# Patent

## Title
Immunogenic composition comprising elements of C. difficile CDTb and/or CDTa proteins

## Abstract

The present invention relates to immunogenic compositions comprising isolated Clostridium difficile CDTb and/or CDTa protein. In particular the isolated Clostridium difficile CDTb protein is suitably a truncated CDTb protein comprising the receptor binding domain or a mutated CDTb protein incapable of binding to CDTa, and the isolated Clostridium difficile CDTa protein is suitably a truncated CDTa protein which does not comprise the C-terminal domain. In particular the invention also relates to fusion proteins comprising a CDTa protein and a CDTb protein and also fusion proteins between an isolated Clostridium difficile toxin A protein and/or an isolated Clostridium difficile toxin B protein fused to a CDTb protein.

## 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/EP2013/077762 filed Dec. 20, 2013, which claims priority to United Kingdom Application No. GB 1223342.5 filed Dec. 23, 2012, and the entire contents of each of the foregoing applications are hereby incorporated by reference.
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 and toxin B. These are both potent cytotoxins (Lyerly et al Current Microbiology 21:29-32 (1990).
It has been demonstrated that fragments of toxin A, in particular fragments of the C-terminal domain, can lead to a protective immune response in hamsters (Lyerly et al Current Microbiology 21:29-32 (1990)), WO96/12802 and WO00/61762. However the present inventors have demonstrated that antibodies against toxin A and toxin B alone are not sufficient in order to prevent disease caused by certain strains, in particular serogroup 078 and 027 strains. For this reason vaccines which are capable of protecting against these strains are still required.
Some strains, but not all, also express the binary toxin (CDT). Similar to many other binary toxins, CDT is composed of two components—an enzymatically active component (CDTa) and a catalytically inert transport component (CDTb). The catalytically inert component facilitates translocation of the CDTa into the target cell.
CDTa has an ADP-ribosylating activity, which transfers the ADP-ribose moiety of NAD/NADPH to the monomeric actin (G-actin) in the target cell and thus preventing its polymerization to F-actin and resulting in disruption of the cytoskeleton and eventual cell death (Sundriyal et al, Protein expression and Purification 74 (2010) 42-48).
WO2013/112867 (Merck) describes vaccines against Clostridium difficile comprising recombinant C. difficile Toxin A and Toxin B and binary toxin A (CDTa) proteins comprising specifically defined mutations relative to the native toxin sequence that are described as substantially reducing or eliminating toxicity, in combination with binary toxin B (CDTb).
The present inventors have found, that binary toxin can be used to provide an improved vaccine against C. difficile particularly providing protection against several of the most concerning C. difficile strains (such as the 027 and 078 strains). Furthermore the present inventors have demonstrated, for the first time, that only CDTa or CDTb (not both) is required in order to generate antibodies which are capable of neutralizing strains expressing binary toxin. In addition the inventors have demonstrated, for the first time, that CDTa proteins comprising mutations which reduce the ADP-ribosylating activity of CDTa, are still capable of raising an immune response. In addition, the inventors have demonstrated that truncated CDTa proteins are capable of raising an immune response. Similarly the inventors have demonstrated that truncated CDTb proteins are capable of raising an immune response, that CDTb can raise an immune response when it is in its monomeric or polymeric form and that fusion proteins comprising CDTa and CDTb or CDTb fused to isolated toxin A and/or isolated toxin B are capable of raising an immune response. Finally, the inventors have demonstrated that an immunogenic composition comprising binary toxin can be improved by adding an adjuvant, in particular an adjuvant comprising an immunologically active saponin presented in the form of a liposome or an oil in water emulsion.
SUMMARY OF INVENTION
In a first aspect of the invention there is provided an immunogenic composition comprising an isolated Clostridium difficile CDTb protein wherein the composition does not further comprise an isolated protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to SEQ ID NO: 1, SEQ ID NO: 31 or SEQ ID NO: 32.
In a second aspect of the invention there is provided an immunogenic composition comprising isolated Clostridium difficile CDTb protein wherein the isolated Clostridium difficile CDTb protein is a truncated CDTb protein comprising the receptor binding domain.
In a third aspect of the invention there is provided an immunogenic composition comprising isolated Clostridium difficile CDTb protein wherein the isolated Clostridium difficile CDTb protein is a mutated CDTb protein incapable of binding to CDTa.
In a fourth aspect of the invention there is provided an immunogenic composition comprising isolated Clostridium difficile CDTa protein wherein the isolated Clostridium difficile CDTa protein is a truncated CDTa protein which does not comprise the C-terminal domain.
In a fifth aspect the present invention provides an immunogenic composition comprising a fusion protein comprising a CDTa protein and a CDTb protein.
In a sixth aspect the present invention provides an immunogenic composition comprising a fusion protein between an isolated Clostridium difficile toxin A protein and/or an isolated Clostridium difficile toxin B protein fused to a CDTb protein.
In a seventh aspect the present invention provides a vaccine comprising the immunogenic composition of any one of the first five aspects and a pharmaceutically acceptable excipient.
In an eighth aspect the present invention provides the immunogenic composition of any one of the first five aspects or the vaccine of the sixth aspect, for use in the treatment or prevention of disease e.g. C. difficile disease.
In a ninth aspect the present invention provides the use of an immunogenic composition of any one of the first five aspects or the vaccine of the sixth aspect in the preparation of a medicament for the prevention or treatment of disease e.g. C. difficile disease.
In a tenth aspect the present invention provides a method of preventing or treating C. difficile disease comprising administering an immunogenic composition of any one of the first six aspects or the vaccine of the seventh aspect to a mammalian subject.
In a further aspect of the invention there is provided an immunogenic composition comprising an isolated Clostridium difficile CDTb protein.
In a further aspect of the invention there is provided an immunogenic composition comprising either an isolated Clostridium difficile CDTb protein or an isolated CDTa protein but does not comprise both an isolated CDTb protein and an isolated CDTa protein.
Novel polypeptides and nucleotides as defined herein also form further aspects of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 (comprising FIGS. 1a-1h)—Graphs describing the size distribution of the different CdtA, CdtB and CdtA-CdtB fusion constructions as determined by sedimentation velocity analytical ultracentrifugation:
FIG. 1a: AUC of C67 (CdtA (aa44-463) mut E428Q-E430Q
FIG. 1b: AUC of C69 CdtA (aa44-463) mut. R345A-Q350A-N385A-R402A-S388F-E428Q-E430Q
FIG. 1c: AUC of C50 (CdtA N-term without linker (aa44-260)
FIG. 1d: AUC of C61 (fusion CdtA N term with linker-CdtBshort)
FIG. 1e: AUC of C62 (fusion CdtA N term without linker-CdtBlong)
FIG. 1f: AUC of C52 (CdtB long)
FIG. 1g: AUC of C53 (CdtB short)
FIG. 1h: AUC of C55 CdtB Δ prodomain (aa. 212-876)
FIG. 2 (comprising FIGS. 2a-2c)—SDS PAGE profiles of CdtA, CdtB and CdtA-CdtB fusion constructions after purification:
FIG. 2a: SDS PAGE of purified CdtA-CdtB fusion constructions. Lane 1: Molecular weight marker Novex sharp prestained. Lane 2: 5 μg of C61 CdtA N-term link (aa. 44-268)-CdtB RBD short (aa. 636-876). Lane 3: 5 μg of C62 CdtA N-term (aa. 44-260)-CdtB RBD long (aa. 621-876).
FIG. 2b: SDS PAGE of purified CdtA constructions. Lane 1: Molecular weight marker Novex sharp prestained. Lane 2: 5 μg of C50 CdtA WO linker (44-260). Lane 3: 5 μg of C67 CdtA full length (aa44-463) mut. E428Q-E430Q. Lane 4: 5 μg of C69 CdtA full length (aa44-463) mut. R345A-Q350A-N385A-R402A-S388F-E428Q-E430Q.
FIG. 2c: SDS PAGE of purified CdtB constructions. Lane 1: Molecular weight marker Novex sharp prestained. Lane 2: 5 μg of C37 CdtB′ Δsignal sequence (aa43-876)+GST N-term after removal of the N-term GST and activation by prodomain cleavage with chymotrypsin. Lane 3: 5 μg of C55 CdtB Δ prodomain (aa. 212-876). Lane 4: 5 μg of C52 CdtB receptor binding domain long (aa. 621-876). Lane 5: Molecular weight marker. Lane 6: 5 μg of C38 CdtB′ Δsignal sequence (aa43-876).
FIG. 3—Graph showing anti-CDTb immunogenicity in mice immunised with C. difficile Binary Toxin component A or C. difficile Binary Toxin component B, in both cases formulated with adjuvant
FIG. 4—Graph showing anti-CDTa immunogenicity in mice immunised with C. difficile Binary Toxin component A or C. difficile Binary Toxin component B, in both cases formulated with adjuvant
FIG. 5—Cytotoxicity inhibition titres in HCT116 cells from mice immunised with C. difficile Binary Toxin component A or C. difficile Binary Toxin component B, in both cases formulated with adjuvant
FIG. 6—Cytotoxicity inhibition titres in HT29 cells from mice immunised with C. difficile Binary Toxin component A or C. difficile Binary Toxin component B, in both cases formulated with adjuvant
FIG. 7—Graph showing anti-CDTb immunogenicity in mice immunised with C. difficile Cdtb (activated or non activated, with and without F2 fusion comprising fragments from Toxin A and Toxin B) formulated with adjuvant
FIG. 8—Graph showing anti-Tox A immunogenicity in mice immunised with C. difficile Cdtb (activated or non activated, with and without F2 fusion comprising fragments from Toxin A and Toxin B) formulated with adjuvant
FIG. 9—Graph showing anti-Tox B immunogenicity in mice immunised with C. difficile Cdtb (activated or non activated, with and without F2 fusion comprising fragments from Toxin A and Toxin B) formulated with adjuvant
FIG. 10—Tox A cytotoxicity inhibition titres in HT29 cells from mice immunised with C. difficile Cdtb (activated or non activated, with and without F2 fusion comprising fragments from Toxin A and Toxin B) formulated with adjuvant
FIG. 11—Tox B cytotoxicity inhibition titres in HCT116 cells from mice immunised with C. difficile Cdtb (activated or non activated, with and without F2 fusion comprising fragments from Toxin A and Toxin B) formulated with adjuvant
FIG. 12—Binary Toxin cytotoxicity inhibition titres in HT29 cells from mice immunised with C. difficile Binary Toxin component A or C. difficile Binary Toxin component B, in both cases formulated with adjuvant
FIG. 13—Graph showing anti-CDTb immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 6 μg/dose in an adjuvant formulation
FIG. 14—Graph showing anti-CDTa immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 6 μg/dose in an adjuvant formulation
FIG. 15—Graph showing anti-Tox B immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 6 μg/dose in an adjuvant formulation
FIG. 16—Graph showing anti-Tox A immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 6 μg/dose in an adjuvant formulation
FIG. 17—Binary Toxin cytotoxicity inhibition titres in HCT116 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 6 μg/dose in an adjuvant formulation
FIG. 18—Binary Toxin cytotoxicity inhibition titres in HT29 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 6 μg/dose in an adjuvant formulation
FIG. 19—Tox A cytotoxicity inhibition titres in HT29 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 6 μg/dose in an adjuvant formulation
FIG. 20—Tox B cytotoxicity inhibition titres in HCT116 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 6 μg/dose in an adjuvant formulation
FIG. 21—Graph showing anti-CDTb immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 2 μg/dose in an adjuvant formulation
FIG. 22—Graph showing anti-CDTa immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 2 μg/dose in an adjuvant formulation
FIG. 23—Graph showing anti-Tox B immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 2 μg/dose in an adjuvant formulation
FIG. 24—Graph showing anti-Tox A immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 2 μg/dose in an adjuvant formulation
FIG. 25—Binary Toxin cytotoxicity inhibition titres in HCT116 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 2 μg/dose in an adjuvant formulation
FIG. 26—Binary Toxin cytotoxicity inhibition titres in HT29 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 2 μg/dose in an adjuvant formulation
FIG. 27—Tox A cytotoxicity inhibition titres in HT29 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 2 μg/dose in an adjuvant formulation
FIG. 28—Tox B cytotoxicity inhibition titres in HCT116 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 2 μg/dose in an adjuvant formulation
FIG. 29—Graph showing anti-CDTb immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 10 μg/dose in a non-adjuvanted formulation
FIG. 30—Graph showing anti-CDTa immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 10 μg/dose in a non-adjuvanted formulation
FIG. 31—Graph showing anti-Tox B immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 10 μg/dose in a non-adjuvanted formulation
FIG. 32—Graph showing anti-Tox A immunogenicity in mice immunized with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 10 μg/dose in a non-adjuvanted formulation
FIG. 33—Binary Toxin cytotoxicity inhibition titres in HCT116 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 10 μg/dose in a non-adjuvanted formulation
FIG. 34—Binary Toxin cytotoxicity inhibition titres in HT29 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 10 μg/dose in a non-adjuvanted formulation
FIG. 35—Tox A cytotoxicity inhibition titres in HT29 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 10 μg/dose in a non-adjuvanted formulation
FIG. 36—Tox B cytotoxicity inhibition titres in HCT116 cells from mice immunised with different binary toxin vaccine candidates (CdtA/CdtB) combined with F2 at 10 μg/dose in a non-adjuvanted formulation



DETAILED DESCRIPTION
Binary Toxin
The Clostridium difficile binary toxin comprises two different proteins, CDTa and CDTb. During infection CDTb is activated by proteolytic cleavage by a chymotrypsin-like protease to produce a CDTb protein lacking the prodomain (also referred to as CDTb′′). Note that CDTb′′ also lacks the CDTb signal sequence, a CDTb protein lacking the signal sequence but not lacking the prodomain is referred to as CDTb′. After proteolytic activation the CDTb oligomerises and binds to CDTa to form the complete ‘binary toxin’. The binding of the binary toxin to the cell receptors leads to receptor-mediated endocytosis. As the endosome acidifies the CDTb binding domain undergoes conformational changes that allow the CDTb oligomer to form a pore, the pore formation triggers translocation of the ADP-ribosyltransferase domain (CDTa) into the target cell.
CDTb
The present invention provides an immunogenic composition comprising an isolated Clostridium difficile CDTb protein. The present invention also provides an immunogenic composition comprising an isolated Clostridium difficile CDTb protein as the sole C. difficile antigen. As used herein the term “as the sole C. difficile antigen” means that the immunogenic composition comprising an isolated Clostridium difficile CDTb protein as the sole C. difficile antigen does not also comprise another antigen from C. difficile e.g. the immunogenic composition does not also comprise a toxin A, toxin B or CDTa protein.
The present invention provides an immunogenic composition comprising an isolated Clostridium difficile CDTb protein wherein the composition does not further comprise an isolated protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to SEQ ID NO: 1, SEQ ID NO: 31 or SEQ ID NO: 32. According to the invention as herein described the term ‘CDTb protein’ encompasses SEQ ID NO:3 or fragments or variants of SEQ ID NO:3.
In one embodiment of this first aspect of the invention, the composition does not comprise an isolated Clostridium difficile CDTa protein.
In one embodiment of this aspect the isolated Clostridium difficile CDTb protein is or comprises
(i) SEQ ID NO: 3; or
(ii) a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO:3; or
(iii) a fragment of CDTb having at least 30, 50, 80, 100, 120, 150, 200, 250 or 300 contiguous amino acids of SEQ ID NO:3.
In one such aspect there is provided an immunogenic composition wherein the isolated Clostridium difficile CDTb protein is a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO:3.
In another aspect there is provided an immunogenic composition wherein the isolated Clostridium difficile CDTb protein is a fragment of CDTb having at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or 850 contiguous amino acids of SEQ ID NO: 3.
CDTb comprises multiple domains, in particular CDTb comprises a signal peptide and a prodomain both of which are cleaved as explained in the section entitled “Binary Toxin” above.
In one embodiment the isolated Clostridium difficile CDTb protein is a truncated CDTb protein with the signal peptide removed. The term ‘truncated CDTb protein with the signal peptide removed’ refers to a fragment or variant of SEQ ID NO: 3 with substantially all of the signal peptide removed (therefore which does not comprise amino acids corresponding to substantially all of the signal peptide), there may be a few amino acids of the signal peptide remaining, for example 2, 5, 10, 15 or 20 amino acids of the signal peptide may remain. The signal peptide corresponds to amino acids 1-48 (encompassing amino acids 1-42) of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a different strain of C. difficile, for example amino acids 1-42 of the amino acid sequence of CDTb from strain CD196 (Perelle, M. et al Infect. Immun., 65 (1997), pp. 1402-1407).
Suitably in this embodiment the isolated Clostridium difficile CDTb protein is or comprises
(i) SEQ ID NO: 7 or SEQ ID NO: 16; or
(ii) a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 7 or SEQ ID NO:16; or
(iii) a fragment of CDTb having at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or 800 contiguous amino acids of SEQ ID NO: 7 or SEQ ID NO:16. In one embodiment the truncated CDTb protein with the signal peptide removed is or comprises a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 7 or SEQ ID NO:16. In a further embodiment the isolated truncated CDTb protein with the signal peptide removed is or comprises a fragment of CDTb having at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 contiguous amino acids of SEQ ID NO: 7 or SEQ ID NO:16.
In one embodiment the isolated Clostridium difficile CDTb protein is a truncated CDTb protein with the prodomain removed. The term ‘truncated CDTb protein with the prodomain removed’ refers to a fragment or variant of SEQ ID NO: 3 with substantially all of the prodomain removed (therefore which does not comprise amino acids corresponding to substantially all of the prodomain), there may be a few amino acids of the prodomain remaining, for example 2, 5, 10, 15 or 20 amino acids of the prodomain may remain. The prodomain corresponds to amino acids 48-211 (encompassing amino acids 48-166) of SEQ ID NO:3 or their equivalents in a binary toxin protein isolated from a different strain of C. difficile. Optionally the truncated CDTb protein with the prodomain removed also lacks the CDTb signal sequence, the CDTb signal sequence corresponds to amino acids 1-48 (encompassing amino acids 1-42) of SEQ ID NO:3 or their equivalents in a different strain. The term ‘truncated CDTb protein with the prodomain removed’ may also refer to a fragment or variant of SEQ ID NO: 3 which is capable of oligomerising and binding to CDTa. In this embodiment of the invention the isolated Clostridium difficile CDTb protein suitably is or comprises
(i) SEQ ID NO: 9 or SEQ ID NO: 51; or
(ii) a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO:9 or SEQ ID NO: 51; or
(iii) a fragment of CDTb having at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or 650 contiguous amino acids of SEQ ID NO:9 or SEQ ID NO: 51.
In one embodiment the truncated CDTb protein with the prodomain removed is or comprises a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO:9. In a further embodiment the isolated truncated CDTb protein with the prodomain removed is or comprises a fragment of CDTb having at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 contiguous amino acids of SEQ ID NO:9.
CDTb also comprises a receptor binding domain. In one embodiment the isolated Clostridium difficile CDTb protein is a truncated CDTb protein comprising the receptor binding domain. The term ‘truncated CDTb protein comprising the receptor binding domain’ refers to a fragment or variant of SEQ ID NO:3 with substantially all but the receptor binding domain removed (therefore which does not comprise amino acids corresponding to substantially all of the protein except for the receptor binding domain), there may be a few amino acids in addition to the receptor binding domain remaining, for example 2, 5, 10, 15 or 20 amino acids except for/in addition to the receptor binding domain. In one version, the receptor binding domain corresponds to amino acids 620-876 of SEQ ID NO:3, or their equivalents in a binary toxin protein isolated from a different strain of C. difficile. In another version, the receptor binding domain corresponds to amino acids 636-876 of SEQ ID NO:3 or their equivalents in a binary toxin protein isolated from a different strain of C. difficile. 
In this embodiment the isolated Clostridium difficile CDTb protein suitably is or comprises
(i) SEQ ID NO: 34 or SEQ ID NO: 36; or
(i) a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 34 or SEQ ID NO: 36; or
(iii) a fragment of CDTb having at least 30, 50, 80, 100, 120, 150 or 200 contiguous amino acids of SEQ ID NO: 34 or SEQ ID NO: 36.
In another embodiment of this aspect of the invention, the isolated Clostridium difficile CDTb protein is a mutated CDTb protein incapable of binding to CDTa.
In this embodiment the isolated Clostridium difficile CDTb protein suitably is or comprises
(i) SEQ ID NO: 50; or
(i) a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 50; or
(iii) a fragment of CDTb having at least 30, 50, 80, 100, 120, 150, 200, 250 or 300 contiguous amino acids of SEQ ID NO: 50.
The CDTb protein varies in amino acid sequence between different strains, for this reason the amino acid numbering may differ between strains. For this reason the term ‘equivalents in a different strain’ refers to amino acids which correspond to those of a reference strain (e.g., C. difficile R20291 from which SEQ ID NO:1 and SEQ ID NO:3 are derived), 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. Example binary toxin producing strains of C. difficile include CD196, CCUG 20309, R8637, IS81, IS93, IS51, IS58, R6786, R7605, R10456 and R5989. The amino acids numbers provided throughout refer to those of reference strain R20291.
In one embodiment the isolated Clostridium difficile CDTb protein is a monomer of CDTb. In a further embodiment the isolated Clostridium difficile CDTb protein is a multimer of CDTb. In a further embodiment the isolated Clostridium difficile CDTb protein is a heptamer of CDTb.
In a second aspect the present invention provides immunogenic compositions comprising isolated Clostridium difficile CDTb protein wherein the isolated Clostridium difficile CDTb protein is a truncated CDTb protein comprising the receptor binding domain. In one embodiment of this aspect the isolated Clostridium difficile CDTb protein suitably is or comprises
(i) SEQ ID NO: 34 or SEQ ID NO: 36; or
(i) a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 34 or SEQ ID NO: 36; or
(iii) a fragment of CDTb having at least 30, 50, 80, 100, 120, 150 or 200 contiguous amino acids of SEQ ID NO: 34 or SEQ ID NO: 36.
In a third aspect the invention provides immunogenic compositions comprising isolated Clostridium difficile CDTb protein wherein the isolated Clostridium difficile CDTb protein is a mutated CDTb protein incapable of binding to CDTa. In one embodiment of this aspect, the isolated Clostridium difficile CDTb protein suitably is or comprises
(i) SEQ ID NO: 50; or
(i) a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 50; or
(iii) a fragment of CDTb having at least 30, 50, 80, 100, 120, 150, 200, 250 or 300 contiguous amino acids of SEQ ID NO: 50.
In one embodiment of the second and third aspects of the invention, the immunogenic composition comprises/further comprises an isolated Clostridium difficile CDTa protein comprising
(i) SEQ ID NO: 1; or
(ii) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO:1; or
(iii) a fragment of CDTa having at least at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 contiguous amino acids of SEQ ID NO:1.
CDTa
The present invention also provides immunogenic compositions comprising an isolated Clostridium difficile CDTa protein. The present invention also provides immunogenic compositions comprising an isolated Clostridium difficile CDTa protein as the sole C. difficile antigen. As used herein the term “as the sole C. difficile antigen” means that the immunogenic composition comprising an isolated Clostridium difficile CDTa protein as the sole C. difficile antigen does not also comprise another antigen from C. difficile e.g. the immunogenic composition does not also comprise a toxin A, toxin B or CDTb protein. According to the invention as herein described the term ‘CDTa protein’ encompasses SEQ ID NO:1 or fragments or variants of SEQ ID NO:1. In one embodiment the isolated Clostridium difficile CDTa protein comprises a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO:1. In a further embodiment the isolated Clostridium difficile CDTa protein comprises a fragment of CDTa having at least at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 contiguous amino acids of SEQ ID NO:1.
CDTa comprises two domains, the C-terminal domain is responsible for the ADP ribosyltransferase activity whilst the N-terminal domain is responsible for interacting with CDTb.
In one embodiment of the first three aspects of the invention, the immunogenic composition comprises/further comprises an isolated Clostridium difficile CDTa protein. Suitably the isolated Clostridium difficile CDTa protein is a truncated CDTa protein. “A truncated CDTa protein” as used herein means a CDTa protein that does not achieve its full length or its proper form, and thus is missing some of the amino acid residues that are present in full length CDTa of SEQ ID NO: 1, and which cannot perform the function for which it was intended because its structure is incapable of doing so, e.g. ADP ribosyltransferase activity and/or interacting with CDTb.
Suitably the isolated Clostridium difficile CDTa protein is a truncated CDTa protein which does not comprise the C-terminal domain. The term ‘truncated CDTa protein which does not comprise the C-terminal domain’ refers to a fragment or variant of SEQ ID NO:1 which does not comprise a substantial portion of the C-terminal domain, there may be a few amino acids of the C-terminal domain remaining, for example, 2, 5, 10, 15, 20, 25, 30, 35 or 50 amino acids of the C-terminal domain may remain. The C-terminal domain corresponds to amino acids 267-463 of SEQ ID NO:1 or their equivalents in a CDTa protein isolated from a different strain of C. difficile. In this embodiment the truncated Clostridium difficile CDTa protein suitably is or comprises
(i) SEQ ID NO: 14 or SEQ ID NO: 15
(i) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or
(iii) a fragment of CDTa having at least 30, 50, 80, 100, 120, 150, or 190 contiguous amino acids of SEQ ID NO: 14 or SEQ ID NO: 15.
In one embodiment the truncated CDTa protein which does not comprise the C-terminal domain is a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO:13. In a further embodiment the truncated CDTa protein which does not comprise the C-terminal domain is a variant of CDTa having at least 30, 50, 80, 100, 120, 150, or 190 contiguous amino acids of SEQ ID NO:13.
In a fourth aspect the invention provides an immunogenic composition comprising isolated Clostridium difficile CDTa protein wherein the isolated Clostridium difficile CDTa protein is a truncated CDTa protein which does not comprise the C-terminal domain. In one embodiment of this aspect, the isolated Clostridium difficile CDTa protein suitably is or comprises
(i) SEQ ID NO: 14 or SEQ ID NO: 15; or
(ii) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO:14 or SEQ ID NO: 15; or
(iii) a fragment of CDTa having at least 30, 50, 80, 100, 120, 150, or 190 contiguous amino acids of SEQ ID NO:14 or SEQ ID NO: 15.
In a further embodiment of any of the aspects of the invention, the isolated Clostridium difficile CDTa protein suitably contains a mutation which reduces its ADP-ribosyltransferase activity. For example the isolated Clostridium difficile CDTa protein has a mutation from glutamate to another amino acid at position 428. The term ‘has a mutation at position 428’ refers to CDTa proteins which have a mutation at this exact location but also to a CDTa protein which is isolated from a different strain and which has a mutation at an equivalent position. The CDTa protein varies in amino acid sequence between different strains, for this reason the amino acid numbering may differ between strains, thus a CDTa protein from a different strain may have a corresponding glutamate which is not number 428 in sequence. In one embodiment the isolated Clostridium difficile CDTa protein has a mutation from glutamate to glutamine at position 428.
In a further embodiment of any of the aspects of the invention, the isolated Clostridium difficile CDTa protein suitably has a mutation from glutamate to a different amino acid at position 430, the term ‘has a mutation at position 430’ refers to proteins which have this exact location but also to a CDTa protein which is isolated from a different strain and which has a mutation at an equivalent position. In one embodiment the isolated Clostridium difficile CDTa protein has a mutation from glutamate to glutamine at position 430.
In a further embodiment of any of the aspects of the invention, the isolated Clostridium difficile CDTa protein suitably is or comprises
(i) SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 52; or SEQ ID NO: 54; or
(ii) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 52; or SEQ ID NO: 54; or
(iii) a fragment of CDTa having at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 contiguous amino acids of SEQ ID ID NO: 46; SEQ ID NO: 48; SEQ ID NO: 52; or SEQ ID NO: 54.
In a further embodiment of any of the aspects of the invention, the isolated Clostridium difficile CDTa protein suitably is or comprises
(i) SEQ ID NO: 48; or
(ii) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 48; or
(iii) a fragment of CDTa having at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 contiguous amino acids of SEQ ID ID NO: 48.
Immunogenic Compositions with CDTa and/or CDTb
In a further embodiment there is provided an immunogenic composition which comprises a CDTb protein but does not comprise a CDTa protein, for example the immunogenic composition does not comprise a variant of CDTa having at least 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1 or a fragment of CDTa having at least at least 250, 400 or 450. contiguous amino acids of SEQ ID NO:1.
In a further embodiment there is provided an immunogenic composition which comprises a CDTa protein but does not comprise a CDTb protein, for example the immunogenic composition does not comprise a variant of CDTb having at least 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3 or a fragment of CDTb having at least 700, 750, or 800 contiguous amino acids of CDTb.
In a further embodiment there is provided an immunogenic composition which comprises either an isolated Clostridium difficile CDTb protein or an isolated CDTa protein but does not comprise both an isolated CDTb protein and an isolated CDTa protein.
In a further embodiment there is provided a fusion protein comprising a CDTa protein and a CDTb protein. In another embodiment there is provided immunogenic compositions comprising a fusion protein comprising a CDTa protein and a CDTb protein.
Fusion Proteins Comprising a CDTa Protein and a CDTb Protein
In a fifth aspect, the invention provides immunogenic compositions comprising a fusion protein comprising a CDTa protein and a CDTb protein. In one embodiment of this aspect, the CDTa protein suitably is truncated. For example, the CDTa protein suitably does not comprise the C-terminal domain. In this aspect, the CDTb protein suitably is truncated. In this embodiment, the CDTb protein suitably comprises the receptor binding domain.
In one embodiment of this aspect of the invention, the fusion protein suitably is or comprises
(i) SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; or SEQ ID NO: 43; or
(ii) a variant having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; or SEQ ID NO: 43; or
(iii) a fragment having at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 contiguous amino acids of SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; or SEQ ID NO: 43.
“Fusion polypeptide” or “fusion protein” refers to a protein having at least two heterologous polypeptides (e.g. at least two Mycobacterium sp. polypeptides) covalently linked, either directly or via an amino acid linker. It may also refer to a protein having at least two heterologous polypeptides linked non-covalently. The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order. This term also refers to conservatively modified variants, polymorphic variants, alleles, mutants, immunogenic fragments, and interspecies homologs of the antigens that make up the fusion protein.
The term “fused” refers to the linkage e.g. covalent linkage between two polypeptides in a fusion protein. The polypeptides are typically joined via a peptide bond, either directly to each other or via an amino acid linker. Optionally, the peptides can be joined via non-peptide covalent linkages known to those of skill in the art.
A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. 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 and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and 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 for example 1, 5, 10, 15, 20, 25, 30, 35 or 40 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
In one embodiment of any of the aspects of the invention, the immunogenic composition elicits antibodies that neutralize CDTa or CDTb or both. In a further embodiment the composition elicits antibodies that neutralize binary toxin. Whether a composition elicits antibodies against a toxin can be measured by immunising mice with the immunogenic composition, collecting sera and analysing the anti-toxin titres of the sera using by ELISA. The sera should be compared to a reference sample obtained from mice which have not been immunised. The composition of the invention elicits antibodies that neutralise CDTa 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 immunogenic compositions of the invention elicits a protective immune response in a mammalian host against strains of C. difficile. In one embodiment the mammalian host is selected from the group consisting of mouse, rabbit, guinea pig, non-human primate, monkey and human. In one embodiment the mammalian host is a mouse. In a further embodiment the mammalian host is a human.
Whether an immunogenic composition 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 immunogenic composition 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 immunogenic composition survives. An immunogenic composition elicits a protective immune response if a mammal immunised with the immunogenic composition survives at least 10%, 20%, 30%, 50%, 80%, 80%, 90%, or 100% longer than a reference mammal which has not been immunised after challenge with C. difficile. 
Toxin A and Toxin B
In one embodiment of any of the aspects of the invention, the immunogenic compositions of the invention further comprise an isolated Clostridium difficile toxin A protein and/or an isolated C. difficile toxin B protein.
The term ‘isolated Clostridium difficile toxin A protein’ refers to a fragment or variant of SEQ ID NO: 31. In one embodiment the isolated Clostridium difficile toxin A protein is a fragment comprising 50, 100, 150, 200, 250, 300, 500, 750, 1000, 1250, 1500, 1750, 2000 or 2500 contiguous amino acids of SEQ ID NO:31. In one embodiment the isolated Clostridium difficile toxin A protein is a variant comprising 80%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity to SEQ ID NO:31.
The term ‘isolated Clostridium difficile toxin B protein’ refers to a fragment or variant of SEQ ID NO: 32. In one embodiment the isolated Clostridium difficile toxin B protein is a fragment comprising 50, 100, 150, 200, 250, 300, 500, 750, 1000, 1250, 1500, 1750 or 2000 SEQ ID NO:32. In one embodiment the isolated Clostridium difficile toxin B protein is a variant comprising 80%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity to SEQ ID NO:32.
In one embodiment the isolated Clostridium difficile toxin A protein comprises a repeating domain fragment. The term ‘toxin A repeating domain’ refers to the C-terminal domain of the toxin A protein from C. difficile, comprising repeated sequences. The toxin A repeating domain refers to amino acids 1832-2710 of toxin A from strain VPI10463 (ATCC43255) and their equivalents in a different strain, the sequence of amino acids 1832-2710 from strain VPI10463 (ATCC43255) corresponds to amino acids 1832-2710 of SEQ ID NO: 31. In a further embodiment the isolated Clostridium difficile toxin A protein comprises a fragment of the toxin A N-terminal domain. The toxin A N-terminal domain refers to amino acids 1-1831 of toxin A from strain VBI10463 (ATCC43255) and their equivalents in a different strain, the sequence of amino acids 1-1831 of SEQ ID NO: 31.
In one embodiment the isolated Clostridium difficile toxin B protein comprises a toxin B repeating domain fragment. The term ‘toxin B repeating domain’ refers to the C-terminal domain of the toxin B protein from C. difficile. This domain refers to amino acids 1834-2366 from strain VPI10463 (ATCC43255) and their equivalents in a different strain, the sequence of amino acids 1834-2366 from strain VPI10463 (ATCC43255) corresponds to amino acids 1834-2366 of SEQ ID NO: 32. In a further embodiment the isolated Clostridium difficile toxin B protein comprises a fragment of the toxin B N-terminal domain. The toxin B N-terminal domain refers to amino acids 1-1833 of toxin B from strain VBI10463 (ATCC43255) and their equivalents in a different strain, the sequence of amino acids 1-1833 of SEQ ID NO: 32.
The 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.
For these reasons the terms toxin A repeating domain and/or toxin B repeating domain to refer to a sequence which is a variant with 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1832-2710 of SEQ ID NO: 31 or a variant with 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1834-2366 of SEQ ID NO:32. Similarly the terms toxin a N-terminal domain and/or toxin B N terminal domain refer to a sequence which is avariant with 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1-1831 of SEQ ID NO:31 or a variant with 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1-1833 of SEQ ID NO:32.
Furthermore the amino acid numbering may differ between the C-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 ‘equivalents in a different strain’ refers to amino acids which correspond to 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.
In a further embodiment of any of the aspects of the invention, the isolated C. difficile toxin A protein and the isolated C. difficile toxin B protein form a fusion protein. In one embodiment the fusion protein is 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 22, 24, 26, 28 and 30. In a further embodiment the fusion protein is a fragment of at least 800, 850, 900 or 950 contiguous amino acids of a sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 22, 24, 26, 28 and 30.
In a further embodiment of any of the aspects of the invention the immunogenic composition comprises/further comprises a fusion protein between an isolated Clostridium difficile toxin A protein and/or an isolated Clostridium difficile toxin B protein fused to a CDTb protein or to a truncated CDTb protein. In one embodiment there is provided a fusion protein comprising a fragment of toxin A, a fragment of toxin B and a CDTb protein, for example the fusion protein may comprise a fragment or variant of SEQ ID NO:18, 19, 20, 21, 22, 24, 26, 28 or 30 fused to a CDTb protein. For example the fusion protein may comprise a fragment or variant of SEQ ID NO:18, 19, 20, 21, 22, 24, 26, 28 or 30 fused to a truncated CDTb protein.
In one embodiment the fusion protein suitably is or comprises
(i) SEQ ID NO: 44 or SEQ ID NO: 45; or
(ii) a variant having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 45; or
(iii) a fragment of at least 800, 850, 900 or 950 contiguous amino acids of a sequence selected from the group consisting of SEQ ID NO: 44 or SEQ ID NO: 45.
Fragments
The term “fragment” as defined herein may refer to a fragment comprising a T cell epitope. T cell epitopes are short contiguous stretches of amino acids which are recognised by T cells (e.g. CD4+ or CD8+ T cells). Identification of T cell epitopes may be achieved through epitope mapping experiments which are well known to the person skilled in the art (see, for example, Paul, Fundamental Immunology, 3rd ed., 243-247 (1993); Beiβbarth et al Bioinformatics 2005 21(Suppl. 1):i29-i37).
Suitably the fragments of the invention are immunogenic fragments. “Immunogenic fragments” according to the present invention will typically comprise at least 9 contiguous amino acids from the full length polypeptide sequence (e.g. at least 10), such as at least 12 contiguous amino acids (e.g. at least 15 or at least 20 contiguous amino acids), in particular at least 50 contiguous amino acids, such as at least 100 contiguous amino acids (for example at least 200 contiguous amino acids). Suitably the immunogenic fragments will be at least 20%, such as at least 50%, at least 70% or at least 80% of the length of the full length polypeptide sequence.
It will be understood that in a diverse out-bred population, such as humans, different HLA types mean that specific epitopes may not be recognised by all members of the population. Consequently, to maximise the level of recognition and scale of immune response to a polypeptide, it is generally desirable that an immunogenic fragment contains a plurality of the epitopes from the full length sequence (suitably all epitopes).
Variants
“Variants” or “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
In respect of variants of a protein sequence, the skilled person will recognise that individual substitutions, deletions or additions to polypeptide, which alters, adds or deletes a single amino acid or a small percentage of amino acids is a “conservatively modified variant” where the alteration(s) results in the substitution of an amino acid with a functionally similar amino acid or the substitution/deletion/addition of residues which do not substantially impact the biological function of the variant.
Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
A polypeptide of the invention (such as a CDTa protein or a CDTb protein) may contain a number of conservative substitutions (for example, 1-50, such as 1-25, in particular 1-10, and especially 1 amino acid residue(s) may be altered) when compared to the reference sequence. In general, such conservative substitutions will fall within one of the amino-acid groupings specified below, though in some circumstances other substitutions may be possible without substantially affecting the immunogenic properties of the antigen. The following eight groups each contain amino acids that are typically conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins 1984).
Suitably such substitutions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen.
Polypeptide variants may also include those wherein additional amino acids are inserted compared to the reference sequence, for example, such insertions may occur at 1-10 locations (such as 1-5 locations, suitably 1 or 2 locations, in particular 1 location) and may, for example, involve the addition of 50 or fewer amino acids at each location (such as 20 or fewer, in particular 10 or fewer, especially 5 or fewer). Suitably such insertions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen. One example of insertions includes a short stretch of histidine residues (e.g. 2-6 residues) to aid expression and/or purification of the antigen in question.
Polypeptide variants include those wherein amino acids have been deleted compared to the reference sequence, for example, such deletions may occur at 1-10 locations (such as 1-5 locations, suitably 1 or 2 locations, in particular 1 location) and may, for example, involve the deletion of 50 or fewer amino acids at each location (such as 20 or fewer, in particular 10 or fewer, especially 5 or fewer). Suitably such deletions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen.
The skilled person will recognise that a particular polypeptide variant may comprise substitutions, deletions and additions (or any combination thereof).
Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity (such as at least about 95%, at least about 98% or at least about 99%) to the associated reference sequence.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using, for example, one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least about 25 to about 50 amino acids or nucleotides in length, or optionally over a region that is 75-100 amino acids or nucleotides in length. Suitably, the comparison is performed over a window corresponding to the entire length of the reference sequence.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, references to a segment in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984).
Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
Polynucleotide Identification and Characterisation
Polynucleotides encoding the Clostridium difficile CDTa, CDTb, Toxin A and Toxin B proteins of the invention may be identified, prepared and/or manipulated using any of a variety of well established techniques. For example, a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs. Such screens may be performed, for example, using a Synteni microarray (Palo Alto, Calif.) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA 93:10614-10619 (1996) and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155 (1997)). Alternatively, polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as M. tuberculosis cells. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesised.
An amplified portion of a polynucleotide may be used to isolate a full length gene from a suitable library (e.g., a M. tuberculosis cDNA library) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5′ and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5′ sequences.
For hybridisation techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32P) using well known techniques. A bacterial or bacteriophage library is then generally screened by hybridising filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual (2000)). Hybridising colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones. The complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences can then be assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
Alternatively, there are numerous amplification techniques for obtaining a full length coding sequence from a partial cDNA sequence. Within such techniques, amplification is generally performed via PCR. Any of a variety of commercially available kits may be used to perform the amplification step. Primers may be designed using, for example, software well known in the art. Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68° C. to 72° C. The amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.
One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16:8186 (1988)), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularised by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as “rapid amplification of cDNA ends” or RACE. This technique involves the use of an internal primer and an external primer, which hybridises to a polyA region or vector sequence, to identify sequences that are 5′ and 3′ of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111-19 (1991)) and walking PCR (Parker et al., Nucl. Acids. Res. 19:3055-60 (1991)). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA sequences may also be obtained by analysis of genomic fragments.
Polynucleotide Expression in Host Cells
Polynucleotide sequences or fragments thereof which encode the Clostridium difficile CDTa, CDTb, Toxin A and Toxin B proteins, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
Moreover, the polynucleotide sequences can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. For example, DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.
Natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognised by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.
Sequences encoding a desired polypeptide may be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980), Horn et al., Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge et al., Science 269:202-204 (1995)) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).
A newly synthesised peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, Proteins, Structures and Molecular Principles (1983)) or other comparable techniques available in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
In order to express a desired polypeptide, the nucleotide sequences encoding the polypeptide, or functional equivalents, may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (2000), and Ausubel et al., Current Protocols in Molecular Biology (updated annually).
A variety of expression vector/host systems may be utilised to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 μlasmids); or animal cell systems.
The “control elements” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 μlasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.
In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509 (1989)); and the like. pGEX Vectors (Promega, Madison, Wis.; GE Healthcare.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
In the yeast, Saccharomyces cerevisiae or Pichia such as Pichia pastoris for example, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. Other vectors containing constitutive or inducible promoters include GAP, PGK, GAL and ADH. For reviews, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544 (1987) and Romas et al. Yeast 8 423-88 (1992).
In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, e.g., Hobbs in McGraw Hill Yearbook of Science and Technology pp. 191-196 (1992)).
An insect system may also be used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard et al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)).
In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. Methods and protocols for working with adenovirus vectors are reviewed in Wold, Adenovirus Methods and Protocols, 1998. Additional references regarding use of adenovirus vectors can be found in Adenovirus: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References, 2004.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf. et al., Results Probl. Cell Differ. 20:125-162 (1994)).
In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-32 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-23 (1990)) genes which can be employed in tk.sup.- or aprt.sup.-cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70 (1980)); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilise indole in place of tryptophan, or hisD, which allows cells to utilise histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51 (1988)). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
Alternatively, host cells which contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridisations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labelled hybridisation or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labelling or PCR amplification using a labelled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.) between the purification domain and the encoded polypeptide may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilised metal ion affinity chromatography) as described in Porath et al., Prot. Exp. Purif. 3:263-281 (1992) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll et al., DNA Cell Biol. 12:441-453 (1993)).
Polypeptide Compositions
Generally, a polypeptide of use in the invention (for example the Clostridium difficile CDTa, CDTb, Toxin A and Toxin B proteins) will be an isolated polypeptide (i.e. separated from those components with which it may usually be found in nature).
For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO. Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.
Polypeptides for use in the invention, immunogenic fragments thereof, and other variants having less than about 100 amino acids, and generally less than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesised using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146 (1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer's instructions.
Within certain specific embodiments, a polypeptide may be a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, e.g., Stoute et al., New Engl. J. Med. 336:86-91 (1997)). A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognised by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.
Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.
A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. 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 and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and 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. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
Adjuvants
In a further embodiment of any of the aspects of the invention, the immunogenic composition further comprises an adjuvant. In one embodiment the adjuvant comprises aluminium hydroxide or aluminium phosphate. Alternatively the immunogenic composition of the invention may comprise an aluminium-free adjuvant, the immunogenic composition is 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 immunogenic composition 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 another 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 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.
When the adjuvant comprises an immunologically active saponin fraction presented in the form of a liposome, the adjuvant may further comprise a sterol. Suitably the sterol is provided at a ratio of saponin:sterol of from 1:1 to 1:100 w/w, such as from 1:1 to 1:10 w/w; or 1:1 to 1:5 w/w.
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 lipsome-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 β-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 about 1-about 70 μg per dose, for example at an amount of about 50 μg.
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.
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.
In compositions comprising a lipopolysaccharide, the lipopolysaccharide may be present at an amount of about 1-about 70 μg per dose, for example at an amount of about 50 μg.
The lipopolysaccharide may be a non-toxic derivative of lipid A, particularly monophosphoryl lipid A or more particularly 3-Deacylated monophoshoryl lipid A (3D-MPL).
3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals S.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-γ (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 0.22 μm 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 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 i.e. the ratio of QS21:3D-MPL is 1:1. 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. In one embodiment the adjuvant comprises an oil in water emulsion, wherein the oil in water emulsion comprises a metabolisable oil, a tocol and an emulsifier. 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, Polysorbate® 80, 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 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 about 2.14 mg per 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 about 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 of the Invention
In one embodiment the immunogenic composition has a volume of 0.5 to 1.5 ml.
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 S. pneumoniae, H. influenzae, N. meningitidis, E. coli, M. catarrhalis, Clostridium tetani (tetanus), Corynebacterium diphtheria (diphtheria), Bordetella pertussis (pertussis), S. epidermidis, enterococci, S. aureus, and Pseudomonas aeruginosa. 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 an immunogenic composition of the invention and a pharmaceutically acceptable excipient.
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 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 host an immunoprotective dose of the immunogenic composition or vaccine or kit 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 host an immunoprotective dose of the immunogenic composition or vaccine or kit of the invention.
In one embodiment of the invention there is provided an immunogenic composition or vaccine of the invention for use in the treatment or prevention of C. difficile disease. In a further embodiment of the invention there is provided an immunogenic composition or vaccine of the invention for use in the treatment or prevention of disease caused by a strain of C. difficile selected from the group consisting of 078, 019, 023, 027, 033, 034, 036, 045, 058, 059, 063, 066, 075, 078, 080, 111, 112, 203, 250 and 571. Preferably the strain is strain 078.
In a further aspect of the invention there is provided a use of an immunogenic composition or vaccine of the invention in the preparation of a medicament for the prevention or treatment of C. difficile disease. In a further embodiment the disease is a disease caused by a strain of C. difficile selected from the group consisting of 078, 019, 023, 027, 033, 034, 036, 045, 058, 059, 063, 066, 075, 078, 080, 111, 112, 203, 250 and 571. Preferably the strain is strain 078.
In a further aspect of the invention there is provided a method of preventing or treating C. difficile disease comprising administering the immunogenic composition of the invention or the vaccine of the invention to a mammalian subject such as a human subject. In a further embodiment the disease is a disease caused by a strain of C. difficile selected from the group consisting of 078, 019, 023, 027, 033, 034, 036, 045, 058, 059, 063, 066, 075, 078, 080, 111, 112, 203, 250 and 571. Preferably the strain is strain 078.
General
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.”
The amino acid numbering used herein is derived from the sequences for CDTa, CDTb, Toxin A and Toxin B presented herein as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 31 and SEQ ID NO: 32 which are to be considered as reference sequences for these proteins.
Embodiments herein relating to “vaccine compositions” 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 approximate, and are provided for description. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, may be approximate.
All references or patent applications cited within this patent specification are incorporated by reference herein in their entirety.
In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.
EXAMPLES
The AS01B adjuvant referred to is an 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 mice contains 5 μg QS21, 5 μg 3D-MPL, 0.025 mg cholesterol and 0.1 mg DOPC.
Example 1: Design of Binary Toxin Antigens
The Binary Toxin (other name: ADP-ribosyltransferase toxin) is composed by two components: the enzymatic component, named CDTa and the transport and binding component, named CDTb.
Based on literature data and the known 3D structure of CDTa (J. Biol. Chem. 2009, vol. 284: 28713-28719), this protein could be divided into two domains. The N-terminal domain binds to CDTb and the C-terminal domain contains the enzymatic activity. Both domains are linked by a flexible peptide.
Based on literature data and information available for other B components of other bacterial binary toxins, CDTb could be divided into five domains. The first one is the prodomain, its cleavage by an enzyme having a chymotrypsin activity allows the heptamerization of the mature protein. The second domain allows the binding to CDTa. The third and fourth ones are involved in the oligomerisation and membrane insertion. Finally, the last domain is the host cell receptor binding domain.
Example 1a: Design of CDTa Antigens
In order to be allowed to work with CDTa and CDTb together, CDTa must be inactivated. Two possibilities of inactivation were evaluated. The first one is the design of CDTa mutants that abolish the enzymatic activity and the second one is the use of the N-terminal domain of CDTa alone. This latter domain allows the binding to CDTb and does not contain residue involved in the enzymatic activity.
The first set of mutants were designed based on literature information (Infection & Immunity, 2001, vol. 69: 6004-6011). Authors demonstrated that CDTa mutant proteins E428Q, E430Q, S388A and R345K have a significant reduced activity. Based on data shown in the publication, two mutations were preferred amongst the four: CDTa mutant E428Q and E430Q. In the publication, these mutants abolish completely the CDTa enzymatic activity. In order to rank these mutants, some structural analyses were performed for these residues: surface-accessibility of the residues glutamate 428 (E428) and glutamate 430 (E430), effect of their mutations on the surrounding 3D structure. Based on these analyses, the CDTa mutant E428Q was chosen as preferred mutation and the CDTa mutant E430Q was selected as second choice. A double mutant E428Q, E430Q was also generated in order to be sure that the enzymatic activity was abolished.
A second set of mutants was designed because the first cytotoxicity results, obtained with the first set of mutants, were not convincing.
In this second set, a CDTa mutant containing 7 mutations (including the two mutations already described) was designed. All these mutations were designed based on literature information (available for CDTa or its Clostridium perfringens homologs Ia) and 3D structure analysis. All mutated residues are located around the catalytic site of CDTa. These residues have been modified in order to avoid ligand or water molecule binding. This CDTa “super mutant” contains the mutations R345A, Q350A, N385A, R402A, S388F, E428Q and E430Q.
Based on this “super mutated” CDTa, two other CDTa mutated variants were evaluated in order to eliminate E428Q and E430Q mutations (construct C108 contains the E430Q mutation but not the mutation E428Q, construct C107 does not contain both mutations).
CDTa Nterminal Domain Alone
It was described in the literature (Infection & Immunity, 2001, vol. 69: 6004-6011) that the CDTa1-240 is the minimal CDTa fragment that still allows a binding to Ib (B component of binary toxin of Clostridium perfringens). This fragment will be tested in the lab but based on known 3D structure, it was suggested that this domain will probably not be optimal in term of correct folding of this domain of CDTa.
Antigen design was performed based on the known 3D structure (Protein Data Bank accession number: 2WN4, J. Biol. Chem., 2009, vol. 284: 28713-28719) to improve the expression and folding of an isolated CDTa N-terminal domain. On the 3D structure, a linker peptide of eight amino acids allows the separation between the N- and C-terminal domains of CDTa. Two isolated CDTa N-terminal domains were designed, the first one contains this flexible peptide and the second one not.
CDTa: Sequences Summary
A summary of all CDTa sequences is presented in table 1.










TABLE 1






Length




Name
(aa)*
Location
Comments







CDTa
463
 1-463
Full length of CDTa coming from 





strain R20291


CDTa′
421
44-463
CDTa without signal peptide (C34)


CDTa_E428Q
421
44-463
CDTa′ with mutation of Glu428 





into Gln (C44)


CDTa_E430Q
421
44-463
CDTa′ with mutation of Glu430 





into Gln (C54)


CDTa_E428 430Q
421
44-463
CDTa′ with the two mutations Glu428 





into Gln and Glu430 into Gln (C67)


CDTa_7mutations
421
44-463
CDTa′ containing 7 mutated amino 





acids (C69)


CDTa_N_litt
198
44-240
Minimum CDTa N-terminal domain 





that still allows binding to lb (C51)


CDTa_NADlink
226
44-268
CDTa N-terminal domain based on 





antigen design work (C49)


CDTa_NAD
218
44-260
CDTa N-terminal domain based on 





antigen design work (C50)





*Length contains additional N-terminal Methionine but not the His-tag






Example 1b: Design of CDTb Antigens
CDTb Mature
In order to avoid the chymotrypsin activation step in the CDTb process, it was tried to express only the mature CDTb protein (without its signal peptide and prodomain).
In the literature (Protein Expression and Purification, 2010, vol. 74: 42-48), the mature CDTb was described as starting at Leucine 210. This mature CDTb was named CDTb′′. After in house experimental data, it seems that the activated CDTb starts at Serine 212. This result was supported by analysis of a 3D modelised structure of CDTb. This model was built using SwissModel (Bioinformatics, 2006, vol. 22: 195-201). The template used for the homology modeling was the B component of Bacillus anthracis, named Protective Antigen or PA (Protein Data Bank accession number: 3TEW).
CDTb Receptor-Binding Domain Alone
Given the fact that a fusion containing only the receptor-binding domains of Toxin A and B is sufficient to induce neutralizing antibodies, it was decided to produce and evaluate the CDTb receptor-binding domain alone.
The 3D structure model obtained for CDTb is accurate for the four first domains of CDTb but not for the receptor-binding domain (these domains of CDTb and PA are too different). To design constructs expressing this domain alone, the C-terminal part of the fourth domain was analysed on the 3D structure model in order to decide where the last domain will start. Two versions of the CDTb-receptor binding domain were designed. In the first one, this domain starts just after the modelised 3D structure of the fourth domain. In this version, the CDTb-receptor-binding domain will probably have a long flexible peptide in its N-terminal part. The second version starts where the 2D predicted structures performed on the C-terminal part of CDTb (predictions done using Psipred program, Bioinformatics, 2000, vol. 16: 404-405) become more compacts after a lack of predicted secondary structures. This could indicate the beginning of a new structural domain. In this second version, no flexible peptide is present at the N-terminal part of the isolated CDTb receptor-binding domain.
CDTb Ca2+ Binding Motif Mutation
Following literature information, mutations in the Ca2+ binding domain of the B component of Iota toxin of Clostridium perfringens (Ib) abolish the binding with the A component of this binary toxin (Ia). These mutations could be very interesting in the case of a vaccine composition containing a mixture of mature CDTb protein and a wild type CDTa protein. Using multiple protein sequence alignment, these mutations were located on the CDTb sequence and mutated. It concerns residues Asp220, Asp222 and Asp224. They were mutated into Ala residues.
CDTb Prodomain
In order to try to decrease the degradation issues observed with C55 in gel, some co-expression tests were evaluated. The working hypothesis of doing that is to improve the folding of the mature CDTb.
Two limits of prodomain were proposed. The first one starts at residue 43 of CDTb (after the signal peptide cleavage) and finishes at residue Met211 (given that the experimentally determined first residue of the mature CDTb is Ser212). The second prodomain was designed based on the predicted 3D structure of CDTb. The linker existing between the prodomain and the first structural domain of the mature CDTb protein is removed in this construct.
CDTb: Sequence Summary
A summary of all CDTb sequences is presented in table 2.










TABLE 2





Name
Length (aa)*
Location
Comments







CDTb
876
 1-876
Full length of CDTb coming from strain





R20291


CDTb′
835
 43-876
CDTb without signal peptide (C38)


CDTb′′
668
210-876
CDTb without signal peptide and prodomain





as defined in the literature


CDTb′′_xp data
666
212-876
CDTb without signal peptide and prodomain,





as demonstrated by in house experimental





results (C55)


CDTbClg
258
620-876
CDTb receptor-binding domain containing





natural flexible peptide in its N-terminal part,





based on antigen design work (C52)


CDTbCsh
242
636-876
CDTb receptor-binding domain, based on





antigen design work (C53)


CDTb Ca2+
666
212-876
Mature CDTb (without signal peptide and


mutated


without prodomain) containing 3 mutations





D220A, D222A and D224A (C97)


CDTbprodomainLg
170
 43-211
CDTb prodomain (C58)


CDTbprodomainSh
145
 43-186
CDTb prodomain without the linker existing





between the prodomain and the first





structural domain of mature CDTb (C59)





*Length contains additional N-terminal Methionine but not the His-tag






Example 1c: Design of CDTa-CDTb Fusions
Background Information
The aim of these constructs is to obtain both components of the Binary Toxin into one process.
A lot of different kinds of fusions could be designed but, as proof of concept, the first fusion evaluated is the combination of CDTa N-terminal domain (named CDTaNADlink and CDTaNAD) with CDTb receptor-binding domain (named CDTbCsh and CDTbClg).
Fusion CDTaNterm-CDTb Receptor-Binding Domain Alone
Without additional experimental data on each partner of the fusion, all possible combinations were initiated but always with the CDTa domain as first partner of the fusion.
In these fusions, the CDTaNADlink and the CDTaNAD domains have two and one residues less than the designed isolated domains, respectively. These CDTa additional amino acids were kept in the isolated designs in order to avoid potential issues during the expression process.
A summary of all CDTa-CDTb fusion sequences is presented in table 3.









TABLE 3








Length
Location










Name
(aa)*
CDTa
CDTb





CDTaNADlink-CDTbCsh 
465
44-266
636-876


(C61)





CDTaNAD-CDTbCsh
458
44-259
636-876


(C63)





CDTaNADlink-CDTbClg
481
44-266
620-876


(C60)





CDTaNAD-CDTbClg
474
44-259
620-876


(C62)








*Length contains additional N-terminal Methionine but not the His-tag






Example 1d: Design of ToxA-ToxB-CDTb Receptor-Binding Domain Fusion
The objective of this fusion is the combination of receptor-binding domains of the three major toxins of Clostridium difficile into one construct.
Given the fact that F2 and CDTb receptor-binding domains are not supposed to adopt the same fold, a linker/spacer must be used between the two partners of the fusion in order to allow a correct independent folding of them. Two fusions were designed.
In the first one (named F2_CDTbCIg), the long designed version of the receptor-binding domain is fused at the C-terminal part of F2. In this version, the long flexible N-terminal peptide of CDTb receptor-binding domain will function as spacer.
In the second fusion (named F2_GG_NVCDTbCsh), the short designed version of the receptor-binding domain is fused at the C-terminal part of F2. In order to allow a correct folding of the two partners, the length of the linker created in this fusion must be increased. In order to do that, the CDTb-receptor binding domain was extended with two natural residues, moreover two exogenous Glycines were added between F2 and the longer version of CDTbCsh.
A summary of all F2-CDTb fusion sequences is presented in table 4.










TABLE 4








Length
Location












Name
(aa)*
F2
CDTb
Comments





F2_CDTbClg
1223
ToxA: 2121-
620-





2686 ToxB: 
876





1968-2366




F2_GG_NVCDTbCsh
1211
ToxA: 2121-
634-
Two exogenous 




2686 ToxB: 
876
Gly residues




1968-2366

added as spacer 






between the two






partners of the fusion





*Length contains additional N-terminal Methionine but not the His-tag






Example 2: Cloning, Expression and Purification of CdtA Protein
Expression Plasmid and Recombinant Strain: CdtA Full Length
Genes encoding the protein of full length without signal peptide of CdtA with and without mutations (see tables below) and a His tag in C-term were cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures. Final constructs were generated by the transformation of E. coli strain HMS174 (DE3) or BLR (DE3) pLysS (C34) with each recombinant expression vector separately 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.).














CdtA




C number









C34
CdtA (aa44-463)



C44
CdtA (aa44-463) mut. E428Q



C49
CdtA linker (44-268)



C50
CdtA WO linker (44-260)



C54
CdtA (aa44-463) mut. E430Q



C67
CdtA (aa44-463) mut. E428Q-E430Q



C68
CdtA (aa44-463) mut. R345A-Q350A-




N385A-R402A-E428Q-E430Q



C69
CdtA (aa44-463) mut. R345A-Q350A-




N385A-R402A-S388F-E428Q-E430Q



C107
CdtA (aa44-463) mut. R345A-Q350A-




N385A-R402A-S388F



C108
CdtA (aa44-463) mut. R345A-Q350A-




N385A-R402A-S388F-E430Q



C110
CdtA (aa44-463) mut. R345A-Q350A-




N385A-R402A-S388F-E428Q










Host Strain:
HMS 174 (DE3). HMS174 strains provide the recA mutation in a K-12 background. 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
Genotype: F− recA1 hsdR(rK12−mK12+) (RifR).
BLR(DE3) pLysS. 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, pLysS strains express T7 lysozyme which further suppress basal expression of the T7 RNA polymerase prior to induction.
Genotype: E. coli BLR::DE3 strain, F− ompT hsdSB(rB− mB−) gal dcm (DE3) Δ(srl-recA)306::Tn10 pLysS (CamR, TetR).
Expression of the Recombinant Proteins:
E. coli transformants were 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.
Each overnight culture were 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 cultures were 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 were evaluated after induction and culture were centrifuged at 14 000 RPM for 15 minutes and pellets were frozen at −20° C. separately.
Expression Plasmid and Recombinant Strain: CdtA-N-Term
Genes encoding protein of N-terminal, without signal peptide of CdtA (see tables below) and a His tag in C-term were cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures. Final constructs were generated by the transformation of E. coli strain HMS174 (DE3) with each recombinant expression vectors separately 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.).










CdtA N-term










C number








C49
CdtA linker (44-268)



C50
CdtA WO linker (44-260)










Host Strain:
HMS 174 (DE3). HMS174 strains provide the recA mutation in a K-12 background. 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
Genotype: F− recA1 hsdR(rK12−mK12+) (RifR).
Expression of the Recombinant Proteins:
E. coli transformants were 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 following procedure was used to purify constructs C34, C44, C49, C50, C54, C67, C69, C107 and C110.
The bacterial pellets were re-suspended in 20 mM or 50 mM bicine buffers (pH 7.5 or pH 8.0), containing 500 mM NaCl, 0 mM or 5 mM TCEP (Thermo Scientific Pierce, (2-carboxyethyl)phosphine hydrochloride) and a mixture of protease inhibitors (Complete, Roche, without EDTA). Bacteria were lysed using a French Press system 3×20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation at 20 000 g for 30 min at 4° C.
The 6-His tagged-proteins were purified under native conditions on IMAC. The soluble components were loaded on a 5 ml GE Histrap column (GE) pre-equilibrated with the same buffer used to bacterial re-suspension. After loading on the column, the column was washed with a 20 mM or 50 mM bicine buffer (pH7.5 or pH8.0), containing 500 mM NaCl, 10 mM imidazole, 5 mM TCEP. Elution was performed using a 50 mM bicine buffer pH7.6, 500 mM NaCl, 1 mM TCEP and imidazole (250 mM or 500 mM).
After desalting (BIORAD Bio-Gel P6 Desalting) and concentration (Amicon Ultra 10 kDa) steps, the product was loaded on SEC chromatography (SUPERDEXTM 75 or 200) in 20 mM or 50 mM bicine buffer (pH7.5 or pH8.0), 150 mM NaCl, 1 mM TCEP, for further purification step.
Fractions containing Cdta antigen were selected on the basis of purity by SDS-PAGE. Protein concentration was determined using Lowry RC/DC Protein Assay of BioRad. The purified bulk was sterile-filtered on 0.22 μm and stored at −80° C.
Example 4: Cloning, Expression and Purification of C. difficile CdtB Protein
Expression Plasmid and Recombinant Strain: CdtB Full Length.
Genes encoding the truncated protein of CdtB without signal peptide (Pro-CdtB′) and a His tag in C-term were cloned into the pGEX-6p1 expression vector (GE Healthcare) using the BamHI/XhoI restriction sites using standard procedures. This vector included GST (Gluthathione-S-transferase) as fusion partner in N-terminal of either CdtB′(GST-Pro-Cdtb′). The final construct was generated by the transformation of E. coli strain BL21 (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.).
Genes encoding the truncated protein of CdtB without signal peptide (Pro-CdtB′: C38) and without signal peptide and prodomain (CdtB′′: C40 or C55) and a His tag in C-term were cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures. Final constructs were generated by the transformation of E. coli B834 (DE3) modified strain for C55 and BL21(DE3) for C38 and C40 with the appropriate recombinant expression vectors 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.).














CdtB




C number









C37
CdtB′ Δ signal sequence (aa43-




876) + GST N-term



C38
CdtB′ Δ signal sequence (aa43-




876)



C40
CdtB′′ Δ signal sequence and




prodomain (aa210-876)



C55
CdtB Δ prodomain (aa. 212-876)










Host Strain
BL21(DE3). BL21(DE3) is a non-methionine auxotroph derivative of B834. 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 BL21::DE3 strain, F− ompT hsdSB(rB−mB−) gal dcm (DE3).
B834 is the parental strain for BL21. These protease-deficient hosts are methionine auxotrophs. λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.
Modification: Including PGL gene to avoid phosphogluconoylation in the biotin locus (Strain is auxotroph for biotin).
Genotype: B834::DE3 strain, F-ompT hsdSB(rB-mB-) gal dcm met (DE3)
Modification: Δ(bioA-bioD)::PGL
Expression of the Recombinant Proteins:
E. coli transformants were stripped from agar plate and used to inoculate 200 ml of LBT broth ±1% (w/v) glucose+/−kanamycin (50 μg/ml) or ampicillin (100 μg/ml) to obtain O.D.600nm between 0.1-0.2. Cultures were incubated overnight at 37° C., 250 RPM.
Overnight culture were diluted to 1:20 in 500 ml of LBT medium containing +/−kanamycin (50 μg/ml) or ampicillin (100 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, cultures were 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 inductions (around 16 hours), O.D. at 600 nm were evaluated after induction and cultures were centrifuged at 14 000 RPM for 15 minutes and pellets were frozen at −20° C. separately.
Expression Plasmid and Recombinant Strain.
Genes encoding the truncated protein of CdtB without prodomain CdtB mature, Knock-out of Ca++ binding site (inhibit binding of CdtA to CdtB) and a His tag in C-term was 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 B834 (DE3) modified strain 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.).










CdtB Ca++ binding domain knock-out










C number








C97
CdtB mature mut Asp 220-222-224 Ala




(aa. 212-876)










Host Strain
B834 is the parental strain for BL21. These protease-deficient hosts are methionine auxotrophs. λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.
Modification: Including PGL gene to avoid phosphogluconoylation in the biotin locus (Strain is auxotroph for biotin).
Genotype: B834::DE3 strain, F-ompT hsdSB(rB-mB-) gal dcm met (DE3)
Modification: Δ(bioA-bioD)::PGL
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
C37
The bacterial pellet was re-suspended in 50 mM bicine buffer (pH 8.0) containing 500 mM NaCl, 5 mM TCEP (Thermo Scientific Pierce, (2-carboxyethyl)phosphine hydrochloride) and a mixture of protease inhibitor (Complete, Roche). Bacteria were lysed using a French Press system
3×20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation 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 5 ml GE Histrap column (GE) pre-equilibrated with the same buffer used to bacterial re-suspension. After loading on the column, the column was washed with a 50 mM bicine buffer pH8.0, containing 150 mM NaCl, 25 mM imidazole, 1 mM TCEP. Elution was performed using a 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 250 mM imidazole, 1 mM TCEP.
After desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM bicine buffer pH8.0 containing
150 mM NaCl and 1 mM TCEP, the product was treated (overnight at 4° C.) with PreScission protease (GE-Healthcare) in order to cleave the GST tag. After overnight treatment, 0.2% Tween 20 was added to the digestion mixture.
Then the protein was passed through a GST affinity column (GE GSTrap FF) pre-equilibrated with buffer 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 1 mM TCEP, 0.2% tween20 and 20 mM reduced glutation, in order to remove the cleaved tag, un-cleaved fusion protein and the PreScission protease.
The GST-free protein was collected in the flow through and loaded again on a 5 ml GE Histrap column (GE) pre-equilibrated with 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 1 mM TCEP, 0.2% tween20. After loading on the column, the column was washed with a 50 mM bicine buffer pH8.0, containing 150 mM NaCl, 0.2% tween20, 1 mM TCEP and 10 mM imidazole. Elution was performed using a 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 0.2% tween20, 1 mM TCEP and 500 mM imidazole.
After desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 1 mM TCEP and 0.2% tween 20, the product was treated with α-chymotrypsin (from bovine pancreas—Sigma), followed by trypsin inhibitor treatment (from egg white—Sigma). The complete activation of Cdtb by chymotrypsin was monitored by SDS-PAGE.
The fully activated product was loaded on SEC chromatography (SUPERDEXTM 75) in 50 mM bicine buffer pH8.0 containing 300 mM NaCl, 1 mM TCEP. Fractions containing CdtB antigen were selected on the basis of purity by SDS-PAGE. Protein concentration was determined using Lowry RC/DC Protein Assay of BioRad. The purified bulk was sterile-filtered on 0.22 μm and stored at −80° C.
C38
The bacterial pellet was re-suspended in 50 mM bicine buffer (pH 8.0) containing 150 mM NaCl, 5 mM TCEP (Thermo Scientific Pierce, (2-carboxyethyl)phosphine hydrochloride), 0.4% empigen and a mixture of protease inhibitor (Complete, Roche). Bacteria were lysed using a French Press system 3×20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation 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 5 ml GE Histrap column (GE) pre-equilibrated with 50 mM bicine buffer (pH 8.0) containing 150 mM NaCl, 1 mM TCEP (Thermo Scientific Pierce, (2-carboxyethyl)phosphine hydrochloride) and 0.15% empigen. After loading on the column, the column was washed with a 50 mM bicine buffer pH8.0, containing 150 mM NaCl, 20 mM imidazole, 1 mM TCEP and 0.2% tween 20. Elution was performed using a 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 500 mM imidazole, 1 mM TCEP and 0.2% tween 20.
After desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 1 mM TCEP and 0.2% tween 20, the product was treated with α-chymotrypsin (from bovine pancreas—Sigma), followed by trypsin inhibitor treatment (from egg white—Sigma). The complete activation of Cdb by chymotrypsin was monitored by SDS-PAGE.
The fully activated product was loaded on SEC chromatography (SUPERDEXTM 75) in 50 mM bicine buffer pH8.0, 300 mM NaCl, 1 mM TCEP. Fractions containing Cdtb protein were selected on the basis of purity by SDS-PAGE and loaded again on a 5 ml GE Histrap column (GE) pre-equilibrated with 50 mM bicine buffer (pH 8.0) containing 300 mM NaCl, 1 mM TCEP. After loading on the column, the column was washed with a 50 mM bicine buffer pH8.0, containing 300 mM NaCl, 10 mM imidazole, 1 mM TCEP. Elution was performed using a 50 mM bicine buffer pH8.0 containing 300 mM NaCl, 500 mM imidazole, 1 mM TCEP.
After desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM bicine buffer pH8.0 containing 300 mM NaCl, 1 mM TCEP the protein concentration was determined using Lowry RC/DC Protein Assay of BioRad. The purified bulk was sterile-filtered on 0.22 μm and stored at −80° C.
C40
The bacterial pellet was re-suspended in 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl, 5 mMCaCl2 and a mixture of protease inhibitor (Complete, Roche). Bacteria were lysed using a French Press system 3×20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation 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 1 ml GE Histrap column (GE) pre-equilibrated with 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl, 5 mMCaCl2. After loading on the column, the column was washed with a 20 mM bicine buffer pH8.0, containing 500 mM NaCl, 5 mM CaCl2 and 5 mM imidazole. Elution was performed using a 20 mM bicine buffer pH8.0 containing 150 mM NaCl, 5 mM CaCl2 and 250 mM imidazole.
After desalting step (BIORAD Bio-Gel P6 Desalting) in 20 mM bicine buffer pH8.0 containing 150 mM NaCl, 1 mM TCEP the product was loaded on SEC chromatography (SUPERDEXTM 75) in the same buffer. Fractions containing Cdtb antigen were selected on the basis of purity by SDS-PAGE. Protein concentration was determined using Lowry RC/DC Protein Assay of BioRad. The purified bulk was sterile-filtered on 0.22 μm and stored at −80° C.
C55
The bacterial pellet was re-suspended in 50 mM bicine buffer (pH 8.0) containing 150 mM NaCl,
5 mM TCEP (Thermo Scientific Pierce, (2-carboxyethyl) phosphine hydrochloride), 0.4% empigen and a mixture of protease inhibitors (Complete, Roche). Bacteria were lysed using a French Press system 3×20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation 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 5 ml GE Histrap column (GE) pre-equilibrated with 50 mM bicine buffer (pH 8.0) containing 150 mM NaCl, 0.15% empigen, 1 mM TCEP. After loading on the column, the column was washed with a 50 mM bicine buffer pH8.0, containing 150 mM NaCl, 0.2% tween 20, 20 mM imidazole and 1 mM TCEP. Elution was performed using a 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 0.2% tween 20, 500 mM imidazole and 1 mM TCEP.
After desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM bicine buffer pH8.0 containing 300 mM NaCl, 1 mM TCEP the product was loaded on SEC chromatography (SUPERDEXTM 75) in the same buffer. Fractions containing Cdtb antigen were selected on the basis of purity by SDS-PAGE. Protein concentration was determined using Lowry RC/DC Protein Assay of BioRad. The purified bulk was sterile-filtered on 0.22 μm and stored at −80° C.
Expression of the Recombinant Proteins: CdtB Receptor Binding Domain:
Expression Plasmid and Recombinant Strain.
Genes encoding the truncated protein of CdtB only recptor binding domain (C52-C53) and a His tag in C-term was cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures. The final constructs were generated by the transformation of E. coli B834 (DE3) modified strain 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
B834 is the parental strain for BL21. These protease-deficient hosts are methionine auxotrophs. λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.
Modification: Including PGL gene to avoid phosphogluconoylation in the biotin locus (Strain is auxotroph for biotin).
Genotype: B834::DE3 strain, F-ompT hsdSB(rB-mB-) gal dcm met (DE3)
Modification: Δ(bioA-bioD)::PGL










CdtB Rec. Bdng domain










C number








C52
CdtB receptor binding domain




long (aa. 620-876)



C53
CdtB receptor binding domain




short (aa. 636-876)










Expression of the Recombinant Proteins:
A E. coli transformants were 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.
These overnight cultures were 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 cultures were 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 inductions (around 16 hours), O.D. at 600 nm were evaluated after induction and cultures were centrifuged at 14 000 RPM for 15 minutes and pellets were frozen at −20° C. separately.
Purification
C52 and C53
The bacterial pellets were re-suspended in 50 mM bicine buffer pH 8.0, containing 500 mM NaCl and a mixture of protease inhibitors (Complete, Roche, without EDTA). Bacteria were lysed using a French Press system 3×20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation at 20 000 g for 30 min at 4° C.
The 6-His tagged-proteins were purified under native conditions on IMAC. The soluble components were loaded on a 5 ml GE Histrap column (GE) pre-equilibrated with the same buffer used to bacterial re-suspension. After loading on the column, the column was washed with a 20 mM bicine buffer
pH7.5 containing 500 mM NaCl, 25 mM imidazole. Elution was performed using a 50 mM bicine buffer pH7.5, 500 mM NaCl, and 250 mM imidazole.
After desalting (BIORAD Bio-Gel P6 Desalting) and concentration (Amicon Ultra 10 kDa) steps, the product was loaded on SEC chromatography (SUPERDEXTM 75) in 20 mM buffer pH7.5,
150 mM NaCl.
Fractions containing Cdtb antigen were selected on the basis of purity by SDS-PAGE. Protein concentration was determined using Lowry RC/DC Protein Assay of BioRad. The purified bulk was sterile-filtered on 0.22 μm and stored at −80° C.
Example 6: Cloning, Expression and Purification of C. difficile CdtA N-Term and CdtB Receptor Binding Domain Fusion Proteins
Expression Plasmid and Recombinant Strain.
Genes encoding the fusion protein of CdtA N-term (C49 or C50) with CdtB receptor binding domain protein long or short version (C61 or C62) and a His tag in C-term were cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures. The final constructs were generated by the transformation of E. coli B834 (DE3) modified strain with the appropriate 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.).










Fusion CdtA N-term/CdtB-receptor binding domain










C number








C61
CdtA N-term link (aa. 44-268)-




CdtB RBD short (aa. 636-876)



C62
CdtA N-term (aa. 44-260)-CdtB




RBD long (aa. 621-876)










Host Strain
Genotype: E. coli BL21::DE3 strain, F− ompT hsdSB(rB−mB−) gal dcm (DE3).
B834 is the parental strain for BL21. These protease-deficient hosts are methionine auxotrophs. λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.
Modification: Including PGL gene to avoid phosphogluconoylation in the biotin locus (Strain is auxotroph for biotin).
[016] Genotype: B834::DE3 strain, F-ompT hsdSB(rB-mB-) gal dcm met (DE3)
Modification: Δ(bioA-bioD)::PGL
Expression of the Recombinant Proteins:
E. coli transformants were stripped from each 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.
These overnight cultures were 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 cultures were 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
C61
The bacterial pellet was re-suspended in 50 mM bicine buffer (pH 8.0) containing 300 mM NaCl, 5 mM TCEP (Thermo Scientific Pierce, (2-carboxyethyl) phosphine hydrochloride), 0.4% empigen and a mixture of protease inhibitors (Complete, Roche). Bacteria were lysed using a French Press system 3×20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation 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 5 ml GE Histrap column (GE) pre-equilibrated with 50 mM bicine buffer (pH 8.0) containing 300 mM NaCl, 0.15% empigen, 1 mM TCEP. After loading on the column, the column was washed with a 50 mM bicine buffer pH8.0, containing 300 mM NaCl, 0.2% tween 20, 25 mM imidazole and 1 mM TCEP. Elution was performed using a 50 mM bicine buffer pH8.0 containing 150 mM NaCl, 0.2% tween 20, 500 mM imidazole and 1 mM TCEP.
After desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM bicine buffer pH8.0 containing 300 mM NaCl, 1 mM TCEP the product was loaded on SEC chromatography (SUPERDEXTM 200) in the same buffer. Fractions containing the recombinant antigen were selected on the basis of purity by SDS-PAGE. Protein concentration was determined using Lowry RC/DC Protein Assay of BioRad. The purified bulk was sterile-filtered on 0.22 μm and stored at −80° C.
Example 7: Cloning and Expression of C. difficile CdtB Mature Co-Expressed (C55) with Prodomain of CdtB C58
Expression Plasmid and Recombinant Strain.
Genes encoding protein pro domain of CdtB without His tag was cloned into the pET21b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures. The final construct was generated by the transformation of E. coli B834 (DE3) modified strain with the recombinant expression vector of prodomain CdtB and CdtB mature protein C55—information about cloning of C55 see example 3 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.).










Pro domain of CdtB alone










C number








C58
Pro-domaine CdtB long (aa. 43-211)










Host Strain
B834 is the parental strain for BL21. These protease-deficient hosts are methionine auxotrophs. λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.
Modification: Including PGL gene to avoid phosphogluconoylation in the biotin locus (Strain is auxotroph for biotin).
[016] Genotype: B834::DE3 strain, F-ompT hsdSB(rB-mB-) gal dcm met (DE3)
Modification: Δ(bioA-bioD)::PGL
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) and ampicillin (100 μg/ml) to obtain O.D.600nm between 0.1-0.2. Culture was 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 ampicillin (100 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
same as C55 produced alone
Example 8: Molecular Weight Evaluation of CdtA, CdtB and CdtA-CdtB Fusion Constructions
Analytical ultracentrifugation was 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 ProteomeLab XL-1 analytical ultracentrifuge at 8000 RPM, 25000 RPM or 42000 RPM depending of the target protein size, after the AN-60Ti rotor had been equilibrated to 15° C.
2. For data collection, 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. Determination of the molecular weight of the different species has been 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.
FIGS. 1a to 1h describe the size distribution of the different CdtA. CdtB and CdtA-CdtB fusion constructions as determined by sedimentation velocity analytical ultracentrifugation.
The calculated molecular weight of the main species for C67 and C69 mutated full length CdtA protein may correspond with a monomer, while the C50 truncated CdtA N-terminal construction is present in solution as a mix of monomer and dimer (FIGS. 1a, 1b and 1c).
Both C61 and C62 fusions of CdtA-CdtB are mainly dimeric, with a minor proportion of monomer (FIGS. 1d and 1e).
Constructions of the CdtB receptor binding domain C52 and C52 are mainly dimeric with presence of small amount of monomer (FIGS. 1f and 1g).
Full length CdtB without prodomain C55 is highly aggregated after purification, presenting a heterogeneous size distribution by AUC (FIG. 1h).
Example 9: SDS PAGE Profile of CdtA, CdtB and CdtA-CdtB Fusion Constructions after Purification
Purified proteins from each construction were separated on denaturing and reducing SDS PAGE in order to assess the sequence integrity.
FIG. 2a shows that CdtA-CdtB fusion constructions C61 and C62 are present in majority at the expected molecular weight. Same observations are made for CdtA constructions on FIG. 2b. 
It is shown on FIG. 2c that chymotrypsin activation of C37 CdtB (aa. 43-876) construction results in the truncation of the prodomain for the obtention of a protein (lane 2) at a molecular weight comparable to the mature CdtB represented at the lane 3 by C55 (aa. 212-876). SDS PAGE profile of C55 contains significant amount of secondary products that couldn't be separated from the complete protein, which is coherent with the highly aggregated profile observed by AUC on FIG. 2h. 
CdtB expressed with prodomain C38 (aa. 43-876) was purified as a an heterogeneous preparation composed at the majority of a doublet of the expected molecular weight containing a significant amount of secondary products.
Example 9: Immunisation of Mice with C. difficile CdtA and CdtB Sub-Units Proteins in a AS01B Formulation
Mice Immunisation
Groups of 25 female Balb/C mice were immunized IM at days 0, 14 and 28 with 5 μg of full CdtA and CdtB binary toxin purified sub-units. These antigens were injected in an AS01B formulation.
Anti-CdtA and anti-CdtB ELISA titers were determined in individual sera collected at day 42 (Post III 14). Results are shown in FIGS. 3-4.
A binary toxin cytotoxicity inhibition assay was also performed on pooled Post III sera (day42). Results are shown in FIGS. 5-6
Anti-CdtA and Anti-CdtB ELISA Response: Protocol
Full CdtA (C34) or full CdtB (C37)sub-units were coated at 1 μg/ml (for CdtA) or 2 μg/ml (for CdtB) 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 1/500 in PBS-BSA0.2%-TWEENTM 0.05%. and then, further twofold dilutions were made in microplates and incubated at RT for min. After washing, bound mouse antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated Anti-Mouse (ref: 110-035-003) diluted 1:5000 in PBS-BSA0.2%-tween 0.05%. The detection antibodies were incubated for 30 min. at room temperature (RT) with agitation. The color was developed using 4 mg O-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-CdtA or anti-CdtB antibodies are expressed in mid-point titers. A GMT was calculated for the 25 samples in each treatment group.
Binary Toxin Cytotoxicity Inhibition Assay
Human colonic eptithelial cells (HT29 or HCT-116 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 black tissue culture plates (Greiner Bio-one, Ref: 655090) at a density of 4.104 cells/well for HT29 and 1.104 cells/well for HCT116. After 24 h, the cell media was removed from the wells.
The mice anti-sera were prediluted 1:50 in cell media and then, further three-fold dilutions were made in microplate (NUNC, Ref: 163320). 50 μl of serial dilutions of mice pooled antisera were added to the black plates. 50 μl of a mix of CdtA (25 ng/ml) and chemotrypsin-activated CdtB (75 ng/ml) were then added and the black plates incubated at 37° C. with 5% CO2 for 6 days.
After 6 days, the mix of antisera and toxin were removed from the wells and 100 μl of Hoescht stain (BD Pharmingen, Ref: 561908) diluted 1:500 in phosphate buffer saline (PBS) was added in each well for 2 hours in the dark at room temperature.
After coloration, the Hoescht stain was removed from the wells and the cells fluorescence cells was measured using an Axiovision microscope.
The surface covered by fluorescent staining was determined in each well and cytotoxicity inhibition titers were defined as the reciprocal dilution inducing a 50% inhibition of the fluorescent signal.
Example 10: Immunisation of Mice with C. difficile CdtB Chemotrypsin-Activated or not, Mixed with F2 or not, Formulated in AS01B
Mice Immunisation
Groups of 25 female Balb/C mice were immunized IM at days 0, 14 and 28 with 5 μg of CdtB binary toxin purified sub-unit chemotrypsin-activated or not, mixed with 5 μg of F2 or not. These antigens were injected in an AS01B formulation.
Anti-CdtB, anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 42 (Post III 14). Results are shown in FIGS. 7-9.
A binary toxin, ToxA and ToxB cytotoxicity inhibition assay was also performed on pooled Post III sera (day42). Results are shown in FIGS. 10-12.
Anti-CdtB, Anti-ToxA and Anti-ToxB ELISA Response: Protocol
Full CdtB (C37)sub-unit, F2 Cter ToxA and F2 Cter ToxB were coated at 0.5 μg/ml (for CdtB), 2 μg/ml (for ToxA F2 Cter) and 1 μg/ml (for ToxB F2 Cter) 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 1/500 in PBS-BSA0.2%-TWEENTM 0.05%. and then, further twofold dilutions were made in microplates and incubated at RT for 30 min. After washing, bound mouse antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated Anti-Mouse (ref: 110-035-003) diluted 1:5000 in PBS-BSA0.2%-tween 0.05%. The detection antibodies were incubated for 30 min. at room temperature (RT) 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-CdtB antibodies are expressed in mid-point titers.
The level of anti-F2Cter ToxA and F2Cter ToxB antibodies present in each individual sera is determined by comparison to a reference serum added on each plate and expressed in μg/ml.
A GMT was calculated for the 25 samples in each treatment group.
Binary Toxin, ToxA and ToxB Cytotoxicity Inhibition Assay
Human colonic eptithelial cells (HT29 or HCT-116 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 black tissue culture plates (Greiner Bio-one, Ref: 655090) at a density of 4.104 cells/well for HT29 and 1.104 cells/well for HCT116.
After 24 h, the cell media was removed from the wells.
The mice anti-sera were prediluted 1:5 for g1 (CdtB non-activated) and g2 (CdtB activated) and 1:20 for g3 (CdtB non-activated+F2) and g4 (Cdtb activated+F2) in cell media, for ToxA inhibition cytotoxicity assay, 1:10 for ToxB inhibition cytotoxicity assay and 1:50 for binary toxin inhibition assay. Then, further three-fold dilutions were made in microplate (NUNC, Ref: 163320). 50 μl of serial dilutions of mice pooled antisera were added to the black plates. 50 μl of ToxA (0.01 μg/ml) on HT29, ToxB (0.022 μg/ml) on HCT116 and a mix of CdtA (25 ng/ml) and chemotrypsin-activated CdtB (75 ng/ml) on HT29 and HCT116 were then added in the black plates and incubated at 37° C. with 5% CO2 for 6 days.
After 6 days, the mix of antisera and toxin were removed from the wells and 100 μl of Hoescht stain (BD Pharmingen, Ref: 561908) diluted 1:500 in phosphate buffer saline (PBS) was added in each well for 2 hours in the dark at room temperature.
After coloration, the Hoescht stain was removed from the wells and the cells fluorescence cells was measured using an Axiovision microscope.
The surface covered by fluorescent staining was determined in each well and cytotoxicity inhibition titers were defined as the reciprocal dilution inducing a 50% inhibition of the fluorescent signal.
Example 11: Immunisation of Mice with Different Binary Toxin Vaccine Candidates (CdtA/CdtB) Combined with F2 at 6 μg/Dose in an AS01B Formulation
Mice Immunisation
Groups of 20 female Balb/C mice were immunized IM at days 0, 14 and 28 with 6 μg of CdtA-CdtB fusion (C61 and C62), or 3 μg of CdtA (C34, C50 or C67) and/or 3 μg of CdtB (C37, C52, C55 or C55/C58) mixed or not with 6 μg of F2. These antigens were injected in an AS01B formulation.
Anti-CdtA, anti-CdtB, anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 42 (Post III 14). Results are shown in FIGS. 13-16.
A binary toxin, ToxA and ToxB cytotoxicity inhibition assay was also performed on pooled Post III sera (day42). Results are shown in FIGS. 17-20.
Anti-CdtA, Anti-CdtB, Anti-ToxA F2Cter and Anti-ToxB F2 Cter ELISA Response: Protocol
CdtA mut E428Q (C44), Full CdtB (C37)sub-units, F2 Cter ToxA and F2 Cter ToxB were coated at 1 μg/ml (for CdtA), 0.5 μg/ml (for CdtB), 2 μg/ml (for ToxA F2 Cter) and 1 μg/ml (for ToxB F2 Cter) 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 were prediluted 1:100 (for CdtA, CdtB, ToxB) or 1:200 (for ToxA) for Post II and 1:500 (for CdtA and ToxA), 1:500 or 1:2000 (for CdtB) and 1:250 (for ToxB) for Post III in PBS-BSA0.2%-TWEENTM 0.05%. Then, further twofold dilutions were made in microplates and incubated at RT for 30 min. After washing, bound mouse antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated Anti-Mouse (ref: 110-035-003) diluted 1:5000 in PBS-BSA0.2%-tween 0.05%. The detection antibodies were incubated for 30 min. at room temperature (RT) with agitation. The color was developed using 4 mg O-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-CdtA, anti-CdtB, anti-F2Cter ToxA and F2Cter ToxB antibodies present in each individual sera is determined by comparison to a reference serum added on each plate and expressed in μg/ml. A GMT was calculated for the 20 samples in each treatment group.
Binary Toxin, ToxA and ToxB Cytotoxicity Inhibition Assay
Human colonic eptithelial cells (HT29 or HCT-116 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 black tissue culture plates (Greiner Bio-one, Ref: 655090) at a density of 4.104 cells/well for HT29 and 1.104 cells/well for HCT116. After 24 h, the cell media was removed from the wells.
The mice anti-sera were prediluted 1:50 in cell media, for ToxA inhibition cytotoxicity assay, 1:10 for ToxB inhibition cytotoxicity assay, 1:50 for binary toxin inhibition assay on HT29 and 1:30 (for Post II) and 1:30 or 1:100 (for Post III) for binary toxin inhibition assay on HCT116. Then, further three-fold dilutions were made in microplate (NUNC, Ref: 163320). 50 μl of serial dilutions of mice pooled antisera were added to the black plates. 50 μl of ToxA (0.025 μg/ml) on HT29, ToxB (0.6 μg/ml) on HCT116 and a mix of CdtA (25 ng/ml) and chemotrypsin-activated CdtB (75 ng/ml) on HT29 and HCT116 were then added in the black plates and incubated at 37° C. with 5% CO2 for 6 days.
After 6 days, the mix of antisera and toxin were removed from the wells and 100 μl of Hoescht stain (BD Pharmingen, Ref: 561908) diluted 1:500 in phosphate buffer saline (PBS) was added in each well for 2 hours in the dark at room temperature.
After coloration, the Hoescht stain was removed from the wells and the cells fluorescence cells was measured using an Axiovision microscope.
The surface covered by fluorescent staining was determined in each well and cytotoxicity inhibition titers were defined as the reciprocal dilution inducing a 50% inhibition of the fluorescent signal.
Example 12: Immunisation of Mice with Different Binary Toxin Vaccine Candidates (CdtA/CdtB) Combined with F2 at 2 m/Dose in an AS01B Formulation
Mice Immunisation
Groups of 20 female Balb/C mice were immunized IM at days 0, 14 and 28 with 2 μg of CdtA-CdtB fusion (C61 and C62), or 1 μg of CdtA (C34, C50 or C67) and/or 1 μg of CdtB (C37, C52, C55 or C55/C58) mixed or not with 2 μg of F2. These antigens were injected in an AS01B formulation.
Anti-CdtA, anti-CdtB, anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 42 (Post III 14). Results are shown in FIGS. 21-24.
A binary toxin, ToxA and ToxB cytotoxicity inhibition assay was also performed on pooled Post III sera (day42). Results are shown in FIGS. 25-28.
Anti-CdtA, Anti-CdtB, Anti-ToxA and Anti-ToxB ELISA Response: Protocol
CdtA mut E428Q (C44), Full CdtB (C37)sub-units, F2 Cter ToxA and F2 Cter ToxB were coated at 1 μg/ml (for CdtA), 0.5 μg/ml (for CdtB), 2 μg/ml (for ToxA F2 Cter) and 1 μg/ml (for ToxB F2 Cter) 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 were prediluted 1:100 (for CdtB, ToxA, ToxB) and 1:100 or 1:250 (for CdtA) for Post II and 1:500 for Post III in PBS-BSA0.2%-TWEENTM 0.05%. Then, further twofold dilutions were made in microplates and incubated at RT for 30 min. After washing, bound mouse antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated Anti-Mouse (ref: 110-035-003) diluted 1:5000 in PBS-BSA0.2%-tween 0.05%. The detection antibodies were incubated for 30 min. at room temperature (RT) with agitation. The color was developed.
Example 13: Immunisation of Mice with Different Binary Toxin Vaccine Candidates (CdtA/CdtB) Combined with F2 at 10 μg/Dose in a Non-Adjuvanted Formulation
Mice Immunisation
Groups of 20 female Balb/C mice were immunized IM at days 0, 14 and 28 with 10 μg of CdtA-CdtB fusion (C61 and C62), or 5 μg of CdtA (C34, C50 or C67) and/or 5 μg of CdtB (C37, C52, C55 or C55/C58) mixed or not with 10 μg of F2. These antigens were injected in a non-adjuvanted formulation.
Anti-CdtA, anti-CdtB, anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 42 (Post III 14). Results are shown in FIGS. 29-32.
A binary toxin, ToxA and ToxB cytotoxicity inhibition assay was also performed on pooled Post III sera (day42). Results are shown in FIGS. 33-36.
Anti-CdtA, Anti-CdtB, Anti-ToxA and Anti-ToxB ELISA Response: Protocol
CdtA mut E428Q (C44), Full CdtB (C37)sub-units, F2 Cter ToxA and F2 Cter ToxB were coated at 1 μg/ml (for CdtA), 0.5 μg/ml (for CdtB), 2 μg/ml (for F2 Cter ToxA) and 1 μg/ml (for F2 Cter ToxB) 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 were prediluted 1:100 (for CdtA, CdtB, ToxA, ToxB) for Post II and 1:100 (for CdtA, ToxA, ToxB), 1:100 or 1:200 (for CdtB) for Post III in PBS-BSA0.2%-TWEENTM 0.05%. Then, further twofold dilutions were made in microplates and incubated at RT for 30 min. After washing, bound mouse antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated Anti-Mouse (ref: 110-035-003) diluted 1:5000 in PBS-BSA0.2%-tween 0.05%. The detection antibodies were incubated for 30 min. at room temperature (RT) 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.
A GMT was calculated for the 20 samples in each treatment group.
Binary Toxin, ToxA and ToxB Cytotoxicity Inhibition Assay: Protocol
Human colonic eptithelial cells (HT29 or HCT-116 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 black tissue culture plates (Greiner Bio-one, Ref: 655090) at a density of 4.104 cells/well for HT29 and 1.104 cells/well for HCT116.
After 24 h, the cell media was removed from the wells.
The mice anti-sera were prediluted 1:50 in cell media, for ToxA inhibition cytotoxicity assay, 1:10 for ToxB inhibition cytotoxicity assay, 1:50 for binary toxin inhibition assay on HT29 and 1:30 (for Post II) and 1:30 or 1:100 (for Post III) for binary toxin inhibition assay on HCT116. Then, further three-fold dilutions were made in microplate (NUNC, Ref: 163320). 50 μl of serial dilutions of mice pooled antisera were added to the black plates. 50 μl of ToxA (0.025 μg/ml) on HT29, ToxB (0.6 μg/ml) on HCT116 and a mix of CdtA (25 ng/ml) and chemotrypsin-activated CdtB (75 ng/ml) on HT29 and HCT116 were then added in the black plates and incubated at 37° C. with 5% CO2 for 6 days.
After 6 days, the mix of antisera and toxin were removed from the wells and 100 μl of Hoescht stain (BD Pharmingen, Ref: 561908) diluted 1:500 in phosphate buffer saline (PBS) was added in each well for 2 hours in the dark at room temperature.
After coloration, the Hoescht stain was removed from the wells and the cells fluorescence cells was measured using an Axiovision microscope.
The surface covered by fluorescent staining was determined in each well and cytotoxicity inhibition titers were defined as the reciprocal dilution inducing a 50% inhibition of the fluorescent signal.
Example 14: Cloning and Expression of C. difficile F2 and CdtB Receptor Binding Domain Fusion Proteins
Expression Plasmid and Recombinant Strain.
Genes encoding the fusion protein of F2 protein with CdtB receptor binding domain protein long or short version (C64 and C65) and a His tag in C-term were cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI restriction sites using standard procedures.










Fusion F2/CdtB-receptor binding domain










C number








C64
CdtA N-term (aa. 44-260)-CdtB RBD long 




(aa. 621-876)



C65
F2-CdtB RBD short (aa. 636-876)




















Sequence Summary (Table A)











Construct

Polynucleotide


Description
reference
Amino acid sequence
sequence





CDTa full length (strain R20291)
N/A
SEQ. I.D. NO: 1
SEQ. I.D. NO: 2


CDTb full length (strain R20291)
N/A
SEQ. I.D. NO: 3
SEQ. I.D. NO: 4


CDTa without signal peptide
C34
SEQ. I.D. NO: 5
SEQ. I.D. NO: 6


CDTb′ (minus signal peptide)
C37
SEQ. I.D. NO: 7
SEQ. I.D. NO: 8


ligated to Glutathione-S-transferase





protein. (GST underlined)





CDTb′′ (minus pro-domain and
C40
SEQ. I.D. NO: 9
N/A


signal peptide)





CDTa mutation E428Q
C44
SEQ. I.D. NO: 10
SEQ. I.D. NO: 11


CDTa mutation E430Q
C54
SEQ. I.D. NO: 12
N/A


CDTa N terminal domain (residue
Gülke et
SEQ. I.D. NO: 13
N/A


44 to residue 240)
al 2001




CDTa without signal peptide, with a
C49
SEQ. I.D. NO: 14
N/A


linker between the N-term domain





and the C-term domain (containing





the enzymatic activity). This





construct covers the fragment from





amino acid 44 to aa 268.





CDTa without signal peptide or
C50
SEQ. I.D. NO: 15



linker. This construct covers the





fragment from aa 44 to aa 260.





CDTb minus signal peptide (CDTb′)
C38
SEQ ID NO: 16
SEQ ID NO: 17


Fusion 1
F1
SEQ ID NO: 18



Fusion 2
F2
SEQ ID NO: 19



Fusion 3
F3
SEQ ID NO: 20



Fusion 4
F4
SEQ ID NO: 21



Fusion 5
F5
SEQ ID NO: 22



Fusion F54 Gly
N/A
SEQ ID NO: 24
SEQ ID NO: 23


Fusion F54 New
N/A
SEQ ID NO: 26
SEQ ID NO: 25


Fusion F5 ToxB
N/A
SEQ ID NO: 28
SEQ ID NO: 27


Fusion F52 New
N/A
SEQ ID NO: 30
SEQ ID NO: 29


Toxin A
N/A
SEQ ID NO: 31



Toxin B
N/A
SEQ ID NO: 32



CDTb′′ (minus pro-domain and
C39
SEQ ID NO: 33
N/A


signal peptide) ligated to





Glutathione-S-transferase protein.





CdtB receptor binding domain with 
C52
SEQ ID NO: 34
SEQ ID NO: 35


linker in N-term of sequence, from





aa 620-876





CdtB receptor binding domain
C53
SEQ ID NO: 36
SEQ ID NO: 37


without linker in N-term of





sequence, from aa 636-876





CDTb with prodomain removed
C55
SEQ ID NO: 51



(CDTb′′, aa212-876)





CDTb prodomain sequence (long,
C58
SEQ ID NO: 38
N/A


aa43-211)





CDTb prodomain sequence (short,
C59
SEQ ID NO: 39
N/A


aa43-186)





Fusion CDTa N-term with linker
C60
SEQ ID NO: 40
N/A


(aa44-268) to CDTb receptor





binding domain with linker in N term





of sequence (aa621-876)





Fusion CDTa N-term with linker
C61
SEQ ID NO: 41
N/A


(aa44-268) to CDTb receptor





binding domain without linker in N





term of sequence (aa636-876)





Fusion CDTa N-term without linker 
C62
SEQ ID NO: 42
N/A


(aa44-260) to CDTb receptor





binding domain with linker in N term





of sequence (aa621-876)





Fusion CDTa N-term without linker 
C63
SEQ ID NO: 43
N/A


(aa44-260) to CDTb receptor





binding domain without linker in N





term of sequence (aa636-876)





Fusion F2-CDTb receptor binding
C64
SEQ ID NO: 44
N/A


domain with linker in N term of





sequence (aa621-876)





Fusion of F2 to CDTb receptor
C65
SEQ ID NO: 45
N/A


binding domain without linker in N





term of sequence (aa636-876) with





2 heterogeneous Gly residues





between F2 and CTDb sequences





CDTa without signal peptide, with
C67
SEQ ID NO: 46
SEQ ID NO: 47


two mutations (E428Q, E430Q, aa





44-463).





CDTa without signal peptide, with
C69
SEQ ID NO: 48
SEQ ID NO: 49


seven mutations (R345A, Q350A,





N385A, R402A, S388F, E428Q,





E430Q, aa 44-463).





CDTb without signal sequence and
C97
SEQ ID NO: 50
N/A


prodomain (mature fragment based





on MS data) with Ca2+ binding





motif mutation (aa212-876, mut





Asp-9-11-13 Ala)





CDTa without signal peptide, with
C107
SEQ ID NO: 52
SEQ ID NO: 53


five mutations (R345A, Q350A,





N385A, R402A, S388F, aa 44-463).





CDTa without signal peptide, with
C108
SEQ ID NO: 54
SEQ ID NO: 55


six mutations (R345A, Q350A,





N385A, R402A, S388F, E430Q, aa





44-463).





CdtA without signal peptide, with six 
C110
SEQ ID NO: 56
N/A


mutations (R345A-Q350A-N385A-





R402A-S388F-E428Q, aa 44-463).

















SEQUENCE LISTING



SEQ ID 1



- CDTa full length polypeptide sequence



MKKFRKHKRISNCISILLILYLTLGGLLPNNIYAQDLQSYSEKVCNTTYKAPIERPEDFLKDKE


 


KAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQTRNYFYDYQIEANSREKEYKEL


 


RNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLEKFNEFKETIQNKLFKQDGFKDIS


 


LYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIEQGYSIKIDKIVRIVIDGKHYIKAE


 


ASVVSSLDFKDDVSKGDSWGKANYNDWSNKLTPNELADVNDYMRGGYTAINNYLISNGPV


 


NNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEFGLTLTSPEYDFNKLENIDAFKSKWE


 


GQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGSPGAYLSAIPGYAGEYEVLLNHGSKFKI


 


NKIDSYKDGTITKLIVDATLIP


 


SEQ ID 2



- CDTa full length polynucleotide sequence



ATGAAAAAATTTAGGAAACATAAAAGGATTAGTAATTGTATATCTATATTGTTGATATTAT


 


ATCTAACTTTAGGTGGTTTGTTACCTAATAACATTTATGCACAAGACTTACAAAGCTATA


 


GTGAAAAAGTTTGCAATACTACTTACAAGGCTCCTATAGAAAGACCAGAAGATTTTCTTA


 


AAGATAAAGAAAAGGCTAAAGAATGGGAAAGAAAAGAAGCAGAAAGAATAGAGCAAAAA


 


CTTGAAAGATCTGAAAAAGAAGCATTAGAATCATATAAAAAAGATTCTGTAGAAATAAGT


 


AAATATTCTCAGACAAGAAATTATTTTTATGATTATCAAATAGAAGCAAATTCTCGAGAAA


 


AAGAATATAAAGAACTTCGAAATGCTATATCAAAAAATAAAATAGATAAACCTATGTATGT


 


CTATTATTTTGAATCTCCAGAAAAATTTGCATTTAATAAAGTAATAAGAACAGAAAATCAA


 


AACGAAATTTCATTAGAAAAATTTAATGAGTTTAAAGAAACTATACAAAACAAATTATTTA


 


AGCAAGATGGATTTAAAGATATTTCTTTATATGAACCTGGAAAAGGTGATGAAAAACCTA


 


CACCATTACTTATGCACTTAAAATTACCTAGAAATACTGGTATGTTACCATATACAAATAC


 


TAACAATGTAAGTACATTAATAGAGCAAGGATATAGTATAAAAATAGATAAAATTGTTCGT


 


ATAGTTATAGATGGGAAGCACTATATTAAAGCAGAAGCATCTGTTGTAAGTAGTCTTGAT


 


TTTAAAGATGATGTAAGTAAGGGGGATTCTTGGGGTAAAGCAAATTATAATGATTGGAG


 


TAATAAATTAACACCTAATGAACTTGCTGATGTAAATGATTATATGCGTGGAGGATATAC


 


TGCAATTAATAATTATTTAATATCAAATGGTCCAGTAAATAATCCTAACCCAGAATTAGAT


 


TCTAAAATCACAAACATTGAAAATGCATTAAAACGTGAACCTATTCCAACTAATTTAACTG


 


TATATAGAAGATCTGGTCCTCAAGAATTTGGTTTAACTCTTACTTCCCCTGAATATGATTT


 


TAACAAACTAGAAAATATAGATGCTTTTAAATCAAAATGGGAAGGACAAGCACTGTCTTA


 


TCCAAACTTTATTAGTACTAGTATTGGTAGTGTGAATATGAGTGCATTTGCTAAAAGAAA


 


AATAGTACTACGTATAACTATACCTAAAGGTTCTCCTGGAGCTTATCTATCAGCTATTCC


 


AGGTTATGCAGGTGAATATGAAGTGCTTTTAAATCATGGAAGCAAATTTAAAATCAATAA


 


AATTGATTCTTACAAAGATGGTACTATAACAAAATTAATTGTTGATGCAACATTGATACCT


 


TAA


 


SEQ ID 3



- CDTb full length polypeptide sequence



MKIQMRNKKVLSFLTLTAIVSQALVYPVYAQTSTSNHSNKKKEIVNEDILPNNGLMGYYFTDE


 


HFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTLSTDRDDVLM


 


QVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELDGMKKIIPEENLFLR


 


DYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFA


 


EQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIISTN


 


EHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNG


 


ESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLS


 


PGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQI


 


VTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKK


 


DGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDY


 


NNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKI


 


KAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEV


 


KIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFK


 


DIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVD


 


SEQ ID 4



- CDTb full length polynucleotide sequence



ATGAAAATACAAATGAGGAATAAAAAGGTATTAAGTTTTTTAACACTTACAGCTATAGTTA


 


GTCAAGCACTAGTATATCCTGTATATGCTCAAACTAGTACAAGTAATCATTCTAATAAGA


 


AAAAAGAAATTGTAAATGAAGATATACTCCCAAACAATGGATTAATGGGATATTATTTCA


 


CAGATGAGCACTTTAAAGATTTAAAATTAATGGCACCCATAAAAGATGGTAATTTAAAAT


 


TTGAAGAAAAGAAAGTAGATAAACTTCTGGATAAAGACAAATCAGATGTAAAATCTATAC


 


GATGGACAGGAAGAATAATTCCTTCTAAGGATGGTGAATATACATTATCAACTGATAGA


 


GATGATGTCTTAATGCAAGTAAATACTGAGAGTACTATATCAAATACACTTAAAGTTAATA


 


TGAAAAAGGGTAAAGAATATAAAGTTAGAATAGAGCTACAAGATAAAAATTTAGGTTCAA


 


TAGATAATTTATCATCACCTAATCTTTATTGGGAATTAGATGGTATGAAGAAAATTATACC


 


AGAAGAAAATTTATTCTTAAGAGATTATTCTAATATAGAAAAAGATGATCCATTTATCCCA


 


AATAACAATTTCTTTGACCCAAAGTTGATGTCTGATTGGGAAGACGAAGATTTGGATACA


 


GATAATGATAATATACCAGATTCATATGAACGAAATGGATATACTATTAAGGACTTAATT


 


GCAGTTAAGTGGGAAGATAGTTTTGCAGAACAAGGCTATAAGAAATATGTATCAAATTAT


 


TTAGAGTCAAATACTGCTGGAGATCCATATACAGATTATGAAAAAGCTTCAGGTTCTTTT


 


GACAAGGCTATAAAGACTGAAGCAAGAGATCCGTTAGTTGCAGCATATCCAATTGTTGG


 


AGTAGGTATGGAAAAATTAATTATATCTACAAATGAACATGCCTCTACTGATCAAGGTAA


 


AACTGTTTCCAGAGCTACTACTAACAGTAAAACTGAATCTAATACAGCTGGTGTGTCTGT


 


TAATGTAGGATATCAAAATGGATTCACAGCTAATGTAACTACAAATTATTCCCATACAAC


 


AGATAATTCAACTGCTGTTCAAGATAGTAATGGAGAATCATGGAATACTGGATTAAGTAT


 


AAACAAAGGAGAATCTGCATATATAAATGCAAATGTTAGATATTACAACACAGGTACTGC


 


ACCTATGTACAAAGTGACACCAACAACAAATTTAGTGTTAGATGGAGATACATTATCAAC


 


TATCAAAGCACAAGAAAATCAAATTGGCAATAATCTATCTCCTGGAGATACTTATCCCAA


 


AAAAGGGCTTTCACCTCTAGCTCTTAACACAATGGATCAATTTAGCTCTAGACTGATTCC


 


TATAAATTATGATCAATTAAAAAAATTAGATGCTGGAAAGCAAATTAAATTAGAAACAACA


 


CAAGTAAGTGGAAATTTTGGTACAAAAAATAGTTCTGGACAAATAGTAACAGAAGGAAAT


 


AGTTGGTCAGACTATATAAGTCAAATTGACAGTATTTCTGCATCTATTATATTAGATACAG


 


AGAATGAATCTTACGAAAGAAGAGTTACTGCTAAAAATTTACAGGATCCAGAAGATAAAA


 


CACCTGAACTTACAATTGGAGAAGCAATTGAAAAAGCTTTTGGCGCTACTAAAAAAGAT


 


GGTTTGTTATATTTTAATGATATACCAATAGATGAAAGTTGTGTTGAACTCATATTTGATG


 


ATAATACAGCCAATAAGATTAAAGATAGTTTAAAAACTTTGTCTGATAAAAAGATATATAA


 


TGTTAAACTTGAAAGAGGAATGAATATACTTATAAAAACACCAACTTACTTTACTAATTTT


 


GATGATTATAATAATTACCCTAGTACATGGAGTAATGTCAATACTACGAATCAAGATGGT


 


TTACAAGGCTCAGCAAATAAATTAAATGGTGAGACGAAGATTAAAATCCCTATGTCTGAG


 


CTAAAACCTTATAAACGTTATGTTTTTAGTGGATATTCAAAGGATCCTTTAACATCTAATT


 


CAATAATTGTAAAGATAAAAGCAAAAGAAGAGAAAACGGATTATTTGGTACCAGAACAA


 


GGATATACAAAATTTAGTTATGAATTTGAAACTACTGAAAAAGATTCTTCTAATATAGAGA


 


TAACATTAATTGGTAGTGGTACAACATACTTAGATAACTTATCTATTACAGAGCTAAATAG


 


TACTCCTGAAATACTTGATGAACCAGAAGTTAAAATTCCAACTGACCAAGAAATAATGGA


 


TGCACATAAAATATATTTTGCAGATTTAAATTTTAATCCAAGTACAGGAAATACTTATATA


 


AATGGTATGTATTTTGCACCAACACAAACTAATAAAGAAGCTCTCGATTATATCCAAAAA


 


TATAGAGTTGAAGCTACTTTACAATATTCTGGATTTAAAGATATTGGAACTAAAGATAAA


 


GAAATGCGTAATTATTTAGGAGATCCAAATCAGCCTAAAACTAATTATGTTAATCTTAGG


 


AGTTATTTTACAGGTGGAGAAAATATTATGACATACAAGAAATTAAGAATATATGCAATTA


 


CTCCAGACGATAGAGAGTTATTAGTTCTTAGTGTTGATTAG


 


SEQ ID 5



- CDTa C34 construct polypeptide sequence



MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLE


 


KFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIE


 


QGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDVSKGDSWGKANYNDWSNKLTPNELAD


 


VNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEFGLT


 


LTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGSPGAYL


 


SAIPGYAGEYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP


 


SEQ ID 6



- CDTb C34 construct polynucleotide sequence



ATGGTTTGCAATACCACCTATAAAGCACCGATTGAACGTCCGGAAGATTTTCTGAAAGA


 


TAAAGAAAAAGCCAAAGAATGGGAACGCAAAGAAGCAGAACGTATTGAACAGAAACTG


 


GAACGTAGCGAAAAAGAAGCACTGGAAAGCTACAAAAAAGATAGCGTGGAAATTTCAAA


 


ATATAGCCAGACCCGCAATTATTTCTATGATTATCAGATTGAAGCCAATAGCCGTGAAAA


 


AGAATATAAAGAACTGCGCAATGCCATTAGCAAAAACAAAATTGATAAACCGATGTATGT


 


GTATTATTTCGAAAGTCCGGAAAAATTTGCCTTTAACAAAGTGATTCGCACCGAAAATCA


 


GAATGAAATTAGCCTGGAAAAATTCAATGAATTTAAAGAAACCATTCAGAATAAACTGTT


 


TAAACAGGATGGCTTTAAAGATATTTCACTGTATGAACCGGGTAAAGGTGATGAAAAAC


 


CGACACCGCTGCTGATGCATCTGAAACTGCCTCGTAATACCGGTATGCTGCCGTATAC


 


CAATACCAATAATGTTAGCACCCTGATTGAACAGGGCTATAGCATCAAAATTGATAAAAT


 


TGTGCGCATTGTGATTGATGGCAAACATTATATCAAAGCCGAAGCCAGCGTTGTTTCAA


 


GCCTGGATTTTAAAGATGATGTGAGCAAAGGCGATAGCTGGGGTAAAGCAAACTATAAT


 


GATTGGAGCAATAAACTGACCCCGAATGAACTGGCAGATGTGAATGATTATATGCGTGG


 


TGGTTATACCGCCATTAACAATTATCTGATTAGCAATGGTCCGGTGAATAATCCGAATCC


 


GGAACTGGATAGCAAAATTACCAATATTGAAAATGCCCTGAAACGCGAACCGATTCCGA


 


CCAATCTGACCGTTTATCGTCGTAGCGGTCCGCAAGAATTTGGTCTGACCCTGACCAGT


 


CCGGAATATGACTTTAACAAACTGGAAAATATTGATGCCTTTAAAAGCAAATGGGAAGG


 


TCAGGCACTGAGCTATCCGAACTTTATTAGCACCAGCATTGGTAGCGTTAATATGAGCG


 


CATTTGCCAAACGTAAAATTGTGCTGCGTATTACCATTCCGAAAGGTAGTCCGGGTGCA


 


TATCTGAGCGCAATTCCGGGTTATGCCGGTGAATATGAAGTTCTGCTGAATCATGGCAG


 


CAAATTCAAAATTAACAAAATTGATAGCTATAAAGATGGCACCATTACCAAACTGATTGT


 


TGATGCAACCCTGATTCCGTAA


 


SEQ ID 7



- CDTb C37 construct. CDTb′ (minus signal peptide)



ligated to Glutathione-S-transferase protein (GST


underlined) polypeptide sequence.


MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD


 


VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL


 


PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIP


 


QIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSHMEIVNEDILPNNGLMG


 


YYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTLSTDR


 


DDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELDGMKKIIPE


 


ENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVK


 


WEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEARDPLVAAYPIVGVGME


 


KLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNSTA


 


VQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQEN


 


QIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGT


 


KNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEK


 


AFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTY


 


FTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPL


 


TSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTP


 


EILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEA


 


TLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDREL


 


LVLSVD


 


SEQ ID 8



- CDTb C37construct. CDTb′ (minus pro-domain) ligated to Glutathione-



S-transferase protein (GST underlined) polynucleotide sequence.


atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaag


 


agcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatatt


 


gatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagag


 


cgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactct


 


caaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgt


 


aacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttt


 


taaaaaacgtattgaagctatcccacaaattgataagtacttaaaatccagcaagtatatagcatggcctttgcagggctggcaa


 


gccacgtttggtggtggcgaccatcctccaaaatcggatctggaagttctgttccaggggcccctgggatcccatatggaaattgtg


 


aatgaagatattctgccgaataatggtctgatgggatactactttaccgatgaacattttaaagatctgaaactgatggcaccgatta


 


aagatggcaatctgaaatttgaagaaaaaaaagtggataaactgctggataaagataaaagtgatgtgaaaagcattcgttgg


 


accggtcgtattattccgagcaaagatggtgaatacaccctgagcaccgatcgtgatgatgttctgatgcaggttaataccgaaag


 


caccattagcaataccctgaaagtgaatatgaaaaaaggcaaagaatataaagtgcgcattgaactgcaggataaaaatctgg


 


gtagcattgataatctgagcagcccgaatctgtattgggaactggatggtatgaaaaaaatcattccggaagaaaacctgtttctg


 


cgcgattatagcaatattgaaaaagatgatccgtttattccgaataataacttttttgatccgaaactgatgagcgattgggaagatg


 


aagatctggataccgataatgataatattccggatagctatgaacgcaatggctataccattaaagatctgattgccgtgaaatgg


 


gaagatagctttgcagaacagggctataagaaatatgtgagcaattatctggaaagcaataccgcaggcgatccgtataccgat


 


tatgaaaaagcaagcggcagctttgataaagccattaaaaccgaagcacgtgatccgctggttgcagcatatccgattgttggtg


 


ttggtatggaaaaactgattattagcaccaatgaacatgcaagcaccgatcagggtaaaaccgttagccgtgcaaccaccaata


 


gcaaaaccgaaagcaatacagccggtgttagcgttaatgttggttatcagaatggttttaccgccaatgtgaccaccaattatagc


 


cataccaccgataatagcaccgcagttcaggatagcaatggtgaaagctggaataccggtctgagcattaacaaaggtgaaa


 


gcgcatatatcaatgccaatgtgcgctattataacaccggcaccgcaccgatgtataaagttaccccgaccaccaatctggttctg


 


gatggtgataccctgagtaccattaaagcacaagaaaatcagattggcaataatctgagtccgggtgatacctatccgaaaaaa


 


ggtctgagtccgctggcactgaataccatggatcagtttagcagccgtctgattccgattaactatgatcagctgaaaaaactggat


 


gccggtaaacaaatcaaactggaaaccacccaggttagcggtaattttggcaccaaaaattcaagcggtcagattgttaccgaa


 


ggtaatagctggtcagattatatcagccagattgatagcattagcgccagcattattctggatacagaaaatgaaagctatgaacg


 


tcgtgtgaccgcaaaaaatctgcaggacccggaagataaaacaccggaactgaccattggtgaagcaattgaaaaagcattt


 


ggtgccaccaaaaaagatggcctgctgtattttaacgatattccgattgatgaaagctgcgtggaactgatttttgatgataataccg


 


ccaataaaatcaaagatagcctgaaaaccctgagcgacaaaaaaatctataatgtgaaactggaacgcggtatgaatattctg


 


attaaaaccccgacctattttaccaattttgatgattataacaattatccgagcacttggagcaatgtgaataccaccaatcaggatg


 


gtctgcagggtagcgcaaataaactgaatggtgaaaccaaaatcaaaattccgatgagcgaactgaaaccgtataaacgttat


 


gtgtttagcggctatagcaaagatccgctgaccagcaatagcattattgtgaaaatcaaagccaaagaagaaaaaaccgattat


 


ctggttccggaacagggttataccaaatttagctatgaatttgaaaccaccgaaaaagatagcagtaatattgaaattaccctgatt


 


ggtagcggcaccacctatctggataatctgagtattaccgaactgaatagcacaccggaaattctggatgaaccggaagtgaaa


 


attccgaccgatcaagaaattatggatgcccataaaatctattttgccgatctgaactttaatccgagcaccggcaatacctatatta


 


acggcatgtattttgcaccgacccagaccaataaagaagccctggattatattcagaaatatcgtgttgaagccaccctgcagtat


 


agcggttttaaagatattggcaccaaagataaagaaatgcgtaattatctgggcgatccgaatcagccgaaaaccaattatgtta


 


atctgcgcagctattttaccggtggcgaaaacattatgacctacaaaaaactgcgcatttatgccattacaccggatgatcgtgaa


 


ctgctggttctgagcgttgattaa


 


SEQ ID 9



- CDTb C40 construct. CDTb′′ (minus pro-domain and



signal peptide) polypeptide sequence.


LMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDP


 


YTDYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTE


 


SNTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANV


 


RYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQ


 


FSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIIL


 


DTENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDN


 


TANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQG


 


SANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSY


 


EFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFN


 


PSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPK


 


TNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVD


 


SEQ ID 10



- C44 construct. CDTa mutation E428Q polypeptide sequence.



MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLE


 


KFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIE


 


QGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDVSKGDSWGKANYNDWSNKLTPNELAD


 


VNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEFGLT


 


LTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGSPGAYL


 


SAIPGYAGqYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP


 


SEQ ID 11



- CDTa mutation E428Q polynucleotide sequence.



atggtttgcaataccacctataaagcaccgattgaacgtccggaagattttctgaaagataaagaaaaagccaaagaatggga


 


acgcaaagaagcagaacgtattgaacagaaactggaacgtagcgaaaaagaagcactggaaagctacaaaaaagatagc


 


gtggaaatttcaaaatatagccagacccgcaattatttctatgattatcagattgaagccaatagccgtgaaaaagaatataaaga


 


actgcgcaatgccattagcaaaaacaaaattgataaaccgatgtatgtgtattatttcgaaagtccggaaaaatttgcctttaacaa


 


agtgattcgcaccgaaaatcagaatgaaattagcctggaaaaattcaatgaatttaaagaaaccattcagaataaactgtttaaa


 


caggatggctttaaagatatttcactgtatgaaccgggtaaaggtgatgaaaaaccgacaccgctgctgatgcatctgaaactgc


 


ctcgtaataccggtatgctgccgtataccaataccaataatgttagcaccctgattgaacagggctatagcatcaaaattgataaa


 


attgtgcgcattgtgattgatggcaaacattatatcaaagccgaagccagcgttgtttcaagcctggattttaaagatgatgtgagca


 


aaggcgatagctggggtaaagcaaactataatgattggagcaataaactgaccccgaatgaactggcagatgtgaatgattat


 


atgcgtggtggttataccgccattaacaattatctgattagcaatggtccggtgaataatccgaatccggaactggatagcaaaatt


 


accaatattgaaaatgccctgaaacgcgaaccgattccgaccaatctgaccgtttatcgtcgtagcggtccgcaagaatttggtct


 


gaccctgaccagtccggaatatgactttaacaaactggaaaatattgatgcctttaaaagcaaatgggaaggtcaggcactgag


 


ctatccgaactttattagcaccagcattggtagcgttaatatgagcgcatttgccaaacgtaaaattgtgctgcgtattaccattccga


 


aaggtagtccgggtgcatatctgagcgcaattccgggttatgccggtCaatatgaagttctgctgaatcatggcagcaaattcaaa


 


attaacaaaattgatagctataaagatggcaccattaccaaactgattgttgatgcaaccctgattccgtaa


 


SEQ ID 12



- C54 construct. CDTa mutation E430Q polypeptide



sequence.


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLE


 


KFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIE


 


QGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDVSKGDSWGKANYNDWSNKLTPNELAD


 


VNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEFGLT


 


LTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGSPGAYL


 


SAIPGYAGEYqVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP


 


SEQ ID 13



- CDTa N terminal domain (residue 44 to residue 240)



polypeptide sequence.


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLE


 


KFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIE


 


QGYSIKIDKI


 


SEQ ID 14



- C49 construct. CDTa Nterminal domain without signal



peptide, with the linker existing between the N-term


domain and the C-term domain (containing the enzymatic


activity). This construct covers the fragment from amino


acid 44 to aa 268 polypeptide sequence.


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLE


 


KFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIE


 


QGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDVS


 


SEQ ID 15



- C50 construct. CDTa without signal peptide and the



linker existing between the N terminal and C terminal


domains of CDTa. This construct covers the fragment


from aa 44 to aa 260 polypeptide sequence.


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLE


 


KFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIE


 


QGYSIKIDKIVRIVIDGKHYIKAEASVVSS


 


SEQ ID NO: 16



- Polypeptide sequence of CDTb with pro-domain removed



(CDTb′)


EIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTG


 


RIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSP


 


NLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIPDS


 


YERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEAR


 


DPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA


 


NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNL


 


VLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGK


 


QIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQD


 


PEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYN


 


VKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELK


 


PYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGS


 


GTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQ


 


TNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIM


 


TYKKLRIYAITPDDRELLVLSVD


 


SEQ ID NO: 17



- Polypeptide sequence of CDTb with pro-domain removed (CDTb′)



catatggaaattgtgaatgaagatattctgccgaataatggtctgatgggatactactttaccgatgaacattttaaagatctgaaac


 


tgatggcaccgattaaagatggcaatctgaaatttgaagaaaaaaaagtggataaactgctggataaagataaaagtgatgtg


 


aaaagcattcgttggaccggtcgtattattccgagcaaagatggtgaatacaccctgagcaccgatcgtgatgatgttctgatgca


 


ggttaataccgaaagcaccattagcaataccctgaaagtgaatatgaaaaaaggcaaagaatataaagtgcgcattgaactgc


 


aggataaaaatctgggtagcattgataatctgagcagcccgaatctgtattgggaactggatggtatgaaaaaaatcattccgga


 


agaaaacctgtttctgcgcgattatagcaatattgaaaaagatgatccgtttattccgaataataacttttttgatccgaaactgatga


 


gcgattgggaagatgaagatctggataccgataatgataatattccggatagctatgaacgcaatggctataccattaaagatctg


 


attgccgtgaaatgggaagatagctttgcagaacagggctataagaaatatgtgagcaattatctggaaagcaataccgcaggc


 


gatccgtataccgattatgaaaaagcaagcggcagctttgataaagccattaaaaccgaagcacgtgatccgctggttgcagca


 


tatccgattgttggtgttggtatggaaaaactgattattagcaccaatgaacatgcaagcaccgatcagggtaaaaccgttagccg


 


tgcaaccaccaatagcaaaaccgaaagcaatacagccggtgttagcgttaatgttggttatcagaatggttttaccgccaatgtga


 


ccaccaattatagccataccaccgataatagcaccgcagttcaggatagcaatggtgaaagctggaataccggtctgagcatta


 


acaaaggtgaaagcgcatatatcaatgccaatgtgcgctattataacaccggcaccgcaccgatgtataaagttaccccgacca


 


ccaatctggttctggatggtgataccctgagtaccattaaagcacaagaaaatcagattggcaataatctgagtccgggtgatacc


 


tatccgaaaaaaggtctgagtccgctggcactgaataccatggatcagtttagcagccgtctgattccgattaactatgatcagctg


 


aaaaaactggatgccggtaaacaaatcaaactggaaaccacccaggttagcggtaattttggcaccaaaaattcaagcggtca


 


gattgttaccgaaggtaatagctggtcagattatatcagccagattgatagcattagcgccagcattattctggatacagaaaatga


 


aagctatgaacgtcgtgtgaccgcaaaaaatctgcaggacccggaagataaaacaccggaactgaccattggtgaagcaatt


 


gaaaaagcatttggtgccaccaaaaaagatggcctgctgtattttaacgatattccgattgatgaaagctgcgtggaactgatttttg


 


atgataataccgccaataaaatcaaagatagcctgaaaaccctgagcgacaaaaaaatctataatgtgaaactggaacgcgg


 


tatgaatattctgattaaaaccccgacctattttaccaattttgatgattataacaattatccgagcacttggagcaatgtgaataccac


 


caatcaggatggtctgcagggtagcgcaaataaactgaatggtgaaaccaaaatcaaaattccgatgagcgaactgaaaccg


 


tataaacgttatgtgtttagcggctatagcaaagatccgctgaccagcaatagcattattgtgaaaatcaaagccaaagaagaaa


 


aaaccgattatctggttccggaacagggttataccaaatttagctatgaatttgaaaccaccgaaaaagatagcagtaatattgaa


 


attaccctgattggtagcggcaccacctatctggataatctgagtattaccgaactgaatagcacaccggaaattctggatgaacc


 


ggaagtgaaaattccgaccgatcaagaaattatggatgcccataaaatctattttgccgatctgaactttaatccgagcaccggca


 


atacctatattaacggcatgtattttgcaccgacccagaccaataaagaagccctggattatattcagaaatatcgtgttgaagcca


 


ccctgcagtatagcggttttaaagatattggcaccaaagataaagaaatgcgtaattatctgggcgatccgaatcagccgaaaa


 


ccaattatgttaatctgcgcagctattttaccggtggcgaaaacattatgacctacaaaaaactgcgcatttatgccattacaccgg


 


atgatcgtgaactgctggttctgagcgttgattaa


 


SEQ ID NO: 18



- sequence of Fusion 1 (F1)



MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN


 


NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYF


 


SYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNK


 


FLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAAT


 


GWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANN


 


IEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY


 


FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRF


 


LYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVF


 


KGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFM


 


PDTAMAAAGGLFEIDGVIYFFGVDGVKAPGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYF


 


AENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWK


 


DLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQN


 


IDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETY


 


TIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNEN


 


GEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLD


 


EKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 19



- sequence of Fusion 2 (F2)



MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN


 


NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYF


 


SYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNK


 


FLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAAT


 


GWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANN


 


IEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY


 


FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRF


 


LYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVF


 


KGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFM


 


PDTAMAAAGGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFY


 


FAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGW


 


KDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQ


 


NIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGET


 


YTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNE


 


NGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDL


 


DEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 20



- sequence of Fusion 3 (F3)



MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN


 


NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYF


 


SYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNK


 


FLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAAT


 


GWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANN


 


IEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY


 


FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRF


 


LYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVF


 


KGSNGFEYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFD


 


EDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDN


 


GIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDME


 


NESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIE


 


DKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYI


 


AATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 21



- sequence of Fusion 4 (F4)



MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN


 


NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYF


 


SYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNK


 


FLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAAT


 


GWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANN


 


IEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY


 


FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRF


 


LYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVF


 


KGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFM


 


PDTAMAAAGGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKL


 


IIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFV


 


SINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEE


 


GEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFND


 


DGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANT


 


VNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLI


 


DDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPD


 


GFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQL


 


VISE


 


SEQ ID NO: 22



- sequence of Fusion 5 (F5)



MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN


 


NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYF


 


SYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNK


 


FLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAAT


 


GWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANN


 


IEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY


 


FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRF


 


LYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVF


 


KGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFM


 


PDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGGGFVSINDNKHYFDDSGVMKVGYTEIDG


 


KHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAV


 


VGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIE


 


SGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYY


 


FGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNY


 


YFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTG


 


WLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE


 


SEQ ID NO: 23



- nucleotide sequence of F54 Gly



ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGC


 


AATTGCAAGCACCGGCTATACCATTATCAACGGCAAACACTTTTATTTTAACACCGACG


 


GCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCA


 


AATACCGATGCCAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGAC


 


CCTGAACGGCAAAAAATACTACTTTGGCAGCGATAGCAAAGCAGTTACCGGTTGGCGC


 


ATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTG


 


TGCACCATTAACAACGACAAATATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTAC


 


ATTACCATCGAACGCAACAACTTTTATTTCGATGCCAACAACGAAAGCAAAATGGTGAC


 


CGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATA


 


ACAACATTGAAGGTCAGGCGATCGTTTATCAGAACAAATTCCTGACGCTGAATGGTAAG


 


AAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGCTGGCAGACAATTGACGGGA


 


AGAAATATTACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGAC


 


GGTAAAAAGTACTACTTCAACCTGAACACAGCCGAAGCAGCCACAGGATGGCAGACTA


 


TTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCATCTACCGGTTATACCA


 


GCATTAACGGTAAACATTTCTACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCA


 


AAGGTCCAAATGGTTTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACATCGAG


 


GGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGGAAAAAATATTACTTT


 


GGTAGCGATTCTAAAGCCGTTACCGGTCTGCGTACCATTGATGGTAAAAAATACTACTT


 


TAATACGAATACAGCCGTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACT


 


ATTTTAACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATTTCGGGTAAACACT


 


TCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCTTTAAAGGACCTGATGGGTTCG


 


AATATTTTGCGCCTGCGAACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTAT


 


CAGAATCGCTTTCTGTATCTGCACGACAACATCTATTATTTTGGCAACAATTCAAAAGCA


 


GCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCGAATACCGCAAT


 


GGGTGCAAATGGCTACAAAACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGC


 


CGCAGATCGGGGTATTTAAAGGTAGCAACGGCTTCGAATACTTCGCTCCAGCGAATAC


 


GGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGC


 


TGGGCAAAATCTACTACTTTGGCAATAACAGTAAAGCAGTTACTGGATGGCAGACAATC


 


AATGGTAAAGTGTACTATTTTATGCCGGATACCGCCATGGCAGCAGCCGGTGGTCTGTT


 


TGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCACCGGGAATAT


 


ACGGTGGTACCGGCTTTGTGACCGTGGGTGATGATAAATACTATTTCAATCCGATTAAC


 


GGTGGTGCAGCGAGCATTGGCGAAACCATCATCGATGACAAAAACTATTATTTCAACCA


 


GAGCGGTGTGCTGCAGACCGGTGTGTTTAGCACCGAAGATGGCTTTAAATATTTTGCG


 


CCAGCGAACACCCTGGATGAAAACCTGGAAGGCGAAGCGATTGATTTTACCGGCAAAC


 


TGATCATCGATGAAAACATCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGG


 


AAAGAACTGGATGGCGAAATGCATTATTTTTCTCCGGAAACCGGTAAAGCGTTTAAAGG


 


CCTGAACCAGATCGGCGATTACAAATACTACTTCAACAGCGATGGCGTGATGCAGAAA


 


GGCTTTGTGAGCATCAACGATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGT


 


GGGCTATACCGAAATTGATGGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGA


 


TTGGCGTGTTCAATACCGAAGATGGTTTCAAATACTTCGCGCACCATAACGAAGATCTG


 


GGTAACGAAGAAGGCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAAT


 


CTACTACTTTGATGATAGCTTTACCGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGC


 


AGCAAATATTATTTCGATGAAGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATTAA


 


CGATGGCCAGTACTATTTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTA


 


ATGATAAAGTGTTCTATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGCAGAACATT


 


GATGATAACTACTTCTACATCGATGATAACGGCATTGTGCAGATCGGCGTTTTTGATAC


 


CAGCGATGGCTACAAATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGCC


 


AGGCGGTGGAATATAGCGGTCTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGA


 


AACCTATACCATCGAAACCGGCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTA


 


CTTTAATCCGGAAACGAAAAAAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAAT


 


ACTATTTTGATGAAAAAGGCATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACT


 


ATTACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATG


 


TTCTACTTCGGCGAAGATGGTGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTT


 


CAAATACTTTGCCCATCAGAATACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTA


 


TACCGGCTGGCTGGATCTGGATGAAAAACGCTACTACTTCACCGATGAATACATTGCGG


 


CGACCGGCAGCGTGATTATTGATGGCGAAGAATACTACTTCGATCCGGATACCGCGCA


 


GCTGGTGATTAGCGAACATCATCATCATCACCAT


 


SEQ ID NO: 24



- amino acid of F54Gly



MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTD


 


ANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY


 


YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ


 


NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYY


 


FNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEY


 


FAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTG


 


WQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIE


 


GQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKT


 


IDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNS


 


KAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGGTGFVTVGDDK


 


YYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFT


 


GKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQK


 


GFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLG


 


NEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYY


 


FNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAP


 


ANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKG


 


INLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFN


 


TPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDT


 


AQLVISEHHHHHH


 


SEQ ID NO: 25



- nucleotide sequence of F54 New



ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGC


 


AATTGCAAGCACCGGCTATACCATTATCAACGGCAAACACTTTTATTTTAACACCGACG


 


GCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCA


 


AATACCGATGCCAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGAC


 


CCTGAACGGCAAAAAATACTACTTTGGCAGCGATAGCAAAGCAGTTACCGGTTGGCGC


 


ATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTG


 


TGCACCATTAACAACGACAAATATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTAC


 


ATTACCATCGAACGCAACAACTTTTATTTCGATGCCAACAACGAAAGCAAAATGGTGAC


 


CGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATA


 


ACAACATTGAAGGTCAGGCGATCGTTTATCAGAACAAATTCCTGACGCTGAATGGTAAG


 


AAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGCTGGCAGACAATTGACGGGA


 


AGAAATATTACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGAC


 


GGTAAAAAGTACTACTTCAACCTGAACACAGCCGAAGCAGCCACAGGATGGCAGACTA


 


TTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCATCTACCGGTTATACCA


 


GCATTAACGGTAAACATTTCTACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCA


 


AAGGTCCAAATGGTTTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACATCGAG


 


GGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGGAAAAAATATTACTTT


 


GGTAGCGATTCTAAAGCCGTTACCGGTCTGCGTACCATTGATGGTAAAAAATACTACTT


 


TAATACGAATACAGCCGTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACT


 


ATTTTAACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATTTCGGGTAAACACT


 


TCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCTTTAAAGGACCTGATGGGTTCG


 


AATATTTTGCGCCTGCGAACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTAT


 


CAGAATCGCTTTCTGTATCTGCACGACAACATCTATTATTTTGGCAACAATTCAAAAGCA


 


GCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCGAATACCGCAAT


 


GGGTGCAAATGGCTACAAAACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGC


 


CGCAGATCGGGGTATTTAAAGGTAGCAACGGCTTCGAATACTTCGCTCCAGCGAATAC


 


GGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGC


 


TGGGCAAAATCTACTACTTTGGCAATAACAGTAAAGCAGTTACTGGATGGCAGACAATC


 


AATGGTAAAGTGTACTATTTTATGCCGGATACCGCCATGGCAGCAGCCGGTGGTCTGTT


 


TGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTTACCGGCTT


 


TGTGACCGTGGGTGATGATAAATACTATTTCAATCCGATTAACGGTGGTGCAGCGAGCA


 


TTGGCGAAACCATCATCGATGACAAAAACTATTATTTCAACCAGAGCGGTGTGCTGCAG


 


ACCGGTGTGTTTAGCACCGAAGATGGCTTTAAATATTTTGCGCCAGCGAACACCCTGGA


 


TGAAAACCTGGAAGGCGAAGCGATTGATTTTACCGGCAAACTGATCATCGATGAAAACA


 


TCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGGAAAGAACTGGATGGCGAA


 


ATGCATTATTTTTCTCCGGAAACCGGTAAAGCGTTTAAAGGCCTGAACCAGATCGGCGA


 


TTACAAATACTACTTCAACAGCGATGGCGTGATGCAGAAAGGCTTTGTGAGCATCAACG


 


ATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTGGGCTATACCGAAATTGAT


 


GGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGATTGGCGTGTTCAATACCGA


 


AGATGGTTTCAAATACTTCGCGCACCATAACGAAGATCTGGGTAACGAAGAAGGCGAA


 


GAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCTACTACTTTGATGATAGC


 


TTTACCGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAGCAAATATTATTTCGATGA


 


AGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATTAACGATGGCCAGTACTATTTTA


 


ACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAAGTGTTCTATTTCA


 


GCGATAGCGGCATTATTGAAAGCGGCGTGCAGAACATTGATGATAACTACTTCTACATC


 


GATGATAACGGCATTGTGCAGATCGGCGTTTTTGATACCAGCGATGGCTACAAATATTT


 


CGCACCGGCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAATATAGCGGT


 


CTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGAAACCTATACCATCGAAACCG


 


GCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTACTTTAATCCGGAAACGAAAA


 


AAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAATACTATTTTGATGAAAAAGGCA


 


TTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACTATTACTTCAACGAAAACGGT


 


GAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATGTTCTACTTCGGCGAAGATGG


 


TGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCATCAGAA


 


TACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATACCGGCTGGCTGGATCTGG


 


ATGAAAAACGCTACTACTTCACCGATGAATACATTGCGGCGACCGGCAGCGTGATTATT


 


GATGGCGAAGAATACTACTTCGATCCGGATACCGCGCAGCTGGTGATTAGCGAACATC


 


ATCATCATCACCAT


 


SEQ ID NO: 26



amino acid sequence of F54 New



MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTD


 


ANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY


 


YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ


 


NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEA


 


ATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDA


 


NNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKK


 


YYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQN


 


RFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIG


 


VFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYF


 


MPDTAMAAAGGLFEIDGVIYFFGVDGVKAVTGFVTVGDDKYYFNPINGGAASIGETIIDDKN


 


YYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVE


 


WKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMK


 


VGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYY


 


FDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFY


 


FSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVR


 


VGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLI


 


SFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFE


 


GESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISEHHHHHH


 


SEQ ID NO: 27



nucleotide sequence of F5 ToxB



ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGC


 


AATTGCAAGCACCGGCTATACCATTATCAACGGCAAACACTTTTATTTTAACACCGACG


 


GCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCA


 


AATACCGATGCCAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGAC


 


CCTGAACGGCAAAAAATACTACTTTGGCAGCGATAGCAAAGCAGTTACCGGTTGGCGC


 


ATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTG


 


TGCACCATTAACAACGACAAATATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTAC


 


ATTACCATCGAACGCAACAACTTTTATTTCGATGCCAACAACGAAAGCAAAATGGTGAC


 


CGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATA


 


ACAACATTGAAGGTCAGGCGATCGTTTATCAGAACAAATTCCTGACGCTGAATGGTAAG


 


AAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGCTGGCAGACAATTGACGGGA


 


AGAAATATTACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGAC


 


GGTAAAAAGTACTACTTCAACCTGAACACAGCCGAAGCAGCCACAGGATGGCAGACTA


 


TTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCATCTACCGGTTATACCA


 


GCATTAACGGTAAACATTTCTACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCA


 


AAGGTCCAAATGGTTTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACATCGAG


 


GGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGGAAAAAATATTACTTT


 


GGTAGCGATTCTAAAGCCGTTACCGGTCTGCGTACCATTGATGGTAAAAAATACTACTT


 


TAATACGAATACAGCCGTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACT


 


ATTTTAACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATTTCGGGTAAACACT


 


TCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCTTTAAAGGACCTGATGGGTTCG


 


AATATTTTGCGCCTGCGAACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTAT


 


CAGAATCGCTTTCTGTATCTGCACGACAACATCTATTATTTTGGCAACAATTCAAAAGCA


 


GCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCGAATACCGCAAT


 


GGGTGCAAATGGCTACAAAACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGC


 


CGCAGATCGGGGTATTTAAAGGTAGCAACGGCTTCGAATACTTCGCTCCAGCGAATAC


 


GGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGC


 


TGGGCAAAATCTACTACTTTGGCAATAACAGTAAAGCAGTTACTGGATGGCAGACAATC


 


AATGGTAAAGTGTACTATTTTATGCCGGATACCGCCATGGCAGCAGCCGGTGGTCTGTT


 


TGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTGAGCGGTC


 


TGATTTATATTAACGATAGCCTGTATTACTTTAAACCACCGGTGAATAACCTGATTACCG


 


GCTTTGTGACCGTGGGTGATGATAAATACTATTTCAATCCGATTAACGGTGGTGCAGCG


 


AGCATTGGCGAAACCATCATCGATGACAAAAACTATTATTTCAACCAGAGCGGTGTGCT


 


GCAGACCGGTGTGTTTAGCACCGAAGATGGCTTTAAATATTTTGCGCCAGCGAACACC


 


CTGGATGAAAACCTGGAAGGCGAAGCGATTGATTTTACCGGCAAACTGATCATCGATGA


 


AAACATCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGGAAAGAACTGGATG


 


GCGAAATGCATTATTTTTCTCCGGAAACCGGTAAAGCGTTTAAAGGCCTGAACCAGATC


 


GGCGATTACAAATACTACTTCAACAGCGATGGCGTGATGCAGAAAGGCTTTGTGAGCAT


 


CAACGATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTGGGCTATACCGAAA


 


TTGATGGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGATTGGCGTGTTCAAT


 


ACCGAAGATGGTTTCAAATACTTCGCGCACCATAACGAAGATCTGGGTAACGAAGAAG


 


GCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCTACTACTTTGATG


 


ATAGCTTTACCGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAGCAAATATTATTTC


 


GATGAAGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATTAACGATGGCCAGTACT


 


ATTTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAAGTGTTCT


 


ATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGCAGAACATTGATGATAACTACTTC


 


TACATCGATGATAACGGCATTGTGCAGATCGGCGTTTTTGATACCAGCGATGGCTACAA


 


ATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAATATA


 


GCGGTCTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGAAACCTATACCATCGA


 


AACCGGCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTACTTTAATCCGGAAAC


 


GAAAAAAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAATACTATTTTGATGAAAA


 


AGGCATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACTATTACTTCAACGAAAA


 


CGGTGAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATGTTCTACTTCGGCGAAG


 


ATGGTGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCATC


 


AGAATACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATACCGGCTGGCTGGAT


 


CTGGATGAAAAACGCTACTACTTCACCGATGAATACATTGCGGCGACCGGCAGCGTGA


 


TTATTGATGGCGAAGAATACTACTTCGATCCGGATACCGCGCAGCTGGTGATTAGCGAA


 


CATCATCATCATCACCAT


 


SEQ ID NO: 28



amino acid sequence of F5 ToxB



MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTD


 


ANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY


 


YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ


 


NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEA


 


ATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDA


 


NNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKK


 


YYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQN


 


RFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIG


 


VFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYF


 


MPDTAMAAAGGLFEIDGVIYFFGVDGVKAVSGLIYINDSLYYFKPPVNNLITGFVTVGDDKYY


 


FNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGK


 


LIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGF


 


VSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNE


 


EGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFN


 


DDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPAN


 


TVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINL


 


IDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTP


 


DGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQ


 


LVISEHHHHHH


 


SEQ ID NO: 29



- nucleotide sequence of F52 new



ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGC


 


AATTGCAAGCACCGGCTATACCATTATCAACGGCAAACACTTTTATTTTAACACCGACG


 


GCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCA


 


AATACCGATGCCAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGAC


 


CCTGAACGGCAAAAAATACTACTTTGGCAGCGATAGCAAAGCAGTTACCGGTTGGCGC


 


ATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTG


 


TGCACCATTAACAACGACAAATATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTAC


 


ATTACCATCGAACGCAACAACTTTTATTTCGATGCCAACAACGAAAGCAAAATGGTGAC


 


CGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATA


 


ACAACATTGAAGGTCAGGCGATCGTTTATCAGAACAAATTCCTGACGCTGAATGGTAAG


 


AAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGCTGGCAGACAATTGACGGGA


 


AGAAATATTACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGAC


 


GGTAAAAAGTACTACTTCAACCTGAACACAGCCGAAGCAGCCACAGGATGGCAGACTA


 


TTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCATCTACCGGTTATACCA


 


GCATTAACGGTAAACATTTCTACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCA


 


AAGGTCCAAATGGTTTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACATCGAG


 


GGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGGAAAAAATATTACTTT


 


GGTAGCGATTCTAAAGCCGTTACCGGTCTGCGTACCATTGATGGTAAAAAATACTACTT


 


TAATACGAATACAGCCGTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACT


 


ATTTTAACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATTTCGGGTAAACACT


 


TCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCTTTAAAGGACCTGATGGGTTCG


 


AATATTTTGCGCCTGCGAACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTAT


 


CAGAATCGCTTTCTGTATCTGCACGACAACATCTATTATTTTGGCAACAATTCAAAAGCA


 


GCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCGAATACCGCAAT


 


GGGTGCAAATGGCTACAAAACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGC


 


CGCAGATCGGGGTATTTAAAGGTAGCAACGGCTTCGAATACTTCGCTCCAGCGAATAC


 


GGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGC


 


TGGGCAAAATCTACTACTTTGGCAATAACAGTAAAGCAGTTACTGGATGGCAGACAATC


 


AATGGTAAAGTGTACTATTTTATGCCGGATACCGCCATGGCAGCAGCCGGTGGTCTGTT


 


TGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTGAAAGGCC


 


TGAACCAGATCGGCGATTACAAATACTACTTCAACAGCGATGGCGTGATGCAGAAAGG


 


CTTTGTGAGCATCAACGATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTGG


 


GCTATACCGAAATTGATGGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGATT


 


GGCGTGTTCAATACCGAAGATGGTTTCAAATACTTCGCGCACCATAACGAAGATCTGGG


 


TAACGAAGAAGGCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCT


 


ACTACTTTGATGATAGCTTTACCGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAG


 


CAAATATTATTTCGATGAAGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATTAACG


 


ATGGCCAGTACTATTTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAAT


 


GATAAAGTGTTCTATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGCAGAACATTGA


 


TGATAACTACTTCTACATCGATGATAACGGCATTGTGCAGATCGGCGTTTTTGATACCA


 


GCGATGGCTACAAATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGCCAG


 


GCGGTGGAATATAGCGGTCTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGAAA


 


CCTATACCATCGAAACCGGCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTACT


 


TTAATCCGGAAACGAAAAAAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAATAC


 


TATTTTGATGAAAAAGGCATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACTAT


 


TACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATGTT


 


CTACTTCGGCGAAGATGGTGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTTCA


 


AATACTTTGCCCATCAGAATACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATA


 


CCGGCTGGCTGGATCTGGATGAAAAACGCTACTACTTCACCGATGAATACATTGCGGC


 


GACCGGCAGCGTGATTATTGATGGCGAAGAATACTACTTCGATCCGGATACCGCGCAG


 


CTGGTGATTAGCGAACATCATCATCATCACCAT


 


SEQ ID NO: 30



- amino acid sequence of F52 New



MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTD


 


ANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY


 


YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ


 


NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEA


 


ATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDA


 


NNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKK


 


YYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQN


 


RFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIG


 


VFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYF


 


MPDTAMAAAGGLFEIDGVIYFFGVDGVKAVKGLNQIGDYKYYFNSDGVMQKGFVSINDNKH


 


YFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYS


 


GILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQV


 


GFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYG


 


QAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYF


 


DEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFA


 


HQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISEHHH


 


HHH


 


SEQ ID NO: 31



- amino acid 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: 32



- amino acid 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: 33



- amino acid sequence of CDTb′′ C39 when expressed in fusion with



GST.


LMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKA


 


IKTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSH


 


TTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLS


 


PGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQ


 


IDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNT


 


ANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIP


 


MSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLD


 


NLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYEADLNENPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEAT


 


LQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVD


 


Remarks:


The protein tested in the cytotoxicity assay was obtained after


cleavage of the GST by PreScission protease


 


Following experimental results, it is demonstrated that the mature


CDTb (without SP and pro-domain) starts at the Ser212 (in red and


underlined in the sequence).


 


SEQ ID NO: 34



- amino acid sequence of CdtB receptor binding domain with linker



in N-term of sequence, from aa 620-876 (C52)


MTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEE


 


KTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIY


 


FADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLR


 


SYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGGHHHHHH


 


SEQ ID NO: 35



- Nucleotide sequence of C52



ATGACCAATTTTGATGATTATAACAATTATCCGAGCACTTGGAGCAATGTGAATACCACCAATCAGGATGGTCTG


 


CAGGGTAGCGCAAATAAACTGAATGGTGAAACCAAAATCAAAATTCCGATGAGCGAACTGAAACCGTATAAACGT


 


TATGTGTTTAGCGGCTATAGCAAAGATCCGCTGACCAGCAATAGCATTATTGTGAAAATCAAAGCCAAAGAAGAA


 


AAAACCGATTATCTGGTTCCGGAACAGGGTTATACCAAATTTAGCTATGAATTTGAAACCACCGAAAAAGATAGC


 


AGTAATATTGAAATTACCCTGATTGGTAGCGGCACCACCTATCTGGATAATCTGAGTATTACCGAACTGAATAGC


 


ACACCGGAAATTCTGGATGAACCGGAAGTGAAAATTCCGACCGATCAAGAAATTATGGATGCCCATAAAATCTAT


 


TTTGCCGATCTGAACTTTAATCCGAGCACCGGCAATACCTATATTAACGGCATGTATTTTGCACCGACCCAGACC


 


AATAAAGAAGCCCTGGATTATATTCAGAAATATCGTGTTGAAGCCACCCTGCAGTATAGCGGTTTTAAAGATATT


 


GGCACCAAAGATAAAGAAATGCGTAATTATCTGGGCGATCCGAATCAGCCGAAAACCAATTATGTTAATCTGCGC


 


AGCTATTTTACCGGTGGCGAAAACATTATGACCTACAAAAAACTGCGCATTTATGCCATTACACCGGATGATCGT


 


GAACTGCTGGTTCTGAGCGTTGATGGCGGTCACCACCATCATCATCATTAA


 


SEQ ID NO: 36



- amino acid sequence of CdtB receptor binding domain without 



linker in N-term of sequence, from aa 636-876 (C55)


MNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSY


 


EFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYEADLNENPSTGNTYIN


 


GMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLR


 


IYAITPDDRELLVLSVDGGHHHHHH


 


SEQ ID NO: 37



- nucleotide sequence of C55



ATGAATACCACCAATCAGGATGGTCTGCAGGGTAGCGCAAATAAACTGAATGGTGAAACCAAAATCAAAATTCCGATGAG


 


CGAACTGAAACCGTATAAACGTTATGTGTTTAGCGGCTATAGCAAAGATCCGCTGACCAGCAATAGCATTATTGTGAAAA


 


TCAAAGCCAAAGAAGAAAAAACCGATTATCTGGTTCCGGAACAGGGTTATACCAAATTTAGCTATGAATTTGAAACCACC


 


GAAAAAGATAGCAGTAATATTGAAATTACCCTGATTGGTAGCGGCACCACCTATCTGGATAATCTGAGTATTACCGAACT


 


GAATAGCACACCGGAAATTCTGGATGAACCGGAAGTGAAAATTCCGACCGATCAAGAAATTATGGATGCCCATAAAATCT


 


ATTTTGCCGATCTGAACTTTAATCCGAGCACCGGCAATACCTATATTAACGGCATGTATTTTGCACCGACCCAGACCAAT


 


AAAGAAGCCCTGGATTATATTCAGAAATATCGTGTTGAAGCCACCCTGCAGTATAGCGGTTTTAAAGATATTGGCACCAA


 


AGATAAAGAAATGCGTAATTATCTGGGCGATCCGAATCAGCCGAAAACCAATTATGTTAATCTGCGCAGCTATTTTACCG


 


GTGGCGAAAACATTATGACCTACAAAAAACTGCGCATTTATGCCATTACACCGGATGATCGTGAACTGCTGGTTCTGAGC


 


GTTGATGGCGGTCACCACCATCATCATCATTAA


 


SEQ ID NO: 38



- amino acid sequence of CDTb prodomain sequence (long, aa43-211)



(C58)


MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTL


 


STDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELDGMKKIIPEENLFLRDYS


 


NIEKDDPFIPNNNFFDPKLM


 


SEQ ID NO: 39



- amino acid sequence of CDTb prodomain sequence (short, aa43-186)



(C59)


MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTL


 


STDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELDGMKKIIPEENLF


 


SEQ ID NO: 40



- amino acid sequence of Fusion CDTa N-term with



linker (aa44-268) to CDTb receptor binding domain


with linker in N term of sequence (aa621-876) (C60)


CDTa part of the fusion is underlined.


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL


 


EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL


 


IEQGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDTNFDDYNNYPSTWSNVNTTNQDGL


 


QGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYT


 


KFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKI


 


YFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYL


 


GDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGGHHHHHH


 


SEQ ID NO: 41



- amino acid sequence of Fusion CDTa N-term with



linker (aa44-268) to CDTb receptor binding domain


without linker in N term of sequence (aa636-876) (C61)


CDTa part of the fusion is underlined.


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL


 


EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL


 


IEQGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDNTTNQDGLQGSANKLNGETKIKIP


 


MSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNI


 


EITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYI


 


NGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSY


 


FTGGENIMTYKKLRIYAITPDDRELLVLSVDGGHHHHHH


 


SEQ ID NO: 42



- amino acid sequence of Fusion CDTa N-term without



linker (aa44-260) to CDTb receptor binding domain


with linker in N term of sequence (aa621-876) (C62)


CDTa part of the fusion is underlined.


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL


 


EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL


 


IEQGYSIKIDKIVRIVIDGKHYIKAEASVVSTNFDDYNNYPSTWSNVNTTNQDGLQGSANKL


 


NGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFE


 


TTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNF


 


NPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPK


 


TNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGGHHHHHH


 


SEQ ID NO: 43



- amino acid sequence of Fusion CDTa N-term without



linker (aa44-260) to CDTb receptor binding domain


without linker in N term of sequence (aa636-876) (C63)


CDTa part of the fusion is underlined.


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL


 


EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL


 


IEQGYSIKIDKIVRIVIDGKHYIKAEASVVSNTTNQDGLQGSANKLNGETKIKIPMSELKPY


 


KRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGS


 


GTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAP


 


TQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENI


 


MTYKKLRIYAITPDDRELLVLSVDGGHHHHHH


 


SEQ ID NO: 44



- amino acid sequence of Fusion F2- CDTb receptor binding domain



with linker in N term of sequence (aa621-876) (C64)


F2 sequence is underlined.


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHEYENTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF


 


LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNEYFDANN


 


ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE


 


AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYENTNTFIASTGYTSINGKHEYENTDGIMQIGVFKGPNGFE


 


YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN


 


TNTSIASTGYTIISGKHEYENTDGIMQIGVFKGPDGFEYEAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS


 


KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNEYERNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN


 


RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFD


 


DSGVMKVGYTEIDGKHFYFAENGEMQIGVENTEDGEKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTA


 


VVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVEYESDSGIIESGVQNIDDNYFY


 


IDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFN


 


PETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPD


 


GFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISETNFDDYNNY


 


PSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQG


 


YTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYEADLNENPST


 


GNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIM


 


TYKKLRIYAITPDDRELLVLSVDGGHHHHHH


 


SEQ ID NO: 45



- amino acid sequence of Fusion of F2 to CDTb receptor binding



domain without linker in N term of sequence (aa636-876) with 2


heterogeneous Gly residues between F2 and CTDb sequences (C65)


F2 sequence is underlined.


MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF


 


LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN


 


ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE


 


AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE


 


YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN


 


TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS


 


KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN


 


RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFD


 


DSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTA


 


VVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFY


 


IDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFN


 


PETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPD


 


GFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISEGGNVNTTNQ


 


DGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTE


 


KDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAP


 


TQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITP


 


DDRELLVLSVDGGHHHHHH


 


SEQ ID NO: 46



- amino acid sequence of CDTa without signal peptide, with two



mutations (E428Q, E430Q, aa 44-463) (C67)


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQTRNYFYDYQIEAN


 


SREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLEKFNEFKETIQNKLFKQDGFKDISLY


 


EPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIEQGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDV


 


SKGDSWGKANYNDWSNKLTPNELADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLT


 


VYRRSGPQEFGLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGSPGA


 


YLSAIPGYAGgYgVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP


 


SEQ ID NO: 47



- nucleotide sequence of C67



ATGGTTTGCAATACCACCTATAAAGCACCGATTGAACGTCCGGAAGATTTTCTGAAAGATAAAGAAAAAGCCAAA


 


GAATGGGAACGCAAAGAAGCAGAACGTATTGAACAGAAACTGGAACGTAGCGAAAAAGAAGCACTGGAAAGCTAC


 


AAAAAAGATAGCGTGGAAATTTCAAAATATAGCCAGACCCGCAATTATTTCTATGATTATCAGATTGAAGCCAAT


 


AGCCGTGAAAAAGAATATAAAGAACTGCGCAATGCCATTAGCAAAAACAAAATTGATAAACCGATGTATGTGTAT


 


TATTTCGAAAGTCCGGAAAAATTTGCCTTTAACAAAGTGATTCGCACCGAAAATCAGAATGAAATTAGCCTGGAA


 


AAATTCAATGAATTTAAAGAAACCATTCAGAATAAACTGTTTAAACAGGATGGCTTTAAAGATATTTCACTGTAT


 


GAACCGGGTAAAGGTGATGAAAAACCGACACCGCTGCTGATGCATCTGAAACTGCCTCGTAATACCGGTATGCTG


 


CCGTATACCAATACCAATAATGTTAGCACCCTGATTGAACAGGGCTATAGCATCAAAATTGATAAAATTGTGCGC


 


ATTGTGATTGATGGCAAACATTATATCAAAGCCGAAGCCAGCGTTGTTTCAAGCCTGGATTTTAAAGATGATGTG


 


AGCAAAGGCGATAGCTGGGGTAAAGCAAACTATAATGATTGGAGCAATAAACTGACCCCGAATGAACTGGCAGAT


 


GTGAATGATTATATGCGTGGTGGTTATACCGCCATTAACAATTATCTGATTAGCAATGGTCCGGTGAATAATCCG


 


AATCCGGAACTGGATAGCAAAATTACCAATATTGAAAATGCCCTGAAACGCGAACCGATTCCGACCAATCTGACC


 


GTTTATCGTCGTAGCGGTCCGCAAGAATTTGGTCTGACCCTGACCAGTCCGGAATATGACTTTAACAAACTGGAA


 


AATATTGATGCCTTTAAAAGCAAATGGGAAGGTCAGGCACTGAGCTATCCGAACTTTATTAGCACCAGCATTGGT


 


AGCGTTAATATGAGCGCATTTGCCAAACGTAAAATTGTGCTGCGTATTACCATTCCGAAAGGTAGTCCGGGTGCA


 


TATCTGAGCGCAATTCCGGGTTATGCCGGTCAATATCAGGTTCTGCTGAATCATGGCAGCAAATTCAAAATTAAC


 


AAAATTGATAGCTATAAAGATGGCACCATTACCAAACTGATTGTTGATGCAACCCTGATTCCGTAA


 


SEQ ID NO: 48



- amino acid sequence of CDTa without signal peptide,



with seven mutations (R345A, Q350A, N385A, R402A,


S388F, E428Q, E430Q, aa 44-463) (C69)


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL


 


EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL


 


IEQGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL


 


ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF


 


GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS


 


PGAYLSAIPGYAGQYQVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP


 


SEQ ID NO: 49



- nucleotide sequence of C69



ATGGTTTGCAATACCACCTATAAAGCACCGATTGAACGTCCGGAAGATTTTCTGAAAGATAA


 


AGAAAAAGCCAAAGAATGGGAACGCAAAGAAGCAGAACGTATTGAACAGAAACTGGAACGTA


 


GCGAAAAAGAAGCACTGGAAAGCTACAAAAAAGATAGCGTGGAAATTTCAAAATATAGCCAG


 


ACCCGCAATTATTTCTATGATTATCAGATTGAAGCCAATAGCCGTGAAAAAGAATATAAAGA


 


ACTGCGCAATGCCATTAGCAAAAACAAAATTGATAAACCGATGTATGTGTATTATTTCGAAA


 


GTCCGGAAAAATTTGCCTTTAACAAAGTGATTCGCACCGAAAATCAGAATGAAATTAGCCTG


 


GAAAAATTCAATGAATTTAAAGAAACCATTCAGAATAAACTGTTTAAACAGGATGGCTTTAA


 


AGATATTTCACTGTATGAACCGGGTAAAGGTGATGAAAAACCGACACCGCTGCTGATGCATC


 


TGAAACTGCCTCGTAATACCGGTATGCTGCCGTATACCAATACCAATAATGTTAGCACCCTG


 


ATTGAACAGGGCTATAGCATCAAAATTGATAAAATTGTGCGCATTGTGATTGATGGCAAACA


 


TTATATCAAAGCCGAAGCCAGCGTTGTTTCAAGCCTGGATTTTAAAGATGATGTGAGCAAAG


 


GCGATAGCTGGGGTAAAGCAAACTATAATGATTGGAGCAATAAACTGACCCCGAATGAACTG


 


GCAGATGTGAATGATTATATGCGTGGTGGTTATACCGCCATTAACAATTATCTGATTAGCAA


 


TGGTCCGGTGAATAATCCGAATCCGGAACTGGATAGCAAAATTACCAATATTGAAAATGCCC


 


TGAAACGCGAACCGATTCCGACCAATCTGACCGTTTATGCACGTAGCGGTCCGGCAGAATTT


 


GGTCTGACCCTGACCAGTCCGGAATATGACTTTAACAAACTGGAAAATATTGATGCCTTTAA


 


AAGCAAATGGGAAGGTCAGGCACTGAGCTATCCGGCATTTATTTTCACCAGCATTGGTAGCG


 


TTAATATGAGCGCATTTGCCAAAGCAAAAATTGTGCTGCGTATTACCATTCCGAAAGGTAGT


 


CCGGGTGCATATCTGAGCGCAATTCCGGGTTATGCCGGTCAGTATCAGGTTCTGCTGAATCA


 


TGGCAGCAAATTCAAAATTAACAAAATTGATAGCTATAAAGATGGCACCATTACCAAACTGA


 


TTGTTGATGCAACCCTGATTCCG


 


SEQ ID NO: 50



- amino acid sequence of CDTb without signal sequence and



prodomain (mature fragment based on MS data) with Ca2+


binding motif mutation (aa212-876, mut Asp-9-11-13 Ala) (C97)


3 mutated residues in this sequence. Three Asp residues were


mutated into Ala. They are highlighted in bold and underlined.


MSDWEDEDLATANANIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAI


 


KTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHT


 


TDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLSP


 


GDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQI


 


DSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEATEKAFGATKKDGLLYENDIPIDESCVELIFDDNTA


 


NKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPM


 


SELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDN


 


LSITELNSTPEILDEPEVKIPTDQEIMDAHKIYEADLNENPSTGNTYINGMYEAPTQTNKEALDYIQKYRVEATL


 


QYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDHHHHHH


 


SEQ ID NO: 51



- amino acid sequence of CDTb with prodomain removed (CDTb′′,



aa212-876) (C55)


MSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAI


 


KTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHT


 


TDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLSP


 


GDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNEGTKNSSGQIVTEGNSWSDYISQI


 


DSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEATEKAFGATKKDGLLYENDIPIDESCVELIFDDNTA


 


NKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPM


 


SELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDN


 


LSITELNSTPEILDEPEVKIPTDQEIMDAHKIYEADLNENPSTGNTYINGMYEAPTQTNKEALDYIQKYRVEATL


 


QYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDHHHHHH


 


SEQ ID NO: 52



- amino acid sequence of CDTa without signal peptide,



with five mutations (R345A, Q350A, N385A, R402A,


S388F, aa 44-463) (C107)


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ


 


TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLE


 


KFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIE


 


QGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDVSKGDSWGKANYNDWSNKLTPNELAD


 


VNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEFGLT


 


LTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGSPGAYL


 


SAIPGYAGEYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH**


 


SEQ ID NO: 53



- Polynucleotide sequence of CDTa without



signal peptide, with five mutations (R345A,


Q350A, N385A, R402A, S388F, aa 44-463) (C107)


ATGGTTTGCAATACCACCTATAAAGCACCGATTGAACGTCCGGAAGATTTTCTGAAAGA


 


TAAAGAAAAAGCCAAAGAATGGGAACGCAAAGAAGCAGAACGTATTGAACAGAAACTG


 


GAACGTAGCGAAAAAGAAGCACTGGAAAGCTACAAAAAAGATAGCGTGGAAATTTCAAA


 


ATATAGCCAGACCCGCAATTATTTCTATGATTATCAGATTGAAGCCAATAGCCGTGAAAA


 


AGAATATAAAGAACTGCGCAATGCCATTAGCAAAAACAAAATTGATAAACCGATGTATGT


 


GTATTATTTCGAAAGTCCGGAAAAATTTGCCTTTAACAAAGTGATTCGCACCGAAAATCA


 


GAATGAAATTAGCCTGGAAAAATTCAATGAATTTAAAGAAACCATTCAGAATAAACTGT


 


TTAAACAGGATGGCTTTAAAGATATTTCACTGTATGAACCGGGTAAAGGTGATGA


 


AAAACCGACACCGCTGCTGATGCATCTGAAACTGCCTCGTAATACCGGTATGCTG


 


CCGTATACCAATACCAATAATGTTAGCACCCTGATTGAACAGGGCTATAGCATCA


 


AAATTGATAAAATTGTGCGCATTGTGATTGATGGCAAACATTATATCAAAGCCGA


 


AGCCAGCGTTGTTTCAAGCCTGGATTTTAAAGATGATGTGAGCAAAGGCGATAG


 


CTGGGGTAAAGCAAACTATAATGATTGGAGCAATAAACTGACCCCGAATGAACT


 


GGCAGATGTGAATGATTATATGCGTGGTGGTTATACCGCCATTAACAATTATCTG


 


ATTAGCAATGGTCCGGTGAATAATCCGAATCCGGAACTGGATAGCAAAATTACC


 


AATATTGAAAATGCCCTGAAACGCGAACCGATTCCGACCAATCTGACCGTTTATG


 


CACGTAGCGGTCCGGCAGAATTTGGTCTGACCCTGACCAGTCCGGAATATGACTT


 


TAACAAACTGGAAAATATTGATGCCTTTAAAAGCAAATGGGAAGGTCAGGCACT


 


GAGCTATCCGGCATTTATTTTCACCAGCATTGGTAGCGTTAATATGAGCGCATTT


 


GCCAAAGCAAAAATTGTGCTGCGTATTACCATTCCGAAAGGTAGTCCGGGTGCA


 


TATCTGAGCGCAATTCCGGGTTATGCCGGTgAaTATgAaGTTCTGCTGAATCATGG


 


CAGCAAATTCAAAATTAACAAAATTGATAGCTATAAAGATGGCACCATTACCAA


 


ACTGATTGTTGATGCAACCCTGATTCCGCACCACCATCATCATCATTAATAA


 


SEQ ID NO: 54



- Amino acid sequence of CDTa without signal



peptide, with six mutations (R345A, Q350A,


N385A, R402A, S388F, E430Q, aa 44-463) (C108)


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEI


 


SKYSQTRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIR


 


TENQNEISLEKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTG


 


MLPYTNTNNVSTLIEQGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDVSKGDSW


 


GKANYNDWSNKLTPNELADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIE


 


NALKREPIPTNLTVYARSGPAEFGLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIF


 


TSIGSVNMSAFAKAKIVLRITIPKGSPGAYLSAIPGYAGEYQVLLNHGSKFKIN


 


KIDSYKDGTITKLIVDATLIPHHHHHH**


 


SEQ ID NO: 55



- Polynucleotide sequence of CDTa without



signal peptide, with six mutations (R345A,


Q350A, N385A, R402A, S388F, E430Q, aa 44-463)


(C108)


ATGGTTTGCAATACCACCTATAAAGCACCGATTGAACGTCCGGAAGATTTTCTGA


 


AAGATAAAGAAAAAGCCAAAGAATGGGAACGCAAAGAAGCAGAACGTATTGAA


 


CAGAAACTGGAACGTAGCGAAAAAGAAGCACTGGAAAGCTACAAAAAAGATAG


 


CGTGGAAATTTCAAAATATAGCCAGACCCGCAATTATTTCTATGATTATCAGATT


 


GAAGCCAATAGCCGTGAAAAAGAATATAAAGAACTGCGCAATGCCATTAGCAAA


 


AACAAAATTGATAAACCGATGTATGTGTATTATTTCGAAAGTCCGGAAAAATTTG


 


CCTTTAACAAAGTGATTCGCACCGAAAATCAGAATGAAATTAGCCTGGAAAAAT


 


TCAATGAATTTAAAGAAACCATTCAGAATAAACTGTTTAAACAGGATGGCTTTAA


 


AGATATTTCACTGTATGAACCGGGTAAAGGTGATGAAAAACCGACACCGCTGCT


 


GATGCATCTGAAACTGCCTCGTAATACCGGTATGCTGCCGTATACCAATACCAAT


 


AATGTTAGCACCCTGATTGAACAGGGCTATAGCATCAAAATTGATAAAATTGTGC


 


GCATTGTGATTGATGGCAAACATTATATCAAAGCCGAAGCCAGCGTTGTTTCAAG


 


CCTGGATTTTAAAGATGATGTGAGCAAAGGCGATAGCTGGGGTAAAGCAAACTA


 


TAATGATTGGAGCAATAAACTGACCCCGAATGAACTGGCAGATGTGAATGATTA


 


TATGCGTGGTGGTTATACCGCCATTAACAATTATCTGATTAGCAATGGTCCGGTG


 


AATAATCCGAATCCGGAACTGGATAGCAAAATTACCAATATTGAAAATGCCCTG


 


AAACGCGAACCGATTCCGACCAATCTGACCGTTTATGCACGTAGCGGTCCGGCA


 


GAATTTGGTCTGACCCTGACCAGTCCGGAATATGACTTTAACAAACTGGAAAATA


 


TTGATGCCTTTAAAAGCAAATGGGAAGGTCAGGCACTGAGCTATCCGGCATTTAT


 


TTTCACCAGCATTGGTAGCGTTAATATGAGCGCATTTGCCAAAGCAAAAATTGTG


 


CTGCGTATTACCATTCCGAAAGGTAGTCCGGGTGCATATCTGAGCGCAATTCCGG


 


GTTATGCCGGTgAaTATcAaGTTCTGCTGAATCATGGCAGCAAATTCAAAATTAAC


 


AAAATTGATAGCTATAAAGATGGCACCATTACCAAACTGATTGTTGATGCAACCC


 


TGATTCCGCACCACCATCATCATCATTAATAA


 


SEQ ID NO: 56



- Amino acid sequence of CDTa without signal



peptide, with six mutations (R345A, Q350A,


N385A, R402A, S388F, E428Q, aa 44-463) (C110


MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEI


 


SKYSQTRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIR


 


TENQNEISLEKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTG


 


MLPYTNTNNVSTLIEQGYSIKIDKIVRIVIDGKHYIKAEASVVSSLDFKDDVSKGDSW


 


GKANYNDWSNKLTPNELADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIE


 


NALKREPIPTNLTVYARSGPAEFGLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIF


 


TSIGSVNMSAFAKAKIVLRITIPKGSPGAYLSAIPGYAGQYEVLLNHGSKFKIN


 


KIDSYKDGTITKLIVDATLIPHHHHHH**

## Claims

1. An immunogenic composition comprising a fusion protein, said fusion protein comprising a Clostridium difficile binary toxin a (CDTa) protein sequence covalently linked to a Clostridium difficile binary toxin b (CDTb) protein sequence, wherein:
(a) the CDTa protein sequence comprises the CDTa C-terminal domain and comprises an amino acid substitution that reduces CDTa enzymatic activity, and
(b) the CDTb protein sequence lacks the CDTb prodomain sequence and comprises a sequence having at least 95% sequence identity to SEQ ID NO:9.

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

3. A method of preventing or treating C. difficile disease comprising administering the vaccine of claim 2 to a human subject.

4. The immunogenic composition of claim 1, wherein said CDTa amino acid substitution is selected from R345K, Q350A, N385A, R402A, S388F, E428Q, and E430Q, where the amino acid numbering corresponds to that of SEQ ID NO: 1.

5. An immunogenic composition comprising a fusion protein, said fusion protein comprising a Clostridium difficile binary toxin a (CDTa) protein sequence covalently linked to a Clostridium difficile binary toxin b (CDTb) protein sequence, wherein:
(a) the CDTa protein sequence comprises the CDTa C-terminal domain and comprises an amino acid substitution that reduces CDTa enzymatic activity, where the CDTa sequence is selected from a sequence having at least 95% sequence identity to SEQ ID NO:46, a sequence having at least 95% sequence identity to SEQ ID NO:48, a sequence having at least 95% sequence identity to SEQ ID NO:52, or a sequence having at least 95% sequence identity to SEQ ID NO:54, and
(b) the CDTb protein sequence lacks the CDTb prodomain sequence and comprises a sequence having at least 95% sequence identity to SEQ ID NO:9.

6. The immunogenic composition of claim 1, wherein in said fusion protein, the CDTa protein sequence and CDTb protein sequence are covalently linked via a peptide linker.

7. The immunogenic composition of claim 5, wherein in said fusion protein, the CDTa protein sequence and CDTb protein sequence are covalently linked via a peptide linker.

8. A vaccine comprising the immunogenic composition of claim 5 and a pharmaceutically acceptable excipient.

9. A method of preventing or treating C. difficile disease comprising administering the vaccine of claim 8 to a human subject.

10. The immunogenic composition of claim 1, wherein the CDTb protein sequence lacks the CDTb prodomain sequence and comprises the sequence of amino acids 212-876 of SEQ ID NO: 3.

11. The immunogenic composition of claim 1, wherein the CDTb protein sequence lacks the CDTb prodomain sequence and comprises SEQ ID NO:9.

12. The immunogenic composition of claim 5, wherein the CDTa protein sequence comprises the CDTa C-terminal domain and comprises an amino acid substitution that reduces CDTa enzymatic activity, where the CDTa sequence is selected from SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 52, or SEQ ID NO: 54.

13. The immunogenic composition of claim 5, wherein the CDTb protein sequence lacks the CDTb prodomain sequence and comprises the sequence of amino acids 212-876 of SEQ ID NO: 3.

14. The immunogenic composition of claim 5, wherein the CDTb protein sequence lacks the CDTb prodomain sequence and comprises SEQ ID NO: 9.