Epsilon toxin from Clostridium perfringens as a vaccine

Abstract

The present invention relates to methods and compositions for detecting, diagnosing, preventing, treating or ameliorating symptoms of demyelinating disorders in human or animal subjects in need thereof, the disorders being selected from: enterotoxemia (ET), multiple sclerosis (MS), clinically confirmed MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica spectrum disorders (NMOSD), optic neuritis (ON), neuromyelitis optica (NMO), myelitis, 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). The method comprises administering to the subject a composition comprising an effective amount of an agent that directly or indirectly interferes with an epsilon toxin (Etx) produced by a type B or D strain of Clostridium perfringens, an Etx-binding receptor, or the interaction of Etx with its binding receptor, thereby inhibiting or suppressing the receptor signaling activity regulated by Etx. The present invention also provides novel polypeptides useful as vaccines against diseases caused by or associated with the epsilon toxin of Clostridium perfringens.

Description

Technical Field

[0001] The present invention relates to methods and compositions for detecting, diagnosing, preventing, treating or ameliorating symptoms of demyelinating disorders. Background Art

[0002] 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 of Clostridium. Clostridium perfringens strains are classified into one of five toxin types (referred to as types A to E) based on their ability to produce four types of toxins (i.e., α-toxin, β-toxin, ε-toxin, and ι-toxin) (Petit et al. (1999) Trends Microbiol. vol. 7. 104-110).

[0003] Epsilon toxin (Etx) is produced by toxin types B and D. These strains are responsible for a serious disease called enterotoxemia, which primarily affects sheep and lambs, but also causes infection 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 toxins of the classification, the bacteria are also capable of producing a variety of so-called secondary toxins, such as β1, β2, δ, θ, λ, μ, v and enterotoxins (Rood (1998) Annu. Rev. Microbiol. vol. 2052. 333-360).

[0004] The most important factor causing disease in sheep and other ruminants is excessive intake of high-calorie foods, which leads to the presence of 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 the subsequent overproduction of Etx. The toxin causes increased intestinal permeability, promotes its entry into the bloodstream and spreads to the main target organs (kidneys and brain) (McDonel (1980) Pharmacol Ther 10 (3): 617-655). Here, due to increased vascular permeability, poisoning can lead to fluid accumulation. Accumulation in the central nervous system leads to neurological disorders, which quickly lead to death (Finnie (2003 Aust. Vet. J. vol. 81, 219-221).

[0005] Recently, Etx has been implicated in the development of multiple sclerosis in humans (Rumah et al. (2013) PLoS One 8:e76359).

[0006] Multiple sclerosis (MS) is a demyelinating disease in which the insulation of nerve cells in the brain and spinal cord is damaged. This damage disrupts the ability of parts of the nervous system to communicate, leading to a range of symptoms including double vision, monocular blindness, muscle weakness, and sensory or coordination impairments. The condition often begins as a clinically isolated syndrome (CIS) for some time before being confirmed as clinically definite MS (CDMS).

[0007] Neuromyelitis optica spectrum disorder (NMOSD) is a rare neurological condition characterized by episodes of optic neuritis (ON) and transverse myelitis (TM), accompanied by one or more other diagnostic criteria, including in some cases the presence of specific antibodies (aquaporin-4 (AQP-4)).

[0008] 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 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) speculated that the gut microbiota, and possibly Clostridium perfringens itself, may be involved in the pathogenesis of NMO.

[0009] Transverse myelitis (TM) is an inflammation of both sides of a segment of the spinal cord and can also lead to myelin damage.

[0010] Acute disseminated encephalomyelitis (ADEM) is characterized by brief but widespread episodes of inflammation in the brain and spinal cord that damage myelin. ADEM usually occurs after a viral or bacterial infection, or less frequently after vaccination with measles, mumps, or rubella. ADEM usually damages white matter, leading to neurological symptoms such as loss of vision in one or both eyes (due to inflammation of the optic nerve).

[0011] Etx is expressed with a signal sequence directing the export of the prototoxin from the bacteria (McDonel (1986) in Pharmacology of bacterial toxins eds. Dorner & Drew, Pergamon Press, 477-517). During the course of the disease, the relatively inactive protoxin is converted into active toxin by proteolytic cleavage in the intestinal lumen (by host digestive proteases such as trypsin and chymotrypsin) (Bhown & Habeerb (1977) Biochem. Biophys. Res. Commun. vol. 78, 889-896) or by Clostridium perfringens λ-protease (Minami et al. (1997) Microbiol. Immun. 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). When both the N-terminus and the C-terminus are cleaved, maximum activation occurs (Worthington & Mulders (1977) Infect. Immun. vol. 18, 549-551).

[0012] The 3D structure of Etx has been determined (Cole et al. (2004) Nature 5 Structural & Molecular Biology vol. 11, 797-798) and reveals a molecule composed primarily of β-sheets that can be divided into three functional domains. Domain I at the N-terminus includes the proposed receptor interaction region. Domain II in the middle includes an amphipathic β-hairpin structure that is predicted to play a role in membrane insertion. Domain III at the C-terminus includes the C-terminal peptide that must be removed for activation to occur.

[0013] Epsilon toxin is an aeromonas lysin-like β-pore-forming toxin (β-PFT) in which an amphipathic β-hairpin loop inserts into the membrane to form a β-barrel structure. The overall fold of Etx is similar to the structures of aeromonas hydrophila from the Gram-negative bacterium 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 sulphurous (Mancheno et al. (2005) J. Biol. Chem. vol. 280, 17251-17259). The structural similarity between these toxins is most pronounced 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).

[0014] In Aeromonas bacteriophages, the 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 domain I of Etx (equivalent to domain II of Aeromonas bacteriophages) is proposed to perform a similar function (Cole et al. (2004)), but this has not been confirmed. Domain II of Aeromonas bacteriophages contains a mannose 6-phosphate binding loop. However, the residues of domain II involved in mannose-6-phosphate binding in Aeromonas bacteriophages are not conserved in domain I of Etx, suggesting that structural variations in the N-terminal receptor binding domains of these toxins may account for the differences between their target cell specificities.

[0015] Etx is unique among β-PFTs because of its potent and highly cell-specific activity. Due to its potent activity, Etx is considered by the U.S. government Centers for Disease Control and Prevention to be a potential bioweapon for international terrorism (Morbidity and Mortality Weekly Report (MMWR) Recommendations and Reports 2000) vol. 49, 1-14). The 50% lethal dose (LD50) of Etx in mice following intravenous injection is 1.5 mmol / l. 50 ) is typically 100 ng / kg (Gill (1982) Microbiol. Rev. vol. 46, 86-94), making Etx the most potent Clostridial toxin after botulinum neurotoxin. Etx also exhibits high cell specificity. Of the many cell lines tested, only four were identified as sensitive to the toxin. These include kidney cell lines of 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 (G-402 (Shortt et al. (2000) Hum. Exp. Toxicol. vol. 19. 108-116) and ACHN (Ivie et al. (2011) PloS ONE vol. 6, e17787) origin). Most in vitro studies on Etx have been performed 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 is reported that the dose of Etx that kills 50% of MDCK cells (CT 50 ) as low as 15ng / ml.

[0016] Binding of Etx to MDCK cells is associated with the formation of a stable, high molecular weight complex (Petit et al. (1997) J. Bacteriol. vol. 179, 6480-6487). Intoxicated cells undergo morphological changes prior to cell death, including swelling and plasma membrane blebbing (Petit et al. (1997) J. Bacteriol. vol. 179, 6480-6487). Rapid toxin-induced cell death and the specificity of epsilon toxin to only some cell lines suggest the presence of specific receptors on target cells. Etx acts by binding to host cells, and there is evidence that the seven monomers of the protein assemble into pores across the cell membrane (Miyata et al. (2002) J Biol Chem. 277: 39463-8.), leading to unregulated ion movement across the membrane and cell death. Toxicity appears to be a result of the formation of pores in the target cell membrane (Petit et al. (2001) J. Biol. Chem. 25 vol. 276. 15736-15740).

[0017] 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). 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 expressing MAL, and MAL knockout mice are reported to be highly resistant to the toxin (Rumah et al. (2015)).

[0018] Many commercial vaccines are available to prevent enterotoxemia. These vaccines are typically produced by treating C. perfringens culture filtrates with formaldehyde to detoxify Etx. These vaccines contain a variety of proteins in addition to Etx, and there can be considerable batch-to-batch variability in the immunogenicity of these preparations. Inflammatory responses following vaccination have been reported to result in reduced feed consumption. These deficiencies have prompted work to design improved vaccines, and a number of 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). Site-directed mutants overcome the problem of batch-to-batch variability in immunogenicity associated with chemical detoxification methods for vaccine production. However, the high potency of the toxin can make it difficult to eliminate toxicity. The toxicity of the mutants has been assessed using MDCK cell culture (Ivie and McClain (2012) Biochemistry 51:7588-95; Kang et al. (2017)) or in mice.

[0019] It was reported that a site-directed mutant of Etx (Y30A-Y196A) reduced cytotoxicity in MDCK.2 cells by more than 430-fold compared to the wild-type toxin and showed reduced but not abolished toxicity in mice (Bokori-Brown et al., (2014) Vaccine vol. 32, 2682-2687).

[0020] There remains a need to identify improved molecules with potential for use as vaccines against diseases caused by or associated with the presence of Etx and / or diseases caused by infection with C. perfringens. DETAILED DESCRIPTION

[0021] The present invention relates to methods and compositions for detecting, diagnosing, preventing, treating or ameliorating symptoms of a demyelinating disorder in a human or animal subject in need thereof, the disorder being selected from the group consisting of enterotoxemia (ET), multiple sclerosis (MS), clinically definite MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica spectrum disorder (NMOSD), optic neuritis (ON), neuromyelitis optica (NMO), myelitis, transverse myelitis (TM), diseases or disorders characterized by increased 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) produced by a type B or type D strain of Clostridium perfringens, an Etx-binding receptor, or the interaction of Etx with its binding receptor, thereby inhibiting or repressing Etx-regulated receptor signaling activity. The present invention relates to novel polypeptides for use as vaccines against diseases caused by or associated with the epsilon toxin of Clostridium perfringens, particularly in animals susceptible to enterotoxemia and in the treatment of demyelinating disorders.

[0022] The present inventors have discovered that subjects with demyelinating disorders, such as enterotoxemia (ET), neuromyelitis optica (NMO), and transverse myelitis (TM), the latter two being examples of neuromyelitis optica spectrum disorders (NMOSD), test positive for the presence of epsilon toxin (Etx) produced by type B or type D strains of Clostridium perfringens and / or test positive for the presence of antibodies against aquaporin-4 (AQP4).

[0023] Demyelinating disorders are characterized by myelin damage and include disorders selected from the group consisting of enterotoxemia (ET), multiple sclerosis (MS), clinically definite MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica spectrum disorder (NMOSD), optic neuritis (ON), neuromyelitis optica (NMO), myelitis, transverse myelitis (TM), diseases or disorders characterized by increased or presence of antibodies to aquaporin-4 (AQP4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0024] According to a first aspect of the present invention, a method for preventing, treating or ameliorating symptoms of a demyelinating disorder in a human or animal subject in need thereof is provided, wherein the disorder is selected from the group consisting of: enterotoxemia (ET), multiple sclerosis (MS), clinically confirmed 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). 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) produced by a type B or type D strain of Clostridium perfringens, an Etx-binding receptor, or the interaction of Etx with its binding receptor, thereby inhibiting or suppressing the receptor signaling activity regulated by Etx.

[0025] According to a second aspect of the present invention, the composition comprises an agent that directly or indirectly interferes with epsilon toxin (ETX) produced by a type B or type D strain of Clostridium perfringens, an ETX-bound receptor, or the interaction of ETX with its bound receptor, thereby inhibiting or suppressing Etx-regulated receptor signaling activity, the agent being a Clostridium perfringens epsilon toxin (Etx) polypeptide having reduced toxicity to cells expressing myelin and lymphocyte (MAL) protein and comprising a modified domain III compared to a wild-type Etx polypeptide having the sequence 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 a sequence represented by SEQ ID NO: 65 and / or SEQ ID NO: 14.

[0026] Instead of or in addition to the ability to bind to an antibody that binds to SEQ ID NO: 13 and / or SEQ ID NO: 14, the polypeptide can bind to at least one antibody that binds to SEQ ID NO: 11 (an inactivated prototoxin form of epsilon toxin).

[0027] The modified domain III can be any modification in the glycan (β-octyl-glucoside) binding site of domain III and / or in the carbohydrate binding capacity of domain III, and / or modifications to domain III such that the Etx polypeptide has reduced ability to bind to CHO cells expressing MAL compared to the corresponding wild-type sequence when activated.

[0028] The modified domain III may include one or more amino acid mutations in the amino acid sequence constituting domain III (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3): VYVGKALLTNDTQQEQKLKSQSFTCK (SEQ ID NO: 1), THNVPSQDILVPANTTVEVIAYLK (SEQ ID NO: 2); and DELIVKVRNLNTNNVQEYVIPVDKKEKSNDSNIVKYRSLYIKAPGIK (SEQ ID NO: 3), wherein the mutation is a substitution or deletion 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 is more than one mutation in domain III, the mutation may optionally be a combination of substitutions and deletions.

[0029] The modified domain III may include one or more of the following mutations in SEQ ID NO: 1 and SEQ ID NO: 2:

[0030] The underlined and bold V and F can be replaced by any other amino acid, or can be deleted. For example, V can be replaced by F (V[F]) and / or F can be replaced by A (F[A]);

[0031] The underlined and bolded H, V and A can be replaced by any other amino acid, or can be deleted. For example, H can be replaced by A (H[A]), V can be replaced by A (V[A]) and / or A can be replaced by F (A[F]).

[0032] The polypeptides of the invention may include mutations at positions as 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 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 contained in SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0033] Table 1: Mutations contained in SEQ ID NO: 4 to SEQ ID NO: 10

[0034]

[0035]

[0036] The mutation contained in any one or more of SEQ ID NO:4 to SEQ ID NO:10 may be contained in a stretch 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 consecutive amino acids from SEQ ID NO:13, which includes one or more of SEQ ID NO:4 to SEQ ID NO:10.

[0037] The present invention is not necessarily limited to the specific mutations of Table 1, and any one or more mutations that affect the carbohydrate binding ability of domain III and / or one or more mutations that reduce the ability of the Etx polypeptide to bind to (CHO) cells expressing MAL equally constitute part of the present invention. Suitable mutations can be readily determined by those skilled in the art using known tools and routine techniques (such as those described herein).

[0038] 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 lacking the first 32 amino acids. The sequence (SEQ ID NO: 12) is the sequence published for the crystal structure (see the Consortium for Structural Bioinformatics (RCSB) database, wwww.rcsb.org / PDB; PDB ID: 1UYJ).

[0039] SEQ ID NO:65 is the trypsin activated wild-type Clostridium perfringens epsilon toxin, which is the portion that remains 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 when determined as described below.

[0040] 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 identical to SEQ ID NO: 13, but with an H>A mutation at position 151 of SEQ ID NO: 13 (referred to as an H149A mutation in the following examples, the difference in residue numbering being explained below). This is a variant of the activated toxin that can be studied in an ACGM Level 2 laboratory (Oyston et al. (1998) Microbiol. vol. 144 (Pt 2), 333-341) and may therefore be more practical to determine antibody binding. The inclusion of the H149A mutation described herein will doubly ensure that the polypeptide according to the invention can be used at ACGM Level 2.

[0041] Table 2: Sequence identity information

[0042] SEQ ID NO Sequence identity information 11 Full-length wild-type native epsilon toxin 12 Sequence used to obtain the crystal structure (PDB ID: 1YUJ) 13 Trypsin-activated recombinant epsilon toxin 14 Trypsin-activated recombinant epsilon toxin with H149A mutation 15 Recombinant epsilon toxin sequence 65 Trypsin-activated wild-type epsilon toxin

[0043] The following mutated positions, i.e., 30, 196, 72, 92, 149, 166, and 168, referred to in Table 1 cited herein are referenced as counting 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 33-45 (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 (a portion of the synthetic signal sequence) of SEQ ID NO:13 and SEQ ID NO:14). Identical 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). One skilled in the art will be able to readily determine mutations equivalent to positions 30, 196, 72, 92, 149, 166, and 168 in any given Etx polypeptide.

[0044] References to the "Y30A-Y196A" and "Y43A-Y209A" double mutants are used interchangeably herein to refer to the same mutation position in the Etx wild-type sequence (represented by SEQ ID NO: 65), with the position numbering depending on whether the position is within the activated toxin or within its inactive precursor. When the double mutant is referred to as Y30A-Y196A, positions 30 and 196 are counted from the beginning of the activated protein (position 1 of the sequence shown in SEQ ID NO: 65), and when the double mutant is referred to as Y43A-Y209A, the equivalent positions 43 and 209 are counted from the beginning of the inactive precursor (i.e., from the beginning of the inactive precursor). Figure 2 The positions of the V72F, F92A, H149A, V166A, A168F mutations are as found in SEQ ID NO:65.

[0045] 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, suggesting that the Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.

[0046] However, previous studies used the MDCK cell line to measure cytotoxicity. Surprisingly, the double mutant did not produce the same results of reduced toxicity in CHO cells expressing MAL. It has now 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, for example, any modification described herein.

[0047] Thus, as described in WO2013 / 144636, modification of domain III improves the known vaccine candidate Y30A-Y196. Most in vitro studies on Etx have been conducted using the MDCK cell line, as 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 vaccine candidates may not have been tested on (CHO) cells expressing MAL, the present invention provides an opportunity to improve existing Etx vaccines and Etx vaccine candidates by modifying domain III as described herein.

[0048] The polypeptide according to the present invention may comprise a modified domain III compared to the wild-type polypeptide SEQ ID NO: 65, and the polypeptide according to the present invention exhibits reduced toxicity compared to an Etx polypeptide comprising SEQ ID NO: 4 and SEQ ID NO: 5 or compared to an Etx polypeptide comprising SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In the present context, 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 capable of binding to SEQ ID NO: 65.

[0049] 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 have also been shown to effectively reduce toxicity to MDCK cells. For example, one or more of the mutated tyrosine residues can be, for example, a mutation (replacement or deletion) of one or more of Y29, Y33, Y42, Y43, Y49 and / or Y209, where the residue numbering is from the beginning of the inactive precursor (i.e., from Figure 2 170, 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.

[0050] According to the present invention, there is also provided an Etx polypeptide having a modified domain III compared to the wild-type polypeptide SEQ ID NO: 65 and showing 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 describes a proposed 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 and H106 in domain 2); Oyston et al. (1998) Microbiology 144: 333-41 describes an 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 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).

[0051] It was also surprisingly found that the Y30A-Y196 double mutant showed different toxicity results depending on the species of MAL source. For example, compared with wild-type Etx, the double mutant was only slightly less toxic to CHO cells expressing sheep MAL, but more toxic to CHO cells expressing human MAL. However, in CHO cells expressing canine MAL, the toxicity of the mutant was significantly lower. This finding suggests that MAL from different species interacts differently with Etx, indicating 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 the test. Therefore, according to another aspect of the present invention, there is provided the use of (CHO) cells expressing MAL as a model in testing the toxicity of ε candidate vaccines.

[0052] Table 3 below shows examples of mutations introduced into domain III of the Y30A-Y196A double mutant.

[0053] Table 3: Further mutations of Y30A-Y196A

[0054]

[0055]

[0056]

[0057] Therefore, the novel sequences described herein (SEQ ID NO: 18 to SEQ ID NO: 50 in Table 3) are suitable genetic toxoids for inclusion in next generation enterotoxemia or demyelinating disease vaccines and vaccines against diseases caused by or associated with C. perfringens and / or Etx. In particular, SEQ ID NO: 21 is suitable.

[0058] 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 the equivalent position in the full-length Etx polypeptide (SEQ ID NO: 11) or the equivalent position in the full-length recombinant Etx polypeptide (SEQ ID NO: 15); or the mutations shown in Table 3 may be included at the equivalent position 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.

[0059] Any mutation described herein may be contained 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] The polypeptides of SEQ ID NO: 18 to SEQ ID NO: 50 may have the same or similar or reduced toxicity as SEQ ID NO: 14 or SEQ ID NO: 14 lacking the first two N-terminal amino acid residues, and / or may be non-toxic.

[0061] The polypeptide according to the present invention may include a polypeptide from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID 65, and a sequence 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, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310 or 315 consecutive amino acids of any one of SEQ ID NO: 65, and a sequence equivalent to SEQ ID NO: 65 comprises a double mutation at positions 30-196 and additionally comprises 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 mutation may comprise a substitution or a deletion.

[0062] , 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 comprises or consists of the mutations represented as the relevant SEQ ID NO in Table 3.

[0063] The polypeptide according to the present invention comprises one of the amino acid sequences SEQ ID NO:4 to SEQ ID NO:10, resulting in the sequence contained in the polypeptide being identical to the equivalent (i.e. corresponding) sequence 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 position comprising the mutation as "X" in one of SEQ ID NO:4 to SEQ ID NO:10.

[0064] 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 may comprise or consist of the mutations indicated for the relevant SEQ ID NO.

[0065] Toxicity levels can be measured in vitro or in vivo as described herein. For example, by using cell-based assays, such as LDH assays based on MDCK.2 cells, ACHN cells, using Chinese hamster ovary (CHO) cells (e.g., CHO cells expressing green fluorescent protein (GFP), human MAL (CHO-hMAL), sheep MAL (CHO-sMAL), dog MAL (CHO-dMAL), etc.). Toxicity studies can also be performed in vivo, for example, in mice or any other suitable animals.

[0066] When administered to a subject, such as a human or non-human animal (e.g., lamb, sheep, goat, pig, cattle, horse or rabbit), the polypeptides of the invention provide protection to the subject from developing a disease caused by infection with Clostridium perfringens and / or caused by the presence of active epsilon toxin (or associated with the presence of Clostridium perfringens or said toxin) (e.g., enterotoxemia) and / or the development of a condition associated with demyelination. This protection may be partial, thereby reducing the probability of an individual subject in the population developing the disease, or it may be complete, such that 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%).

[0067] The term "subject" as used throughout this specification, for any aspect of the invention, refers to any human or animal individual, including but not limited to cats, dogs or horses, or ruminants, cattle, sheep, goats or pigs. The human or animal may 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 condition characterized by increased or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0068] As further outlined below, sequence identity can be determined using the Needleman-Wunsch global sequence alignment tool, available from the National Center for Biotechnology Information (NCBI) in Bethesda, Maryland, USA, for example, at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using default parameter settings. When comparing sequence identity levels to, for example, SEQ ID NO:65, it should generally be done relative to the entire length of SEQ ID NO:65 to avoid short regions of high identity that overlap, thereby resulting in a high overall assessed identity (i.e., using a global alignment approach). For example, a short polypeptide fragment of, for example, five amino acids may have a 100% identical sequence to a five amino acid region throughout 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 identical 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: 65, and positions 1-45 and positions 306-328 of SEQ ID NO: 11 are not present in SEQ ID NO: 65. SEQ ID NO: 65 is a sequence identical to (or corresponding to) positions 46-305 of SEQ ID NO: 11, and positions 46-305 of SEQ ID NO: 11 are identical to (or corresponding to) SEQ ID NO: 65.

[0069] Thus, the skilled artisan can readily determine equivalent positions between two sequences by aligning the sequences to maximize amino acid identity at as many positions as possible, for example by using a global sequence alignment program such as available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, discussed further below.

[0070] The present invention also encompasses polypeptides comprising the polypeptide variants and methods of using the variant polypeptides. As used herein, a "variant" refers to a polypeptide whose amino acid sequence is different from the base sequence from which it is derived, and one or more amino acids in the sequence are replaced by other amino acids. The variant is a functional variant because the functional characteristics of the polypeptide from which the variant is derived are maintained. For example, the variant polypeptide may have a similar ability to bind to an antibody that can bind to a non-variant polypeptide (e.g., as a non-limiting example, any one of SEQ ID NO: 18 to SEQ ID NO: 50 in Table 3). In particular, any amino acid substitution, addition or deletion does not necessarily change or significantly change the tertiary structure of one or more epitopes contained in the polypeptide from which the variant is derived, so 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 technician can easily determine a suitable functional variant and determine the tertiary structure of the epitope and any changes thereto without inventive effort.

[0071] Amino acid substitutions may be considered "conservative" where one amino acid is replaced with a different amino acid having generally similar properties. Non-conservative substitutions are where an amino acid is replaced with an amino acid of a different type.

[0072] A "conservative substitution" is one in which an amino acid is replaced by another amino acid of the same class, wherein the classes are defined as follows:

[0073]

[0074] As is well known to those skilled in the art, altering the primary structure of a polypeptide by conservative substitutions may not significantly alter the activity of the polypeptide, because the amino acid side chains in the inserted sequence may be able to form similar bonds and contacts as the replaced amino acid side chains, even when the substitutions are in key regions that determine the conformation of the polypeptide.

[0075] As noted above, non-conservative substitutions are possible so long as such substitutions do not disrupt the tertiary structure of the epitope within the polypeptide, eg, do not disrupt the immunogenicity (eg, antigenicity) of the polypeptide.

[0076] In a broad sense, less non-conservative substitutions are possible without changing the biological activity of the polypeptide. As mentioned above, suitably, the variant can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the basic sequence.

[0077] As briefly mentioned, the sequence identity between amino acid sequences can be determined by comparing the sequences. When the equivalent position in the compared sequences is occupied by the same amino acid, the molecules are identical at that position. The comparison score is a function of the number of identical amino acids at the positions common to the compared sequences as a percentage of identity. When comparing sequences, the best alignment may need to introduce a gap into one or more sequences to take into account possible insertions and deletions in the sequence. The sequence comparison method can use a gap penalty so that for the same number of identical molecules in the compared sequences, a sequence alignment with as few gaps as possible (reflecting the higher correlation between the two compared sequences) obtains a higher score than a sequence alignment with many gaps. The calculation of the maximum percentage identity involves the generation of the best alignment taking into account the gap penalty. As mentioned above, the Needleman-Wunsch global sequence alignment tool should be used to determine the sequence identity percentage, which can be publicly available through http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using default parameter settings. The Needleman-Wunsch algorithm was published in J. Mol. Biol. (1970) vol. 48: 443-553.

[0078] Another aspect of the present invention provides a polynucleotide having a nucleic acid sequence encoding a polypeptide according to the first aspect of the present invention. The present invention also includes variant nucleic acids encoding polypeptides of the present invention. The term "variant" with respect to a nucleic acid sequence refers to any replacement, variation, modification, deletion or any addition of one or more nucleic acids to a polynucleotide sequence relative to a polynucleotide sequence, so that the resulting polypeptide sequence encoded by the polynucleotide provided exhibits at least the same properties as the polypeptide encoded by the basic sequence. Therefore, the term includes allelic variants, and also includes polynucleotides ("probe sequences") that substantially hybridize with the polynucleotide sequence of the present invention. This hybridization can occur under low stringency conditions and high stringency conditions or between low stringency conditions and high stringency conditions. In general, low stringency conditions can be defined as a washing step in a 0.330M to 0.825M NaCl buffer at a temperature lower than the calculated melting temperature or actual melting temperature (T m ) at a temperature 40°C to 48°C lower (e.g., from about ambient laboratory temperature to about 55°C), while high stringency conditions involve hybridization in a 0.0165M to 0.0330M NaCl buffer at a temperature lower than the calculated T of the probe sequence. m Or actual T mThe washing is carried out at a temperature of about 5°C to 10°C lower (e.g., about 65°C). The buffer solution can be, for example, SSC buffer (0.15M NaCl and 0.015M 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: alaboratory manual", 3rd Edition, Cold Spring Harbor Laboratory Press. 30 New York).

[0079] The polypeptides and nucleic acids of the present invention can be synthesized using conventional synthesizers. Alternatively, they can be produced using recombinant DNA technology and can be incorporated into a suitable expression vector, which is then used to transform a suitable host cell, such as a prokaryotic cell such as Escherichia coli (E. coli). The transformed host cell is cultured and the polypeptide is isolated therefrom.

[0080] Therefore, the present invention also provides vectors comprising such polynucleotides. This includes recombinant constructs containing one or more of the above-mentioned nucleic acid molecules. The construct includes a vector, such as a plasmid vector or a viral vector, into which the nucleic acid molecule of the present invention is inserted in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct also includes a regulatory sequence, including, for example, a promoter operably connected 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 describe suitable cloning vectors and expression vectors for prokaryotic and eukaryotic hosts.

[0081] 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, in some embodiments from a serotype Salmonella typhimurium. Salmonella can be an attenuated strain. Strains x8914 and x9241 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, in particular not a human stem cell, such as a human embryonic stem cell.

[0082] Another aspect of the present invention provides an affinity agent that is capable of binding to one of the polypeptides according to the first aspect of the present invention and promoting an immune response in an individual to which the affinity agent is administered. For example, the affinity agent may be an antibody, which may be a monoclonal antibody or a synthetic antibody, Molecules or other antibody mimetics, aptamers, protein scaffolds or major histocompatibility complex (MHC) proteins or parts thereof. The affinity agent may be an antibody raised against a polypeptide according to the first aspect of the invention.

[0083] Another aspect of the present invention provides a subunit vaccine or a combined vaccine containing 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 a recombinant virus vaccine.

[0084] Another aspect of the present invention provides a method for preparing a vaccine, comprising altering an Etx polypeptide or a vaccine or candidate vaccine 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.

[0085] The vaccine or candidate vaccine for improvement (by modification of domain III compared to the wild-type polypeptide) can be selected from any of the following, which shows reduced toxicity after modification of domain III compared to the original vaccine or candidate vaccine. 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 with 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. (2014) Hum Vaccin 25 Immunother 9: 2386-92 describes F199E and H106P mutants (F199 in domain 1, H106 in domain 2); al. (1998) Microbiology 144: 333-41 described the H106P mutant and also mentioned that the H149P mutant was non-toxic (H106 was in domain 2); Dorca-Arévalo et al. (2014) PLoS One 9: e102417 described that the V56C / F118C and H106P mutations were non-toxic to MDCK cells (V56 was in domain 2 and F118 was in domain 2).

[0086] Another aspect of the present invention provides an immunotherapeutic composition comprising: a polypeptide, a polynucleotide, a vector, an affinity reagent, a subunit vaccine and / or a conjugate vaccine according to the aforementioned aspects of the present invention, in the form of a pharmaceutically acceptable formulation. For example, the immunotherapeutic composition may be a vaccine composition comprising a polypeptide according to the first aspect of the present invention and an adjuvant. The composition may also comprise an excipient and / or a diluent suitable for administering the composition to a subject in need of treatment or immunization against a disease caused by Clostridium perfringens and / or Etx disease. The selection of appropriate components is within the routine capabilities of those skilled in the art and does not require creative effort.

[0087] 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 (e.g., salts), preservatives, buffers, and the like. 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, saline solutions, parenteral vehicles (e.g., sodium chloride, Ringer's dextrose), and the like. Preservatives include antimicrobials, antioxidants, chelating agents, and inert gases. The pH and exact concentrations of the various components in the vaccine composition are adjusted according to conventional techniques.

[0088] Optionally, the immunotherapy preparation may include 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 easily available. Methods for coupling peptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-nitrobenzidine.

[0089] In some cases, it may be necessary to formulate an immunotherapeutic composition containing 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. Montanide, provided by SEPPIC Inc (New Jersey, USA) TM Adjuvants of adjuvants, such as Montanide TM ISA 61VG may also be a suitable adjuvant. The inventors have found that the use of Montanide has a significantly better effect than the use of alhydrogel adjuvant. TMISA 61VG adjuvant will result in the induction of better antibody responses. In addition to the adjuvant, it may be desirable to co-administer a biological response modifier (BRM) with the peptide or variant or derivative to downregulate suppressive T cell activity.

[0090] Possible carriers for administration of immunotherapeutic preparations 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, are generally safe to administer and are biodegradable. The technology for preparing liposomes and the technology for preparing (e.g., encapsulating) various molecules (including peptides and oligonucleotides) with liposomes is well known.

[0091] Depending on the method of preparation, liposomes can be monolayer or multilayer and can vary in size, ranging from 0.02 μm to greater than 10 μm in diameter. Liposomes can also be adsorbed onto almost any type of cell and then release the encapsulated agent. Alternatively, the liposomes fuse with the target cells, thereby emptying the contents of the liposomes into the target cells. Alternatively, the adsorbed liposomes can be endocytosed by phagocytes. Endocytosis is followed by intralysosomal degradation of the liposomal lipids and release of the encapsulated agent. In this article, the polypeptide according to the present invention can be positioned on the surface of the liposome to promote antigen presentation without destroying the liposomes or endocytosis. However, regardless of the mechanism or delivery, the result is the intracellular placement of the relevant polypeptide.

[0092] Liposome carriers can be anionic or cationic. Anionic liposome carriers include pH-sensitive liposomes that disrupt or fuse with the endosomal membrane after 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 drugs, such as peptides.

[0093] Other suitable liposomes for use in the methods of the invention include multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium-sized unilamellar vesicles (MIN), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multilamellar vesicles (MVV), unilamellar vesicles or oligolamellar vesicles (REV) prepared by reverse phase evaporation, multilamellar vesicles (MLV-REV) prepared by reverse phase evaporation, stable multilamellar vesicles (plurilamellar vesicles) (SPLV), freeze-thaw MLV (FATMLV), liposomes prepared by extrusion (VET), liposomes prepared by French press (FPV), liposomes prepared by fusion (FUV), dehydration-rehydration liposomes (DRV) and bubblesomes (BSV). The techniques for preparing these liposomes are well known in the art.

[0094] Other forms of delivery particles, such as microspheres, have also been considered for the delivery of peptide epitopes or multiple epitopes.

[0095] Alternatively, nucleic acid-based vaccines can be produced comprising nucleic acids, such as DNA or RNA, encoding an immunologically active peptide epitope or polytopes and cloned into a suitable vector, such as a vector of vaccinia, canarypox, adenovirus or other eukaryotic virus.

[0096] Alternatively, the polypeptide can be administered in the form of a cellular vaccine by administering autologous or allogeneic APC cells or dendritic cells that have been treated in vitro so that the peptide is presented on its surface. Mutations carried by Salmonella enterica or Escherichia coli strains can reduce their virulence and allow them to colonize in host animals without causing disease, and can be used to deliver vaccine antigens, especially for non-human animal administration. The bacteria used may include strains that have been used as vaccines in livestock, where attenuated lesions are not fully characterized. In addition, as described in WO2013 / 144636, strains whose virulence has been reasonably weakened by intentionally introducing mutations into bacteria can be used to deliver polypeptides. Antigens can also be delivered as naked DNA vaccines, in which the gene encoding the ε toxoid is cloned into a mammalian expression vector and expressed from a eukaryotic promoter.

[0097] The most widely studied class of attenuated Salmonella used as carriers of foreign antigens is auxotrophy. Genetically defined mutants of the aroA gene encoding 5-enolpyruvate-3-phosphate synthase have been constructed in S. enterica var. Typhimurium and S. 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 group of well-studied attenuated Salmonella is a mutant with a defined deletion in a gene involved in the regulation of Salmonella virulence. For example, mutations in genes encoding adenylate cyclase (CyA) and cyclic adenosine monophosphate receptor protein (CRP) can affect the expression of the genes involved.

[0098] If the vaccine composition is for administration to human subjects, it can be in a form suitable for oral administration (e.g., in a dietary supplement) and / or parenteral administration (e.g., by injection, inhalation, or transdermal administration via a patch, lotion, or gel). The specific forms outlined above are also generally useful for administration to human subjects.

[0099] The polypeptides, polynucleotides, vectors, subunit vaccines, conjugate vaccines, antibodies, affinity agents, vaccine compositions and / or immunotherapeutic compositions according to the present invention can be used in methods for treating or vaccinating a subject against a disease caused by Clostridium perfringens and / or Etx, the disease, for example, involving the accumulation of epsilon toxin (particularly active epsilon toxin lacking the N-terminus and C-terminus of the full-length protoxin) released by Clostridium perfringens in the blood of the subject; and / or the polypeptides, polynucleotides, vectors, subunit vaccines, conjugate vaccines, antibodies, affinity agents, vaccine compositions and / or immunotherapeutic compositions according to the present invention can be used in methods for treating or vaccinating a subject against a disease associated with Clostridium perfringens infection or associated with the presence of Etx or the development of a disease associated with demyelination. According to one embodiment, the disease is enterotoxemia or a demyelinating disease, such as multiple sclerosis, neuromyelitis optica (NMO), optic neuritis (ON) or myelitis.

[0100] The present invention also provides a method for vaccinating a subject against a disease caused by Clostridium perfringens and / or caused by ε toxin (particularly active toxin) or a disease associated with Clostridium perfringens infection or associated with the presence of Etx, or the occurrence of a condition associated with demyelination, the method comprising administering to the subject a polypeptide, polynucleotide, vector, subunit vaccine, conjugate vaccine, affinity reagent, vaccine composition and / or immunotherapeutic agent (e.g., in a protective amount) according to the present invention. The subject can be a human or a non-human animal. The non-human animal can be a horse or a ruminant (e.g., a sheep, a pig or a goat), or a bovine animal (e.g., a domesticated cattle) or a companion animal (e.g., a dog, a cat or a rabbit). Young animals, such as lambs, piglets, children and calves are also included.

[0101] A "protective amount" is an amount sufficient to induce an immune response in a subject, thereby reducing or eliminating the probability of the subject developing a disease caused by Clostridium perfringens (e.g., caused by (or associated with the presence of) epsilon toxin, particularly active toxin). For example, an antibody capable of binding to SEQ ID NO: 65 and / or SEQ ID NO: 14 may be detectable after administration, wherein such an antibody is not detectable before administration, or is detectable only at a lower concentration than after administration.

[0102] The present invention also provides a kit comprising a polypeptide, polynucleotide, vector, subunit vaccine, conjugate vaccine, affinity agent, vaccine composition and / or immunotherapeutic agent according to the present invention, the kit having, for example, use in a method of treating or vaccinating a subject to combat a disease caused by infection with Clostridium perfringens and / or Etx; or the kit having use in a method of treating or vaccinating a subject to combat a disease associated with infection with Clostridium perfringens or a disease associated with the presence of Etx or a condition associated with the presence of demyelination. The kit may include a means for administering the polypeptide, polynucleotide, vector, subunit vaccine, conjugate vaccine, affinity agent, vaccine composition and / or immunotherapeutic agent to an individual. For example, the kit may include one or more buffers or diluents and / or one or more administration devices, such as a syringe or other injection device. The kit may alternatively or additionally include instructions that enable a user to perform a method of treating or vaccinating a subject against a disease caused by or associated with Clostridium perfringens and / or Etx and / or 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 condition characterized by increased or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0103] As described herein, the disease caused by or associated with Clostridium perfringens and / or (active) epsilon toxin can be, for example, enterotoxemia, including pre-disease symptoms (such as systemic lesions and enterocolitis). Other symptoms can include edema of the main target organs kidney and brain and damage to vascular endothelial cells. The terminal stage of enterotoxemia is characterized by severe neurological diseases, including opisthotonos, epilepsy and painful struggles. The disease can be a demyelinating disease, such as multiple sclerosis (MS), clinically diagnosed MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica spectrum disease (NMOSD), optic neuritis (ON), neuromyelitis optica (NMO), myelitis, transverse myelitis (TM), with aquaporin-4 (AQP-4) antibodies increase or presence and / or astrocyte damage as a disease or illness characterized by, and acute disseminated encephalomyelitis (ADEM).

[0104] In the description and claims of the present application, the words "comprise" and "include" and variations of the words, such as "comprising" and "comprises", mean "including but not limited to", and do not exclude other parts, additives, components, integers or steps.

[0105] In the description and claims of this specification, the singular includes the plural, unless the context requires otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating both the plural and the singular, unless the context requires otherwise.

[0106] Preferred features of each aspect of the invention may be as described in connection with any other aspect.

[0107] 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 text of this application (including the attached claims and drawings). Therefore, the features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or embodiment of the present invention should be understood to be applicable to any other aspect, embodiment or embodiment described herein, unless incompatible therewith.

[0108] Furthermore, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or similar purpose. Sequence Listing <110> Yikang Joint Venture Co., Ltd. <120> Epsilon toxin from Clostridium perfringens as a vaccine <130> P3260PC00 <150> GB1803401.7 <151> 2018-03-02 <150> GB1900361.5 <151> 2019-01-10 <160> 68 <170> PatentIn version 3.5 <210> 1 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Protein fragments <400> 1 Val Tyr Val Gly Lys Ala Leu Leu Thr Asn Asp Thr Gln Gln Glu Gln 1 5 10 15 Lys Leu Lys Ser Gln Ser Phe Thr Cys Lys 20 25 <210> 2 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Protein fragments <400> 2 Thr His Asn Val Pro Ser Gln Asp Ile Leu Val Pro Ala Asn Thr Thr 1 5 10 15 Val Glu Val Ile Ala Tyr Leu Lys 20 <210> 3 <211> 47 <212> PRT <213> Artificial sequence <220> <223> Protein fragments <400> 3 Asp Glu Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln 1 5 10 15 Glu Tyr Val Ile Pro Val Asp Lys Lys Glu Lys Ser Asn Asp Ser Asn 20 25 30 Ile Val Lys Tyr Arg Ser Leu Tyr Ile Lys Ala Pro Gly Ile Lys 35 40 45 <210> 4 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Protein fragment mutant <220> <221> XA <222> (6) <223> Any amino acid except Tyr, or missing <400> 4 Arg Met Glu Lys Tyr Xaa Pro Asn Ala Met 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Protein fragment mutant <220> <221> XA <222> (6) <223> Any amino acid except Tyr, or missing <400> 5 Gly Glu Ile Pro Ser Xaa Leu Ala Phe Pro 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Protein fragment mutant <220> <221> XA <222> (6) <223> Any amino acid except His, or missing <400> 6 Ser Lys Glu Ile Thr Xaa Asn Val Pro Ser 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Protein fragment mutant <220> <221> XA <222> (6) <223> Any amino acid except Val, or missing <400> 7 Leu Glu Asp Val Tyr Xaa Gly Lys Ala Leu 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Protein fragment mutant <220> <221> XA <222> (6) <223> Any amino acid except Phe, or its absence <400> 8 Leu Lys Ser Gln Ser Xaa Thr Cys Lys Asn 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Protein fragment mutant <220> <221> XA <222> (6) <223> Any amino acid except Val, or missing <400> 9 Asn Thr Thr Val Glu Xaa Ile Ala Tyr Leu 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Protein fragment mutant <220> <221> XA <222> (6) <223> Any amino acid except Ala, or its absence <400> 10 Thr Val Glu Val Ile Xaa Tyr Leu Lys Lys 1 5 10 <210> 11 <211> 328 <212> PRT <213> Clostridium perfringens <400> 11 Met Lys Lys Asn Leu Val Lys Ser Leu Ala Ile Ala Ser Ala Val Ile 1 5 10 15 Ser Ile Tyr Ser Ile Val Asn Ile Val Ser Pro Thr Asn Val Ile Ala 20 25 30 Lys Glu Ile Ser Asn Thr Val Ser Asn Glu Met Ser Lys Lys Ala Ser 35 40 45 Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly Arg Tyr Asn Thr Lys 50 55 60 Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr Pro Asn Ala Met Ala 65 70 75 80 Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly Asn Asp Phe Tyr Ile 85 90 95 Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro Ser Met Asn Tyr Leu 100 105 110 Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr Asn Asp Thr Gln Gln 115 120 125 Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys Lys Asn Thr Asp Thr 130 135 140 Val Thr Ala Thr Thr Thr His Thr Val Gly Thr Ser Ile Gln Ala Thr 145 150 155 160 Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly Val Ser Leu Thr Thr 165 170 175 Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr Asn Ser Lys Glu Ile 180 185 190 Thr His Asn Val Pro Ser Gln Asp Ile Leu Val Pro Ala Asn Thr Thr 195 200 205 Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn Val Lys Gly Asn Val 210 215 220 Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp Gly Glu Ile Pro Ser 225 230 235 240 Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe Ser Leu Ser Asp Thr 245 250 255 Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr Ile Asn Ile Asn Gly 260 265 270 Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu Leu Ile Val Lys Val 275 280 285 Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr Val Ile Pro Val Asp 290 295 300 Lys Lys Glu Lys Ser Asn Asp Ser Asn Ile Val Lys Tyr Arg Ser Leu 305 310 315 320 Ser Ile Lys Ala Pro Gly Ile Lys 325 <210> 12 <211> 296 <212> PRT <213> Artificial sequence <220> <223> Constructs used for crystal structure analysis <400> 12 Lys Glu Ile Ser Asn Thr Val Ser Asn Glu Met Ser Lys Lys Ala Ser 1 5 10 15 Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly Arg Tyr Asn Thr Lys 20 25 30 Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr Pro Asn Ala Met Ala 35 40 45 Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly Asn Asp Phe Tyr Ile 50 55 60 Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro Ser Met Asn Tyr Leu 65 70 75 80 Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr Asn Asp Thr Gln Gln 85 90 95 Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys Lys Asn Thr Asp Thr 100 105 110 Val Thr Ala Thr Thr Thr His Thr Val Gly Thr Ser Ile Gln Ala Thr 115 120 125 Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly Val Ser Leu Thr Thr 130 135 140 Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr Asn Ser Lys Glu Ile 145 150 155 160 Thr His Asn Val Pro Ser Gln Asp Ile Leu Val Pro Ala Asn Thr Thr 165 170 175 Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn Val Lys Gly Asn Val 180 185 190 Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp Gly Glu Ile Pro Ser 195 200 205 Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe Ser Leu Ser Asp Thr 210 215 220 Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr Ile Asn Ile Asn Gly 225 230 235 240 Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu Leu Ile Val Lys Val 245 250 255 Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr Val Ile Pro Val Asp 260 265 270 Lys Lys Glu Lys Ser Asn Asp Ser Asn Ile Val Lys Tyr Arg Ser Leu 275 280 285 Ser Ile Lys Ala Pro Gly Ile Lys 290 295 <210> 13 <211> 263 <212> PRT <213> Clostridium perfringens <400> 13 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 14 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 14 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 15 <211> 315 <212> PRT <213> artificial sequence <220> <223> recombinant polypeptide <400> 15 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Met Gly Lys Ala Ser Tyr Asp Asn Val Asp 20 25 30 Thr Leu Ile Glu Lys Gly Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys 35 40 45 Arg Met Glu Lys Tyr Tyr Pro Asn Ala Met Ala Tyr Phe Asp Lys Val 50 55 60 Thr Ile Asn Pro Gln Gly Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val 65 70 75 80 Glu Leu Asp Gly Glu Pro Ser Met Asn Tyr Leu Glu Asp Val Tyr Val 85 90 95 Gly Lys Ala Leu Leu Thr Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys 100 105 110 Ser Gln Ser Phe Thr Cys Lys Asn Thr Asp Thr Val Thr Ala Thr Thr 115 120 125 Thr His Thr Val Gly Thr Ser Ile Gln Ala Thr Ala Lys Phe Thr Val 130 135 140 Pro Phe Asn Glu Thr Gly Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala 145 150 155 160 Asn Thr Asn Thr Asn Thr Asn Ser Lys Glu Ile Thr His Asn Val Pro 165 170 175 Ser Gln Asp Ile Leu Val Pro Ala Asn Thr Thr Val Glu Val Ile Ala 180 185 190 Tyr Leu Lys Lys Val Asn Val Lys Gly Asn Val Lys Leu Val Gly Gln 195 200 205 Val Ser Gly Ser Glu Trp Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro 210 215 220 Arg Asp Gly Tyr Lys Phe Ser Leu Ser Asp Thr Val Asn Lys Ser Asp 225 230 235 240 Leu Asn Glu Asp Gly Thr Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser 245 250 255 Ala Val Met Gly Asp Glu Leu Ile Val Lys Val Arg Asn Leu Asn Thr 260 265 270 Asn Asn Val Gln Glu Tyr Val Ile Pro Val Asp Lys Lys Glu Lys Ser 275 280 285 Asn Asp Ser Asn Ile Val Lys Tyr Arg Ser Leu Tyr Ile Lys Ala Pro 290 295 300 Gly Ile Lys Leu Glu His His His His His His 305 310 315 <210> 16 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 16 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 17 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 17 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 18 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 18 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 19 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 19 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 20 <211> 263 <212> PRT <213> artificial sequence <220> <223> Mutant polypeptide <400> 20 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 21 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 21 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 22 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 22 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 23 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 23 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 24 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 24 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 25 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 25 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 26 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 26 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 27 <211> 263 <212> PRT <213> artificial sequence <220> <223> Mutant polypeptide <400> 27 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 28 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 28 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 29 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 29 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 30 <211> 263 <212> PRT <213> Artificial sequence <220> <223> Mutant polypeptide <400> 30 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 31 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 31 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 32 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 32 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 33 <211> 263 <212> PRT <213> Artificial Sequence <220> <223> Mutant Polypeptide <400> 33 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 34 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 34 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 35 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 35 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 36 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 36 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 37 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 37 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 38 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 38 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 39 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 39 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 40 <211> 263 <212> PRT <213> artificial sequence <220> <223> Mutant polypeptide <400> 40 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 41 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 41 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 42 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 42 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 43 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 43 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 44 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 44 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 45 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 45 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Ala 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Ala Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 46 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 46 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Phe Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 47 <211> 263 <212> PRT <213> artificial sequence <220> <223> Mutant polypeptide <400> 47 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Ala Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 48 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 48 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr Ala Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 49 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 49 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Ala Ile Ala Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 50 <211> 263 <212> PRT <213> artificial sequence <220> <223> mutant polypeptide <400> 50 Met Gly Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly 1 5 10 15 Arg Tyr Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr 20 25 30 Pro Asn Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly 35 40 45 Asn Asp Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro 50 55 60 Ser Met Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr 65 70 75 80 Asn Asp Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys 85 90 95 Lys Asn Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr 100 105 110 Ser Ile Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly 115 120 125 Val Ser Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr 130 135 140 Asn Ser Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val 145 150 155 160 Pro Ala Asn Thr Thr Val Glu Val Ile Phe Tyr Leu Lys Lys Val Asn 165 170 175 Val Lys Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp 180 185 190 Gly Glu Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe 195 200 205 Ser Leu Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr 210 215 220 Ile Asn Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu 225 230 235 240 Leu Ile Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr 245 250 255 Val Ile Pro Val Asp Lys Lys 260 <210> 51 <211> 83 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 51 gaaaggaaga tataatacaa aatataatta cttaaagaga atggaaaaat atgcgcctaa 60 tgctatggca tattttgata agg 83 <210> 52 <211> 83 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 52 ccttatcaaa atatgccata gcattaggcg catatttttc cattctcttt aagtaattat 60 attttgtatt atatcttcct ttc 83 <210> 53 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 53 gtgaatgggg agagatacct agtgcgttag cttttcctag ggatggtta 49 <210> 54 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 54 taaccatccc taggaaaagc taacgcacta ggtatctctccccattcac 49 <210> 55 <211> 65 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 55 caaatacaaa tacaaatact aattcaaaag aaattactgc taatgtccct tcacaagata 60 tacta 65 <210> 56 <211> 65 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 56 tagtatatct tgtgaaggga cattagcagt aatttctttt gaattagtat ttgtatttgt 60 atttg 65 <210> 57 <211> 54 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 57 agaaccatca atgaattatc ttgaagatgt ttattttgga aaagctctct taac 54 <210> 58 <211> 54 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 58 gttaagagag cttttccaaa ataaacatct tcaagataat tcattgatgg ttct 54 <210> 59 <211> 76 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 59 tcttaactaa tgatactcaa caagaacaaa aattaaaatc acaatcagcg acttgtaaaa 60 atactgatac agtaac 76 <210> 60 <211> 76 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 60 gttactgtat cagtattttt acaagtcgct gattgtgatt ttaatttttg ttcttgttga 60 gtatcattag ttaaga 76 <210> 61 <211> 64 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 61 atactagtac cagctaatac tactgtagaa gcgatagcat atttaaaaaa agttaatgtt 60 aaag 64 <210> 62 <211> 64 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 62 ctttaacatt aacttttttt aaatatgcta tcgcttctac agtagtatta gctggtacta 60 gtat 64 <210> 63 <211> 82 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 63 gatatactag taccagctaa tactactgta gaagtaatattttatttaaa aaaagttaat 60 gttaaaggaa atgtaaagtt ag 82 <210> 64 <211> 82 <212> DNA <213> Artificial sequence <220> <223> Primer sequences <400> 64 ctaactttac atttccttta acattaactt tttttaaata aaatattact tctacagtag 60 tattagctgg tactagtata tc 82 <210> 65 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Protein Fragment <400> 65 Lys Ala Ser Tyr Asp Asn Val Asp Thr Leu Ile Glu Lys Gly Arg Tyr 1 5 10 15 Asn Thr Lys Tyr Asn Tyr Leu Lys Arg Met Glu Lys Tyr Tyr Pro Asn 20 25 30 Ala Met Ala Tyr Phe Asp Lys Val Thr Ile Asn Pro Gln Gly Asn Asp 35 40 45 Phe Tyr Ile Asn Asn Pro Lys Val Glu Leu Asp Gly Glu Pro Ser Met 50 55 60 Asn Tyr Leu Glu Asp Val Tyr Val Gly Lys Ala Leu Leu Thr Asn Asp 65 70 75 80 Thr Gln Gln Glu Gln Lys Leu Lys Ser Gln Ser Phe Thr Cys Lys Asn 85 90 95 Thr Asp Thr Val Thr Ala Thr Thr Thr His Thr Val Gly Thr Ser Ile 100 105 110 Gln Ala Thr Ala Lys Phe Thr Val Pro Phe Asn Glu Thr Gly Val Ser 115 120 125 Leu Thr Thr Ser Tyr Ser Phe Ala Asn Thr Asn Thr Asn Thr Asn Ser 130 135 140 Lys Glu Ile Thr His Asn Val Pro Ser Gln Asp Ile Leu Val Pro Ala 145 150 155 160 Asn Thr Thr Val Glu Val Ile Ala Tyr Leu Lys Lys Val Asn Val Lys 165 170 175 Gly Asn Val Lys Leu Val Gly Gln Val Ser Gly Ser Glu Trp Gly Glu 180 185 190 Ile Pro Ser Tyr Leu Ala Phe Pro Arg Asp Gly Tyr Lys Phe Ser Leu 195 200 205 Ser Asp Thr Val Asn Lys Ser Asp Leu Asn Glu Asp Gly Thr Ile Asn 210 215 220 Ile Asn Gly Lys Gly Asn Tyr Ser Ala Val Met Gly Asp Glu Leu Ile 225 230 235 240 Val Lys Val Arg Asn Leu Asn Thr Asn Asn Val Gln Glu Tyr Val Ile 245 250 255 Pro Val Asp Lys 260 <210> 66 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Protein fragments <400> 66 Met Lys Lys Asn Leu Val Lys Ser Leu Ala Ile Ala Ser Ala Val Ile 1 5 10 15 Ser Ile Tyr Ser Ile Val Asn Ile Val Ser Pro Thr Asn Val Ile Ala 20 25 30 <210> 67 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Protein fragments <400> 67 Lys Glu Ile Ser Asn Thr Val Ser Asn Glu Met Ser Lys 1 5 10 <210> 68 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Protein fragments <400> 68 Lys Glu Lys Ser Asn Asp Ser Asn Ile Val Lys Tyr Arg Ser Leu Tyr 1 5 10 15 Ile Lys Ala Pro Gly Ile Lys 20 BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Now refer to Figures 1 to 5 Embodiments of the present invention are described by way of example only, in which:

[0110] Figure 1 is a schematic diagram of a recombinant epsilon protoxin (P-Etx) in which the N-terminal PeIB leader peptide replaces the 13 amino acid N-terminal peptide sequence and has a C-terminal His-tag to aid purification (the amino acid sequence surrounding the processing site is also shown);

[0111] Figure 2 The Etx amino acid sequence is shown with the signal sequence (residues 1-32), N-terminal propeptide (residues 33-45), 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 acid shown below the mutated position;

[0112] Figure 3A Five residues flanking the glycan (β-octyl-glucoside) binding site in Etx domain III identified for mutagenesis (proposed second receptor binding site): V72, F92, H149, V166, A168. Residue positions previously mutated (shown in dark grey and circled (top); Y30 and Y196) and residues in the β-octyl-glucoside binding cleft (V72, F92, H149, V166, A168; shown in light grey and circled (bottom);

[0113] Figure 3B SDS-PAGE gels of purified proteins tested in this study are shown. 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 in E. coli and purified;

[0114] Figure 4 The ability of rabbit sera against WHO standard antitoxin (5 IU / ml) or against genotoxin to displace neutralizing monoclonal antibody binding in a competitive ELISA is shown. Sera were diluted as indicated in the figure legend prior to testing. Data shown are the average of two assays, with SEM bars shown.

[0115] Figure 5 The melting temperatures (T) of the wild-type and variant proteins are shown. m ). 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.

[0116] Figure 6 The results of human erythrocytes treated with epsilon toxin (wild-type Etx; Y30AY196A; Y30AY196A+H149A; Y30AY196A+A168F; Y30AY196A+F92A; Y30AY196A+V166A; and quad mutation: Y30AY196+A168F+H149A) are shown. Y30AY196A+A168F, Y30AY196A+H149A and quad mutation are not hemolytic even upon trypsin activation.

[0117] project

[0118] The present invention will now be described with reference to the following items, wherein:

[0119] 1. A method for preventing or treating a demyelinating disorder in a human or animal subject in need thereof, the disorder being selected from the group consisting of: enterotoxemia (ET), multiple sclerosis (MS), clinically definite MS (CDMS), clinically isolated syndrome (CIS), neuromyelitis optica spectrum disorder (NMOSD), optic neuritis (ON), neuromyelitis optica (NMO), myelitis, myelitis, transverse myelitis (TM), a disease or disorder characterized by increased or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM), comprising: administering to the subject a composition comprising an effective amount of an agent that directly or indirectly interferes with epsilon toxin (Etx) produced by a type B or D strain of Clostridium perfringens, an Etx-binding receptor, or the interaction of Etx with its binding receptor, thereby inhibiting or repressing Etx-regulated receptor signaling activity.

[0120] 2. The method according to item 1, wherein the agent is an inhibitor of Etx, such as an antibody or a functional component thereof.

[0121] 3. The method according to item 1, wherein the agent is an inhibitor or antagonist of Etx binding receptor.

[0122] 4. The method according to item 3, wherein the Etx binding receptor is myelin and lymphocyte protein (MAL) or hepatitis A virus cell receptor 1 protein (HAVCR1).

[0123] 5. The method according to item 1, wherein the agent is a vaccine against Clostridium perfringens type B or type D strains or against epsilon toxin (Etx) produced thereby.

[0124] 6. The method according to any of the preceding claims, wherein the agent comprises an epsilon toxin (Etx) polypeptide having reduced toxicity to cells expressing myelin and lymphocyte protein (MAL) and comprising 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 a sequence represented by SEQ ID NO: 65 and / or SEQ ID NO: 14.

[0125] 7. The method according to item 6, wherein the modified domain III is a modification in the glycan (β-octyl-glucoside) binding site of domain III.

[0126] 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 of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 constituting domain III.

[0127] 9. The method according to any one of items 6 to 8, comprising 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.

[0128] 10. The method according to any one of items 6 to 9, comprising SEQ ID NO: 4 and / or SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0129] 11. The method according to any one of items 6 to 10, comprising at least the following sequences: i) SEQ ID NO: 4 and SEQ ID NO: 5;

[0130] and optionally, in addition to (i), also includes

[0131] ii) SEQ ID NO: 6; and

[0132] iii) one or more of the following: SEQ ID NO:10, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0133] 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.

[0134] 13. The method according to any one of items 6 to 12, having at least 60% sequence identity with any one of SEQ ID NO: 18 to SEQ ID NO: 50, and comprising or consisting of one or more mutations indicated in Table 3 for the relevant SEQ ID NO.

[0135] 14. A polynucleotide having a nucleic acid sequence encoding the polypeptide according to any one of the preceding items.

[0136] 15. A vector comprising the polynucleotide according to item 14 or the agent according to any one of items 1 to 5.

[0137] 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.

[0138] 17. A subunit vaccine or conjugate vaccine comprising the agent according to any one of items 1 to 5, and the polypeptide according to any one of items 6 to 13.

[0139] 18. An affinity agent capable of binding to the agent according to any one of items 1 to 5, or one of the polypeptides according to any one of items 6 to 13, and promoting an immune response in an individual to which the affinity agent has been administered.

[0140] 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 a wild-type Etx polypeptide.

[0141] 20. An immunotherapeutic composition or vaccine composition prepared by the method according to item 19.

[0142] 21. An immunotherapeutic composition or vaccine composition comprising 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 14.

[0143] 22. 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 or a conjugate vaccine according to item 17 and / or an affinity reagent according to item 18 and / or an immunotherapeutic composition or a vaccine composition according to any one of items 20 to 21, for use in a method for treating or immunizing 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.

[0144] 23. A 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), diseases or conditions characterized by increased or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0145] 24. A method of 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 treating a subject against a demyelinating disease, or a method of immunizing a subject against suffering from 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. The vaccine and / or affinity agent according to item 18 and / or the 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 increased or presence of antibodies to aquaporin-4 (AQP-4) and / or astrocyte damage, and acute disseminated encephalomyelitis (ADEM).

[0146] 25. An agent, polypeptide, polynucleotide, vector, cell, affinity reagent, vaccine composition or immunotherapeutic composition according to any one of items 20 to 21, or a method according to item 24, wherein the subject is a ruminant, a horse, a companion animal or a human.

[0147] 26. Use of MAL cells as a model to test the toxicity of epsilon vaccine candidates.

[0148] 27. A kit comprising 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 immunotherapy composition according to any one of items 20 to 21.

[0149] 28. A polypeptide, polynucleotide, vector, cell, subunit vaccine, conjugate vaccine, affinity agent, vaccine composition or immunotherapeutic composition or method substantially as described herein.

[0150] Example

[0151] Materials and methods

[0152] Chemicals

[0153] All chemicals were obtained from Sigma Chemical Co. (St Louis, MO) unless otherwise stated.

[0154] Expression and purification of recombinant ε toxin wild type and mutants

[0155] The etxD gene encoding epsilon protoxin D from Clostridium perfringens type D strain NCTC 8346 was cloned into the expression vector pET-26b(+) (Merck, Darmstadt, Germany) with an N-terminal PelB leader peptide replacing the 13 amino acid N-terminal peptide sequence (residues KEISNTVSNEMSK) and 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). The amino acid numbering corresponds to the protoxin without the 13 amino acid N-terminal peptide sequence. 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) 3534 Vaccine 32: 2682-7) using the QuickChange Lightning Site-Directed mutagenesis kit (Agilent Technologies, Inc. Santa Clara, US) according to the manufacturer's instructions (amino acid numbering corresponds to the original toxin without the 13 amino acid N-terminal peptide sequence). The primers used for site-directed mutagenesis are shown in Table 4 below. Recombinant proteins were expressed in E. coli Rosetta 2 (DE3) cells (Merck, Darmstadt, Germany) and grown in ZYM-5052 auto-induction 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 hours and then cultured at 20°C, 300 rpm for another 24 hours.

[0156] Table 4 - Primers used for site-directed mutagenesis

[0157]

[0158]

[0159] a The underlined bases are the codons used for the substitution. All primer sequences are shown in the 5' to 3' direction. The amino acid numbering corresponds to the protoxin without the N-terminal peptide sequence.

[0160] Protein purification

[0161] For protein purification, cells were harvested by centrifugation and purified using BugBuster TM Protein extraction reagent (Merck, Darmstadt, Germany) was used for enzymatic cleavage, and Y30A-Y196A and its derivatives were purified by Ni-NTA column (GE Healthcare Life Sciences, Little Chalfont, UK) according to the manufacturer's instructions. For buffer exchange and further sample cleanup, the eluate containing the original toxin was added 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 the BCA assay (Fisher Scientific UK Ltd, Loughborough, UK).

[0162] For studies in mice, rabbits, or sheep, a commercially available kit containing a high-capacity endotoxin removal resin (Pierce TM The protein was treated with 5% paraformaldehyde (HPLC) to remove endotoxin. The residual level of endotoxin was measured using a quantitative chromogenic assay (Pierce LAL Chromogenic Endotoxin Quantitation kit, Thermo Scientific).

[0163] Trypsin activation

[0164] 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. EDTA-free protease inhibitor cocktail (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.

[0165] SDS-PAGE analysis

[0166] Using XCell SureLock TM Protein purity was analyzed by SDS-PAGE on 4-12% Bis-Tris NuPAGE gels (Invitrogen Ltd..Paisley, UK) using a Mini-Cell electrophoresis instrument (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) before loading. The gel was typically run at 200V for 45 minutes. After electrophoretic separation, proteins were displayed by staining with SimplyBlue (Invitrogen Ltd., Paisley, UK). Perfect protein molecular weight standards (Merck, Darmstadt, Germany) were used as markers.

[0167] Thermal stability test

[0168] 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.

[0169] Cell culture

[0170] 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-LGCStandards, 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. Cells were routinely detached by incubation in trypsin / EDTA and split appropriately (usually at a 1:6 dilution).

[0171] Chinese hamster ovary (CHO) cells or CHO cells expressing green fluorescent protein (GFP)-tagged human MAL (CHO-hMAL), sheep MAL (CHO-sMAL) or canine MAL (CHO-dMAL) 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. The cells were routinely detached by incubation in trypsin / EDTA and split appropriately (usually at a 1:6 dilution).

[0172] Cytotoxicity assay

[0173] 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 assay 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 4cells / well). After incubation for 3 hours at 37°C, cell culture medium (50 μL) was collected from the cell monolayer, transferred to a fresh 96-well ELISA plate, and 50 μL of the reconstituted substrate mixture was added to each well. The plate was incubated at room temperature for 30 minutes in the dark. The absorbance was read at 490 nm using a 680 microplate reader (Bio-Rad). The absorbance value of each sample was normalized by subtracting the absorbance value obtained from the culture medium of untreated cells. The toxin dose (CT) required to kill 50% of the cell monolayer was determined by nonlinear regression analysis (GraphPad). 50 ). All experiments were performed three times, each with three technical replicates.

[0174] Immunization of rabbits

[0175] Groups of three New Zealand white rabbits were immunized subcutaneously with 100 μg 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 third booster immunization (day 49) and 7 days after the fourth booster immunization (day 63).

[0176] The WHO international standard Clostridium perfringens epsilon equine antitoxin serum (CPEPAT) was obtained from the National Institute for Biological Products Control (NIBSC, South Mimms, UK).

[0177] Immunization of sheep with Y30AY196A+A168F toxoid

[0178] Lambs were raised without immunization against the epsilon toxin of C. perfringens and tested at intervals for the presence of anti-epsilon toxin antibodies using Western blotting. After 12 months, groups of 5 lambs received no treatment and groups of 6 lambs received 200 μg of Montanide TM Y30AY196A+A168F toxoid (1:1) adjuvanted with ISA 61VG (Seppic, Paris, France), and one group of 5 lambs received 200 μg of Y30AY196A+A168F toxoid adjuvanted with aluminum hydroxide (alhydrogel; Sigma-Aldrich, Poole UK; 0.25% weight / volume final concentration). The inventors also used 200 μg of Y30AY196A+A168F toxoid with Montanide TMA group of 4 lambs with significant levels of pre-existing reactivity to epsilon toxin were immunized with a mixture of ISA 61VG adjuvant (1:1). All adjuvanted mixtures were given subcutaneously in 6 x 0.5 ml doses. Three weeks later, the lambs were given a second dose of adjuvanted protein. Blood samples were collected at the start of the study and at weeks 3, 7 and 12. Immunization of the lambs was performed by Orygen Antibodies Ltd, Penicuik, Scotland.

[0179] Competition ELISA assay for measuring neutralizing antibodies

[0180] Competitive ELISA was performed to measure neutralizing antibodies using the Monoscreen ELISA kit (BioX Diagnostics, BIO K222 / 2) according to the manufacturer's instructions. The absorbance was read at 450 nm and the inhibition was calculated using the following formula:

[0181] Inhibition sample % = [(OD 450nm Negative serum-OD sample) / OD 450nm Negative serum] × 100

[0182] Inhibition positive % = [(OD 450nm Negative serum-OD positive serum) / OD 450nm Negative serum] × 100

[0183] The assay is valid only if the ODnegative-ODpositive is greater than 0.7 and the inhibition of the positive control is greater than 30%.

[0184] Neutralization of cell culture toxicity

[0185] CHO cells expressing human MAL were cultured at 3 × 10 4 The cells were seeded in 96-well plates and left to stand overnight. Pure rabbit serum and 20 IU / ml standard ε antitoxin (National Institute for the Control of Biological Products) were diluted in DPBS in serial dilutions and mixed with an equal volume of trypsin-activated wild-type ε toxin (5×CT 50 ) were incubated at room temperature for 1 hour. CHO hMAL cells were washed twice with serum-free DMEM / F12 and then the toxin:antibody / standard antitoxin mixture was added, along with DPBS-only and toxin-only controls (5×CT 50 After incubation at 37°C in a humidified environment for 3 hours, the medium was replaced with 100 μl of fresh serum-free DMEM / F12 and 10 μl of WST-1 cell proliferation reagent (Abcam). After incubation at 37°C in a humidified environment for 1 hour, the absorbance at 420 nm was read.

[0186] Toxicity in mice

[0187] Groups of six female BALB / c mice were challenged with 100 μl of unactivated or trypsin-activated protein via intraperitoneal (ip) or subcutaneous (sc) routes. The experiment was terminated 24 hours after ip challenge or 7 days after sc challenge. The study was conducted with the approval of the on-site animal ethics committee. Animals were evaluated for neurological symptoms, changes in appearance, or changes in behavior at intervals according to a predetermined scoring matrix. Animals with a combined score of 5 or higher were excluded.

[0188] result

[0189] Mutagenesis of residues flanking the β-octyl-glucoside binding site

[0190] In a previous study, the present inventors identified a glycan (β-octyl-glucoside) binding site in domain III of Etx and proposed that this site could be a second receptor binding site (Bokori-Brown (2013)). In the present study, five residues flanking this site were identified for mutagenesis (V72, F92, H149, V166, A168; Figure 3A ) to assess their role in toxicity. Using a plasmid encoding the Y30A-Y196A variant 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 protein encoded by the mutant gene was expressed in Escherichia coli and purified. The Y30A-Y196A variant of Etx and a mutant containing H149A and A168A in addition to Y30A-Y196A were also expressed and purified. The authenticity of the protein was verified in two ways. First, the gene encoding the mutant gene was sequenced to verify the presence of the expected mutation. Secondly, the purified protein was 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 protein were less than 1 endotoxin unit (EU) / ml for mice and rabbits, and less than 40 endotoxin units (EU) / ml for sheep.

[0191] Thermal stability of proteins

[0192] Thermostability assays were used to assess the structural stability of wild-type ε toxin, Y30A-Y196A variant protein, and four mutants of Y30A-Y196A. This showed that the melting temperature (T m, in °C) is lower than the melting temperature of the wild-type toxin. However, the introduction of additional mutations (V72, F92, H149, V166, and A168) into the Y30A-Y196A variant protein resulted in only minor changes in the thermal stability of the protein ( Figure 5 ), indicating that these substitutions do not destabilize the tertiary structure of the protein. Y30A-Y196A-H149A-A1 68F has the lowest melting temperature, indicating that it is the most unstable of the mutants tested.

[0193] Toxicity of variant proteins in cell culture

[0194] The toxicity of the purified Etx protein activated by trypsin was tested against MDCK.2 cells, CHO cells, and CHO cells expressing hMAL, sMAL, or dMAL (Table 5). As previously found, the Y30AY196A mutation resulted in a more than 400-fold reduction in toxicity to MDCK cells. However, compared to the wild-type toxin, the mutant showed a 57-fold increase in toxicity to CHO cells expressing hMAL, a 12-fold reduction in toxicity to CHO cells expressing sMAL, and a 180-fold reduction in toxicity to CHO cells expressing dMAL. The additional introduction of H149A, A1 68F, F92A, or V72F mutations into the β-octyl-glucoside binding site reduced the toxicity to CHO cells expressing hMAL.

[0195]

[0196] Variant protein toxicity in mice

[0197] Variant proteins with reduced toxicity in CHO-hMAL cell culture (Y30A-Y196A-H149A, Y30A-Y196A-A1 68F and Y30A-Y196A-H149A-A168F) were tested for toxicity in mice (Table 5). When given by ip route, the MLD dose of trypsin-activated Y30A-Y196A-H149A was 2 μg to 20 μg, while the MLD dose of trypsin-activated Y30A-Y1 96A-A1 68F or Y30A-Y196A-H149A-A168F was higher than the highest dose tested (20 μg). Y30A-Y196A-A168F protein was selected and further toxicity tests were performed by sc route before or after trypsin activation. The MLD doses of the trypsin-activated protein ranged from 20 μg to 200 μg, whereas the inactivated protein was non-toxic at the doses tested.

[0198] Antibody responses to variant proteins

[0199] Groups of 3 rabbits were immunized with Y30A-Y196A, Y30A-Y196A-A168F or Y30A-Y196A-H149A-A168F genetic toxins administered with Freund's incomplete adjuvant. This work was performed by Cambridge Research Biochemicals (Cleveland, UK). One week after the fourth immunization, the serum was tested for antibodies that could replace neutralizing monoclonal antibodies against epsilon toxin, indicating the presence of neutralizing antibodies in the serum. For comparison, the inventors included the WHO international standard epsilon toxin antitoxin, diluted to 5 IU / ml. The results showed that all rabbits produced antibodies that reacted with wild-type epsilon toxin when tested with a competitive ELISA. The titers in the sera from each rabbit in each immunization group were roughly similar, so the inventors combined the sera for subsequent testing.

[0200] The inventors found that undiluted sera or sera diluted 10-fold in PBS were similar to each other in their ability to displace neutralizing antibodies and were similar to the international standard ε toxin antitoxin. 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 displacing neutralizing antibodies ( Figure 4 ).

[0201] Vaccination of lambs

[0202] The inventor used Montanide TM Groups of 5 or 6 lambs were immunized with 2 doses of Y30A-Y196A-A1 68F protoxin administered with either ISA 61VG or aluminum hydroxide adjuvant (Table 6). Another group of lambs had pre-existing anti-epsilon toxin antibodies (detected by Western blot) at the start of the study and these lambs were immunized with 2 doses of Y30A-Y196A-A1 68F protein and Montanide TM ISA 61VG adjuvant immunization. The presence of antibodies capable of neutralizing epsilon toxin in sera from these animals and control sera from non-immunized lambs was tested using two different assays. First, the inventors used a competitive ELISA to measure the ability of sheep sera to displace neutralizing monoclonal antibodies. The inventors also tested the ability of the sera to neutralize the toxicity of epsilon toxin to CHO-hMAL cells. In both assays, the inventors included dilutions of standard sera containing known concentrations of neutralizing antibodies, expressed in international units (IU / ml).

[0203] We did not detect antibodies in control lambs using either assay. Using competition ELISA, we detected low levels of antibodies in the group immunized with toxoid and aluminum hydroxide adjuvant, but using the CHO-hMAL assay, we were unable to detect any neutralizing antibodies. In contrast, using Montanide TM Lambs immunized with toxoid in 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 competition ELISA assay (Table 6). TM Lambs without pre-existing anti-epsilon toxin antibodies immunized with toxoid in ISA 61VG adjuvant, using Montanide TM Lambs immunized with toxoid in ISA 61VG adjuvant but with pre-existing anti-epsilon toxin antibodies produced lower levels of neutralizing antibodies after immunization.

[0204] Table 6. Neutralizing antibodies in lambs immunized with Y30A-Y196A-A168F

[0205]

[0206] hMAL = human MAL

[0207] sMAL=Sheep MAL

[0208] discuss

[0209] In previous studies, the 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 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 an in vitro neutralization assay, polyclonal antibodies against Y30A-Y196A provided protection against wild-type toxin.

[0210] However, previous studies used MDCK cell lines to measure cytotoxicity, and subsequently showed that CHO cells expressing MAL were also highly sensitive to the toxin. The possibility that MAL is a receptor for the toxin is supported by the discovery that MAL knockout mice become resistant to the effects of Etx. In the present study that 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, while being 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, indicating that MDCK cells as well as CHO cells expressing MAL should be used in parallel in future studies.

[0211] The inventor introduced additional mutations to reduce the toxicity of Y30A-Y196A to CHO cells expressing MAL. The mutation was introduced into the region related to sugar binding in domain 3. Many of these mutants show reduced toxicity in CHO-hMAL cell culture and to MDCK cells and in mice. The data confirm the role of this region in toxicity. The inventor was able to produce all these proteins, and based on thermal stability measurements, the protein does not seem to show a significant change in stability, indicating that the conformation of the protein is roughly similar to that of the wild-type ε toxin. The Y30AY196A+A168F mutant was selected for testing in sheep because it induced a strong antibody response in rabbits.

[0212] Livestock vaccines containing aluminum hydroxide or saponin as adjuvants usually induce short-term antibody responses (Khorasani et al., (2016) Iranian journal of veterinary research 17: 8-12). This requires booster immunizations at regular intervals, sometimes as short as 4 months. Montanide TM ISA 61VG is a new, ready-to-use mineral oil-based adjuvant for livestock that offers the potential to induce high-level and long-lasting responses in animals (Khorasani et al., (2016)). Compared with the use of aluminum hydroxide adjuvant, the use of Montanide TM ISA 61VG adjuvant resulted in the induction of better antibody responses, a finding similar to previous findings using 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 in inducing antibody responses. TMGel 01 (aqueous polymer) adjuvant (Petermann et al., (2017) Exp Appl Acarol. 72: 303-315). Montanide-containing TM No evidence of local side effects was seen following administration of the protein with ISA 61VG adjuvant, although others have reported evidence of local side effects with this adjuvant (Petermann et al., (2017) Exp Appl Acarol. 72: 303-315). In Europe, compliance with the European Pharmacopoeia (Ph. Eur.) monograph on Clostridium perfringens vaccines for veterinary use (0363) is required for the licensure of epsilon toxoid vaccines. The toxoid produced showed a residual toxicity below that required, and the inventors used Montanide TM The level of neutralizing antibodies achieved with ISA 61VG adjuvant was at least 10 times higher than the threshold requirement (5 IU / ml).

[0213] Lambs with pre-existing anti-epsilon toxin antibodies responded less well to vaccination, a finding consistent with studies in other species. For example, antibodies inhibit responses to a variety of 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 result in clearance of antigen or formation of antigen-antibody complexes that limit B cell activation or by physically masking epitopes from B cells (Zarnitsyna et al., (2016)).

[0214] The vaccine designed by the present invention will be used for livestock susceptible to enterotoxemia caused by the epsilon toxin of Clostridium perfringens. The vaccine has many advantages over existing vaccines because it does not require detoxification before use. The purity of the antigen and the adjuvant (such as Montanide TMThe use of ISA 61VG) should promote long-term immunity and reduce or eliminate the need for booster vaccinations. In addition, it can be used as a protein carrier for polysaccharides that induce protective antibodies against other diseases in livestock (Peterman 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). Glycoconjugates will promote T cell responses to polysaccharide moieties (Avci et al., (2011) Nature medicine 17: 1602-9), increase the strength of antibody responses and the induction of memory responses to polysaccharides (Avci (2013) Current Topics in Medicinal Chemistry 13: 2535-40); Pace (2013) Expert opinion on biological therapy 13: 11-33). In addition, the connection of polysaccharides to epsilon toxin carriers will allow the vaccine to be used in young animals (PACE (2013)). The Y30AY1 96A+A1 68F protein can be chemically coupled to polysaccharides, or it can be further modified to serve as an acceptor for recombinant glycoconjugates produced using the glycosylation system naturally present in bacteria (Valguarnera et al., (2016) J Mol Biol. 428: 3206-20; Cuccui et al., (2015) The Journal of pharmacy and pharmacology 67: 338-50).

[0215] Finally, the Y30AY1 96A+A1 68F protein could be developed for use as a human vaccine in the future. Clostridium perfringens epsilon toxin is considered a potential biothreat agent (Greenfield et al., (2002) Am J Med Sci. 323: 326-40; Berger et al., (2016) Disaster and military medicine 2: 7), and vaccination of individuals at risk would protect them from disease. In addition, epsilon toxin has recently been suggested to play a role 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, vaccination against this toxin could be a potential preventive or therapeutic option.

[0216] Longevity of protective antibody responses

[0217] Lambs were reared without vaccination against C. perfringens epsilon toxin and after 12 months, groups of 5 lambs were given 200 μg of Montanide TM Y30AY196A+A1 68F toxoid (1:1) adjuvanted with ISA 61VG (Seppic, Paris, France) was administered subcutaneously in 6×0.5 ml doses. Three weeks later, lambs were given a second dose of adjuvanted protein. Blood samples were collected at the beginning of the study and at intervals of up to 12 months after immunization. Competitive ELISA was performed using a MonoscreenELISA 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 / m1), to enable them to calculate antibody levels of neutralizing antibodies expressed in IU / l. The results are shown in Table 7 below.

[0218] Table 7. Montanide TM Neutralizing antibodies in sera at week 0 and week 3 after immunization with Y30AY196A+A168F adjuvanted with ISA 61VG

[0219]

[0220]

[0221] The neutralizing antibody titers found in sheep administered the toxoid of the present invention and the Montanide ISA 61VG adjuvant far exceed the minimum protective titers reported in sheep (0.1 IU / ml to 0.3 IU / ml (de la Rosa et al. 1997 (JAnim Sci 75(9):2328-2334); Uzal and Kelly 1998 (Veterinary Record 142(26):722-725); or in goats (1 IU / ml (Uzal, Bodero et al. 1998 (Vet Rec 143(17):472-474), Uzal and Kelly 1998 (Veterinary Record 144(26):722-725). 142(26):722-725)), and remained above this threshold one year after immunization. In addition, the level of neutralizing antibodies exceeded the protective titer after one dose of the vaccine of the present invention (i.e., at week 3), indicating that a single-dose vaccine for livestock can be achieved.

[0222] Testing of NMO, ON and TM patient samples

[0223] Table 8 below shows Western blot data for samples taken from subjects with NMO, almost all of whom initially developed ON and / or TM. 15 of 30 samples (50%) showed very strong positive (3), strong positive (2), positive (8), or weak positive (2) reactivity to Etx compared to controls, of which 2 / 25 (8%) were positive.

[0224] All subjects who tested positive for AQP-4 antibodies and 11 of 15 subjects who tested positive for Etx initially showed features of transverse myelitis (TM) and / or optic neuritis (ON), TM and ON being examples of neuromyelitis optica spectrum disorder (NMOSD).

[0225] Table 8: Western blot data

[0226]

[0227]

[0228] Controls in bold italics: Gender and age matched + / - 2 years old human red blood cells (RBC) treated with epsilon toxin

[0229] Hemolysis of human erythrocytes exposed to wild-type Etx, Y30AY196A, Y30AY196A+H149A, Y30AY196A+A168F, Y30AY196A+F92A, Y30AY196A+V166A, and the four mutants Y30AY196+A168F+H149A was tested. 10 μM of the original toxin and 10 μM and 1 μM of the trypsin-activated toxin were tested with 3.3% of RBCs. Each toxin was tested in triplicate at each dose. Results are expressed relative to the 1% Tx100 control (which resulted in 100% hemolysis); negative control = PBS. Results are shown in Table 1. Figure 6 As shown, it is demonstrated that Y30AY196A+H149A, Y30AY196A+A168F and Y30AY196+A168F+H149A are non-hemolytic even upon trypsin activation.

[0230] Sequence (mutation position is shown in bold)

[0231] SEQ ID NO: 1

[0232]

[0233] SEQ ID NO: 2

[0234]

[0235] SEQ ID NO: 3

[0236]

[0237] SEQ ID NO: 4 Y30A mutation

[0238]

[0239] SEQ ID NO: 5 Y196A mutation

[0240]

[0241] SEQ ID NO: 6 H149A mutation

[0242]

[0243] SEQ ID NO: 7 V72F mutation

[0244]

[0245] SEQ ID NO: 8 F92A mutation

[0246]

[0247] SEQ ID NO: 9 V166A mutation

[0248]

[0249] SEQ ID NO: 10 A168F mutation

[0250]

[0251] SEQ ID NO: 11 Full length wild type native epsilon toxin

[0252]

[0253] SEQ ID NO: 12 sequence used to obtain the crystal structure (PDB ID: 1YUJ)

[0254]

[0255]

[0256] SEQ ID NO: 13 Trypsin-activated wild-type recombinant epsilon toxin

[0257]

[0258] SEQ ID NO: 14 Trypsin-activated recombinant epsilon toxin with H149A mutation

[0259]

[0260] SEQ ID NO: 15 Full-length recombinant epsilon toxin

[0261]

[0262] SEQ ID NO: 16 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A)

[0263]

[0264] SEQ ID NO: 17 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A)

[0265]

[0266] SEQ ID NO: 18 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F)

[0267]

[0268] SEQ ID NO: 19 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+F92A)

[0269]

[0270] SEQ ID NO: 20 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V166A)

[0271]

[0272] SEQ ID NO: 21 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+A168F)

[0273]

[0274] SEQ ID NO: 22 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F)

[0275]

[0276] SEQ ID NO: 23 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+F92A)

[0277]

[0278] SEQ ID NO: 24 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V166A)

[0279]

[0280] SEQ ID NO: 25 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+A168F)

[0281]

[0282] SEQ ID NO: 26 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+F92A)

[0283]

[0284] SEQ ID NO: 27 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+V166A)

[0285]

[0286] SEQ ID NO: 28 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+A168F)

[0287]

[0288] SEQ ID NO: 29 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+F92A+V166A)

[0289]

[0290] SEQ ID NO: 30 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+F92A+A168F)

[0291]

[0292] SEQ ID NO: 31 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V166A+A168F)

[0293]

[0294] SEQ ID NO: 32 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+F92A)

[0295]

[0296] SEQ ID NO: 33 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+V166A)

[0297]

[0298] SEQ ID NO: 34 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+A168F)

[0299]

[0300] SEQ ID NO: 35 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+F92A+V166A)

[0301]

[0302] SEQ ID NO: 36 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+F92A+A168F)

[0303]

[0304] SEQ ID NO: 37 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V166A+A168F)

[0305]

[0306] SEQ ID NO: 38 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+F92A+V166A)

[0307]

[0308] SEQ ID NO: 39 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+F92A+A168F)

[0309]

[0310] SEQ ID NO: 40 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+V166A+A168F)

[0311]

[0312] SEQ ID NO: 41 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+F92A+V166A+A168F)

[0313]

[0314] SEQ ID NO: 42 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+F92A+V166A)

[0315]

[0316] SEQ ID NO: 43 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+H149A+V72F+F92A+A168F)

[0317]

[0318] SEQ ID NO: 44 Trypsin-activated recombinant epsilon toxin (Y30A+Y196A+V72F+F92A+V166A+A168F)

[0319]

[0320] SEQ ID NO: 45 Trypsin-activated recombinant ε toxin (Y30A+Y196A+H149A+V72F+F92A+V166A+ A168F)

[0321]

[0322] SEQ ID NO: 46 Trypsin-activated recombinant epsilon toxin (V72F)

[0323]

[0324] SEQ ID NO: 47 Trypsin-activated recombinant epsilon toxin (F92A)

[0325]

[0326] SEQ ID NO: 48 Trypsin-activated recombinant epsilon toxin (H149A)

[0327]

[0328] SEQ ID NO: 49 Trypsin-activated recombinant epsilon toxin (V166A)

[0329]

[0330] SEQ ID NO: 50 Trypsin-activated recombinant epsilon toxin (A168F)

[0331]

[0332] SEQ ID NO: 51 Y43A forward

[0333]

[0334] SEQ ID NO: 52 Y43A reverse

[0335]

[0336] SEQ ID NO: 53 Y209A forward

[0337]

[0338] SEQ ID NO: 54 Y209A reverse

[0339]

[0340] SEQ ID NO: 55 H149A forward

[0341]

[0342] SEQ ID NO: 56 H149A reverse

[0343]

[0344] SEQ ID NO: 57 V72F forward

[0345]

[0346] SEQ ID NO: 58 V72F reverse

[0347]

[0348] SEQ ID NO: 59 F92A forward

[0349]

[0350] SEQ ID NO: 60 F92A reverse

[0351]

[0352] SEQ ID NO: 61 V166A forward

[0353]

[0354] SEQ ID NO: 62 V166A reverse

[0355]

[0356] SEQ ID NO: 63 A168F forward

[0357]

[0358] SEQ ID NO: 64 A168F reverse

[0359]

[0360] SEQ ID NO: 65 ( Figure 2 Residues 1-260 of the unmutated sequence; trypsin-activating polypeptide)

[0361]

[0362]

[0363] SEQ ID NO: 66 ( Figure 2 signal sequence residues 1-32)

[0364]

[0365] SEQ ID NO: 67 ( Figure 2 N-terminal propeptide residues 33-45) to 45)

[0366]

[0367] SEQ ID NO: 68 ( Figure 2 C-terminal propeptide residues 305-328)

[0368]

Claims

1. An epsilon toxin polypeptide having reduced toxicity to cells expressing myelin and lymphocyte protein and comprising a modified domain III compared to the wild-type epsilon toxin 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 epsilon toxin polypeptide is capable of binding to at least one antibody that binds to a sequence represented by SEQ ID NO: 65 and / or SEQ ID NO: 14; wherein the epsilon toxin polypeptide is represented by SEQ ID NO: 21 or SEQ ID NO:

25.

2. A polynucleotide having a nucleic acid sequence encoding the epsilon toxin polypeptide according to claim 1. A vector comprising the polynucleotide according to claim 2.

4. A cell comprising the epsilon toxin polypeptide according to claim 1 and / or the polynucleotide according to claim 2 and / or the vector according to claim 3.

5. A subunit vaccine or conjugate vaccine comprising the epsilon toxin polypeptide according to claim 1.

6. A method for preparing an immunotherapeutic composition comprising altering an epsilon toxin polypeptide by modifying domain III relative to a wild-type epsilon toxin polypeptide of SEQ ID NO: 65; wherein the epsilon toxin polypeptide is represented by SEQ ID NO: 21 or SEQ ID NO:

25.

7. The method of claim 6, wherein the immunotherapeutic composition is a vaccine composition.

8. An immunotherapeutic composition prepared by the method according to claim 6 or 7.

9. An immunotherapeutic composition comprising the epsilon toxin polypeptide according to claim 1, the polynucleotide according to claim 2, the vector according to claim 3, the cell according to claim 4, or the subunit vaccine or combined vaccine according to claim 5.

10. The immunotherapeutic composition according to claim 8 or 9, which is in a form suitable for oral administration.

11. The immunotherapeutic composition according to claim 8 or 9, wherein the immunotherapeutic composition is a vaccine composition.

12. A kit comprising: The epsilon toxin polypeptide according to claim 1, the polynucleotide according to claim 2, the vector according to claim 3, the cell according to claim 4, the subunit vaccine or the combined vaccine according to claim 5, or the immunotherapeutic composition according to any one of claims 8 to 11, and one or more buffers, or diluents and / or one or more administration devices.

Images