagent according to the fifth aspect of the invention, or an immunotherapy composition according to the sixth aspect of the invention, for use in a method for protecting an individual at risk of developing, or treating an individual suffering from, a condition associated with demyelination, the method comprising administering the composition or affinity reagent or immunotherapy composition to the individual. Any step of administering to the individual, as referred to herein in relation to any aspect of the invention, may involve delivery of a composition by peroral, topical, or transmucosa l delivery, or delivery via inhalation, or transdermal delivery such as via injection or by use of a nanoneedle array. Delivery by injection may be especially suitable.

A ninth aspect of the invention provides a method for identifying an individual at risk of a condition associated with demyelination, comprising the steps of: a) contacting a biological sample obtained from the individual with an epsilon toxin polypeptide, as defined above, and determining whether a response is observed in a first immunoassay; and b) contacting a biological sample obtained from the individual with sequentially overlapping portions of an epsilon toxin sequence (SEQ ID NO: l, 18, 19, 20 or 21) and determining whether a response is observed in a second immunoassay; wherein detection of a response in the first and/or the second immunoassay is indicative of the individual being at risk of a condition associated with demyelination.

"Sequentially overlapping peptides" indicates a set of synthetic peptides each peptide of which is a portion of any of SEQ ID NOs: l or 18-21 and each of which overlaps with at least one other peptide in the set. In one embodiment, all of the peptides in the set between them encompass the whole length of SEQ ID NO: 1, 18, 19, 20 or 21. Such a set of peptides may be prepared in accordance with the principles set out in Geysen et al. (14) and the second immunoassay conducted generally as described therein. For example, step (b) may be conducted by the company Pepscan (8243 RC Lelystad, Netherlands).

A tenth aspect of the invention provides a composition comprising one or more of: a) a first immunising polypeptide consisting of 9-200 amino acids and comprising the sequence LKRMEK (SEQ ID NO:22); b) a second immunising polypeptide consisting of 10-200 amino acids and

comprising the sequence LKRMEKYYPN (SEQ ID NO: 23); c) a third immunising polypeptide consisting of 9-200 amino acids and comprising the sequence FDKVTINPQ (SEQ ID NO: 24); d) a fourth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence NNPKVELDGEP (SEQ ID NO:25); e) a fifth immunising polypeptide consisting of 9-200 amino acids and comprising the sequence EDVYVGKAL (SEQ ID NO:26); f) a sixth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence VPSQDILVPAN (SEQ ID NO: 27); g) a seventh immunising polypeptide consisting of 10-200 amino acids and

comprising the sequence YKFSLSDTVN (SEQ ID NO: 28); h) an eighth immunising polypeptide consisting of 11-200 amino acids and

comprising the sequence DLNEDGTININ (SEQ ID NO: 29); i) a ninth immunising polypeptide consisting of 12-200 amino acids and comprising the sequence MGDELIVKVRNL (SEQ ID NO:30); and j) a tenth immunising polypeptide consisting of 9-200 amino acids and comprising the sequence EYVIPVDKK (SEQ ID NO: 31).

As described in relation to the first aspect of the invention, the tenth aspect of the invention does not encompass a composition comprising SEQ ID NO: 1, or 18-21, or any of these sequences comprising one or more amino acid substitutions at any position. Therefore, the invention does not encompass a composition in which any of the immunising polypeptides is contained within a naturally occurring epsilon toxin polypeptide selected from SEQ ID NO: l or 18-21, or contained within any of these sequence comprising one or more amino acid substitutions at any position. That is, each immunising polypeptide may be "isolated" in that it is not contained within a polypeptide having amino acid sequence SEQ ID NO: l or 18-21, or within any of these sequence comprising one or more amino acid substitutions at any position.

An eleventh aspect of the invention provides affinity reagent capable of binding to any one of: a) a first immunising polypeptide consisting of 9-200 amino acids and comprising the sequence YDNVDTLIE (SEQ ID NO: 22); b) a second immunising polypeptide consisting of 10-200 amino acids and

comprising the sequence YKFSLSDTVN (SEQ ID NO: 28); h) an eighth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence DLNEDGTININ (SEQ ID NO: 29); i) a ninth immunising polypeptide consisting of 12-200 amino acids and comprising the sequence MGDELIVKVRNL (SEQ ID NO:30); and j) a tenth immunising polypeptide consisting of 9-200 amino acids and comprising the sequence EYVIPVDKK (SEQ ID NO: 31).

In the tenth and eleventh aspects of the invention, each immunising polypeptide consists of an amino acid sequence which is not contiguously linked to a further sequence of contiguous amino acids from within SEQ ID NO: 1 which is not one of SEQ ID NOs: 22-31, i.e., it does not form part of a longer portion of any of SEQ ID NOs: 1 or 18-21. However, any one of immunising polypeptides may be formed as a fusion protein with any further one or more of immunising polypeptides. That is, the composition may comprise a primary polypeptide formed as a fusion protein with a secondary polypeptide, each of the primary and secondary polypeptides being independently selected from the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth immunising

polypeptides. The term "fusion protein" may indicate that the primary and secondary polypeptides are contiguous with one another, or that they may be linked by an intervening linker sequence of one or more amino acids. The fusion protein may also comprise one or more further polypeptides which may be any amino acid sequence required for successful translation of a fusion protein, and or one or more further polypeptides each independently selected from the immunising polypeptides described above. In some embodiments, the immunising polypeptide is not directly joined at its N- terminal end to an amino acid (i.e., to a carboxylic group) and/or is not directly joined at its C-terminal end to an amino acid (i.e., to an amine group).

Any of the immunising polypeptides may be up to 19 amino acids in length. The first, third, fifth or tenth immunising polypeptide may consist of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. The second or seventh immunising polypeptide may consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. The fourth, sixth or eighth immunising polypeptide may consist of 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. The ninth immunising polypeptide may consist of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids.

Any of the immunising polypeptides may be about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 amino acids in length. An immunising polypeptide may comprise any one of SEQ ID NOs:22-31 joined to one or more sequences of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids which are not found in SEQ ID NO: l.

The composition according to the tenth aspect of the invention may be a liquid composition or a lyophilised form of a liquid composition.

When the composition is a liquid, it may consist of one or more of the immunising polypeptides in a diluent, carrier or vehicle. The diluent, carrier or vehicle may be any liquid suitable for carrying the polypeptide components of the composition, such as, for example, an aqueous solvent, non-aqueous solvent, non-toxic excipient, such as a salt, preservative, buffer and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous solvents include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.

Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the composition are adjustable according to the routine ability of the skilled person.

When the composition is a lyophilised form of a liquid composition, it may consist of one or more of the immunising polypeptides in a lyophilised diluent, carrier or vehicle.

A twelfth aspect of the invention provides an immunotherapy composition comprising a composition according to the tenth aspect of the invention, or comprising an affinity reagent according to the eleventh aspect of the invention, in a pharmaceutically acceptable formulation. For example, the immunotherapy composition may be a vaccine composition comprising a composition according to the tenth aspect of the invention and an adjuvant. Suitable adjuvants may be any as described above in relation to the sixth aspect of the invention. Likewise, suitable vaccine carriers and/or vehicles for administration of the vaccine composition may be any as described in relation to the sixth aspect of the invention.

A thirteenth aspect of the invention provides a method for protecting an individual at risk of developing, or treating an individual suffering from, a condition associated with the presence of C. perfringens epsilon toxin (for example, an epsilon toxin polypeptide having amino acid sequence SEQ ID NOs: 1 or 18-21), and/or at risk of developing, or suffering from, a condition associated with demyelination, comprising administering to the individual a vaccine composition according to the twelfth aspect of the invention.

A fourteenth aspect of the invention provides a kit comprising a composition according to the first aspect of the invention and one or more reagents useful for (i.e., suitable for) conducting an immunoassay to detect a response when the composition is contacted with a biological sample. For example, the kit may be used to conduct a method according to any of the second, third or fourth aspects of the invention . For example, the kit may comprise one or more ELISA reagents or one or more reagents for conducting a CMI assay.

A fifteenth aspect of the invention provides a kit comprising a liquid composition according to the first aspect of the invention, or a composition according to the tenth aspect of the invention, or an affinity reagent according to the fifth or eleventh aspects of the invention, or an immunotherapy composition according to the sixth or twelfth aspects of the invention, and means for administering the composition, affinity reagent or immunotherapy composition to an individual. For example, the kit may comprise one or more buffer reagents or diluents and/or one or more administration devices such as a syringe or other injection device.

A sixteenth aspect of the invention provides a polynucleotide encoding for one or more of the following :

(a) a first polypeptide consisting of 10-200 amino acids and comprising the

sequence YLKKVNVKGN (SEQ ID NO : 2) ;

(b) a second polypeptide of 11-200 amino acids and comprising the sequence RYNTKYNYLKR (SEQ ID NO: 5);

(c) a third polypeptide consisting of 6-200 amino acids and comprising the

sequence IVKYRS (SEQ ID NO: 7) ;

(d) a fourth polypeptide consisting of 14-200 amino acids and comprising the sequence QEQKLKSQSFTCKN (SEQ ID NO : 10);

(e) a fifth polypeptide consisting of 6-200 amino acids and comprising at least 6 contiguous amino acids from within the sequence TGVSLTTSYSFANTN (SEQ ID NO : 11);

(f) a sixth polypeptide consisting of 13-200 amino acids and comprising the

sequence TVGTSIQATAKFT (SEQ ID NO: 17) ;

(g) a first immunising polypeptide consisting of 9-200 amino acids and comprising the sequence YDNVDTLIE (SEQ ID NO : 22);

(h) a second immunising polypeptide consisting of 10-200 amino acids and

comprising the sequence LKRMEKYYPN (SEQ ID NO : 23); (i) a third immunising polypeptide consisting of 9-200 amino acids and comprising the sequence FDKVTINPQ (SEQ ID NO: 24) ;

(j) a fourth immunising polypeptide consisting of 11-200 amino acids and

comprising the sequence NNPKVELDGEP (SEQ ID NO: 25);

(k) a fifth immunising polypeptide consisting of 9-200 amino acids and comprising the sequence EDVYVGKAL (SEQ ID NO: 26) ;

(L) a sixth immunising polypeptide consisting of 11-200 amino acids and

comprising the sequence VPSQDILVPAN (SEQ ID NO : 27);

(m) a seventh immunising polypeptide consisting of 10-200 amino acids and

comprising the sequence YKFSLSDTVN (SEQ ID NO : 28);

(n) an eighth immunising polypeptide consisting of 11-200 amino acids and

comprising the sequence DLNEDGTININ (SEQ ID NO : 29);

(o) a ninth immunising polypeptide consisting of 12-200 amino acids and

comprising the sequence MGDELIVKVRNL (SEQ ID NO : 30); and

(p) a tenth immunising polypeptide consisting of 9-200 amino acids and

comprising the sequence EYVIPVDKK (SEQ ID NO : 31).

The first, second, third, fourth, fifth or sixth polypeptides may be as described above in relation to the first aspect of the invention; the first, second, third, fourth, fifth sixth, seventh, eighth, ninth or tenth immunising polypeptides may be as described above in relation to the tenth and eleventh aspects of the invention.

A seventeenth aspect of the invention provides a polynucleotide encoding an antibody or antibody fragment which is an affinity reagent according to the fifth or eleventh aspects of the invention.

Polypeptides and polynucleotides of the invention may be prepared synthetically using conventional synthesisers. Alternatively, they may be produced using recombinant DNA technology and may be incorporated into a suitable expression vector, which is then used to transform a suitable host cell, such as a prokaryotic cell such as E. coli. The transformed host cells are cultured and the polypeptide isolated therefrom.

Therefore, the polynucleotide of the sixteenth or seventeenth aspects of the invention may ta ke the form of a vector comprising a polynucleotide as described above. This includes recombinant constructs comprising one or more of the polynucleotides described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a polynucleotide as described above has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter operably linked to the polynucleotide sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook et at. (2001 ; "Molecular Cloning : a laboratory manual", 3^rd Edition, Cold Spring Harbor Laboratory Press, New York)

A further aspect of the invention provides a cell comprising any of the polypeptide, polynucleotide or vector according to the invention . For example, a suitable cell may be a Salmonella cell, such as a Salmonella enterica cell, in some embodiments from the serovar typhimurium. The Salmonella may be an attenuated strain. Strains c8914 and c9241 may optionally be employed. For example, a suitable system is described in Kulkarni et at. (2008, Vaccine vol. 26, 4194-4203). Preferably the host cell is not a stem cell, especially not a human stem cell.

The term "individual" as used throughout this specification, in relation to any aspect of the invention, indicates a ny human or animal individual, including (but not limited to) a cat, dog or horse, or a ruminant animal such as a cow, sheep, goat or pig . The human or animal may be a human or animal exhibiting symptoms of a demyelinating disease, for example of enterotoxemia (ET), multiple sclerosis (MS), clinically definite MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica spectrum disorder (NMOSD), optic neuritis (ON), neuromyelitis optica (NMO), myelitis, transverse myelitis (TM), a disease or condition characterised by the increase or presence of antibodies against aquaporin-4 (AQP4) and /or astrocyte damage, and acute disseminated

encephalomyelitis (ADEM) .

The term "biological sample" as used throughout this specification indicates a sample which has been obtained from an individual such as (but not limited to) may be a blood, plasma, serum, tissue, saliva or milk sample.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean (and are to be considered interchangeable with) "including but not limited to" a nd do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and figures). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

EXAMPLES

The invention is further described, by way of non-limiting example, with reference to the following Figures 1-4 in which :

Figure 1 shows a western blot indicating immunoreactivity to epsilon toxin (SEQ ID NO: 18) from different sera, with Lane A showing epsilon toxin (arrowed) reacted with a strongly positive serum (BUH00226), lane B showing epsilon toxin reacted with a weakly positive serum (BLT00139) and lane C shows an example of a serum (BUH00239) which did not react with epsilon toxin (molecular size markers (kDa) are in Lane M);

Figure 2 shows the proportion of CDMS and control sera that reacted with epsilon toxin by Western blotting or by Pepscan alone or in combination;

Figure 3 shows results of a competition ELISA to measure toxin-neutralising antibodies (sera BLT00139, BLT00143 and BUH00117 were obtained from CDMS patients, sera CIS 309, CIS 310, CIS 312 and CIS 313 from patients diagnosed with CIS, sera ON YB6, ON BG5, ON MF6 and ONWV1 from patients diagnosed with ON, with PEG14, PEG16 and PEG17 being control sera, and BUH00226 being from a non-MS control found to have detectable anti-epsilon toxin antibodies (Figure 1)); and

Figure 4 shows the location of recognised peptides by each sample in Table 6 underlined in the sequence of full-length epsilon toxin amino acid sequence (SEQ ID NO: l), with SEQ ID NOs: 2, 5, 7, 10, 11 and 17 marked in underlined bold in the top sequence marked "Etx Sequence" (Figure 4A is amino acids 1-100, Figure 4B is amino acids 101- 200, Figure 4C is amino acids 201-300, Figure 4D is amino acids 301-328). Materials and methods

Patients and samples

Sera from patients with CDMS (n = 65), CIS (N = 20) or ON (n=44) were obtained from UK centres. Patients were adults (age> 18 years old) and disease assessment had been carried out by a MS specialist. McDonalds 2010 Criteria (revised) was used for the diagnosis of CIS and MS (15). Sera from CDMS, CIS or ON patients were obtained at UCL and Sheffield from London-South East UK Research and Ethics Committee

(ethics:2011-003475-l l) and Basildon NHS trusts collected under the ALS biomarkers study (ethics: 09/H0703/27) or at the Charing Cross Hospital in accordance with guidelines approved by 05/MRE12/8 NRES Committee South Central - Berkshire. Optic neuritis patient samples were collected as part of the trial. Age- and gender-matched samples from control patients were collected as part of the Exeter 10,000 project (ethics:09/H0106/75). Consent was obtained from subjects. Data on clinical subtypes, occurrence or absence of disease activity and/or progression, disease duration, the occurrence of MRI activity, use of disease modifying therapy and use of high dose steroids is summarised in Tables 2-4 below.

Toxins

Epsilon protoxin (16) was activated with TPCK-treated trypsin from bovine pancreas (Sigma-Aldrich Company Ltd., Gillingham, UK) for 1 hour at room temperature. Bacillus anthracis protective antigen (PA83) was kindly provided by Dr ED Williamson, dstl Porton Down.

Cell culture

Chinese hamster ovary (CHO) cells, CHO cells expressing green fluorescent protein (CHO-GFP) and CHO cells expressing human myelin and lymphocyte protein (CHO-hMAL) were cultured in Dulbecco's Modified Eagle's Medium / Ham's F12 (DMEM/F12) medium (Life Technologies) supplemented with 10% foetal bovine serum at 37°C in 95% air/5% C0₂.

Construction of CHO stable cell line expressing human myelin and lymphocyte protein

(MALI

The human MAL gene (NCBI reference NP_002362.1) was synthesised (GeneArt, Thermo Fisher Scientific) and cloned into pEFlaAcGFP-N l (Clontech). After sequencing, the plasmid was transfected into CHO cells using Turbofect (Thermo Fisher Scientific).

Transfectants were selected in media containing 400 pg/ml G418 for three weeks.

Individual clones were analysed under a fluorescence microscope (Olympus X81) to confirm membrane associated MAL-GFP expression.

Neutralisation Assay Rabbit polyclonal antibody against Y43A-Y209A mutant epsilon-toxin (17) (SEQ ID NO: 19 with Y®A substitution at each of positions 30 and 196), pre-immune rabbit sera, MS patient sera or control sera were serially diluted in an equal volume of PBS containing 5xCT₇s of activated epsilon toxin. After 1 hour, the mixtures were added to CHO-hMAL cells to a final toxin concentration of lxCT₇5 of epsilon toxin. Control CHO- hMAL cells were treated with; PBS; lxCT₇5 of toxin; 0.1% Triton X-100. Following incubation for 3 hours at 37°C, the media was replaced with 100 pi of serum-free DMEM/F12 and 10 pi of WST-1 reagent (Abeam). Metabolic activity of cells was measured as the conversion of WST-1 into a coloured product. Absorbance at 420 nm was read following incubation for 1 hour at 37°C and normalised with respect to the Triton X-100 treated controls.

Competitive ELISA

A competitive ELISA to measure neutralising antibodies was carried out using a

Monoscreen ELISA kit (BioX Diagnostics, BIO K 222/2), according to the manufacturers' instructions.

Western blotting

Toxins (3-6 μg) having sequence SEQ ID NO: 18 were separated using NuPAGE 4-12% Bis-Tris gels and morpholineethanesulfonic acid (MES)-SDS running buffer (Life

Technologies) and blotted onto nitrocellulose membranes which were blocked in phosphate buffered saline-tween (PBST) containing 3% (w/v) dry milk powder. Toxin was detected after adding sera diluted 1000-fold in PBST with 3% (w/v) milk and incubating overnight at 4°C. This was followed by incubation with HRP-conjugated donkey anti-human IgG 1 : 10,000 (Jackson Immunoresearch) in PBST with 3% (w/v) milk for 1 hour at room temperature followed by three 15 min washes in PBST. Signals were detected by using Pierce ECL Western Blotting substrate (Thermo Scientific) and a Chemidoc imaging system equipped with QuantityOne software (Bio-Rad). Samples that were immunoreactive with epsilon toxin were Western blotted with a molar equivalent amount of Bacillus anthracis protective antigen (PA83). Serum samples reactive with epsilon toxin and PA83 were excluded from further analysis.

Epitope scanning

Overlapping peptides spanning translated epsilon toxin SEQ ID NO: l were synthesised and reacted by Pepscan (8243 RC Lelystad, Netherlands) with from sera from MS patents, sera from non-MS controls and sera from rabbits immunised with Y43A-Y209A epsilon toxin . Antibody binding to peptides was tested using an ELISA. Peptides arrays were incubated with primary antibody (overnight at 4°C). After washing, the arrays were incubated with a 1/1000 dilution of an antibody peroxidase conjugate for 1 hour at 25°C. Colour development after adding 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 mI/ml of 3% (v/v) H2O2I hour was quantified with a charge coupled device - camera and an image processing system. To verify the quality of the synthesized peptides, a separate set of positive and negative control peptides was synthesized in parallel . These were screened with a ntibody 57.9 (18).

The same method is followed to scan binding of antibodies, in sera obtained from patients suffering from neuromyelitis optica (NMO), to peptides spanning epsilon toxin SEQ ID NO: 1.

Molecular modelling

Epsilon toxin (PDB ID : 1UYJ) and epitopes were visualised using PyMOL42.

BLAST searches

We searched the US National Center for Biotechnology Information with the query epsilon toxin peptide "TGVSLTTSYSFANTN" using BLASTP and TBLASTN algorithms using default values but with the E value set to 1000.

Western blotting of sera

Western blotting was used to detect antibodies in human sera, based on the method described by Rumah et at. (18) but we used native epsilon toxin (with signal sequence removed but not protease-activated, i.e. toxin having sequence SEQ ID NO : 18) in place of recombinant His-tagged protein, and diluted sera 1000-fold, rather than 10,000-fold, before testing . Figure 1 shows a typical blot with human sera which reacted strongly to epsilon toxin, weakly to epsilon toxin or did not react with epsilon toxin under the test conditions.

Samples that reacted with epsilon toxin were subsequently screened for reactivity with Bacillus anthracis PA83, a pore forming toxin which shares a hydrophobicity map similar to epsilon toxin (12) . Seroconversion to PA83 is rare and would only occur in those individuals who were vaccinated against it or upon exposure to B. anthracis. Rumah et al. ( 12) suggested that a positive PA83 result could suggest nonspecific antibody responses.

Antibodies to epsilon toxin identified bv Western blotting

We assessed the prevalence of antibodies to epsilon toxin in patients with demyelination, the subgroups of which were CDMS, CIS or ON . The latter 2 groups may develop CDMS but a re early in the disease course. Age- and gender-matched controls were then examined (Table 1 and Figure 2) . We repeated the Western blotting at least three times for each sample. Table 5 shows the demographic features of the different groups we tested in this study. In total, we identified 34 serum samples in the combined CDMS, CIS, ON groups (n = 129) that reacted with epsilon toxin, of which 4 also reacted with PA83, and therefore were excluded from further analysis. In the combined control groups (n = 129) we identified 17 serum samples that reacted with epsilon toxin, of which 4 also reacted with PA83, and were therefore excluded from further analysis outlined below.

We found that 23% of the CDMS patient sera had antibodies to epsilon toxin, while the matched control group for this cohort showed reactivity in 8% of samples (Table 1 and Figure 2). The proportion of patients diagnosed with CIS/MS, relapsing remitting MS (RRMS), secondary progressive MS (SPMS) and showing reactivity with epsilon toxin was broadly similar (31%, 21%, 18%). None of the primary progressive MS (PPMS) samples we tested were reactive though the number tested (n= 5) may be too low to be representative of this group.

In CIS patients we found that 35% of the sera had antibodies to epsilon toxin, while in the matched control group 10% of sera was reactive. Follow up patient data on the 7 positive epsilon toxin samples from the CIS cohort showed that 5 of the 7 patients were subsequently diagnosed with MS. We found that 20% of the sera from ON patients were positive for epsilon toxin antibodies, while the matched control group showed that 14% of the sera samples had antibodies to epsilon toxin. All of the Western blotting positive sera from CDMS, CIS and ON patients reacted weakly with epsilon toxin by Western blotting (data not shown). One control serum sample reacted strongly with epsilon toxin by Western blotting (Figure 1, lane A) but the other control sera reacted weakly.

Epitope scanning of sera

Where sufficient sera from CDMS patients (n=43) or control sera (n = 32) was available we analysed them for reactivity with linear overlapping peptides spanning the amino acid sequence of epsilon toxin (Pepscan). We included negative and positive control sera from rabbits before and after immunisation with Y43A-Y209A, an epsilon-toxoid vaccine (17). We used a stringent signal/noise cut-off of ³2.0 to identify positive samples. Rabbit sera before immunisation did not react with the peptide array. Sera from rabbits immunised with the epsilon toxoid recognised several peptides (Table 6).

Of 43 CDMS sera tested, 14 (33%) reacted with at least one peptide, whereas in the matched control group 5 of 32 (16%) reacted (Figure 2). Most sera recognised multiple peptides (Table 6). Only 3 (5%) CDMS sera (309, BLT00139 and BLT00143) were positive by both Western blotting and Pepscan and 1 control (2%) was positive by both testing methods (Figure 2). Conversely, when the Western blotting and Pepscan results were considered together, 43% of CDMS samples and 16% of control sera were positive by at least one assay (Figure 2).

Most of the peptides recognised by CDMS sera and control sera were identical (data not shown). However, one peptide (TGVSLTTSYSFANTN) was recognised by sera from CDMS patients that were positive by Western blotting but not by sera that were negative by Western blotting nor by control sera. When the NCBI database was searched using this peptide, the only complete matches identified were towards C. perfringens epsilon toxin. The next closest match, with a Mycobacterium heraklionense hypothetical protein, showed 11/15 residues matched. There were no complete or partial matches with human proteins.

Within this peptide region, several samples recognised peptides which overlapped with this region. A neighbouring region was recognised by two of the four control samples (one of which was positive for epsilon toxin antibodies in the Western blot). However, the amino acid sequence 170-179 of SEQ ID NO: 1 was not recognised by controls and was included in peptides recognised by 6 samples from MS patients.

It was also noted that several other peptides were consistently recognised by multiple sera from MS patients (including those which were negative in the Western blot), but not recognised by sera from non-MS controls (including those which were positive in the Western blot). Seven samples recognised RYNTKYNYLKR (SEQ ID NO: 5; positions 60-70 of SEQ ID NO: 1) and AYLKKVNVKGN (SEQ ID NO: 3; positions 213-223 of SEQ ID NO: l) and KEKSNDSNIVKYRS (SEQ ID NO: 8; positions 306-319 of SEQ ID NO: 1). A further sample recognised peptides closely overlapping one of the above, IEKGRYNTKYNYLKR (SEQ ID NO: 6; positions 56-70 of SEQ ID NO: l) and YLKKVNVKGNVK (SEQ ID NO:4; positions 214-225 of SEQ ID NO: l) and a further sample recognised KEKSNDSNIVKYRS (SEQ ID NO: 8; positions 306-319 of SEQ ID NO: l). Of these samples, in three samples a further peptide, QEQKLKSQSFTCKN (positions 128-141 of SEQ ID NO: l) was recognised. TVGTSIQATAKFT (SEQ ID NO: 17; positions 152-164 of SEQ ID NO: l) and IVKYRSLSIKAPGIK (SEQ ID NO:9; positions 314-328 of SEQ ID NO: l) were also recognised each by one sample from an MS patient but not by the non-MS controls.

We mapped epitopes recognised by sera onto the molecular structure of epsilon toxin. The antisera raised against an epsilon-toxoid in rabbits recognised a range of epitopes, mainly located in domain 1 and domain 3 of the protein. Control and CDMS sera also recognised domains 1 and 3, as well as additional peptides in domain 2. We found antibodies directed against the membrane insertion loop of domain 2, and especially against the TGVSLTTSYSFANTN peptide which is located there, in CDMS patients but not in sera from controls. Ability of sera to neutralise toxicity

We tested serum neutralisation of epsilon toxin in two ways. First, using a competitive ELISA kit to measure competition between antisera and a neutralising monoclonal antibody for binding to C. perfringens epsilon toxin. The neutralising polyclonal serum and monoclonal antibody caused 90% and 75% signal inhibition respectively (Figure 4). Rabbit sera from animals vaccinated against epsilon toxoid (17) caused 95% inhibition of the signal, whereas pre-immune rabbit sera caused only 6% signal inhibition.

Surprisingly, none of the sera tested from CDMS, CIS or ON patients caused significant inhibition of the signal, even though the sera were reactive in Western blots or as tested by Pepscan. None of the control sera, including the strongly positive BUH00226 sample, caused significant inhibition of the signal, even though these sera were reactive in Western blots or as tested by Pepscan. Without being bound by theory, these results suggest that the types of antibodies raised by immunisation as described differ from the types of antibodies detecta ble by Western blotting or as tested by Pepscan.

We also tested the ability of sera to directly neutralise the cytotoxicity of epsilon toxin towards CHOhMAL cells. Rabbit sera against an epsilon toxoid ( 17) neutralised toxin at 0.25 mg/ml antibody. Sera from non-immune rabbits or the strongly positive BUH00226 sample did not neutralise toxicity (data not shown) .

MS is a pro-inflammatory demyelinating disease of the central nervous system, the aetiology of which involves contribution from genetic and environmental factors. More recently Rumah et al. (12) showed that antibodies to epsilon toxin, were more preva lent in MS patients than in healthy controls, suggesting a role for epsilon toxin in the development of MS (12) . In support of this, many pathophysiological consequences of the exposure of animals to epsilon toxin are consistent with a role of the toxin in MS. Epsilon toxin targets synaptosomes (4), myelinic structures (5, 6), glial cells (7) and oligodendrocytes (8) and causes demyelination (6) . The toxin has been shown to recognise cells expressing myelin and lymphocyte protein (MAL) (19) including human T- cells (20). Against this background, we investigated whether antibodies to epsilon toxin are more frequently found in MS patients. We used Western blotting, because this methodology was used to screen sera in the study from the USA ( 12).

Our data suggests that sero-reactivity towards epsilon-toxin, measured using Western blotting or as tested by Pepscan, was more frequent in CDMS, CIS or ON patients than in controls. Using Western blotting the overall incidence of immunoreactivity in CDMS patients in the UK (24%) was higher than the incidence reported in the USA (10%) . Sero-reactivity to epsilon toxin also occurred in the control group, also at a higher incidence ( 10%) than previously reported ( 1%). These findings are consistent with the increased sensitivity of the assay we have used ( 12) . MS patients often show elevated levels of a ntibodies in sera, though it is not clear what these antibodies are directed against (21). Our finding that antibody reactivity occurred with some control sera, albeit at a lower frequency, indicates that the responses we detected in CDMS patients were not simply due to the elevated level of antibodies as a consequence of MS disease (21) .

Reactivity in the control group also suggests that exposure to epsilon toxin does not necessarily result in the development of MS. In an attempt to understand whether exposure to epsilon toxin is associated with the subsequent development of disease, we looked at patients with CIS. Between 30% to 70% individuals with CIS develop MS (22, 23) . In this small study, we found that 71% (5 out of 7) CIS individuals with a ntibody to epsilon toxin went on to develop CDMS.

The intensity of the responses we saw in CDMS patients and in controls were broadly similar by Western blotting, although, with the exception of control BUH0226, the responses we saw were weak. Different serotypes of epsilon toxin have not been described, but we cannot discount the possibility of this being the cause of the weak response. Another possible explanation for the weak responses is that antibodies are directed against a different structural form of epsilon toxin. During insertion into host cell membranes, the protein would undergo structural changes, altering epitopes (2).

Antibodies directed against a different structural form might be unable to neutralise the toxin . Small differences in the epitope recognised by antibodies can profoundly influence their abilities to neutralise other toxins (24).

There was little overlap between results obtained using Western blotting and peptide sca nning . This is surprising since both methods should primarily detect linea r epitopes. However, Western blots can be sensitive to the denaturation state of the electrophoresed antigen (25). Additionally, since the Western blotting study and Pepscan's testing were carried out in different laboratories it is possible that differences in sample handling affected the results. During this study we also carried out some preliminary work to peptide map sera from 8 patients with CIS/ON. In this pilot study we found that 5 sera showed reactivity with peptides. Further studies should go on to investigate a larger cohort of CIS/ON sera .

Several previous studies have found that dysbiosis of C. perfringens in the gut is not associated with MS (26, 27, 28, 29, 30), though one study did find that C. perfringens levels were elevated in patients diagnosed with neuromyelitis optica (26). However, these studies characterised the population at the species level . It is known that C.

perfringens stra ins which normally colonise the human gut are not able to produce epsilon toxin. One hypothesis is that replacement with strains producing the toxin triggers MS (18); such replacement would not be apparent from changes in the gut microbiome. During this project we searched human gut metagenome datasets at the NCBI human microbiome project (70 healthy volunteers) for the presence of the epsilon toxin gene. We identified the gene encoding alpha-toxin, which is common to all C. perfringens strains, but we did not identify matches with the epsilon toxin gene.

A key question is whether immune-reactivity towards epsilon toxin is indicative of toxin exposure and whether the toxin plays a role in the development of MS. Some elements of our study support this suggestion, especially when viewed in combination with the previous findings (12). Of CDMS samples, tested by Western Blotting or by Pepscan, 26 (43%) were positive under one or the other methods whereas only 10 (16%) of the control sera were positive. This indicates that MS patients are more than twice as likely to possess antibodies to epsilon toxin.

o

Table 6: Peptides recognised by sera tested by Pepscan, with s/n ³ 2.0.

REFERENCES

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2 Bokori-Brown M, Savva CG, Fernandes da Costa SP, Naylor CE, Basak AK and Titball RW. Molecular basis of toxicity of Clostridium perfringens epsilon toxin. FEBS J . 2011 ; 278: 4589-601.

3 Finnie JW. Pathogenesis of brain damage produced in sheep by Clostridium

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Claims

1. A composition comprising one or more of: a. a first polypeptide consisting of 10-200 amino acids and comprising the sequence YLKKVNVKGN; b. a second polypeptide of 11-200 amino acids and comprising the sequence GRYNTKYNYLKR; c. a third polypeptide consisting of 6-200 amino acids and comprising the sequence IVKYRS; d. a fourth polypeptide consisting of 14-200 amino acids and comprising the sequence QEQKLKSQSFTCKN ; e. a fifth polypeptide consisting of 6-200 amino acids and comprising at least 6 contiguous amino acids from within the sequence TGVSLTTSYSFANTN; f. a sixth polypeptide consisting of 13-200 amino acids and comprising the sequence TVGTSIQATAKFT.

2. The composition according to claim 1 wherein any of the first, second, third, fourth, fifth or sixth polypeptides consists of 15, 16, 17, 18, 19, 20, 21, 22, 23,

24 or 25 amino acids.

3. The composition according to claim 1 or 2 comprising at least the first and the second polypeptide or at least the first and the third polypeptide or at least the first, second and third polypeptide.

4. The composition according to any of claims 1-3 comprising the first polypeptide, the second polypeptide and the third polypeptide.

5. The composition according to claim 3 or 4 comprising the fourth polypeptide.

6. The composition according to any preceding claim which is a liquid composition consisting of AYLKKVNVKGN (SEQ ID NO:3) and RYNTKYNYLKR (SEQ ID NO: 5) and KEKSNDSNIVKYRS (SEQ ID NO:8) and QEQKLKSQSFTCKN (SEQ ID NO: 10) in a diluent, carrier or vehicle.

7. The composition according to any of claims 1-5 which is a surface having

AYLKKVNVKGN (SEQ ID NO: 3) and RYNTKYNYLKR (SEQ ID NO: 5) and

KEKSNDSNIVKYRS (SEQ ID NO: 8) and QEQKLKSQSFTCKN (SEQ ID NO: 10) adhered thereto.

8. A method of identifying an individual at risk of a condition associated with demyelination, comprising the steps of: a . contacting a biological sample obtained from the individua l with the

composition according to any preceding claim; and b. determining whether a response is observed in an immunoassay.

9. The method according to claim 8 wherein the immunoassay is an antibody

binding assay or a cell-mediated immunity (CMI) assay.

10. The method according to claim 9 wherein the CMI assay detects interferon-y (IFN-Y) .

11. A method according to claim 8 wherein a sample obtained from the individual has previously been determined by a different method not to comprise an a ntibody to an epsilon toxin polypeptide.

12. A method of identifying an individual at risk of a condition associated with

demyelination, comprising the steps of: a . obtaining a biological sample from an individual suspected of having the condition ; b. contacting a biological sample obtained from the individua l with the

composition according to any preceding claim; c. determining whether a response is observed in a n immunoassay; wherein, if a response is observed in step (c), the individual is identified as at risk of having a condition associated with de-myelination.

13. The composition according to any of claims 1-7 for use in the method according to claim 12.

14. An affinity reagent capable of binding to a polypeptide selected from : a . a first polypeptide consisting of 10-200 amino acids and comprising the sequence YLKKVNVKGN (SEQ ID NO : 2) ; b. a second polypeptide of 11-200 amino acids and comprising the sequence RYNTKYNYLKR (SEQ ID NO : 5) ; c. a third polypeptide consisting of 6-200 amino acids and comprising the sequence IVKYRS (SEQ ID NO: 7); d . a fourth polypeptide consisting of 14-200 amino acids and comprising the sequence QEQKLKSQSFTCKN (SEQ ID NO: 10) ; e. a fifth polypeptide consisting of 6-200 amino acids and comprising at least 6 contiguous amino acids from within the sequence TGVSLTTSYSFANTN (SEQ ID NO : 11); f. a sixth polypeptide consisting of 13-200 amino acids and comprising the sequence TVGTSIQATAKFT (SEQ ID NO : 17).

15. An immunotherapy composition comprising the composition according to any of claims 1-6, or comprising an affinity reagent according to claim 14.

16. A method for protecting an individual at risk of developing, or treating an

individual suffering from, a condition associated with demyelination comprising administering to the individual an immunotherapy composition according to claim 15.

17. The composition according to any of claims 1-6, or the affinity reagent according to claim 14, or a immunotherapy composition according to claim 15 for use in a method for protecting an individual at risk of developing, or treating an individual suffering from, a condition associated with demyelination, the method comprising administering the composition or affinity reagent or immunotherapy composition to the individual.

18. A method for identifying an individual at risk of a condition associated with

demyelination, comprising the steps of: a . contacting a biological sa mple obtained from the individual with an epsilon toxin polypeptide and determining whether a response is observed in a first immunoassay; and b. contacting a biological sample obtained from the individual with

sequentially overlapping portions of an epsilon toxin and determining whether a response is observed in a second immunoassay; wherein detection of a response in the first and/or the second immunoassay is indicative of the individual being at risk of a condition associated with demyelination.

19. A composition comprising one or more of: a. a first immunising polypeptide consisting of 9-200 amino acids and comprising the sequence YDNVDTLIE (SEQ ID NO:22); b. a second immunising polypeptide consisting of 10-200 amino acids and comprising the sequence LKRMEKYYPN (SEQ ID NO:23); c. a third immunising polypeptide consisting of 9-200 amino acids and comprising the sequence FDKVTINPQ (SEQ ID NO: 24); d. a fourth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence NNPKVELDGEP (SEQ ID NO: 25); e. a fifth immunising polypeptide consisting of 9-200 amino acids and comprising the sequence EDVYVGKAL (SEQ ID NO: 26); f. a sixth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence VPSQDILVPAN (SEQ ID NO:27); g. a seventh immunising polypeptide consisting of 10-200 amino acids and comprising the sequence YKFSLSDTVN (SEQ ID NO:28); h. an eighth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence DLNEDGTININ (SEQ ID NO:29); i. a ninth immunising polypeptide consisting of 12-200 amino acids and comprising the sequence MGDELIVKVRNL (SEQ ID NO:30); and j. a tenth immunising polypeptide consisting of 9-200 amino acids and comprising the sequence EYVIPVDKK (SEQ ID NO:31).

20. An affinity reagent capable of binding to any one of: a. a first immunising polypeptide consisting of 9-200 amino acids and comprising the sequence YDNVDTLIE (SEQ ID NO:22); b. a second immunising polypeptide consisting of 10-200 amino acids and comprising the sequence LKRMEKYYPN (SEQ ID NO:23); c. a third immunising polypeptide consisting of 9-200 amino acids and comprising the sequence FDKVTINPQ (SEQ ID NO: 24); d. a fourth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence NNPKVELDGEP (SEQ ID NO: 25); e. a fifth immunising polypeptide consisting of 9-200 amino acids and

comprising the sequence EDVYVGKAL (SEQ ID NO: 26); f. a sixth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence VPSQDILVPAN (SEQ ID NO:27); g. a seventh immunising polypeptide consisting of 10-200 amino acids and comprising the sequence YKFSLSDTVN (SEQ ID NO:28); h. an eighth immunising polypeptide consisting of 11-200 amino acids and comprising the sequence DLNEDGTININ (SEQ ID NO:29); i. a ninth immunising polypeptide consisting of 12-200 amino acids and comprising the sequence MGDELIVKVRNL (SEQ ID NO:30); and j. a tenth immunising polypeptide consisting of 9-200 amino acids and comprising the sequence EYVIPVDKK (SEQ ID NO:31).

21. An immunotherapy composition comprising the composition according to claim 19, or the affinity reagent according to claim 20.

22. A method for protecting an individual at risk of developing a condition associated with the presence of C. perfringens epsilon toxin, or for treating an individual suffering from a condition associated with the presence of C. perfringens epsilon toxin, comprising administering to the individual an immunotherapy composition according to claim 21.

23. A method according to claim 22 which is a method for protecting an individual at risk of developing a condition associated with demyelination, or a method for treating an individual suffering from a condition associated with demyelination.

24. The composition according to claim 19 or the affinity reagent according to claim 20 or the immunotherapy composition according to claim 21, for use in a method for protecting an individual at risk of developing a condition associated with the presence of C. perfringens epsilon toxin, or for treating an individual suffering from a condition associated with the presence of C. perfringens epsilon toxin, the method comprising administering the composition or affinity reagent or immunotherapy composition to the individual.

25. A kit comprising a composition according to any of claims 1-7 and one or more reagents useful for conducting an immunoassay to detect a response when the composition is contacted with a biological sample.

26. A kit comprising a composition according to any of claims 1-6, or 19, or an

affinity reagent according to claim 14 or 20, or an immunotherapy composition according to claim 15 or 21, and means for administering the composition, affinity reagent or vaccine composition to an individual.