Epsilon toxins from clostridium perfringens as vaccines

Abstract

The present invention relates to methods and compositions for detecting, diagnosing, preventing, treating or ameliorating symptoms of a demyelination disorder in a human or animal subject in need thereof, the present invention relates to a method for treating a disease or condition selected from the group consisting of enterotoxemia (ET), multiple sclerosis (MS), clinically definite MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica lineage disease (NMOSD), neuromyelitis (ON), neuromyelitis optica (NMO), myelitis, myelitis, transverse myelitis (TM), a disease or condition characterized by an increase or presence of an antibody to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM). The method comprises administering to a subject a composition comprising an effective amount of an agent that directly or indirectly interferes with epsilon toxin (Etx) produced by a Clostridium perfringens type B or type D strain, Etx binding receptor, or interaction of Etx with its binding receptor, to inhibit or repress Etx modulated receptor signaling activity.

Description

[0001] This divisional patent application is a divisional application of the patent application with the application number 2019800160707 and the invention title "Epsilon toxin from Clostridium perfringens as a vaccine", which was filed on March 1, 2019. Technical Field

[0002] The present invention relates to methods and compositions for detecting, diagnosing, preventing, treating or ameliorating the symptoms of demyelinating disorders. Background Art

[0003] Clostridium perfringens is a rod-shaped, spore-forming, Gram-negative, anaerobic bacterium that is capable of producing at least 17 toxins, making it one of the most pathogenic species in the genus Clostridium. Based on its ability to produce four types of toxins (i.e., alpha-toxin, beta-toxin, epsilon-toxin, and iota-toxin), Clostridium perfringens strains are classified as one of five toxin types (referred to as types A to E) (Petit et al. (1999) Trends Microbiol. vol. 7. 104-110).

[0004] Epsilon toxin (Etx) is produced by toxin types B and D. These strains are the cause of a severe disease called enterotoxemia, which mainly affects sheep and lambs, but also causes infections in ruminant species (including goats and calves) (Songer (1996) Clin. Microbiol. Rev. vol. 9, 216-234). Enterotoxemia in naturally infected animals is usually characterized by systemic lesions in sheep and enterocolitis in goats. In addition to the classified toxins, the bacterium is also capable of producing a variety of so-called minor toxins, such as beta1, beta2, delta, theta, lambda, mu, v, and enterotoxin (Rood (1998) Annu. Rev. Microbiol. vol. 2052. 333-360).

[0005] The most important factor triggering disease in sheep and other ruminants is overeating high-calorie foods, resulting in a large amount of carbohydrates in the intestine. This leads to a disruption of the microbial balance in the intestine, resulting in the proliferation of Clostridium perfringens and subsequent overproduction of Etx. The toxin causes an increase in intestinal permeability, facilitating its entry into the bloodstream and its spread to the main target organs (kidneys and brain) (McDonel (1980) Pharmacol Ther 10(3): 617-655). Here, due to the increase in vascular permeability, intoxication leads to fluid accumulation. Accumulation in the central nervous system results in neurological disorders and rapidly leads to death (Finnie (2003 Aust. Vet. J. vol. 81, 219-221).

[0006] Recently, Etx has been suggested to play a role in the development of human multiple sclerosis (Rumah et al. (2013) PLoS One 8: e76359).

[0007] Multiple sclerosis (MS) is a demyelinating disease in which the insulating layer of nerve cells in the brain and spinal cord is damaged. This damage disrupts the communication ability of parts of the nervous system, leading to a range of symptoms, including double vision, blindness in one eye, muscle weakness, sensory disturbances, or problems with coordination. This condition usually starts as a clinically isolated syndrome (CIS) for some time before being confirmed as clinically definite MS (CDMS).

[0008] Neuromyelitis optica spectrum disorder (NMOSD) is a rare neurological condition characterized by episodes of optic neuritis (ON) and transverse myelitis (TM), along with one or more other diagnostic criteria, including the presence of specific antibodies (aquaporin-4 (AQP-4)) in some cases.

[0009] Optic neuritis (ON) is a demyelinating inflammation of the optic nerve. It is often associated with multiple sclerosis. The autoimmune disease neuromyelitis optica (NMO) is a heterogeneous condition consisting of simultaneous inflammation and demyelination of the optic nerve (optic neuritis) and the spinal cord (myelitis). Approximately 80% of patients diagnosed with NMO test positive for aquaporin-4 (AQP-4) antibodies (http: / / www.nmouk.nhs.uk / healthcare-professionals / aqp4-antibodies). Zamvil et al. (Neurotherapeutics (2018) 15: 92 - 101) hypothesized that the gut microbiota and possibly Clostridium perfringens itself may be involved in the pathogenesis of NMO.

[0010] Transverse myelitis (TM) is an inflammation on both sides of a segment of the spinal cord and can also cause myelin damage.

[0011] Acute disseminated encephalomyelitis (ADEM) is characterized by a transient but widespread inflammatory episode in the brain and spinal cord that damages myelin. ADEM usually occurs after a viral or bacterial infection, or less commonly after vaccination against measles, mumps, or rubella. ADEM usually damages white matter, resulting in neurological symptoms such as loss of vision in one or both eyes (due to inflammation of the optic nerve).

[0012] Etx is expressed with a signal sequence that directs the export of the prototoxin from the bacterium (McDonel (1986) in Pharmacology of bacterial toxins eds. Dorner & Drew, Pergamon Press, 477 - 517). During disease development, the relatively inactive prototoxin is converted to the active toxin by proteolytic cleavage in the intestinal lumen, either by host digestive proteases such as trypsin and chymotrypsin (Bhown & Habeerb (1977) Biochem. Biophys. Res. Commun. vol. 78, 889 - 896) or by Clostridium perfringens lambda - protease (Minami et al. (1997) Microbiol. Immunol. vol. 41, 527 - 535). Proteolytic activation of Etx can also be achieved in vitro by controlled proteolysis (Hunter et al. (1992) Infect. Immun. vol. 60, 102 - 110). Depending on the protease, proteolytic cleavage results in the removal of 10 - 13 amino - terminal amino acids and 22 - 29 carboxyl - terminal amino acids (Bhown & Habeerb (1977); Minami et al. (1997)). Maximal activation occurs when both the N - terminus and C - terminus are cleaved (Worthington & Mulders (1977) Infect. Immun. vol. 18, 549 - 551).

[0013] The 3D structure of Etx has been determined (Cole et al. (2004) Nature Structural & Molecular Biology vol. 11, 797 - 798) and reveals a molecule composed mainly of β - sheets that can be divided into three functional domains. The N - terminal domain I includes the proposed receptor - interaction region. The middle domain II includes amphipathic β - hairpin structures that are expected to play a role in membrane insertion. The C - terminal domain III includes the C - terminal peptide that must be removed for activation to occur.

[0014] Epsilon toxin is an aerolysin-like β-pore forming toxin (β-PFT), in which an amphiphilic β-hairpin loop inserts into the membrane to form a β-barrel structure. The overall fold of Etx is similar to that of aerolysin from Gram-negative Aeromonas hydrophila (Parler et al. (1994) Nature vol. 367, 292-295), parasporin-2 (PS) from Bacillus thuringiensis (Akiba et al. (2009) J. Mol. Biol. vol. 386, 121-133) and the pore-forming lectin (LSL) from Laetiporus sulphureus (Mancheno et al. (2005) J. Biol. Chem. vol. 280, 17251-17259). The structural similarity among these toxins is most prominent in their two C-terminal domains. Their N-terminal domains show greater structural variation, which may account for the differences in their target cell specificity and potency (Bokori-Brown et al. (2011) FEBS J. vol. 278. 45894601).

[0015] In aerolysin, two amino-terminal domains (domain I and domain II) are thought to play a role in binding to the cell surface with overlapping functions (MacKenzie et al. 25 (1999) J. Biol. Chem. vol, 274, 22604-22609), and it has been proposed that domain I of Etx (equivalent to domain II of aerolysin) performs a similar function (Cole et al. (2004)), but this has not been confirmed. Domain II of aerolysin contains a mannose 6-phosphate binding loop. However, the residues of domain II involved in mannose-6-phosphate binding in aerolysin are not conserved in domain I of Etx, indicating that the structural changes in the N-terminal receptor-binding domains of these toxins may account for the differences in their target cell specificities.

[0016] Etx is unique among β-PFTs because of its high potency and high cell specificity. Because of its high potency, Etx is considered a potential biological weapon for international terrorism by the Centers for Disease Control and Prevention of the US government (Morbidity and Mortality Weekly Report (MMWR) Recommendations and Reports 2000) vol.49, 1-14). The 50% lethal dose (LD 50 ) of Etx in mice after intravenous injection is usually 100 ng / kg (Gill (1982) Microbiol. Rev. Vol.46, 86-94), making Etx the most potent clostridial toxin after botulinum neurotoxin. Etx also shows high cell specificity. Among many cell lines tested, only four cell lines were identified as sensitive to the toxin. These cell lines include canine (MDCK (Knight et al. (1990) Biologicals vol.18, 263-270)), mouse (mpkCCDcl4 (Chassin et al. (2007) Am. J. Physiol. Renal Physiol. vol.293, F927-937)) and human (renal cell lines derived from G-402 (Shortt et al. (2000) Hum. Exp. Toxicol. vol.19.108-1l6) and ACHN (Ivie et al. (2011) PloS ONE vol.6, e17787)). Most in vitro studies on Etx have been conducted using the Madin-Darby canine kidney (MDCK) cell line because this cell line is the most sensitive to the toxin (Payne et al. (1994) FEMS Microbiol. Lett. Vol.116, 161-167). It has been reported that the dose (CT 50 ) of Etx that kills 50% of MDCK cells is as low as 15 ng / ml.

[0017] The binding of Etx to MDCK cells is associated with the formation of stable high molecular weight complexes (Petit et al. (1997) J. Bacteriol. vol. 179, 6480 - 6487). Poisoned cells undergo morphological changes before cell death, including swelling and plasma membrane blebbing (Petit et al. (1997) J. Bacteriol. Vol. 179, 6480 - 6487). The rapid toxin-induced cell death and the specificity of ε-toxin for only some cell lines suggest the presence of specific receptors on the target cells. Etx acts by binding to host cells, and there is evidence that the seven monomers of this protein assemble into pores that span the cell membrane (Miyata et al. (2002) J Biol Chem. 277:39463 - 8.), leading to unregulated ion transmembrane movement and cell death. Toxicity appears to be the result of pore formation in the target cell membrane (Petit et al. (2001) J. Biol. Chem. 25 vol. 276. 15736 - 15740).

[0018] The identity of the cell surface receptor for the toxin remains incompletely elucidated. There is evidence that the toxin binds to the hepatitis A virus cellular receptor 1 protein (HAVCR1) on MDCK.2 cells (Ivie et al. (2011) PLoS One 6: e17787). More recently, there has been evidence that the receptor is myelin and lymphocyte protein (MAL) (Rumah et al. (2015) PLoS Pathog. 11: e1004896). CHO cells, which are normally highly resistant to the toxin, become sensitive when MAL is expressed, and MAL knockout mice are reported to be highly resistant to the toxin (Rumah et al. (2015)).

[0019] Many commercial vaccines are available for the prevention of enterotoxemia. These vaccines are typically produced by detoxifying Etx by treating the culture filtrate of Clostridium perfringens with formaldehyde. These vaccines contain many proteins in addition to Etx, and there can be considerable batch-to-batch variation in the immunogenicity of these preparations. Inflammatory responses following vaccination have been reported to result in reduced feed consumption. These deficiencies have prompted work on the design of improved vaccines, and many recombinant immunogens have been reported, including formaldehyde-treated Etx produced by Escherichia coli (Lobato et al. (2010) Vaccine 28:6125-7) and site-directed mutants of Etx with reduced toxicity (genetic toxoids) (Kang et al. (2017) Human vaccines & immunotherapeutics 13:1598-608). The site-directed mutants overcome the problem of batch-to-batch variation in immunogenicity associated with the chemical detoxification methods of vaccine production. However, the high potency of the toxin may make it difficult to eliminate toxicity. The toxicity of the mutants has been evaluated using MDCK cell cultures (Ivie and McClain (2012) Biochemistry 51:7588-95; Kang et al. (2017)) or in mice.

[0020] It has been reported that a site-directed mutant of Etx (Y30A-Y196A) has over 430-fold reduced cytotoxicity against MDCK.2 cells compared to the wild-type toxin and shows reduced but not eliminated toxicity in mice (Bokori-Brown et al., (2014) Vaccine vol.32, 2682-2687).

[0021] There remains a need to identify improved molecules with the potential to serve as vaccines against diseases caused by or associated with the presence of Etx and / or diseases caused by Clostridium perfringens infection. DETAILED DESCRIPTION

[0022] The present invention relates to methods and compositions for detecting, diagnosing, preventing, treating or ameliorating the symptoms of a demyelinating disorder in a human or animal subject in need thereof, said disorder being selected from: enterotoxaemia (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 disorder characterized by an increase or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM). The method comprises administering to the subject a composition comprising an effective amount of an agent that directly or indirectly interferes with epsilon toxin (Etx), an Etx binding receptor, or the interaction of Etx with its binding receptor produced by Clostridium perfringens type B or D strains, thereby inhibiting or repressing Etx-regulated receptor signaling activity. The present invention relates to novel polypeptides useful as vaccines against diseases caused by or associated with the epsilon toxin of Clostridium perfringens, particularly in animals susceptible to enterotoxaemia and in the treatment of demyelinating disorders.

[0023] The inventors have found that subjects suffering from demyelinating disorders such as enterotoxaemia (ET), neuromyelitis optica (NMO) and transverse myelitis (TM), the latter two being examples of neuromyelitis optica spectrum disorder (NMOSD), test positive for the presence of epsilon toxin (Etx) produced by Clostridium perfringens type B or D strains and / or test positive for the presence of antibodies against aquaporin-4 (AQP4).

[0024] Demyelinating disorders are characterized by myelin damage and include disorders selected from: enterotoxaemia (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 disorder characterized by an increase or presence of antibodies to aquaporin-4 (AQP4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0025] According to a first aspect of the present invention, there is provided a method of preventing, treating or ameliorating the symptoms of a demyelinating disorder in a human or animal subject in need thereof, said disorder being selected from: enterotoxaemia (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 disorder characterized by an increase or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM). The method comprises administering to the subject a composition comprising an effective amount of an agent that directly or indirectly interferes with epsilon toxin (Etx), an Etx-binding receptor, or the interaction of Etx with its binding receptor produced by Clostridium perfringens type B or D strains, thereby inhibiting or repressing Etx-regulated receptor signaling activity.

[0026] According to a second aspect of the present invention, the composition comprises an agent that directly or indirectly interferes with epsilon toxin (ETX), an ETX-binding receptor, or the interaction of ETX with its binding receptor produced by Clostridium perfringens type B or D strains, thereby inhibiting or repressing Etx-regulated receptor signaling activity, said agent being a Clostridium perfringens epsilon toxin (Etx) polypeptide that has reduced toxicity to cells expressing myelin and lymphocyte (MAL) protein and comprises a modified domain III compared to the wild-type Etx polypeptide having sequence SEQ ID NO: 65, wherein said reduced toxicity is relative to SEQ ID NO: 65 and / or SEQ ID NO: 14, and wherein the Etx polypeptide is capable of binding to at least one antibody that binds to the sequence represented by SEQ ID NO: 65 and / or SEQ ID NO: 14.

[0027] In place of, or on the basis of, the ability to bind to an antibody that binds to SEQ ID NO: 13 and / or SEQ ID NO: 14, the polypeptide may bind to at least one antibody that binds to SEQ ID NO: 11 (epsilon toxin in the unactivated prototoxin form).

[0028] The modified domain III may be any modification in the glycosyl (beta-octyl-glucoside) binding site of domain III and / or in the sugar-binding ability of domain III, and / or a modification of domain III that results in the Etx polypeptide having a reduced ability to bind to CHO cells expressing MAL compared to the corresponding wild-type sequence when activated.

[0029] The modified domain III may include one or more amino acid mutations in the amino acid sequences (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) that make up domain III: VYVGKALLTNDTQQEQKLKSQSFTCK (SEQ ID NO: 1), THNVPSQDILVPANTTVEVIAYLK (SEQ ID NO: 2); and DELIVKVRNLNTNNVQEYVIPVDKKEKSNDSNIVKYRSLYIKAPGIK (SEQ ID NO: 3), where the mutations are substitutions or deletions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or substantially all amino acid residues in domain III (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3). When there are more than one mutation in domain III, the mutations can optionally be a combination of substitutions and deletions.

[0030] The modified domain III may include one or more of the following mutations in SEQ ID NO: 1 and SEQ ID NO: 2:

[0031] (SEQ ID NO: 1), where the underlined and bold V and F can be replaced with any other amino acid, or can be deleted. For example, V can be replaced with F (V[F]) and / or F can be replaced with A (F[A]);

[0032] (SEQ ID NO: 2), where the underlined and bold H, V, and A can be replaced with any other amino acid, or can be deleted. For example, H can be replaced with A (H[A]), V can be replaced with A (V[A]) and / or A can be replaced with F (A[F]).

[0033] The polypeptides of the present invention may include mutations at the positions shown in Table 1 below, which gives examples of suitable mutations in domain III (SEQ ID NO: 6 to SEQ ID NO: 10). The mutations shown in SEQ ID NO: 4 and SEQ ID NO: 5 are domain I mutations. Thus, the Etx polypeptides of the present invention may include one or more of the following sequences representing mutations in domain III: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10; or one or more mutations included in SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0034] Table 1: Mutations included in SEQ ID NO: 4 to SEQ ID NO: 10

[0035]

[0036]

[0037] Mutations included in any one or more of SEQ ID NO: 4 to SEQ ID NO: 10 may be included in a segment of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 255, 260, 261, 262, 263 contiguous amino acids from SEQ ID NO: 13, which includes one or more of SEQ ID NO: 4 to SEQ ID NO: 10.

[0038] The present invention is not necessarily limited to the specific mutations of Table 1, and any one or more mutations that affect the sugar-binding ability of Domain III and / or any one or more mutations that reduce the ability of the Etx polypeptide to bind to (CHO) cells expressing MAL equally constitute a part of the present invention. Those skilled in the art can readily determine suitable mutations using known tools and conventional techniques (such as those described herein).

[0039] As shown in Table 2 below, SEQ ID NO: 11 is the full-length wild-type Clostridium perfringens epsilon toxin and SEQ ID NO: 12 is the same sequence but lacks the first 32 amino acids. The sequence (SEQ ID NO: 12) is the sequence published for the crystal structure (see the Research Collaboratory for Structural Bioinformatics (RCSB) database, wwww.rcsb.org / PDB; PDB ID: 1UYJ).

[0040] SEQ ID NO: 65 is the trypsin-activated wild-type Clostridium perfringens epsilon toxin, which is the portion remaining after trypsin cleavage, with the N-terminus and C-terminus removed. SEQ ID NO: 11 and SEQ ID NO: 65 have 79% identity at the global alignment level as determined below.

[0041] SEQ ID NO: 13 is a recombinant toxin comprising SEQ ID NO: 65 and two additional amino acid residues at the N-terminus. SEQ ID NO: 14 is a sequence equivalent to SEQ ID NO: 13 but having an H>A mutation at position 151 of SEQ ID NO: 13 (referred to as the H149A mutation in the following examples, the difference in residue numbering is explained below). This is a variant of the activated toxin which can be studied in a containment level 2 ACGM laboratory (Oyston et al. (1998) Microbiol. vol. 144 (Pt 2), 333 - 341) and may thus be actually more convenient for determining antibody binding. Inclusion of the H149A mutation described herein will double ensure that the polypeptides according to the invention can be used at containment level 2 ACGM.

[0042] Table 2: Identity information of sequences

[0043] SEQ ID NO Identity information of the sequence 11 Full-length wild-type native ε-toxin 12 Sequence for obtaining the crystal structure (PDB ID: 1YUJ) 13 Trypsin-activated recombinant ε-toxin 14 Trypsin-activated recombinant ε-toxin with H149A mutation 15 Recombinant ε-toxin sequence 65 Trypsin-activated wild-type ε-toxin

[0044] The designations of the following mutation positions mentioned in Table 1 cited herein, namely 30, 196, 72, 92, 149, 166 and 168, are counted from position 1 of SEQ ID NO: 65. The same residue positions can be found in SEQ ID NO: 11 (counting from residue 46, i.e., SEQ ID NO: 65 lacks residues 1 - 32 (signal sequence) and residues 33 - 4's (N-terminal propeptide) of SEQ ID NO: 11). The same residue positions can be found in SEQ ID NO: 12 (counting from residue 14, i.e., SEQ ID NO: 65 lacks residues 1 - 13 (N-terminal propeptide) of SEQ ID NO: 12). The same residue positions can be found in SEQ ID NO: 13 and SEQ ID NO: 14 (counting from residue 3, i.e., SEQ ID NO: 65 lacks residues 1 - 2 (part of the synthetic signal sequence) of SEQ ID NO: 13 and SEQ ID NO: 14). The same residue positions can be found in SEQ ID NO: 15 (counting from residue 25, i.e., SEQ ID NO: 65 lacks residues 1 - 24 (synthetic signal sequence) of SEQ ID NO: 15). Those skilled in the art will be able to readily determine the mutations equivalent to those at positions 30, 196, 72, 92, 1's, 166 and 168 in any given Etx polypeptide.

[0045] The "Y30A - Y196A" and "Y43A - Y209A" double mutants mentioned herein are used interchangeably and refer to the same mutation positions in the wild - type Etx sequence (represented by SEQ ID NO: 65). The position numbers depend on whether the position is within the activated toxin or its inactive precursor. When the double mutant is referred to as Y30A - Y196A, positions 30 and 196 are counted from the start of the activated protein (position 1 of the sequence shown in SEQ ID NO: 65). When the double mutant is referred to as Y43A - Y209A, the equivalent positions 43 and 209 are counted from the start of the inactive precursor (i.e., starting from Figure 2 position - 13 of the sequence shown or from position 1 of SEQ ID NO: 12). The positions of the V72F, F92A, H149A, V166A, A168F mutations are as found in SEQ ID NO: 65.

[0046] In a previous study (WO2013 / 144636), it was demonstrated that the Y30A - Y196 double mutant (located in domain I) significantly reduced the ability of the toxin to bind to and kill MDCK cells and had reduced toxicity in mice, indicating that the Y30A - Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.

[0047] However, the previous study used the MDCK cell line to measure cytotoxicity. Surprisingly, the double mutant did not produce the same result of reduced toxicity in CHO cells expressing MAL. Now, it has been surprisingly found that modifying domain III of the Etx polypeptide (e.g., by introducing one or more mutations into domain III) reduces toxicity in CHO cells expressing MAL. The modification of domain III can be any modification described herein.

[0048] Thus, as described in WO2013 / 144636, the modification of domain III improves the known candidate vaccine Y30A - Y196. Most in vitro studies on Etx have been conducted using the MDCK cell line because this cell line is considered to be the most sensitive to the toxin (Payne et al. (1994) FEMS Microbiol. Lett. vol. 116, 161 - 167). Considering that Etx candidate vaccines may not have been tested on (CHO) cells expressing MAL, the present invention provides an opportunity to improve existing Etx vaccines and Etx candidate vaccines by modifying domain III as described herein.

[0049] The polypeptide according to the invention may comprise a modified domain III compared to the wild-type polypeptide SEQ ID NO: 65, and the polypeptide according to the invention shows reduced toxicity compared to the Etx polypeptide comprising SEQ ID NO: 4 and SEQ ID NO: 5 or compared to the Etx polypeptide comprising SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Herein, the "Etx polypeptide" may be a polypeptide having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% sequence identity with SEQ ID NO: 65 at the global level, and the polypeptide is capable of binding to at least one antibody that can bind SEQ ID NO: 65.

[0050] SEQ ID NO: 4 and SEQ ID NO: 5 are present in the Y30A - Y196A double mutant, but mutations of other tyrosine residues in domain I also show effective reduction of toxicity to MDCK cells. For example, one or more mutated tyrosine residues may be mutations (substitutions or deletions) of one or more of, for example, Y29, Y33, Y42, Y43, Y49 and / or Y209, where the residue numbers are counted starting from the beginning of the inactive precursor (i.e., starting from Figure 2 position -13 of the shown sequence or from position 1 of SEQ ID NO: 12). One or more tyrosine mutations may be included in a segment of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 255, 256, 257, 258, 259 or 260 consecutive amino acids from SEQ ID NO: 65.

[0051] According to the present invention, there is also provided an Etx polypeptide which has a modified domain III compared to the wild-type polypeptide SEQ ID NO: 65 and shows reduced toxicity compared to other Etx vaccine polypeptides and polypeptides used as candidate vaccines. For example, the following publications disclose polypeptides that show reduced toxicity to MCDK cells and can be further improved by modifying domain III: Kang J et al. (2017) Hum Vaccin Immunother 13: 1598-1608 describe a proposed vaccine with a mutation (F199) in domain 1; Yao et al. (2016) Sci Rep. 6: 24162 describe a Y196 mutation in domain 1; Li et al. (2013) Hum Vaccin 25 Immunother 9: 2386-92 describe F199E and H106P mutants (F199 is in domain 1, H106 is in domain 2); Oyston et al. (1998) Microbiology 144: 333-41 describe the H106P mutant and also mention that the H149P mutant is non-toxic (H106 is in domain 2); Dorca-Aré valo et al. (2014) PLoS One 9: e102417 describe that the V56C / F118C and H106P mutations are non-toxic to MDCK cells (V56 is in domain 2 and F118 is in domain 2).

[0052] It has also been surprisingly found that the Y30A-Y196 double mutant shows different toxicity results depending on the species from which the MAL is derived. For example, compared to wild-type Etx, the double mutant is only slightly less toxic to CHO cells expressing ovine MAL, but is more toxic to CHO cells expressing human MAL. However, in CHO cells expressing canine MAL, the toxicity of the mutant is significantly lower. This finding indicates that the interaction between MAL from different species and Etx is different, suggesting that MDCK cells may not be a good model for testing the toxicity of Etx candidate vaccines, while CHO cells expressing MAL may be a better model for this test. Accordingly, on the other hand, the present invention provides the use of (CHO) cells expressing MAL as a model in testing the toxicity of ε candidate vaccines.

[0053] Table 3 below shows examples of mutations introduced into domain III of the Y30A-Y196A double mutant.

[0054] Table 3: Further mutations of Y30A-Y196A

[0055]

[0056]

[0057]

[0058] Thus, the novel sequences described herein (SEQ ID NO: 18 to SEQ ID NO: 50 in Table 3) are genetic toxoids suitable for inclusion in next-generation vaccines against enterotoxemia or demyelinating diseases and against diseases caused by or associated with Clostridium perfringens and / or Etx. In particular, SEQ ID NO: 21 is suitable.

[0059] The mutations shown in Table 3 are described as being included in trypsin-activated recombinant Etx (SEQ ID NO: 13 or SEQ ID NO: 65), but the same mutations may also be included at equivalent positions in the full-length Etx polypeptide (SEQ ID NO: 11) or in the full-length recombinant Etx polypeptide (SEQ ID NO: 15); or the mutations shown in Table 3 may be included at equivalent positions in a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 65.

[0060] Any mutation described herein may be included in a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 65.

[0061] The polypeptides of SEQ ID NO: 18 to SEQ ID NO: 50 may have the same, similar or reduced toxicity as SEQ ID NO: 14 or SEQ ID NO: 14 lacking the first 2 N-terminal amino acid residues, and / or may be non-toxic.

[0062] The polypeptides according to the invention may comprise at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310 or 315 consecutive amino acids from any one of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 65, and comprise a double mutation at positions equivalent to positions 30 - 196 of SEQ ID NO: 65, and further comprise at least one, two or more mutations at positions equivalent to positions 72, 92, 149, 166 and 168. The numbering of the mutation positions is as described herein. The mutations may include substitutions or deletions.

[0063] The polypeptides according to the invention may comprise at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 261, 262 or 263 consecutive amino acids from any one of SEQ ID NO: 18 to SEQ ID NO: 50 in Table 3, and comprise or consist of the mutations indicated as the relevant SEQ ID NO in Table 3.

[0064] The polypeptides according to the invention comprise one of the amino acid sequences SEQ ID NO: 4 to SEQ ID NO: 10, resulting in the sequences comprised in the polypeptides being identical to the equivalent (i.e., corresponding) sequences from any one of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 65, except for the positions of the mutations designated as "X" in one of SEQ ID NO: 4 to SEQ ID NO: 10.

[0065] The polypeptide according to the present invention may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NO: 18 to SEQ ID NO: 50 in Table 3, and comprises or consists of the mutations indicated for the relevant SEQ ID NO.

[0066] The toxicity level can be determined in vitro or in vivo as described herein. For example, by using cell-based assays such as the LDH assay based on MDCK.2 cells, ACHN cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells expressing green fluorescent protein (GFP), human MAL (CHO-hMAL), sheep MAL (CHO-sMAL), canine MAL (CHO-dMAL), etc.). Toxicity studies can also be conducted in vivo, for example, in mice or any other suitable animal.

[0067] When administered to a subject such as a human or non-human animal (e.g., lamb, sheep, goat, pig, cow, horse or rabbit), the polypeptide of the present invention provides protection to the subject against diseases (such as enterotoxemia) caused by Clostridium perfringens infection and / or caused by the presence of active epsilon toxin (or associated with the presence of Clostridium perfringens or said toxin) and / or the occurrence of a demyelination-related disorder. Such protection can be partial, thereby reducing the probability of affected individual subjects in the population, or can be complete, whereby the subject will not develop the disease (i.e., the probability of developing a disease caused by or associated with Clostridium perfringens and / or Etx is 0%).

[0068] As used throughout this specification, the term "subject" in any aspect of the present invention refers to any individual human or animal, including (but not limited to) cats, dogs or horses, or ruminants, cows, sheep, goats or pigs. The human or animal can be a human or animal showing symptoms of a demyelinating disease 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 disorder characterized by an increase or presence of antibodies against aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0069] As further outlined below, sequence identity can be determined using the Needleman-Wunsch global sequence alignment tool, which is available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA, e.g., via http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using default parameter settings. When comparing the level of sequence identity to (e.g.) SEQ ID NO: 65, it should generally be done relative to the full length of SEQ ID NO: 65 to avoid short regions of high identity overlap that could result in a high overall assessed identity (i.e., using a global alignment method). For example, a short polypeptide fragment of, e.g., five amino acids might have 100% identical sequence to a five amino acid region in the entire SEQ ID NO: 65, but this does not provide 100% amino acid identity unless the fragment forms part of a longer sequence that also has the same amino acids at other positions equivalent to those in SEQ ID NO: 65. For example, SEQ ID NO: 11 is 79% identical to SEQ ID NO: 65 at the global level; positions 46 - 305 of SEQ ID NO: 11 are 100% identical to positions 1 - 260 of SEQ ID NO: 6, and positions 1 - 45 and 306 - 328 of SEQ ID NO: 11 are not present in SEQ ID NO: 65. SEQ ID NO: 65 is the sequence equivalent to (or corresponding to) positions 46 - 305 of SEQ ID NO: 11, and positions 46 - 305 of SEQ ID NO: are equivalent to (or corresponding to) SEQ ID NO: 65.

[0070] Thus, one of ordinary skill in the art can readily determine the equivalent positions between two sequences by aligning the sequences to maximize amino acid identity at as many positions as possible, e.g., by using a global sequence alignment program such as that available via http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, which will be discussed further below.

[0071] The present invention also encompasses polypeptides comprising the polypeptide variants and methods of using these variant polypeptides. As used herein, a "variant" refers to a polypeptide whose amino acid sequence is different from the underlying sequence from which it is derived, and in which one or more amino acids in that sequence have been replaced by other amino acids. The variant is a functional variant in that the functional characteristics of the polypeptide from which the variant is derived are maintained. For example, a variant polypeptide may have a similar ability to bind to an antibody that can bind to the non-variant polypeptide (e.g., as non-limiting examples, any of SEQ ID NOs: 18 to SEQ ID NO: 50 in Table 3). In particular, any amino acid substitution, addition, or deletion need not alter or need not significantly alter the tertiary structure of one or more epitopes contained within the polypeptide from which the variant is derived such that the variant polypeptide retains the ability to bind to an antibody that binds to SEQ ID NO: 65 or SEQ ID NO: 14. A person skilled in the art can readily identify suitable functional variants and determine the tertiary structure of the epitopes and any changes thereto without creative effort.

[0072] In cases where one amino acid is replaced by a different amino acid having substantially similar properties, the amino acid substitution can be considered "conservative". A non-conservative substitution is one in which an amino acid is replaced by a different type of amino acid.

[0073] "Conservative substitution" means that an amino acid is replaced by another amino acid of the same classification, where the classifications are defined as follows:

[0074]

[0075] As is well known to those skilled in the art, changing the primary structure of a polypeptide by conservative substitution may not significantly alter the activity of the polypeptide because the amino acid side chains in the inserted sequence may be able to form bonds and contacts similar to those of the replaced amino acid side chains. This is the case even when the substitution is in a critical region that determines the conformation of the polypeptide.

[0076] As noted above, non-conservative substitutions are possible provided that these substitutions do not disrupt the tertiary structure of the epitopes within the polypeptide, e.g., do not disrupt the immunogenicity (e.g., antigenicity) of the polypeptide.

[0077] Broadly speaking, fewer non-conservative substitutions are possible without altering the biological activity of the polypeptide. As noted above, suitably, the variant can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the underlying sequence.

[0078] As briefly mentioned already, sequence identity between amino acid sequences can be determined by comparing sequence alignments. When equivalent positions in the sequences being compared are occupied by the same amino acid, the molecules are identical at that position. Scoring an alignment as a percentage identity is a function of the number of identical amino acids at positions shared by the sequences being compared. When comparing sequences, the optimal alignment may require introducing gaps into one or more of the sequences to account for possible insertions and deletions in the sequences. Sequence comparison methods can employ gap penalties such that, for the same number of identical molecules in the sequences being compared, a sequence alignment with as few gaps as possible (reflecting a higher relatedness between the two sequences being compared) receives a higher score than a sequence alignment with many gaps. Calculation of the maximum percentage identity involves generating the optimal alignment taking into account the gap penalties. As described above, the Needleman-Wunsch global sequence alignment tool should be used to determine the percentage sequence identity, which is publicly available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using the default parameter settings. The Needleman-Wunsch algorithm was published in J. Mol. Biol. (1970) vol. 48: 443-553.

[0079] Another aspect of the invention provides a polynucleotide having a nucleic acid sequence encoding a polypeptide according to the first aspect of the invention. The invention also includes variant nucleic acids encoding the polypeptides of the invention. The term "variant" with respect to a nucleic acid sequence refers to any substitution, variation, modification, deletion, or addition of one or more nucleic acids to a polynucleotide sequence such that the resulting polypeptide sequence encoded by the polynucleotide exhibits at least the same properties as the polypeptide encoded by the basic sequence. Thus, the term includes allelic variants and also includes polynucleotides that hybridize substantially to the polynucleotide sequences of the invention ("probe sequences"). Such hybridization can occur under low stringency conditions and high stringency conditions or between low stringency conditions and high stringency conditions. Generally, low stringency conditions can be defined as hybridization where the wash step is carried out in a 0.330 M to 0.825 M NaCl buffer at a temperature 40 °C to 48 °C lower than the calculated or actual melting temperature (T m ) of the probe sequence (e.g., from about laboratory ambient temperature to about 55 °C), while high stringency conditions involve a wash in a 0.0165 M to 0.0330 M NaCl buffer at a temperature higher than the calculated T m or actual T mWashing at a temperature about 5°C to 10°C lower (e.g., about 65°C). The buffer solution can be, for example, SSC buffer (0.15 M NaCl and 0.015 M trisodium citrate). Low-stringency washing is carried out in 3× SSC buffer, and high-stringency washing is carried out in 0.1× SSC buffer. The steps involved in nucleic acid sequence hybridization have been described by, for example, Sambroket et al. (2001; "Molecular Cloning: a laboratory manual", 3rd Edition, Cold Spring Harbor Laboratory Press. 30 New York).

[0080] The polypeptides and nucleic acids of the present invention can be prepared by synthesis using a conventional synthesizer. Alternatively, they can be produced using recombinant DNA techniques, incorporated into a suitable expression vector, and then the appropriate host cells, such as prokaryotic cells like Escherichia coli (E. coli), are transformed with the expression vector. The transformed host cells are cultured and the polypeptides are isolated therefrom.

[0081] Accordingly, the present invention also provides a vector comprising such a polynucleotide. This includes recombinant constructs containing one or more of the above nucleic acid molecules. The constructs include vectors, such as plasmid vectors or viral vectors, into which the nucleic acid molecule of the present invention is inserted in the 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 sequence. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available. Sambrook et al. also described suitable cloning vectors and expression vectors for prokaryotic and eukaryotic hosts.

[0082] Another aspect of the present invention provides a cell comprising any one of the polypeptide, polynucleotide, or vector according to the present invention. For example, a suitable cell can be a Salmonella cell, such as a Salmonella enterica cell, which in some embodiments is from the serovar Typhimurium. The Salmonella can be a attenuated strain. Strains χ8914 and χ9241 can be optionally used. For example, a suitable system described by Kulkami et al. (2008, Vaccine vol. 26, 4194 - 4203). Preferably, the host cell is not a stem cell, especially not a human stem cell, such as a human embryonic stem cell.

[0083] Another aspect of the present invention provides an affinity reagent that can bind to one of the polypeptides according to the first aspect of the present invention and promote an immune response in an individual to whom the affinity reagent is administered. For example, the affinity reagent can be an antibody, which can be a monoclonal antibody or a synthetic antibody. A molecule or other antibody mimetic, aptamer, protein scaffold, or major histocompatibility complex (MHC) protein or a portion thereof. The affinity reagent can be an antibody generated against the polypeptide according to the first aspect of the present invention.

[0084] Another aspect of the present invention provides a subunit vaccine or conjugate vaccine comprising the polypeptide according to the first aspect of the present invention. For example, it can be in the form of a fusion protein and / or in the form of a recombinant viral vaccine.

[0085] Another aspect of the present invention provides a method for preparing a vaccine, comprising altering an Etx polypeptide or a vaccine or vaccine candidate comprising an Etx polypeptide by modifying domain III relative to the wild-type Etx polypeptide SEQ ID NO: 65. Another aspect provides a vaccine composition prepared by a method comprising the above method.

[0086] A vaccine or vaccine candidate for improvement (by modification of domain III compared to the wild-type polypeptide) can be selected from any of the following, which shows a reduced toxicity after modification of domain III compared to the original vaccine or vaccine candidate. For example, the following publications disclose polypeptides that show reduced toxicity to MCDK cells and can be further improved by modifying domain III: Kang J et al. (2017) Hum Vaccin Immunother 13: 1598 - 1608 describes a proposed human vaccine having a mutation (F199) in domain 1; Yao et al. (2016) Sci Rep. 6: 24162 describes a Y196 mutation in domain 1; Li et al. (2013) Hum Vaccin 25 Immunother 9: 2386 - 92 describes F199E and H106P mutants (F199 in domain 1, H106 in domain 2); Oyston et al. (1998) Microbiology 144: 333 - 41 describes the H106P mutant and also mentions that the H149P mutant is non-toxic (H106 in domain 2); Dorca - Aré valo et al. (2014) PLoS One 9: e102417 describes that the V56C / F118C and H106P mutations are non-toxic to MDCK cells (V56 in domain 2 and F118 in domain 2).

[0087] Another aspect of the present invention provides an immunotherapeutic composition, which comprises: a polypeptide, a polynucleotide, a vector, an affinity reagent, a subunit vaccine, and / or a conjugate vaccine according to the foregoing aspects of the present invention, in the form of a pharmaceutically acceptable preparation. For example, the immunotherapeutic composition may be a vaccine composition comprising a polypeptide and an adjuvant according to the first aspect of the present invention. The composition may further comprise excipients and / or diluents suitable for administering the composition to a subject in need of treatment or vaccination against a disease caused by Clostridium perfringens and / or Etx disease. Selecting appropriate components is within the routine capabilities of those skilled in the art without undue experimentation.

[0088] For example, the immunotherapeutic compositions of the present invention can be conveniently formulated using pharmaceutically acceptable excipients or diluents such as aqueous solvents, non-aqueous solvents, non-toxic excipients (such as salts), preservatives, buffers, etc. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate. Aqueous solvents include water, alcohol / water solutions, salt solutions, parenteral carriers (such as sodium chloride, Ringer's dextrose), etc. Preservatives include antimicrobial agents, antioxidants, chelating agents, and inert gases. The pH and exact concentration of the various components in the vaccine composition are adjusted according to conventional techniques.

[0089] Optionally, the immunotherapeutic formulation may comprise a carrier. Commonly used carrier molecules are bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin, mouse serum albumin, rabbit serum albumin, etc. Synthetic carriers can be used and are readily available. Methods for conjugating peptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and diazotized benzidine.

[0090] In certain cases, it may also be necessary to formulate an immunotherapeutic composition comprising an adjuvant to enhance the immune response. Such adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions. Commonly used adjuvants include aluminum hydroxide, aluminum phosphate, calcium phosphate, Freund's adjuvant, and Quil-A saponin. An adjuvant provided by SEPPIC Inc (New Jersey, USA) as Montanide TM adjuvant, such as Montanide TM ISA 61VG, may also be a suitable adjuvant. The inventors have found that, compared to using an aluminum hydroxide (alhydrogel) adjuvant, using Montanide TMThe ISA 61VG adjuvant results in the induction of a better antibody response. In addition to the adjuvant, it may also be necessary to co-administer a biological response modifier (BRM) with the peptide or variant or derivative to down-regulate the activity of inhibitory T cells.

[0091] Possible carriers for the administration of immunotherapeutic agents include liposomes. Liposomes are microscopic vesicles composed of one or more lipid bilayers surrounding an aqueous compartment. Liposomes are similar in composition to cell membranes and, therefore, can generally be administered safely and are biodegradable. Techniques for preparing liposomes and for formulating (e.g., encapsulating) various molecules, including peptides and oligonucleotides, with liposomes are well known.

[0092] Depending on the method of preparation, liposomes can be unilamellar or multilamellar and can vary in size, with diameters ranging from 0.02 μm to greater than 10 μm. Liposomes can also adsorb to almost any type of cell and then release the encapsulated agent. Alternatively, liposomes fuse with the target cell, thereby emptying the contents of the liposome into the target cell. Or, the adsorbed liposomes can be endocytosed by phagocytes. Following endocytosis is lysosomal degradation of the liposome lipids and release of the encapsulated agent. In the present invention, the polypeptides can be located on the surface of the liposomes to facilitate antigen presentation without disrupting the liposomes or endocytosis. However, regardless of the mechanism or delivery, the result is an intracellular disposition of the relevant polypeptides.

[0093] Liposome carriers can be anionic or cationic. Anionic liposome carriers include pH-sensitive liposomes that disrupt or fuse with the endosomal membrane following endocytosis and endosomal acidification. Cationic liposomes are preferably used to mediate in vitro mammalian cell transfection or general delivery of nucleic acids, but can also be used to deliver other therapeutic agents, such as peptides.

[0094] Other suitable liposomes for the methods of the present invention include multilamellar liposomes (MLV), oligolamellar liposomes (OLV), unilamellar liposomes (UV), small unilamellar liposomes (SUV), medium-sized unilamellar liposomes (MIN), large unilamellar liposomes (LUV), giant unilamellar liposomes (GUV), porous liposomes (MVV), unilamellar or oligolamellar liposomes prepared by the reverse-phase evaporation method (REV), multilamellar liposomes prepared by the reverse-phase evaporation method (MLV-REV), stable multilamellar vesicles (SPLV), freeze-thawed MLV (FATMLV), liposomes prepared by the extrusion method (VET), liposomes prepared by the French press (FPV), liposomes prepared by fusion (FUV), dehydrated-rehydrated liposomes (DRV), and bubblesomes (BSV). Techniques for preparing these liposomes are well known in the art.

[0095] Other forms of delivery particles, such as microspheres, etc., are also considered for the delivery of peptide epitopes or multi-epitopes.

[0096] Alternatively, nucleic acid-based vaccines can be produced that contain nucleic acids encoding immunologically active peptide epitopes or multi-epitopes and cloned into a suitable vector (such as a vector of vaccinia, canarypox, adenovirus or other eukaryotic virus), such as DNA or RNA.

[0097] Alternatively, polypeptides can be administered in the form of a cellular vaccine by administering autologous or allogeneic APC cells or dendritic cells that have been processed in vitro so as to present the peptide on their surface. Mutations carried by Salmonella enterica or Escherichia coli strains that reduce their virulence and allow them to colonize in a host animal without causing disease can be used to deliver vaccine antigens, particularly for administration to non-human animals. The bacteria used may include strains that have been used as vaccines in livestock, where the attenuating lesions have not been fully characterized. In addition, as described in WO2013 / 144636, strains in which virulence has been rationally attenuated by deliberately introducing mutations into the bacteria can be used to deliver polypeptides. Antigens can also be delivered as a naked DNA vaccine, where the gene encoding the epsilon toxin is cloned into a mammalian expression vector and expressed from a eukaryotic promoter.

[0098] One of the most widely studied classes of attenuated Salmonella used as a carrier for foreign antigens is auxotrophs. Genetically defined mutants of the aroA gene encoding 5-enolpyruvyl-3-phosphate synthase have been constructed in Salmonella enterica var. Typhimurium and Salmonella enterica var. Typhi. These mutants are attenuated and immunogenic in mice. Examples of other auxotrophic mutants include Salmonella with deletions in genes involved in the purine biosynthetic pathway. Another well-studied group of attenuated Salmonella are mutants with defined deletions in genes involved in Salmonella virulence regulation. For example, mutations in the genes encoding adenylate cyclase (CyA) and cyclic AMP receptor protein (CRP) affect the expression of the genes involved.

[0099] If the vaccine composition is for administration to a human subject, it can be in a form suitable for oral administration (such as in a dietary supplement) and / or parenteral administration (such as by injection, inhalation or transdermal administration via a patch, lotion or gel). The specific forms outlined above can generally also be used for administration to human subjects.

[0100] The polypeptides, polynucleotides, vectors, subunit vaccines, conjugate vaccines, antibodies, affinity reagents, vaccine compositions and / or immunotherapeutic compositions according to the invention can be used in methods for treating or vaccinating a subject against a disease caused by Clostridium perfringens and / or Etx, such as a disease involving the accumulation in the subject's blood of epsilon toxin (in particular the active epsilon toxin lacking the N-terminal and C-terminal ends of the full-length protoxin) that can be released by Clostridium perfringens; and / or the polypeptides, polynucleotides, vectors, subunit vaccines, conjugate vaccines, antibodies, affinity reagents, vaccine compositions and / or immunotherapeutic compositions according to the invention can be used in methods for treating or vaccinating a subject against demyelination-related disorders. The polypeptides, polynucleotides, vectors, subunit vaccines, conjugate vaccines, antibodies, affinity reagents, vaccine compositions and / or immunotherapeutic compositions according to the invention can be used in methods for treating or vaccinating a subject against a disease associated with Clostridium perfringens infection or with the presence of Etx or for the occurrence of a demyelination-related disorder. According to one embodiment, the disease is enterotoxemia or a demyelinating disease, such as multiple sclerosis, neuromyelitis optica (NMO), optic neuritis (ON) or myelitis.

[0101] The invention also provides a method for vaccinating a subject against a disease caused by Clostridium perfringens and / or caused by epsilon toxin (in particular the active toxin) or a disease associated with Clostridium perfringens infection or with the presence of Etx, or for the occurrence of a demyelination-related disorder, the method comprising administering to the subject a polypeptide, polynucleotide, vector, subunit vaccine, conjugate vaccine, affinity reagent, vaccine composition and / or immunotherapeutic agent according to the invention (e.g., in a protective amount). The subject can be a human or a non-human animal. The non-human animal can be a horse or a ruminant (e.g., sheep, pig or goat), or a bovine (e.g., domestic cattle) or a companion animal (e.g., dog, cat or rabbit). Young animals, such as lambs, piglets, children and calves are also included.

[0102] A "protective amount" is an amount sufficient to induce an immune response in the subject, thereby reducing or eliminating the probability that the subject will develop a disease caused by Clostridium perfringens (e.g., caused by epsilon toxin, in particular the active toxin (or associated with its presence)). For example, antibodies capable of binding to SEQ ID NO: 65 and / or SEQ ID NO: 14 can be detectable after administration, where such antibodies are not detectable before administration or are only detectable at a lower concentration than after administration.

[0103] The present invention also provides a kit comprising a polypeptide, polynucleotide, vector, subunit vaccine, conjugate vaccine, affinity reagent, vaccine composition, and / or immunotherapeutic reagent according to the present invention, said kit having use, for example, in a method of treating or vaccinating a subject against a disease caused by and / or infection with Clostridium perfringens and / or Etx; or said kit having use in a method of treating or vaccinating a subject against a disease associated with Clostridium perfringens infection or a disease associated with the presence of Etx or the occurrence of a demyelination-related disorder. The kit may include means for administering the polypeptide, polynucleotide, vector, subunit vaccine, conjugate vaccine, affinity reagent, vaccine composition, and / or immunotherapeutic reagent to an individual. For example, the kit may include one or more buffers or diluents and / or one or more administering devices, such as a syringe or other injection device. The kit may alternatively or additionally include instructions that enable a user to implement a method of treating or vaccinating a subject against a disease caused by and / or associated with Clostridium perfringens and / or Etx and / or a demyelination disease, said diseases being, for example, 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 disorder characterized by an increase or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0104] As described herein, a disease caused by and / or associated with Clostridium perfringens and / or (active) epsilon toxin may be, for example, enterotoxemia, including pre-disease symptoms (such as systemic lesions and enterocolitis). Other symptoms may include edema of the major target organs, the kidney and the brain, and damage to vascular endothelial cells. The terminal stage of enterotoxemia is characterized by severe neurological diseases, including opisthotonus, seizures, and agonizing struggles. The disease may be a demyelination disease, such as 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 disorder characterized by an increase or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0105] In the description and claims of the present application text, the words "comprise" and "include" and variations of those words, such as "comprising" and "comprises", mean "including but not limited to" and do not exclude other parts, additives, components, integers or steps.

[0106] In the description and claims of the present application text, the singular includes the plural unless the context otherwise requires. In particular, in the case of using an indefinite article, unless the context otherwise requires, the present application text should be understood to contemplate both the plural and the singular.

[0107] Preferred features of each aspect of the present invention may be as described in connection with any other aspect.

[0108] Other features of the present invention will become apparent from the following examples. In general, the present invention extends to any new feature or any new combination of features disclosed in the present application text (including the appended claims and drawings). Accordingly, features, integers, characteristics, compounds or chemical moieties described in connection with a particular aspect, embodiment or example of the present invention should be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0109] Furthermore, unless otherwise stated, any feature disclosed herein may be replaced by an alternative feature for the same or a similar purpose. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Reference will now be made to Figures 1 to 5 describe embodiments of the present invention by way of example only, wherein:

[0111] Figure 1 is a schematic diagram of recombinant epsilon-protoxin (P-Etx), in which the N-terminal PelB leader peptide replaces the 13-amino acid N-terminal peptide sequence and has a C-terminal His-tag to assist in purification (the amino acid sequence around the processing site is also shown);

[0112] Figure 2 shows the Etx amino acid sequence, which has a signal sequence (residues 1-32), an N-terminal propeptide (residues 33-45), a C-terminal propeptide (residues 305-328), and the positions of the mutated residues Y30A (Y43), V72f, F92a, H129a, V166a, A168f, Y196A (Y209), with the original unmutated amino acids shown below the mutated positions;

[0113] Figure 3AShows five residues (proposed secondary receptor binding sites) flanking the glycan (β-octyl-glucoside) binding site in Etx domain III for mutagenesis identification: V72, F92, H149, V166, A168. Positions of previously mutated residues (shown in dark gray and circled (top); Y30 and Y196) and residues in the β-octyl-glucoside binding cleft (V72, F92, H149, V166, A168; shown in light gray and circled (bottom);

[0114] Figure 3B Shows an SDS-PAGE gel of the purified proteins tested in this study. Using a plasmid encoding the Y30A - Y196A variant form of Etx, additional mutations V72, F92, H149, V166, and A168 were introduced. These residues were mutated to alanine (H149, F92, V166) or phenylalanine (A168, V72), and the His-tagged proteins encoded by the mutant genes were expressed and purified in E. coli;

[0115] Figure 4 Shows the ability of WHO standard antitoxin (5 IU / ml) or rabbit serum against genetic toxoid to displace the binding of neutralizing monoclonal antibodies in a competitive ELISA. The sera were diluted as shown in the legend before testing. The data shown are the mean of two assays, with SEM bars shown.

[0116] Figure 5 Shows the melting temperature (T m ) of wild-type and variant proteins. The thermal stability of Etx protoxin was determined by the Boltzmann method using Protein Thermal Shift software (Applied Biosystems). The results represent the mean and standard deviation of three samples.

[0117] Figure 6 Shows the results of treating human red blood cells with ε-toxin (wild-type Etx; Y30AY196A; Y30AY196A + H149A; Y30AY196A + A168F; Y30AY196A + F92A; Y30AY196A + V166A; and quadruple mutant (quad): Y30AY196 + A168F + H149A). Y30AY196A + A168F, Y30AY196A + H149A, and the quadruple mutant do not cause hemolysis even when activated by trypsin.

[0118] Item

[0119] The present invention will now be described with reference to the following items, in which:

[0120] 1. A method for preventing or treating demyelinating disorders in a human or animal subject in need thereof, said disorders selected from: 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), diseases or disorders characterized by increased or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM), comprising: administering to said subject a composition comprising an effective amount of an agent that directly or indirectly interferes with epsilon toxin (Etx), an EtX binding receptor, or the interaction of Etx with its binding receptor produced by Clostridium perfringens type B or D strains, thereby inhibiting or repressing Etx-regulated receptor signaling activity.

[0121] 2. The method according to item 1, wherein said agent is an inhibitor of Etx, such as an antibody or a functional component thereof.

[0122] 3. The method according to item 1, wherein said agent is an inhibitor or antagonist of an Etx binding receptor.

[0123] 4. The method according to item 3, wherein said Etx binding receptor is myelin and lymphocyte protein (MAL) or hepatitis A virus cellular receptor 1 protein (HAVCR1).

[0124] 5. The method according to item 1, wherein said agent is a vaccine against Clostridium perfringens type B or D strains or against epsilon toxin (Etx) produced by them.

[0125] 6. The method according to any one of the foregoing, wherein said agent comprises an epsilon toxin (Etx) polypeptide that has reduced toxicity to cells expressing myelin and lymphocyte protein (MAL) and comprises a modified domain III compared to the wild-type Etx polypeptide SEQ ID NO: 65, wherein said reduced toxicity is relative to SEQ ID NO: 65 and / or SEQ ID NO: 14, and wherein said Etx polypeptide is capable of binding to at least one antibody that binds to the sequence represented by SEQ ID NO: 65 and / or SEQ ID NO: 14.

[0126] 7. The method according to item 6, wherein said modified domain III is a modification at the glycan (β-octyl-glucoside) binding site of domain III.

[0127] 8. The method according to item 6 or 7, wherein the modified domain III comprises one or more mutations of the amino acids in the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 that constitute domain III.

[0128] 9. The method according to any one of items 6 to 8, which comprises one or more of the following: SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0129] 10. The method according to any one of items 6 to 9, which comprises SEQ ID NO: 4 and / or SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0130] 11. The method according to any one of items 6 to 10, which comprises at least the following sequences:

[0131] i) SEQ ID NO: 4 and SEQ ID NO: 5;

[0132] And optionally in addition to (i), also comprises

[0133] ii) SEQ ID NO: 6; and

[0134] iii) one or more of the following: SEQ ID NO: 10, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0135] 12. The method according to any one of items 6 to 11, wherein the reduced toxicity is reduced compared to an Etx polypeptide comprising SEQ ID NO: 4 and SEQ ID NO: 5, or an Etx polypeptide comprising SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or compared to a known Etx vaccine or Etx vaccine candidate.

[0136] 13. The method according to any one of items 6 to 12, having at least 60% sequence identity with any one of SEQ ID NOs: 18 to 50 and comprising or consisting of one or more mutations indicated in Table 3 for the relevant SEQ ID NO.

[0137] 14. A polynucleotide having a nucleic acid sequence encoding a polypeptide according to any one of the foregoing.

[0138] 15. A vector comprising the polynucleotide according to item 14 or the agent according to any one of items 1 - 5.

[0139] 16. A cell comprising the agent according to any one of items 1 to 5, the polypeptide according to any one of items 6 to 13, and / or the polynucleotide according to item 14, and / or the vector according to item 15.

[0140] 17. A subunit vaccine or conjugate vaccine comprising the agent according to any one of items 1 - 5 and the polypeptide according to any one of items 6 - 13.

[0141] 18. An affinity reagent capable of binding to one of the agent according to any one of items 1 to 5 and the polypeptide according to any one of items 6 to 13, and promoting an immune response in an individual to whom the affinity reagent has been administered.

[0142] 19. A method for preparing an immunotherapeutic composition, optionally a vaccine composition, comprising altering an Etx polypeptide or a vaccine comprising an Etx polypeptide by modifying domain III relative to the wild - type Etx polypeptide.

[0143] 20. An immunotherapeutic composition or vaccine composition prepared by the method according to item 19.

[0144] 21. An immunotherapeutic composition or vaccine composition comprising the agent according to any one of items 1 to 5, the polypeptide according to any one of items 6 to 13, and / or the polynucleotide according to item 14, and / or the vector according to item 15, and / or the cell according to item 16, and / or the subunit vaccine according to item 17, and / or the affinity reagent according to item 18.

[0145] 22. Use of the agent according to any one of items 1 to 5, the polypeptide according to any one of items 6 to 13, and / or the polynucleotide according to item 14, and / or the vector according to item 15, and / or the cell according to item 16, and / or the subunit vaccine or conjugate vaccine according to item 17, and / or the affinity reagent according to item 18, and / or the immunotherapeutic composition or vaccine composition according to any one of items 20 to 21, for treating or vaccinating a subject against a disease caused by or associated with Clostridium perfringens and / or a disease caused by or associated with (active) epsilon - toxin and / or for use in a method of counteracting a demyelinating disease.

[0146] 23. The polypeptide according to item 22, wherein the disease is selected from: 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 disorder characterized by an increase or presence of antibodies against aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0147] 24. A method for treating a subject suffering from a disease caused by or associated with the presence of Clostridium perfringens and / or a disease caused by or associated with the presence of (active) epsilon toxin and / or for treating a subject against a demyelinating disease, or a method for vaccinating a subject against developing said disease, the method comprising administering to the subject an agent according to any one of items 1 to 5, a polypeptide according to any one of items 6 to 13, and / or a polynucleotide according to item 14, and / or a vector according to item 15, and / or a cell according to item 16, and / or a subunit vaccine according to item 17, and / or an affinity reagent according to item 18, and / or a vaccine composition or immunotherapeutic composition according to any one of items 20 to 21, wherein the demyelinating disorder is selected from: 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 disorder characterized by an increase or presence of antibodies against aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0148] 25. The agent, polypeptide, polynucleotide, vector, cell, affinity reagent, vaccine composition or immunotherapeutic composition according to any one of items 20 to 21, or the method according to item 24, wherein the subject is a ruminant, a horse, a companion animal or a human.

[0149] 26. Use of MAL cells as a model in testing the toxicity of an epsilon candidate vaccine.

[0150] 27. A kit, which comprises an agent according to any one of items 1 to 5, a polypeptide according to any one of items 6 to 13 and / or a polynucleotide according to item 14 and / or a vector according to item 15 and / or a cell according to item 16 and / or a subunit vaccine according to item 17 and / or an affinity reagent according to item 18 and / or a vaccine composition or an immunotherapeutic composition according to any one of items 20 to 21.

[0151] 28. A polypeptide, polynucleotide, vector, cell, subunit vaccine, conjugate vaccine, affinity reagent, vaccine composition or immunotherapeutic composition or method substantially as described herein.

[0152] Example

[0153] Materials and Methods

[0154] Chemicals

[0155] All chemicals were obtained from Sigma Chemical Co. (St Louis, MO) unless otherwise specified.

[0156] Expression and purification of recombinant epsilon toxin wild-type and mutants

[0157] The etxD gene encoding ε-protoxin D from Clostridium perfringens type D strain NCTC 8346 was cloned into the expression vector pET-26b(+) (Merck, Darmstadt, Germany), where the N-terminal PelB leader peptide replaced the 13-amino acid N-terminal peptide sequence (residues KEISNTVSNEMSK), and had a C-terminal polyhistidine (6×His) tag to facilitate affinity purification of the recombinant protoxin (Bokori-Brown et al., (2013) Protein science: a publication of the Protein Society 22: 650-9). Amino acid numbering corresponds to the protoxin without the 13-amino acid N-terminal peptide sequence. Using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc. Santa Clara, US), the mutations H149A, A168F, F92A, V166A, and V72F (residues flanking the β-octyl-glucoside binding site) were introduced into the previously generated Y30A-Y196A mutant (Bokori-Brown et al. (2014) Vaccine 32: 2682-7) according to the manufacturer's instructions (amino acid numbering corresponds to the protoxin without the N-terminal peptide sequence). The primers used for site-directed mutagenesis are shown in Table 4 below. The recombinant protein was expressed in Escherichia coli Rosetta 2(DE3) cells (Merck, Darmstadt, Germany) and grown in ZYM-5052 autoinduction medium (Studier FW (2005) Protein expression and purification 41: 207-34) supplemented with 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. Cells (100 mL) were grown at 37 °C for 3 h and then incubated at 20 °C and 300 rpm for an additional 24 h.

[0158] Table 4 - Primers for site-directed mutagenesis

[0159]

[0160]

[0161] a The underlined bases are the codons for replacement. All primer sequences are shown in the 5′ to 3′ direction. Amino acid numbering corresponds to the protoxin without the N-terminal peptide sequence.

[0162] Protein purification

[0163] For purified proteins, cells were collected by centrifugation and lysed enzymatically using BugBuster TM Protein Extraction Reagent (Merck, Darmstadt, Germany), and Y30A - Y196A and its derivatives were purified by Ni - NTA chromatography column (GE Healthcare Life Sciences, Little Chalfont, UK) according to the manufacturer's instructions. For buffer exchange and further sample purification, the eluate containing the protoxin was applied to a PD - 10 desalting column (GE Healthcare Life Sciences, Little Chalfont, UK) and eluted with 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM NaCl (pH 7.4). Protein concentration was determined using BCA assay (Fisher Scientific UK Ltd, Loughborough, UK).

[0164] For studies in mice, rabbits or sheep, the proteins were treated using a commercially available kit (Pierce TM High Capacity Endotoxin Removal Spin Columns, Thermo Scientific) containing high - capacity endotoxin - removing resin according to the manufacturer's instructions. Residual levels of endotoxin were measured using a quantitative chromogenic assay (Pierce LAL Chromogenic Endotoxin Quantitation kit, Thermo Scientific).

[0165] Trypsin activation

[0166] Purified recombinant ε-protoxin and its derivatives were activated with TPCK - treated trypsin from bovine pancreas (Sigma - Aldrich Company Ltd., Gillingham, UK) to remove the C - terminal peptide sequence. Trypsin was prepared in PBS and added to recombinant P - Etx at a ratio of 1:100 (weight / weight) and incubated at room temperature for 1 hour. A protease inhibitor mixture without EDTA (Fisher Scientific UK Ltd, Loughborough, UK) was added to the digest to inhibit trypsin in the sample. Removal of the C - terminal peptide sequence was evaluated by SDS - PAGE.

[0167] SDS - PAGE analysis

[0168] Using XCell SureLockTM Protein purity was analyzed by SDS-PAGE on 4-12% Bis-Tris NuPAGE gels (Invitrogen Ltd., Paisley, UK) using a Mini-Cell electrophoresis apparatus (Invitrogen Ltd., Paisley, UK) and NuPAGE MESSDS running buffer (Invitrogen Ltd., Paisley, UK). All samples were heated at 70°C for 10 minutes in NuPAGE LDS sample buffer (Invitrogen Ltd., Paisley, UK). Gels were typically run at 200V for 45 minutes. After electrophoretic separation, proteins were visualized by staining with SimplyBlue (Invitrogen Ltd., Paisley, UK). Perfect Protein Molecular Weight Standards (Merck, Darmstadt, Germany) were used as markers.

[0169] Thermal stability test

[0170] Thermal stability was assessed by mixing purified protein (0.25 mg / mL) with 240× SYPRO Orange protein gel stain (Sigma-Aldrich Company Ltd., UK). Fluorescence was monitored using a StepOnePlus quantitative PCR instrument (Applied Biosystems, US) with a 1% thermal gradient from 25°C to 99°C. The obtained fluorescence data were analyzed using Protein Thermal Shift software (Applied Biosystems) to calculate the melting temperature (T) using the Boltzmann method. m ). All measurements were performed three times.

[0171] Cell culture

[0172] MDCK.2 cells (ATCC-LGC Standards. Teddington, UK) and ACHN cells (ECACC, Salisbury, UK) were routinely cultured in Eagle's Minimum Essential Medium (EMEM; ATCC-LGC Standards, Teddington, UK) supplemented with 10% premium fetal bovine serum (PAA, Pasching, Austria) at 37°C in a humidified atmosphere of 95% air / 5% CO2. The medium was changed every 2 to 3 days. The cells were routinely detached by incubation in trypsin / EDTA and split appropriately (usually at a 1:6 dilution).

[0173] Chinese hamster ovary (CHO) cells expressing green fluorescent protein (GFP)-tagged human MAL (CHO-hMAL), sheep MAL (CHO-sMAL), or canine MAL (CHO-dMAL), or CHO cells were routinely cultured in Dulbecco's Modified Eagle's Medium / Ham's F12 (DMEM / F12) medium (Life Technologies) supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 95% air / 5% CO2. The medium was changed every 2 to 3 days. Cells were routinely detached by incubation in trypsin / EDTA and split appropriately (usually at a 1:6 dilution).

[0174] Cytotoxicity assay

[0175] The cytotoxicity of trypsin-activated toxins against MDCK.2 cells was determined by measuring the amount of lactate dehydrogenase (LDH) released from the cytosol of lysed cells into the cell culture medium using the CytoTox96 non-radioactive cytotoxicity detection kit (Promega UK, Southampton, UK) according to the manufacturer's protocol. Briefly, a two-fold dilution series of each activated toxin (ranging from 10 μM to 0.15 nM) was prepared in PBS and added to cells seeded in 96-well plates (3 × 10 4 cells / well). After incubation at 37 °C for 3 h, the cell culture medium (50 μL) was harvested from the cell monolayer, transferred to a fresh 96-well microtiter plate, and 50 μL of the reconstituted substrate mixture was added to each well. The plate was incubated in the dark at room temperature for 30 min. Absorbance was read at 490 nm using a Model 680 microplate reader (Bio-Rad). The absorbance value of each sample was normalized by subtracting the absorbance value obtained from the medium of untreated cells. The toxin dose required to kill 50% of the cell monolayer (CT 50 ) was determined by non-linear regression analysis (GraphPad). All experiments were performed three times with three technical replicates each.

[0176] Immunization of rabbits

[0177] Multiple groups (three rabbits per group) of New Zealand white rabbits were immunized subcutaneously with 100 μg of Y30A-Y196A, Y30A-Y196A-A168F, or Y30A-Y196A-H149A-A168F (Cambridge Research Biochemicals). Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for 4 subsequent immunizations at 14-day intervals. Blood was collected 7 days after the 3rd booster immunization (day 49) and 7 days after the 4th booster immunization (day 63).

[0178] The WHO international standard Clostridium perfringens epsilon antitoxin serum (CPEPAT) was obtained from the National Institute for Biological Standards and Control (NIBSC, South Mimms, UK).

[0179] Lambs were immunized with Y30AY196A+A168F toxoid

[0180] Lambs were raised without immunization against Clostridium perfringens epsilon toxin vaccine, and the presence of anti-epsilon toxin antibodies was tested by Western blotting at intervals. After 12 months, a group of 5 lambs received no treatment, a group of 6 lambs received 200 μg of Y30AY196A+A168F toxoid (1:1) containing Montanide TM ISA 61VG (Seppic, Paris, France) adjuvant, and a group of 5 lambs received 200 μg of Y30AY196A+A168F toxoid containing aluminum hydroxide (alhydrogel; Sigma-Aldrich, Poole UK; 0.25% weight / volume final concentration) adjuvant. The inventors also immunized a group of 4 lambs with a mixture of 200 μg of Y30AY196A+A168F toxoid and Montanide TM ISA 61VG adjuvant (1:1), which had a significant level of pre-existing reactivity with epsilon toxin. All adjuvant-containing mixtures were administered subcutaneously in 6×0.5 ml doses. Three weeks later, the lambs were given a second dose of adjuvant-containing protein. Blood samples were collected at the start of the study and at weeks 3, 7, and 12. The immunization of the lambs was carried out by Orygen Antibodies Ltd, Penicuik, Scotland.

[0181] Competitive ELISA assay for measuring neutralizing antibodies

[0182] Competitive ELISA was performed to measure neutralizing antibodies using a Monoscreen ELISA kit (BioX Diagnostics, BIO K222 / 2) according to the manufacturer's instructions. Absorbance was read at 450 nm, and inhibition was calculated using the following formula:

[0183] Inhibition sample % = [(OD 450nm negative serum - OD sample) / OD 450nm negative serum] × 100

[0184] Inhibition positive % = [(OD 450nm negative serum - OD positive serum) / OD 450nm negative serum] × 100

[0185] The test is only valid when OD negative - OD positive is greater than 0.7 and the inhibition of the positive control is greater than 30%.

[0186] Neutralization of cytotoxicity to cell cultures

[0187] CHO cells expressing human MAL were seeded at 3×10 4 cells per well in a 96-well plate and allowed to stand overnight. Pure rabbit sera and 20 IU / ml standard ε-antitoxin (National Institute for the Control of Pharmaceutical and Biological Products) were serially diluted in DPBS and incubated with an equal volume of trypsin-activated wild-type ε-toxin (5×CT 50 ) for 1 hour at room temperature. CHO hMAL cells were washed twice with serum-free DMEM / F12 and then the toxin:antibody / standard antitoxin mixtures were added, together with controls of only DPBS and only toxin (5×CT 50 ). After incubation for 3 hours in a humidified atmosphere at 37 °C, the medium was replaced with 100 μl of fresh serum-free DMEM / F12 and 10 μl of WST-1 cell proliferation reagent (Abcam). The absorbance at 420 nm was read after incubation for 1 hour in a humidified atmosphere at 37 °C.

[0188] Toxicity in mice

[0189] Multiple groups (six per group) of female BALB / c mice were challenged with 100 μl volumes of unactivated or trypsin-activated protein by the intraperitoneal (i.p.) or subcutaneous (s.c.) route. The experiments were terminated 24 hours after i.p. challenge or 7 days after s.c. challenge. The study was conducted with the approval of the local animal ethics committee. The animals were evaluated at intervals for neurological symptoms, appearance changes, or behavior changes according to a predefined scoring matrix. Animals with a combined score of 5 or higher were excluded.

[0190] Results

[0191] Mutagenesis of residues flanking the β-octyl-glucoside binding site

[0192] In previous studies, the inventors identified the glycan (β-octyl-glucoside) binding site in domain III of Etx and proposed that this site might be the second receptor binding site (Bokori-Brown (2013)). In the present study, 5 residues flanking this site were identified for mutagenesis (V72, F92, H149, V166, A168; Figure 3A) to evaluate their role in toxicity. Using plasmids encoding variant forms of Y30A - Y196A of Etx, additional mutations were introduced at V72, F92, H149, V166, and A168. These residues were mutated to alanine (H149, F92, V166) or phenylalanine (A168, V72), and the His - tagged proteins encoded by the mutant genes were expressed and purified in Escherichia coli. The Y30A - Y196A variant form of Etx and mutants containing H149A and A168A in addition to Y30A - Y196A were also expressed and purified. The authenticity of the proteins was verified in two ways. First, the genes encoding the mutant genes were sequenced to verify the presence of the expected mutations. Second, the purified proteins were analyzed by mass spectrometry to confirm that the experimentally determined mass matched the expected molecular mass of the protein. For studies in mice, rabbits, or sheep, the endotoxin levels of the proteins were less than 1 endotoxin unit (EU) / ml for mice and rabbits and less than 40 endotoxin units (EU) / ml for sheep.

[0193] Thermal stability of the protein

[0194] The structural stability of wild - type epsilon toxin, Y30A - Y196A variant protein, and four mutants of Y30A - Y196A was evaluated using thermal stability assays. This showed that the melting temperature (T m , in °C) of Y30A - Y196A was lower than that of the wild - type toxin. However, introducing additional mutations (V72, F92, H149, V166, and A168) into the Y30A - Y196A variant protein only resulted in minor changes in the thermal stability of the protein ( Figure 5 ), indicating that these substitutions did not disrupt the stability of the protein's tertiary structure. Y30A - Y196A - H149A - A168F had the lowest melting temperature, indicating that it was the most unstable among the mutants tested.

[0195] Toxicity of variant proteins in cell cultures

[0196] The toxicity of trypsin - activated purified Etx proteins against MDCK.2 cells, CHO cells, and CHO cells expressing hMAL, sMAL, or dMAL was tested (Table 5). As previously found, the Y30AY196A mutation led to a more than 400 - fold decrease in toxicity against MDCK cells. However, compared with the wild - type toxin, this mutant showed a 57 - fold increase in toxicity against CHO cells expressing hMAL, a 12 - fold decrease in toxicity against CHO cells expressing sMAL, and a 180 - fold decrease in toxicity against CHO cells expressing dMAL. Introducing additional mutations of H149A, A168F, F92A, or V72F into the β - octyl - glucoside - binding site decreased the toxicity against CHO cells expressing hMAL.

[0197]

[0198] Toxicity of Variant Proteins in Mice

[0199] The toxicity of variant proteins (Y30A - Y196A - H149A, Y30A - Y196A - A168F, and Y30A - Y196A - H149A - A168F) with reduced toxicity in CHO - hMAL cell cultures was tested in mice (Table 5). When administered via the i.p. route, the MLD dose of trypsin - activated Y30A - Y196A - H149A was 2 μg to 20 μg, while the MLD dose of trypsin - activated Y30A - Y196A - A168F or Y30A - Y196A - H149A - A168F was higher than the highest dose tested (20 μg). The Y30A - Y196A - A168F protein was selected for further toxicity testing via the s.c. route, before or after trypsin activation. The MLD dose of trypsin - activated protein was 20 μg to 200 μg, while the unactivated protein was non - toxic at the doses tested.

[0200] Antibody Response to Variant Proteins

[0201] Multiple groups (n = 3 per group) of rabbits were immunized with Y30A - Y196A, Y30A - Y196A - A168F, or Y30A - Y196A - H149A - A168F genetic toxoids administered with Freund's incomplete adjuvant. This work was carried out by Cambridge Research Biochemicals (Cleveland, UK). One week after the fourth immunization, antibodies in the serum that could replace the neutralizing monoclonal antibody against ε - toxin were tested, indicating the presence of neutralizing antibodies in the serum. For comparison, the inventors included the WHO international standard ε - toxin antitoxin diluted to 5 IU / ml. The results showed that all rabbits produced antibodies reactive with wild - type ε - toxin when tested by competitive ELISA. The titers in the serum from each rabbit in each immunized group were approximately similar, so the inventors pooled the sera for subsequent testing.

[0202] The inventors found that undiluted serum or serum diluted 10 - fold in PBS was similar to each other and to the international standard ε - toxin antitoxin in their ability to replace neutralizing antibodies. When diluted 100 - fold, sera against Y30A - Y196A, Y30A - Y196A - A168F, and Y30A - Y196A - H149A - A168F were more effective than the international standard ε - toxin antitoxin in replacing neutralizing antibodies ( Figure 4 ).

[0203] Immunization of Lambs

[0204] The inventors immunized multiple groups (5 or 6 animals per group) of lambs with two doses of the Y30A-Y196A-A168F protoxin administered with Montanide TM ISA 61VG or aluminum hydroxide adjuvant (Table 6). Another group of lambs had pre-existing anti-ε toxin antibodies (detected by Western blot) at the start of the study, and these lambs were immunized with two doses of the Y30A-Y196A-A168F protein and Montanide TM ISA 61VG adjuvant. Using two different assays, the inventors tested for the presence of antibodies in the sera from these animals and control sera from non-immunized lambs that were able to neutralize ε toxin. First, the inventors used a competitive ELISA to measure the ability of ovine sera to displace a neutralizing monoclonal antibody. The inventors also tested the ability of this serum to neutralize the toxicity of ε toxin to CHO-hMAL cells. In both assays, the inventors included dilutions of a standard serum containing known concentrations of neutralizing antibody, expressed in international units (IU / ml).

[0205] The inventors detected no antibodies in the control lambs using either assay. Using the competitive ELISA, the inventors detected low levels of antibodies in the group immunized with the toxoid and aluminum hydroxide adjuvant, but using the CHO-hMAL assay, the inventors were unable to detect any neutralizing antibodies. In contrast, lambs immunized with the toxoid in Montanide TM ISA 61VG adjuvant produced high levels of neutralizing antibodies in both assays, exceeding 64 IU / ml in the CHO-hMAL assay and 200 IU / ml in the competitive ELISA assay (Table 6). Relative to lambs without pre-existing anti-ε toxin antibodies immunized with the toxoid in Montanide TM ISA 61VG adjuvant, lambs immunized with the toxoid in Montanide TM ISA 61VG adjuvant but with pre-existing anti-ε toxin antibodies produced lower levels of neutralizing antibodies after immunization.

[0206] Table 6. Neutralizing antibodies in lambs immunized with Y30A-Y196A-A168F

[0207]

[0208] hMAL = human MAL

[0209] sMAL = sheep MAL

[0210] Discussion

[0211] In previous studies, the present inventors investigated the potential of site-directed mutants of Etx with mutations in the putative receptor-binding domain (domain 1) and demonstrated that the combination of the Y30A and Y196A mutations significantly reduced the ability of the toxin to bind to and kill MDCK cells. The present inventors also previously demonstrated that Y30A-Y196A had reduced toxicity in mice, suggesting that the Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia. In in vitro neutralization assays, polyclonal antibodies against Y30A-Y196A provided protection against wild-type toxin.

[0212] However, previous studies used the MDCK cell line to measure cytotoxicity and subsequently showed that CHO cells expressing MAL were also highly sensitive to the toxin. The possibility that MAL is the receptor for the toxin is supported by the discovery that MAL knockout mice become resistant to the effects of Etx. In the present study, which forms the basis of the present application, it was found that the Y30A-Y196A mutant was only slightly less toxic to CHO cells expressing sheep MAL than wild-type Etx, but more toxic to CHO cells expressing human MAL. However, in CHO cells expressing canine MAL, the toxicity of the mutant was significantly reduced. This finding suggests that MAL from different species interacts differently with Etx, suggesting that in future studies both MDCK cells and CHO cells expressing MAL should be used in parallel.

[0213] The inventors introduced additional mutations to reduce the toxicity of Y30A-Y196A to CHO cells expressing MAL. This mutation was introduced into the region involved in sugar binding in domain 3. Many of these mutants showed reduced toxicity in CHO-hMAL cell cultures, as well as on MDCK cells and in mice. The data confirmed the role of this region in toxicity. The inventors were able to produce all of these proteins, and based on thermal stability measurements, the protein did not seem to show significant changes in stability, indicating that the conformation of the protein is roughly similar to that of the wild-type epsilon toxin. The Y30AY196A+A168F mutant was selected for testing in sheep because it induced a strong antibody response in rabbits.

[0214] Livestock vaccines containing aluminum hydroxide or saponin as adjuvants generally induce short-term antibody responses (Khorasani et al., (2016) Iranian Journal of Veterinary Research 17: 8-12). This requires booster vaccinations at regular intervals, sometimes as short as 4 months. TMISA 61VG is a new ready-to-use mineral oil-based adjuvant for livestock that offers the potential to induce high levels and long-lasting responses in animals (Khorasani et al., (2016)). Compared to the use of aluminum hydroxide adjuvant, the use of Montanide TM ISA 61VG adjuvant resulted in the induction of a better antibody response, a finding similar to that previously reported with foot-and-mouth disease vaccines (Khorasani et al., (2016)). Previous reports have also shown that ISA 61VG is superior to ISA 201VG (water-in-oil) adjuvant or Montanide TM Gel 01 (aqueous polymer) adjuvant (Petermann et al., (2017) Exp Appl Acarol. 72:303-315) in inducing antibody responses. In lambs, no evidence of local side effects was seen after the use of a protein containing Montanide TM ISA 61VG adjuvant, although others have reported evidence of local side effects with the use of this adjuvant (Petermann et al., (2017) Exp Appl Acarol. 72:303-315). In Europe, for the licensing of epsilon-toxoid vaccines, compliance with the European Pharmacopoeia (Ph.Eur.) monograph on veterinary Clostridium perfringens vaccines (0363) is required. The toxoid produced showed residual toxicity below the required level, and the inventors used Montanide TM ISA 61VG adjuvant to achieve neutralizing antibody levels at least 10 times the threshold requirement (5 IU / ml).

[0215] Lambs with pre-existing anti-epsilon toxin antibodies responded less well to vaccination, a finding consistent with studies in other species. For example, antibodies can inhibit responses to multiple vaccines (Voysey et al.,) 2017) JAMA paediatrics 171:637-46; Edwards et al., (2015) Vaccine 33:6469-72; Idoko et al., (2014) Vaccine 32:4220-7; Zarnitsyna et al., (2016) PLoS Pathog. 12:e1005692). These pre-existing antibodies may lead to the clearance of antigen or the formation of antigen-antibody complexes that limit B cell activation or physically mask epitopes from B cells (Zarnitsyna et al., (2016)).

[0216] The vaccine designed by the present invention will be used for livestock susceptible to enterotoxemia caused by Clostridium perfringens epsilon toxin. This vaccine has many advantages compared to existing vaccines, as it does not require detoxification before use. The purity of the antigen and the use of adjuvants (such as Montanide TM ISA 61VG) should promote long-term immunity and reduce or eliminate the need for booster vaccinations. In addition, it can serve as a protein carrier for polysaccharides that induce protective antibodies against other diseases in livestock (Petermann et al., (2017) Exp Appl Acarol. 72: 303-315.; Voysey et al., (2017); Edwards et al., (2015) Idoko et al., (2014); Zamitsyna et al., (2016); 35Byrd et al., (1992) Veterinary immunology and immunopahology 34: 307-24). The glycoconjugate will promote the T cell response to the polysaccharide moiety (Avci et al., (2011) Nature medicine 17: 1602-9), increasing the intensity of the antibody response and the induction of memory response to the polysaccharide (Avci (2013) Current Topics in Medicinal Chemistry 13: 2535-40); Pace (2013) Expert opinion on biological therapy 13: 11-33). In addition, the linkage of the polysaccharide to the epsilon toxin carrier will allow the vaccine to be used in young animals (PACE (2013)). The Y30AY196A+A168F protein can be chemically conjugated to the polysaccharide, or it can be further modified to serve as a receptor for recombinant glycoconjugates produced using the naturally occurring glycosylation system in bacteria (Valguarnera et al., (2016) J Mol Biol. 428: 3206-20; Cuccui et al., (2015) The Journal of pharmacy and 10 pharmacology 67: 338-50).

[0217] Finally, the Y30AY196A+A168F protein could potentially be developed as a human vaccine in the future. Clostridium perfringens epsilon toxin is considered a potential biological threat agent (Greenfield et al., (2002) Am J Med Sci. 323:326-40; Berger et al., (2016) Disaster and military medicine 2:7), and vaccinating individuals at risk would protect them from the disease. Additionally, epsilon toxin has recently been implicated in the development of multiple sclerosis (Rumah et al., (2013) PLoS One 8:e76359; Rumah et al., (2015) PLoS Pathog. 11:e1004896; Linden et al., (2015) mBio. 6). If this link is established, then vaccination against the toxin could potentially be a preventive or therapeutic option.

[0218] Duration of the protective antibody response

[0219] Lambs were raised without vaccination against Clostridium perfringens epsilon toxin. After 12 months, a group of 5 lambs received 200 μg of Y30AY196A+A168F toxoid (1:1) containing Montanide TM ISA 61VG (Seppic, Paris, France) adjuvant subcutaneously in 6 doses of 0.5 ml. Three weeks later, the lambs received a second dose of the adjuvanted protein. Blood samples were collected at the start of the study and at intervals up to 12 months after vaccination. Competitive ELISA was performed using the Monoscreen ELISA kit (BioX Diagnostics, BIO K 222 / 2) according to the manufacturer's instructions to measure neutralizing antibodies. The inventors included standardized serum dilutions containing known concentrations of neutralizing antibodies, expressed in international units (IU / ml), to enable them to calculate the antibody levels of neutralizing antibodies expressed in IU / l. The results are shown in Table 7 below.

[0220] Table 7. Neutralizing antibodies in sera at week 0 and week 3 after vaccination with Y30AY196A+A168F containing Montanide TM ISA 61VG adjuvant

[0221]

[0222]

[0223] The neutralizing antibody titers found in sheep administered the toxoid of the present invention and Montanide ISA 61VG adjuvant far exceed the reported minimum protective titers in sheep (0.1 IU / ml to 0.3 IU / ml (de la Rosa et al. 1997 (J Anim Sci 75(9): 2328-2334); Uzal and Kelly 1998 (Veterinary Record 142(26): 722-725)); or the minimum protective titers in goats (1 IU / ml (Uzal, Bodero et al. 1998 (Vet Rec 143(17): 472-474), Uzal and Kelly 1998 (Veterinary Record 142(26): 722-725)), and remain above this threshold one year after immunization. Additionally, the level of neutralizing antibodies exceeded the protective titer after a single dose of the vaccine of the present invention (i.e., at week 3), indicating that a single-dose vaccine for livestock can be achieved.

[0224] Testing of patient samples from NMO, ON, and TM

[0225] Table 8 below shows the Western blot data of samples taken from subjects with NMO, almost all of whom initially presented with ON and / or TM. Compared to the control group (where 2 / 25 (8%) were positive), 15 out of 30 samples (50%) showed very strong positive (3), strong positive (2), positive (8), or weak positive (2) reactivity to Etx.

[0226] All subjects who tested positive for AQP-4 antibodies and 11 out of 15 subjects who tested positive for Etx initially showed characteristics of transverse myelitis (TM) and / or optic neuritis (ON), which are examples of neuromyelitis optica spectrum disorders (NMOSD).

[0227] Table 8: Western blot data

[0228]

[0229]

[0230] Control indicated in bold italics: sex and age matched + / - 2 years

[0231] Treatment of human red blood cells (RBC) with epsilon toxin

[0232] Hemolysis of human red blood cells (RBCs) exposed to wild-type Etx, Y30AY196A, Y30AY196A+H149A, Y30AY196A+A168F, Y30AY196A+F92A, Y30AY196A+V166A, and the quadruple mutant Y30AY196+A168F+H149A. Pro-toxins at 10 μM and trypsin-activated toxins at 10 μM and 1 μM were tested against 3.3% RBCs. Each toxin was tested in triplicate at each dose. Results are expressed relative to a 1% Tx100 control (which causes 100% hemolysis); negative control = PBS. Results are as Figure 6 shown and demonstrate that even upon trypsin activation, Y30AY196A+H149A, Y30AY196A+A168F, and Y30AY196+A168F+H149A do not cause hemolysis.

[0233] Sequence (mutation positions shown in bold)

[0234] SEQ ID NO: 1

[0235]

[0236] SEQ ID NO: 2

[0237]

[0238] SEQ ID NO: 3

[0239]

[0240] SEQ ID NO: 4 Y30A mutation

[0241]

[0242] SEQ ID NO: 5 Y196A mutation

[0243]

[0244] SEQ ID NO: 6 H149A mutation

[0245]

[0246] SEQ ID NO: 7 V72F mutation

[0247]

[0248] SEQ ID NO: 8 F92A mutation

[0249]

[0250] SEQ ID NO: 9 V166A mutation

[0251]

[0252] SEQ ID NO: 10 A168F mutation

[0253]

[0254] SEQ ID NO: ii Full-length wild-type native ε-toxin

[0255]

[0256] SEQ ID NO: 12 sequence for obtaining the crystal structure (PDB ID: 1YUJ)

[0257]

[0258]

[0259] SEQ ID NO: 13 Trypsin-activated wild-type recombinant ε-toxin

[0260]

[0261] SEQ ID NO: 14 Trypsin-activated recombinant ε-toxin with H149A mutation

[0262]

[0263] SEQ ID NO: 15 Full-length recombinant ε-toxin

[0264]

[0265] SEQ TD NO: 16 Trypsin-activated recombinant ε-toxin (Y30A + Y196A)

[0266]

[0267] SEQ ID NO: 17 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + H149A)

[0268]

[0269] SEQ ID NO: 18 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + V72F)

[0270]

[0271] SEQ ID NO: 19 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + F92A)

[0272]

[0273] SEQ ID NO: 20 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + V166A)

[0274]

[0275] SEQ ID NO: 21 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + A168F)

[0276]

[0277] SEQ ID NO: 22 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + H149A + V72F)

[0278]

[0279] SEQ ID NO: 23 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + H149A + F92A)

[0280]

[0281] SEQ ID NO: 24 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + H149A + V166A)

[0282]

[0283] SEQ ID NO: 25 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + H149A + A168F)

[0284]

[0285] SEQ ID NO: 26 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + V72F + F92A)

[0286]

[0287] SEQ ID NO: 27 Trypsin-activated recombinant ε-toxin (Y30A + Y196A + V72F + V166A)

[0288]

[0289] SEQ ID NO: 28 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+A168F)

[0290]

[0291] SEQ ID NO: 29 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+F92A+V166A)

[0292]

[0293] SEQ ID NO: 30 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+F92A+A168F)

[0294]

[0295] SEQ ID NO: 31 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V166A+A168F)

[0296]

[0297] SEQ ID NO: 32 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+F92A)

[0298]

[0299] SEQ ID NO: 33 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+V166A)

[0300]

[0301] SEQ ID NO: 34 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+A168F)

[0302]

[0303] SEQ ID NO: 35 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+F92A+V166A)

[0304]

[0305] SEQ ID NO: 36 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+F92A+A168F)

[0306]

[0307] SEQ ID NO: 37 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V166A+A168F)

[0308]

[0309] SEQ ID NO: 38 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+F92A+V166A)

[0310]

[0311] SEQ ID NO: 39 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+F92A+A168F)

[0312]

[0313] SEQ ID NO: 40 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+V166A+A168F)

[0314]

[0315] SEQ ID NO: 41 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+F92A+V166A+A168F)

[0316]

[0317] SEQ ID NO: 42 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+F92A+V166A)

[0318]

[0319] SEQ ID NO: 43 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+F92A+A168F)

[0320]

[0321] SEQ ID NO: 44 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+F92A+V166A+A168F)

[0322]

[0323] SEQ ID NO: 45 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+F92A+V166A+ A168F)

[0324]

[0325] SEQ ID NO: 46 Trypsin-activated recombinant epsilon toxin (V72F)

[0326]

[0327] SEQ ID NO: 47 Trypsin-activated recombinant epsilon toxin (F92A)

[0328]

[0329] SEQ ID NO: 48 Trypsin-activated recombinant epsilon toxin (H149A)

[0330]

[0331] SEQ ID NO: 49 Trypsin-activated recombinant epsilon toxin (V166A)

[0332]

[0333] SEQ ID NO: 50 Trypsin-activated recombinant epsilon toxin (A168F)

[0334]

[0335] SEQ ID NO: 51 Y43A forward

[0336]

[0337] SEQ ID NO: 52 Y43A reverse

[0338]

[0339] SEQ ID NO: 53 Y209A forward

[0340]

[0341] SEQ ID NO: 54 Y209A reverse

[0342]

[0343] SEQ ID NO: 55 H149A forward

[0344]

[0345] SEQ ID NO: 56 H149A reverse

[0346]

[0347] SEQ ID NO: 57 V72F forward

[0348]

[0349] SEQ ID NO: 58 V72F reverse

[0350]

[0351] SEQ ID NO: 59 F92A forward

[0352]

[0353] SEQ ID NO: 60 F92A reverse

[0354]

[0355] SEQ ID NO: 61 V166A forward

[0356]

[0357] SEQ ID NO: 62 V166A reverse

[0358]

[0359] SEQ ID NO: 63 A168F forward

[0360]

[0361] SEQ ID NO: 64 A168F reverse

[0362]

[0363] SEQ ID NO: 65( Figure 2 Residues 1 - 260 of the unmutated sequence: Trypsin activation polypeptide)

[0364]

[0365]

[0366] SEQ ID NO: 66( Figure 2 Signal sequence residues 1-32)

[0367]

[0368] SEQ ID NO: 67( Figure 2 N-terminal propeptide residues 33 - 45) to 45)

[0369]

[0370] SEQ ID NO: 68( Figure 2 C-terminal propeptide residues 305 - 328 (305 to 328)

[0371]

Claims

1. A method for preventing or treating a demyelinating disorder in a human or animal subject in need thereof, said disorder being selected from: 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 disorder characterized by an increase or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM), comprising: Administer to the subject a composition comprising an effective amount of an agent that directly or indirectly interferes with epsilon toxin (Etx), an Etx-binding receptor, or the interaction of Etx with its binding receptor produced by Clostridium perfringens type B or D strains, thereby inhibiting or repressing Etx-regulated receptor signaling activity.

2. The method according to claim 1, wherein the agent is an inhibitor of Etx, such as an antibody or a functional component thereof.

3. The method according to claim 1, wherein the agent is an inhibitor or antagonist of an Etx-binding receptor.

4. The method according to claim 3, wherein the Etx-binding receptor is myelin and lymphocyte protein (MAL) or hepatitis A virus cellular receptor 1 protein (HAVCR1).

5. The method according to claim 1, wherein the agent is a vaccine against Clostridium perfringens type B or D strains or a vaccine against epsilon toxin (Etx) produced by Clostridium perfringens type B or D strains.

6. The method according to any one of the preceding claims, wherein the agent comprises an epsilon toxin (Etx) polypeptide that has reduced toxicity to cells expressing myelin and lymphocyte (MAL) protein and comprises a modified domain III compared to the wild-type Etx polypeptide SEQ ID NO: 65, wherein the reduced toxicity is relative to SEQ ID NO: 65 and / or SEQ ID NO: 14, and wherein the Etx polypeptide is capable of binding to at least one antibody that binds to the sequence represented by SEQ ID NO: 65 and / or SEQ ID NO:

14.

7. The method according to claim 6, wherein the modified domain III is a modification at the glycan (β-octyl-glucoside) binding site of domain III.

8. The method according to claim 6 or 7, wherein the modified domain III comprises one or more mutations of the amino acids in the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 that constitute domain III.

9. The method according to any one of claims 6 to 8, comprising one or more of: SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

10. The method according to any one of claims 6 to 9, comprising SEQ ID NO: 4 and / or SEQ ID NO: 5 and / or SEQ ID NO:

6.

11. The method according to any one of claims 6 to 10, comprising at least the following sequences: a. SEQ ID NO: 4 and SEQ ID NO: 5; and optionally in addition to (i) b. SEQ ID NO: 6; and c. one or more of the following: SEQ ID NO: 10, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

12. The method according to any one of claims 6 to 11, wherein the reduced toxicity is reduced compared to an Etx polypeptide comprising SEQ ID NO: 4 and SEQ ID NO: 5, or an Etx polypeptide comprising SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or compared to a known Etx vaccine or Etx vaccine candidate.

13. The method according to any one of claims 6 to 12, having at least 60% sequence identity with any one of SEQ ID NOs: 18 to 50 and comprising or consisting of one or more mutations indicated for the relevant SEQ ID NO in Table 3.

14. A polynucleotide having a nucleic acid sequence encoding an agent or polypeptide according to any one of the preceding claims.

15. A vector comprising the polynucleotide according to claim 14.

16. A cell comprising an agent according to any one of claims 1 to 5, a polypeptide according to any one of claims 6 to 13, and / or a polynucleotide according to claim 14 and / or a vector according to claim 15.

17. A subunit vaccine or conjugate vaccine comprising an agent according to any one of claims 1 to 5, a polypeptide according to any one of claims 6 to 13.

18. An affinity reagent capable of binding to one of an agent according to any one of claims 1 to 5, a polypeptide according to any one of claims 6 to 13, and promoting an immune response in an individual to whom the affinity reagent has been administered.

19. A method of preparing an immunotherapeutic composition, optionally a vaccine composition, comprising altering an Etx polypeptide or a vaccine comprising an Etx polypeptide by modifying domain III relative to the wild-type Etx polypeptide.

20. An immunotherapeutic composition or vaccine composition prepared by the method according to claim 19.

21. An immunotherapeutic composition or vaccine composition comprising an agent according to any one of claims 1 to 5, a polypeptide according to any one of claims 6 to 13, and / or a polynucleotide according to claim 14, and / or a vector according to claim 15, and / or a cell according to claim 16, and / or a subunit vaccine according to claim 17, and / or an affinity reagent according to claim 14.

22. An immunotherapeutic composition according to claim 20 or 21, optionally a vaccine composition, which is a food for humans or animals.

23. The use of the polypeptide according to any one of claims 6 to 13 and / or the polynucleotide according to claim 14 and / or the vector according to claim 15 and / or the cell according to claim 16 and / or the subunit vaccine or conjugate vaccine according to claim 17 and / or the affinity reagent according to claim 18 and / or the immunotherapeutic composition or vaccine composition according to any one of claims 20 to 22, or the agent according to any one of claims 1 to 5, in a method for treating or vaccinating a subject against a disease caused by or associated with Clostridium perfringens and / or a disease caused by or associated with (active) epsilon toxin and / or against a demyelinating disease.

24. The polypeptide according to claim 23, wherein the disease is selected from: 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 disorder characterized by an increase or presence of antibodies against aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

25. A method for treating a subject having a disease caused by or associated with the presence of Clostridium perfringens and / or a disease caused by or associated with the presence of (active) epsilon toxin and / or treating a subject against a demyelinating disease, or a method for vaccinating a subject against developing said disease, the method comprising administering to the subject the agent according to any one of claims 1 to 5, the polypeptide according to any one of claims 6 to 13 and / or the polynucleotide according to claim 14 and / or the vector according to claim 15 and / or the cell according to claim 16 and / or the subunit vaccine according to claim 17 and / or the affinity reagent according to claim 18 and / or the vaccine composition or immunotherapeutic composition according to any one of claims 20 to 22, wherein the demyelinating disorder is selected from: 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 disorder characterized by an increase or presence of antibodies against aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

26. The polypeptide, polynucleotide, vector, cell, affinity reagent, vaccine composition or immunotherapeutic composition according to any one of claims 20 to 22, or the method according to claim 25, wherein the subject is a ruminant, a horse, a companion animal or a human.

27. Use of MAL cells as a model in testing the toxicity of an epsilon candidate vaccine.

28. A kit comprising the agent according to any one of claims 1 to 5, the polypeptide according to any one of claims 6 to 13 and / or the polynucleotide according to claim 14 and / or the vector according to claim 15 and / or the cell according to claim 16 and / or the subunit vaccine according to claim 17 and / or the affinity reagent according to claim 18 and / or the vaccine composition or immunotherapeutic composition according to any one of claims 20 to 22.

29. A polypeptide, polynucleotide, vector, cell, subunit vaccine, conjugate vaccine, affinity reagent, vaccine composition or immunotherapeutic composition or method substantially as described herein.

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