IMMUNOGENIC COMPOSITION

专利摘要:
The present invention relates to immunogenic compositions comprising CDTb isolated from Clostridium difficile. In particular, the CDTb protein isolated from Clostridium difficile has been mutated to modify the ability to form pores, to modify the capacity for heptamerization, or is a truncated CDTb protein with the signal peptide and prodomain removed and also the binding domain. at the receptors removed and / or the CDTα binding domain removed. The invention also relates to fusion proteins comprising a CDTa protein and a CDTb protein. Vaccines comprising such immunogenic compositions and their therapeutic uses also form part of the invention.
公开号:BE1022949B1
申请号:E2015/5390
申请日:2015-06-25
公开日:2016-10-21
发明作者:Cindy Castado
申请人:Glaxosmithkline Biologicals S.A.；
IPC主号:

专利说明:

IMMUNOGENIC COMPOSITION
Context
Clostridium difficile (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 C. difficile-infected individuals was calculated as 5.99% within 3 months of diagnosis, with higher mortality associated with older age, 13.5% in elderly patients over 80 years old (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, strains that are resistant to these antibiotics have been identified (Shah et al., Expert Rev. Anti Infect Ther 8 (5), 555-564 (2010)). Therefore, there is a need for immunogenic compositions capable of inducing antibodies against, and / or a protective immune response against, C. difficile. The enterotoxicity of C. difficile is primarily due to the action of two toxins, toxin A and toxin B. Both are potent cytotoxins (Lyerly et al., Current Microbiology 21: 29-32 (1990) .
It has been demonstrated that fragments of toxin A, particularly fragments of the C-terminal domain, can lead to a protective immune response in hamsters (Lyerly et al, Current Microbiology 21: 29-32 (1990)), WO 96/12802 and WO 00/61762.
Some, but not all, strains also express the binary toxin Clostridium difficile (CDT). Similar to many other binary toxins, CDT is composed of two components - an enzymatically active component ("binary toxin A" or "CDTa") and a catalytically inert, transport and binding component ( "Binary toxin B" or "CDTb"). The catalytically inert component facilitates the translocation of CDTa into the target cell.
CDTa is endowed with ADP-ribosylating activity, which transfers the ADP-ribose radical of NAD / NADPH to actin monomer (actin G) in the target cell and thus prevents its polymerization in actin F and leading to cytoskeletal rupture. and eventual death of the cell (Sundriyal et al., Protein expression and Purification 74 (2010) 42-48).
WO 2013/112867 (Merck) discloses vaccines directed against Clostridium difficile comprising recombinant toxin A, toxin B and CDTa proteins of C. difficile, all including mutations defined specifically with respect to the native sequences of the toxins which are described as Substantially reducing or eliminating toxicity, in combination with B-toxin (CDTb).
The present inventors have found that CDTb proteins comprising mutations designed to modify the ability to form CDTb pores or to modify the heptamerization capacity of CDTb exhibit improved characteristics. Summary of the invention
In a first aspect of the invention there is provided an immunogenic composition comprising a CDTb protein isolated from Clostridium difficile, wherein the CDTb protein isolated from Clostridium difficile has been mutated to modify the ability to form pores.
In a second aspect of the invention there is provided an immunogenic composition comprising a CDTb protein isolated from Clostridium difficile which is a truncated CDTb protein with deleted signal peptide and prodomain and also the eliminated receptor binding domain and / or the CDTa binding domain eliminated.
In a third aspect of the invention, there is provided an immunogenic composition comprising a Clostridium difficile CDTb protein, wherein the CDTb protein isolated from Clostridium difficile has been mutated to modify the heptamerization capacity.
In a fourth aspect of the invention, there is provided an immunogenic composition comprising a fusion protein comprising a full-length CDTa protein and a CDTb protein.
In a fifth aspect, the present invention provides a vaccine comprising the immunogenic composition of any one of the first four aspects and a pharmaceutically acceptable excipient.
In a sixth aspect, the present invention provides the immunogenic composition of any one of the first four aspects or the vaccine of the fifth aspect, for use in the treatment or prevention of a disease, e.g. . difficult.
In a seventh aspect, the present invention provides the use of the immunogenic composition of any one of the first four aspects or the fifth aspect vaccine in the preparation of a medicament for the prevention or treatment of a disease, for example, a C. difficile disease.
In an eighth aspect, the present invention provides a method of preventing or treating a C. difficile disease comprising administering an immunogenic composition of any one of the first four aspects or the vaccine of the fifth aspect to a mammal subject.
In a ninth aspect, the present invention provides novel polypeptides and nucleotides as defined herein.
In another aspect, the present invention provides a method for treating or preventing an ailment or disease caused in whole or in part by C. difficile. The method comprises administering to a subject in need of a therapeutically effective amount of the proteins as described herein, the immunogenic composition of the invention or vaccines of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 - Cytotoxicity of candidate CDTb alone on HT29 cells: data of example 7. The C37, C123, C124, C126 and C128 referred to in FIG. 1 are the CDTb proteins as described in examples 2 , 4 and 6, whose sequences are presented in the sequence summary (Table A).
Figure 2 - Cytotoxicity of candidate CDTb mixed with whole CDTa on HT29 cells: data of Example 7. The C37, C123, C124, C126 and C128 referred to in Figure 1 are CDTb proteins such as described in Examples 2, 4 and 6, the sequences of which are presented in the sequence summary (Table A). The "whole CDTa" is the CDTa C34 protein whose sequence is presented in the sequence summary (Table A).
Figure 3 - Dynamic light scattering measurements of hydrodynamic radius of CDTb KO constructs for pore formation C123, C126 and CDTb KO construct for C128 heptamerization. The mean radius gives an indication of the homogeneity of the sample, while the peak of radius 1 is an estimate of the hydrodynamic radius of the protein of interest.
Figure 4 - Diagram showing anti-CDTb immunogenicity in mice immunized with C. difficile CDTa or C. difficile CDTb, both formulated with adjuvant.
Figure 5 - Diagram showing anti-CDTa immunogenicity in mice immunized with C. difficile CDTa or C. difficile CDTb, both formulated with adjuvant.
Figure 6 - Titers for inhibition of cytotoxicity in HCT116 cells from mice immunized with C. difficile CDTa or C. difficile CDTb, both formulated with adjuvant.
Figure 7 - Titers for inhibition of cytotoxicity in HT29 cells from mice immunized with C. difficile CDTa or C. difficile CDTb, both formulated with adjuvant.
FIG. 8 - Cytotoxicity of candidate CDTbs alone (FIG. 8a) or mixed with the entire CDTa (FIG. 8b) on HT29 cells: data from example 7. The C37, C123, C124, C126 and C128 are the CDTb proteins such as as described in Examples 2, 4 and 6, whose sequences are presented in the sequence summary (Table A). The "whole CDTa" is the CDTa C34 protein whose sequence is presented in the sequence summary (Table A). C149, C152, C164, C166, C116, C117 are the CDTb proteins as described in Examples 4 and 5.
Figure 9 - Cytotoxicity of candidate CDTb alone (Figure 9a) or mixed with the entire CDTa (Figure 9b) on HCT116 cells (as described in Example 12).
Figure 10 - Cytotoxicity of CDTa-CDTb fusion proteins mixed with whole CDTa activated on HT29 cells (as described in Example 13).
Figure 11 - Cytotoxicity of CDTa-CDTb fusion proteins mixed with whole CDTb on HT29 cells (as described in Example 13).
Figure 12 - Diagram showing anti-CDTa immunogenicity in mice immunized with CDTb proteins or CDTa-CDTb fusions (see Example 14).
Figure 13 - Diagram showing anti-CDTb immunogenicity in mice immunized with CDTb proteins or CDTa-CDTb fusions (see Example 14).
Figure 14 - Titers of inhibition of cytotoxicity in HT29 and HCT116 cells, mice immunized with CDTb proteins or CDTa-CDTb fusions (see Example 15).
detailed description
The binary toxin Clostridium difficile comprises two different proteins, CDTa and CDTb. CDTa comprises two domains, the C-terminal domain which is responsible for ADP-ribosyl-transferase activity while the N-terminal domain is responsible for the interaction with CDTb.
During infection, CDTb is activated by proteolytic cleavage with a chymotrypsin-like protease to produce a CDTb protein lacking the prodomain (termed CDTb-) Please note that CDTb also lacks the signal sequence. of the CDTb. A CDTb protein which lacks the signal sequence but which does not miss the prodomaine is called CDTb '. After proteolytic activation, CDTb becomes oligomerized and binds to CDTa to form the complete C. difficile (CDT) binary toxin. The binding of binary toxin to cellular receptors leads to receptor-mediated endocytosis. As the endosome becomes acidic, the CDTb binding domain undergoes conformational changes that allow the CDTb oligomer to form a pore, and pore formation triggers the translocation of the ADP-ribosyl transferase domain. (CDTa) in the target cell.
In one aspect, the present invention provides an immunogenic composition comprising a CDTb protein isolated from Clostridium difficile that has been mutated to alter the pore-forming ability.
The term "CDTb protein that has been mutated to alter the pore-forming ability" refers to a CDTb protein that has been mutated to alter the ability of the CDTb protein to form a pore.
In one embodiment, the CDTb protein isolated from Clostridium difficile has been mutated to reduce the pore-forming ability.
The ability of pore formation can be analyzed according to the methods described in The J. of Biol. Chem. 2001, vol. 276: 8371-8376, PNAS 2004, vol. 101: 16756-16761, or The J. of Biol. Chem. 2008, vol. 283: 3904-3914
In this aspect, the CDTb protein isolated from Clostridium difficile may have been mutated to avoid formation of the beta-barrel structure which is composed of beta strands shared by each of the CDTb monomers contained in the heptamer that forms in 1 oligomerization of CDTb. In this aspect, the CDTb protein may be a truncated CDTb protein with the deleted signal peptide. In this aspect, the CDTb protein may be a truncated CDTb protein with the signal peptide removed and the prodomaine removed.
The term "truncated CDTb protein with deleted signal peptide" refers to a CDTb protein with substantially all of the signal peptide removed (therefore, which does not include amino acids corresponding to substantially all of the signal peptide). For example, the term "truncated CDTb protein with the deleted signal peptide" refers to a CDTb protein with at least 25 amino acids of the signal peptide removed. There may be a few amino acids in the signal peptide. For example, 2, 5, 10, 15 or 20 amino acids of the signal peptide may remain.
The term "truncated CDTb protein with deleted signal peptide and deleted prodomain" refers to a CDTb protein with essentially all of the signal peptide and prodomain removed (therefore, which does not include amino acids corresponding to substantially all of the peptide signal and essentially the entire prodomaine). For example, the term "truncated CDTb protein with deleted signal peptide and deleted prodomain" refers to a CDTb protein with all of the deleted signal peptide and at least 85 of the prodomain amino acids removed. There may be a few amino acids of the signal peptide and / or prodomain. For example, 2, 5, 10, 15 or 20 amino acids of the signal peptide may remain. For example, 2, 5, 10, 15 or 20 amino acids of the prodomaine may remain. The signal peptide of CDTb corresponds to amino acids 1 to 48 (encompassing amino acids 1 to 42) of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a strain different from C. difficile, for example amino acids 1 to 42 of the CDTb amino acid sequence from strain CD196 (Perelle, M. et al., Infect Immun., 65 (1997), pp. 1402-1407). The CDTb prodomain corresponds to amino acids 48 to 211 (encompassing amino acids 48 to 166) of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a strain different from C. difficile.
In one embodiment, the CDTb protein isolated from Clostridium difficile comprises a mutation at position 455 of SEQ ID NO: 3 or its equivalent in a different strain of C. difficile. In another embodiment of this aspect, the CDTb protein isolated from Clostridium difficile comprises a mutation at position 426 of SEQ ID NO: 3 or its equivalent in a different strain of C. difficile. In another embodiment of this aspect, the CDTb protein isolated from Clostridium difficile comprises a mutation at position 453 of SEQ ID NO: 3 or its equivalent in a different strain of C. difficile. In another embodiment of this aspect, the CDTb protein isolated from Clostridium difficile comprises a mutation at position 426 and a mutation at position 453 of SEQ ID NO: 3 or its equivalent in a strain different from C . difficult.
In one embodiment, the CDTb protein isolated from Clostridium difficile comprises at least one mutation selected from the group consisting of F455R, F455G, E426A and D453A. "F455R" means that phenylalanine (F) at position 455 of the CDTb sequence in SEQ ID NO: 3 is mutated to arginine (R). "F455G" means that phenylalanine (F) at position 455 of the CDTb sequence in SEQ ID NO: 3 is mutated to glycine (G). "E426A" means that glutamate (E) at position 426 of the CDTb sequence in SEQ ID NO: 3 is mutated to alanine (A). "D453A" means that aspartate (D) at position 453 of the CDTb sequence in SEQ ID NO: 3 is mutated to alanine (A). In one embodiment of this aspect, the CDTb protein isolated from Clostridium difficile comprises the F455R mutation or its equivalent in a strain different from C. difficile. In one embodiment of this aspect, the CDTb protein isolated from Clostridium difficile comprises the F455G mutation or its equivalent in a strain different from C. difficile. In one embodiment of this aspect, the CDTb protein isolated from Clostridium difficile comprises mutations E426A and D453A or their equivalents in a different strain of C. difficile.
In one embodiment, the CDTb protein isolated from
Clostridium difficile is or comprises (i) SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 25 or SEQ ID NO: 26; or (ii) a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 25 or SEQ ID NO: 26; or (iii) a fragment of CDTb comprising at least 30, 50, 80,
100, 120, 150, 200, 250 or 300 consecutive amino acids of SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 25 or SEQ ID NO: 26.
In one embodiment, the CDTb protein isolated from
Clostridium difficile is or comprises (i) SEQ ID NO: 43 or SEQ ID NO: 44; or (ii) a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 43 or SEQ ID NO: 44; or (iii) a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250 or 300 consecutive amino acids of SEQ ID NO: 43 or SEQ ID NO: 44.
In such an aspect, there is provided an immunogenic composition in which the CDTb protein isolated from Clostridium difficile is a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 25 or SEQ ID NO: 26.
In such an aspect, there is provided an immunogenic composition in which the CDTb protein isolated from Clostridium difficile is a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 43 or SEQ ID NO: 44.
In another aspect, there is provided an immunogenic composition wherein the CDTb protein isolated from Clostridium difficile is a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400. , 450, 500, 550, 600, 650, 700, 750, 800 or 850 consecutive amino acids of SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 25 or SEQ ID NO: 26.
In another aspect, there is provided an immunogenic composition wherein the CDTb protein isolated from Clostridium difficile is a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400. , 450, 500, 550, 600 or 650 consecutive amino acids of SEQ ID NO: 43 or SEQ ID NO: 44.
The present invention also provides an immunogenic composition comprising a CDTb protein isolated from Clostridium difficile which is a truncated CDTb protein with deleted signal peptide and prodomain and also the eliminated receptor binding domain and / or CDTa binding domain removed. .
The term "deleted truncated CDTb protein with the signal peptide and prodomain and also the deleted receptor binding domain and / or CDTa binding domain" means a truncated CDTb protein with the signal peptide and prodomain removed, in which also the receptor binding domain and / or the CDTa binding domain have been eliminated. It refers to SEQ ID NO: 3 or a fragment or variant thereof with essentially all of the signal peptide and prodomain removed (therefore, which does not include amino acids corresponding to substantially all of the signal peptide and essentially all of the prodomain) and also substantially all of the receptor binding domain removed and / or substantially all of the deleted CDTa binding domain (therefore, which does not include amino acids corresponding essentially to the entire domain receptor binding and / or does not include amino acids corresponding essentially to the entire CDTa binding domain). For example, the term "truncated CDTb protein with deleted signal peptide and deleted prodomain" refers to a CDTb protein with all of the deleted signal peptide and at least 85 of the prodomain amino acids removed. There may be a few amino acids of the signal peptide, prodomain, receptor binding domain, or CDTa binding domain. For example, 2, 5, 10, 15 or 20 amino acids of the signal peptide may remain. For example, 2, 5, 10, 15 or 20 amino acids of the prodomaine may remain. For example, 2, 5, 10, 15 or 20 amino acids of the receptor binding domain may remain. For example, 2, 5, 10, 15 or 20 amino acids of the CDTa binding domain may remain.
The signal peptide of CDTb corresponds to amino acids 1 to 48 (encompassing amino acids 1 to 42) of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a strain different from C. difficile, for example amino acids 1 to 42 of the CDTb amino acid sequence from strain CD196 (Perelle, M. et al., Infect Immun., 65 (1997), pp. 1402-1407).
The CDTb prodomain corresponds to amino acids 48 to 211 (encompassing amino acids 48 to 166) of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a strain different from C. difficile.
In one embodiment, the CDTb receptor binding domain corresponds to amino acids 620 to 876 of SEQ ID NO: 3, or their equivalents in a binary toxin protein isolated from a strain different from C. difficile. . In another embodiment, the receptor binding domain corresponds to amino acids 636 to 876 of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a strain different from C. difficile.
The CDTa binding domain of CDTb corresponds to amino acids 212 to 296 of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a strain different from C. difficile.
In one embodiment of this aspect, the immunogenic composition comprises a CDTb protein isolated from Clostridium difficile which is a truncated CDTb protein with deleted signal peptide and prodomain and also either the deleted receptor binding domain or the CDTa eliminated.
In another embodiment of this aspect, the immunogenic composition comprises a CDTb protein isolated from Clostridium difficile which is a truncated CDTb protein with deleted signal peptide and prodomain and also the eliminated receptor binding domain and the binding domain. CDTa eliminated.
Suitably, the CDTb protein isolated from Clostridium difficile is or comprises (i) SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 37; (ii) a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 37; or (iii) a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or 800 acids consecutive amines of SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 37.
Suitably, the CDTb protein isolated from Clostridium difficile is or comprises (i) SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 37; (ii) a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 37; or (iii) a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 consecutive amino acids of SEQ ID NO: 8 or at least 30, 50, 80 , 100, 120, 150, 200, 250, 300, 350, 400, 450, 500 or 550 consecutive amino acids of SEQ ID NO: 9 or at least 30, 50, 80, 100, 120, 150, 200, 250 or 300 consecutive amino acids of SEQ ID NO: 37.
In such an aspect, there is provided an immunogenic composition in which the CDTb protein isolated from Clostridium difficile is a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 37.
In another aspect, there is provided an immunogenic composition wherein the CDTb protein isolated from Clostridium difficile is a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400. , 450, 500, 550 consecutive amino acids of SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 37.
In a third aspect of the invention, there is provided an immunogenic composition comprising a CDTb protein isolated from Clostridium difficile where the CDTb protein isolated from Clostridium difficile has been mutated to modify the heptamerization capacity.
The CDTb protein that has been mutated to alter the heptamerization capacity means that the CDTb protein has been mutated to alter its ability to self-assemble into a heptamer fold. This heptamerization is enabled by sharing the beta strands from each monomer of the CDTb to form a beta barrel structure. In this aspect, the CDTb protein may be a truncated CDTb protein with the deleted signal peptide. In this aspect, the CDTb protein may be a truncated CDTb protein with the signal peptide removed and the prodomaine removed.
In one embodiment, the CDTb protein isolated from Clostridium difficile has been mutated to reduce the heptamerization capacity.
The ability of the CDTb protein to heptamerize can be measured using a technique such as dynamic light scattering (DLS). DLS can be used to evaluate the hydrodynamic radius in solution of purified CDTb proteins, in addition to providing information on homogeneity and for detecting the presence of high molecular weight aggregates (such as heptamers) within a sample of proteins. This is based on the calculation of the diffusion coefficient of the different species that are obtained by measuring the fluctuation of light scattering, which depends on the molecular size and shape of the proteins, and the other minor constituents of the sample.
Based on a deep analysis of a three-dimensional structural model obtained for CDTb, a loop in the oligomerization domain may be potentially involved in the heptamerization of CDTb. In this loop, four amino acids were identified with potential electrostatic interactions with amino acids contained in other monomers of the heptamer CDTb. These residues are Asp537, Glu539, Asp540 and Lys541 of SEQ ID NO: 3.
In one embodiment, the CDTb protein isolated from Clostridium difficile comprises at least one mutation and / or truncation in the oligomerization domain that modifies the heptamerization capacity.
The oligomerization domain corresponds to amino acids 297 to 619 of SEQ ID NO: 3, or their equivalents in a CDTb protein isolated from a strain different from C. difficile.
In one embodiment, the CDTb protein isolated from Clostridium difficile comprises at least one mutation and / or truncation in the region of CDTb comprising amino acids 533 to 542 of SEQ ID NO: 3 or in the equivalent region in a strain. different from C. difficile.
In one embodiment, the CDTb protein isolated from Clostridium difficile comprises at least one mutation at Asp537, Glu539, Asp540 and Lys541 of SEQ ID NO: 3 or their equivalents in a different strain of C. difficile. In one embodiment, suitably any one or more of Asp537, Glu539, Asp540 and / or Lys541 is / are mutated to Gly or other small residue.
In another embodiment, the oligomerization domain is truncated. For example, the amino acids 533 to 542 of SEQ ID NO: 3, or their equivalents in a CDTb protein isolated from a different strain of C. difficile, can be deleted and possibly replaced by other residues, for example by 3 , 4, 5 Gly and / or Ala residues or more. In one embodiment, the amino acids 533 to 542 of SEQ ID NO: 3, or their equivalents in a CDTb protein isolated from a different strain of C. difficile, can be deleted and possibly replaced by 3 residues Gly or 3 residues. To the.
In one embodiment, the CDTb protein isolated from Clostridium difficile comprises mutations in four amino acids. For example, the CDTb protein comprises the D537G-E539G-D540G-K541G mutation or their equivalents in a strain different from C. difficile. "D537G" means that aspartate at position 537 of the CDTb sequence in SEQ ID NO: 3 is mutated to glycine. "E539G" means that glutamate at position 539 of the CDTb sequence in SEQ ID NO: 3 is mutated to glycine. "D540G" means that the aspartate at position 540 of the CDTb sequence in SEQ ID NO: 3 is mutated to glycine. "K541G" means that the lysine at position 541 of the CDTb sequence in SEQ ID NO: 3 is mutated to glycine.
In another embodiment of this aspect, the CDTb protein isolated from Clostridium difficile comprises a mutation in which amino acids 533 to 542 of SEQ ID NO: 3, or their equivalents in a CDTb protein isolated from a different strain of C difficult, are replaced by 3 Gly residues.
In one embodiment, the CDTb protein isolated from Clostridium difficile is or comprises (i) SEQ ID NO: 12 or SEQ ID NO: 13; or (ii) a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13; or (iii) a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or 850 consecutive amino acids of SEQ ID NO: 12 or SEQ ID NO: 13.
In such an aspect, there is provided an immunogenic composition in which the CDTb protein isolated from Clostridium difficile is a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 13.
In another aspect, there is provided an immunogenic composition wherein the CDTb protein isolated from Clostridium difficile is a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400. , 450, 500, 550, 600, 650, 700, 750, 800 or 850 consecutive amino acids of SEQ ID NO: 12 or SEQ ID NO: 13.
The CDTb and CDTa proteins vary in the amino acid sequence between the different strains, for this reason the numbering of amino acids may differ between strains. For this reason, the term "equivalents in a different strain" refers to amino acids that correspond to those of a reference strain (for example, 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 can thus be numbered differently. An "equivalent" amino acid region can be determined by the alignment of toxin sequences from different strains. Examples of C. difficile strains producing binary toxins include CD196, CCUG 20309, R8637, IS81, IS93, IS51, IS58, R6786, R7605, R10456 and R5989. Unless otherwise indicated, the amino acid numbers provided herein refer to those of the reference strain R20291, as represented by SEQ ID NO: 1 (CDTa) and SEQ ID NO: 3 (CDTb).
In one embodiment, the CDTb protein isolated from Clostridium difficile is a CDTb monomer. In another embodiment, the CDTb protein isolated from Clostridium difficile is a CDTb multimer. In another embodiment, the CDTb protein isolated from Clostridium difficile is a heptamer of CDTb.
In one embodiment, the immunogenic composition does not further include / understand a CDTa protein isolated from Clostridium difficile.
The present invention provides an immunogenic composition comprising a CDTb protein isolated from Clostridium difficile as the only C. difficile antigen. As used herein, the term "as the only C. difficile antigen" means that the immunogenic composition also does not include another antigen from C. difficile, for example an immunogenic composition also not comprising a toxin A protein, toxin B or CDTa.
In one embodiment, the immunogenic composition further comprises a CDTa protein isolated from Clostridium difficile.
Suitably, the CDTa protein isolated from Clostridium difficile is a truncated CDTa protein. "Truncated CDTa protein" as used herein means a CDTa protein that does not reach its full length or correct form, and thus lacks some of the amino acid residues that are present in the full-length CDTA. SEQ ID NO: 1 or an equivalent in a different strain, and which can not perform the function for which it was intended because its structure is incapable, for example the ADP-ribosyl-transferase activity and / or the interaction with the CDTb. An appropriate assay for ADP-ribosyl transferase activity is described in PLOS pathogens 2009, vol 5 (10): el000626.
Suitably, the CDTa protein isolated from Clostridium difficile is a truncated CDTa protein that does not include the C-terminal domain. The term "truncated CDTa protein that does not include the C-terminal domain" refers to a fragment or variant of SEQ ID NO: 1 that does not comprise a substantial portion of the C-terminal domain, there may remain some amino acids of the C-terminal domain, 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 269 to 463 of SEQ ID NO: 1 or their equivalents in a CDTa protein isolated from a strain different from C. difficile. In this embodiment, the truncated CDTa protein of Clostridium difficile is or suitably comprises (i) SEQ ID NO: 18 or SEQ ID NO: 19; (i) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 19; or (iii) a fragment of CDTa comprising at least 30, 50, 80, 100, 120, 150, or 190 consecutive amino acids of SEQ ID NO: 18 or SEQ ID NO: 19.
In one embodiment, the truncated CDTa protein that does not include the C-terminal domain is a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99 %, 100% sequence identity with SEQ ID NO: 20. In another embodiment, the truncated CDTa protein that does not include the C-terminal domain is a variant of CDTa comprising at least 30, 50, 80 , 100, 120, 150, or 190 consecutive amino acids of SEQ ID NO: 20.
In another embodiment of any aspect of the invention, the CDTa protein isolated from Clostridium difficile suitably contains a mutation that reduces its ADP-ribosyl transferase activity. For example, CDTa protein isolated from Clostridium difficile has a glutamate mutation at another amino acid at position 428 of SEQ ID NO: 1 or an equivalent mutation in another strain of C. difficile. The term "mutation at position 428" refers to CDTa proteins that contain a mutation at that exact location but also to a CDTa protein that is isolated from a different strain and that has a mutation at the an equivalent position. The CDTa protein varies in the amino acid sequence between different strains, for this reason the amino acid numbering may differ between strains, so a CDTa protein from a different strain may have a corresponding glutamate that is not the same. number 428 in the sequence. In one embodiment, the CDTa protein isolated from Clostridium difficile has a glutamine to glutamine mutation at position 428 of SEQ ID NO: 1.
In another embodiment of any aspect of the invention, the CDTa protein isolated from Clostridium difficile suitably has a glutamate mutation to a different amino acid at position 430 of SEQ ID NO: 1. or an equivalent mutation in another strain of C. difficile. The term "mutation at position 430" refers to proteins that have this exact location but also to a CDTa protein that is isolated from a different strain and that has a mutation at an equivalent position. In one embodiment, the CDTa protein isolated from Clostridium difficile has a glutamine to glutamine mutation at position 430 of SEQ ID NO: 1.
In another embodiment of any aspect of the invention, the CDTa protein isolated from Clostridium difficile is or suitably comprises (i) SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; or SEQ ID NO: 24; or (ii) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; or SEQ ID NO: 24; or (iii) a fragment of CDTa comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 consecutive amino acids of SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; or SEQ ID NO: 24.
In another embodiment of any aspect of the invention, the CDTa protein isolated from Clostridium difficile is or suitably comprises (i) SEQ ID NO: 22; or (ii) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 22; or (iii) a fragment of CDTa comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 consecutive amino acids of SEQ ID NO: 22.
Fusion Proteins Comprising a CDTa Protein and a CDTb Protein
In another aspect, the invention provides immunogenic compositions comprising a fusion protein comprising a full-length CDTa protein and a CDTb protein. In this aspect, "full-length CDTa" means the full-length CDTa of SEQ ID NO: 36 or the equivalent in another strain of C. difficile, or a CDTa variant exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 36
A "fusion polypeptide" or "fusion protein" refers to a protein having at least two heterologous polypeptides (e.g., at least two Clostridium difficile polypeptides) covalently linked, either directly or via a linker ("linker") of amino acids. This can also refer to a protein comprising at least two non-covalently linked heterologous polypeptides. Polypeptides forming the fusion protein are generally C-terminally linked at the N-terminus, although they may be C-terminal to C-terminally related to the C-terminus. N-terminus at the N-terminus, or N-terminus at the C-terminus. The polypeptides of the fusion protein may be in any order. This term also refers to conservatively modified variants, polymorphic variants, alleles, mutants, immunogenic fragments, and inter-species homologs of the antigens that make up the fusion protein.
The term "fused" refers to the linkage, for example the covalent linkage between two polypeptides in a fusion protein. The polypeptides are generally joined via a peptide bond, either directly to each other or via a linker ("linker") of amino acids. Optionally, the peptides may be joined via nonpeptide covalent bonds known to those skilled in the art.
A peptide linker sequence can be employed to separate the first and second peptide components a sufficient distance 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. The appropriate sequences of the peptide linkers can be selected 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 could react with the functional epitopes of the polypeptides. The sequences of the preferred peptide linkers contain Gly, Asn and Ser residues. Other near-neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that can 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. Patent No. 4,935,233 and US Patent 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 functional domains and prevent steric interference.
In this aspect, the fusion protein may suitably include a linker. An example of a suitable linker comprises or consists of six consecutive Gly residues.
In this aspect, the CDTa protein is suitably first in fusion. This means that the CDTa protein is placed in the N-terminal position of the fusion protein with respect to the position of the CDTb protein. This improves the processability of the CDTa-CDTb fusion.
In one embodiment, the CDTa protein comprises a mutation of cysteine to another amino acid at position 45 of SEQ ID NO: 1 or the equivalent amino acid in another strain of C. difficile. The term "mutation at position 45" refers to CDTa proteins that have a mutation at this exact location of SEQ ID NO: 1 but also to a CDTa protein that is isolated from a different strain and that has a mutation at an equivalent position. In one embodiment, the CDTa protein has a cysteine tyrosine mutation at position 45. This mutation is performed to prevent / avoid the formation of a disulfide bridge between two CDTa proteins.
In one embodiment, the full-length CDTa protein comprises one or more mutations that reduce its ADP-ribosyl transferase activity.
For example, the CDTa protein may comprise any of the sets of mutations disclosed in WO 2013/112867. For example, the CDTa protein may comprise at least two mutations selected from the following mutations at positions 2, 67, 69, 253, 255, 258, 302, 307, 342, 345, 356, 359, 382, 385 and 387 (corresponding to positions 3, 68, 70, 254, 256, 259, 303, 308, 343, 346, 357, 360, 383, 386 and 388 of the sequence of SEQ ID NO: 36 of the present application :) C2A, Y67A, Y69A, Y253A, R255A, Y258A, R302A, Q307E, N342A, S345F, F356A, R359A, Y382A, E385Q, E385A, E387Q, and E387D as disclosed in WO2013 / 112867. "C2A" means that cysteine at position 2 of the CDTb protein sequence disclosed in WO 2013/112867 (corresponding to position 3 of SEQ ID NO: 36 of the present application) is mutated to alanine, and the other mutations listed in this group will have to be understood in a similar way. In some embodiments, CDTa mutations are selected from: C2A, R302A, S345F, E385Q, E385A, E387Q, and E387D. In embodiments of this aspect of the invention, the CDTa protein comprises a set of mutations selected from the group consisting of: (a) S345F, E385Q and E387Q; (b) R302A, E385A, E387D; (c) C2A, S345F, E385Q and E387Q; (d) R302A, S345F, E385Q and E387Q; (e) Y67A, Y69A, and R255A; (f) R359A, Y67A, and Q307E; (g) Y258A, F356A, S345F; and (h) N342A, Y253A and Y382A.
In one embodiment, the CDTa protein comprises a mutation that reduces its ADP-ribosyl transferase activity.
The term "the CDTa protein comprises a mutation that reduces its ADP-ribosyl transferase activity" refers to a CDTa protein that includes a mutation at a single position that is only intended to reduce its ADP-ribosyl transferase activity. The CDTa protein may optionally include other mutations not specifically intended to reduce its ADP-ribosyl transferase activity. Mutations specifically intended to reduce ADP-ribosyl transferase activity include E428Q and E430Q (amino acid numbering with reference to SEQ ID NO: 1) or C2A, Y67A, Y69A, Y253A, R255A, Y258A, R302A, Q307E, N342A. , S345F, F356A, R359A, Y382A, E385Q, E385A, E387Q, and E387D as disclosed in WO 2013/112867 (amino acid numbering corresponding to positions 3, 68, 70, 254, 256, 259, 303, 308 , 343, 346, 357, 360, 383, 386 and 388 with reference to SEQ ID NO: 36).
For example, the CDTa protein has a mutation of glutamate to another amino acid at position 428 of SEQ ID NO: 1 or the equivalent amino acid in another strain of C. difficile. The term "mutation at position 428" refers to CDTa proteins that have a mutation at this exact location of SEQ ID NO: 1 but also to a CDTa protein that is isolated from a different strain and that has a mutation at an equivalent position. The CDTa protein varies in the amino acid sequence between the different strains, for this reason the amino acid numbering may differ between strains, so a CDTa protein from a different strain may have a corresponding glutamate that is not the number 428 in the sequence. In one embodiment, the CDTa protein has a mutation of glutamate to glutamine at position 428. In another embodiment, the CDTa protein suitably includes a mutation of glutamate to a different amino acid at the position 430 of SEQ ID NO: 1 or the equivalent amino acid in another strain of C. difficile. The term "mutation at position 430" refers to proteins that have this exact location but also to a CDTa protein that is isolated from a different strain and that has a mutation at an equivalent position. In one embodiment, the CDTa protein has a mutation of glutamate to glutamine at position 430.
In one embodiment, the full-length CDTa protein is or suitably comprises (i) SEQ ID NO: 39; or (ii) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 39; or (iii) a fragment of CDTa comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 consecutive amino acids of SEQ ID NO: 39.
In another embodiment, the full-length CDTa protein is or suitably comprises. (i) an amino acid sequence as represented by SEQ ID NO: 40, 42 or 67 in WO 2013/112867; or (ii) a variant of CDTa having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with any of the sequences in (i); or (iii) a fragment of CDTa comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 consecutive amino acids of any of the sequences in (i).
In one embodiment, the CDTb protein is a full-length CDTb protein. In this aspect, "full-length CDTb" means the full-length CDTb of SEQ ID NO: 7, i.e. CDTb with the eliminated prodomaine.
In this embodiment of this aspect, the fusion protein comprising a full-length CDTa protein and a CDTb protein is or suitably comprises (i) SEQ ID NO: 14; or (ii) a variant fusion protein having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 14; or (iii) a moiety of at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or 850 amino acids consecutive of SEQ ID NO: 14.
In another embodiment, the CDTb protein is a truncated CDTb protein with the CDTa binding domain removed. The term "with the CDTa binding domain removed" refers to a fragment or variant of SEQ ID NO: 3, or its equivalent in another strain of C. difficile, with essentially the entire binding domain at the same time. CDTa eliminated (therefore, which does not include amino acids corresponding to essentially the entire CDTa binding domain). For example, the term "truncated CDTb protein with deleted CDTa binding domain" refers to a CDTb protein having at least 65 amino acids of the CDTa binding domain removed. There may still be a few amino acids in the CDTa binding domain. For example, 2, 5, 10, 15 or 20 amino acids of the CDTa binding domain may remain.
The CDTa binding domain of CDTb corresponds to amino acids 212 to 295 of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a strain different from C. difficile.
In this embodiment, the truncated CDTb protein with the removed CDTa binding domain is or suitably comprises (i) SEQ ID NO: 9; or (ii) a variant fusion protein having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 9; or (iii) a fragment comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 consecutive amino acids of SEQ ID NO: 9.
In this embodiment, the fusion protein comprising a full length CDTa protein and a truncated CDTb protein with the removed CDTa binding domain is or suitably comprises (i) SEQ ID NO: 15; or (ii) a variant fusion protein having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 15; or (iii) a moiety of at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or 850 amino acids consecutive of SEQ ID NO: 15.
In another embodiment, the 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, or its equivalents in another strain of C. difficile, with essentially all but the binding domain. at the eliminated receptors (therefore, which does not include amino acids corresponding to essentially all of the protein except the receptor binding domain), there may remain a few amino acids in addition to the receptor binding domain, for example, 2, 5, 10, 15 or 20 amino acids except / in addition to the receptor binding domain. In one version, the receptor binding domain corresponds to amino acids 620 to 876 of SEQ ID NO: 3, or their equivalents in a binary toxin protein isolated from a strain different from C. difficile. In another version, the receptor binding domain corresponds to amino acids 636 to 876 of SEQ ID NO: 3 or their equivalents in a binary toxin protein isolated from a strain different from C. difficile.
In this embodiment, the CDTb protein is or suitably comprises (i) SEQ ID NO: 27 or SEQ ID NO: 28; or (i) a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 27 or SEQ ID NO: 28; or (iii) a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150 or 200 consecutive amino acids of SEQ ID NO: 27 or SEQ ID NO: 28.
In this embodiment, the fusion protein comprising a full length CDTa protein and a truncated CDTb protein comprising the receptor binding domain is or suitably comprises (i) SEQ ID NO: 16 or SEQ ID NO: 17; or (ii) a variant fusion protein having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 16 or SEQ ID NO: 17; or (iii) a fragment comprising at least 30, 50, 80, 100, 120, 150, 200, 250, 300, 350 or 400 consecutive amino acids of SEQ ID NO: 16 or SEQ ID NO: 17.
In another aspect, the invention provides novel polypeptides and nucleotides as defined herein.
In one embodiment, the invention provides a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 25, 26, 37, 43, 44, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 66, 67 or 71.
In one embodiment of any aspect of the invention, the immunogenic composition elicits antibodies that neutralize CDTa or CDTb or both. In another embodiment, the composition triggers antibodies that neutralize the binary toxin. If a composition triggers antibodies against a toxin, these can be measured by immunizing mice with the immunogenic composition, collecting the sera and analyzing the anti-toxin titers of the sera using an ELISA. The sera should be compared to a reference sample obtained from mice that have not been immunized. The composition of the invention elicits antibodies which neutralize CDTa if the sera directed against the polypeptide give an ELISA result of more than 10%, 20%, 30%, 50%, 70%, 80%, 90%, or 100%. % higher than the reference sample.
In another embodiment, the immunogenic composition of the invention elicits a protective immune response in a mammalian host against C. difficile strains. In one embodiment, the mammalian host is selected from the group consisting of mice, rabbits, guinea pigs, non-human primates, monkeys, and humans. In one embodiment, the mammalian host is a mouse. In another embodiment, the mammalian host is a human being.
If an immunogenic composition elicits a protective immune response in a mammalian host against C. difficile strains, it can be determined using a challenge test. In such a test, the mammalian host is vaccinated with the immunogenic and stimulated composition ("challenge") by exposure to C. difficile, the time during which the mammal survives after stimulation ("challenge") is compared to the time during which the reference mammal that has not been immunized with the immunogenic composition survives. An immunogenic composition elicits a protective immune response if a mammal immunized with the immunogenic composition survives at least 10%, 20%, 30%, 50%, 80%, 80%, 90%, or 100% longer than a mammal. reference that was not immunized after stimulation ("challenge") with C. difficile.
Toxin A and Toxin B
In one embodiment of any aspect of the invention, the immunogenic compositions of the invention further comprise toxin A protein isolated from Clostridium difficile and / or toxin B protein isolated from C. difficile.
The term "toxin A protein isolated from Clostridium difficile" refers to a protein with the amino acid sequence of SEQ ID NO: 29, or a fragment or variant of SEQ ID NO: 29. In one embodiment, the toxin A protein isolated from Clostridium difficile is a fragment comprising 50, 100, 150, 200, 250, 300, 500, 750, 1000, 1250, 1500, 1750, 2000 or 2500 consecutive amino acids of SEQ ID NO: 29. In one embodiment, toxin A protein isolated from Clostridium difficile is a variant having 80%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity with SEQ ID. NO: 29.
The term "toxin B protein isolated from Clostridium difficile" refers to a protein with the amino acid sequence of SEQ ID NO: 30, or a fragment or variant of SEQ ID NO: 30. In one embodiment , toxin B protein isolated from Clostridium difficile is a fragment comprising 50, 100, 150, 200, 250, 300, 500, 750, 1000, 1250, 1500, 1750 or 2000 consecutive amino acids of SEQ ID NO: 30. one embodiment, toxin B protein isolated from Clostridium difficile is a variant having 80%, 85%, 90%, 92%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 30 .
In one embodiment, toxin A protein isolated from Clostridium difficile comprises a repetitive domain fragment. The term "repetitive domain of toxin A" refers to the C-terminal domain of toxin A protein from C. difficile, including repeats. The repetitive domain of toxin A refers to amino acids 1832 to 2710 of toxin A from strain VPI10463 (ATCC43255) and their equivalents in a different strain. The amino acid sequence 1832 to 2710 from strain VPI10463 (ATCC43255) corresponds to amino acids 1832 to 2710 of SEQ ID NO: 29.
In another embodiment, the toxin A protein isolated from Clostridium difficile comprises a fragment of the N-terminal domain of toxin A. The N-terminal domain of toxin A relates to amino acids 1 to 1831 of toxin A from strain VPI10463 (ATCC43255) and their equivalents in a different strain. The amino acid sequence 1 to 1831 of toxin A from strain VPI10463 (ATCC43255) corresponds to amino acids 1 to 1831 of SEQ ID NO: 29.
In one embodiment, toxin B protein isolated from Clostridium difficile comprises a repetitive domain fragment of toxin B. The term "repetitive domain of toxin B" refers to the C-terminal domain of toxin B protein from from C. difficile. This field relates to amino acids 1834 to 2366 from strain VPI10463 (ATCC43255) and their equivalents in a different strain. The amino acid sequence 1834 to 2366 from strain VPI10463 (ATCC43255) corresponds to amino acids 1834 to 2366 of SEQ ID NO: 30.
In another embodiment, the toxin B protein isolated from Clostridium difficile comprises a fragment of the N-terminal domain of toxin B. The N-terminal domain of toxin B refers to amino acids 1 to 1833 of toxin B from strain VBI10463 (ATCC43255) and their equivalents in a different strain. The amino acid sequence 1 to 1833 of toxin B from strain VBI10463 (ATCC43255) corresponds to amino acids 1 to 1833 of SEQ ID NO: 30.
C. difficile toxins A and B are conserved proteins, however, the sequence differs in a small amount between strains, in addition the amino acid sequence for toxins A and B in different strains may differ in the number amino acids.
For these reasons, the terms repetitive domain of toxin A and / or repetitive domain of toxin B may refer to a sequence which is a variant having 90%, 95%, 98%, 99% or 100% identity of sequence with amino acids 1832 to 2710 of SEQ ID NO: 29 or a variant having 90%, 95%, 98%, 99% or 100% sequence identity with amino acids 1834 to 2366 of SEQ ID NO: 30 Similarly, the terms N-terminal domain of toxin A and / or N-terminal domain of toxin B refer to a sequence which is a variant having 90%, 95%, 98%, 99% or 100% sequence identity with amino acids 1 to 1831 of SEQ ID NO: 29 or a variant having 90%, 95%, 98%, 99% or 100% sequence identity with amino acids 1 to 1833 of SEQ ID NO: 30
In addition, 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 that correspond to those of a reference strain (eg, C. difficile VPI10463), but found in a toxin from a strain different and which can thus be numbered differently. An "equivalent" amino acid region can be determined by aligning toxin sequences from different strains. Amino acid numbers provided herein refer to those of strain VPI10463 (and are as represented by SEQ ID NO: 29 and SEQ ID NO: 30).
In another embodiment of any aspect of the invention, the toxin protein A isolated from C. difficile and the toxin protein B isolated from C. difficile form a fusion protein. In one embodiment, the fusion protein is 80%, 85%, 90%, 95%, 98%, 99% or 100% identical with a sequence selected from the group consisting of SEQ ID NO: 31, 32, 33, 34, and 35. In another embodiment, the fusion protein is a fragment of at least 800, 850, 900 or 950 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 31 , 32, 33, 34, and 35.
fragments
The term "fragment" as defined herein may refer to a fragment comprising a T cell epitope. T cell epitopes are short consecutive sequences of amino acids that are recognized by T cells (e.g., T cells). CD4 + or CD8 +). The identification of T cell epitopes can be obtained using epitope mapping experiments which are well known to those skilled in the art (see, for example, Paul, Fundamental Immunology, 3rd ed., 243-247 (1993), Beipbarth et al., Bioinformatics 2005 21 (Suppl 1): 29-37).
The term "fragment" when used in connection with a polypeptide comprising a histidine tag comprises the polypeptide without the histidine tag, for example from which the histidine tag has been cleaved. For example, suitable fragments of polypeptides comprising an amino acid sequence represented by any of ID NO: 5, 7-28 or 36-42 (each of which comprises a histidine tag) include the corresponding polypeptide without the marker histidine represented by sequences ID NO: 47 to 76, as presented in the following agreement:
Suitably, the fragments of the invention are immunogenic fragments. The "immunogenic fragments" according to the present invention will generally comprise at least 9 consecutive amino acids from the full-length (e.g., at least 10) polypeptide sequence, such as at least 12 consecutive amino acids (e.g., at least 15 or more 20 consecutive amino acids), in particular at least 50 consecutive amino acids, such as at least 100 consecutive amino acids (for example, at least 200 consecutive amino acids). Suitably, the immunogenic fragments will comprise 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 should be understood that in a diverse non-consanguineous population, such as humans, the different HLA types mean that specific epitopes may not be recognized by all members of the population. Therefore, to maximize the recognition rate and scale of the immune response against a polypeptide, it is generally desirable that an immunogenic fragment contain a plurality of epitopes from the full-length sequence (suitably all epitopes).
Variants "Variants" or "conservatively modified variants" apply to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, the conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences. When the nucleic acid does not encode an amino acid sequence, "conservatively modified variants" refer to essentially identical sequences.
With regard to variants of a protein sequence, those skilled in the art will understand that individual substitutions, deletions, or additions to the polypeptide that modify, add, or delete a single amino acid or a small percentage of acids is a "conservatively modified variant" where the modification (s) result (s) in the substitution of an amino acid by a functionally similar amino acid or the substitution / deletion / addition of residues that do not have substantial impact on the biological function of the variant.
Conservative substitution tables providing functionally similar amino acids are well known in the state of the art. Such conservatively modified variants are in addition to and do not exclude the polymorphic variants, interspecies homologues, 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 to 50, such as 1 to 25, especially 1 to 10, and especially 1 acid residue amine can be modified) when compared to the reference sequence. In general, such conservative substitutions are within one of the amino acid groups specified below, although under certain 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 to 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 therefore do not have a significant impact on the immunogenic properties of the antigen.
Polypeptide variants may also include those in which additional amino acids are inserted relative to the reference sequence, for example, such insertions may occur at 1 to 10 locations (such as 1 to 5 locations, suitably 1 or 2 locations, particularly 1 location) and may, for example, involve the addition of at least 50 amino acids at each location (such as 20 or less, especially 10 or less, especially 5 or less). Conveniently, such insertions do not occur in the region of an epitope, and therefore do not have a significant impact on the immunogenic properties of the antigen. An exemplary insertion includes a short sequence of histidine residues (eg, 2 to 6 residues) to assist the expression and / or purification of the antigen in question.
Polypeptide variants include those in which amino acids have been deleted compared to the reference sequence, for example, such deletions may occur at 1 to 10 locations (such as 1 to 5 locations, suitably 1 or 2 locations in particular 1 location) and may, for example, involve deletion of 50 amino acids or less at each location (such as 20 or less, especially 10 or less, especially 5 or less). Suitably, such deletions do not occur in the region of an epitope, and therefore do not have a significant impact on the immunogenic properties of the antigen. It will be understood by those skilled in the art that a particular polypeptide variant may include substitutions, deletions, and additions (or any combination thereof).
The variants preferably have at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity (as at least about 95%, at least about 98% or at least about 99%) with the associated reference sequence.
The terms "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are identical or that contain a specified percentage of Nucleic acid or nucleotide residues that are identical (ie, 70% identity, possibly 75%, 80%, 85%, 90%, 95%, 98% or 99% identity over a specified region), when comparing and aligning for maximum match on a comparison window, or a designated region as measured using, for example, sequence comparison algorithms or manual alignment and a visual examination. Such sequences are then said to be "essentially identical". This definition also relates to the complement of a sequence to be tested. Optionally, the identity exists over a region that is at least about 25 to about 50 amino acids or nucleotides in length, or optionally at a region that is 75 to 100 amino acids or nucleotides in length. Suitably, the comparison is made on a window corresponding to the entire length of the reference sequence.
For sequence comparison, generally a sequence acts as a reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the sub-sequence coordinates are designated, if necessary, and the program parameters of the sequence algorithm. are designated. The program's default settings can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the sequence identity percentages for the test sequences relative to the reference sequence, based on the program parameters.
A "comparison window" as used herein refers to a segment in which a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. The sequence alignment methods for comparison are well known in the state of the art. The optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the Needleman & homology alignment algorithm. Wunsch, J. Mol. Biol. 48: 443 (1970), by the 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, WI), or by a manual alignment and visual examination (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds., 1995 supplement)).
An example of a useful algorithm is PILEUP. PILEUP creates an alignment of multiple sequences from a group of related sequences using progressive pairwise alignments to show the relationship and percentage of sequence identity. It also traces a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment process of Feng & Doolittle, J. Mol. Evol. 35: 351-360 (1987). The process used is similar to the process described by Higgins & Sharp, CABIOS 5: 151-153 (1989). The program can align up to 300 sequences, each with a maximum length of 5000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a group of two aligned sequences. This group is then aligned with the next most related sequence or group of aligned sequences. Two groups of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is obtained by a series of progressive alignments in pairs. The program is executed by designating specific sequences and their amino acid or nucleotide coordinates for sequence comparison regions and designating the parameters of the program. Using PILEUP, a reference sequence is compared to other test sequences to determine the relationship of percent sequence identity using the following parameters: default gap weight (3.00), gap length weight default (0.10), and weighted extremity gaps. PILEUP can be obtained from the GCG sequence analysis package, for example, version 7.0 (Devereaux et al., Acid Res., 12: 387-395 (1984).
Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nue. Acids Res. 25: 33893402 (1977) and Altschul et al., J. Mol. Biol. 215: 403-410 (1990), respectively. The BLAST analysis software is available to the public through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov/). This algorithm first involves identifying high score sequence pairs (HSPs) by identifying short words of length W in the request sequence, which either match or satisfy a certain positive value threshold score T when they are aligned with a word of the same length in a sequence of the database. T is referred to as the score threshold of the neighboring word (Altschul et al., Supra). These initial matches of neighboring words act like seeds to initiate searches to find longer HSPs containing them. The word matches are extended in both directions along each sequence as far as the cumulative alignment score can be increased. The cumulative scores are calculated using, for the nucleotide sequences, the parameters M (reward score for a pair of corresponding residues, always> 0) and N (penalty score for the residues that do not correspond, always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word matches in each direction is stopped when: the cumulative alignment score is an amount X outside its maximum value obtained; the cumulative score goes to zero or below because of the accumulation of one or more negative-scoring residue alignments; or the end of one or the other sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The .BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4 and a comparison of the two strands. For amino acid sequences, the BLASTP program uses by default a word length of 3, and an expectation (E) of 10, and the score matrix BLOSUM62 (see Henikoff & Henikoff, Proc Natl Acad Sci USA 89: 10915 (1989)), alignments (B) of 50, expectation (E) of 10, M = 5, N = -4, and comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc Natl Acad Sci USA 90: 5873-5787 (1993)). A measure of similarity provided by the BLAST algorithm is the smallest sum of the probabilities (P (N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences might occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum of the probabilities in a comparison of the nucleic acid to be tested with the reference nucleic acid is less than about 0.2, more preferably preferred less than about 0.01, and most preferably less than about 0.001.
IDENTIFICATION AND CHARACTERIZATION OF POLYNUCLEOTIDES
Polynucleotides encoding CDTa, CDTb, toxin A and Clostridium difficile 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 can be identified, as described in more detail below, by screening a cDNA microarray. Such screens may be performed, for example, using a Synteni microchip (Palo Alto, CA) 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, the polynucleotides can be amplified from cDNA prepared from cells expressing the proteins described herein, such as M. tuberculosis cells. These polynucleotides can be amplified via a polymerase chain reaction (PCR). For this approach, sequence-specific primers can be designed based on the sequences provided herein, and can be purchased or synthesized.
An amplified portion of a polynucleotide can be used to isolate a full length gene from a suitable library (eg, a M. tuberculosis cDNA library) using well known techniques. In these techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is chosen by size to include larger molecules. Randomly initiated libraries may also be preferred for identifying regions 5 'and upstream of the genes.
Genomic libraries are preferred for obtaining introns and extending sequences in 5 '.
For hybridization techniques, a partial sequence may be labeled (eg by cleavage displacement or end labeling by 32P) using well known techniques. A bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or mats containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual (2000)). . The hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. The cDNA clones can be analyzed to determine the amount of additional sequence, for example, by PCR using a primer derived from the partial sequence and a primer derived from the vector. Restriction maps and partial sequences can be generated to identify one or more overlapping clones. The complete sequence can then be determined using standard techniques, which may involve the production of 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 produced by ligation of the appropriate fragments, using well known techniques.
Alternatively, there are many amplification techniques for obtaining a full-length coding sequence from a partial cDNA sequence. In these techniques, amplification is generally performed via PCR. Any of a variety of commercially available kits can be used to perform the amplification step. The primers can be designed using, for example, well-known software of the state of the art. The primers are preferably 22 to 30 nucleotides long, have a GC content of at least 50% and hybridize to the target sequence at temperatures of about 68 ° C to 72 ° C. The amplified region can be sequenced as described above, and the overlapping sequences are assembled into a contiguous sequence.
One such amplification technique is reverse PCR (see Triglia et al., Nucl Acids Res 16: 8186 (1988)), which uses restriction enzymes to produce a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. In this approach variant, the sequences adjacent to a partial sequence can be recovered by amplification with a primer specific for a linker sequence and a primer specific for a known region. The amplified sequences are generally subjected to a second amplification series with the same linker primer ("linker") and a second specific primer of the known region. A variation of this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another technique of this type is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an internal primer and an outer primer, which hybridizes to a polyA region or a vector sequence, to identify sequences that are 5 'and 3' of a known sequence. Other techniques include capture PCR (Lagerstrom et al., PCR Methods Applicant 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 some cases, it is possible to obtain a full-length cDNA sequence by sequence analysis provided in an expressed sequence marker (EST) database, such as that available from GenBank. Overlapping EST searches may be generally performed using well-known programs (eg, NCBI BLAST searches), and these ESTs may be used to generate a contiguous full-length sequence. Full length DNA sequences can also be obtained by genomic fragment analysis.
EXPRESSION OF POLYNUCLEOTIDES IN HOST CELLS
Polynucleotide sequences or fragments thereof that encode clostridium difficile CDTa, CDTb, toxin A, and toxin B proteins, or fusion proteins or their functional equivalents, may be used in recombinant DNA molecules. to direct the expression of a polypeptide in appropriate host cells. Due to the intrinsic degeneracy of the genetic code, other DNA sequences that encode substantially the same amino acid sequence or a functionally equivalent amino acid sequence can be produced and these sequences can be used to clone and express a given polypeptide.
As will be appreciated by those skilled in the art, it may be advantageous in some cases to produce nucleotide sequences encoding polypeptides that have non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host may be selected to increase the level of expression of the protein or to produce a recombinant RNA transcript with desirable properties, such as a half-life that is longer than that of a transcript produced from the sequence existing in the natural state.
In addition, the polynucleotide sequences may be modified using methods generally known in the art to modify the polypeptide coding sequences for a variety of reasons, including but not limited to, changes that alter cloning. , the treatment, and / or the expression of the gene product. For example, DNA rearrangement by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to modify nucleotide sequences. In addition, site-directed mutagenesis can be used to insert new restriction sites, modify glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so on.
Natural, modified, or recombinant nucleic acid sequences can be ligated into a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to code for a chimeric protein that can be recognized by a commercially available antibody. A fusion protein may also be modified to contain a cleavage site located between the coding sequence of the polypeptide and the sequence of the heterologous protein, so that the polypeptide may be cleaved and purified away from the heterologous radical.
Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using well-known chemical methods of the state of the art (see Caruthers, MH et al., Nucl Acids Res, Symp Ser. pp. 215223 (1980), Horn et al., Nucl Acids Res Symp., Ser., pp. 225232 (1980)). Alternatively, the protein itself can be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or part thereof. For example, peptide synthesis can be performed using various solid phase techniques (Roberge et al., Science 269: 202-204 (1995)) and automated synthesis can be achieved, for example, using the peptide synthesizer ABI 431A (Perkin Elmer, Palo Alto, CA).
A newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (eg Creighton, Proteins, Structures and Molecular Principles (1983)) or other comparable techniques available in the state of the art. The composition of the synthetic peptides can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Alternatively, the amino acid sequence of a polypeptide, or any part thereof, may be modified during direct and / or combined synthesis 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 coding for the polypeptide, or functional equivalents, may be inserted into an appropriate expression vector, i.e., a vector that contains the elements necessary for transcription and translating the inserted coding sequence. The methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences containing the coding sequences for a polypeptide of interest and the appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. These techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (2000), and Ausubel et al., Current Protocols in Molecular Biology (updated annually).
Various expression vector / host systems can be used to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with bacteriophage, plasmid or cosmid recombinant DNA expression vectors; yeasts transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculoviruses); plant cell systems transformed with virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or by bacterial expression vectors (eg for example, Ti or pBR322 plasmids); or animal cell systems.
The "control elements" or "regulatory sequences" present in an expression vector are those untranslated regions of the vector - amplifiers, promoters, 5 'and 3' untranslated regions - which interact with the cellular proteins of the host for transcription and translation. These elements can vary in strength and specificity. Depending on the vector system and host used, any number of suitable transcriptional and translational control elements, including constitutive and inducible promoters, may be used. For example, upon cloning into bacterial systems, inducible promoters, such as the PBLUESCRIPT phagemid lacZ hybrid promoter (Stratagene, La Jolla, Calif.) Or the PSP0RT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like may be used. In mammalian cell systems, promoters from mammalian genes or mammalian viruses are generally preferred. If it is necessary to produce a cell line that contains multiple copies of the polypeptide coding sequence, vectors based on SV40 or EBV can be advantageously used with an appropriate selectable marker.
In bacterial systems, a number of expression vectors may be selected according to the intended use for the expressed polypeptide. For example, when large amounts are needed, for example for induction of antibodies, vectors that direct high-level expression of fusion proteins that are easily purified, can be used. These vectors include, but are not limited to, multifunctional cloning and expression vectors of E. coli such as BLUESCRIPT (Stratagene), in which the coding sequence for the polypeptide of interest can be ligated into the vector in the reading frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase, although that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster, J. Biol Chem 264: 5503-5509 (1989)); and the like. The pGEX vectors (Promega, Madison, Wis, GE Healthcare) can also be used to express foreign polypeptides in the form of fusion proteins with glutathione S-transferase (GST). In general, these fusion proteins are soluble and can be easily purified from lysed cells by adsorption on glutathione-agarose beads followed by elution in the presence of free glutathione. The proteins made in these systems may be designed to include heparin, thrombin, factor XA protease cleavage sites so that the cloned polypeptide of interest may be released from the GST moiety at will.
In yeasts, Saccharomyces cerevisiae or Pichia as Pichia pastoris for example, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. Other vectors containing constitutive or inducible promoters include GAP, PGK, GAL and ADH. For descriptions, see Ausubel et al. (above) 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 the coding sequences for the polypeptides may be directed by any one of a number of promoters. For example, viral promoters such as 35S and 19S promoters of CaMV may be used alone or in combination with the TMV-derived omega leader (Takamatsu, EMBO J. 6: 307-311 (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can 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. These techniques are described in a number of generally available descriptions (see, for example, 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 such a 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 coding sequences for the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the coding sequence for the polypeptide will render the polyhedrin gene inactive and produce a recombinant virus lacking the envelope protein. The recombinant viruses can then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest can be expressed (Engelhard et al., Proc Natl Acad Sci USA 91 3224-3227 (1994)).
In mammalian host cells, a number of virus-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, the sequences coding for a polypeptide of interest can be ligated into a transcription / translation complex of the adenovirus consisting of the late promoter and the sequence tripartite head. Insertion into an E1 or E3 non-essential region of the viral genome can be used to obtain a viable virus that is capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc Natl Acad Sci USA 81: 3655-3659 (1984)). In addition, transcriptional enhancers, such as the Rous Sarcoma Virus (RSV) enhancer, can be used to increase expression in mammalian host cells. Methods and protocols for working with adenovirus vectors are described in Wold, Adenovirus Methods and Protocols, 1998. Additional references regarding the use of adenovirus vectors can be found in Adenovirus: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References, 2004.
Specific initiation signals can also be used to obtain a more efficient translation of the sequences encoding a polypeptide of interest. These signals include the ATG initiation codon and the 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 translation control signal is required. However, in cases where only a coding sequence, or a portion thereof, is inserted, exogenous translation control signals including the ATG initiation codon will have to be provided. In addition, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. The elements of the exogenous translation and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers that are appropriate for the particular cellular system that is used, such as those described in the literature (Scharf, et al., Results Probl Cell Differ. 125-162 (1994)).
In addition, a host cell strain may be chosen for its ability to modulate the expression of inserted sequences or to process the expressed protein in the desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves a "prepro" form of the protein can also be used to facilitate proper insertion, folding and / or function. Different host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which include specific cell machinery and mechanisms characteristic of 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 that stably express a polynucleotide of interest can be transformed using expression vectors that can contain viral origins of replication and / or endogenous expression elements and a selectable marker gene on the same vector or on a separate vector. Following the introduction of the vector, the cells can be left to grow for 1 to 2 days in enriched medium before passing them to a selective medium. The objective of the selectable marker is to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, herpes simplex virus thymidine kinase genes (Wigler et al., Cell 11: 223-32 (1977)) and adenine phosphoribosyl transferase (Lowy et al. al., Cell 22: 817-23 (1990)) which can be employed in tk.sup cells. or after, respectively. In addition, resistance to antimetabolites, antibiotics or herbicides 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 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). Other selectable genes have been described, for example, trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol instead of histidine. (Hartman & Mulligan, Proc Natl Acad Sci USA 85: 8047-51 (1988)). Recently, the use of visible markers has gained some popularity with markers such 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 expression of a transient or stable protein attributable to a specific vector system (Rhodes et al., Mol Mol Methods 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 coding sequence for a polypeptide is inserted within the sequence of a marker gene, the recombinant cells containing sequences can be identified by the absence of marker gene function. Alternatively, a marker gene may be placed in tandem with a coding sequence for a polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates both gene expression in tandem.
Alternatively, host cells that contain and express a desired polynucleotide sequence can be identified by various procedures known to those skilled in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and biological or immunological protein assay techniques that include membrane-based technologies, solutions or chips for detection and / or or the quantification of nucleic acids or proteins.
Various protocols for detecting and measuring the expression of products encoded by polynucleotides, using either polyclonal or monoclonal antibodies specific for the product, are known from the state of the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). An immunoassay based on two-site monoclonal antibodies using monoclonal antibodies reactive against 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 tests are described, inter alia, in Hampton et al., Serological Methods, Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158: 1211-1216 (1983).
A wide variety of markers and conjugation techniques are known to those skilled in the art and can be used in various assays for nucleic acids and amino acids. The means for producing hybridization probes or labeled PCRs for the detection of polynucleotide-related sequences include oligolabeling, cleavage displacement, end labeling, or PCR amplification using a labeled 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 state of the art, are commercially available, and can be used to synthesize RNA probes in vitro by the addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures can be conducted using various 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 can be cultured under conditions suitable for expression and recovery of the protein from the cell culture. The protein produced by a recombinant cell can be secreted or contained intracellularly according to the sequence and / or the vector used. As will be understood by those skilled in the art, expression vectors containing polynucleotides may be designed to contain signal sequences that direct the secretion of the encoded polypeptide through the membrane of a prokaryotic or eukaryotic cell. Other recombinant constructs can be used to join sequences encoding a polypeptide of interest to a nucleotide sequence encoding a polypeptide domain that will facilitate the 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 immobilized immunoglobulins. and the domain used 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, California) between the purification domain and the encoded polypeptide can be used to facilitate purification. Such an expression vector allows the expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or enterokinase cleavage site. Histidine residues facilitate purification on IMIAC (immobilized 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 of purifying the desired polypeptide from the fusion protein. A discussion of vectors that contain fusion proteins is provided in Kroll et al., DNA Cell Biol. 12: 441453 (1993)).
POLYPEPTIDE COMPOSITIONS
In general, a polypeptide for use in the invention (eg, CDTa, CDTb, toxin A, and Clostridium difficile toxin B proteins) will be an isolated polypeptide (i.e., separated from these components with which it can be usually 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 isolated if, for example, it is cloned into a vector that is not part of the natural environment.
The polypeptides can be prepared using any of a variety of well-known techniques. Recombinant polypeptides encoded by DNA sequences as described above can be readily prepared from the DNA sequences using any of a variety of expression vectors known to those skilled in the art. Expression may be obtained in any suitable 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, yeasts, 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 the appropriate host / vector systems that secrete the recombinant protein or polypeptide into the culture medium can 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 may be employed to further purify a recombinant polypeptide.
The polypeptides for use in the invention, their immunogenic fragments, and other variants having less than about 100 amino acids, and generally less than about 50 amino acids, may also be produced by synthetic means, using techniques well known to those skilled in the art. For example, such polypeptides can be synthesized using any of the commercially available solid phase techniques, such as the Merrifield solid phase synthesis method, wherein amino acids are added sequentially to an amino acid chain growing. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146 (1963). Apparatus for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division (Foster City, CA), and can operate according to the manufacturer's instructions.
In 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 comprising tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al., New Engl J. Med 336: 86-91 (1997)). A fusion partner can, for example, help provide T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or it can help express the protein (an expression enhancer ) at higher yields compared to the native recombinant protein. Some preferred fusion partners are both immunological and expression enhancer fusion partners. Other fusion partners may be chosen to increase the solubility of the protein or to allow the protein to be targeted to desired intracellular compartments. Still other fusion partners include affinity markers, which facilitate the purification of the protein.
Fusion proteins can be generally 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 an unfused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components can be assembled separately, and ligated into an appropriate expression vector. The 3 'end of the DNA sequence encoding a polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component of the polypeptide. in such a way that the reading frames of the sequences are in phase. This allows translation into a single fusion protein that retains the biological activity of both polypeptide components.
A peptide linker sequence may be employed to separate the first and second polypeptide components a sufficient distance 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 selected 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 could react with the functional epitopes of the polypeptides. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other nearly neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Amino acid sequences that can 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. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. The linker sequence can generally be from 1 to about 50 amino acids in length. Linker sequences are not needed when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric interference.
admixtures
In another embodiment of any aspect of the invention, the immunogenic composition further comprises an adjuvant. In one embodiment, the adjuvant comprises aluminum hydroxide or aluminum phosphate. Alternatively, the immunogenic composition of the invention may comprise an aluminum-free adjuvant, the immunogenic composition is formulated with an adjuvant that is free of aluminum or aluminum salts, i.e., an adjuvant or an aluminum-free adjuvant system.
In some embodiments, the immunogenic composition is formulated with an adjuvant comprising an immunologically active saponin fraction presented as a liposome. The adjuvant may further comprise a lipopolysaccharide. The adjuvant may comprise QS21. For example, in one embodiment, the adjuvant contains QS21 in a liposomal formulation. In one embodiment, the adjuvant system comprises 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 a specific embodiment, the adjuvant comprises QS21 and cholesterol. Optionally, the adjuvant system contains 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). For example, a specific adjuvant system contains cholesterol, DOPC, 3D-MPL, and QS21.
In a specific example, the immunogenic composition comprises an adjuvant formulated in a dose that comprises: from about 0.1 to about 0.5 mg cholesterol; from about 0.25 to about 2 mg of DOPC; from about 10 μg to about 100 μg of 3D-MPL; and from about 10 μg to about 100 μg of QS21. In another specific example, the immunogenic composition comprises an adjuvant formulated in a dose that comprises: from about 0.1 to about 0.5 mg cholesterol; from about 0.25 to about 2 mg of DOPC; from about 10 μg to about 70 μg of 3D-MPL; and from about 10 μg to about 70 μg of QS21. In a specific formulation, the adjuvant is formulated in a single dose that contains: about 0.25 mg cholesterol; about 1.0 mg of DOPC; about 50 μg of 3D-MPL; and about 50 μg of QS21. In other embodiments, the immunogenic composition is formulated with a fractionated dose (i.e., a dose that is a fraction of the previous single dose formulations, such as one-half of the previous amount of the components (cholesterol , DOPC, 3D-MPL and QS21), 1/4 of the previous amount of the components, or another fractionated dose (e.g. 1/3, 1/6, etc.) of the previous amount of the components.
In one embodiment, the immunogenic compositions according to the invention comprise an adjuvant containing combinations of lipopolysaccharide and Quillaja saponins which have been disclosed previously, for example in the document EP 0671948. This patent has demonstrated a strong synergy when Lipopolysaccharide (3D-MPL) was combined with a Quillaja saponin (QS21). The adjuvant may further comprise immunostimulatory oligonucleotides (eg, 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 has been described for the first time by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv für die gesamte Virusforschung, Vol 44, Springer Verlag, Berlin, p243-254) as having an adjuvant activity. Quil A was isolated by HPLC from purified fragments which retain adjuvant activity without Quil A-associated toxicity (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 lymphocytes (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 in a ratio of saponin / sterol of 1/1 to 1/100 w / w, such as 1/1 to 1/10 w / w; or 1/1 to 1/5 p / p.
In a specific embodiment, QS21 is provided in its least reactogenic composition where it is neutralized by an exogenous sterol, such as cholesterol for example. There are several particular forms of less reactogenic compositions in which QS21 is neutralized by exogenous cholesterol. In a specific embodiment, the saponin / sterol is in the form of a liposomal 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 egg yolk phosphatidylcholine, dioleoylphosphatidylcholine (DOPC) or dilauryl-phosphatidylcholine. The liposomes may also contain a charged lipid which enhances the stability of the liposome-QS21 structure for liposomes composed of saturated lipids. In these cases, the amount of lipid loaded is suitably 1 to 20% w / w, preferably 5 to 10%. The ratio of sterol to phospholipid is 1 to 50% (mol / mol), suitably 20 to 25%.
Suitable sterols include β-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In a particular embodiment, the composition of the adjuvant comprises cholesterol as sterol. These sterols are well known in the state of the art, for example cholesterol is disclosed in the Merck Index, 11th Edition, page 341, as a naturally occurring sterol found in animal fat.
When the active saponin fraction is QS21, the ratio of QS21 / sterol will generally be in the range of 1/100 to 1/1 (w / w), suitably 1/10 to 1/1 (w / w) ), and preferably 1/5 to 1/1 (w / w). Suitably, an excess of 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). Sterol is appropriately 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 to about 70 μg per dose, for example in an amount of about 50 pg.
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 lower, for example between 1 and 60 μg. In one embodiment, the dose of the immunogenic composition comprises QS21 at a rate of approximately around 50 μg, for example between 45 and 55 μg, suitably between 46 and 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 around 25 μg, for example between 20 and 30 μg, suitably between 21 and 29 μg. or between 22 and 28 pg or between 23 and 27 pg or between 24 and 26 pg, or 25 pg.
In another embodiment, the dose of the immunogenic composition comprises QS21 at a level 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.
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 in an amount of from about 1 to about 70 μg per dose, for example in an amount of about 50 μg.
The lipopolysaccharide may be a nontoxic derivative of lipid A, particularly monophosphoryl lipid A or more particularly monophosphoryl lipid A 3-deacylated (3D-MPL).
3D-MPL is sold under the name of MPL by GlaxoSmithKline Biologicals S.A. and reference is made to it by MPL or 3D-MPL. See, for example, U.S. Patent 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. From the chemical point of view, it is a mixture of monophosphoryl lipid A 3-deacylated with 3, 4, 5 or 6 acylated chains. Preferably, in the compositions of the present invention, small particle 3D-MPL is used. The small particle 3D-MPL has a particle size such that it can be sterilized by filtration through a 0.22 μιη filter. Such preparations are described in WO 94/21292.
Therefore, the invention provides a dose of an immunogenic composition comprising lipopolysaccharide, preferably 3D-MPL, at a level of 75 μg or lower, for example between 1 and 60 μg.
In one embodiment, the dose of the immunogenic composition comprises 3D-MPL at a rate around 50 μg, for example between 45 and 55 μg, suitably between 46 and 54 μg or between 47 and 53 μg or between 48 and 50 μg. 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 rate of around 25 μg, for example between 20 and 30 μg, suitably between 21 and 29 μg or between 22 and 28 μg or between 23 and 25 μg. 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 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 pg or between 9 and 11 pg, or 10 pg.
In one embodiment, the dose volume is 0.5 ml. In another embodiment, the immunogenic composition is in a volume suitable for a dose having a volume greater than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In another 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 is suitably related to a human dose. By the term "human dose" is meant a dose that is in a volume suitable for human use. In general, this is between 0.3 and 1.5 ml. In one embodiment, a human dose is 0.5 ml. In another embodiment, a human dose is greater than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In another embodiment, a human dose is between 1 ml and 1.5 ml.
Suitable compositions of the invention are those in which liposomes are initially prepared without MPL (as described in WO 96/33739), and the MPL is then suitably added in the form of small particles less 100 nm or particles capable of being sterilized by filtration through a 0.22 μm membrane. Therefore, MPL is not contained within the vesicular membrane (known as the MPL on the outside). Compositions in which MPL is contained within the vesicular membrane (known as MPL on the inside) also form one aspect of the invention. The polypeptide comprising a fragment of C. difficile toxin A and / or a C. difficile toxin B fragment may be contained within the vesicular membrane or contained outside the vesicular 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 3D-MPL and 25 μg QS21 or 50 μg 3D-MPL and 50 μg QS21.
In one embodiment, the adjuvant comprises an oil-in-water emulsion. In one embodiment, the adjuvant comprises an oil-in-water emulsion, wherein the oil-in-water emulsion comprises a metabolizable oil, a tocol, and an emulsifier. For example, the oil-in-water emulsion may include an oily phase that incorporates a metabolizable oil, and an additional component of the oily phase, such as a tocol. The oil-in-water emulsion may also contain an aqueous component, such as buffered saline (e.g., phosphate buffer solution). In addition, the oil-in-water emulsion generally 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 polyoxyethylene sorbitan monooleate, Polysorbate® 80, TWEEN80 ™). In exemplary embodiments, the oil-in-water emulsion contains squalene and alpha-tocopherol in a ratio that is equal to or less than 1 (w / w). The metabolizable oil in the oil-in-water emulsion can be present in an amount of 0.5 to 10 mg. The tocol in the oil-in-water emulsion may be present in an amount of 0.5 to 11 mg. The emulsifying agent may be present in an amount of 0.4 to 4 mg.
In order for any oil-in-water composition to be suitable for administration to a human, the oily phase of the emulsion system must include a metabolizable oil. The meaning of the term metabolizable oil is well known in the state of the art. Metabolizable can be defined as "metabolizable by metabolism" (Dorland's Illustrated Medical Dictionary, W. B. Sanders Company, 25th edition (1974)). The oil can be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and can be metabolized. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and may include commercially available oils such as NEOBEE® (caprylic / capric triglycerides made using glycerol from vegetable oil sources and medium chain fatty acids (MCT). from coconut oils or palm oil) and others. A particularly suitable metabolizable oil is squalene. Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracoshehexaene) is an unsaturated oil found in large amounts in shark liver oil and in lower amounts in olive oil, wheat germ oil, rice bran oil, and yeasts, and is a particularly preferred oil for use in this invention. Squalene is a metabolizable oil because it is an intermediate in the biosynthesis of cholesterol (Index Merck Index, 10th Edition, entry No. 8619).
Suitably, the metabolizable oil is present in the adjuvant composition in an amount of 0.5 to 10 mg, preferably 1 to 10, 2 to 10, 3 to 9, 4 to 8.5 to 7, or 5 to 6 mg (eg 2 to 3, 5 to 6, or 9 to 10 mg), typically about 5.35 mg or about 2.14 mg per dose.
The tocols are well known in the state of the art and are described in EP 0382271. 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 an amount of 0.5 to 11 mg, preferably 1 to 11, 2 to 10, 3 to 9, 4 to 8.5 to 7.5 to 6 mg (for example 10 to 11, 5 to 6, 2, 5 to 3.5 or 1 to 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 emulsifier may suitably be polyoxyethylene sorbitan monooleate. In a particular embodiment, the emulsifying agent may be the
Polysorbate® 80 (polyoxyethylene (20) sorbitan monooleate) or Tween® 80.
Said emulsifying agent is suitably present in the composition of the adjuvant in an amount of 0.1 to 5, 0.2 to 5, 0.3 to 4, 0.4 to 3 or 2 to 3 mg (e.g. 0.4 to 1.2, 2 to 3 or 4 to 5 mg) of emulsifier. In a specific embodiment, the emulsifier is present in an amount of about 0.97 mg or about 2.425 mg.
In one embodiment, the amounts of the specific components present in the composition are the amounts present in a human dose of 0.5 ml. In another embodiment, the immunogenic composition is in a volume suitable for a human dose, which volume is greater than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. . In another embodiment, the human dose is between 1 ml and 1.5 ml.
When the adjuvant is in liquid form and is to be combined with a liquid form of a polypeptide composition, the composition of the adjuvant in a human dose will be a fraction of the expected final volume of the human dose, for example approximately half of the expected final volume of the human dose, for example a volume of 350 μΐ for a predicted human dose of 0.7 ml, or a volume of 250 μΐ for a predicted human dose of 0.5 ml. The composition of the adjuvant is diluted when combined with the composition of the polypeptide antigen to provide the final human dose of the vaccine. The final volume of such a dose will naturally depend on the initial volume of the adjuvant composition and the volume of the composition of the polypeptide antigen added to the adjuvant composition. In an alternative embodiment, a liquid adjuvant is used to reconstitute a freeze-dried polypeptide composition. In this embodiment, the human dose of the adjuvant composition is approximately equal to the final volume of the human dose. The composition of the liquid adjuvant is added to the vial containing the freeze-dried polypeptide composition. The final human dose may vary between 0.5 and 1.5 ml.
The process for producing oil-in-water emulsions is well known to those skilled in the art. Commonly, the process comprises mixing the oily phase containing the tocol with a surfactant such as a PBS / polyoxyethylene sorbitan monooleate solution followed by homogenization using a homogenizer. It will be clear to those skilled in the art that a process comprising passing the mixture through a syringe needle twice will be suitable for homogenizing small volumes of liquid. Also, the emulsification process in a microfluidizer (machine M110S Microfluidics, maximum of 50 passages, for a period of 2 minutes at a maximum inlet pressure of 6 bar (outlet pressure of about 850 bar)) can be adapted by those skilled in the art to produce lower or higher volumes of emulsions. The adaptation may be obtained by routine experimentation including measuring the resulting emulsion until a preparation with oil droplets of the required diameter is obtained.
In an oil-in-water emulsion, the oil and the emulsifier should be in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffer solution.
Preferably, the oil-in-water emulsion systems of the present invention have a small oil droplet size in the submicron range. Suitably, the droplet sizes will be in the range of 120 to 750 nm, more preferably sizes of 120 to 600 nm in diameter. Most preferably, the oil-in-water emulsion contains oil droplets of which at least 70% in intensity are less than 500 nm in diameter, more preferably at least 80% in intensity are less than 300 nm. in diameter, more preferably at least 90% in 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 NO: 1 to 7 combined with an adjuvant comprising an oil-in-water emulsion comprising 0.125 ml of SB62 emulsion. (total volume), 5,35 mg of squalene, 5,94 mg of DL-α-tocopherol and 2,425 mg of polysorbate 80 per dose of 0.5 ml dose. In one embodiment, the immunogenic composition is not 3 μg or 10 μg of any of SEQ ID NO: 1 to 7 combined with an adjuvant comprising an oil-in-water emulsion comprising 5.35 mg of squalene 5.94 mg of DL-α-tocopherol and 2.425 mg of polysorbate 80 per 0.5 ml dose. In one embodiment, the immunogenic composition does not contain an adjuvant comprising an oil-in-water emulsion comprising 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. diphtheriae (diphtheria), Bordetella pertussis (whooping cough), S. epidermidis, enterococci, S. aureus, and Pseudomonas aeruginosa.
In another embodiment, the immunogenic composition of the invention may comprise other C. difficile antigens, for example, the S-layer proteins (WO 01/73030). Optionally, the immunogenic composition further comprises a C. difficile saccharide.
There is further provided a vaccine comprising an immunogenic composition of the invention and a pharmaceutically acceptable excipient.
Immunogenic preparations containing immunogenic compositions of the present invention can be used to protect a mammal susceptible to C. difficile infection or to treat a mammal suffering from C. difficile infection, by administering said vaccine by systemic or mucosal route. Such administrations may include intramuscular, intraperitoneal, intradermal or subcutaneous injection; or by mucosal administration to the oral / food, respiratory, urogenital systems. Although the vaccine of the invention may be administered as a single dose, its components may also be coadministered together at the same time or at different times (for example, pneumococcal saccharide conjugates may be administered separately, at the same time time or 1 to 2 weeks after administration of any of the bacterial protein components of the vaccine for coordination of immune responses relative to each other). In addition to a single route of administration, two different routes of administration may be used. For example, saccharides or saccharide conjugates can be administered intramuscularly (IM) or intradermally (ID) and the bacterial proteins can be administered intranasally (IN) or intradermally (ID). In addition, the vaccines of the invention can be administered by IM for sensitization doses and IN for booster doses.
The toxin content in the vaccine will generally be in the range of 1 to 250, preferably 5 to 50, most usually in the range of 5 to 25. Following an initial vaccination, subjects may receive one or more booster immunizations spaced appropriately. 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). The encapsulation within liposomes is described by Fullerton, U.S. Patent 4,235,877.
In one aspect of the invention there is provided a vaccine kit, comprising a vial containing an immunogenic composition of the invention, optionally in lyophilized form, and further comprising a vial containing an adjuvant as described herein. It is contemplated in this aspect of the invention that the adjuvant is used to reconstitute the freeze-dried immunogenic composition.
Another aspect of the invention is a method of preventing or treating a 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 episodes and / or recurrence of C. difficile infection comprising administering to the host an immunoprotective dose of the composition. immunogen 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 or disease. "C. difficile disease" means disease caused in whole or in part by C. difficile. In another embodiment of the invention, there is provided an immunogenic composition or vaccine of the invention for use in the treatment or prevention of an ailment or disease caused in whole or in part by a C. difficile strain 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.
As used herein, "treatment" means preventing the occurrence of the symptoms of the condition or disease in a subject, preventing the recurrence of the symptoms of the condition or disease in a subject, the delay in the recurrence of the symptoms of the condition or illness in a subject, decrease in the severity or frequency of the symptoms of the condition or disease in a subject, slowing or elimination of progression of the condition and the partial or total elimination of the symptoms of the disease or condition in a subject.
In another 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 another embodiment, the disease is a disease caused by a C. difficile strain 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 another aspect of the invention there is provided a method of preventing or treating a C. difficile disease comprising administering the immunogenic composition of the invention or vaccine of the invention to a mammalian subject as a human subject. In another embodiment, the disease is a disease caused by a C. difficile strain 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 being within plus or minus 10% of the given figure for objectives of the invention.
The term "includes" means "includes". Thus, unless the context requires otherwise, the word "includes", and variations such as "include" and "comprising" will be understood to imply the inclusion of a specified compound or composition (eg, acid nucleic acid, polypeptide, antigen) or a step, or a group of compounds or steps, but not the exclusion of any other compound, composition, step, or group thereof. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used here 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: 29 and SEQ ID. NO: 30, respectively, which should be considered as reference sequences for these proteins.
The 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 indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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 1-56081569-8).
The terms singular "one", "one", "the" and "the" include plural articles unless the context clearly indicates otherwise. Similarly, the term "or" is meant to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more. It should further be understood that all base sizes or amino acid sizes, and all molecular weight or molecular weight values, given for the nucleic acids or polypeptides are approximate, and are provided for the description. In addition, the numerical limitations given with respect to the concentrations or levels of a substance, such as an antigen, may be approximate.
All references or patent applications cited in this patent specification are incorporated by reference herein in their entirety.
For this invention to be better understood, the following examples are presented. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.
EXAMPLES The AS01B adjuvant referred to is an adjuvant containing 50 μg of QS21 presented as a liposome, 50 μg of 3D-MPL, 0.25 mg of cholesterol and 1.0 mg of DOPC per dose of 0.5 ml. A 50 μl dose suitable for immunizing mice contains 5 μg of QS21, 5 μg of 3D-MPL, 0.025 mg of cholesterol and 0.1 mg of DOPC.
Example 1 - Design of Binary Toxin Antigens
The binary toxin (other name: ADP-ribosyl-transferase toxin) is composed of two components: the enzymatic component, named CDTa, and the transport and binding component, named CDTb.
Based on literature data and available information for components of other bacterial binary toxins, CDTb could be divided into five domains. The first is the prodomain, its cleavage by an enzyme with chymotrypsin activity allows the heptamerization of the mature protein. The second domain allows linking to CDTa. The third and fourth are involved in oligomerization and membrane insertion. Finally, the last domain is the receptor binding domain of the host cell. Mature CDTb
In order to avoid the activation step by chymotrypsin in the treatment of CDTb, it has been attempted 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), mature CDTb was described as starting at Leucine 210 of SEQ ID NO: 3. This mature CDTb was named CDTb ". Other experimental data suggest that CDTb starts at Serine 212 of SEQ ID NO: 3. This result was supported by the analysis of a modeled three-dimensional structure of CDTb.This model was constructed using SwissModel (Bioinformatics, 2006, vol.22: 195-201) The template used for homology modeling was component B of Bacillus anthracis, named protective antigen or PA (accession number of the protein database: 3TEW ).
Single receptor binding domain of CDTb
The three-dimensional homology structure model obtained for CDTb using PA antigen from B. anthraxis is accurate for the first four domains of CDTb but not for the receptor binding domain (these domains of CDTb and PA are too different). To design constructs expressing the receiver-only domain, the C-terminal portion of the fourth domain was analyzed on the three-dimensional structure model to decide where the last domain will start.
Two versions of the CDTb receptor binding domain have been designed. In the first, this domain starts just after the modeled three-dimensional structure of the fourth domain. In this version, the receptor binding domain of CDTb will likely have a long, flexible peptide in its N-terminus. The second version of the receptor binding domain starts where predicted two-dimensional structures on the C-terminal part of the CDTb (predictions made using the Psipred program, Bioinformatics, 2000, vol 16: 404-405) become more compact 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 portion of the receptor binding domain isolated from CDTb.
Mutations of the Ca2 + binding motif of CDTb
By following the literature, mutations in the Ca2 + binding domain of component B of the Iota toxin of Clostridium perfringens (Ib) abolish the binding with component A 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 wild-type CDTa protein.
Using multiple protein sequence alignment, these mutations were localized to the CDTb sequence and mutated. This concerns the Asp220, Asp222 and Asp224 residues of SEQ ID NO: 3. They were mutated on the Ala residues.
Prodomaine of the CDTb
In an attempt to reduce the degradation problems observed with the C55 construct (CDTb with the prodomaine removed) in gel, some coexpression tests were evaluated. The working hypothesis in doing this is to improve the folding of the mature CDTb.
Two limits of the prodomaine have been proposed. The first starts at residue 43 of the CDTb of SEQ ID NO: 3 (after cleavage of the signal peptide) and ends at the Met211 residue (since the first experimentally determined residue of the mature CDTb is Ser212). The second prodomain was designed based on the predicted three-dimensional structure of CDTb. The linker between the prodomain and the first structural domain of the mature CDTb protein is eliminated in this construct.
Example 2 Cloning, Expression and Purification of C. difficile CDTb Protein
Expression plasmid and recombinant strain: C37 and C55 (as shown in Table A).
The genes encoding the truncated CDTb protein without the signal peptide (Pro-CDTb ', C37) and a His-tag at the C-terminus were cloned into the pGEX-6pl expression vector (GE Healthcare) using restriction sites
BamHI / XhoI using standard procedures. This vector included GST (glutathione-S-transferase) as a fusion partner at the N-terminus of CDTb '(GST-Pro-CDTb'). The final construction was produced by the transformation of the strain of E. coli BL21 (DE3) with the recombinant expression vector according to the conventional method with CaCl2-treated cells (Hanahan D. "Plasmid transformation by Simanis." In Glover, DM (Ed), DNA cloning, IRL Press London. (1985) ): pp. 109-135.).
The genes encoding the truncated CDTb protein without signal peptide nor prodomain (CDTb-: C55) and a His-tag at the C-terminus were cloned into the pET24b (+) expression vector (Novagen) using the NdeI / XhoI restriction sites using standard procedures The final constructs were produced by transforming the modified strain of E. coli B834 (DE3) with the appropriate recombinant expression vectors according to the conventional method with treated cells. to CaCl2 (Hanahan D. "Plasmid transformation by Simanis." In Glover, DM (Ed), DNA cloning, IRL Press London (1985): pp. 109-135.).
Host strain BL21 (DE3). BL21 (DE3) is a non-auxotrophic derivative for B834 methionine. Strains with the designation (DE3) are lysogenic for λ prophage which contains an IPTG-inducible T7 RNA polymerase. Λ DE3 lysogens are designed for the expression of proteins from pET vectors. This strain is also deficient in proteases Ion and ompT. Genotype: E. coli strain BL21 :: DE3, F "ompT hsdSB (rB_ mB ~) gai dcm (DE3) B834 is the parent strain for BL21 These protease deficient hosts are auxotrophic for methionine. designed for the expression of proteins from pET vectors.This strain is also deficient in proteases Ion and ompT.
Modification: inclusion of the PGL gene to avoid phosphogluconoylation in the biotin locus (the strain is auxotrophic for biotin). Genotype: strain B834 :: DE3, F- ompT hsdSB {xB - mB-) gai dcm met (DE3)
Modification: Δ (bioA-bioD):: PGL Expression of recombinant proteins:
Transformants of E. coli were taken from an agar plate and used to inoculate 200 ml LBT broth ± 1% (w / v) glucose +/- kanamycin (50 μg / ml) or ampicillin (100 μg / ml) to obtain a D.0.6 cm between 0.1 and 0.2. The cultures were incubated overnight at 37 ° C, 250 rpm.
Overnight cultures were diluted 1:20 in 500 ml LBT medium containing +/- kanamycin (50 μg / ml) or ampicillin (100 μg / ml) and cultured at 37 ° C at a stirring rate. 250 rpm to reach an OD620 of 0.5 / 0.6. At OD at 600 nm around 0.6, cultures were cooled prior to induction of recombinant protein expression by the addition of 1 mM isopropyl-pD1-thiogalactopyranoside (IPTG; EMD Chemicals Inc. catalog number 5815) and incubated overnight at 23 ° C, 250 rpm.
After overnight inductions (around 16 hours), the OD at 600 nm were evaluated after induction and the cultures were centrifuged at 14,000 rpm for 15 minutes and the pellets were frozen at -20 ° C. C separately.
Purification C37 (SEQ ID NO: 5)
The bacterial pellet was resuspended 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 inhibitors (Completed, Roche). The bacteria were lysed using a French 3 X 20 000 PSI press system. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation at 20,000 xg for 30 min at 4 ° C.
The 6-His tagged protein was purified under native conditions on IMAC. The soluble components were loaded onto a 5 ml GE Histrap (GE) column pre-equilibrated with the same buffer used for resuspension of the bacteria. After loading on the column, the column was washed with 50 mM bicine buffer pH 8.0, containing 150 mM NaCl, 25 mM imidazole, 1 mM TCEP. Elution was performed using a 50 mM bicine buffer pH 8.0 containing 150 mM NaCl, 250 mM imidazole, 1 mM TCEP.
After a desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM bicine buffer pH 8.0 containing 150 mM NaCl and 1 mM TCEP, the product was treated (overnight at 4 ° C) with PreScission proteases (GE-Healthcare) in order to cleave the GST marker. 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 50 mM bicine buffer pH 8.0 containing 150 mM NaCl, 1 mM TCEP, 0.2% Tween 20 and 20 mM reduced glutathione to remove the cleaved marker, uncleaved fusion protein and PreScission proteases.
The GST-free protein was collected in the non-retained fraction and refilled on a 5 ml GE Histrap (GE) column pre-equilibrated with 50 mM bicine buffer pH 8.0 containing 150 mM NaCl, 1 mM. of TCEP, 0.2% Tween20. After loading onto the column, the column was washed with 50 mM bicine buffer pH 8.0, containing 150 mM NaCl, 0.2% Tween 20, 1 mM TCEP and 10 mM imidazole. Elution was performed using a 50 mM bicine buffer pH 8.0 containing 150 mM NaCl, 0.2% Tween 20, 1 mM TCEP and 500 mM imidazole.
After a desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM of bicine buffer pH 8.0 containing 150 mM NaCl, 1 mM TCEP and 0.2% Tween 20, the product was treated with water. a-chymotrypsin (from bovine pancreas - Sigma), followed by treatment with a trypsin inhibitor (from egg white - Sigma). Complete activation of CDTb by chymotrypsin was followed by SDS-PAGE.
The fully activated product was loaded on SEC (SUPERDEX ™ 75) chromatography in 50mM bicine buffer pH 8.0 containing 300mM NaCl, 1mM TCEP. Fractions containing the CDTb antigen were selected on the basis of purity by SDS-PAGE. The concentration of the protein was determined using the Bio-Rad Lowry RC / DC protein assay. The purified bulk was sterilized by filtration over 0.22 μπι and stored at -80 ° C. C55 (SEQ ID NO: 7)
The bacterial pellet was resuspended in 50 mM bicine buffer (pH 8.0) containing 150 mM NaCl, 5 mM TCEP (Thermo Scientific Pierce, (2-carboxyethyl) -phosphine hydrochloride), 0.4% of Empigen and a mixture of protease inhibitors (Completed, Roche). The bacteria were lysed using a French 3 X 20 000 PSI press system. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation at 20,000 xg for 30 min at 4 ° C.
The 6-His tagged protein was purified under native conditions on IMAC. The soluble components were loaded onto a 5 ml GE Histrap (GE) column pre-equilibrated with 50 mM bicine buffer (pH 8.0) containing 150 mM NaCl, 0.15% Empigen, 1 mM TCEP. . After loading onto the column, the column was washed with 50 mM bicine buffer pH 8.0, containing 150 mM NaCl, 0.2% Tween 20, 20 mM imidazole and 1 mM TCEP. Elution was performed using a 50 mM bicine buffer pH 8.0 containing 150 mM NaCl, 0.2% Tween 20, 500 mM imidazole and 1 mM TCEP.
After the desalting step (BIORAD Bio-Gel P6 Desalting) in 50 mM bicine buffer pH 8.0 containing 300 mM NaCl, 1 mM TCEP, the product was loaded on SEC (SUPERDEX ™ 75) chromatography. in the same buffer. Fractions containing the CDTb antigen were selected on the basis of purity by SDS-PAGE. The concentration of the protein was determined using the Bio-Rad Lowry RC / DC protein assay. The purified bulk was sterilized by filtration over 0.22 μm and stored at -80 ° C.
Example 3 Cloning, Expression and Purification of the CDTa Protein
Expression Plasmid and Recombinant Strain: Full-length CDTa (C34, C44, C49, C50, C54, C67, C68, C69, C107, C108, C110 as shown in Table A)
The genes encoding the full-length protein without CDTa signal peptide with and without mutations (see Table A) and a His-tag at the C-terminus were cloned into the pET24b (+) expression vector (Novagen ) using the NdeI / XhoI restriction sites using standard procedures. The final constructions were produced by the transformation of the strain dΈ. coli HMS174 (DE3) or BLR (DE3) pLysS (C34) with each recombinant expression vector according to the conventional method with CaCl2-treated cells (Hanahan D. "Plasmid transformation by Simanis." In Glover, DM (Ed), DNA cloning, IRL Press London (1985): pp. 109-135.
Host strain: HMS 174 (DE3). HMS174 strains provide the recA mutation in a K-12 context. Strains with the designation (DE3) are lysogenic for λ prophage which contains an IPTG-inducible T7 RNA polymerase. Λ DE3 lysogens are designed for the expression of proteins from pET vectors. Genotype: F "recAl hsdR (rKi2 'mKi2 +) (Rif R). BLR (DE3) pLysS. BLR is a recA derivative of BL21. Strains with the designation (DE3) are lysogenic for λ prophage which contains an IPTG-inducible T7 RNA polymerase. Λ DE3 lysogens are designed for the expression of proteins from pET vectors. This strain is also deficient in proteases Ion and ompT, pLysS strains express T7 lysozyme which further suppresses the basic expression of T7 RNA polymerase before induction. Genotype: E. coli strain BLR :: DE3, F 'ompT hsdSB (rs ~ me-) gal dcm (DE3) Δ (srl-recA) 306:: TnI 0 pLysS (CamR, TetR).
Expression of recombinant proteins:
The transformants of E. coli were collected on an agar plate and used to inoculate 200 ml of LBT broth ± 1% (w / v) glucose + kanamycin (50 μg / ml) to obtain a D. 0.6oonm between 0.1 and 0.2. The cultures were incubated overnight at 37 ° C, 250 rpm.
Each overnight culture was diluted 1/20 in 500 ml of LBT medium containing kanamycin (50 μg / ml) and cultured at 37 ° C at a stirring speed of 250 rpm until the reaction was complete. obtaining an OD 620 of 0.5 / 0.6. At a D0.6oo around 0.6, the cultures were cooled prior to induction of recombinant protein expression by the addition of 1 mM isopropyl-pDl-thiogalactopyranoside (IPTG; EMD Chemicals Inc. catalog number 5815) and incubated overnight at 23 ° C, 250 rpm. '
After overnight induction (around 16 hours), the D. 0.6oonm was evaluated after induction and the cultures were centrifuged at 14,000 rpm for 15 minutes and the pellets were frozen at -20 ° C. C separately.
Expression plasmid and recombinant strain: N-terminal end of CDTa (C49 and C50 as shown in Table A)
The genes encoding the N-terminus protein, without the CDTa signal peptide (see Table A) and a His-tag at the C-terminus were cloned into the pET24b expression vector (+ ) (Novagen) using the NdeI / XhoI restriction sites using standard procedures. The final constructions were produced by the transformation of the strain of E. coli HMS174 (DE3) with each recombinant expression vector separately according to the conventional method with CaCl2-treated cells (Hanahan D. "Plasmid transformation by Simanis." In Glover, DM (Ed), DNA cloning, IRL Press London. 1985): pp. 109-135.).
Host strain:
HMS 174 (DE3). HMS174 strains provide the recA mutation in a K-12 context. Strains with the designation (DE3) are lysogenic for λ prophage which contains an IPTG-inducible T7 RNA polymerase. Λ DE3 lysogens are designed for the expression of proteins from pET vectors. Genotype: F-recAl hsdR (ΐκΐ2 ~ πΐκΐ2 +) (Rif R).
Expression of recombinant proteins:
The transformants of E. coli were taken from an agar plate and used to inoculate 200 ml LBT broth ± 1% (w / v) glucose + kanamycin (50 μg / ml) to obtain an OD 600nm between 0.1 and 0. 2. The cultures were incubated overnight at 37 ° C, 250 rpm.
This overnight culture was diluted 1/20 in 500 ml of LBT medium containing kanamycin (50 μg / ml) and cultured at 37 ° C at a stirring speed of 250 rpm until the reaction was complete. obtaining an OD 620 of 0.5 / 0.6. At an OD 600nm around 0.6, the culture was cooled prior to induction of recombinant protein expression by the addition of 1 mM isopropyl-D1-thiogalactopyranoside (IPTG; EMD Chemicals Inc., catalog number 5815) and incubated overnight at 23 ° C, 250 rpm.
After overnight incubation (around 16 hours), D. 0.6oonm was evaluated after induction and the culture was centrifuged at 14,000 rpm for 15 minutes and the pellets were frozen at -20 ° C. separately.
Purification
The following procedure was used to purify constructions C34, C44, C49, C50, C54, C67, C69, C107 and C110. The bacterial pellets were resuspended 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, Hydrochloride). 2-carboxyethyl) phosphine)) and a mixture of protease inhibitors (Complete, Roche, EDTA-free). The bacteria were lysed using a French 3 X 20 000 PSI press system. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation at 20,000 xg for 30 min at 4 ° C.
The 6-His-tagged proteins were purified under native conditions on IMAC. The soluble components were loaded onto a 5 ml GE Histrap (GE) column pre-equilibrated with the same buffer used for resuspension of the bacteria. After loading onto the column, the column was washed with 20 mM or 50 mM bicine buffer (pH 7.5 or pH 8.0), containing 500 mM NaCl, 10 mM imidazole, 5 mM TCEP . Elution was performed using a 50 mM bicine buffer pH 7.6, 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), the product was loaded on SEC (SUPERDEX ™ 75 or 200) chromatography in 20 mM or 50 mM bicine buffer ( pH 7.5 or pH 8.0), 150 mM NaCl, 1 mM TCEP, for another purification step.
Fractions containing the CDTa antigen were selected on the basis of purity by SDS-PAGE. Protein concentration was determined using the Bio-Rad Lowry RC / DC protein assay. The purified bulk was sterilized by 0.22 μm filtration and stored at -80 ° C.
Example 4 - Design, Cloning, Expression and Purification of C. difficile CDTb Protein with Modified Pore Capability
A first strategy was evaluated in order to reduce the cytotoxicity observed for CDTb alone: to avoid the possibility for CDTb to modify its structure in an endosome in order to form a pore in the membrane of this endosome. The two strategies followed in this approach were based on available literature data for B components from the binary toxin Bacillus anthracis (C123 and C149) and Clostridium botulinum (C126, C152, C164 and C166). The following CDTb proteins have been designed:
* Takes into account the starting codon Met and the His Cloning and Production tag
His tag at the C-terminus / cloning in the pGEX-6pl vector in the BamH1-XhoI / B834 strain (DE3): genotype: E. coli BL21 :: DE3, F-ompT hsdSB (rB- mB-) gd dcm (DE3).
B834 is the parental strain for BL2. These protease deficient hosts are auxotrophic for methionine. Λ DE3 lysogens are designed for the expression of proteins from pET and pGEX vectors. This strain is also deficient in proteases Ion and ompT. Antibiotic selection: ampicillin: 50 μg / ml / overnight production at 16 ° C
Purification (C123, C126, C149 and C152):
French press with lysis buffer: 50 mM Bicine - 500 mM NaCl - 5 mM TCEP - Completed - pH 8.0 Ni-NTA GE Histrap 5 ml (profinia) + desalting
Equilibrated: 50 mM Bicine - 500 mM NaCl, 1 mM TCEP -pH 8.0
Wash: 50 mM Bicine - 150 mM NaCl, 1 mM TCEP - Imidazole 0 mM - pH 8.0
Elution: 50 mM Bicine - 150 mM NaCl, 1 mM TCEP - Imidazole 250 mM - pH 8.0
Desalting: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP -pH 8, 0
Prescission cleavage (GST marker): + 375 μΐ (500 IU / 250 μΐ): 750 IU
One night at 4 ° C + addition of Tween 20 at 0.2% final concentration. GSTrap FF (1 ml)
Equilibrated in the buffer: 50 mM Bicine - 150 mM NaCl -1 mM TCEP - 0.2% Tween 20 - pH 8.0 Maintain the FT
Wash Buffer: Same as Balancing Buffer
Dilution buffer: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - 0.2% Tween 20 - 250 mM imidazole - pH 8.0 IMAC GE 5 ml + desalting
Balanced in buffer: 50 mM Bicine - 150 mM NaCl -1 mM TCEP - 0.2% Tween 20 - pH 8.0
Wash Buffer: 50mM Bicine - 150mM NaCl - 1mM TCEP - 10mM Imidazole - 0.2% Tween20 - pH 8.0
Dilution buffer: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - 250 mM imidazole - 0.2% Tween 20 - pH 8.0
Desalting Buffer: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - 0.2% Tween 20 - pH 8.0
RC / DC dosing
Activation: chymotrypsin treatment:
100 μg of chymotrypsin (1 μg / μΐ) activate 1 mg of protein (42 mg + 3.6 mg of chymotrypsin in 12.5 ml of final volume) 50 min at RT + inactivation: chymotrypsin inhibitor 12.6 mg SEC Superdex 200
Equilibrated: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 8.0
Concentration on IMAC GE 1 ml + desalting
Equilibrated in buffer: 50 mM Bicine - 150 mM NaCl -1 mM TCEP - pH 8.0
Wash buffer: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 8.0
Buffer dilution: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - 250 mM imidazole - pH 8.0
Desalting buffer: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 8.0
0.22 μm filtration, RC / DC dosing
The purification of C164 was carried out in accordance with the following scheme 1:
French Press with lysis buffer: 50 mM Bicine - 500 mM NaCl - 5 mM TCEP - Completed - pH 8.0 -Benzonase
Ni-NTA GE Histrap 5ml
Equilibrated: 50 mM Bicine -150 mM NaCl, 1 mM TCEP - pH 8.0 Wash: 50 mM Bicine -150 mM NaCl -1 mM TCEP - imidazole 10 mM - pH 8.0 Elution: 50 mM Bicine -150 mM NaCl, 1 mM TCEP - Imidazole 250 mM - pH 8.0
Group A = washing 10 mM Group B = elution 250 mM
SEC Superdex 200 SEC Superdex 200
Balanced: 50 mM Bicine - Equilibrated: 50 mM Bicine -
150 mM NaCl -1 mM TCEP 150 mM NaCl -1 mM TCEP - pH 8.0 - pH 8.0
0.22 μπι filtration, RC / DC dosing 0.22 μm filtration, RC / DC dosing
Diagram 1
Purification of C166 was performed according to Scheme 2 below:
French press with lysis buffer: 50 mM Bicine - 500 mM NaCl - 5 mM TCEP - pH 7.5
Nr-NTA GE Histraplml
Equilibrated: 50 mM Bicine - 500 mM NaCl, 1 mM TCEP - pH 7.5 Wash: 50 mM Bicine - 500 mM NaCl, 1 mM TCEP - Imidazole 10 mM - pH 7.5 Elution: 50 mM Bicine -150 mM NaCl -1 mM TCEP - 500 mM Imidazole - pH 7.5
SEC: Superdex 200 xk16 / 60 120ml 50mM Bicine -150mM NaCl -1 mM TCEP - pH 7.5
0,22μπι
* Lowry RC / DC dosing
Figure 2
Characterization :
Dynamic light scattering is used to evaluate the hydrodynamic radius in solution of purified CDTb proteins, in addition to providing information on homogeneity and detecting the presence of high molecular weight aggregates within a protein sample. . This is based on the calculation of the diffusion coefficient of the different species that are obtained by measuring the fluctuation of light scattering, which depends on the molecular size and shape of the proteins, and the other minor constituents of the sample.
Protein samples were analyzed on a Dynapro plate reader (Wyatt technology), using five 15-second acquisitions at 25 ° C.
Buffer a: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 8.0
The results are shown in Figure 3.
Example 5 - Design, cloning, expression and purification of C. difficile CDTb protein with removed signal peptide and prodomain and also eliminated receptor binding domain and / or CDTa binding domain removed
The reason for expressing the different structural domains of CDTb alone has been to understand which leads to the problem of degradation and aggregation. Two domains were evaluated using this strategy: the receptor binding domain and the CDTa binding domain were removed on the C116 and C117 constructs, respectively. The "oligomerization" and "membrane insertion" domains of CDTb have not been expressed separately because they are potentially associated at the structural level. The following CDTb proteins have been designed:
* Takes into account the start codon of Met and the His Cloning and Production counter
His tag at the C-terminus / cloning in the pET24b vector in the NdeI-XhoI site / B834 strain (DE3): genotype: strain of E. coli coli BL21 :: DE3, F-ompT hsdSB (rB- mB-) gd dcm (DE3). B834 is the parental strain for BL2. These protease deficient hosts are auxotrophic for methionine. Λ DE3 lysogens are designed for the expression of proteins from pET vectors. This vector is also deficient in proteases
Ion and ompT. Antibiotic selection: kanamycin: 50 μg / ml / overnight production at 16 ° C.
Purification and characterization C116
French Press
Lysis buffer: 50 mM Bicine - 500 mM NaCl - 5 mM TCEP - Completed - pH 8.0 Ni-NTA GE Histrap 5 ml
Equilibrated: 8 M urea - 50 mM Bicine - 500 mM NaCl - 1 mM TCEP - pH 8.0.
Wash: 8 M urea - 50 mM Bicine - 500 mM NaCl - 1 mM TCEP - 5 mM imidazole - pH 8.0 Fold over Histrap column
Gradient: 100 min at 1 ml / min: 8M at 50 mM Bicine - 150 mM NaCl - 1 mM TECP - pH 8.0 1 mM TCEP at 0 M urea 50 mM Bicine - 150 mM NaCl - 1 mM TECP - pH 8.0
Elution of 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - Imidazole 500 mM - pH 7.5 SEC: Superdex 200
50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 7.5 0.22 μτη - Lowry RC / DC assay C117
French press with buffer, lysis: 50 mM Bicine - 500 mM NaCl - 5 mM TCEP, Completed - pH 8.0 Ni-NTA GE Histrap 5 ml (profinia)
Equilibrated: 50 mM Bicine - 500 mM NaCl - 1 mM TCEP - pH 8, 0
Wash: 50 mM Bicine - 500 mM NaCl - 1 mM TCEP - Imidazole 0 mM - pH 8.0
Elution: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - Imidazole 500 mM - pH 7.5 SEC Superdex 200
Balanced: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP -pH 7.5
0.22 μm filtration, RC / DC dosing
Example 6 - Design, Cloning, Expression and Purification of C. difficile CDTb Protein with Modified Heptamerization Capability
A second strategy was used to reduce the cytotoxicity observed for CDTb alone: avoid heptamerization of CDTb. A three-dimensional model of a heptamer complex of CDTb was designed. Using this model, a loop has been identified as potentially interacting between the monomers. Two approaches have been evaluated: the elimination of this loop (C127) or the mutation of the residues contained in this loop which are probably involved in the interaction between the monomers (C128). The following proteins have been designed:
* Takes into account the start codon of Met and the His marker
Cloning and production
His tag at the C-terminus / cloning in the pET24b vector in the NdeI-XhoI site / B834 strain (DE3): genotype: strain of E. coli coli BL21 :: DE3, F-ompT hsdSB (rB- mB-) gd dcm (DE3). B834 is the parental strain for BL2. These protease deficient hosts are auxotrophic for methionine. Λ DE3 lysogens are designed for the expression of proteins from pET vectors. This strain is also deficient in proteases Ion and ompT. Antibiotics of Selection: Kanamycin: 50 μg / ml / overnight production at 16 ° C.
Purification and characterization French press with lysis buffer: 50 mM Bicine - 150 mM NaCl - 0.4% Empigen - 5 mM TCEP - Completed - pH 8.0 Ni-NTA GE Histrap 1 ml (profinia )
Equilibrated: 50 mM Bicine - 150 mM NaCl - 0.15% Empigen - 1 mM TCEP - pH 8.0
Wash: 50 mM Bicine - 150 mM NaCl - 0.2% Tween 20 -1 mM TCEP - Imidazole 10 mM - pH 8.0
Elution: 50 mM Bicine - 150 mM NaCl - 0.2% Tween 20 -1 mM TCEP - Imidazole 500 mM - pH 8.0
Desalting G-25: 50 mM Bicine - 150 mM NaCl - 0.2% Tween 20 - 1 mM TCEP - pH 8.0
Activation with chymotrypsin SEC: Superdex 200 XK16 / 60 (120 ml)
Equilibrated: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 8.0
Concentration: GE-HisTrap (1 ml) + desalting 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 8.0 0,22 μιη - Lowry RC / DC assay Characterization:
Dynamic light scattering is used to evaluate the hydrodynamic radius in solution of purified CdtB proteins, in addition to providing information on homogeneity and detecting the presence of high molecular weight aggregates within a protein sample. . This is based on the calculation of the diffusion coefficient of the different species that are obtained by measuring the fluctuation of light scattering, which depends on the molecular size and shape of the proteins, and the other minor constituents of the sample.
Protein samples were analyzed on a Dynapro plate reader (Wyatt technology), using five 15-second acquisitions at 25 ° C
Buffer a: 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 8.0.
Example 7 Cytotoxicity of CDTb Proteins on HT29 Cells
Chymotrypsin (C37) activated CDTb subunits alone appear cytotoxic on HT29 cells. This suggests that pore formation could be responsible for cytotoxicity.
A second generation of antigen design has been produced to inhibit the pore formation step by disrupting hydrophobic interactions between beta sheets of individual proteins.
This B-subunit of the binary toxin (cell-binding part) of the binary toxins was added alone or mixed with the subunit A (enzymatic part) on human colon epithelial cells (HT29 cells) to evaluate their residual cytotoxicity. .
The cytotoxicity of the candidate C123, C126, C128, C164, C166, C116, C117, C149 and C152 was tested on HT2 9 cell lines.
Cytotoxicity test of the binary toxin
Human colon epithelial cells (HT29 cells) were cultured at 37 ° C with 5% CO 2 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, reference: 655090) at a density of 4 x 103 cells / well for HT29.
After 24 h, 50 μl of the cell medium were taken from the wells.
The candidate binary toxins to be characterized were diluted to achieve 2 μg / ml CDTa and 6 μg / ml CDTb. In addition, 1/3 dilutions were made in the microplate (NUNC, reference: 163320). 50 μl of the serial dilutions of the binary toxin preparations were added to the black plates and the microplates were incubated at 37 ° C with 5% CO2 for 6 days.
After 6 days, the mixture of the binary toxin and the medium was taken from the wells and 100 μl of Hoechst dye (BD Pharmingen, reference: 561908) diluted 1/500 in phosphate buffer solution (PBS) were added to each well for 2 hours in the dark at room temperature.
After staining, the Hoechst stain was removed from the wells and the fluorescence of the cells was measured using an Axiovision microscope.
The cytotoxicity is expressed as a percentage of surface area covered by fluorescent staining in each well. No cytotoxic effect (100% recovery) is obtained with cells alone. And a higher level of cytotoxicity is obtained with fully activated binary toxin (CDTa C34 + CDTb C37). The results are shown in Figure 1, Figure 2 and Figure 8.
Example 8 - Analysis of CDTb Proteins by DLS
Dynamic Light Diffusion (DLS) was used to evaluate the hydrodynamic radius in solution of the purified CDTb C123, C126 and C128 proteins, in addition to providing information on homogeneity and detecting the presence of molecular weight aggregates. elevated within a protein sample. This is based on the calculation of the diffusion coefficient of the different species that are obtained by measuring the fluctuation of light scattering, which depends on the molecular size and shape of the proteins, and the other minor constituents of the sample.
The following protein samples were analyzed on a Dynapro plate reader (Wyatt technology), using five 15-second acquisitions at 25 ° C.
All proteins are in a buffer composed of 50 mM Bicine - 150 mM NaCl - 1 mM TCEP - pH 8.0. The measurements were made on the purified bulk kept at 4 ° C which had not been frozen.
As shown in Fig. 3, both the C126 KO construct for pore formation and the C128 KO construct for heptamerization have a hydrodynamic radius of the main population around 3 nm which could be consistent with a monomer . The C123 KO construct for heptamerization is found as a high molecular homogeneous oligomer or aggregate with a hydrodynamic radius of 15.7 nm.
The fact that the average hydrodynamic radii of the total samples are close to the radius of the main populations, together with their low polydispersity, suggest that purified proteins labeled with his C123, C126 and C128 have a uniform size distribution.
Example 9 - Design, Cloning, Expression and Purification of a C. difficile CDTb Fusion Protein Comprising a Full-Length CDTa Protein and a CDTb Protein
There are two major advantages for CDTa and CDTb fusion: the first is a single treatment to obtain 2 proteins. The second: CDTa is easier than the CDTb to produce -> placing CDTa as the first fusion partner could potentially have a positive effect on the overall merger processing ability. In C139, the two mature proteins CDTa (without its signal peptide) and CDTb (without its signal peptide and without its prodomaine) were fused.
In C139, the CDTa partner is mutated at position 428 (a glutamate is mutated to glutamine) to mutate the cytotoxic activity of CDTa, and the CDTa partner of this fusion is mutated at position 45 (a Cys). was mutated to a Tyr residue) to avoid observed dimerization of CDTa.
In C145, the CDTa portion of the fusion is the same as for C139. CDTb's CDTa binding domain has been removed to potentially avoid interaction between this domain and the CDTa partner. Between the two partners, a linker / spacer (6 Gly residues) was added to improve the structural flexibility between the partners and to allow an independent correct folding of the two proteins.
The CDTb receptor binding domain has a very good expression efficiency, is homogeneous but less immunogenic than mature CDTb. We have tried to increase the immunogenicity of this domain by fusing it to the mature CDTa partner. Two different limits were designed for this receptor binding domain so that 2 fusions were evaluated. In C155 and C156, the CDTa partner is the same as for C139 and C145. In C156, between the two partners of the fusion, a spacer / linker ("linker") was added (6 Gly residues). This linker was not needed in construct C155 because the CDTb receptor binding domain used in C155 begins with a long unstructured and flexible peptide that can be used as a linker ( "Linker") / spacer.
The following fusion proteins have been designed:
* Takes into account the start codon of Met and the His marker
Cloning and production As for Example 5.
Example 10 - Evaluation of the molecular weight of CDTb and CDTa-CDTb fusion constructs Analytical ultracentrifugation can be used to determine the homogeneity and size distribution of different species in a protein sample by measuring the the rate at which molecules move in response to centrifugal force. This is based on the calculation of the sedimentation coefficients of the different species that are obtained by a sedimentation rate experiment, which depend on their shape and molecular mass. 1. The protein samples are centrifuged in a Beckman-Coulter ProteomeLab XL-1 analytical ultracentrifuge at 8000 rpm, 25000 rpm or 42000 rpm depending on the size of the target protein, after the AN-βθΤί centrifuge has was equilibrated at 15 ° C. 2. For data collection, scans are recorded at 280 nm every 5 minutes. 3. The data analysis is performed using the SEDFIT program for the determination of the C (S) distribution. Determination of the specific partial volume of the proteins is performed with the SEDNTERP software for their amino acid sequence. Sednterp can also be used to determine the viscosity and density of the buffer. 4. The molecular weight of the different species can be determined from the plot of the C (S) distribution (concentration versus sedimentation coefficient), considering that it is a better representation of the raw data than the C (M) distribution (concentration versus molecular weight) to characterize the size distribution of a mixture.
Example 11 Immunization of mice with C. difficile difficile CDTa and CDTb subunit proteins in an ASOlB formulation
Immunization of mice
Groups of 25 female Balb / C mice were immunized with IM at days 0, 14 and 28 with 5 μg of the purified subunits of the CDTa and CDTb binary toxin. These antigens were injected into an ASOlB formulation.
Anti-CDTa and anti-CDTb ELISA titers were determined in individual sera taken at day 42 (14 Post-III). The results are shown in Figures 4 and 5.
A cytotoxicity inhibition test of the binary toxin was also performed on the pooled Post-III sera (day 42). The results are shown in Figures 6 and 7. Anti-CDTa and anti-CDTb ELISA response: Protocol Whole CDTa (C34) or full CDTb (C37) subunits were deposited at 1 μg / ml (for CDTa) or 2 μg / ml (for CDTb) in phosphate buffer solution (PBS) on high-binding microtiter plates (Nunc MAXISORP ™) overnight at 4 ° C. Plates were blocked with 1% PBS-BSA for 30 min at RT with shaking. The antisera of the mice are prediluted at 1/500 in 0.2% PBS-BSA-TEWEEN ™ at 0.05% and then other half dilutions were made in the microplates and incubated at RT for 30 min. . After washing, the bound mouse antibodies were detected using peroxidase-conjugated anti-mouse antibodies of Jackson ImmunoLaboratories Inc. (ref .: 115-035-003) diluted 1/5000 in PBS-BSA at 0, 2% -Tween at 0.05%. The detection antibodies were incubated for 30 min at room temperature (RT) with shaking. The color was developed using 4 mg of O-phenylenediamine (OPD) + 5 μl of H2O2 per 10 ml of 0.1 M citrate buffer pH 4.5 for 15 minutes in the dark at room temperature. The reaction was stopped with 50 μl of HCl, and the optical density (OD) was read at 490 nm against 620 nm.
The levels of anti-CDTa or anti-CDTb antibodies are expressed as intermediate titres. A GMT was calculated for the 25 samples in each treatment group.
Test for inhibition of the cytotoxicity of the binary toxin Human colon epithelial cells (HT29 or HCT-116 cells) were cultured at 37 ° C. with 5% CO 2 in DMEM + 10% fetal bovine serum + 1% of glutamine + 1% of antibiotics (penicillin-streptomycin amphotericin) and were seeded into 96-well black tissue culture plates (Greiner Bio-one, reference: 655090) at a density of 4.104 cells / well for HT29 and 1.104 cells / well for HCT116.
After 24 h, the cell medium was removed from the wells.
The antisera of the mice were prediluted at 1:50 in the cell medium and then further third dilutions were made in the microplate (NUNC, reference: 163320). 50 μl serial dilutions of pooled mouse antisera were added to the black plates. 50 μl of a mixture of CDTa (25 ng / ml) and chymotrypsin-activated CDTb (75 ng / ml) were then added and the black plates were incubated at 37 ° C with 5% CO2 for 6 days.
After 6 days, the mixture of antisera and toxin was taken from the wells and 100 μl of Hoescht dye (BD Pharmingen, reference: 561908) diluted 1/500 in phosphate buffer solution (PBS) were added to each well for 2 hours in the dark at room temperature.
After staining, the Hoescht dye was removed from the wells and the fluorescence of the cells was measured using an Axiovision microscope.
The area covered by the fluorescent staining was determined in each well and the cytotoxicity inhibition titers were defined as the reciprocal of the dilution inducing a 50% inhibition of the fluorescent signal.
Example 12 Cytotoxicity of CDTb Proteins on HCT116 Cells
The chymotrypsin (C37) activated CDTb subunits alone appear cytotoxic on HCT116 cells. This suggests that pore formation could be responsible for cytotoxicity.
A second generation of antigen design has been produced to inhibit the pore formation step by disrupting hydrophobic interactions between beta sheets of individual proteins.
This B-subunit of the binary toxin (cell binding portion) of the binary toxins was added alone or mixed with the binary toxin subunit A (enzymatic portion) on human colon epithelial cells (HCT116) to evaluate their residual cytotoxicity.
The cytotoxicity of the candidate C123, C126, C128, C164, C166, C116, C117, C149 and C152 was tested on HCT116 cell lines.
Cytotoxicity test of the binary toxin
Human colon epithelial cells (HCT116 cells) were cultured at 37 ° C with 5% CO 2 in DMEM + 10% fetal bovine serum + 1% glutamine + 1% antibiotics (penicillin-streptomycin amphotericin) and seeded in 96-well black tissue culture plates (Greiner Bio-one, reference: 655090) at a density of 103 cells / well for HCT116.
After 24 h, 50 μl of the cell medium were taken from the wells.
Candidate binary toxins to be characterized were diluted to achieve 2 μg / ml CDTα and 6 μg / ml CDTb. Other 1/3 dilutions were made in the microplate (NUNC, reference: 163320). 50 μl of the serial dilutions of the binary toxin preparations were added to the black plates and the microplates were incubated at 37 ° C with 5% CO2 for 6 days.
After 6 days, the mixture of binary toxin and medium were taken from the wells and 100 μl of Hoechst dye (BD Pharmingen, reference: 561908) diluted 1/500 in phosphate buffer solution (PBS) were added to each well for 2 hours in the dark at room temperature.
After staining, the Hoechst stain was removed from the wells and the fluorescence of the cells was measured using an Axiovision microscope.
The cytotoxicity is expressed as a percentage of surface area covered by fluorescent staining in each well. No cytotoxic effect (100% recovery) is obtained with cells alone. And a higher level of cytotoxicity is obtained with the activated whole binary toxin (CDTa C34 + CDTb C37). The results are shown in Figure 9.
Example 13 Cytotoxicity of CDTa-CDTb Fusion Proteins on HT29 Cells
Cytotoxicity of C139, C145, C155 and C156 fusions was tested on HT29 cell lines. Potential residual cytotoxicity associated with the CdtB subunit was evaluated by overloading with whole CdtA subunit. The potential residual cytotoxicity associated with the CdtA subunit was evaluated by overload with the entire CdtB subunit. Full-length CDTa and full-length CDTb were used as controls.
Cytotoxicity test of the binary toxin
Human colon epithelial cells (HT29 cells) were cultured at 37 ° C with 5% CO 2 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, reference: 655090) at a density of 4 x 103 cells / well for HT29.
After 24 h, 50 μl of the cell medium were taken from the wells.
The candidate binary toxins to be characterized were diluted in order to reach 2 μg / ml of CDTa and 6 μg / ml of CDTb. Other 1/3 dilutions were made in the microplate (NUNC, reference: 163320). 50 μl of the serial dilutions of the binary toxin preparations were added to the black plates and the microplates were incubated at 37 ° C with 5% CO2 for 6 days.
After 6 days, the mixture of the binary toxin and the medium was taken from the well and 100 μl of Hoechst dye (BD Pharmingen, reference: 561908) diluted 1/500 in phosphate buffer solution (PBS) was added to each well for 2 hours in the dark at room temperature.
After staining, the Hoechst stain was removed from the wells and the fluorescence of the cells was measured using an Axiovision microscope.
The cytotoxicity is expressed as a percentage of surface area covered by fluorescent staining in each well. No cytotoxic effect (100% recovery) is obtained with cells alone. And a higher level of cytotoxicity is obtained with the activated whole binary toxin (CDTa C34 + CDTb C37). None of the fusions were cytotoxic and no residual cytotoxicity associated with the CDTb subunit of fusions was observed. No residual cytotoxicity associated with the CDTa subunit was observed in the whole CdtA-whole CDTb (C139) fusion. A very low residual cytotoxicity associated with the CDTa subunit was observed with the 3 fusions not including the full length CdtB (C145, C155, C156).
The results are shown in Figures 10 and 11.
Example 14 - Immunization of mice with CDTb proteins or CDTa-CDTb fusions of C. difficile in an ASOlB formulation
Immunization of mice
Groups of 12 female Balb / C mice were immunized with IM at days 0, 14 and 28 with 1 μg of the purified subunits of the CDTa and CDTb binary toxin but with 2 μg of CDTa-CDTb fusions. These antigens were injected into an ASOlB formulation.
Anti-CDTa and anti-CDTb ELISA titers were determined in individual sera taken at day 42 (14 Post-III). The results are shown in Figures 12 and 13.
A cytotoxicity inhibition test of the binary toxin was also performed on pooled Post-III sera (day 42) on HT29 and HCT116 cell lines. The results are shown in FIG. 14. Anti-CDTa and anti-CDTb ELISA response: Protocol The mutated CDTa subunits E-428 (C44) or non-activated CDTb (C46) were deposited at 1 μg / ml in phosphate buffer solution (PBS) on high-binding microtiter plates (Nunc MAXISORP ™) overnight at 4 ° C. Plates were blocked with 1% PBS-BSA for 30 min at RT with shaking. The antisera of the mice are prediluted at 1/500 in 0.2% PBS-BSA-TEWEEN ™ at 0.05% and then other half dilutions were made in the microplates and incubated at RT for 30 min. . After washing, bound mouse antibody was detected using peroxidase-conjugated anti-mouse antibody from Jackson ImmunoLaboratories Inc. (ref .: 115035-003) diluted 1/5000 in PBS-BSA at 0, 2% -Tween at 0.05%. The detection antibodies were incubated for 30 min at room temperature (RT) with shaking. The color was developed using 4 mg of O-phenylenediamine (OPD) + 5 μl of H2O2 per 10 ml of 0.1 M citrate buffer pH 4.5 for 15 minutes in the dark at room temperature. The reaction was stopped with 50 μl of HCl, and the optical density (OD) was read at 490 nm against 620 nm.
The level of anti-CDTa or anti-CDTb 'antibodies is expressed as intermediate titres. A GMT was calculated for the 12 samples in each treatment group.
The anti-CdtA antibody titers obtained are shown in FIG.
The anti-CdtB antibody titres obtained are shown in FIG. 13.
Example 15 - Inhibition Test for the Cytotoxicity of CDTb and CDTa-CDTb Fusion
Test for inhibition of the cytotoxicity of the binary toxin Human colon epithelial cells (HT29 or HCT-116 cells) were cultured at 37 ° C. with 5% CO 2 in DMEM + 10% fetal bovine serum + 1% of glutamine + 1% of antibiotics (penicillin-streptomycin amphotericin) and were seeded in 96-well black tissue culture plates (Greiner Bio-one, reference: 655090) at a density of 4.103 cells / well for HT29 and 1.103 cells / well for HCT116.
After 24 h, the cell medium was removed from the wells.
The antisera of the mice were prediluted at 1:50 in cell medium and then further third dilutions were made in the microplate (NUNC, reference: 163320). 50 μl serial dilutions of pooled mouse antisera were added to the black plates. 50 μl of a mixture of CDTa (25 ng / ml) and chymotrypsin-activated CDTb (75 ng / ml) were then added and the black plates were incubated at 37 ° C with 5% CO2 for 6 days.
After 6 days, the mixture of antisera and toxin was taken from the wells and 100 μl of Hoescht dye (BD Pharmingen, reference: 561908) diluted 1/500 in phosphate buffer solution (PBS) were added to each well for 2 hours in the dark at room temperature.
After staining, the Hoescht dye was removed from the wells and the fluorescence of the cells was measured using an Axiovision microscope.
The area covered by fluorescent staining was determined in each well and the cytotoxicity inhibition titers were defined as the reciprocal of the dilution inducing a 50% inhibition of the fluorescent signal.
The results are shown in Figure 14. Sequence Summary (Table A)
SEQ IDENTIFICATION SEQ ID 1 - Full length polypeptide sequence of CDTa
MKKFRKHKRISNCISILLILYLTLGGLLPNNIYAQDLQSYSEKVCNTTYKAPIERPEDFLKD
KEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQTRNYFYDYQIEANSREKEYK
ELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISLEKFNEFKETIQNKLFKQDGF
KDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTLIEQGYSIKIDKIVRIVIDGK
HYIKAEASVVSSLDFKDDVSKGDSWGKANYNDWSNKLTPNELADVNDYMRGGYTAINNYLIS
NGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEFGLTLTSPEYDFNKLENIDAF
KSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGSPGAYLSAIPGYAGEYEVLLN
HGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID 2 - Full length polynucleotide sequence of CDTa
ATGAAAAAATTTAGGAAACATAAAAGGATTAGTAATTGTATATCTATATTGTTGATATTATA
TCTAACTTTAGGTGGTTTGTTACCTAATAACATTTATGCACAAGACTTACAAAGCTATAGTG
AAAAAGTTTGCAATACTACTTACAAGGCTCCTATAGAAAGACCAGAAGATTTTCTTAAAGAT
AAAGAAAAGGCTAAAGAATGGGAAAGAAAAGAAGCAGAAAGAATAGAGCAAAAACTTGAAAG
ATCTGAAAAAGAAGCATTAGAATCATATAAAAAAGATTCTGTAGAAATAAGTAAATATTCTC
AGACAAGAAATTAT T T TAT GAT TATCAAATAGAAGCAAAT TC TC GAGAAAAAGAATATAAA
GAACTTCGAAATGCTATATCAAAAAATAAAATAGATAAACCTATGTATGTCTATTATTTTGA
ATCTCCAGAAAAATTTGCATTTAATAAAGTAATAAGAACAGAAAATCAAAACGAAATTTCAT
TAGAAAAATTTAATGAGTTTAAAGAAACTATACAAAACAAATTATTTAAGCAAGATGGATTT
AAAGATATTTCTTTATATGAACCTGGAAAAGGTGATGAAAAACCTACACCATTACTTATGCA
CTTAAAATTACCTAGAAATACTGGTATGTTACCATATACAAATACTAACAATGTAAGTACAT TAATAGAGCAAGGATATAGTATAAAAATAGATAAAATTGTTCGTATAGTTATAGATGGGAAG,
CACTATATTAAAGCAGAAGCATCTGTTGTAAGTAGTCTTGATTTTAAAGATGATGTAAGTAA
GGGGGATTCTTGGGGTAAAGCAAATTATAATGATTGGAGTAATAAATTAACACCTAATGAAC
TTGCTGATGTAAATGATTATATGCGTGGAGGATATACTGCAATTAATAATTATTTAATATCA
AATGGTCCAGTAAATAATCCTAACCCAGAATTAGATTCTAAAATCACAAACATTGAAAATGC
ATTAAAACGTGAACCTATTCCAACTAATTTAACTGTATATAGAAGATCTGGTCCTCAAGAAT
TTGGTTTAACTCTTACTTCCCCTGAATATGATTTTAACAAACTAGAAAATATAGATGCTTTT
AAATCAAAATGGGAAGGACAAGCACTGTCTTATCCAAACTTTATTAGTACTAGTATTGGTAG
TGTGAATATGAGTGCATTTGCTAAAAGAAAAATAGTACTACGTATAACTATACCTAAAGGTT
CTCCTGGAGCTTATCTATCAGCTATTCCAGGTTATGCAGGTGAATATGAAGTGCTTTTAAAT
CATGGAAGCAAATTTAAAATCAATAAAATTGATTCTTACAAAGATGGTACTATAACAAAATT
AATTGTTGATGCAACATTGATACCTTAA SEQ ID 3 - Full length polypeptide sequence of CDTb
MKIQMRNKKVLSFLTLTAIVSQALVYPVYAQTSTSNHSNKKKEIVNEDILPNNGLMGYYFTD EHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTLSTDRDDVL MQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELDGMKKIIPEENLF LRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDS FAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIIS TNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNG ESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLS PGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQI VTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGAT KKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFT NFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSN 'SIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNST PEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRV EATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDR ELLVLSVD SEQ ID 4 - polynucleotide sequence full length of the cdtB
AT GAAAATACAAAT GAGGAATAAAAAGGTAT TAAGT T TT T TTAACAC TACAGCTATAGT TAG TCAAGCACTAGTATATCCTGTATATGCTCAAACTAGTACAAGTAATCATTCTAATAAGAAAA AAGAAATTGTAAATGAAGATATACTCCCAAACAATGGATTAATGGGATATTATTTCACAGAT GAGCACTTTAAAGATTTAAAATTAATGGCACCCATAAAAGATGGTAATTTAAAATTTGAAGA AAAGAAAG TAGATAAAC T T C T C GGATAAAGACAAAT CAGATG TAAAAT TATAC GAT GGACAG GAAGAATAATTCCTTCTAAGGATGGTGAATATACATTATCAACTGATAGAGATGATGTCTTA ATGCAAGTAAATACTGAGAGTACTATATCAAATACACTTAAAGTTAATATGAAAAAGGGTAA AGAATATAAAGTTAGAATAGAGC TACAAGATAAAAAT T TAGGTTCAATAGATAATT TATCAT CACCTAATCTTTATTGGGAATTAGATGGTATGAAGAAAATTATACCAGAAGAAAATTTATTC TTAAGAGATTATTCTAATATAGAAAAAGATGATCCATTTATCCCAAATAACAATTTCTTTGA CCCAAAGTTGATGTCTGATTGGGAAGACGAAGATTTGGATACAGATAATGATAATATACCAG
ATTCATATGAACGAAATGGATATACTATTAAGGACTTAATTGCAGTTAAGTGGGAAGATAGT TTTGCAGAACAAGGCTATAAGAAATATGTATCAAATTATTTAGAGTCAAATACTGCTGGAGA TCCATATACAGATTATGAAAAAGCTTCAGGTTCTTTTGACAAGGCTATAAAGACTGAAGCAA GAGATCCGTTAGTTGCAGCATATCCAATTGTTGGAGTAGGTATGGAAAAATTAATTATATCT ACAAATGAACATGCCTCTACTGATCAAGGTAAAACTGTTTCCAGAGCTACTACTAACAGTAA AACTGAATCTAATACAGCTGGTGTGTCTGTTAATGTAGGATATCAAAATGGATTCACAGCTA AT GTAAC TACAAAT CCCATACAACAGATAAT TAT T T T GTT TGC CAAC CAAGATAGTAATGGA GAATCATGGAATACTGGATTAAGTATAAACAAAGGAGAATCTGCATATATAAATGCAAATGT TAGATATTACAACACAGGTACTGCACCTATGTACAAAGTGACACCAACAACAAATTTAGTGT TAGATGGAGATACATTATCAACTATCAAAGCACAAGAAAATCAAATTGGCAATAATCTATCT CCTGGAGATACTTATCCCAAAAAAGGGCTTTCACCTCTAGCTCTTAACACAATGGATCAATT TAGCTCTAGACTGATTCCTATAAATTATGATCAATTAAAAAAATTAGATGCTGGAAAGCAAA TTAAATTAGAAACAACACAAGTAAGTGGAAATTTTGGTACAAAAAATAGTTCTGGACAAATA GTAACAGAAGGAAATAGTTGGTCAGACTATATAAGTCAAATTGACAGTATTTCTGCATCTAT TATATTAGATACAGAGAATGAATCTTACGAAAGAAGAGTTACTGCTAAAAATTTACAGGATC CAGAAGATAAAACACCTGAACTTACAATTGGAGAAGCAATTGAA AAAGCTTTTGGCGCTACT AAAAAAGATGGTTTGTTATATTTTAATGATATACCAATAGATGAAAGTTGTGTTGAACTCAT ATTTGATGATAATACAGCCAATAAGATTAAAGATAGTTTAAAAACTTTGTCTGATAAAAAGA TATATAATGTTAAACTTGAAAGAGGAATGAATATACTTATAAAAACACCAACTTACTTTACT AATTTTGATGATTATAATAATTACCCTAGTACATGGAGTAATGTCAATACTACGAATCAAGA TGGTTTACAAGGCTCAGCAAATAAATTAAATGGTGAGACGAAGATTAAAATCCCTATGTCTG AGCTAAAACCTTATAAACGTTATGTTTTTAGTGGATATTCAAAGGATCCTTTAACATCTAAT TCAATAATTGTAAAGATAAAAGCAAAAGAAGAGAAAACGGATTATTTGGTACCAGAACAAGG ATATACAAAAT tagt T T T GAAAC GAAT TAT TAC GAAAAAGAT T T C T T C TAATATAGAGATAA CATTAATTGGTAGTGGTACAACATACTTAGATAACTTATCTATTACAGAGCTAAATAGTACT CCTGAAATACTTGATGAACCAGAAGTTAAAATTCCAACTGACCAAGAAATAATGGATGCACA TAAAATATATTTTGCAGATTTAAATTTTAATCCAAGTACAGGAAATACTTATATAAATGGTA TGTATTTTGCACCAACACAAACTAATAAAGAAGCTCTCGATTATATCCAAAAATATAGAGTT GAAGCTACTTTACAATATTCTGGATTTAAAGATATTGGAACTAAAGATAAAGAAATGCGTAA TTATTTAGGAGATCCAAATCAGCCTAAAACTAATTATGTTAATCTTAGGAGTTATTTTACAG GTGGAGAAAATATTATGACATACAAGAAATTAAGAATATATGCAATTACTCCAGACGATAGA GAGTTATTAGTTCTTAGTGT TGATTAG SEQ ID 5 - Construction of CDTb C37. Polypeptide sequence of CDTb '(minus signal peptide) ligated to glutathione-S-transferase protein (GST underlined)
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD νΚΕΤΟΞΜΑΙΙΕΥΙΑΡΚΗΝΜΙ ^ ΟΡΚΕΡΑΕΙΕΜΕΕΰΑνΕΡΙΡΥΰνΒΕΙΑΥΞΚΡΡΕΤΈΚνΡΓΕΞ KLPEMLKMFEPRLCHKTYLNGDHVTHPPFMLYDALPWLYMDPMCLPAFPKLVCFKKRIEAI PQIDKYLKSSKYIAWPLQGWQATFGGGPHPPKSPLEVLFQGPLGSHMEIVNEDILPNNGLMG YYFTDEHFKDLKLMAPIKPGNLKFEEKKVDKLLPKPKSPVKSIRWTGRIIPSKDGEYTLSTP RDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELPGMKKIIP EENLFLRPYSNIEKDPPFIPNNNFFPPKLMSDWEPEDLPTDNDNIPPSYERNGYTIKDLIAV KWEPSFAEQGYKKYVSNYLESNTAGPPYTPYEKASGSFPKAIKTEARPPLVAAYPIVGVGME KLIISTNEHASTPQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTPNSTAV QDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQI GNNLSPGPTYPKKGLSPLALNTMPQFSSRLIPINYPQLKKLPAGKQIKLETTQVSGNFGTKN SSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEK AFGATKKDGLLYFNDIPIPESCVELIFDDNTANKIKPSLKTLSPKKIYNVKLERGMNILIKT PTYFTNFPPYNNYPSTWSNVNTTNQPGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKD PLTSNSIIVKIKAKEEKTPYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLS IT ELNSTPEILPEPEVKIPTPQEIMPAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALPYI QKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAI TPDPRELLVLSVPHHHHHH SEQ ID 6 - Construction of CDTb C37. polynucleotide sequence of the cdtB '(minus the prodomain) ligated to the protein glutathione-S-transferase (GST underlined) atgtcccctatactaqgttattqqaaaattaaqgqccttqtqcaacccactcqacttctttt qqaatatcttqaaqaaaaatatqaagagcatttqtatqaqcqcqatqaaqqtqataaatqqc gaaacaaaaagtttgaattgggtttgqaqtttcccaatcttccttattatattgatqqtqat gttaaattaacacagtctatggccatcatacgttatataqctgacaagcacaacatqttggg tqqttqtccaaaaqaqcqtgcaqagatttcaatgcttqaaqqaqcggttttgqatattaqat acqqtqtttcqagaattgcatatagtaaagactttqaaactctcaaagttgattttcttagc aaqctacctqaaatqctgaaaatqttcgaagatcqtttatqtcataaaacatatttaaatgg tgatcatgtaacccatcctgacttcatgttgtatgacqctcttgatgttgttttatacatgg acccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatc ccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggca agccacgtttggtggtggcgaccatcctccaaaatcggatctggaagttctgttccaggggc ccctgggatcccatatggaaattgtgaatgaagatattctgccgaataatggtctgatggga tactactttaccgatgaacattttaaagatctgaaactgatggcaccgattaaagatggcaa tctgaaatttgaagaaaaaaaagtggataaactgctggataaagataa aagtgatgtgaaaa gcattcgttggaccggtcgtattattccgagcaaagatggtgaatacaccctgagcaccgat cgtgatgatgttctgatgcaggttaataccgaaagcaccattagcaataccctgaaagtgaa tatgaaaaaaggcaaagaatataaagtgcgcattgaactgcaggataaaaatctgggtagca ttgataatctgagcagcccgaatctgtattgggaactggatggtatgaaaaaaatcattccg gaagaaaacctgtttctgcgcgattatagcaatattgaaaaagatgatccgtttattccgaa taataacttttttgatccgaaactgatgagcgattgggaagatgaagatctggataccgata atgataatattccggatagctatgaacgcaatggctataccattaaagatctgattgccgtg aaatgggaagatagctttgcagaacagggctataagaaatatgtgagcaattatctggaaag caataccgcaggcgatccgtataccgattatgaaaaagcaagcggcagctttgataaagcca ttaaaaccgaagcacgtgatccgctggttgcagcatatccgattgttggtgttggtatggaa aaactgattattagcaccaatgaacatgcaagcaccgatcagggtaaaaccgttagccgtgc aaccaccaatagcaaaaccgaaagcaatacagccggtgttagcgttaatgttggttatcaga atggttttaccgccaatgtgaccaccaattatagccataccaccgataatagcaccgcagtt caggatagcaatggtgaaagctggaataccggtctgagcattaacaaaggtgaaagcgcata tatcaatgccaatgtgcgctattataacaccggcaccgcaccgatgtataaagttaccccga ccaccaatctggttctggatggtgataccctgagtaccat taaagcacaagaaaatcagatt ggcaataatctgagtccgggtgatacctatccgaaaaaaggtctgagtccgctggcactgaa taccatggatcagtttagcagccgtctgattccgattaactatgatcagctgaaaaaactgg atgccggtaaacaaatcaaactggaaaccacccaggttagcggtaattttggcaccaaaaat tcaagcggtcagattgttaccgaaggtaatagctggtcagattatatcagccagattgatag cattagcgccagcattattctggatacagaaaatgaaagctatgaacgtcgtgtgaccgcaa aaaatctgcaggacccggaagataaaacaccggaactgaccattggtgaagcaattgaaaaa gcatttggtgccaccaaaaaagatggcctgctgtattttaacgatattccgattgatgaaag ctgcgtggaactgatttttgatgataataccgccaataaaatcaaagatagcctgaaaaccc tgagcgacaaaaaaatctataatgtgaaactggaacgcggtatgaatattctgattaaaacc ccgacctattttaccaattttgatgattataacaattatccgagcacttggagcaatgtgaa taccaccaatcaggatggtctgcagggtagcgcaaataaactgaatggtgaaaccaaaatca aaattccgatgagcgaactgaaaccgtataaacgttatgtgtttagcggctatagcaaagat ccgctgaccagcaatagcattattgtgaaaatcaaagccaaagaagaaaaaaccgattatct ggttccggaacagggttataccaaatttagctatgaatttgaaaccaccgaaaaagatagca gtaatattgaaattaccctgattggtagcggcaccacctatctggataatctgagtattacc gaactgaatagcacaccggaaattctggatga accggaagtgaaaattccgaccgatcaaga aattatggatgcccataaaatctattttgccgatctgaactttaatccgagcaccggcaata cctatattaacggcatgtattttgcaccgacccagaccaataaagaagccctggattatatt cagaaatatcgtgttgaagccaccctgcagtatagcggttttaaagatattggcaccaaaga taaagaaatgcgtaattatctgggcgatccgaatcagccgaaaaccaattatgttaatctgc gcagctattttaccggtggcgaaaacattatgacctacaaaaaactgcgcatttatgccatt acaccggatgatcgtgaactgctggttctgagcgttgatcaccaccatcatcatcattaa SEQ ID NO: 7 - Amino acid sequence of the cdtB removed with the prodomain (cdtB ', aa 212-876) (C55) MSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYT DYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTES NTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYY NTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSR LIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILD TENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDD NTANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQ GSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSI IVKIKAKEEKTDYLVPEQGYTK FSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIY FADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLG DPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDHHHHHH 'SEQ ID NO: 8 - C116
MSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYT DYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTES NTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYY NTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSR LIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILD
TENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDD
NTANKIKDSLKTLSDKKIYNVKLERGMNILIKTPGGHHHHHH SEQ ID NO: 9 - C117
MAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTN
YSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDT
LSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLET
TQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKT
PELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVK
LERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPY
KRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGS
GTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAP
TQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENI
MTYKKLRIYAITPDDRELLVLSVDHHHHHH SEQ ID NO: 10 - CDTb C123 sequence with the F455R mutation.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLS KLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDWLYMDPMCLDAFPKLVCFKKRIEAI PQIDKYl · KSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSHMEIVNEDILPNNGLMG YYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTLSTD RDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELDGMKKIIP EENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAV KWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEARDPLVAAYPIVGVGME KLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAV QDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQI GNNLSPGDTYPKKGLSPLALNTMDQRSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKN SSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEK AFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNILIKT PTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKD PLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSIT ELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQT NKEALDYI
QKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAI
TPDDRELLVLSVDHHHHHH SEQ ID NO: 11 - C126 CDTb sequence in which residues 426 (Glutamate) and 453 (Aspartate) were both mutated to alanine.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD
VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFl · S
KLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDWLYMDPMCLDAFPKLVCFKKRIEAI
· PQIDKYl KSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSHMEIVNEDILPNNGLMG
YYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTLSTD rddvlmqvntestisntlkvnmkkGkeykvrielqdknlgsidnlsspnlyweldgmkkiip
EENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAV KWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEARDPLVAAYPIVGVGME KLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAV QDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQANQI GNNLS PGDTYPKKGLS PLALNTMAQFS SRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKN SSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEK AFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNILIKT PTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKD PLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSIT ELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYI QKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAI TPDDRELLVLSVDHHHHHH SEQ ID NO: 12 - C127
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT GRIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA VTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVL DGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQI
'KLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAgggPEL TIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLER GMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRY VFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTT YLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQT NKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTY KKLRIYAITPDDRELLVLSVDHHHHHH SEQ ID NO: 13 - C128
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT
GRIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS
SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP
DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA
RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA
NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLV
LDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQ IKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQg
PgggTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKK
IYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMS ELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEI.
TLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYING
MYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFT
GGENIMTYKKLRIYAITPDDRELLVLSVDHHHHHH SEQ ID NO: 14 - C139
MVYNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPSDWEDEDLDTDND
NIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIK
TEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNG
FTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTT
NLVLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDA
GKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKN
LQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLS
DKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKI
PMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSN
IEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTY
INGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRS
YFTGGENIMTYKKLRIYAITPDDRELLVLSVDGGHHHHHH SEQ ID NO: 15 - C145
MVYNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPGGGGGGAYPIVGV GMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNS TAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQE NQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFG TKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEA IEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNIL IKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGY SKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNL SITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEAL DYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMT YKKLRI YAITPDDRELLVLSVDGGHHHHHH SEQ ID NO: 16 - C155
MVYNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPTNFDDYNNYPSTW
SNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEK
TDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIP
TDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDI
GTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGGHH
HHHH SEQ ID NO: 17 - C156
MVYNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADWDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPGGGGGGNTTNQDG
LQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGY
TKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHK
IYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNY
LGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGGHHHHHH SEQ ID NO: 18 - C49
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSHHHHHH SEQ ID NO: 19 - C50
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSHHHHHH SEQ ID NO: 20 - Polypeptide sequence of the N-terminal domain of CDTa (residue 44 to residue 240).
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIHHHHHH SEQ ID NO: 21 - C67
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADWDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAG3Y3VLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 22 - C69
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS
PGAYLSAIPGYAGQYQVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 23 - C107
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS
PGAYLSAIPGYAGEYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 24 - C108
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS
PGAYLSAIPGYAGEYQVLLNHGSKFKIN
KIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 25 - C149
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT GRIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLV LDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQRSSRLIPINYDQLKKLDAGKQ IKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQD PEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKK IYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMS ELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEI TLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYING MYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFT GGENIMTYKKLRIYAITPDDRELLVLSVDHHHHHH SEQ ID NO: 26 - C152
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT
GRIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS
SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP
DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA
RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA
NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLV
LDGDTLSTIKAQANQIGNNLSPGDTYPKKGLSPLALNTMAQFSSRLIPINYDQLKKLDAGKQ
IKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQD
PEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKK
IYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMS
ELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEI
TLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYING
MYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFT
GGENIMTYKKLRIYAITPDDRELLVLSVDHHHHHH SEQ ID NO: 27 - C52
MTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLT
SNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELN
STPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKY
RVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPD
DRELLVLSVDGGHHHHHH SEQ ID NO: 28 - C53
MNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTD
YLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTD
QEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGT
KDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGGHHHH
HH
SEQ ID NO: 29 - 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 EDDKILVPIDDLVISEIDFNNNS IKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIP SLSIYSAIGIETENLDFSKKIMMLPNAPSRVFWWETGAVPGLRSLENDGTRLLDSIRDLY PGKFYWRFYAFFDYAITTLKPVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGA GGTYSLLLSSYPISTNINLSKDDLWIFNIDNEVREISIENGTIKKGKLIKDVLSKIDINK NKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSLLLSGDKNYLISN LSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNDST LEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNES VYSSYLDFVKNSDGHHNTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVE FICDNNKNIDIYFGEWKTSSSKSTIFSGNGRNVWEPIYNPDTGEDISTSLDFSYEPLYG IDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHKKVNINLDSSSFEYKWS TEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFKSF NSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNLVTGWQTINGK KYYFDINTGAALTSYKIINGKHFYFNNDGVMQLGVFKGPDGFEYFAPANTQNNNIEGQAI VYQSKFLTLNGKKYYFDNNSKAVTGWRIINNEKYYFNPNNAIAAVGLQVIDNNKYYFNPD TAIISKGWQTVNGSRYYFDTDTAIAFNGYKTIDGKHFYFDSDCWKIGVFSTSNGFEYFA PANTYNNNIEGQAIVYQSKFLTLNGKKYYFDNNSKAVTGLQTIDSKKYYFNTNTAEAATG WQTIDGKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAIASTGYTIINGK HFYFNTDGIMQ
IGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNK
KYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKG
PNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNL
NTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFY
FNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVT
GLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIM
QIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDG
NRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAI
RYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGV
DGVKAPGIYG
SEQ ID NO: 30 - Amino acid sequence of toxin B
MSLVNRKQLEKMANVRFRTQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYI DTYKKSGRNKALKKFKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKD VNSDYNVNVFYDSNAFLINTLKKTVVESAINDTLESFRENLNDPRFDYNKFFRKRMEIIY DKQKNFINYYKAQREENPELIIDDIVKTYLSNEYSKEIDELNTYIEESLNKITQNSGNDV RNFEEFKNGESFNLYEQELVERWNLAAASDILRISALKEIGGMYLDVDMLPGIQPDLFES IEKPSSVTVDFWEMTKLEAIMKYKEYIPEYTSEHFDMLDEEVQSSFESVLASKSDKSEIF SSLGDMEASPLEVKIAFNSKGIINQGLISVKDSYCSNLIVKQIENRYKILNNSLNPAISE DNDFNTTTNTFIDSIMAEANADNGRFMMELGKYLRVGFFPDVKTTINLSGPEAYAAAYQD LLMFKEGSMNIHLIEADLRNFEISKTNISQSTEQEMASLWSFDDARAKAQFEEYKRNYFE GSLGEDDNLDFSQNIVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYEAACNLFAK TPYDSVLFQKNIEDSEIAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNT DIFAGFDVDSLSTEIEAAIDLAKEDISPKSIEINLLGCNMFSYSINVEETYPGKLLLKVK DKISELMPSISQDSIIVSANQYEVRINSEGRRELLDHSGEWINKEESIIKDISSKEYISF NPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDTQIVEER IEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRF INKETGESIFVETEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLN AAFFIQSLIEYNSSKESLSNLSVA MKVQVYAQLFSTGLNTITDAAKWELVSTALDETID LLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLRQEIEAKIGIMAVNLTTATTAIIT SSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKWDYFKHVSLVETEGVFTLLD DKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHL SIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEG
EFYWRYFAFIADALITTLKPRYEDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSG GTYALSLSQYNMGINIELSESDVWIIDVDNWRDVTIESDKIKKGDLIEGILSTLSIEEN KIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLISGELKILMLNS NHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDWLISKVYMD DSKPSFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDES GVAEILKFMNRKGNTNTSDSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQ FEFICDENDNIQPYFIKFNTLETNYTLYVGNRQNMIVEPNYDLDDSGDISSTVINFSQKY LYGIDSCVNKWISPNIYTDEINITPVYETNNTYPEVIVLDANYINEKINVNINDLSIRY VWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFT PSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNLITGFVTVG DDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEG EAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFN SDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFA HHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAWGWKDLEDGSKYYFDEDTAEAYIG LSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIG VFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESD KYYFNPETKKACKGINLIDDIKYYFDEK GIMRTGLISFENNNYYFNENGEMQFGYINIED KMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYI AATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 31 - Fl
MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN
NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY
YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ
NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAA
TGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN
NIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKY
YFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQN
RFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGV
FKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFM
PDTAMAAAGGLFEIDGVIYFFGVDGVKAPGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFA
ENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAWGWKDL
EDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNI
DDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYT
IETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNEN
GEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDE
KRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 32 - F2
MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAA TGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN NIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKY YFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQN RFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGV FKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFM PDTAMAAAGGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFA ENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAWGWKDL EDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNI DDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYT IETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNEN GEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDE KRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 33 - F3
MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAA TGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN NIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKY YFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQN RFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGV
FKGSNGFEYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAWGWKDLEDGSKYYFD EDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDD NGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDM ENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYIN IEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEY IAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 34 - F4
MGWQTIDGKKYYF.NTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAA TGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN NIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKY YFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQN RFLYLHDNIYY FGNN S KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGV FKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFM PDTAMAAAGGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKL IIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVS INDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEE ISYSGILNFNNKIYYFDDSFTAWGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDG IMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTV NDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLID DIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVM QIGVFNTPDG FKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLV ISE SEQ ID NO: 35 - F5
MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN
NIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKY
YFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQ
NKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAA
TGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDAN
NIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKY YFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQN RFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGV FKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFM PDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGGGFVSINDNKHYFDDSGVMKVGYTEIDGK HFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVV GWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIES GVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYF GETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNY YFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGW LDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 36 - Sequence of polypeptide CDTA C34 construction
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGEYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 37 - Construction of CDTb C118. CdtB without the signal peptide, without the prodomain, without the binding domain to the CDTA and without the binding domain receptor MAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTN YSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDT LSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLET TQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKT PELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVK LERGMNILIKTPGGHHHHHH - SEQ ID NO: 38 - Construction of cdtB C46. Polypeptide sequence of CDTb '(minus signal peptide)
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT GRIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLV LDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQ IKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQD PEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKK IYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMS ELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEI TLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYING MYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFT GGENIMTYKKLRIYAITPDDRELLVLSVDHHHHHH SEQ ID NO: 39 - Construction C44. Polypeptide sequence of CDTa with the E428Q mutation.
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGqYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 40 - Construction C54. Polypeptide sequence of CDTa with the E430Q mutation.
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGEYqVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 41 - C68
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFISTSIGSVNMSAFAKAKIVLRITIPKGS
PGAYLSAIPGYAGgYgVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 42 - Amino acid sequence of CDTa without the signal peptide, with six mutations (R345A, Q350A, N385A, R402A, S388F, E428Q, aa 44-463) (C110)
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFES PEKFAFNKVIRTENQNEISL EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPHHHHHH SEQ ID NO: 43 - Amino acid sequence of C164 (KO for pore formation with the F455R mutation in the CdtB without prodomain)
MSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYT
DYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTES
NTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYY
NTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQRSSR
LÏPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILD
TENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDD
NTANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQ
GSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTK
FSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIY
FADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLG
DPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVD SEQ ID NO: 44 - Amino acid sequence of C166 (KO for pore formation with E426A-D453A mutations on CdtB without the prodomain)
MSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYT DYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTES NTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYY NTGTAPMYKVTPTTNLVLDGDTLSTIKAQANQIGNNLSPGDTYPKKGLSPLALNTMAQFSSR LIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILD TENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDD NTANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQ GSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTK FSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIY FADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLG DPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVD SEQ ID NO: 45 - Nucleic acid sequence of C164 (KO for pore formation with the F455R mutation in the CdtB without prodomain)
ATGAGCGATTGGGAAGATGAAGATCTGGATACCGATAATGATAATATTCCGGATAGCTATGA
ACGCAATGGCTATACCATTAAAGATCTGATTGCCGTGAAATGGGAAGATAGCTTTGCAGAAC
AGGGCTATAAGAAATATGTGAGCAATTATCTGGAAAGCAATACCGCAGGCGATCCGTATACC
GATTATGAAAAAGCAAGCGGCAGCTTTGATAAAGCCATTAAAACCGAAGCACGTGATCCGCT
GGTTGCAGCATATCCGATTGTTGGTGTTGGTATGGAAAAACTGATTATTAGCACCAATGAAC
ATGCAAGCACCGATCAGGGTAAAACCGTTAGCCGTGCAACCACCAATAGCAAAACCGAAAGC
AATACAGCCGGTGTTAGCGTTAATGTTGGTTATCAGAATGGTTTTACCGCCAATGTGACCAC
CAATTATAGCCATACCACCGATAATAGCACCGCAGTTCAGGATAGCAATGGTGAAAGCTGGA
ATACCGGTCTGAGCATTAACAAAGGTGAAAGCGCATATATCAATGCCAATGTGCGCTATTAT
AACACCGGCACCGCACCGATGTATAAAGTTACCCCGACCACCAATCTGGTTCTGGATGGTGA
TACCCTGAGTACCATTAAAGCACAAGAAAATCAGATTGGCAATAATCTGAGTCCGGGTGATA
CCTATCCGAAAAAAGGTCTGAGTCCGCTGGCACTGAATACCATGGATCAGCGCAGCAGCCGT
CTGATTCCGATTAACTATGATCAGCTGAAAAAACTGGATGCCGGTAAACAAATCAAACTGGA
AACCACCCAGGTTAGCGGTAATTTTGGCACCAAAAATTCAAGCGGTCAGATTGTTACCGAAG
GTAATAGCTGGTCAGATTATATCAGCCAGATTGATAGCATTAGCGCCAGCATTATTCTGGAT
ACAGAAAATGAAAGCTATGAACGTCGTGTGACCGCAAAAAATCTGCAGGACCCGGAAGATAA
AACACCGGAACTGACCATTGGTGAAGCAATTGAAAAAGCATTTGGTGCCACCAAAAAAGATG GCCTGCTGTATTTTAACGATATTCCGATTGATGAAAGCTGCGTGGAACTGATTTTTGATGAT '
AATACCGCCAATAAAATCAAAGATAGCCTGAAAACCCTGAGCGACAAAAAAATCTATAATGT
GAAATTAGAACGCGGTATGAATATTCTAATTAAAACCCCGACCTATTTTACCAATTTTGATG
ATTATAACAATTATCCGAGCACTTGGAGCAATGTGAATACCACCAATCAGGATGGTCTGCAG
GGTAGCGCAAATAAACTGAATGGTGAAACCAAAATCAAAATTCCGATGAGCGAACTGAAACC
GTATaAACGTTATGTGTTTAGCGGCTATAGCAAAGATCCGCTGACCAGCAATAGCATTATTG
TGAAAATCAAAGCCAAAGAAGAAAAAACCGATTATCTGGTTCCGGAACAGGGTTATACCAAA
TTTAGCTATGAATTTGAAACCACCGAAAAAGATAGCAGTAATATTGAAATTACCCTGATTGG
TAGCGGCACCACCTATCTGGATAATCTGAGTATTACCGAACTGAATAGCACACCGGAAATTC
TGGATGAACCGGAAGTGAAAATTCCGACCGATCAAGAAATTATGGATGCCCATAAAATCTAT
TTTGCCGATCTGAACTTTAATCCGAGCACCGGCAATACCTATATTAACGGCATGTATTTTGC
ACCGACCCAGACCAATAAAGAAGCCCTGGATTATATTCAGAAATATCGTGTTGAAGCCACCC TGCAGTATAGCGGTTTTAAAGATATTGGCACCAAAGATAAAGAAATGCGTAATTATCTGGGC.
GATCCGAATCAGCCGAAAACCAATTATGTTAATCTGCGCAGCTATTTTACCGGTGGCGAAAA
CATTATGACCTACAAAAAACTGCGCATTTATGCCATTACACCGGATGATCGTGAACTGCTGG
TTCTGAGCGTTGATCA SEQ ID NO: 46 - C166 nucleic acid sequence (KO for pore formation with E426A-D453A mutations on CdtB without prodomain)
ATGAGCGATTGGGAAGATGAAGATCTGGATACCGATAATGATAATATTCCGGATAGCTATGA
ACGCAATGGCTATACCATTAAAGATCTGATTGCCGTGAAATGGGAAGATAGCTTTGCAGAAC
AGGGCTATAAGAAATATGTGAGCAATTATCTGGAAAGCAATACCGCAGGCGATCCGTATACC
GATTATGAAAAAGCAAGCGGCAGCTTTGATAAAGCCATTAAAACCGAAGCACGTGATCCGCT
GGTTGCAGCATATCCGATTGTTGGTGTTGGTATGGAAAAACTGATTATTAGCACCAATGAAC
ATGCAAGCACCGATCAGGGTAAAACCGTTAGCCGTGCAACCACCAATAGCAAAACCGAAAGC
AATACAGCCGGTGTTAGCGTTAATGTTGGTTATCAGAATGGTTTTACCGCCAATGTGACCAC
CAATTATAGCCATACCACCGATAATAGCACCGCAGTTCAGGATAGCAATGGTGAAAGCTGGA
ATACCGGTCTGAGCATTAACAAAGGTGAAAGCGCATATATCAATGCCAATGTGCGCTATTAT
AACACCGGCACCGCACCGATGTATAAAGTTACCCCGACCACCAATCTGGTTCTGGATGGTGA
TACCCTGAGTACCATTAAAGCACAAGCRAATCAGATTGGCAATAATCTGAGTCCGGGTGATA
CCTATCCGAAAAAAGGTCTGAGTCCGCTGGCACTGAATACCATGGCGCAGTTTAGCAGCCGT
CTGATTCCGATTAACTATGATCAGCTGAAAAAACTGGATGCCGGTAAACAAATCAAACTGGA
AACCACCCAGGTTAGCGGTAATTTTGGCACCAAAAATTCAAGCGGTCAGATTGTTACCGAAG
GTAATAGCTGGTCAGATTATATCAGCCAGATTGATAGCATTAGCGCCAGCATTATTCTGGAT
ACAGAAAATGAAAGCTATGAACGTCGTGTGACCGCAAAAAATCTGCAGGACCCGGAAGATAA
AACACCGGAACTGACCATTGGTGAAGCAATTGAAAAAGCATTTGGTGCCACCAAAAAAGATG
GCCTGCTGTATTTTAACGATATTCCGATTGATGAAAGCTGCGTGGAACTGATTTTTGATGAT
AATACCGCCAATAAAATCAAAGATAGCCTGAAAACCCTGAGCGACAAAAAAATCTATAATGT
GAAATTAGAACGCGGTATGAATATTCTAATTAAAACCCCGACCTATTTTACCAATTTTGATG
ATTATAACAATTATCCGAGCACTTGGAGCAATGTGAATACCACCAATCAGGATGGTCTGCAG
GGTAGCGCAAATAAACTGAATGGTGAAACCAAAATCAAAATTCCGATGAGCGAACTGAAACC
GTATAAACGTTATGTGTTTAGCGGCTATAGCAAAGATCCGCTGACCAGCAATAGCATTATTG
TGAAAATCAAAGCCAAAGAAGAAAAAACCGATTATCTGGTTCCGGAACAGGGTTATACCAAA
TTTAGCTATGAATTTGAAACCACCGAAAAAGATAGCAGTAATATTGAAATTACCCTGATTGG
TAGCGGCACCACCTATCTGGATAATCTGAGTATTACCGAACTGAATAGCACACCGGAAATTC
TGGATGAACCGGAAGTGAAAATTCCGACCGATCAAGAAATTATGGATGCCCATAAAATCTAT
TTTGCCGATCTGAACTTTAATCCGAGCACCGGCAATACCTATATTAACGGCATGTATTTTGC
ACCGACCCAGACCAATAAAGAAGCCCTGGATTATATTCAGAAATATCGTGTTGAAGCCACCC
TGCAGTATAGCGGTTTTAAAGATATTGGCACCAAAGATAAAGAAATGCGTAATTATCTGGGC
GATCCGAATCAGCCGAAAACCAATTATGTTAATCTGCGCAGCTATTTTACCGGTGGCGAAAA
CATTATGACCTACAAAAAACTGCGCATTTATGCCATTACACCGGATGATCGTGAACTGCTGG
TTCTGAGCGTTGATCAC SEQ ID 47 - CDTb C37 construction. Polypeptide sequence of CDTb '(minus signal peptide) ligated to glutathione-S-transferase protein (GST underlined), without the his tag.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD
VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLS
KLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDWLYMDPMCLDAFPKLVCFKKRIEAI
PQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSHMEIVNEDILPNNGLMG YYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTLSTD RDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELDGMKKIIP EENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAV KWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEARDPLVAAYPIVGVGME KLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAV QDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQI GNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKN SSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEK AFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNILIKT PTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKD PLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSIT ELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYI QKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAI TPDDRELLVLSVD SEQ ID NO: 48 - Amino acid sequence of the cdtB removed with the prodomain (cdtB "aa 212-87 6) (C55), without the marker his
MSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYT DYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTES NTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYY NTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSR LIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILD TENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDD NTANKIKDSLKTLSDKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQ GSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTK FSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIY FADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLG DPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVD SEQ ID NO: 49 - C116 without his tag
MSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYT DYEKASGSFDKAIKTEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTES NTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYY NTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSR LIPINYDQLKKLDAGKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILD TENESYERRVTAKNLQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDD NTANKIKDS LKTLS DKKIYNVKLERGMNILIKT PGG SEQ ID NO: 50 - C117 without his tag
MAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTN YSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDT LSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLET TQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKT PELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVK LERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPY KRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGS GTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAP TQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENI MTYKKLRIYAITPDDRELLVLSVD SEQ ID NO: 51 - Sequence of the cdtB C123 with the F455R mutation without the His tag.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFl · S KLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDWLYMDPMCLDAFPKLVCFKKRIEAI PQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSHMEIVNEDILPNNGLMG YYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWTGRIIPSKDGEYTLSTD RDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLSSPNLYWELDGMKKIIP EENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIPDSYERNGYTIKDLIAV KWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEARDPLVAAYPIVGVGME KLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNSTAV QDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQENQI GNNLSPGDTYPKKGLSPLALNTMDQRSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFGTKN
SSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEAIEK
AFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNILIKT
PTYFTNFPPYNNYPSTWSNVNTTNQPGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKP
PLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSIT
ELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYI
QKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAI
TPDDRELLVLSVD SEQ ID NO: 52 - C126 CDTb sequence in which residues 426 (Glutamate) and 453 (Aspartate) were both mutated to alanine without the his tag.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD νΚΣ, ΤαΞΜΑΙΙΕΥΙΑΡΚΗΝΜΕΰΰΟΡΚΕΡΑΕΙΕΜΕΕΰΑνΕΡΙΡΥΰνΞΕΙΑΥΕΙ ^ ΕΤΕΚνΡΓΙΕ KLPEMLKMFEPRLCHKTYLNGPHVTHPPFMLYPALPWLYMPPMCLPAFPKLVCFKKRIEAI PQIPKYLKSSKYIAWPLQGWQATFGGGPHPPKSPLEVLFQGPLGSHMEIVNEPILPNNGLMG YYFTPEHFKPLKLMAPIKPGNLKFEEKKVPKLLPKPKSPVKSIRWTGRIIPSKPGEYTLSTP RPPVLMQVNTESTISNTLKVNMKKGKEYKVRIELQPKNLGSIPNLSSPNLYWELPGMKKIIP EENLFLRPYSNIEKPPPFIPNNNFFPPKLMSPWEPEPLPTPNPNIPPSYERNGYTIKPLIAV KWEPSFAEQGYKKYVSNYLESNTAGPPYTPYEKASGSFPKAIKTEARPPLVAAYPIVGVGME KLIISTNEHASTPQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTPNSTAV QPSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLPGPTLSTIKAQANQI GNNLSPGPTYPKKGLSPLALNTMAQFSSRLIPINYPQLKKLPAGKQIKLETTQVSGNFGTKN SSGQIVTEGNSWSPYISQIPSISASIILPTENESYERRVTAKNLQPPEPKTPELTIGEAIEK AFGATKKPGLLYFNPIPIPESCVELIFPPNTANKIKPSLKTLSPKKIYNVKLERGMNILIKT PTYFTNFPPYNNYPSTWSNVNTTNQPGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKP PLTSNSIIVKIKAKEEKTPYLVPEQGYTKFSYEFETTEKPSSNIEITLIGSGTTYLPNL SIT ELNSTPEILPEPEVKIPTPQEIMPAHKIYFAPLNFNPSTGNTYINGMYFAPTQTNKEALPYI QKYRVEATLQYSGFKPIGTKPKEMRNYLGPPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAI TPPPRELLVLSVP SEQ ID NO: 53 - C127 without the his marker
MEIVNEPILPNNGLMGYYFTPEHFKPLKLMAPIKPGNLKFEEKKVPKLLPKPKSPVKSIRWT
GRIIPSKPGEYTLSTPRPPVLMQVNTESTISNTLKVNMKKGKEYKVRIELQPKNLGSIPNLS
SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP
DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA
RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA
VTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVL
DGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQI
KLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAgggPEL
TIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLER
GMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRY
VFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTT
YLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQT
NKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTY
KKLRIYAITPDDRELLVLSVD SEQ ID NO: 54 - C128 without the his marker
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT
GRIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS
SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP
DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA
RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA
NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLV
LDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQ IKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQg
PgggTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKK
IYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMS
ELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEI
TLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYING
MYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFT
GGENIMTYKKLRIYAITPDDRELLVLSVD SEQ ID NO: 55 - C139 without the his marker
MVYNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPSDWEDEDLDTDND
NIPDSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIK
TEARDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNG
FTANVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTT
NLVLDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDA
GKQIKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKN
LQDPEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLS
DKKIYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKI
PMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSN
IEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTY
INGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRS
YFTGGENIMTYKKLRIYAITPDDRELLVLSVDGG SEQ ID NO: 56 - C145 without the his marker
MVYNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPGGGGGGAYPIVGV
GMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTNYSHTTDNS
TAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDTLSTIKAQE
NQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLETTQVSGNFG
TKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKTPELTIGEA
IEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVKLERGMNIL
IKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGY
SKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNL
SITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEAL
DYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRI
YAITPDDRELLVLSVDGG SEQ ID NO: 57 - C155 without the his marker
MVYNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPTNFDDYNNYPSTW SNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEK TDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIP TDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDI GTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGG SEQ ID NO: 58 - C156 without his tag
MVYNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIPGGGGGGNTTNQDG LQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGY TKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHK IYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNY LGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGG SEQ ID NO: 59 - C49 without his tag
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVS SEQ ID NO: 60 - C50 without the his marker
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSS SEQ ID NO: 61 - Polypeptide sequence of the N-terminal domain of CDTa (residue 44 to residue 240) without the his tag.
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKI SEQ ID NO: 62 - C67 without the marker his
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAG3Y3VLLNHGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID NO: 63 - C69 without the his marker
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL IEQGYSIKIDKIVRIVIDGKHYIKAEASWS SLDFKDDVSKGDSWGKANYNDWSNKLTPNEL ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS PGAYLSAIPGYAGQYQVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID NO: 64 - C107 without his tag
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS
PGAYLSAIPGYAGEYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID NO: 65 - Cl08 without the his marker
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS
PGAYLSAIPGYAGEYQVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID NO: 66 - Cl49 without the his marker
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT
GRIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS
SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP
DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA
RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA
NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLV
LDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQRSSRLIPINYDQLKKLDAGKQ
IKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQD
PEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKK
IYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMS
ELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEI
TLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYING
MYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFT
GGENIMTYKKLRIYAITPDDRELLVLSVD SEQ ID NO: 67 - C152 without the his marker
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT
GRIIPSKDGEYTLS TDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS
SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP
DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA
RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA
NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLV
LDGDTLSTIKAQANQIGNNLSPGDTYPKKGLSPLALNTMAQFSSRLIPINYDQLKKLDAGKQ IKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQD '
PEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKK
IYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMS
ELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEI
TLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYING
MYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFT
GGENIMTYKKLRIYAITPDDRELLVLSVD SEQ ID NO: 68 - C52 without the His tag MTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLT SNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELN STPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKY RVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPD DRELLVLSVDGG. SEQ ID NO: 69 - C53 without the marker his
MNTTNQDGLQGSANKLNGETKIKIPMSELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTD
YLVPEQGYTKFSYEFETTEKDSSNIEITLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTD
QEIMDAHKIYFADLNFNPSTGNTYINGMYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGT
KDKEMRNYLGDPNQPKTNYVNLRSYFTGGENIMTYKKLRIYAITPDDRELLVLSVDGG SEQ ID NO: 70 - Polypeptide sequence of the construction of CDTa C34 without the marker his
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGEYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID NO: 71 - Construction of CDTb C118. CDTb without the signal peptide, without the prodomain, without the CDTα binding domain and without the receptor binding domain, without the his tag
MAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTANVTTN YSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLVLDGDT LSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQIKLET TQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQDPEDKT PELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKKIYNVK LERGMNILIKTPGG SEQ ID NO: 72 - Construction of cdtB C46. Polypeptide sequence of CDTb '(minus signal peptide) without his tag.
MEIVNEDILPNNGLMGYYFTDEHFKDLKLMAPIKDGNLKFEEKKVDKLLDKDKSDVKSIRWT GRIIPSKDGEYTLSTDRDDVLMQVNTESTISNTLKVNMKKGKEYKVRIELQDKNLGSIDNLS SPNLYWELDGMKKIIPEENLFLRDYSNIEKDDPFIPNNNFFDPKLMSDWEDEDLDTDNDNIP DSYERNGYTIKDLIAVKWEDSFAEQGYKKYVSNYLESNTAGDPYTDYEKASGSFDKAIKTEA RDPLVAAYPIVGVGMEKLIISTNEHASTDQGKTVSRATTNSKTESNTAGVSVNVGYQNGFTA NVTTNYSHTTDNSTAVQDSNGESWNTGLSINKGESAYINANVRYYNTGTAPMYKVTPTTNLV LDGDTLSTIKAQENQIGNNLSPGDTYPKKGLSPLALNTMDQFSSRLIPINYDQLKKLDAGKQ IKLETTQVSGNFGTKNSSGQIVTEGNSWSDYISQIDSISASIILDTENESYERRVTAKNLQD PEDKTPELTIGEAIEKAFGATKKDGLLYFNDIPIDESCVELIFDDNTANKIKDSLKTLSDKK IYNVKLERGMNILIKTPTYFTNFDDYNNYPSTWSNVNTTNQDGLQGSANKLNGETKIKIPMS ELKPYKRYVFSGYSKDPLTSNSIIVKIKAKEEKTDYLVPEQGYTKFSYEFETTEKDSSNIEI TLIGSGTTYLDNLSITELNSTPEILDEPEVKIPTDQEIMDAHKIYFADLNFNPSTGNTYING MYFAPTQTNKEALDYIQKYRVEATLQYSGFKDIGTKDKEMRNYLGDPNQPKTNYVNLRSYFT GGENIMTYKKLRIYAITPDDRELLVLSVD 'SEQ ID NO: 73 - Construction C44. Polypeptide sequence of CDTa with the E428Q mutation, without the his tag.
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGqYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID NO: 74 - Construction C54. Polypeptide sequence of CDTa with the E430Q mutation, without the his tag.
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYRRSGPQEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPNFISTSIGSVNMSAFAKRKIVLRITIPKGS
PGAYLSAIPGYAGEYqVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID NO: 75 - C68 without the his marker
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFISTSIGSVNMSAFAKAKIVLRITIPKGS
PGAYLSAIPGYAGQYQVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP SEQ ID NO: 76 - Amino acid sequence of CDTa without the signal peptide, with six mutations (R345A, Q350A, N385A, R402A, S388F, E428Q, aa 44-463) (C110 without the his tag)
MVCNTTYKAPIERPEDFLKDKEKAKEWERKEAERIEQKLERSEKEALESYKKDSVEISKYSQ
TRNYFYDYQIEANSREKEYKELRNAISKNKIDKPMYVYYFESPEKFAFNKVIRTENQNEISL
EKFNEFKETIQNKLFKQDGFKDISLYEPGKGDEKPTPLLMHLKLPRNTGMLPYTNTNNVSTL
IEQGYSIKIDKIVRIVIDGKHYIKAEASWSSLDFKDDVSKGDSWGKANYNDWSNKLTPNEL
ADVNDYMRGGYTAINNYLISNGPVNNPNPELDSKITNIENALKREPIPTNLTVYARSGPAEF
GLTLTSPEYDFNKLENIDAFKSKWEGQALSYPAFIFTSIGSVNMSAFAKAKIVLRITIPKGS
PGAYLSAIPGYAGQYEVLLNHGSKFKINKIDSYKDGTITKLIVDATLIP

权利要求:
Claims (19)
[1]
An immunogenic composition comprising a CDTb protein isolated from Clostridium difficile wherein the CDTb protein isolated from Clostridium difficile was mutated by a mutation at position 426 and a mutation at position 453 to modify the ability to form pores wherein the CDTb protein isolated from Clostridium difficile comprises (i) SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 25 or SEQ ID NO: 26; or (ii) a variant of CDTb exhibiting at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 25 or SEQ ID NO: 26; or (iii) a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250 or 300 consecutive amino acids of SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 25 or SEQ ID NO: 26; or (iv) SEQ ID NO: 43 or SEQ ID NO: 44; or (V) a variant of CDTb having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 43 or SEQ ID NO: 44; or (vi) a fragment of CDTb comprising at least 30, 50, 80, 100, 120, 150, 200, 250 or 300 consecutive amino acids of SEQ ID NO: 43 or SEQ ID NO: 44.
[2]
The immunogenic composition according to claim 1, wherein the CDTb protein isolated from Clostridium difficile comprises at least one mutation selected from the group consisting of F455R, F455G, E426A and D453A.
[3]
An immunogenic composition according to claim 1, wherein the CDTb protein isolated from Clostridium difficile comprises mutations E426A and D453A.
[4]
An immunogenic composition according to any one of the preceding claims further comprising a CDTa protein isolated from Clostridium difficile
[5]
An immunogenic composition according to any one of the preceding claims wherein the composition elicits antibodies that neutralize CDTa or CDTb or both.
[6]
An immunogenic composition according to any one of the preceding claims wherein the composition elicits antibodies that neutralize the binary toxin.
[7]
An immunogenic composition according to any one of the preceding claims, wherein the immunogenic composition further comprises toxin A protein isolated from Clostridium difficile and / or toxin B protein isolated from C. difficile.
[8]
The immunogenic composition of claim 7, wherein the immunogenic composition comprises toxin A protein isolated from Clostridium difficile and toxin B protein isolated from C. difficile where toxin A protein isolated from Clostridium difficile and toxin B protein. isolated from C. difficile form a fusion protein and wherein the fusion protein is (i) SEQ ID NO: 32; or (ii) a variant having at least 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity with SEQ ID NO: 32; or (iii) a fragment of at least 800, 850, 900 or 950 consecutive amino acids of SEQ ID NO: 32.
[9]
An immunogenic composition according to any one of the preceding claims, wherein the immunogenic composition further comprises an adjuvant.
[10]
An immunogenic composition according to any one of the preceding claims, further comprising additional antigens.
[11]
Immunogenic composition according to any one of the preceding claims, further comprising a saccharide of C. difficile.
[12]
A vaccine comprising the immunogenic composition of any one of the preceding claims and a pharmaceutically acceptable excipient.
[13]
An immunogenic composition according to any one of claims 1 to 11 or a vaccine according to claim 12 for use in the treatment or prevention of C. difficile disease.
[14]
An immunogenic composition according to any one of claims 1 to 11 or a vaccine according to claim 12 for use in the treatment or prevention of 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.
[15]
15. An immunogenic composition according to any one of claims 1 to 11 or vaccine according to claim 12 for use in the treatment or prevention of a disease caused by C. difficile strain 078.
[16]
16. Use of the immunogenic composition according to any one of claims 1 to 11 or the vaccine of claim 12 in the preparation of a medicament for the prevention or treatment of C. difficile disease.
[17]
The use according to claim 16, wherein the disease is a disease caused by a C. difficile strain 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.
[18]
18. Use according to claim 16, wherein the disease is a disease caused by C. difficile strain 078.
[19]
19. CDTb protein isolated from Clostridium difficile as defined in any one of claims 1 to 4.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO2005004791A2|2002-11-08|2005-01-20|President And Fellows Of Harvard College|Compounds and methods for the treatment and prevention of bacterial infection|

---

WO2013112867A1|2012-01-27|2013-08-01|Merck Sharp & Dohme Corp.|Vaccines against clostridium difficile comprising recombinant toxins|

---

WO2014086787A1|2012-12-05|2014-06-12|Glaxosmithkline Biologicals S.A.|Immunogenic composition|

---

WO2014096393A1|2012-12-23|2014-06-26|Glaxosmithkline Biologicals S.A.|Immunogenic composition comprising elements of c. difficile cdtb and/or cdta proteins|

---

US4235877A|1979-06-27|1980-11-25|Merck & Co., Inc.|Liposome particle containing viral or bacterial antigenic subunit|

---

US4866034A|1982-05-26|1989-09-12|Ribi Immunochem Research Inc.|Refined detoxified endotoxin|

---

US4436727A|1982-05-26|1984-03-13|Ribi Immunochem Research, Inc.|Refined detoxified endotoxin product|

---

US4751180A|1985-03-28|1988-06-14|Chiron Corporation|Expression using fused genes providing for protein product|

---

US4935233A|1985-12-02|1990-06-19|G. D. Searle And Company|Covalently linked polypeptide cell modulators|

---

US4877611A|1986-04-15|1989-10-31|Ribi Immunochem Research Inc.|Vaccine containing tumor antigens and adjuvants|

---

WO1988009797A1|1987-06-05|1988-12-15|The United States Of America, As Represented By Th|Autocrine motility factors in cancer diagnosis and management|

---

US4912094B1|1988-06-29|1994-02-15|Ribi Immunochem Research Inc.|Modified lipopolysaccharides and process of preparation|

---

EP0382271B1|1989-02-04|1994-12-21|Akzo Nobel N.V.|Tocols as adjuvant in vaccine|

---

PL320214A1|1994-10-24|1997-09-15|Ophidian Pharmaceuticals|Vaccine and antitoxin for treating and preventing a disease caused by c.difficile|

---

CA2138997C|1992-06-25|2003-06-03|Jean-Paul Prieels|Vaccine composition containing adjuvants|

---

EP0812593B8|1993-03-23|2010-11-10|SmithKline Beecham Biologicals S.A.|Vaccine compositions containing 3-0 deacylated monophosphoryl lipid a|

---

DE69637254T2|1995-04-25|2008-06-19|Glaxosmithkline Biologicals S.A.|Vaccines containing a saponin and a sterol|

---

AU5972996A|1995-06-02|1996-12-18|Incyte Pharmaceuticals, Inc.|Improved method for obtaining full-length cdna sequences|

---

WO2000061762A1|1999-04-09|2000-10-19|Techlab, Inc.|RECOMBINANT TOXIN A/TOXIN B VACCINE AGAINST $i|

---

WO2001073030A2|2000-03-28|2001-10-04|Diadexus, Inc.|Compositions and methods of diagnosing, monitoring, staging, imaging and treating colon cancer|

---

AU2012349753A1|2011-12-08|2014-06-19|Novartis Ag|Clostridium difficile toxin-based vaccine|US6331537B1|1998-06-03|2001-12-18|Gpi Nil Holdings, Inc.|Carboxylic acids and carboxylic acid isosteres of N-heterocyclic compounds|

---

RU2019132111A3|2017-03-15|2021-06-29|

---

CN112703006A|2018-06-19|2021-04-23|葛兰素史密丝克莱恩生物有限公司|Immunogenic compositions|

法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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GB1411306.2|2014-06-25|

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GBGB1411306.2A|GB201411306D0|2014-06-25|2014-06-25|Immunogenic composition|

---

GB201411371A|GB201411371D0|2014-06-26|2014-06-26|Immunogenic composition|

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