[go: up one dir, main page]

WO2019166832A1 - Détection de maladie - Google Patents

Détection de maladie Download PDF

Info

Publication number
WO2019166832A1
WO2019166832A1 PCT/GB2019/050590 GB2019050590W WO2019166832A1 WO 2019166832 A1 WO2019166832 A1 WO 2019166832A1 GB 2019050590 W GB2019050590 W GB 2019050590W WO 2019166832 A1 WO2019166832 A1 WO 2019166832A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acids
sequence
polypeptide
individual
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2019/050590
Other languages
English (en)
Inventor
Richard William Titball
Nicholas Peter LEWIS
Sariqa WAGLEY
Helen MORCRETTE
Monika BOKORI-BROWN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
One Health Ventures Ltd
Original Assignee
One Health Ventures Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by One Health Ventures Ltd filed Critical One Health Ventures Ltd
Publication of WO2019166832A1 publication Critical patent/WO2019166832A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis

Definitions

  • the invention relates to compositions and methods for identification of a demyelinating condition .
  • 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).
  • 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).
  • MS Multiple sclerosis
  • CIS clinically isolated syndrome
  • CDMS Clinically Definite MS
  • Optic neuritis is a demyelinating inflammation of the optic nerve. It is frequently associated with multiple sclerosis.
  • the autoimmune disease neuromyelitis optica is a heterogeneous condition consisting of the simultaneous inflammation and demyelination of the optic nerve (optic neuritis) and the spinal cord (myelitis).
  • SEQ ID NO : l 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.
  • 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
  • SEQ ID NO:21 being the consequence of processing by l-protease; each protease has a slightly d ifferent cleavage site.
  • epsilon toxin polypeptide 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.
  • a first aspect of the invention provides a composition comprising one or more of:
  • 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 .
  • 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.
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • SEQ ID NO: 6 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.
  • SEQ ID NO : 12 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) .
  • SEQ ID NO: 11 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 may be a surface having one or more of the first, second, third, fourth, fifth and sixth polypeptide adhered thereto.
  • the composition 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.
  • AYLKKVNVKGN SEQ ID NO : 3
  • RYNTKYNYLKR SEQ ID NO : 5
  • KEKSNDSNIVKYRS SEQ ID NO: 8
  • 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.
  • aqueous solvent examples include 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.
  • composition 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.
  • 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.
  • composition 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.
  • the composition 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.
  • composition 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.
  • AYLKKVNVKGN SEQ ID NO:3
  • RYNTKYNYLKR SEQ ID NO: 5
  • KEKSNDSNIVKYRS SEQ ID NO: 8
  • adhered to the surface It may consist of AYLKKVNVKGN (SEQ ID NO: 3) and
  • 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.
  • 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.
  • 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).
  • EEM enterotoxemia
  • MS multiple sclerosis
  • CDMS clinically definite MS
  • CIS clinically isolated syndrome
  • NMOSD neuromyelitis optica spectrum disorder
  • ON optic neuritis
  • NMO neuromyelitis optica
  • TM transverse my
  • An assay utilising the polypeptides is capable of distinguishing between :
  • 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
  • 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.
  • a second aspect of the invention provides a method of identifying an individual at risk of a condition associated with demyelination, comprising the steps of:
  • 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.
  • the immunoassay may be an antibody binding assay or a cell-mediated immunity (CMI) assay.
  • CMI cell-mediated immunity
  • immunoassay is a CMI assay, it may be one which detects interferon-g (IFN-y).
  • 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
  • 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.
  • EEM enterotoxemia
  • MS multiple sclerosis
  • CDMS clinically definite MS
  • CIS clinically isolated syndrome
  • NMOSD neuromyelitis optica spectrum disorder
  • ON optic neuritis
  • NMO neuromyelitis optica
  • TM transverse myelitis
  • TM transverse myelitis
  • 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.
  • control individual 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
  • 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.
  • the method according to the second aspect of the invention 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.
  • 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;
  • step (c) 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:
  • step (c) wherein, if a response is observed in step (c), the individual is identified as being at risk of a condition associated with de-myelination .
  • 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.
  • 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
  • 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 :
  • affinity reagent 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.
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • 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).
  • the affinity reagent may be capable of binding to any one of SEQ ID NOs: 2, 3 or 4.
  • 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 : 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) .
  • the affinity reagent may be capable of binding to any one of SEQ ID NOs: 7, 8 or 9.
  • 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 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.
  • SEQ ID NO: 12 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) .
  • SEQ ID NO: 11 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.
  • 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 MontanideTM adjuvant, for example MontanideTM 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.
  • BRM biologic response modifiers
  • an immunotherapy composition such as a vaccine composition, described herein may include a vaccine carrier.
  • 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.
  • 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
  • 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.
  • Suitable liposomes 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
  • MLV multilamellar ves
  • delivery particle for example, microspheres and the like, also are contemplated for delivery of the immunotherapy composition .
  • 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).
  • a suitable vector e.g ., vaccinia, canarypox, adenovirus, or other eukaryotic virus vector.
  • 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 .
  • 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.
  • 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.
  • a foodstuff i.e., a food material suitable for consumption by a human or an animal
  • 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 .
  • 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.
  • 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 .
  • 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 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
  • 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.
  • 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
  • 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.
  • any one of immunising polypeptides may be formed as a fusion protein with any further one or more of immunising polypeptides.
  • 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
  • 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.
  • 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,
  • 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.
  • composition according to the tenth aspect of the invention may be a liquid composition or a lyophilised form of a liquid composition.
  • the composition when it 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.
  • 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.
  • composition when it 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.
  • 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.
  • 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.
  • C. perfringens epsilon toxin for example, an epsilon toxin polypeptide having amino acid sequence SEQ ID NOs: 1 or 18-21
  • 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.
  • the kit may be used to conduct a method according to any of the second, third or fourth aspects of the invention .
  • 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.
  • 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 :
  • VPSQDILVPAN SEQ ID NO : 27
  • 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.
  • a suitable host cell such as a prokaryotic cell such as E. coli.
  • the transformed host cells are cultured and the polypeptide isolated therefrom.
  • 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.
  • 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.
  • 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.
  • a further aspect of the invention provides a cell comprising any of the polypeptide, polynucleotide or vector according to the invention .
  • 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.
  • a suitable system is described in Kulkarni et at. (2008, Vaccine vol. 26, 4194-4203).
  • the host cell is not a stem cell, especially not a human stem cell.
  • 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
  • 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
  • ADAM encephalomyelitis
  • biological sample 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.
  • any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
  • 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
  • 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.
  • CHO cells 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% C0 2 .
  • DMEM/F12 Dulbecco's Modified Eagle's Medium / Ham's F12
  • 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.
  • a competitive ELISA to measure neutralising antibodies was carried out using a
  • Toxins (3-6 ⁇ g) having sequence SEQ ID NO: 18 were separated using NuPAGE 4-12% Bis-Tris gels and morpholineethanesulfonic acid (MES)-SDS running buffer (Life
  • PBST phosphate buffered saline-tween
  • 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.
  • 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.
  • NMO neuromyelitis optica
  • Epsilon toxin (PDB ID : 1UYJ) and epitopes were visualised using PyMOL42.
  • Antibodies to epsilon toxin identified bv Western blotting
  • QEQKLKSQSFTCKN positions 128-141 of SEQ ID NO: l
  • TVGTSIQATAKFT SEQ ID NO: 17; positions 152-164 of SEQ ID NO: l
  • IVKYRSLSIKAPGIK SEQ ID NO:9; positions 314-328 of SEQ ID NO: l
  • 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).
  • MAL myelin and lymphocyte protein
  • 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).
  • 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.
  • 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.
  • perfringens type B in an individual at first clinical presentation of multiple sclerosis provides clues for environmental triggers of the disease. PLoS One.
  • the myelin and lymphocyte protein MAL is required for binding and activity of Clostridium perfhngens epsilon-toxin. PLoS Pathog. 2015; 11 : el004896.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une composition comprenant un ou plusieurs d'un premier polypeptide constitué de 10 à 200 acides aminés et comprenant la séquence YLKKVNVKGN; un deuxième polypeptide de 11 à 200 acides aminés et comprenant la séquence GRYNTKYNYLKR; un troisième polypeptide constitué de 6 à 200 acides aminés et comprenant la séquence IVKYRS; un quatrième polypeptide constitué de 14 à 200 acides aminés et comprenant la séquence QEQKLKSQSFTCKN; un cinquième polypeptide constitué de 10 à 200 acides aminés et comprenant au moins 6 acides aminés contigus de la séquence TGVSLTTSYSFANTN; et/ou un sixième polypeptide constitué de 13 à 200 acides aminés et comprenant la séquence TVGTSIQATAKFT. L'invention concerne également des procédés d'identification d'un individu présentant un risque d'une affection associée à une démyélinisation, comprenant l'utilisation de la composition.
PCT/GB2019/050590 2018-03-02 2019-03-01 Détection de maladie Ceased WO2019166832A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1803387.8 2018-03-02
GBGB1803387.8A GB201803387D0 (en) 2018-03-02 2018-03-02 Disease detection

Publications (1)

Publication Number Publication Date
WO2019166832A1 true WO2019166832A1 (fr) 2019-09-06

Family

ID=61903714

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2019/050590 Ceased WO2019166832A1 (fr) 2018-03-02 2019-03-01 Détection de maladie

Country Status (2)

Country Link
GB (1) GB201803387D0 (fr)
WO (1) WO2019166832A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022032166A1 (fr) * 2020-08-07 2022-02-10 Othair Prothena Limited Vaccin multi-épitopique pour le traitement de la maladie d'alzheimer
WO2024173889A1 (fr) * 2023-02-17 2024-08-22 Cornell University Diagnostic et thérapie dépendants de l'etx de la sclérose en plaques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014127258A2 (fr) * 2013-02-14 2014-08-21 Cornell University Méthodes de protection contre la sclérose en plaques et méthodes pour la traiter

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014127258A2 (fr) * 2013-02-14 2014-08-21 Cornell University Méthodes de protection contre la sclérose en plaques et méthodes pour la traiter

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUILHERME GUERRA ALVES ET AL: "Mapping of the continuous epitopes displayed on the Clostridium perfringens type D epsilon-toxin", BRAZILIAN JOURNAL OF MICROBIOLOGY, vol. 48, no. 3, 1 July 2017 (2017-07-01), BR, pages 570 - 575, XP055585221, ISSN: 1517-8382, DOI: 10.1016/j.bjm.2016.10.023 *
RACIBORSKA D ET AL: "Epsilon toxin as a causative agent in MS", JOURNAL OF NEUROLOGICAL SCIENCES, vol. 381, 31 December 2017 (2017-12-31), pages 798, XP085295539, ISSN: 0022-510X, DOI: 10.1016/J.JNS.2017.08.2248 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022032166A1 (fr) * 2020-08-07 2022-02-10 Othair Prothena Limited Vaccin multi-épitopique pour le traitement de la maladie d'alzheimer
WO2024173889A1 (fr) * 2023-02-17 2024-08-22 Cornell University Diagnostic et thérapie dépendants de l'etx de la sclérose en plaques

Also Published As

Publication number Publication date
GB201803387D0 (en) 2018-04-18

Similar Documents

Publication Publication Date Title
US9068007B2 (en) Methods and compositions for chlamydial antigens for diagnosis and treatment of chlamydial infection and disease
US20250381258A1 (en) Epsilon toxin from clostridium perfringens as a vaccine
US20130251749A1 (en) Methods and compositions for chlamydial antigens as reagents/strategies for diagnosis and treatment of chlamydial infection and disease
JP4785014B2 (ja) 蛋白質
US10261092B2 (en) Cross-reactive determinants and methods for their identification
US8703432B2 (en) Recombinant Treponema spp. proteins for use in vaccine, antibodies against said proteins, and diagnostic and therapeutic methods including the same
WO2019166832A1 (fr) Détection de maladie
US7169393B2 (en) Antigenic peptide fragments of VapA protein, and uses thereof
KR20060129229A (ko) 표면에 위치한 빈창자캄필로박터 폴리펩티드
Bulashev et al. Validation of a chimeric proteins-based ELISA for the diagnosis of brucellosis in Kazakhstan
CA2682623A1 (fr) Peptides, anticorps et fragments de ceux-ci destines au traitement et au diagnostic de la maladie coeliaque
HK1149770B (en) Compositions, methods and kits
US20140248272A1 (en) Recombinant proteins for use in vaccine, antibodies against said proteins, and diagnostic and therapeutic methods including the same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19715188

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19715188

Country of ref document: EP

Kind code of ref document: A1