# Patent

## Title
Immunogenic composition

## Abstract

The present invention relates to immunogenic compositions comprising a Clostridium difficile (C. difficile) polypeptide and an aluminum-free adjuvant.

## Background
This application is filed pursuant to 35 U.S.C. §371 as a United States National Phase Application of International Patent Application Serial No. PCT/EP2013/075405 filed Dec. 3, 2013, which claims priority to United Kingdom Application No. GB 1221875.6 filed Dec. 5, 2012, and the entire contents of each of the foregoing applications are hereby incorporated by reference.
TECHNICAL FIELD
The present invention relates to immunogenic compositions comprising a Clostridium difficile (C. difficile) polypeptide and an aluminum-free adjuvant. The invention also relates to vaccine compositions and the use of the vaccines and immunogenic compositions of the invention in prophylaxis or therapy or in the manufacture of a medicament.
BACKGROUND
C. difficile is the most important cause of nosocomial intestinal infections and is the major cause of pseudomembranous colitis in humans (Bartlett et al Am. J. Clin. Nutr. 11 suppl: 2521-6 (1980)). The overall associated mortality rate for individuals infected with C. difficile was calculated to be 5.99% within 3 months of diagnosis, with higher mortality associated with advanced age, being 13.5% in patients over 80 years (Karas et al Journal of Infection 561:1-9 (2010)). The current treatment for C. difficile infection is the administration of antibiotics (metronidazole and vancomycin), however there has been evidence of strains which are resistant to these antibiotics (Shah et al., Expert Rev. Anti Infect. Ther. 8(5), 555-564 (2010)). Accordingly there is a need for immunogenic compositions capable of inducing antibodies to, and/or a protective immune response to, C. difficile. 
The enterotoxicity of C. difficile is primarily due to the action of two toxins, toxin A (‘ToxA’) and toxin B (‘ToxB’). The C-terminal domains of toxin A and toxin B comprise repeating units, for example the C-terminal domain of toxin A is made up of contiguous repeating units (Dove et al Infect. Immun. 58:480-499 (1990)). For this reason the C-terminal domain may be referred to as the ‘repeating domain’. These repeat portions can be separated further into short repeats (SRs) and long repeats (LRs) as described in Ho et al (PNAS 102:18373-18378 (2005)).
Immunogenic compositions comprising antigens from C. difficile have been described. WO96/12802 and WO00/61762 and Lyerly et al (Current Microbiology 21:29-32 (1990)) relate to fragments of toxin A, in particular fragments of the C-terminal domain, for inducing a protective immune response in hamsters. WO9920304 relates to a mixture of co-purified toxin A and toxin B inactivated by incubation in formaldehyde. WO00/61762 relates to immunogenic compositions comprising either the full length C-terminal domain or fragments of the C-terminal domain of toxin A and toxin B of C. difficile. Similarly WO2012/163817, WO2012/163811 and WO2012/163810 provide disclosures of possible fragments of toxin A and toxin B as well as fusion proteins thereof.
New compositions or vaccines with improved immunogenicity are needed. As one strategy, adjuvants have been used to try and improve the immune response raised to any given antigen. For example, WO2009035707 describes a composition comprising a toxoid of C. difficile toxins A and B and an adjuvant such as aluminum hydroxide compound.
Adjuvants containing combinations of lipopolysaccharide and Quillaja saponins have been disclosed previously, for example in EP0671948. Oil in water emulsions per se are well known in the art, and have been suggested to be useful as adjuvant compositions (EP 399843; WO 95/17210).
There is still a need for vaccine and immunogenic compositions that provide a suitable immune response against C. difficile. 
SUMMARY
In one aspect, the invention relates to an immunogenic composition comprising a polypeptide comprising a Clostridium difficile (C. difficile) toxin A fragment and/or a C. difficile toxin B fragment and an adjuvant comprising an immunologically active saponin fraction presented in the form of a liposome. The immunologically active saponin fraction may be QS21 and the lipopolysaccharide may be 3D-MPL. Suitably, the immunogenic composition may comprise about 50 μg 3D-MPL, and about 50 μg QS21.
In one aspect, the invention relates to an immunogenic composition comprising a polypeptide comprising a C. difficile toxin A fragment and/or a C. difficile toxin B fragment and an adjuvant comprising an oil in water emulsion, wherein said oil in water emulsion comprises a metabolisable oil, a tocol and an emulsifier. The metabolisable oil may be present at an amount of about 5.35 mg. The tocol may be present at an amount of about 5.94 mg. Suitably, the emulsifying agent may be present at an amount of about 2.425 mg. Suitably, the metabolisable oil is squalene, the tocol is alpha-tocopherol and the emulsifying agent is polyoxyethylene sorbitan monooleate.
Suitably, the immunogenic composition according to any aspect of the invention comprises a C. difficile toxin A repeating domain fragment and a C. difficile toxin B repeating domain fragment and elicits antibodies that neutralize toxin A and toxin B. In a further embodiment of the invention, the immunogenic composition comprises a C. difficile toxin A fragment and a C. difficile toxin B fragment wherein one of the fragments is a N-terminal fragment and the other is a repeating domain fragment.
Suitably, the immunogenic composition according to any aspect of the invention comprises a polypeptide variant of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27.
Suitably, the immunogenic composition according to any aspect of the invention comprises a polypeptide comprising SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35 or a variant or fragment of any of these sequences.
The immunogenic compositions according to the invention may comprise additional antigens such as antigens derived from a bacterium selected from the group consisting of Streptococcus pneumoniae (S. pneumoniae), Haemophilus influenzae (H. influenzae), Neisseria meningitidis (N. meningitidis), Escherichia coli (E. coli), Moraxella catarrhalis (M. cattarhalis), Clostridium tetani (C. tetani), Corynebacterium diptherieriae (C. diptherieriae), Bordetella pertussis (B. pertussis), Staphylococcus epidermidis (S. epidermidis), enterococci, and Staphylococcus aureus (S. aureus).
In one aspect, the invention relates to a vaccine comprising the immunogenic composition according to the invention and a pharmaceutically acceptable excipient.
In one aspect, the invention relates to a use of the immunogenic composition or the vaccine according to the invention in the treatment or prevention of C. difficile disease.
In one aspect, the invention relates to the immunogenic composition or the vaccine according to the invention for use in the treatment or prevention of C. difficile disease.
In one aspect, the invention relates to use of the immunogenic composition or the vaccine of according to the invention in the preparation of a medicament for the prevention or treatment of C. difficile disease.
In one aspect, the invention relates to a method of preventing or treating C. difficile disease comprising administering the immunogenic composition or the vaccine according to the invention to a subject.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1—Graphs showing anti-ToxA and anti-ToxB ELISA results for hamsters immunised with F2 formulated with Adjuvant A; Adjuvant A without QS21 and Adjuvant A without MPL.
FIG. 2—Graphs showing anti-ToxA and anti-ToxB ELISA results for hamsters immunised with non-adjuvanted F2 or F2 formulated with Adjuvant A, Adjuvant B and Alum.
FIG. 3—Graphs showing anti-ToxA and anti-ToxB ELISA results for Post I sera of male and female hamsters immunised with a dose-range of F2 fusion proteins formulated in Adjuvant B, AS04D, Adjuvant A or Adjuvant C.
FIG. 4—Graphs showing anti-ToxA and anti-ToxB ELISA results for Post II sera of male and female hamsters immunised with a dose-range of F2 fusion proteins formulated in Adjuvant B, AS04D, Adjuvant A or Adjuvant C.
FIG. 5—Graphs showing anti-ToxA and anti-ToxB ELISA results for Post III sera of male and female hamsters immunised with a dose-range of F2 fusion proteins formulated in Adjuvant B, AS04D, Adjuvant A or Adjuvant C.
FIG. 6—Graphs showing anti-ToxA and anti-ToxB ELISA results for Post III day 87 sera of male and female hamsters immunised with a dose-range of F2 fusion proteins formulated in Adjuvant B, AS04D, Adjuvant A or Adjuvant C.
FIG. 7—Graphs showing anti-ToxA and anti-ToxB ELISA results for male hamsters immunised with a dose-range of F2 fusion proteins formulated in Adjuvant B, AS04D, Adjuvant A or Adjuvant C on Post I, II and III sera.
FIG. 8—Graphs showing anti-ToxA and anti-ToxB ELISA results for male and female hamsters immunised with a Mix (ToxA+ToxB) or with the F2 fusion protein formulated in Alum or Adjuvant A.
FIG. 9—Graph showing anti-ToxA immunogenicity in mice immunised with a fragment of the C-terminus of toxin A (aa 2387-2706), a fragment of the C-terminus of toxin B (aa 1750-2360), or fusions 1, 2, 3, 4 or 5 formulated in Adjuvant B.
FIG. 10—Graph showing hemagglutination inhibition in mice immunised with a fragment of the C-terminus of toxin A (aa 2387-2706), a fragment of the C-terminus of toxin B (aa 1750-2360), or fusions 1, 2, 3, 4 or 5 formulated in Adjuvant B.
FIG. 11—Graph showing anti-ToxB immunogenicity in mice immunised with a fragment of the C-terminus of toxin A (aa 2387-2706), a fragment of the C-terminus of toxin B (aa 1750-2360), or fusions 1, 2, 3, 4 or 5 formulated in Adjuvant B.
FIG. 12—Cytotoxicity inhibition titres from mice immunised with a fragment of the C-terminus of toxin A (aa 2387-2706), a fragment of the C-terminus of toxin B (aa 1750-2360), or fusions 1, 2, 3, 4 or 5 formulated in Adjuvant B.
FIG. 13—Graph showing anti-ToxA ELISA results for mice immunised with the F2, F52New, F54Gly, G54New or F5 ToxB fusions formulated in Adjuvant B.
FIG. 14—Graph showing anti-ToxB ELISA results for mice immunised with the F2, F52New, F54Gly, F54New or F5ToxB fusions formulated in Adjuvant B.
FIG. 15—Graph showing hemagglutination inhibition in mice immunised with the F2, F52New, F54Gly, F54New or F5ToxB fusions formulated in Adjuvant B.
FIG. 16—Graph showing cytotoxicity titres in HT29 cells from mice immunised with the F2, F52New, F54Gly, F54New or F5ToxB fusions formulated in Adjuvant B.
FIG. 17—Graph showing cytotoxicity titres in IMR90 cells from mice immunised with the F2, F52New, F54Gly, F54New or F5ToxB fusions formulated in Adjuvant B.
FIG. 18—Graph showing anti-ToxA ELISA results for mice immunised with C. difficile ToxA-Cter (aa 2387-2706), ToxB-Cter (aa 1750-2360) and fusion proteins (non-adjuvanted).
FIG. 19—Graph showing anti-ToxB ELISA results for mice immunised with C. difficile ToxA-Cter (aa 2387-2706), ToxB-Cter (aa 1750-2360) and fusion proteins (non-adjuvanted)
FIG. 20—Graph showing anti-ToxA ELISA results for mice immunised with C. difficile ToxA-Cter (aa 2387-2706), ToxB-Cter (aa 1750-2360) and fusion proteins formulated in Adjuvant B: Post II, III, Pre-Boost, Post IV sera.
FIG. 21—Graph showing anti-ToxB ELISA results for mice immunised with C. difficile ToxA-Cter (aa 2387-2706), ToxB-Cter (aa 1750-2360) and fusion proteins formulated in Adjuvant B: Post II, III, Pre-Boost, Post IV sera.
FIG. 22—Graph describing the Far-UV spectrum of Fusions, 2, 3, 4, and 5 measured using circular dichroism. The spectrum for fusion 2 is represented by a line with the points depicted as small squares, the spectrum for fusion 3 is represented by a line with the points depicted as small diamond shapes, fusion 4 is represented by a line with the points depicted as circles, and fusion 5 is represented by a line with the points depicted as cross shapes.
FIG. 23—Graph describing the near-UV spectrum of Fusions 2, 3, 4, and 5 measured using circular dichroism. The spectrum for fusion 2 is represented by a line with the points depicted as cross shapes, the spectrum for fusion 3 is represented by a line with the points depicted as circles, the spectrum for fusion 4 is represented by a line with the points depicted as triangles, and the spectrum for fusion 5 is represented by a line with the points depicted as small diamond shapes.
FIG. 24—Graph describing the Far-UV spectrum of fusions F52New, F54Gly, F54New and F5ToxB measured using circular dichroism. The spectrum for F52New is represented by a line with the points depicted as double crosses, the spectrum for F54Gly is represented by a line with the points depicted as triangles, F54New is represented by a line with the points depicted as squares, and F5ToxB is represented by a line with the points depicted as cross shapes.
FIG. 25—Graph describing the Near-UV spectrum of fusions F52New, F54Gly, F54New and F5ToxB measured using circular dichroism. The spectrum for F52New is represented by a line with the points depicted as double crosses, the spectrum for F54Gly is represented by a line with the points depicted as triangles, F54New is represented by a line with the points depicted as squares, and F5ToxB is represented by a line with the points depicted as cross shapes.



DETAILED DESCRIPTION
The inventors have shown immunogenic compositions comprising a polypeptide comprising a C. difficile toxin A fragment and/or a C. difficile toxin B fragment formulated with alum free adjuvants, particularly adjuvants such as Adjuvant A and Adjuvant B described herein, provide an improved immune response against C. difficile as compared to non-adjuvanted C. difficile polypeptide containing compositions. Immunogenic compositions according to the invention may also provide an improved immune response against C. difficile as compared to C. difficile polypeptides formulated with alum.
Polypeptides
The polypeptide comprising a C. difficile toxin A fragment and/or a C. difficile toxin B fragment may comprise purified toxin A or toxin B which have been inactivated, suitably by incubation in formaldehyde, such as those described in WO9920304.
The polypeptide may comprise a C. difficile toxin A N-terminal fragment and a C. difficile toxin B repeating domain fragment. The polypeptide may contain a C. difficile toxin A repeating domain fragment and a C. difficile toxin B N-terminal fragment. The polypeptide may comprise a C. difficile toxin A repeating domain fragment and a C. difficile toxin B repeating domain fragment.
The polypeptide may comprise either the full length N-terminal and/or C-terminal domain of toxin A and toxin B of C. difficile or fragments thereof.
In one embodiment, the polypeptide in the immunogenic compositions according to the invention does not contain the full length N-terminal domain of toxin A and/or toxin B of C. difficile. In one embodiment, the polypeptide in the immunogenic compositions according to the invention does not contain an N-terminal domain fragment of toxin A and/or toxin B of C. difficile. 
The polypeptide may be a fusion protein. For example, the fusion protein may comprise a fusion sequence such as Seq ID NOS: 36 and 37 or other sequences described in WO2012/028741.
The polypeptide may comprise a first fragment and a second fragment, wherein
(i) the first fragment is a toxin A repeating domain fragment;
(ii) the second fragment is a toxin B repeating domain fragment;
(iii) the first fragment comprises a first proximal end within a first repeat portion;
(iv) the second fragment comprises a second proximal end within a second repeat portion; and
wherein the first fragment and the second fragment are adjacent to one another and wherein the first repeat portion and the second repeat portion have structural similarity.
The term polypeptide refers to a contiguous sequence of amino acids.
The term ‘toxin A repeating domain’ refers to the C-terminal domain of the toxin A protein from C. difficile comprising repeated sequences. For example, the C-terminal domain of the toxin A protein may be amino acids 1832-2710 of toxin A from strain VPI10463 (ATCC43255) and/or their equivalent in a different strain. Amino acids 1832-2710 of toxin A from strain VPI10463 (ATCC43255) corresponds to amino acids 1832-2710 of SEQ ID NO:1.
The term ‘toxin B repeating domain’ refers to the C-terminal domain of the toxin B protein from C. difficile. For example, the C-terminal domain of the toxin B protein may be amino acids 1834-2366 of toxin B from strain VPI10463 (ATCC43255) and/or their equivalent in a different strain. Amino acids 1834-2366 of toxin B from strain VPI10463 (ATCC43255) corresponds to amino acids 1834-2366 of SEQ ID NO:2.
The full length N-terminal domain of toxin A may be amino acids 1-542 of toxin A from strain VPI10463 (ATCC43255) and/or their equivalent in a different strain. Thus the term “a C. difficile toxin A N-terminal fragment” or “an N-terminal domain fragment of toxin A” refers to a fragment of amino acids 1-542 of toxin A from strain VPI10463 (ATCC43255) and/or their equivalent in a different strain. Amino acids 1-542 of toxin A from strain VPI10463 (ATCC43255) corresponds to amino acids 1-542 of SEQ ID NO: 1.
The full length N-terminal domain of toxin B may be amino acids 1-543 of toxin B from strain VPI10463 (ATCC43255) and/or their equivalent in a different strain. Thus the term “a C. difficile toxin B N-terminal fragment” or “an N-terminal domain fragment of toxin B” refers to a fragment of amino acids 1-543 of toxin B from strain VPI10463 (ATCC43255) and/or their equivalent in a different strain. Amino acids 1-543 of toxin B from strain VPI10463 (ATCC43255) corresponds to amino acids 1-543 of SEQ ID NO: 2.
C. difficile toxins A and B are conserved proteins. However the sequence differs a small amount between strains. Moreover the amino acid sequence for toxins A and B in different strains may differ in number of amino acids.
In one embodiment, the toxin A repeating domain and/or toxin B repeating domain may be a sequence which is a variant with at least 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1832-2710 of SEQ ID NO:1 or a variant with at least 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1834-2366 of SEQ ID NO:2.
In one embodiment, the toxin A N-terminal domain may be a sequence which is a variant with at least 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1-542 of SEQ ID NO:1. In one embodiment, the toxin B N-terminal domain may be a sequence which is a variant with at least 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1-543 of SEQ ID NO: 2.
A ‘variant’ is a polypeptide that varies from the referent polypeptides by conservative amino acid substitutions, whereby a residue is substituted by another with the same physico-chemical properties. Typically such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln, and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
Furthermore the amino acid numbering may differ between the C-terminal domains or N-terminal domains of toxin A (or toxin B) from one strain and toxin A (or toxin B) from another strain. For this reason the term ‘equivalent in a different strain’ refers to amino acids which correspond those of a reference strain (e.g., C. difficile VPI10463), but which are found in a toxin from a different strain and which may thus be numbered differently. A region of ‘equivalent’ amino acids may be determined by aligning the sequences of the toxins from the different strains. The amino acids numbers provided throughout refer to those of strain VPI10463.
The term ‘fragment’ of a polypeptide or protein refers to a contiguous portion, such as at least 100, 150, 180, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600 amino acids, from that polypeptide or protein.
“N-terminal domain fragment of toxin A” of C. difficile refers to a contiguous portion, such as at least 100, 150, 180, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500 or 530 amino acids, from the full length N-terminal domain of toxin A. “N-terminal domain fragment of toxin B” of C. difficile refers to a contiguous portion, such as at least 100, 150, 180, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500 or 530 amino acids, from the full length N-terminal domain of toxin B.
Toxin A repeating domain fragment may be a contiguous portion of at least 100, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600 amino acids from the toxin A repeating domain. Toxin A repeating domain fragment may be a contiguous portion of less than 750, less than 700, less than 650, less than 600 or less than 580 amino acids. Toxin B repeating domain fragment may be a contiguous portion of at least 100, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600 amino acids from the toxin B repeating domain. Toxin B repeating domain fragment may be a contiguous portion of less than 530, less than 500, less than 480, or less than 450 amino acids.
The term first fragment′ refers to a contiguous portion of at least 100, such as 150, 180, 250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600, amino acids of the toxin A repeating domain. The term ‘second fragment’ refers to a contiguous portion of at least 100, such as 200, 230, 250, 280, 300, 350, 400, 450 or 500, amino acids of the toxin B repeating domain.
The term ‘first proximal end’ refers to the end of the first fragment (Tox A fragment) which is covalently linked to the second fragment (Tox B fragment) or covalently linked to a linker sequence between the first and second fragment. The term ‘second proximal end’ refers to the end of the second fragment which is closest to the first fragment in primary structure (amino acid sequence).
The C-terminal domain of toxin A is made up of 8 repeat portions (designated repeat portion I, repeat portion II, repeat portion III, repeat portion IV, repeat portion V, repeat portion VI, repeat portion VII and repeat portion VIII). Each of these repeat portions can be further divided into short repeats (SRs) and long repeats (LRs) (except for Tox A repeat portion VIII which does not have a long repeat). Each of the long repeats has some structural and sequence similarity to the other long repeats. Similarly the short repeats have some sequence and structural similarity to one another. Similarly the C-terminal domain of toxin B is made up of 5 repeat portions subdivided into SRs and LRs. Each repeat portion contains one LR and between 2 and 5 SRs (except for Tox B repeat portion V which does not have a long repeat). For the purposes of the disclosure a ‘repeat portion’ refers to one of the eight repeat portions of ToxA (designated I, II, III, IV, V, VI, VII or VIII) or one of the five repeat portions of ToxB (designated I, II, III, IV or V) or a partial repeat portion from the toxin A or toxin B repeating domain. As used herein the term first repeat portion′ refers to a repeat portion (or partial repeat portion) from the toxin A repeating domain. The term ‘second repeat portion’ refers to a repeat portion (or partial repeat portion) from the toxin B repeating domain.
For example, repeat portion I of ToxA contains three SRs and one LR, which can be referred to as the first SRI of ToxA, the second SRI of ToxA, the third SRI of ToxA and the LRI of ToxA, respectively.
The first proximal end is considered to be within a ‘repeat portion’ if the first fragment ends in an amino acid that is within that repeat portion (i.e., the first proximal end contains only part of the repeat portion sequence). Similarly the second proximal end is considered to be within a ‘repeat portion’ if the second fragment ends in an amino acid that is within that repeat portion. For example the first proximal end is within ‘repeat portion I of ToxA if the first fragment ends with any one of amino acids 1833-1923 (inclusive) of VPI10463 or their equivalent in another strain. The first proximal end is within a ‘long repeat’ or a ‘short repeat’ if the first fragment ends in an amino acid that is within a ‘long repeat’ or a ‘short repeat’, similarly the second proximal end is within a ‘long repeat’ or a ‘short repeat’ if the second fragment ends in an amino acid that is within a ‘long repeat’ or a ‘short repeat’.
The amino acid positions of each domain has been defined for toxin A and toxin B from strain VPI10463 (ATCC43255). These are as follows (Table 1)
















Start 
End



Name
position
position





















ToxA_I
SR1
1832
1852




SR2
1853
1873




SR3
1874
1893




LR
1894
1924



ToxA_II
SR1
1925
1944




SR2
1945
1965




SR3
1966
1986




SR4
1987
2007




SR5
2008
2027




LR
2028
2058



ToxA_III
SR1
2059
2078




SR2
2079
2099




SR3
2100
2120




SR4
2121
2141




SR5
2142
2161




LR
2162
2192



ToxA_IV
SR1
2193
2212




SR2
2213
2233




SR3
2234
2253




SR4
2254
2275




LR
2276
2306



ToxA_V
SR1
2307
2326




SR2
2327
2347




SR3
2348
2368




SR4
2369
2389




SR5
2390
2409




LR
2410
2440



ToxA_VI
SR1
2441 
2460




SR2
2461 
2481




SR3
2482 
2502




SR4
2503 
2522




LR
2523 
2553



ToxA_VII
SR1
2554 
2573




SR2
2574 
2594




SR3
2595 
2613




LR
2614 
2644



ToxA_VIII
SR1
2645
2664




SR2
2665 
2686




SR3
2687 
2710



ToxB_I
SR1
1834 
1854




SR2
1855 
1876




SR3
1877 
1896




LR
1897 
1926



ToxB_II 
SR1
1927 
1946




SR2
1947 
1967




SR3
1968 
1987




SR4
1988 
2007




SR5
2008 
2027




LR
2028 
2057



ToxB_III
SR1
2058 
2078




SR2
2079 
2099




SR3
2100 
2119




SR4
2120 
2139




SR5
2140 
2159




LR
2160 
2189



ToxB_IV
SR1
2190 
2212




SR2
2213 
2233




SR3
2234 
2253




SR4
2254 
2273




SR5
2274 
2293




LR
2294 
2323



ToxB_V
SR1
2324 
2343




SR2
2344
2366










In one embodiment, the repeat portion of toxin A refers to amino acids 1832-1924, 1925-2058, 2059-2192, 2193-2306, 2307-2440, 2441-2553, 2554-2644 or 2645-2710 of toxin A (SEQ ID NO:1) or an equivalent in a different strain of C. difficile. In another embodiment, the repeat portion of toxin B refers to amino acids 1834-1926, 1927-2057, 2058-2189, 2190-2323 or 2324-2366 of toxin B (SEQ ID NO:2) or an equivalent in a different strain of C. difficile. 
The term ‘short repeat’ may refer to amino acids 1832-1852, 1853-1873, 1874-1893, 1925-1944 1945-1965, 1966-1986, 1987-2007, 2008-2027, 2059-2078, 2079-2099, 2100-2120, 2121-2141, 2142-2161, 2193-2212, 2213-2233, 2234-2253, 2254-2275, 2307-2326, 2327-2347, 2348-2368, 2369-2389, 2390-2409, 2441-2460, 2461-2481, 2482-2502, 2503-2522, 2554-2573, 2574-2594, 2595-2613, 2645-2664, 2665-2686 or 2687-2710 of toxin A (SEQ ID NO:1) or amino acids 1834-1854, 1855-1876, 1877-1896, 1927-1946, 1947-1967, 1968-1987, 1988-2007, 2008-2027, 2058-2078, 2079-2099, 2100-2119, 2120-2139, 2140-2159, 2190-2212, 2213-2233, 2234-2253, 2254-2273, 2274-2293, 2324-2343 or 2344-2366 of toxin B (SEQ ID NO:2) or their equivalents in a different strain of C. difficile. 
Similarly the term ‘long repeat’ may refer to amino acids 1894-1924, 2028-2058, 2162-2192, 2276-2306, 2410-2440, 2523-2553 or 2614-2644 of toxin A (SEQ ID NO:1) or amino acids 1897-1926, 2028-2057, 2160-2189 or 2294-2323 of toxin B (SEQ ID NO:2) or their equivalents in a different strain of C. difficile. 
The polypeptides of the invention may be part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.
The word ‘adjacent’ means separated by less than or exactly 20, 15, 10, 8, 5, 2, 1 or 0 amino acids in the primary structure.
The fragments may be positioned such that the N-terminus of the first fragment is adjacent to the C-terminus of the second fragment, alternatively the C-terminus of the first fragment may be adjacent to the N-terminus of the second fragment, or the C-terminus of the first fragment may be adjacent to the C-terminus of the second fragment, or the N-terminus of the first fragment may be adjacent to the N-terminus of the second fragment.
Two sequences will have ‘sequence similarity to one another’ if they have greater than 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity.
The term ‘identity’ is known in the art. Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the Needle program, BLASTP, BLASTN (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990)), and FASTA Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988)). The BLAST family of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)). The well known Smith Waterman algorithm may also be used to determine identity.
In one example, parameters for polypeptide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
Gap Penalty: 10
Gap extension penalty: 0.5
A program useful with these parameters is publicly available as the ‘Needle’ program from EMBOSS package (Rice P. et al, Trends in Genetics 2000 col. 16(6):276-277). The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
In one embodiment the first repeat portion and the second repeat portion have high structural similarity to one another. Two sequences can be considered to have high structural similarity when their sequence identity is higher than 40%, 45%, 50% or 60%. The presence of high sequence identity can be determined by using softwares that allow sequences alignment (EMBOSS, Blast, . . . ). 3D model structure could be faithfully built, using the SwissModel web server, for a protein if it shares more than 40%, 45%, 50% or 60% sequence identity with a protein having a known 3D structure.
In one embodiment the polypeptide of the invention elicits antibodies that neutralise toxin A or toxin B. In a further embodiment the polypeptide elicits antibodies that neutralise toxin A. In a further embodiment the polypeptide elicits antibodies that neutralise toxin B. In a further embodiment the polypeptide elicits antibodies that neutralise toxin A and toxin B. The phrase ‘elicits neutralising antibodies’ means that the when the compositions are used to immunise a mammal, for example a mouse, a guinea pig or a human, the mammal generates neutralising antibodies.
Whether a polypeptide elicits neutralizing antibodies against a toxin can be measured by immunising mice with an immunogenic composition comprising the polypeptide, collecting sera and analysing the anti-toxin titres of the sera using by enzyme-linked immunosorbent assay (ELISA). The sera could be compared to a reference sample obtained from mice which have not been immunised. An example of this technique can be found in example 6. The polypeptide of the invention elicits antibodies that neutralise toxin A if the sera against the polypeptide gives an ELISA readout more than 10%, 20%, 30%, 50%, 70%, 80%, 90% or 100% higher than the reference sample.
In a further embodiment the polypeptide of the invention elicits a protective immune response in a mammalian host against strains of C. difficile. The phrase ‘elicit a protective immune response’ means that when the immunogenic composition of the invention is used to immunise a mammal such as a mouse, guinea pig or human, the mammal generates antibodies capable of protecting the mammal from death caused by C. difficile. In one embodiment the mammalian host is selected from the group consisting of mouse, rabbit, guinea pig, monkey, non-human primate or human. In one embodiment the mammalian host is a mouse. In a further embodiment the mammalian host is a human.
Whether a polypeptide elicits a protective immune response in a mammalian host against strains of C. difficile can be determined using a challenge assay. In such an assay the mammalian host is vaccinated with the polypeptide and challenged by exposure to C. difficile. The time which the mammal survives after challenge is compared with the time which a reference mammal that has not been immunised with the polypeptide survives. A polypeptide elicits a protective immune response if a mammal immunised with the polypeptide survives at least 10%, 20%, 30%, 50%, 70%, 80%, 90%, or 100% longer after challenge with C. difficile than a reference mammal which has not been immunised with the polypeptide. In one embodiment the polypeptide of the invention elicits a protective immune response against C. difficile in a mammal selected from the group consisting of mouse, guinea pig, monkey or human. In one embodiment the mammal is a mouse, in a further embodiment the mammal is a human.
The native structure of the C-terminal (repeat) domains from toxins A and B consist of an extended β solenoid-like structure. This structure consists of primarily β sheet structures, with a minority of a helical structures as seen in Ho et al (PNAS 102:18373-18378 (2005)). The secondary structures present can be determined using circular dichroism (CD). For example, the secondary structure may be determined by measuring the shape and the magnitude of the CD spectra in the far-UV region (190-250 nm) and comparing the results with those of known structures. This can be carried out using an optical path of 0.01 cm from 178 to 250 nm, with a 1 nm resolution and bandwidth on a Jasco J-720 spectropolarimeter, for example as seen in example 5 below.
In one embodiment the first fragment comprises less than 28%, 25%, 23%, 20%, 18%, 15%, 10%, or 7% alpha helical secondary structure. In one embodiment the second fragment comprises less than 28%, 25%, 23%, 20%, 18%, 15%, 10%, or 7% alpha helical secondary structure. In a further embodiment both the first fragment and the second fragment comprise less than 28%, 25%, 23%, 20%, 18%, 15%, 10%, or 7% alpha helical secondary structure.
In one embodiment the first fragment comprises more than 20%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, or 42% beta sheet structure. In one embodiment the second fragment comprises more than 20%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, or 42% beta sheet structure. In a further embodiment both the first fragment and the second fragment comprises more than 20%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, or 42% beta sheet structure.
In one embodiment the first proximal end is within repeat portion V (amino acids 2307-2440 of SEQ ID NO:1 or their equivalent in a different strain), repeat portion VI (amino acids 2441-2553 of SEQ ID NO:1 or their equivalent in a different strain), repeat portion VII (amino acids 2554-2644 of SEQ ID NO:1 or their equivalent in a different strain) or repeat portion VIII (amino acids 2645-2710 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A. In a further embodiment the first proximal end is within repeat portion VII (amino acids 2554-2644 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A. In a further embodiment the first proximal end repeat portion VIII (amino acids 2645-2710 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A.
In one embodiment the second proximal end is within repeat portion I (amino acids 1834-1926 of SEQ ID NO:2 or their equivalent in a different strain), repeat portion II (amino acids 1927-2057 of SEQ ID NO:2 or their equivalent in a different strain), or repeat portion III (amino acids 2058-2189 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B. In a further embodiment the second proximal end is within repeat portion II (amino acids 1927-2057 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B. In a further embodiment the second proximal end is within repeat portion I (amino acids 1834-1926 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B.
In one embodiment the first proximal end is within a long repeat. The first proximal end may be within long repeat V of toxin A (amino acids 2410-2440 of SEQ ID NO:1 or their equivalent in a different strain), or within long repeat VI of toxin A (amino acids 2523-2553 of SEQ ID NO:1 or their equivalent in a different strain), or within long repeat VII of toxin A (amino acids 2614-2644 of SEQ ID NO:1 or their equivalent in a different strain).
In one embodiment the second proximal end is within a long repeat. The second proximal end may be within long repeat I of toxin B (amino acids 1897-1926 of SEQ ID NO:2 or their equivalent in a different strain), or within long repeat II of toxin B (amino acids 2028-2057 of SEQ ID NO:2 or their equivalent in a different strain), or within long repeat III of toxin B (amino acids 2160-2189 of SEQ ID NO:2 or their equivalent in a different strain).
In a further embodiment the first proximal end and the second proximal end are both within long repeats. In one embodiment the first proximal end is within long repeat V of toxin A (amino acids 2410-2440 of SEQ ID NO:1 or their equivalent in a different strain), or within long repeat VI of toxin A (amino acids 2523-2553 of SEQ ID NO:1 or their equivalent in a different strain), or within long repeat VII of toxin A (amino acids 2614-2644 of SEQ ID NO:1 or their equivalent in a different strain) and the second proximal end is within long repeat I of toxin B (amino acids 1897-1926 of SEQ ID NO:2 or their equivalent in a different strain), or within long repeat II of toxin B (amino acids 2028-2057 of SEQ ID NO:2 or their equivalent in a different strain), or within long repeat III of toxin B (amino acids 2160-2189 of SEQ ID NO:2 or their equivalent in a different strain). In one embodiment the first proximal end is within long repeat VII of toxin A (amino acids 2614-2644 of SEQ ID NO:1 or their equivalent in a different strain) and the second proximal end is within long repeat II of toxin B (amino acids 2028-2057 of SEQ ID NO:2 or their equivalent in a different strain).
In one embodiment the first proximal end is within amino acids 2620-2660 of toxin A. In one embodiment the second proximal end is within amino acids 2030-2050 of toxin B. In a further embodiment the first proximal end is within amino acids 2620-2660 of toxin A and the second proximal end is within amino acids 2030-2050 of toxin B.
In one embodiment the first fragment comprises at least 100, 150, 180, 200, 240, 250, 280, 300, 330, 350, 380, 400, 430, 450, 480, 500 or 530 amino acids. In one embodiment the second fragment comprises at least 100, 130, 150, 180, 200, 230, 250, 270, 300, 330, 350, 390, or 400 amino acids.
In one embodiment the polypeptide further comprises a linker. This linker may be between the first proximal end and the second proximal end, alternatively the linker may link the distal ends of the first fragment and/or the second fragment to a further sequence of amino acids.
A peptide linker sequence may be employed to separate the first fragment and second fragment. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first fragment and/or the second fragments; and (3) the lack of hydrophobic or charged residues that might react with the ToxA and/or ToxB functional epitopes. Peptide linker sequences may contain Glycine (Gly), Asparagine (Asn) and Serine (Ser) residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length.
In one embodiment the linker comprises 1-20, 1-15, 1-10, 1-5, 1-2, 5-20, 5-15, 5-15, 10-20, or 10-15 amino acids. In one embodiment the linker is a glycine linker, the linker may comprise multiple contiguous glycine residues (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, or 20), or alternatively the linker may comprise some glycine residues and some residues of other amino acids such as alanine. In a further embodiment the linker comprises a single glycine residue.
In an embodiment the polypeptide of the invention is part of a larger fusion protein. The fusion proteins may further comprise amino acids encoding an immunogenic portion of a further protein antigen. For example the fusion protein may further comprise an immunogenic portion of a protein antigen obtained or derived from a bacterium selected from the group consisting of S. pneumoniae, H. influenzae, N. meningitidis, E. coli, M. cattarhalis, C. tetanii, C. diphtheriae, B. pertussis, S. epidermidis, enterococci, S. aureus, and Pseudomonas aeruginosa. In this case the linker may be between the first fragment or the second fragment and a further immunogenic portion of a protein antigen.
The term “immunogenic portion thereof” or ‘immunogenic fragment’ refers to a fragment of a polypeptide wherein the fragment comprises an epitope that is recognized by cytotoxic T lymphocytes, helper T lymphocytes or B cells. Suitably, the immunogenic portion will comprise at least 30%, suitably at least 50%, especially at least 75% and in particular at least 90% (e.g. 95% or 98%) of the amino acids in the reference sequence. In one embodiment, the immunogenic portion will comprise all of the epitope regions of the reference sequence.
Immunogenic compositions according to the invention comprising the specific fusion proteins of C. difficile toxin A repeating domain fragment and a C. difficile toxin B repeating domain fragment described herein are also expected to provide an improved immune response against C. difficile as compared to compositions comprising known C. difficile polypeptides or fragments.
Adjuvants
The immunogenic compositions disclosed herein include at least one C. difficile polypeptide in combination with an aluminum-free adjuvant. The polypeptides are formulated with an adjuvant that is free of aluminum or aluminum salts, that is, an aluminum-free adjuvant or adjuvant system.
In certain embodiments, the C. difficile polypeptide is formulated with an adjuvant comprising an immunologically active saponin fraction presented in the form of a liposome. The adjuvant may further comprise a lipopolysaccharide. The adjuvant may include QS21. For example, in one embodiment, the adjuvant contains QS21 in a liposomal formulation. In one embodiment, the adjuvant system includes 3D-MPL and QS21. For example, in one embodiment, the adjuvant contains 3D-MPL and QS21 in a liposomal formulation. Optionally, the adjuvant system also contains cholesterol. In one specific embodiment, the adjuvant includes QS21 and cholesterol. Optionally, the adjuvant system contains 1, 2-Dioleoyl-sn-Glycero-3-phosphocholine (DOPC). For example, in one specific adjuvant system contains cholesterol, DOPC, 3D-MPL and QS21.
In one specific example, the immunogenic composition includes an adjuvant formulated in a dose that includes: from about 0.1 to about 0.5 mg cholesterol; from about 0.25 to about 2 mg DOPC; from about 10 μg to about 100 μg 3D-MPL; and from about 10 μg to about 100 μg QS21. In a further specific example the immunogenic composition includes and adjuvant formulated in a dose that includes: from about 0.1 to about 0.5 mg cholesterol, from about 0.25 to about 2 mg DOPC, from about 10 μg to about 100 μg 3D-MPL, and from about 10 μg to about 100 μg QS21. In a further specific formulation the adjuvant is formulated in a dose comprising from about 0.1 to about 0.5 mg cholesterol, from about 0.25 to about 2 mg DOPC, from about 10 μg to about 70 μg 3D-MPL, and from about 10 μg to about 70 μg QS21. In one specific formulation, the adjuvant is formulated in a single dose that contains: about 0.25 mg cholesterol; about 1.0 mg DOPC; about 50 μg 3D-MPL; and about 50 μg QS21. In other embodiments, the immunogenic composition is formulated with a fractional dose (that is a dose, which is a fraction of the preceding single dose formulations, such as one half of the preceding quantity of components (cholesterol, DOPC, 3D-MPL and QS21), 1⁄4 of the preceding quantity of components, or another fractional dose (e.g., 1⁄3, 1⁄6, etc.) of the preceding quantity of components.
In one embodiment, the immunogenic compositions according to the invention include an adjuvant containing combinations of lipopolysaccharide and Quillaja saponins that have been disclosed previously, for example in EP0671948. This patent demonstrated a strong synergy when a lipopolysaccharide (3D-MPL) was combined with a Quillaja saponin (QS21).
The adjuvant may further comprise immunostimulatory oligonucleotides (for example, CpG) or a carrier.
A particularly suitable saponin for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quillaja Saponaria Molina and was first described by Dalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. für die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p 243-254) to have adjuvant activity. Purified fragments of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS21 (also known as QA7 and QA21). QS21 is a natural saponin derived from the bark of Quillaja saponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant IgG2a antibody response and is a preferred saponin in the context of the present invention.
In a specific embodiment, QS21 is provided in its less reactogenic composition where it is quenched with an exogenous sterol, such as cholesterol for example. Several particular forms of less reactogenic compositions wherein QS21 is quenched with an exogenous cholesterol exist. In a specific embodiment, the saponin/sterol is in the form of a liposome structure (WO 96/33739, Example 1). In this embodiment the liposomes suitably contain a neutral lipid, for example phosphatidylcholine, which is suitably non-crystalline at room temperature, for example eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilauryl phosphatidylcholine. The liposomes may also contain a charged lipid which increases the stability of the liposome-QS21 structure for liposomes composed of saturated lipids. In these cases the amount of charged lipid is suitably 1-20% w/w, preferably 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), suitably 20-25%.
Suitable sterols include 3-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In one particular embodiment, the adjuvant composition comprises cholesterol as sterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 11th Edn., page 341, as a naturally occurring sterol found in animal fat.
Where the active saponin fraction is QS21, the ratio of QS21:sterol will typically be in the order of 1:100 to 1:1 (w/w), suitably between 1:10 to 1:1 (w/w), and preferably 1:5 to 1:1 (w/w). Suitably excess sterol is present, the ratio of QS21:sterol being at least 1:2 (w/w). In one embodiment, the ratio of QS21:sterol is 1:5 (w/w). The sterol is suitably cholesterol.
In one embodiment, the invention provides a dose of an immunogenic composition comprising immunologically active saponin, preferably QS21, at a level of 60 μg or less, for example between 1 and 60 μg. In one embodiment, the dose of the immunogenic composition comprises QS21 at a level of approximately around 50 μg, for example between 45 and 55 μg, suitably between 46-54 μg or between 47 and 53 μg or between 48 and 52 μg or between 49 and 51 μg, or 50 μg per dose.
In another embodiment the dose of the immunogenic composition comprises QS21 at a level of around 25 μg, for example between 20-30 μg, suitably between 21-29 μg or between 22 and 28 μg or between 23 and 27 μg or between 24 and 26 μg, or 25 μg.
In another embodiment, the dose of the immunogenic composition comprises QS21 at a level of around 10 μg per, for example between 5 and 15 μg, suitably between 6 and 14 μg, for example between 7 and 13 μg or between 8 and 12 μg or between 9 and 11 μg, or 10 μg.
Specifically, a 0.5 ml vaccine dose volume contains 25 μg or 50 μg of QS21 per dose. Specifically, a 0.5 ml vaccine dose volume contains 50 μg of QS21 per dose.
The lipopolysaccharide may be a non-toxic derivative of lipid A, particularly monophosphoryl lipid A or more particularly 3-Deacylated monophosphoryl lipid A (3D-MPL).
3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A. and is referred throughout the document as MPL or 3D-MPL. See, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL primarily promotes CD4+ T cell responses with an IFN-□ (Th1) phenotype. 3D-MPL can be produced according to the methods disclosed in GB 2 220 211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. Preferably in the compositions of the present invention small particle 3D-MPL is used. Small particle 3D-MPL has a particle size such that it may be sterile-filtered through a 0.24 m filter. Such preparations are described in WO 94/21292.
The invention therefore provides a dose of an immunogenic composition comprising lipopolysaccharide, preferably 3D-MPL, at a level of 75 μg or less, for example between 1 and 60 μg. In one embodiment the lipopolysaccharide is present at an amount of about 50 μg per dose.
In one embodiment, the dose of the immunogenic composition comprises 3D-MPL at a level of around 50 μg, for example between 45-55 μg, suitably between 46-54 μg or between 47 and 53 μg or between 48 and 52 μg or between 49 and 51 μg, or 50 μg.
In one embodiment, the dose of the immunogenic composition comprises 3D-MPL at a level of around 25 μg, for example between 20-30 μg, suitably between 21-29 μg or between 22 and 28 μg or between 23 and 27 μg or between 24 and 26 μg, or 25 μg.
In another embodiment, the dose of the immunogenic composition comprises 3D-MPL at a level of around 10 μg, for example between 5 and 15 μg, suitably between 6 and 14 μg, for example between 7 and 13 μg or between 8 and 12 μg or between 9 and 11 μg, or 10 μg.
In one embodiment, the volume of the dose is 0.5 ml. In a further embodiment, the immunogenic composition is in a volume suitable for a dose which volume is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In a further embodiment, the human dose is between 1 ml and 1.5 ml.
Specifically, a 0.5 ml vaccine dose volume contains 25 μg or 50 μg of 3D-MPL per dose. Specifically, a 0.5 ml vaccine dose volume contains 50 μg of 3D-MPL per dose.
The dose of the immunogenic composition according to any aspect of the invention suitably refers to human dose. By the term “human dose” is meant a dose which is in a volume suitable for human use. Generally this is between 0.3 and 1.5 ml. In one embodiment, a human dose is 0.5 ml. In a further embodiment, a human dose is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In a further embodiment, a human dose is between 1 ml and 1.5 ml.
Suitable compositions of the invention are those wherein liposomes are initially prepared without MPL (as described in WO 96/33739), and MPL is then added, suitably as small particles of below 100 nm particles or particles that are susceptible to sterile filtration through a 0.22 μm membrane. The MPL is therefore not contained within the vesicle membrane (known as MPL out). Compositions where the MPL is contained within the vesicle membrane (known as MPL in) also form an aspect of the invention. The polypeptide comprising a C. difficile toxin A fragment and/or a C. difficile toxin B fragment can be contained within the vesicle membrane or contained outside the vesicle membrane.
In a specific embodiment, QS21 and 3D-MPL are present in the same final concentration per dose of the immunogenic composition. In one aspect of this embodiment, a dose of immunogenic composition comprises a final level of 25 μg of 3D-MPL and 25 μg of QS21 or 50 μg of 3D-MPL and 50 μg of QS21.
In one embodiment, the adjuvant includes an oil-in-water emulsion. For example, the oil-in-water emulsion can include an oil phase that incorporates a metabolisable oil, and an additional oil phase component, such as a tocol. The oil-in-water emulsion may also contain an aqueous component, such as a buffered saline solution (e.g., phosphate buffered saline). In addition, the oil-in-water emulsion typically contains an emulsifier. In one embodiment, the metabolizable oil is squalene. In one embodiment, the tocol is alpha-tocopherol. In one embodiment, the emulsifier is a nonionic surfactant emulsifier (such as polyoxyethethylene sorbitan monooleate, TWEEN80TM). In exemplary embodiments, the oil-in-water emulsion contains squalene and alpha tocopherol in a ratio which is equal or less than 1 (w/w).
The metabolisable oil in the oil-in-water emulsion may be present in an amount of 0.5-10 mg. The tocol in the oil-in-water emulsion may be present in an amount of 0.5-11 mg. The emulsifying agent in the oil-in-water emulsion may be present in an amount of 0.4-4 mg.
In order for any oil in water composition to be suitable for human administration, the oil phase of the emulsion system has to comprise a metabolisable oil. The meaning of the term metabolisable oil is well known in the art. Metabolisable can be defined as ‘being capable of being transformed by metabolism’ (Dorland's Illustrated Medical Dictionary, W.B. Sanders Company, 25th edition (1974)). The oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE® (caprylic/capric triglycerides made using glycerol from vegetable oil sources and medium-chain fatty acids (MCTs) from coconut or palm kernel oils) and others. A particularly suitable metabolisable oil is squalene. Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil for use in this invention. Squalene is a metabolisable oil by virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no. 8619).
Suitably the metabolisable oil is present in the adjuvant composition in an amount of 0.5-10 mg, preferably 1-10, 2-10, 3-9, 4-8, 5-7, or 5-6 mg (e.g. 2-3, 5-6, or 9-10 mg), specifically about 5.35 mg or around 2.14 mg/dose.
Tocols are well known in the art and are described in EP0382271. Suitably the tocol is alpha-tocopherol or a derivative thereof such as alpha-tocopherol succinate (also known as vitamin E succinate). Said tocol is suitably present in in an amount of 0.5-11 mg, preferably 1-11, 2-10, 3-9, 4-8, 5-7, 5-6 mg (e.g. 10-11, 5-6, 2.5-3.5 or 1-3 mg). In a specific embodiment the tocol is present in an amount of about 5.94 mg or around 2.38 mg per dose.
The oil in water emulsion further comprises an emulsifying agent. The emulsifying agent may suitably be polyoxyethylene sorbitan monooleate. In a particular embodiment the emulsifying agent may be Polysorbate® 80 (Polyoxyethylene (20) sorbitan monooleate) or Tween® 80.
Said emulsifying agent is suitably present in the adjuvant composition in an amount of 0.1-5, 0.2-5, 0.3-4, 0.4-3 or 2-3 mg (e.g. 0.4-1.2, 2-3 or 4-5 mg) emulsifying agent. In a specific embodiment the emulsifying agent is present in an amount of about 0.97 mg or about 2.425 mg.
In one embodiment, the amounts of specific components present in the composition are the amounts present in a 0.5 ml human dose. In a further embodiment, the immunogenic composition is in a volume suitable for a human dose which volume is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In a further embodiment, the human dose is between 1 ml and 1.5 ml.
Where the adjuvant is in a liquid form and is to be combined with a liquid form of a polypeptide composition, the adjuvant composition in a human dose will be a fraction of the intended final volume of the human dose, for example approximately half of the intended final volume of the human dose, for example a 350 μl volume for an intended human dose of 0.7 ml, or a 250 μl volume for an intended human dose of 0.5 ml. The adjuvant composition is diluted when combined with the polypeptide antigen composition to provide the final human dose of vaccine. The final volume of such dose will of course vary dependent on the initial volume of the adjuvant composition and the volume of polypeptide antigen composition added to the adjuvant composition. In an alternative embodiment, a liquid adjuvant is used to reconstitute a lyophilised polypeptide composition. In this embodiment, the human dose of the adjuvant composition is approximately equal to the final volume of the human dose. The liquid adjuvant composition is added to the vial containing the lyophilised polypeptide composition. The final human dose can vary between 0.5 and 1.5 ml.
The method of producing oil-in-water emulsions is well known to the person skilled in the art. Commonly, the method comprises mixing the tocol-containing oil phase with a surfactant such as a PBS/polyoxyethylene sorbitan monooleate solution, followed by homogenisation using a homogenizer. It would be clear to a man skilled in the art that a method comprising passing the mixture twice through a syringe needle would be suitable for homogenising small volumes of liquid. Equally, the emulsification process in microfluidiser (M110S Microfluidics machine, maximum of 50 passes, for a period of 2 minutes at maximum pressure input of 6 bar (output pressure of about 850 bar)) could be adapted by the man skilled in the art to produce smaller or larger volumes of emulsion. The adaptation could be achieved by routine experimentation comprising the measurement of the resultant emulsion until a preparation was achieved with oil droplets of the required diameter.
In an oil in water emulsion, the oil and emulsifier should be in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.
Preferably the oil-in-water emulsion systems of the present invention have a small oil droplet size in the sub-micron range. Suitably the droplet sizes will be in the range 120 to 750 nm, more preferably sizes from 120 to 600 nm in diameter. Most preferably the oil-in water emulsion contains oil droplets of which at least 70% by intensity are less than 500 nm in diameter, more preferably at least 80% by intensity are less than 300 nm in diameter, more preferably at least 90% by intensity are in the range of 120 to 200 nm in diameter.
In one embodiment, the immunogenic composition is not 3 μg or 10 μg of any of SEQ ID Nos. 1 to 7 combined with an adjuvant comprising an oil in water emulsion having 0.125 mL SB62 emulsion (Total volume), 5.35 mg squalene, 5.94 mg DL-α-tocopherol and 2.425 mg polysorbate 80 per 0.5 ml dose. In one embodiment, the immunogenic composition is not 3 μg or 10 μg of any of SEQ ID Nos. 1 to 7 combined with an adjuvant comprising an oil in water emulsion 5.35 mg squalene, 5.94 mg DL-α-tocopherol and 2.425 mg polysorbate 80 per 0.5 ml dose. In one embodiment, the immunogenic composition does not contain an adjuvant comprising a oil in water emulsion having squalene, DL-α-tocopherol and polysorbate 80.
Immunogenic Compositions and Vaccines
In one embodiment the immunogenic composition further comprises additional antigens. In one embodiment the additional antigens are antigens derived from a bacterium selected from the group consisting of Streptococcus pneumoniae (S. pneumoniae), Haemophilus influenzae (H. influenzae), Neisseria meningitidis (N. meningitidis), Escherichia coli (E. coli), Moraxella catarrhalis (M. cattarhalis), Clostridium tetani (C. tetani), Corynebacterium diptherieriae (C. diptherieriae), Bordetella pertussis (B. pertussis), Staphylococcus epidermidis (S. epidermidis), enterococci, and Staphylococcus aureus (S. aureus). In a further embodiment the immunogenic composition of the invention may comprise further antigens from C. difficile for example the S-layer proteins (WO01/73030). Optionally the immunogenic composition further comprises a saccharide from C. difficile. 
There is further provided a vaccine comprising the immunogenic composition. This vaccine may further comprise a pharmaceutically acceptable excipient. In a further aspect of the invention there is provided a vaccine comprising the immunogenic composition of the invention and an adjuvant.
The vaccine preparations containing immunogenic compositions of the present invention may be used to protect a mammal susceptible to C. difficile infection or treat a mammal with a C. difficile infection, by means of administering said vaccine via a systemic or mucosal route. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts. Although the vaccine of the invention may be administered as a single dose, components thereof may also be co-administered together at the same time or at different times (for instance pneumococcal saccharide conjugates could be administered separately, at the same time or 1-2 weeks after the administration of the any bacterial protein component of the vaccine for coordination of the immune responses with respect to each other). In addition to a single route of administration, 2 different routes of administration may be used. For example, saccharides or saccharide conjugates may be administered intramuscularly (IM) or intradermally (ID) and bacterial proteins may be administered intranasally (IN) or intradermally (ID). In addition, the vaccines of the invention may be administered IM for priming doses and IN for booster doses.
The content of toxins in the vaccine will typically be in the range 1-250 μg, preferably 5-50 μg, most typically in the range 5-25 μg. Following an initial vaccination, subjects may receive one or several booster immunizations adequately spaced. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
In one aspect of the invention is provided a vaccine kit, comprising a vial containing an immunogenic composition of the invention, optionally in lyophilised form, and further comprising a vial containing an adjuvant as described herein. It is envisioned that in this aspect of the invention, the adjuvant will be used to reconstitute the lyophilised immunogenic composition.
A further aspect of the invention is a method of preventing or treating C. difficile infection comprising administering to the subject an immunoprotective dose of the immunogenic composition or vaccine of the invention. In one embodiment there is provided a method of preventing or treating primary and/or recurrence episodes of C. difficile infection comprising administering to the subject an immunoprotective dose of the immunogenic composition or vaccine of the invention. In one embodiment the subject is selected from the group consisting of mouse, rabbit, guinea pig, monkey, non-human primate or human. In one embodiment the subject is a mouse. In a further embodiment the subject is a human
A further aspect of the invention is an immunogenic composition or vaccine or kit of the invention for use in the treatment or prevention of infection or disease caused by C. difficile. In one embodiment there is provided an immunogenic composition or vaccine or kit of the invention for use in the treatment or prevention of primary and/or recurrence episodes of C. difficile disease.
A further aspect of the invention is use of the immunogenic composition or vaccine or kit of the invention in the manufacture of a medicament for the treatment or prevention of C. difficile disease. In one embodiment there is provided an immunogenic composition or vaccine or kit of the invention for use in the manufacture of a medicament for the treatment or prevention of primary and/or recurrence episodes of C. difficile disease.
“C. difficile disease” refers to any infection or disease caused by toxins released by C. difficile. Examples of C. difficile disease are antibiotic-associated diarrhea (AAD), pseudomembranous colitis and toxic megacolon, which can be life-threatening.
“Around” or “approximately” are defined as within 10% more or less of the given figure for the purposes of the invention.
The terms “comprising”, “comprise” and “comprises” herein are intended by the inventors to be optionally substitutable with the terms “consisting of”, “consist of” and “consists of”, respectively, in every instance. The term “comprises” means “includes.” Thus, unless the context requires otherwise, the word “comprises,” and variations such as “comprise” and “comprising” will be understood to imply the inclusion of a stated compound or composition (e.g., nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps, or groups thereof. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
Embodiments herein relating to “vaccine” of the invention are also applicable to embodiments relating to “immunogenic compositions” of the invention, and vice versa.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “plurality” refers to two or more. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are estimates, and are provided for description. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as a polypeptide antigen, may be approximate.
All references or patent applications cited within this patent specification are incorporated by reference herein in their entirety.
These examples set forth below are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.
It will be understood that particular aspects and embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. All documents referred to herein are incorporated by reference to the fullest extent permissible.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
The invention will be further described by reference to the following, non-limiting, figures and examples.
EXAMPLES
Adjuvants Used in the Experiments:
Adjuvant A
Adjuvant having 50 μg QS21 presented in the form of a liposome, 50 μg 3D-MPL, 0.25 mg cholesterol and 1.0 mg DOPC per 0.5 ml dose. A dose of 50 μl suitable for immunizing hamsters contains 5 μg QS21, 5 μg 3D-MPL, 0.025 mg cholesterol and 0.1 mg DOPC.
Adjuvant B
Adjuvant consisting of an oil in water emulsion having 0.125 mL SB62 emulsion (Total volume), 5.35 mg squalene, 5.94 mg DL-α-tocopherol and 2.425 mg polysorbate 80 per 0.5 ml dose. A 5011 dose contains 0.0125 ml SB62 emulsion (Total volume), 0.535 mg squalene, 0.594 mg DL-α-tocopherol and 0.2425 mg polysorbate 80
Adjuvant C
Adjuvant having 25 μg QS21 presented in the form of a liposome, 25 μg 3D-MPL, 0.25 mg cholesterol and 1.0 mg DOPC. A 50 μl dose contains 2.5 μg QS1′, 2.5 μg 3D-MPL, 0.025 mg cholesterol and 0.1 mg DOPC.
GSK Adjuvant AS04D
Adjuvant Having 50 μg MPL and 0.5 mg (Al3+ Total) Per 0.5 ml Dose. A Dose of 50 μl Contains 514 MPL and 50 μg (AL3+ Total).
Alum
Adjuvant Having Aluminium Hydroxide, 500 μg Per 0.5 ml Dose. A Dose of 50 μl Suitable of Hamster Immunisation Contains 50 μg Per 50 μg Dose.
Protocols
Anti-ToxA and Anti-ToxB ELISA Response: Protocol for Examples 2 to 4, 7 and 8
ToxA or ToxB fragments (for the examples 2 and 4-ToxA (2121-2686) and ToxB (1968-2366) for the examples 3, 5, 6, 7 and 8-ToxA (2387-2706) and ToxB (1750-2360)) were coated at 2 μg/ml (for ToxA) or 1 μg/ml (for ToxB) in phosphate buffered saline (PBS) on high-binding microtitre plates (Nunc MAXISORPTM), overnight at 4° C. The plates were blocked with PBS+1% Bovine serum albumin (BSA) for 30 min at RT with agitation. Hamster or mice sera are prediluted 1/100 (for post dose I or II samples) or 1/500 (for post dose III samples) in PBS-BSA 0.2%-TWEENTM 0.05% and then, 2 further twofold dilutions were made in microplates and incubated at room temperature (RT) for 30 min. After washing, bound hamster or mouse antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated Anti-Mouse (ref: 110-035-003) or Anti-Hamster (ref: 107-035-142) diluted 1:5000 in PBS-BSA 0.2%-tween 0.05% (‘detection antibody’). The detection antibodies were incubated for 30 min at 20° C. with agitation. The color was developed using 4 mg 0-phenylenediamine (OPD)+5 μl H2O2 per 10 ml pH 4.5 0.1M citrate buffer for 15 minutes in the dark at room temperature. The reaction was stopped with 50 μl HCl, and the optical density (OD) was read at 490 nm relative to 620 nm.
The level of anti-ToxA or anti-ToxB antibodies present in each individual sera is determined by comparison to a reference serum (calibrated against Jackson Global IgG) added on each plate and expressed in μg/ml. A geometric mean titer GMT was calculated using Excel software for the samples in each treatment group for the hamster or mice immunizations.
Example 1
Design of Five C. difficile ToxA-ToxB Fusion Proteins
Fusion proteins (also called ‘fusions’) containing fragments of the C-terminal repeating domains of ToxA and ToxB were designed. These fusion proteins contained a fragment of the C-terminal repeating domain of ToxA and a fragment of the C-terminal repeating domain of ToxB, and a junction between the C-terminal end of the ToxA fragment and the N terminal end of the ToxB fragment. Two strategies were devised, in the first strategy; the fusion was designed such that the long solenoid structure was maintained at the junction between the two fragments. In the second strategy, the two fragments of the fusions are separated by a linker to allow their independent correct folding.
The C-terminal part of ToxA and ToxB is composed of repeated sequences: short repeats (SR) and long repeats (LR) (PNAS 2005 vol 102: 18373-18378).
The partial known 3D structure for the C-terminal domain of ToxA has been described (PNAS 2005 Greco et al., vol 102: 18373-18378; Nature Structural & Molecular biology 2006 vol 13(5): 460-461; PDB codes: 2F6E, 2G7C and 2QJ6).
The inventors predicted that there are two kinds of important interactions between residues of the C-terminal part of ToxA and ToxB. The first interaction occurs between residues contained in a LR and its preceding SR and is important to maintain the solenoid-like structure. The second type of interaction occurs between residues contained in a LR and the following SR and this interaction is mediating the carbohydrate-binding function of the toxin.
A new “structural-functional” repeat SR-LR-SR was defined. The structure of this repeat was maintained intact in the designed fusions.
The positions of the short (SR) and long repeats (LR) of ToxA and ToxB repeats are presented in Table 1.
A list of the “SR-LR-SR” boxes contained in the C-terminal domain of ToxA and ToxB is presented in Table 2 below.













Start
End


Name
position
position







ToxA_1
1874
1944


ToxA_2
2008
2078


ToxA_3
2142
2212


ToxA_4
2254
2326


ToxA_5
2390
2460


ToxA_6
2503
2573


ToxA_7
2595
2664


ToxB_1
1877
1946


ToxB_2
2008
2078


ToxB_3
2140
2212


ToxB_4
2274
2343









Finally, the number of SRs between two LRs were maintained in the designed fusions to keep the long solenoid-like structure.
Before the design of junctions for the fusions, two working hypotheses were defined: first hypothesis, the shorter the fusions, the better the probability for the fusions to be stably over expressed; second hypothesis, according to the concept of “SR-LR-SR” boxes, the start position has to be chosen in order to ensure a correct folding of the first SR of this previously defined SR-LR-SR box. Thus the fusions start at the beginning of the SR that precedes the SR-LR-SR box. Using these two hypotheses, three start positions were analysed: residue 2370, 2234 and 2121 of ToxA.
The start position 2370 was excluded. The start position 2234 was also excluded because one of the residues involved in interactions important for the protein structural stability is not conserved. So, it was decided that all the designed fusion will begin at residue 2121 of ToxA.
All fusions will end at the last residue of ToxB.
Four fusions (F1-4) were designed in order to maintain the entire fusion in a long solenoid-like structure between the two fusion fragments.
Fusions 1 (F1) and 2 (F2) were designed using the same hypothesis. All SR protein sequences of ToxA and ToxB had been compared using a multiple alignment software (ClustalW—Thompson J D et al. (1994) Nucleic Acids Res., 22, 4673-4680). The more similar sequences were the third SR VIII of ToxA and the third SR II of ToxB and third SR III of ToxB. In order to make a choice between these two SR of ToxB, a structural homology modelling (using the SwissModel interface—Arnold K et al. (2006) Bioinformatics, 22, 195-201) was performed on the C-terminal part of ToxB using the known 3D structure of partial ToxA C-terminal domain (PDB code: 2QJ6). Using the third SR VIII of ToxA, the best local structural superposition (performed using SwissPDBViewer—Guex N et al. (1997), Electrophoresis 18, 2714-2723) was obtained with the third SR II of ToxB. So, two junctions were designed: the first one is between the third SR VIII of ToxA and the fourth SR II of ToxB (F1) and the second one is between the second SR VIII of ToxA and the third SR II of ToxB (F2).
To design fusion 3 (F3), a global structural superposition was performed between both the known structure of the partial C-terminal domain of ToxA and the predicted structure of C-terminal domain of ToxB (using SwissModel and SwissPDBViewer softwares). The best superposition was found between LR VII of ToxA and LR II of ToxB. So, it was decided to make a junction in this similar LR. The junction was performed firstly in a region where the sequence is conserved between ToxA and ToxB, after that in order to keep in the ToxA part of the fusion, the residues in interaction with the preceding SR and lastly, in order to keep in the ToxB part, the residues in interaction with the following SR.
For the design of fusion 4 (F4), the C-terminal domain of ToxB was divided in 4 fragments and a more precise homology modelling (SwissModel) was performed on them. The split was realised in order to keep intact the “SR-LR-SR” boxes (each domain finishes at the end of the SR that follows a LR). A structural superposition between the predicted structures of these fragments and the known 3D structure of ToxA was made and the best structural superposition was obtained for the third SR of ToxB (SR I) and the last SR of ToxA (third SR VIII). So, the junction was done between the second SR VIII of ToxA and the third SRI of ToxB.
The last fusion (F5) was designed in order to allow an independent correct folding of the two fragments of the fusion. A linker was added between the last residue of the ToxA protein sequence and the beginning of the fourth SR II of ToxB (always taking into account the importance of an intact “SR-LR-SR” box). Only one exogenous residue (Glycine) was added as a linker and located between two existing Glycines. Thus, the linker can also be described as composed of 3 Glycines surrounding by known (for ToxA) and predicted (for ToxB) beta-strand.
Example 2
Immunisation of Hamsters with F2 Fusion Protein: Adjuvant A, Adjuvant A without QS21 or Adjuvant A without MPL and Comparison with Adjuvant B, Alum or Non Adjuvanted Formulations.
Groups of 16 hamsters (mix of male and female) were immunized IM at days 0, 14 and 28 with 1 μg of the F2 fusion protein adjuvanted with 50 μl of Adjuvant A, Adjuvant A without MPL, Adjuvant A without QS21, Adjuvant B, Alum or non adjuvanted.
Anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 41/42 (Post III). The results are described in tables 3 and 4 and FIGS. 1 and 2.







TABLE 3







ELISA anti-ToxA: concentrations (μg/ml) on individual day 42 sera









Groups














1
2
3
4
5
6



F2
F2
F2
F2
F2
F2



1 μg/
1 μg/
1 μg/
1 μg/
1 μg/
1 μg/dose



dose
dose
dose
dose
dose
non-


ADJUVANT
A
A-QS21
A-MPL
B
Al(OH)3 
adjuvanted
















1
1472
29
935
457
577
3


2
1828
39
414
197
329
23


3
4928
35
747
375
401
64


4
2966
28
550
60
769
25


5
2025
19
388
408
652
3


6
1116
155
1498
173
449
37


7
2579
43
879
732
360
179


8
2463
33
780
196
1342
31


9
1425
52
1183
681
817
7


10
1984
307
696
190
534
199


11
1093
198
572
344
2665
106


12
2080
126
972
508
1944
22


13
3541
403
1823
193
1603
221


14
1635
152
813
1142
2209
680


15
4688
630
1046
494
855
359


16
3360
21
1119
163
664
100


Geomean 
2213
77
830
312
815
49


CI−
1717
40
655
202
563
14


CI+
2802
124
1032
454
1129
104


St dev min
496
37
174
111
251
36


St dev max
589
47
202
141
314
55





CI = confidence interval


St Dev = standard deviation













TABLE 4







ELISA anti-ToxB: concentrations (μg/ml) on individual day 42 sera









Groups














1
2
3
4
5
6



F2
F2
F2
F2
F2
F2



1 μg/
1 μg/
1 μg/
1 μg/
1 μg/
1 μg/dose



dose
dose
dose
dose
dose
non-


ADJUVANT
A
A-QS21
A-MPL
B
Al(OH)3
adjuvanted
















1
811
33
817
65
63
5


2
1186
88
1144
107
177
5


3
4388
90
943
207
216
92


4
1158
66
1224
464
239
26


5
1992
53
1088
212
91
5


6
1518
305
1070
193
138
44


7
5437
54
1021
244
44
57


8
1493
74
899
266
313
28


9
1488
281
1506
189
135
30


10
2637
346
1588
214
318
256


11
1568
215
1125
251
863
95


12
1879
50
1890
549
534
93


13
1920
237
1724
147
355
57


14
1709
266
2450
606
543
57


15
1553
492
2033
301
251
178


16
1556
95
744
167
115
88


Geomean
1789
125
1254
225
207
41


CI−
1387
76
1039
162
128
16


CI+
2264
186
1498
301
308
76


St dev min
402
49
215
63
79
25


St dev max 
475
61
244
75
101
34









FIGS. 1 and 2

Results for FIG. 1:

The F2 was injected with the full Adjuvant A, a similar formulation without QS21, or without MPL. For the anti-ToxA response, both MPL and QS21 have an added value even if the QS21 seems the most effective.
For the anti-ToxB response, the effect of the MPL is less clear while the QS21 removal has a drastic effect on the IgG titers.
Results for FIG. 2:
Regarding the adjuvant comparison, the adjuvant A induced significant higher anti-ToxA and anti-ToxB antibodies compared to Alum, Adjuvant B or non adjuvanted formulations. Adjuvant B induced higher anti-ToxA and anti-ToxB antibodies compared to the non adjuvanted formulations.
Example 3
Pims 20110544—Immunisation of Male Hamsters with a Dose-Range of F2 Fusion Protein Formulated in Adjuvant B, AS04D, Adjuvant A or Adjuvant C.
Groups of 10 male hamsters were immunized IM at days 0, 14 and 28 with 0.3 μg, 1 μg or 3 μg of the F2 fusion protein adjuvanted with 50 μl doses of Adjuvant B, AS04D, Adjuvant A or Adjuvant C.
Anti-ToxA and anti-ToxB ELISA titers were measured from individual sera collected at day 13 (post I), day 27 (Post II), day 42 (Post III 14) and day 84 (Post III 56)
Pims 20110545—Immunisation of Female Hamsters with a Dose-Range of F2 Fusion Protein Formulated in Adjuvant B, AS04D, Adjuvant A or Adjuvant C.
Groups of 10 female hamsters were immunized IM at days 0, 14 and 28 with 0.3 μg, 1 μg or 3 μg of the F2 fusion protein adjuvanted with Adjuvant B, AS04D, Adjuvant A or Adjuvant C.
Anti-ToxA and anti-ToxB ELISA titers were measured from individual sera collected at day 13 (post I), day 27 (Post II), day 42 (Post III 14) and day 84 (Post III 56)
FIGS. 3 to 7
Pims 20110544 and 20110545







TABLE 5







Post I Anti-ToxA ELISA titers (Geometric mean titre or GMT)



















Group
1
2
3
4
5
6
7
8
9
10
11
12





Ag/animal
3 μg 
1 μg 
0.3 μg
3 μg
1 μg
0.3 μg 
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg


Adj. 
C
C
C
A
A
A
B
B
B
AS04D
AS04D
AS04D


544
28
17
 6
 84
 17
 6
44
27
 3
 68
 74
33


545
50
36
12
109 
111
26
85
20
16
194 
107
36
















TABLE 6







Post I Anti-ToxB ELISA titers (GMT)



















Group
1
2
3
4
5
6
7
8
9
10
11
12





Ag/animal
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg


Adj
C
C
C
A
A
A
B
B
B
AS04D
AS04D
AS04D


544
115
82
52
412
121
79
189
119
34
7
5
5


545 
178
257
150
391
383
224
196
164
69
27
10
3


(female)




























TABLE 7







Post II Anti-ToxA ELISA titers (GMT)



















Groups
1
2
3
4
5
6
7
8
9
10
11
12





Ag/animal
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg


Adjuvant
C
C
C
A
A
A
B
B
B
AS04D
AS04D
AS04D


544
475
416
191
974
530
277
312
332
138
487
597
401


545
762
834
428
1466
1424
688
818
423
650
803
844
849
















TABLE 8







Post II Anti-ToxB ELISA titers (GMT)



















Groups
1
2
3
4
5
6
7
8
9
10
11
12





Ag/animal
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg


Adjuvant
C
C
C
A
A
A
B
B
B
AS04D
AS04D
AS04D


544 
319
686
488
1181
1120
1049
435
401
340
64
56
28


(male)














545 
974
1555
1419
1552
1871
2386
585
649
1486
215
109
91


(female)
















TABLE 9







Post III day 42 Anti-ToxA ELISA titers (GMT)



















Groups
1
2
3
4
5
6
7
8
9
10
11
12





Ag/animal
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg


Adjuvant
C
C
C
A
A
A
B
B
B
AS04D
AS04D
AS04D


544
449
674
471
1462
720
688
247
486
266
662
599
566


545
642
814 
667
1654
1020
1500
800
539
388
923
1005
794
















TABLE 10







Post III day 42 Anti-ToxB ELISA titers (GMT)



















Groups
1
2
3
4
5
6
7
8
9
10
11
12





Ag/animal
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg


Adjuvant
C
C
C
A
A
A
B
B
B
AS04D
AS04D
AS04D


544
630
612
594
1259
1116
1172
335
426
512
67
86
51


545
568
831
550
1213
828
1273
556
695
944
182
145
134
















TABLE 11







Post III day 84 Anti-ToxA ELISA titers (GMT)



















Groups
1
2
3
4
5
6
7
8
9
10
11
12





Ag/animal
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg


Adjuvant
C
C
C
A
A
A
B
B
B
AS04D
AS04D
AS04D


544
213
239
169
357
356
307
106
70
154
182
257
266


545
311
309
401
595 
578 
717
311
251 
417
353
414
337
















TABLE 12







Post III day 84 Anti-ToxB ELISA titers (GMT)



















Groups
1
2
3
4
5
6
7
8
9
10
11 
12





Ag/animal
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg
3 μg
1 μg
0.3 μg


Adjuvant
C
C
C
A
A
A
B
B
B
AS04D
AS04D
AS04D


544
291
195
145
299
360
440
78
70
315
24
25
15


(male)














545 
250
299
328
318
374
435
134
211
392
60
41
41


(female)





















Example 4
Pims 20120011 and 20120008—Immunisation of Hamsters with F2 Fusion Protein or a Mixture of Toxin A and Toxin B Fragments, Formulated in Alum, Adjuvant A or Non Adjuvanted.
Groups of 8 female and 8 male hamsters were immunized IM at days 0, 14 and 28 with 1 μg of the F2 fusion protein, an F2 fusion with a poly His tag (F2-His tagged), or a mix of C-terToxA (2387-2706) (0.6 μg)+C-terToxB (1750-2360) (0.4 μg) formulated in Alum, Adjuvant A or non adjuvanted.
Anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 13, day 27 and day 42 (Post III). FIG. 8







TABLE 13







20120008 and 20120011: Female and Male hamster immunization.


Comparison of the immunogenicity of a Mix (ToxA + ToxB) with the F2 fusion protein formulated in


Alum or Adjuvant A 


ELISA anti-ToxA: concentrations (μg/ml) on individual Post III sera









Groups














1
2
3
4
5
6



Mix ToxA 
F2
Mix ToxA 
F2
F2-His
F2 (1 μg)



(0.6 μg) + ToxB(0.4 μg)
(1 μg)
(0.6 μg) + ToxB(0.4 μg)
(1 μg)
tagged (1 μg)
non-



Al(OH)3
Al(OH)3
Adj. A
Adj. A
Adj. A
adjuvanted
















1
62
1156
41
3075
4006
195


2
77
871
105
1385
2463
127


3
104
833
239
3119
1202
31


4
66
506
143
3806
3903
574


5
179
736
340
3180
6852
116


6
641
1239
48
Dead
5571
32


7
128
328
345
5327
3174
10


8
48
3998
63
3542
4990
47


9
59
681
296
888
6769
13


10
352
491
164
978
1192
2


11
Dead
604
363
2913
1791
3


12
47
1524
87
1231
4362
68


13
88
486
77
3473
1647
123


14
23
1185
62
1341
4929
35


15
254
602
644
2794
3085
136


16
206
1094
24
2975
3354
90


Geomean
101
823
144
2310
3234
42


CI−
65
612
78
1727
2398
20


CI+
172
1148
213
3196
4381
99
















TABLE 14







20120008 and 20120011: Female and Male hamster immunization.


Comparison of the immunogenicity of a Mix (ToxA + ToxB) with the F2 fusion protein formulated in


Alum or Adjuvant A 


ELISA anti-ToxB: concentrations (μg/ml) on individual Post III sera









Groups














1
2
3
4
5
6



Mix ToxA 
F2
Mix ToxA 
F2
F2-His
F2 (1 μg)



(0.6 μg) + ToxB(0.4 μg)
(1 μg)
(0.6 μg) + ToxB(0.4 μg)
(1 μg)
tagged (1 μg)
non-



Al(OH)3
Al(OH)3
ADJUVANT A
ADJUVANT A
ADJUVANT A
adjuvanted
















1
58
591
2510
2364
2647
304


2
172
302
4981
952
1021
302


3
14
167
1409
2630
804
125


4
53
186
4158
2261
1090
299


5
107
408
3046
2553
2682
296


6
925
373
1190
Dead
3808
193


7
40
65
1673
2938
2820
170


8
112
1112
1809
1404
3186
51


9
160
652
2701
1385
2837
128


10
24
75
1210
2252
1676
38


11
Dead
242
546
1749
2174
12


12
61
1081
5746
1623
2557
74


13
15
193
1975
1507
2020
130


14
56
344
3499
1018
4735
14


15
15
237
2897
2554
2673
80



332
309
2489
2894
2148
127


Geomean
61
297
2228
1834
2210
100


CI−
35
194
1626
1542
1708
59


CI+
132
457
3094
2319
2850
176





Geomean = Geometric Mean Titre






Results:

Anti-ToxA and anti-ToxB antibodies were induced after immunization with the F2 fusion protein but also with the mix of ToxA and ToxB. Adjuvant A induces significantly higher ELISA titers compared to other formulations.
Example 5
Immunisation of Mice with Tox A or Tox B Fragments and ToxA-ToxB Fusions
FIGS. 9 to 12
Balb/C mice were immunized with the constructs described in example 1.
Groups of 15 female Balb/c mice were immunized IM at days 0, 14 and 28 with 3 μg or 10 μg of the separate fragments of ToxA and ToxB (ToxA C-term (2387-2706) (0.6 μg)+C-terToxB (1750-2360) as well as with ToxA-ToxB fusions proteins adjuvanted with Adjuvant B. A control group of 10 mice was vaccinated with Adjuvant B alone.
Anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 42 (post III).
Hemagglutination inhibition titers were determined in pooled Post III sera.
Anti-ToxA and Anti-ToxB ELISA Response: Protocol
Samples of the ToxA or ToxB fragments were coated at 1 μg/ml for ToxB fragments or 2 μg/ml for ToxA fragments in phosphate buffered saline (PBS) on high-binding microtitre plates (Nunc MAXISORPTM), overnight at 4° C. The plates were blocked with PBS-BSA 1% for 30 min at RT with agitation. The mice anti-sera are prediluted 1/1000 in PBS-BSA 0.2%-TWEENTM 0.05%. and then, further twofold dilutions were made in microplates and incubated at RT for 30 min with agitation. After washing, bound murine antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated affiniPure Goat Anti-Mouse IgG (H+L) (ref: 115-035-003) diluted 1:5000 in PBS-BSA 0.2%-tween 0.05%. The detection antibodies were incubated for 30 min. at 20° C. with agitation. The color was developed using 4 mg 0-phenylenediamine (OPD)+5 μl H2O2 per 10 ml pH 4.5 0.1M citrate buffer for 15 minutes in the dark at room temperature. The reaction was stopped with 50 μl HCl, and the optical density (OD) was read at 490 nm relative to 620 nm.
The level of anti-ToxA or anti-ToxB antibodies present in the sera was expressed in mid-point titers by comparison with a reference serum. A GMT was calculated for the 15 samples in each treatment group (10 for the control group).
Hemagglutination Inhibition Assay: Protocol
8 serial twofold dilutions of mice pooled antisera (25 μl) were performed in phosphate buffered saline (PBS) in 96-well U-bottom microplates.
25 μl of native Toxin A (0.2 μg/well) were then added and the plates were incubated at room temperature for 30 minutes.
After incubation, 50 μl of purified rabbit erythrocytes diluted at 2% were added to each well. The plates were incubated at 37° C. for 2 hours.
Plates were analysed visually, with hemagglutination presenting as diffuse red cells in the well and the inhibition of hemagglutination observed as a red point settled in the well.
The inhibition titers were defined as the reciprocal of the highest dilution of the serum inhibiting hemagglutination.
Cytotoxicity Assay
IMR90 fibroblast cells were cultured at 37° C. with 5% CO2, in Eagle's Minimal Essential Medium EMEM+10% fetal calf serum+1% glutamine+1% antibiotics (penicillin-streptomycin-amphotericin) and were seeded in 96-well tissue culture plates at a density of 5.10 cells/well.
After 24 h, 100 μl of the cell media was removed from the wells.
8 (for the experiment in example 5) or 12 (for the experiment in example 6) serial twofold dilutions of mice pooled antisera (50 μl) were performed in cell media.
50 μl of native Toxin B (0.5 ng/ml) is then added and the plates incubated at 37° C. with 5% CO2 for 24 hours.
Cells were observed after 24 hours, and the proportion of rounded cells was determined.
The inhibition titers were defined as the reciprocal of the highest dilution of the serum inhibiting 50% cell rounding.
Results:
Anti-Tox A antibodies were induced after immunization with the ToxA fragment alone but also with each of the 5 fusions.
The functional properties of these antibodies were tested in the hemagglutination assay. This assay is only adapted for Tox A evaluation as no hemagglutination is observed with ToxB.
Haemaglutination inhibition was observed with the anti-Tox A fragment sera or sera directed against each of the ToxA-ToxB fusions.
An ELISA using ToxB antibodies was also performed. Anti-Tox B antibodies were induced after immunization with the ToxB fragment alone but also with the F2, F3 and F4 fusions.
Inhibition titers obtained using sera from mice immunised with the ToxB fragment or the ToxA-ToxB fusions were greater than that obtained using control sera.
Example 6
Immunisation of Mice with Tox A-Tox B Fusions
Groups of 25 female mice were immunized IM at days 0, 14 and 28 with 3 μg of the ToxA-ToxB fusion proteins formulated in Adjuvant B or 10 μg of a non-adjuvanted mix of ToxA (6 μg)+ToxB (4 μg). In addition to F2, four other fusion protein constructs were used, there were F54 Gly (SEQ ID NO:21), F54 New (SEQ ID NO:23), F5 ToxB (SEQ ID NO:25) and F52 New (SEQ ID NO:27).
An ELISA was carried out using the anti-ToxA and anti-ToxB ELISA response: protocol described in example 5 except here the samples of the toxA or toxB fragments were coated at 1 μg/ml in phosphate buffered saline on high-binding microtitre plates. A hemagglutination inhibition assay was performed as described in example 5 except that the rabbit erythrocytes were diluted at 2.5%. A toxB cytotoxicity assay was performed as described in example 5 except that 50 μl of Toxin B at a concentration of 0.015 μg/ml was used. A further toxA cytotoxicity assay was performed as described below.
Anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 42 (Post III 14).
FIGS. 13 and 14







TABLE 15







20110350: Mice immunization with C. difficile fusion proteins formulatd in Adjuvant B


ELISA a-ToxA: concentrations (μg/ml) on Post III sera









Groups















2
3
4
5
6



1
Fusion A
Fusion B
Fusion C
Fusion D
Mix ToxA +



F2
(F52new)
(F54Gly)
(F54new)
(F5 ToxB)
ToxB



3 μg
3 μg
3 μg
3 μg
3 μg
10 μg 



Ag/animal
Ag/animal
Ag/animal
Ag/animal
Ag/animal
(6 μg A + 4 μg B)



ADJUVANT 
ADJUVANT 
ADJUVANT 
ADJUVANT 
ADJUVANT 
Ag/animal



B
B
B
B
B
non-adjuvanted



IM
IM
IM
IM
IM
IM
















1
518
681
735
436
586
328


2
761
1058
975
455
1252
22


3
557
718
977
684
1108
327


4
1415
992
862
577
555
152


5
782
457
1202
861
622
135


6
563
724
611
575
569
54


7
615
585
1330
824
807
147


8
994
878
611
576
1108
169


9
412
623
Dead
389
887
124


10
495
993
Dead
809
1146
219


11
366
1039
923
872
965
12


12
1075
1089
1454
748
950
122


13
882
754
1176
809
725
196


14
1028
1550
1164
1578
696
312


15
738
1268
1170
1352
678
188


16
519
721
1028
781
930
224


17
927
890
805
346
791
209


18
946
992
647
1255
410
32


19
715
1346
1142
670
634
92


20
546
Dead
1061
Dead
Dead
166


21
1107
1604
826
1038
791
112


22
1351
1289
1223
708
778
66


23
889
1319
61
567
759
102


24
976
824
597
414
456
281


25
1153
1212
995
868
758
242


Geomean
764
937
841
704
761
126


CI−
656
818
641
597
674
89


CI+
889
1074
1103
830
858
179
















TABLE 16







20110350: Mice immunization with C. difficile fusion proteins formulatd in Adjuvant B


ELISA a-ToxB: concentrations (μg/ml) on Post III sera









Groups















2
3
4
5
6



1
Fusion A
Fusion B
Fusion C
Fusion D
Mix ToxA +



F2
(F52new)
(F54Gly)
(F54new)
(F5 ToxB)
ToxB



3 μg
3 μg
3 μg
3 μg
3 μg
10 μg 



Ag/animal
Ag/animal
Ag/animal
Ag/animal
Ag/animal
(6 μg A + 4 μg B)



ADJUVANT 
ADJUVANT 
ADJUVANT 
ADJUVANT 
ADJUVANT 
Ag/animal



B
B
B
B
B
ADJUVANT B
















1
916
749
736
454
581
1823


2
1090
551
491
517
872
575


3
687
1178
574
549
529
1386


4
940
868
501
536
828
854


5
837
522
653
420
866
1170


6
836
609
397
757
690
2132


7
309
702
405
792
732
1466


8
569
381
598
737
856
1539


9
382
450
Dead
914
632
1461


10
552
693
Dead
465
639
2267


11
679
832
1273
520
505
1037


12
1123
575
809
551
431
2273


13
764
659
532
1077
930
1405


14
944
970
909
1065
870
1978


15
812
628
222
727
1052
1414


16
502
552
736
560
1001
1269


17
983
639
551
431
728
1732


18
1133
628
604
561
1085
1211


19
938
1058
805
470
435
2017


20
519
Dead
857
Dead
Dead
2140


21
887
1025
319
670
310
2342


22
861
769
596
555
1025
2897


23
903
1161
552
694
260
2343


24
1327
536
431
737
313
3074


25
674
518
680
472
663
2697


Geomean
765
688
581
611
653
1661


CI−
663
607
495
545
550
1411


CI+
884
781
682
686
776
1957









FIGS. 15 to 17

A hemagglutination inhibition assay was performed as described in example 5.
A toxB cytotoxicity assay was performed as described in example 5 except that 12 serial twofold dilutions of mice pooled antisera were performed rather than 8, and the plates were incubated overnight rather than for 24 hours before cells were observed. A further toxA cytotoxicity assay was performed as described below.
ToxA Cytotoxicity Assay
HT29 cells were cultured at 37° C. with 5% CO2 in DMEM+10% fetal bovine serum+1% glutamine+1% antibiotics (penicillin-streptomycin-amphotericin) and were seeded in 96-well tissue culture plates at a density of 5·104 cells/well. After 24 h, the cell media was removed from the wells. Serial twofold dilutions of mice pooled antisera (50 μl) were performed in cell media.
50 μl of native Toxin A (0.15 ng/ml) is then added and the plates incubated at 37° C. with 5% CO2 for 48 hours. Cells were observed after 48 hours and the proportion of rounded cells were determined. The results of the anti-ToxA ELISA, anti-ToxB Elisa, Hemagglutination inhibition and cytotoxicity assays are described in the aforementioned FIGS. 13 to 17.
Example 7
Immunisation of Mice with C. difficile ToxA-Cter, ToxB-Cter or Fusion Proteins in a Non-Adjuvanted Formulation
Groups of 15 female mice were immunized IM at days 0, 14 28 and 120 with 1 or 3 μg of ToxA C-ter, ToxB C-ter or ToxA-ToxB fusion proteins. All these antigens were injected in a non-adjuvanted formulation. A control group (10 mice) was injected with the NaCl alone.
Anti-ToxA and anti-ToxB ELISA titers were measured from individual sera collected at day 28 (Post II), day 42 (Post III 14), day 120 (Pre-Boost 87) and day 134 (Post IV 14). FIGS. 18 and 19.







TABLE 17







20100723: Mice immunization with C. difficile ToxA-Cter, ToxB-Cter and fusion proteins non-adjuvanted


ELISA a-ToxA: GMT concentrations ( μg/ml)









Bleeding























1
2





8
9









ToxA
ToxB





ToxA
ToxB









(aa
(aa
3
4
5
6
7
(aa
(aa
10
11
12
13
14




2387-
1750-
Fusion
Fusion
Fusion
Fusion
Fusion
2387-
1750-
Fusion
Fusion
Fusion
Fusion
Fusion




2706)
2360)
F1
F2
F3
F4
F5
2706)
2360)
F1
F2
F3
F4
F5
15









Ag/ml




































3 μg 




10 μg
10 μg
10 μg
10 μg
10 μg
10 μg
10 μg
3 μg
3 μg
3 μg
3 μg
3 μg
3 μg
Ag/ml










Adjuvant























Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 




adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
NaCl



IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM

























Post II
178
3
16
60
74
44
269
84
3
10
49
26
25
60
3


Post III
222
2
40
94
192
109
366
137
1.25
51
110
88
67
137
1.25


Pre-
128
4
28
70
120
58
172
165
4
48
62
63
44
107
3


Boost

















Post IV
539
5
90
174
249
177
353
466
3
168
147
163
139
188
3
















TABLE 18







20100723: Mice immunization with C. difficile ToxA-Cter, ToxB-Cter and fusion proteins non-adjuvanted


ELISA a-ToxB: GMT concentrations ( μg/ml)









Bleeding























1
2





8
9









ToxA
ToxB





ToxA
ToxB









(aa
(aa
3
4
5
6
7
(aa
(aa
10
11
12
13
14




2387-
1750-
Fusion
Fusion
Fusion
Fusion
Fusion
2387-
1750-
Fusion
Fusion
Fusion
Fusion
Fusion




2706)
2360)
F1
F2
F3
F4
F5
2706)
2360)
F1
F2
F3
F4
F5
15









Ag/ml




































3 μg 




10 μg
10 μg
10 μg
10 μg
10 μg
10 μg
10 μg
3 μg
3 μg
3 μg
3 μg
3 μg
3 μg
Ag/ml










Adjuvant























Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 
Non 




adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
adj.
NaCl



IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM

























Post II
2
62
2
110
58
283
18
2
7
2
101
29
125
8
2


Post III
3
316
7
200
199
455
65
4
105
13
242
126
257
44
2


Pre-
3
125
5
81
58
157
18
4
38
8
68
42
101
14
3


Boost

















Post IV
5
856
27
258
227
380
125
6
632
79
232
175
293
126
4









Example 8
Immunisation of Mice with C. difficile ToxA-Cter, ToxB-Cter or Fusion Proteins Formulated in Adjuvant B.
FIGS. 20 and 21
Groups of 15 female mice were immunized IM at days 0, 14 and 28 and 120 with 1 or 3 μg of ToxA C-term (amino acids 2387-2706) and ToxB C-term (amino acids 1750-2360) or ToxA-ToxB fusion proteins. These proteins were injected in an Adjuvant B formulation.
A control group (10 mice) was injected with the Adjuvant B alone.
Anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 28 (Post II), day 42 (Post III 14), day 120 (Pre Boost III 87 or pre boost) and day 134 (Post IV 14).







TABLE 19







Mice immunization with C. difficile ToxA-Cter, ToxB-Cter and fusion proteins 


formulated in Adjuvant B 


ELISA a-ToxA: concentrations (μg/ml) on Post II, III, Pre-Boost, Post IV sera









Bleeding























1
2





8
9









ToxA 
ToxB 





ToxA 
ToxB 









(aa 
(aa 





(aa 
(aa









2387-
1750-
3
4
5
6
7
2387-
1750-
10
11
12
13
14
15



2706)
2360)
F1
F2
F3
F4
F5
2706)
2360)
F1
F2
F3
F4
F5 n
/









Ag/ml























10 μg
10 μg
10 μg
10 μg
10 μg
10 μg
10 μg
3 μg
3 μg
3 μg
3 μg
3 μg
3 μg
3 μg
/









ADJUVANT























B
B
B
B
B
B
B
B
B
B
B
B
B
B
B



IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM

























Post II
213
2.3
465
332
312
158
672
534
1.7
620
498
578
248
925
2.1


Post III
852
3
1144
859
695
644
1137
1376
4
1356
768
950
470
1537
2


Pre-Boost
738
4
762
484
417
394
674
1091
3
725
513
598
324
1197
3


Post IV
2764
9
1450
1423
1036
986
1626
301
26
990
1000
1099
732
1465
1
















TABLE 20







20100798: Mice immunization with C. difficile ToxA-Cter, ToxB-Cter and fusion proteins 


formulated in Adjuvant B 


ELISA a-ToxA: concentrations (μg/ml) on Post II, III, Pre-Boost, Post IV sera









Bleeding























1
2





8
9









ToxA 
ToxB 





ToxA 
ToxB 









(aa 
(aa 





(aa 
(aa









2387-
1750-
3
4
5
6
7
2387-
1750-
10
11
12
13
14
15



2706)
2360)
F1
F2
F3
F4
F5
2706)
2360)
F1
F2
F3
F4
F5
/









Ag/ml























10 μg
10 μg
10 μg
10 μg
10 μg
10 μg
10 μg
3 μg
3 μg
3 μg
3 μg
3 μg
3 μg
3 μg
/









ADJUVANT























B
B
B
B
B
B
B
B
B
B
B
B
B
B
B



IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM
IM

























Post II
2
246
10
424
284
794
25
3
338
17
619
471
753
93
2


Post III
2
1762
285
793
671
1204
253
4
1888
232
951
834
1026
365
2


Pre-Boost
3
989
180
512
396
761
129
4
984
108
479
441
581
205
3


Post IV
3
4212
911
1422
1353
1799
1046
78
657
738
1272
747
1572
812
2









Example 9
Cloning Expression and Purification of the Fusion Proteins
Expression Plasmid and Recombinant Strain
Genes encoding the fusion proteins of partial C-terminal domains of ToxA and ToxB (SEQ ID NO:3, 4, 5, 6 and 7) and a His tag were cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures. The final construct was generated by the transformation of E. coli strain BLR (DE3) with the recombinant expression vector according to standard method with CaCl2-treated cells (Hanahan D. <<Plasmid transformation by Simanis.>> In Glover, D. M. (Ed), DNA cloning. IRL Press London. (1985): p. 109-135.).
Host Strain:
BLR(DE3). BLR is a recA derivative of BL21. Strains having the designation (DE3) are lysogenic for a λ prophage that contains an IPTG inducible T7 RNA polymerase. λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.
Genotype: E. coli BLR::DE3 strain, F− ompT hadSB(rB−mB−) gal dcm (DE3) Δ(srl-recA)306::Tn10 (TetR)
Expression of the Recombinant Proteins:
An E. coli transformant was stripped from agar plate and used to inoculate 200 ml of LBT broth ±1% (w/v) glucose+kanamycin (50 μg/ml) to obtain O.D.600 nm between 0.1-0.2. Cultures were incubated overnight at 37° C., 250 RPM.
This overnight culture was diluted to 1:20 in 500 ml of LBT medium containing kanamycin (50 μg/ml) and grown at 37° C. at a stirring speed of 250 rpm until O.D.620 reached 0.5/0.6.
At O.D.600 nm around 0.6, the culture was cooled down before inducing the expression of the recombinant protein by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; EMD Chemicals Inc., catalogue number: 5815) and incubated overnight at 23° C., 250 RPM.
After overnight induction (around 16 hours), O.D.600nm was evaluated after induction and culture was centrifuged at 14 000 RPM for 15 minutes and pellets were frozen at −20° C. separately.
Purification:
The bacterial pellet was resuspended in 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl and a mixture of protease inhibitor (Complete, Roche). Bacteria were lysed using a French Press system 20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation for example at 20 000 g for 30 min at 4° C.
The 6-His tagged-protein was purified under native conditions on IMAC. The soluble components were loaded on a GE column (15 ml for example) (Ni loaded) preequilibrated with the same buffer used to bacterial resuspension. After loading on the column, the column was washed with the same buffer. Elution was performed using a 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl and different concentrations of imidazole (5-600 mM). After gel analysis, more pure fractions were selected, concentrated and loaded on SEC chromatography for further purification step.
Fractions containing the fusion proteins were selected on the basis of purity by SDS-PAGE and dialyzed against bicine buffer (20 mM Bicine, 150 mM NaCl, with or without 5 mM EDTA pH8.0), Protein concentration was determined using DC Protein Assay of BioRad. Proteins were thus pooled, sterile-filtered on 0.22 μm, stored at −80° C.
Alternatively, IMAC purification was preceded by a DEAE purification step using 2 mM bicine buffer (pH 8.0) for loading and washing, and eluted using a gradient with the same buffer but with 1M NaCl added.
Example 10
Cloning Expression and Purification of the Separate C. difficile Tox A and Tox B Fragments
Expression Plasmid and Recombinant Strain.
Genes encoding the protein fragments of ToxA and ToxB (SEQ ID NO:8 and SEQ ID NO:9) and a His tag were cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures. The final construct was generated by the transformation of E. coli strain BLR (DE3) with the recombinant expression vector according to standard method with CaCl2-treated cells (Hanahan D. <<Plasmid transformation by Simanis.>> In Glover, D. M. (Ed), DNA cloning. IRL Press London. (1985): p. 109-135.).
Host Strain:
BLR(DE3). BLR is a recA derivative of BL21. Strains having the designation (DE3) are lysogenic for a λ prophage that contains an IPTG inducible T7 RNA polymerase. λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.
Genotype: E. coli BLR::DE3 strain, F− ompT hsdSB(rB−mB−) gal dcm (DE3) Δ(srl-recA)306::Tn10 (TetR)
Expression of the Recombinant Proteins:
A E. coli transformant was stripped from agar plate and used to inoculate 200 ml of LBT broth ±1% (w/v) glucose+kanamycin (50 μg/ml) to obtain O.D.600nm between 0.1-0.2. Cultures were incubated overnight at 37° C., 250 RPM.
This overnight culture was diluted to 1:20 in 500 ml of LBT medium containing kanamycin (50 μg/ml) and grown at 37° C. at a stirring speed of 250 rpm until O.D.620 reached 0.5/0.6.
At an O.D. at 600 nm of around 0.6, the culture was cooled down before inducing the expression of the recombinant protein by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; EMD Chemicals Inc., catalogue number: 5815) and incubated overnight at 23° C., 250 RPM.
After the overnight induction (around 16 hours), O.D. at 600 nm was evaluated after induction and culture was centrifuged at 14 000 RPM for 15 minutes and pellets were frozen at −20° C. separately.
Purification:
The bacterial pellet was resuspended in 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl supplemented by a mixture of protease inhibitor (Complete without EDTA, Roche cat 11873580001) and benzonase. (Roche cat 1.01695.0001). Bacteria were lysed using a French Press system 2×20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation at 34 000 g or 48 000 g for 25-30 min at 4° C. Supernatant was harvested and filtrated on 0.22 μm filter.
The 6-His tagged-protein was purified under native conditions on IMAC. The soluble components were loaded on a GE column (for example 15 ml) (Ni loaded) pre-equilibrated with the same buffer used to bacterial resuspension. After loading, the column was washed with the same buffer.
For ToxA:
Elution was performed using a 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl and different concentrations of imidazole (5-100 mM). After gel analysis, more pure fractions were selected, concentrated and loaded on SEC chromatography (SUPERDEXTM 75) for further purification step in the same buffer without imidazole.
For ToxB:
A second wash was performed with 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl and 0.5% deoxycholate or same buffer with 150 mM NaCl. Elution was performed using a 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl and different concentrations of imidazole (10-500 mM). After gel analysis, more pure fractions were selected, supplemented with 5 mM EDTA and loaded on SEC chromatography (SUPERDEXTM 200) for further purification step in same buffer with 5 mM EDTA.
Fractions containing ToxA or ToxB fragments were selected on the basis of purity by SDS-PAGE and dialyzed against bicine buffer (20 mM Bicine, 150 mM NaCl, pH8.0), protein concentration was determined using RCDC Protein Assay of BioRad. Proteins were thus pooled, sterile-filtered on 0.22 μm, stored at −80° C.
Example 11
Molecular Weight Evaluation of the Five C. difficile ToxA-ToxB Fusions
Analytical ultracentrifugation is used to determine the homogeneity and size distribution in solution of the different species within a protein sample by measuring the rate at which molecules move in response to a centrifugal force. This is based on the calculation of the coefficients of sedimentation of the different species that are obtained by sedimentation velocity experiment, which depend on their molecular shape and mass.
1. Protein samples are spun in a Beckman-Coulter PROTEOMELABTM XL-1 analytical ultracentrifuge at 42 000 RPM after the AN-60Ti rotor had been equilibrated to 15° C.

    
    
        a. F1 fusion protein, 500 μg/ml, 20 mM Bicine, 150 mM NaCl, pH8.0
        b. F2 fusion protein, 500 μg/ml, 20 mM Bicine, 150 mM NaCl, pH8.0
        c. F3 fusion protein, 500 μg/ml, 20 mM Bicine, 150 mM NaCl, pH8.0
        d. F4 fusion protein, 500 μg/ml, 20 mM Bicine, 150 mM NaCl, pH8.0
        e. F5 fusion protein, 500 μg/ml, 20 mM Bicine, 150 mM NaCl, pH8.0
    
    


2. For data collection, 160 scans were recorded at 280 nm every 5 minutes.
3. Data analysis was performed using the program SEDFIT for determination of the C(S) distribution. Determination of the partial specific volume of the proteins was performed with the SEDNTERP software from their amino acid sequence. SEDNTERP was also used to determine the viscosity and the density of the buffer.
4. The molecular weight of the different species was determined from the C(S) distribution plot (concentration vs sedimentation coefficient), considering that it's a better representation of the raw data than the C(M) distribution (concentration vs molecular weight) to characterize the size distribution of a mixture. The molecular weight of the major species detected from the C(S) distribution of all five ToxA-ToxB fusion proteins corresponds to their monomeric form. The best fit frictional ratios determined for the five fusions are all between 2 and 2.2. This may indicate that the proteins are present in solution as an elongated form, which would be consistent with the protein structure.
Example 12
Evaluation of Secondary and Tertiary Structures of C. difficile ToxA-ToxB Fusions by Circular Dichroism and Fluorescence Spectroscopy
Circular dichroism is used to determine the secondary structure composition of a protein by measuring the difference in the absorption of left-handed polarized light versus right-handed polarized light which is due to structural asymmetry. The shape and the magnitude of the CD spectra in the far-UV region (190-250 nm) are different whether a protein exhibits a beta-sheet, alpha-helix or random coil structure. The relative abundance of each secondary structure type in a given protein sample can be calculated by comparison to reference spectra.
The tertiary structure of a protein sample can be assessed by the evaluation of the immobilisation of the aromatic amino acids. The observation of a CD signal in the near-UV region (250-50 nm) may be attributable to the polarization of phenylalanine, tyrosine and tryptophane residues and is a good indication that the protein is folded into a well defined structure.
The following protocol was used:
1. Far UV spectra are measured using an optical path of 0.01 cm from 178 to 250 nm, with a 1 nm resolution and bandwidth on a Jasco J-720 spectropolarimeter. Temperature of the cell is maintained at 23° C. by a Peltier thermostated RTE-111 cell block. A nitrogen flow of 10 L/min is maintained during the measurements.
2. Near-UV spectra are measured using an optical path of 0.01 cm from 250 to 300 nm, with a 1 nm resolution and bandwidth on a Jasco J-720 spectropolarimeter. Temperature of the cell is maintained at 23° C. by a Peltier thermostated RTE-111 cell block. A nitrogen flow of 6 L/min is maintained during the measurements.
The observation of the far-UV spectra (FIG. 22) for all five ToxA-ToxB fusion proteins suggests a weak content of alpha helix structures and a high content of beta sheet structures. Also, all proteins exhibited a maximum at 230 nm, which is unusual for soluble globular proteins. This particularity has been well characterized in the literature and is associated with a small group of proteins known for their absence of alpha helix and their high content in beta sheet and aromatic amino acids (Zsila, Analytical Biochemistry, 391(2009) 154-156). Those particularities are coherent with the structure that is expected for the ToxA-ToxB fusion proteins. Crystal structures of 13 proteins exhibiting the characteristic CD spectra with a positive signal at 230 nm were compared (Protein Data Bank). The average secondary structure content of those proteins is 42% beta sheet ±9% and 7% alpha helix ±6%. This strongly indicates that the spectral signature of the ToxA-ToxB fusion proteins is diagnostic of a high beta sheet and low alpha helix containing protein.
The observation of the shape of the near-UV spectra (FIG. 23) for all five fusion proteins indicates that at least some of the aromatic amino acids are immobilised, which is a strong indication of a compact and specific tertiary structure. Moreover, the treatment of the protein with a denaturing concentration of urea caused the disappearance of the near-UV signal, which is an additional indication that this characteristic spectra was due to protein folding.
Example 13
Design, Cloning, Expression and Purification of 4 Further Fusion Proteins
Four further fusion proteins were designed using the design principles described in example 1, these were named F54 Gly (SEQ ID NO:21), F54 New (SEQ ID NO:23), F5 ToxB (SEQ ID NO:25) and F52 New (SEQ ID NO:27).
These fusion proteins were expressed according to the procedure described in example 9.
Example 14
Molecular Weight Evaluation of the C. difficile ToxA-ToxB Fusions Described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO: 27
The molecular weight of the fusions described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27 were determined as described in example 11.
The molecular weight of the main species determined from the C(S) distribution of all four protein fusions described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27 corresponds to their monomeric form and all proteins exhibit sedimentation properties similar to F1 to F5 fusions.
Example 15
Evaluation of Secondary and Tertiary Structures of C. difficile ToxA-ToxB Fusions Described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:1525 and SEQ ID NO:27
The secondary and tertiary structures of the fusions described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27 were assessed according to the method described in example 5. The far UV CD for these fusion proteins can be found in FIG. 24, and the near UV spectra for these fusions can be found in FIG. 25.
Analysis of the near and far UV CD spectra of the proteins described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27 shows that all four have the same high beta sheet structure than F1 to F5 fusions. In addition, observation of the near UV spectra show no significant difference in the position of the aromatic amino acids in the tertiary structure compared to F1 to F5 fusions.
SEQUENCES








SEQ ID NO: 1 - sequence of toxin A



MSLISKEELIKLAYSIRPRENEYKTILTNLDEYNKLTTNNNENKYLQLKKLNESIDVFMN


 


KYKTSSRNRALSNLKKDILKEVILIKNSNTSPVEKNLHFVWIGGEVSDIALEYIKQWADI


 


NAEYNIKLWYDSEAFLVNTLKKAIVESSTTEALQLLEEEIQNPQFDNMKFYKKRMEFIYD


 


RQKRFINYYKSQINKPTVPTIDDIIKSHLVSEYNRDETVLESYRTNSLRKINSNHGIDIR


 


ANSLFTEQELLNIYSQELLNRGNLAAASDIVRLLALKNFGGVYLDVDMLPGIHSDLFKTI


 


SRPSSIGLDRWEMIKLEAIMKYKKYINNYTSENFDKLDQQLKDNFKLIIESKSEKSEIFS


 


KLENLNVSDLEIKIAFALGSVINQALISKQGSYLTNLVIEQVKNRYQFLNQHLNPAIESD


 


NNFTDTTKIFHDSLFNSATAENSMFLTKIAPYLQVGFMPEARSTISLSGPGAYASAYYDF


 


INLQENTIEKTLKASDLIEFKFPENNLSQLTEQEINSLWSFDQASAKYQFEKYVRDYTGG


 


SLSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDISYEATCNLF


 


SKNPKNSIIIQRNMNESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEF


 


NTSEFARLSVDSLSNEISSFLDTIKLDISPKNVEVNLLGCNMFSYDFNVEETYPGKLLLS


 


IMDKITSTLPDVNKNSITIGANQYEVRINSEGRKELLAHSGKWINKEEAIMSDLSSKEYI


 


FFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDYIYY


 


EKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSV


 


RFINKSNGESVYVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNT


 


LNAAFFIQSLIDYSSNKDVLNDLSTSVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDT


 


INVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLKKELEAKVGVLAINMSLSIAAT


 


VASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESKKYGPLKT


 


EDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIP


 


SLSIYSAIGIETENLDFSKKIMMLPNAPSRVFWWETGAVPGLRSLENDGTRLLDSIRDLY


 


PGKFYWRFYAFFDYAITTLKPVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGA


 


GGTYSLLLSSYPISTNINLSKDDLWIFNIDNEVREISIENGTIKKGKLIKDVLSKIDINK


 


NKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSLLLSGDKNYLISN


 


LSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNDST


 


LEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNES


 


VYSSYLDFVKNSDGHHNTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVE


 


FICDNNKNIDIYFGEWKTSSSKSTIFSGNGRNVVVEPIYNPDTGEDISTSLDFSYEPLYG


 


IDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHKKVNINLDSSSFEYKWS


 


TEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFKSF


 


NSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNLVTGWQTINGK


 


KYYFDINTGAALTSYKIINGKHFYFNNDGVMQLGVFKGPDGFEYFAPANTQNNNIEGQAI


 


VYQSKFLTLNGKKYYFDNNSKAVTGWRIINNEKYYFNPNNAIAAVGLQVIDNNKYYFNPD


 


TAIISKGWQTVNGSRYYFDTDTAIAFNGYKTIDGKHFYFDSDCVVKIGVFSTSNGFEYFA


 


PANTYNNNIEGQAIVYQSKFLTLNGKKYYFDNNSKAVTGLQTIDSKKYYFNTNTAEAATG


 


WQTIDGKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQ


 


IGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNK


 


KYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKG


 


PNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNL


 


NTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFY


 


FNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVT


 


GLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIM


 


QIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDG


 


NRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAI


 


RYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGV


 


DGVKAPGIYG


 


SEQ ID NO: 2 - sequence of toxin B


MSLVNRKQLEKMANVRFRTQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYI


 


DTYKKSGRNKALKKFKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKD


 


VNSDYNVNVFYDSNAFLINTLKKTVVESAINDTLESFRENLNDPRFDYNKFFRKRMEIIY


 


DKQKNFINYYKAQREENPELIIDDIVKTYLSNEYSKEIDELNTYIEESLNKITQNSGNDV


 


RNFEEFKNGESFNLYEQELVERWNLAAASDILRISALKEIGGMYLDVDMLPGIQPDLFES


 


IEKPSSVTVDFWEMTKLEAIMKYKEYIPEYTSEHFDMLDEEVQSSFESVLASKSDKSEIF


 


SSLGDMEASPLEVKIAFNSKGIINQGLISVKDSYCSNLIVKQIENRYKILNNSLNPAISE


 


DNDFNTTTNTFIDSIMAEANADNGRFMMELGKYLRVGFFPDVKTTINLSGPEAYAAAYQD


 


LLMFKEGSMNIHLIEADLRNFEISKTNISQSTEQEMASLWSFDDARAKAQFEEYKRNYFE


 


GSLGEDDNLDFSQNIVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYEAACNLFAK


 


TPYDSVLFQKNIEDSEIAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNT


 


DIFAGFDVDSLSTEIEAAIDLAKEDISPKSIEINLLGCNMFSYSINVEETYPGKLLLKVK


 


DKISELMPSISQDSIIVSANQYEVRINSEGRRELLDHSGEWINKEESIIKDISSKEYISF


 


NPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDTQIVEER


 


IEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRF


 


INKETGESIFVETEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLN


 


AAFFIQSLIEYNSSKESLSNLSVAMKVQVYAQLFSTGLNTITDAAKVVELVSTALDETID


 


LLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLRQEIEAKIGIMAVNLTTATTAIIT


 


SSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKHVSLVETEGVFTLLD


 


DKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHL


 


SIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEG


 


EFYWRYFAFIADALITTLKPRYEDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSG


 


GTYALSLSQYNMGINIELSESDVWIIDVDNVVRDVTIESDKIKKGDLIEGILSTLSIEEN


 


KIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLISGELKILMLNS


 


NHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMD


 


DSKPSFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDES


 


GVAEILKFMNRKGNTNTSDSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQ


 


FEFICDENDNIQPYFIKFNTLETNYTLYVGNRQNMIVEPNYDLDDSGDISSTVINFSQKY


 


LYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYINEKINVNINDLSIRY


 


VWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFT


 


PSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNLITGFVTVG


 


DDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEG


 


EAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFN


 


SDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFA


 


HHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIG


 


LSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIG


 


VFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESD


 


KYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIED


 


KMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYI


 


AATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 3 - sequence of Fusion 1


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVI


 


YFFGVDGVKAPGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGN


 


EEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVG


 


FVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLV


 


RVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYY


 


FNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFT


 


DEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 4 - sequence of Fusion 2


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLNQIGDYK


 


YYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGN


 


EEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVG


 


FVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLV


 


RVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYY


 


FNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFT


 


DEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 5 - sequence of Fusion 3


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQY


 


YFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNI


 


YGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRT


 


GLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGW


 


LDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 6 - sequence of Fusion 4


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGETIIDDKN


 


YYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHY


 


FSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGV


 


FNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGL


 


SLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAP


 


ANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYF


 


DEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEG


 


ESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 7 - sequence of Fusion 5


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVI


 


YFFGVDGVKAPGIYGGGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHN


 


EDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDG


 


IMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVE


 


YSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFE


 


NNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEK


 


RYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 8 sequence of individual toxin A fragment


MASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSK


 


AVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPD


 


GFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDN


 


KNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKV


 


YYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAP


 


SEQ ID NO: 9 - sequence of individual toxin B fragment


MILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFTPSYYEDGLIGYDLGLVSLYN


 


EKFYINNFGMMVSGLIYINDSLYYFKPPVNNLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSG


 


VLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGK


 


AFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGF


 


KYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQ


 


YYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDN


 


IYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMR


 


TGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTG


 


WLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTA


 


SEQ ID NO: 10 - sequence of toxin A fragment from fusion 1


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVI


 


YFFGVDGVKAP


 


SEQ ID NO: 11 - sequence of toxin A fragment from fusion 2


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAG


 


SEQ ID NO: 12 - sequence of toxin A fragment from fusion 3


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


Y


 


SEQ ID NO: 13 - sequence of toxin A fragment from fusion 4


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAG


 


SEQ ID NO: 14 - sequence of toxin A fragment from fusion 5


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQ


 


NEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNF


 


YFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKK


 


YYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIM


 


QIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVA


 


VTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRY


 


QNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFE


 


YFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVI


 


YFFGVDGVKAPGIYG


 


SEQ ID NO: 15 - sequence of toxin B fragment from fusion 1


GFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGI


 


LNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYF


 


SDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGE


 


TYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGY


 


INIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVI


 


IDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 16 - sequence of toxin B fragment from fusion 2


GLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYF


 


AHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYF


 


NDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYG


 


QAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGL


 


ISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLD


 


LDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 17 - sequence of toxin B fragment from fusion 3


FAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYY


 


FNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIY


 


GQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTG


 


LISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWL


 


DLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 18 - sequence of toxin B fragment from fusion 4


GETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEW


 


KELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFA


 


ENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDE


 


DTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDT


 


SDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGIN


 


LIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQ


 


NTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 19 - sequence of toxin B fragment from fusion 5


GFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGI


 


LNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYF


 


SDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGE


 


TYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGY


 


INIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVI


 


IDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 20 - nucleotide sequence of F54 Gly fusion protein


ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGCAATTGCAAGCACCGGCTATACCATTATCAAC


 


GGCAAACACTTTTATTTTAACACCGACGGCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCAAAT


 


ACCGATGCCAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGACCCTGAACGGCAAAAAATACTACTTTGGCAGCGAT


 


AGCAAAGCAGTTACCGGTTGGCGCATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTGTGCACC


 


ATTAACAACGACAAATATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTACATTACCATCGAACGCAACAACTTTTATTTCGATGCC


 


AACAACGAAAGCAAAATGGTGACCGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATAACAACATT


 


GAAGGTCAGGCGATCGTTTATCAGAACAAATTCCTGACGCTGAATGGTAAGAAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGC


 


TGGCAGACAATTGACGGGAAGAAATATTACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGACGGTAAAAAGTAC


 


TACTTCAACCTGAACACAGCCGAAGCAGCCACAGGATGGCAGACTATTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCA


 


TCTACCGGTTATACCAGCATTAACGGTAAACATTTCTACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCAAAGGTCCAAATGGT


 


TTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACATCGAGGGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGG


 


AAAAAATATTACTTTGGTAGCGATTCTAAAGCCGTTACCGGTCTGCGTACCATTGATGGTAAAAAATACTACTTTAATACGAATACAGCC


 


GTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACTATTTTAACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATT


 


TCGGGTAAACACTTCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCTTTAAAGGACCTGATGGGTTCGAATATTTTGCGCCTGCG


 


AACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTATCAGAATCGCTTTCTGTATCTGCACGACAACATCTATTATTTTGGCAAC


 


AATTCAAAAGCAGCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCGAATACCGCAATGGGTGCAAATGGCTACAAA


 


ACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGCCGCAGATCGGGGTATTTAAAGGTAGCAACGGCTTCGAATACTTCGCTCCA


 


GCGAATACGGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGCTGGGCAAAATCTACTACTTTGGC


 


AATAACAGTAAAGCAGTTACTGGATGGCAGACAATCAATGGTAAAGTGTACTATTTTATGCCGGATACCGCCATGGCAGCAGCCGGTGGT


 


CTGTTTGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCACCGGGAATATACGGTGGTACCGGCTTTGTGACCGTG


 


GGTGATGATAAATACTATTTCAATCCGATTAACGGTGGTGCAGCGAGCATTGGCGAAACCATCATCGATGACAAAAACTATTATTTCAAC


 


CAGAGCGGTGTGCTGCAGACCGGTGTGTTTAGCACCGAAGATGGCTTTAAATATTTTGCGCCAGCGAACACCCTGGATGAAAACCTGGAA


 


GGCGAAGCGATTGATTTTACCGGCAAACTGATCATCGATGAAAACATCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGGAAA


 


GAACTGGATGGCGAAATGCATTATTTTTCTCCGGAAACCGGTAAAGCGTTTAAAGGCCTGAACCAGATCGGCGATTACAAATACTACTTC


 


AACAGCGATGGCGTGATGCAGAAAGGCTTTGTGAGCATCAACGATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTGGGCTAT


 


ACCGAAATTGATGGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGATTGGCGTGTTCAATACCGAAGATGGTTTCAAATACTTC


 


GCGCACCATAACGAAGATCTGGGTAACGAAGAAGGCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCTACTACTTT


 


GATGATAGCTTTACCGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAGCAAATATTATTTCGATGAAGATACCGCGGAAGCGTATATT


 


GGCCTGAGCCTGATTAACGATGGCCAGTACTATTTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAAGTGTTC


 


TATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGCAGAACATTGATGATAACTACTTCTACATCGATGATAACGGCATTGTGCAGATC


 


GGCGTTTTTGATACCAGCGATGGCTACAAATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAATATAGC


 


GGTCTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGAAACCTATACCATCGAAACCGGCTGGATTTATGATATGGAAAACGAAAGC


 


GATAAATATTACTTTAATCCGGAAACGAAAAAAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAATACTATTTTGATGAAAAAGGC


 


ATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACTATTACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAACATCGAA


 


GATAAAATGTTCTACTTCGGCGAAGATGGTGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCATCAGAAT


 


ACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATACCGGCTGGCTGGATCTGGATGAAAAACGCTACTACTTCACCGATGAATAC


 


ATTGCGGCGACCGGCAGCGTGATTATTGATGGCGAAGAATACTACTTCGATCCGGATACCGCGCAGCTGGTGATTAGCGAACATCATCAT


 


CATCACCAT


 


SEQ ID NO: 21 - amino acid of F54Gly fusion protein


MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLN


 


GKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVF


 


KGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYY


 


FNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILY


 


QNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDG


 


IMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKT


 


IDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMP


 


DTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGGTGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFS


 


TEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFN


 


SDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGIL


 


NFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESG


 


VQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESD


 


KYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPD


 


GFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISEHHHHHH


 


SEQ ID NO: 22 - nucleotide sequence of F54 New fusion protein


ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGCAATTGCAAGCACCGGCTATACCATTATCAAC


 


GGCAAACACTTTTATTTTAACACCGACGGCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCAAAT


 


ACCGATGCCAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGACCCTGAACGGCAAAAAATACTACTTTGGCAGCGAT


 


AGCAAAGCAGTTACCGGTTGGCGCATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTGTGCACC


 


ATTAACAACGACAAATATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTACATTACCATCGAACGCAACAACTTTTATTTCGATGCC


 


AACAACGAAAGCAAAATGGTGACCGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATAACAACATT


 


GAAGGTCAGGCGATCGTTTATCAGAACAAATTCCTGACGCTGAATGGTAAGAAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGC


 


TGGCAGACAATTGACGGGAAGAAATATTACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGACGGTAAAAAGTAC


 


TACTTCAACCTGAACACAGCCGAAGCAGCCACAGGATGGCAGACTATTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCA


 


TCTACCGGTTATACCAGCATTAACGGTAAACATTTCTACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCAAAGGTCCAAATGGT


 


TTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACATCGAGGGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGG


 


AAAAAATATTACTTTGGTAGCGATTCTAAAGCCGTTACCGGTCTGCGTACCATTGATGGTAAAAAATACTACTTTAATACGAATACAGCC


 


GTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACTATTTTAACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATT


 


TCGGGTAAACACTTCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCTTTAAAGGACCTGATGGGTTCGAATATTTTGCGCCTGCG


 


AACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTATCAGAATCGCTTTCTGTATCTGCACGACAACATCTATTATTTTGGCAAC


 


AATTCAAAAGCAGCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCGAATACCGCAATGGGTGCAAATGGCTACAAA


 


ACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGCCGCAGATCGGGGTATTTAAAGGTAGCAACGGCTTCGAATACTTCGCTCCA


 


GCGAATACGGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGCTGGGCAAAATCTACTACTTTGGC


 


AATAACAGTAAAGCAGTTACTGGATGGCAGACAATCAATGGTAAAGTGTACTATTTTATGCCGGATACCGCCATGGCAGCAGCCGGTGGT


 


CTGTTTGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTTACCGGCTTTGTGACCGTGGGTGATGATAAATAC


 


TATTTCAATCCGATTAACGGTGGTGCAGCGAGCATTGGCGAAACCATCATCGATGACAAAAACTATTATTTCAACCAGAGCGGTGTGCTG


 


CAGACCGGTGTGTTTAGCACCGAAGATGGCTTTAAATATTTTGCGCCAGCGAACACCCTGGATGAAAACCTGGAAGGCGAAGCGATTGAT


 


TTTACCGGCAAACTGATCATCGATGAAAACATCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGGAAAGAACTGGATGGCGAA


 


ATGCATTATTTTTCTCCGGAAACCGGTAAAGCGTTTAAAGGCCTGAACCAGATCGGCGATTACAAATACTACTTCAACAGCGATGGCGTG


 


ATGCAGAAAGGCTTTGTGAGCATCAACGATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTGGGCTATACCGAAATTGATGGC


 


AAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGATTGGCGTGTTCAATACCGAAGATGGTTTCAAATACTTCGCGCACCATAACGAA


 


GATCTGGGTAACGAAGAAGGCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCTACTACTTTGATGATAGCTTTACC


 


GCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAGCAAATATTATTTCGATGAAGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATT


 


AACGATGGCCAGTACTATTTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAAGTGTTCTATTTCAGCGATAGC


 


GGCATTATTGAAAGCGGCGTGCAGAACATTGATGATAACTACTTCTACATCGATGATAACGGCATTGTGCAGATCGGCGTTTTTGATACC


 


AGCGATGGCTACAAATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAATATAGCGGTCTGGTGCGTGTG


 


GGCGAAGATGTGTATTATTTCGGCGAAACCTATACCATCGAAACCGGCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTACTTT


 


AATCCGGAAACGAAAAAAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAATACTATTTTGATGAAAAAGGCATTATGCGTACCGGT


 


CTGATTAGCTTCGAAAACAACAACTATTACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATGTTCTAC


 


TTCGGCGAAGATGGTGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCATCAGAATACCCTGGATGAAAAT


 


TTCGAAGGTGAAAGCATTAACTATACCGGCTGGCTGGATCTGGATGAAAAACGCTACTACTTCACCGATGAATACATTGCGGCGACCGGC


 


AGCGTGATTATTGATGGCGAAGAATACTACTTCGATCCGGATACCGCGCAGCTGGTGATTAGCGAACATCATCATCATCACCAT


 


SEQ ID NO: 23 amino acid sequence of F54 New fusion protein


MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN


 


EFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESK


 


MVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTI


 


DGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIE


 


GQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHF


 


YFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMG


 


ANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGK


 


VYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAVTGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVF


 


STEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYF


 


NSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGI


 


LNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIES


 


GVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENES


 


DKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTP


 


DGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISEHHHHHH


 


SEQ ID NO: 24 nucleotide sequence of F5 ToxB fusion protein


ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGCAATTGCAAGCACCGGCTATACCATTATCAAC


 


GGCAAACACTTTTATTTTAACACCGACGGCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCAAAT


 


ACCGATGCCAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGACCCTGAACGGCAAAAAATACTACTTTGGCAGCGAT


 


AGCAAAGCAGTTACCGGTTGGCGCATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTGTGCACC


 


ATTAACAACGACAAATATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTACATTACCATCGAACGCAACAACTTTTATTTCGATGCC


 


AACAACGAAAGCAAAATGGTGACCGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATAACAACATT


 


GAAGGTCAGGCGATCGTTTATCAGAACAAATTCCTGACGCTGAATGGTAAGAAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGC


 


TGGCAGACAATTGACGGGAAGAAATATTACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGACGGTAAAAAGTAC


 


TACTTCAACCTGAACACAGCCGAAGCAGCCACAGGATGGCAGACTATTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCA


 


TCTACCGGTTATACCAGCATTAACGGTAAACATTTCTACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCAAAGGTCCAAATGGT


 


TTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACATCGAGGGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGG


 


AAAAAATATTACTTTGGTAGCGATTCTAAAGCCGTTACCGGTCTGCGTACCATTGATGGTAAAAAATACTACTTTAATACGAATACAGCC


 


GTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACTATTTTAACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATT


 


TCGGGTAAACACTTCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCTTTAAAGGACCTGATGGGTTCGAATATTTTGCGCCTGCG


 


AACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTATCAGAATCGCTTTCTGTATCTGCACGACAACATCTATTATTTTGGCAAC


 


AATTCAAAAGCAGCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCGAATACCGCAATGGGTGCAAATGGCTACAAA


 


ACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGCCGCAGATCGGGGTATTTAAAGGTAGCAACGGCTTCGAATACTTCGCTCCA


 


GCGAATACGGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGCTGGGCAAAATCTACTACTTTGGC


 


AATAACAGTAAAGCAGTTACTGGATGGCAGACAATCAATGGTAAAGTGTACTATTTTATGCCGGATACCGCCATGGCAGCAGCCGGTGGT


 


CTGTTTGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTGAGCGGTCTGATTTATATTAACGATAGCCTGTAT


 


TACTTTAAACCACCGGTGAATAACCTGATTACCGGCTTTGTGACCGTGGGTGATGATAAATACTATTTCAATCCGATTAACGGTGGTGCA


 


GCGAGCATTGGCGAAACCATCATCGATGACAAAAACTATTATTTCAACCAGAGCGGTGTGCTGCAGACCGGTGTGTTTAGCACCGAAGAT


 


GGCTTTAAATATTTTGCGCCAGCGAACACCCTGGATGAAAACCTGGAAGGCGAAGCGATTGATTTTACCGGCAAACTGATCATCGATGAA


 


AACATCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGGAAAGAACTGGATGGCGAAATGCATTATTTTTCTCCGGAAACCGGT


 


AAAGCGTTTAAAGGCCTGAACCAGATCGGCGATTACAAATACTACTTCAACAGCGATGGCGTGATGCAGAAAGGCTTTGTGAGCATCAAC


 


GATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTGGGCTATACCGAAATTGATGGCAAACATTTCTACTTCGCGGAAAACGGC


 


GAAATGCAGATTGGCGTGTTCAATACCGAAGATGGTTTCAAATACTTCGCGCACCATAACGAAGATCTGGGTAACGAAGAAGGCGAAGAA


 


ATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCTACTACTTTGATGATAGCTTTACCGCGGTGGTGGGCTGGAAAGATCTGGAA


 


GATGGCAGCAAATATTATTTCGATGAAGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATTAACGATGGCCAGTACTATTTTAACGAT


 


GATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAAGTGTTCTATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGCAGAAC


 


ATTGATGATAACTACTTCTACATCGATGATAACGGCATTGTGCAGATCGGCGTTTTTGATACCAGCGATGGCTACAAATATTTCGCACCG


 


GCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAATATAGCGGTCTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGAA


 


ACCTATACCATCGAAACCGGCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTACTTTAATCCGGAAACGAAAAAAGCGTGCAAA


 


GGCATTAACCTGATCGATGATATCAAATACTATTTTGATGAAAAAGGCATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACTAT


 


TACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATGTTCTACTTCGGCGAAGATGGTGTTATGCAGATT


 


GGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCATCAGAATACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATACC


 


GGCTGGCTGGATCTGGATGAAAAACGCTACTACTTCACCGATGAATACATTGCGGCGACCGGCAGCGTGATTATTGATGGCGAAGAATAC


 


TACTTCGATCCGGATACCGCGCAGCTGGTGATTAGCGAACATCATCATCATCACCAT


 


SEQ ID NO: 25 amino acid sequence of F5 ToxB fusion protein


MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN


 


EFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESK


 


MVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTI


 


DGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIE


 


GQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHF


 


YFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMG


 


ANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGK


 


VYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAVSGLIYINDSLYYFKPPVNNLITGFVTVGDDKYYFNPINGGAASIGE


 


TIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYF


 


SPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKY


 


FAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQ


 


VGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDV


 


YYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINI


 


EDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPD


 


TAQLVISEHHHHHH


 


SEQ ID NO: 26 - nucleotide sequence of F52 new fusion protein


ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGCAATTGCAAGCACCGGCTATACCATTATCAAC


 


GGCAAACACTTTTATTTTAACACCGACGGCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCAAAT


 


ACCGATGCCAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGACCCTGAACGGCAAAAAATACTACTTTGGCAGCGAT


 


AGCAAAGCAGTTACCGGTTGGCGCATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTGTGCACC


 


ATTAACAACGACAAATATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTACATTACCATCGAACGCAACAACTTTTATTTCGATGCC


 


AACAACGAAAGCAAAATGGTGACCGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATAACAACATT


 


GAAGGTCAGGCGATCGTTTATCAGAACAAATTCCTGACGCTGAATGGTAAGAAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGC


 


TGGCAGACAATTGACGGGAAGAAATATTACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGACGGTAAAAAGTAC


 


TACTTCAACCTGAACACAGCCGAAGCAGCCACAGGATGGCAGACTATTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCA


 


TCTACCGGTTATACCAGCATTAACGGTAAACATTTCTACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCAAAGGTCCAAATGGT


 


TTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACATCGAGGGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGG


 


AAAAAATATTACTTTGGTAGCGATTCTAAAGCCGTTACCGGTCTGCGTACCATTGATGGTAAAAAATACTACTTTAATACGAATACAGCC


 


GTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACTATTTTAACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATT


 


TCGGGTAAACACTTCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCTTTAAAGGACCTGATGGGTTCGAATATTTTGCGCCTGCG


 


AACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTATCAGAATCGCTTTCTGTATCTGCACGACAACATCTATTATTTTGGCAAC


 


AATTCAAAAGCAGCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCGAATACCGCAATGGGTGCAAATGGCTACAAA


 


ACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGCCGCAGATCGGGGTATTTAAAGGTAGCAACGGCTTCGAATACTTCGCTCCA


 


GCGAATACGGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGCTGGGCAAAATCTACTACTTTGGC


 


AATAACAGTAAAGCAGTTACTGGATGGCAGACAATCAATGGTAAAGTGTACTATTTTATGCCGGATACCGCCATGGCAGCAGCCGGTGGT


 


CTGTTTGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTGAAAGGCCTGAACCAGATCGGCGATTACAAATAC


 


TACTTCAACAGCGATGGCGTGATGCAGAAAGGCTTTGTGAGCATCAACGATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTG


 


GGCTATACCGAAATTGATGGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGATTGGCGTGTTCAATACCGAAGATGGTTTCAAA


 


TACTTCGCGCACCATAACGAAGATCTGGGTAACGAAGAAGGCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCTAC


 


TACTTTGATGATAGCTTTACCGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAGCAAATATTATTTCGATGAAGATACCGCGGAAGCG


 


TATATTGGCCTGAGCCTGATTAACGATGGCCAGTACTATTTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAA


 


GTGTTCTATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGCAGAACATTGATGATAACTACTTCTACATCGATGATAACGGCATTGTG


 


CAGATCGGCGTTTTTGATACCAGCGATGGCTACAAATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAA


 


TATAGCGGTCTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGAAACCTATACCATCGAAACCGGCTGGATTTATGATATGGAAAAC


 


GAAAGCGATAAATATTACTTTAATCCGGAAACGAAAAAAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAATACTATTTTGATGAA


 


AAAGGCATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACTATTACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAAC


 


ATCGAAGATAAAATGTTCTACTTCGGCGAAGATGGTGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCAT


 


CAGAATACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATACCGGCTGGCTGGATCTGGATGAAAAACGCTACTACTTCACCGAT


 


GAATACATTGCGGCGACCGGCAGCGTGATTATTGATGGCGAAGAATACTACTTCGATCCGGATACCGCGCAGCTGGTGATTAGCGAACAT


 


CATCATCATCACCAT


 


SEQ ID NO: 27 - amino acid sequence of F52 New fusion protein


MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLN


 


GKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVF


 


KGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYY


 


FNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILY


 


QNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDG


 


IMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKT


 


IDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMP


 


DTAMAAAGGLFEIDGVIYFFGVDGVKAVKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFY


 


FAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAY


 


IGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVN


 


DNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISF


 


ENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDE


 


YIAATGSVIIDGEEYYFDPDTAQLVISEHHHHHH


 


SEQ ID NO: 28 - sequence of toxin A fragment of F54 Gly


MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAIL


 


YQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERN


 


NFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDG


 


KKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDG


 


IMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTA


 


VAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAI


 


RYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNG


 


FEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDG


 


VIYFFGVDGVKAPGIYG


 


SEQ ID NO: 29 - sequence of toxin A fragment of F54 New


MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAIL


 


YQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERN


 


NFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDG


 


KKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDG


 


IMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTA


 


VAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAI


 


RYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNG


 


FEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDG


 


VIYFFGVDGVKAV


 


SEQ ID NO: 30 - sequence of toxin A fragment of F5 ToxB


MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAIL


 


YQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERN


 


NFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDG


 


KKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDG


 


IMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTA


 


VAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAI


 


RYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNG


 


FEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDG


 


VIYFFGVDGVKAV


 


SEQ ID NO: 31 - sequence of toxin A fragment of F52 New


MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAIL


 


YQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERN


 


NFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDG


 


KKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDG


 


IMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTA


 


VAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAI


 


RYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNG


 


FEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDG


 


VIYFFGVDGVKAV


 


SEQ ID NO: 32 - sequence of toxin B fragment of F54Gly


TGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDF


 


TGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKH


 


YFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYF


 


DDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGV


 


QNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIY


 


DMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYF


 


GEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDP


 


DTAQLVISE


 


SEQ ID NO: 33 - sequence of toxin B fragment of F54 New


TGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDF


 


TGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKH


 


YFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYF


 


DDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGV


 


QNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIY


 


DMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYF


 


GEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDP


 


DTAQLVISE


 


SEQ ID NO: 34 - sequence of toxin B fragment of F5 ToxB


SGLIYINDSLYYFKPPVNNLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDG


 


FKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYK


 


YYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGN


 


EEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVG


 


FVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLV


 


RVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYY


 


FNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFT


 


DEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 35 - sequence of toxin B fragment of F52 New


KGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKY


 


FAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYY


 


FNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIY


 


GQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTG


 


LISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWL


 


DLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 36 - C-TAB.G5 fusion protein


MVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE


 


AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPN


 


GFEYFAPANTHNNNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTING


 


KKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHD


 


NIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDA


 


NNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVK


 


APGIYGRSMHNLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPAN


 


TLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGV


 


MQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISY


 


SGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKV


 


FYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYY


 


FGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNEN


 


GEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYI


 


AATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 37 C-TAB.G5.1 fusion protein


VTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEA


 


ATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNG


 


FEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGK


 


KYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDN


 


IYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDAN


 


NIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKA


 


PGIYGRSMHNLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANT


 


LDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVM


 


QKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYS


 


GILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVF


 


YFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYF


 


GETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQF


 


GYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGS


 


VIIDGEEYYFDPDTAQLVISE

## Claims

1. An immunogenic composition comprising:
a) a fusion polypeptide comprising a Clostridium difficile toxin A fragment and a C. difficile toxin B fragment; and
b) an adjuvant comprising an immunologically active saponin fraction presented in the form of a liposome.

2. An immunogenic composition according to claim 1 in which said adjuvant further comprises any one or more of a sterol, a lipopolysaccharide and 1, 2-Dioleoyl-sn-Glycero-3-phosphocholine (DOPC).

3. An immunogenic composition according to claim 2 wherein said sterol is cholesterol.

4. An immunogenic composition according to claim 1 wherein said immunologically active saponin fraction is QS21.

5. An immunogenic composition according to claim 2 wherein said lipopolysaccharide is a lipid A derivative.

6. The immunogenic composition according to claim 1 wherein said adjuvant comprises cholesterol, DOPC, 3D-MPL and QS21.

7. The immunogenic composition of claim 1, wherein the adjuvant is formulated in a dose comprising:
from about 0.1 to about 0.5 mg cholesterol,
from about 0.25 to about 2 mg DOPC,
from about 10 μg to about 70 μg 3D-MPL, and
from about 10 μg to about 70 μg QS21.

8. The immunogenic composition of claim 7, wherein the adjuvant is formulated in a dose comprising:
about 0.25 mg cholesterol,
about 1.0 mg DOPC,
about 50 μg 3D-MPL, and
about 50 μg QS21.

9. An immunogenic composition according to claim 1 wherein the polypeptide is a fusion polypeptide comprising a first fragment and a second fragment, wherein
(i) the first fragment is a toxin A repeating domain fragment;
(ii) the second fragment is a toxin B repeating domain fragment;
(iii) the first fragment has a first proximal end;
(iv) the second fragment has a second proximal end; and
wherein the first fragment and the second fragment are adjacent to one another.

10. An immunogenic composition according to claim 9 wherein the first proximal end is within a short repeat and/or the second proximal end is within a short repeat.

11. An immunogenic composition according to claim 9 wherein the first proximal end and the second proximal end do not disrupt short repeat-long repeat-short repeat (SR-LR-SR) portions.

12. An immunogenic composition according to claim 9 wherein the polypeptide comprises an immunogenic fragment of SEQ ID NO:4.

13. An immunogenic composition according to claim 1 wherein the polypeptide is a polypeptide comprising a first fragment and a second fragment, wherein
(i) the first fragment is a toxin A repeating domain fragment;
(ii) the second fragment is a toxin B repeating domain fragment;
(iii) the first fragment comprises a first proximal end within a first repeat portion;
(iv) the second fragment comprises a second proximal end within a second repeat portion; and
wherein the first fragment and the second fragment are adjacent to one another and wherein the first repeat portion and the second repeat portion have high structural similarity to one another.

14. An immunogenic composition according to claim 13 wherein the first proximal end is within repeat portion VII of toxin A and the second proximal end is within repeat portion II of toxin B.

15. An immunogenic composition according to claim 13 wherein the first proximal end is within a long repeat and/or the second proximal end is within a long repeat.

16. A vaccine comprising the immunogenic composition of claim 1 and a pharmaceutically acceptable excipient.

17. A method of preventing or treating C. difficile disease comprising administering the immunogenic composition of claim 1 to a patient.

18. An immunogenic composition according to claim 1 wherein the polypeptide is a polypeptide comprising a first fragment and a second fragment, wherein
i. the first fragment is a toxin A repeating domain fragment as set forth in SEQ ID NO:11;
ii. the second fragment is a toxin B repeating domain fragment as set forth in SEQ ID NO:16;
iii. the first fragment has a first proximal end; and
iv. the second fragment has a second proximal end; and
wherein the first fragment and the second fragment are adjacent to one another.

19. An immunogenic composition according to claim 2 wherein said lipopolysaccharide is 3D-MPL.