HK1242375B - Compositions relating to a mutant clostridium difficile toxin and methods thereof - Google Patents

Description

Compositions and methods involving mutant Clostridium difficile toxins

This application is a divisional application of Chinese patent application No. 201280030656.7, filed on April 20, 2012, entitled “Compositions and methods involving mutant Clostridium difficile toxins”.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application 61/478,474, filed April 22, 2011, and U.S. Provisional Patent Application 61/478,899, filed April 25, 2011. The entire contents of the above applications are incorporated herein by reference in their entirety.

Field of the Invention

The present invention is directed to compositions and methods involving mutant Clostridium difficile toxins.

Background of the Invention

Clostridium difficile (C. difficile) is a Gram-positive, anaerobic bacterium associated with gastrointestinal disease in humans. Colonization by C. difficile typically occurs in the colon if the native intestinal flora is reduced due to treatment with antibodies. Infection can lead to antibiotic-associated diarrhea and sometimes pseudomembranous colitis through the secretion of glucosylating toxins, toxin A and toxin B (308 and 270 kDa, respectively), which are the main virulence factors of C. difficile.

Toxin A and toxin B are encoded by the genes tcdA and tcdB, respectively, within a 19 kb pathogenicity locus (PaLoc). In non-pathogenic strains of C. difficile, this locus is replaced by an additional 115 base pair sequence.

Both toxins A and B are potent cytotoxins. These proteins are homologous glucosyltransferases that inactivate small GTPases of the Rho/Rac/Ras family. The resulting signaling disruption leads to loss of cell-cell junctions, dysregulation of the actin cytoskeleton, and/or apoptosis, resulting in the profound secretory diarrhea associated with Clostridium difficile infection (CDI).

The number and severity of Clostridium difficile outbreaks in hospitals, nursing homes, and other long-term care facilities have increased significantly over the past decade. Major factors contributing to this increase include the emergence of highly virulent pathogenic strains, increased antibiotic use, improved detection methods, and increased exposure to airborne spores in healthcare facilities.

Metronidazole and vancomycin are currently accepted standards of care for antibiotic treatment of Clostridium difficile-associated disease (CDAD). However, approximately 20% of patients receiving such treatment experience recurrence of infection after an initial episode of CDI, and up to approximately 50% of those patients suffer additional relapses. Treatment of relapses is a significant challenge, with most relapses typically occurring within a month of the previous episode.

Therefore, there is a need for immunogenic and/or therapeutic compositions and methods involving C. difficile.

SUMMARY OF THE INVENTION

The present invention herein provides these and other objectives.

In one aspect, the present invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin A. The mutant Clostridium difficile toxin A comprises a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation relative to the corresponding wild-type Clostridium difficile toxin A. In one embodiment, at least one amino acid of the mutant Clostridium difficile toxin A is chemically cross-linked.

In one aspect, the present invention relates to an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, and wherein the polypeptide comprises at least one amino acid side chain chemically modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

In one embodiment, at least one amino acid of the mutant C. difficile toxin is chemically cross-linked.

In one embodiment, the at least one amino acid is chemically cross-linked via formaldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinate, or a combination of EDC and NHS.

In one embodiment, the immunogenic composition is recognized by the respective antitoxin neutralizing antibody or binding fragment thereof

In one embodiment, the immunogenic composition exhibits reduced cytotoxicity relative to a corresponding wild-type Clostridium difficile toxin.

In another aspect, the present invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin A, wherein the mutant Clostridium difficile toxin A comprises a glucosyltransferase domain having SEQ ID NO: 29, having amino acid substitutions at positions 285 and 287 (relative to the corresponding wild-type Clostridium difficile toxin A), and a cysteine protease domain having SEQ ID NO: 32, having an amino acid substitution at position 158 (relative to the corresponding wild-type Clostridium difficile toxin A), wherein at least one amino acid of the mutant Clostridium difficile toxin A is chemically cross-linked.

In an additional aspect, the invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin A comprising SEQ ID NO: 4, wherein at least one amino acid of the mutant Clostridium difficile toxin A is chemically cross-linked.

In another aspect, the invention relates to an immunogenic composition comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

In one aspect, the invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin B. The mutant Clostridium difficile toxin B comprises a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation relative to a corresponding wild-type Clostridium difficile toxin B.

In another aspect, the present invention relates to an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, and wherein the polypeptide comprises at least one amino acid side chain chemically modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

In another aspect, the present invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin B, wherein the mutant Clostridium difficile toxin B comprises a glucosyltransferase domain having SEQ ID NO: 31, having amino acid substitutions at positions 286 and 288 (relative to the corresponding wild-type Clostridium difficile toxin B), and a cysteine protease domain having SEQ ID NO: 33, having an amino acid substitution at position 155 (relative to the corresponding wild-type Clostridium difficile toxin B), wherein at least one amino acid of the mutant Clostridium difficile toxin B is chemically cross-linked.

In an additional aspect, the invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin B comprising SEQ ID NO: 6, wherein at least one amino acid of the mutant Clostridium difficile toxin B is chemically cross-linked.

In one aspect, the present invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin A comprising SEQ ID NO: 4 and a mutant Clostridium difficile toxin B comprising SEQ ID NO: 6, wherein at least one amino acid of each of said mutant Clostridium difficile toxins is chemically cross-linked.

In additional aspects, the invention relates to a recombinant cell, or progeny thereof, comprising a polynucleotide encoding any of the aforementioned mutant C. difficile toxins, wherein the cell lacks an endogenous polynucleotide encoding the toxin.

In another aspect, the invention relates to antibodies or antibody-binding fragments thereof that are specific for an immunogenic composition comprising a mutant C. difficile toxin.

In one aspect, the present invention relates to a method for treating a Clostridium difficile infection in a mammal. The method comprises administering to the mammal an immunogenic composition comprising a mutant Clostridium difficile toxin A comprising SEQ ID NO: 4 and a mutant Clostridium difficile toxin B comprising SEQ ID NO: 6, wherein at least one amino acid of each of the mutant Clostridium difficile toxins is cross-linked with formaldehyde.

In one aspect, the present invention relates to a method for treating a Clostridium difficile infection in a mammal. The method comprises administering to the mammal an immunogenic composition comprising a mutant Clostridium difficile toxin A comprising SEQ ID NO: 4 and a mutant Clostridium difficile toxin B comprising SEQ ID NO: 6, wherein at least one amino acid of each of the mutant Clostridium difficile toxins is cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and/or N-hydroxysuccinimide (NHS).

In one aspect, the present invention relates to a method for inducing an immune response to a Clostridium difficile infection in a mammal. The method comprises administering to the mammal an immunogenic composition comprising a mutant Clostridium difficile toxin A comprising SEQ ID NO: 4 and a mutant Clostridium difficile toxin B comprising SEQ ID NO: 6, wherein at least one amino acid of each of the mutant Clostridium difficile toxins is cross-linked with formaldehyde.

In one aspect, the present invention relates to a method for inducing an immune response to a Clostridium difficile infection in a mammal. The method comprises administering to the mammal an immunogenic composition comprising a mutant Clostridium difficile toxin A comprising SEQ ID NO: 4 and a mutant Clostridium difficile toxin B comprising SEQ ID NO: 6, wherein at least one amino acid of each of the mutant Clostridium difficile toxins is cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and/or N-hydroxysuccinimide (NHS).

In one embodiment, the method of treating or inducing an immune response is in a mammal in need thereof.

In one embodiment, the method of treating or inducing an immune response involves a mammal that has relapsed C. difficile infection.

In one embodiment, the method of treating or inducing an immune response comprises parenteral administration of the composition.

In one embodiment, the method of treatment or method of inducing an immune response comprises an immunogenic composition further comprising an adjuvant.

In one embodiment, the adjuvant comprises aluminum hydroxide gel and CpG oligonucleotides. In another embodiment, the adjuvant comprises ISCOMATRIX.

In one embodiment, the isolated polypeptide comprises at least one aspartic acid residue side chain of the polypeptide or at least one glutamic acid residue side chain of the polypeptide chemically cross-linked via glycine.

In one embodiment, the isolated polypeptide comprises at least one cross-link between a side chain of an aspartic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide; and at least one cross-link between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide.

In one embodiment, the isolated polypeptide comprises a β-alanine moiety attached to the side chain of at least one lysine residue of the polypeptide.

In one embodiment, the isolated polypeptide comprises a glycine moiety attached to the side chain of an aspartic acid residue of the polypeptide or to the side chain of a glutamic acid residue of the polypeptide.

In one embodiment, the isolated polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, and wherein at least one lysine residue of the polypeptide is side-chain-linked to a β-alanine moiety.

In one embodiment, the isolated polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, and wherein at least one lysine residue of the polypeptide is side-chain-linked to a β-alanine moiety.

In one embodiment, the isolated polypeptide comprises the side chain of a second lysine residue linked to the side chain of an aspartic acid residue or to the side chain of a glutamic acid residue.

In one embodiment, the isolated polypeptide comprises an aspartic acid residue side chain or a glutamic acid residue side chain of the polypeptide linked to a glycine moiety.

In one embodiment, the isolated polypeptide has an _EC50 of at least about 100 μg/ml.

In one aspect, the immunogenic composition comprises an isolated polypeptide having the amino acid sequence shown in SEQ ID NO: 4 (wherein the methionine residue at position 1 is optionally absent), and an isolated polypeptide having the amino acid sequence shown in SEQ ID NO: 6 (wherein the methionine residue at position 1 is optionally absent), and wherein the polypeptide has at least one amino acid side chain chemically modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

In one embodiment, the polypeptide comprises at least one of any of the following: a) at least one β-alanine moiety attached to a lysine side chain of the polypeptide; b) at least one cross-link between a side chain of an aspartic acid residue and a side chain of a lysine residue of the polypeptide; and c) at least one cross-link between a side chain of a glutamic acid residue and a side chain of a lysine residue of the polypeptide.

In one embodiment, the isolated polypeptide has an _EC50 of at least about 100 μg/ml.

In one aspect, the immunogenic composition comprises an isolated polypeptide having the amino acid sequence of SEQ ID NO: 4 (wherein the methionine residue at position 1 is optionally absent), and an isolated polypeptide having the amino acid sequence of SEQ ID NO: 6 (wherein the methionine residue at position 1 is optionally absent), and a) wherein at least one lysine residue of SEQ ID NO: 4 is side chain linked to a β-alanine moiety, and b) wherein at least one lysine residue of SEQ ID NO: 6 is side chain linked to a β-alanine moiety.

In one embodiment, the immunogenic composition comprises the second lysine residue of SEQ ID NO: 4 linked to an aspartic acid residue or to a glutamic acid residue, and wherein the second lysine residue of SEQ ID NO: 6 is linked to an aspartic acid residue or to a glutamic acid residue.

In one embodiment, the immunogenic composition comprises an aspartic acid residue side chain or a glutamic acid residue side chain of a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 (wherein the methionine residue at position 1 is optionally absent) linked to a glycine moiety.

In one embodiment, the immunogenic composition comprises an aspartic acid residue side chain or a glutamic acid residue side chain of a polypeptide having the amino acid sequence of SEQ ID NO: 6 (wherein the methionine residue at position 1 is optionally absent) linked to a glycine moiety.

In one embodiment, the isolated polypeptide has an _EC50 of at least about 100 μg/ml.

In one aspect, the immunogenic composition comprises: an isolated polypeptide having the amino acid sequence of SEQ ID NO: 84 and an isolated polypeptide having the amino acid sequence of SEQ ID NO: 86, wherein each polypeptide comprises: a) at least one cross-link between a side chain of an aspartic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide; b) at least one cross-link between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide; c) a β-alanine moiety attached to the side chain of at least one lysine residue of the polypeptide; and d) a glycine moiety attached to the side chain of at least one aspartic acid residue of the polypeptide or at least one glutamic acid residue of the polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Sequence alignment of wild-type C. difficile toxin A and mutant toxin A having SEQ ID NO: 4 from strains 630, VPI10463, R20291, CD196, aligned using CLUSTALW with default parameters.

Figure 2: Sequence alignment of wild-type C. difficile toxin A and mutant toxin B having SEQ ID NO: 6 from strains 630, VPI10463, R20291, CD196, aligned using CLUSTALW with default parameters.

Figure 3: Graph showing the identification of wild-type toxin-negative C. difficile strains. Culture media from 13 C. difficile strains were tested by ELISA for toxin A. As shown, seven strains expressed toxin A, while six did not (strains 1351, 3232, 7322, 5036, 4811, and VPI 11186).

Figures 4A and B: SDS-PAGE results showing that triple mutant A (SEQ ID NO: 4), double mutant B (SEQ ID NO: 5), and triple mutant B (SEQ ID NO: 6) did not glucosylate Rac1 or RhoA GTPase in an in vitro glucosylation assay using UDP- ^14C -glucose; whereas 10 μg to 1 ng of wild-type toxin B did glucosylate Rac1.

Figure 5: Western blot showing ablation of cysteine protease activity in mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively) compared to cleaved fragments of wild-type toxins A and B (SEQ ID NOs: 1 and 2, respectively).

Figure 6: Graph showing that triple mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively) exhibit residual cytotoxicity when tested at high concentrations (e.g., approximately 100 μg/ml) as determined by in vitro cytotoxicity assays in IMR-90 cells.

Figure 7: Graph showing similar _EC50 values for triple mutant toxin B (SEQ ID NO: 6) and sevenfold mutant toxin B (SEQ ID NO: 8).

Figure 8: Graph representing the results of an in vitro cytotoxicity assay, wherein ATP levels (RLU) are plotted against increasing concentrations of triple mutant TcdA (SEQ ID NO: 4) (upper panel) and triple mutant TcdB (SEQ ID NO: 6) (lower panel). The residual cytotoxicity of mutant toxins A and B can be completely eliminated by neutralizing antibodies specific for mutant toxin A (upper panel - pAb A and mAb A3-25 + A60-22) and for mutant toxin B (lower panel - pAb B).

Figure 9: Images of IMR-90 cell morphology 72 hours after treatment. Part A shows mock-treated control cells. Part B shows cell morphology after treatment with a formalin-inactivated mutant TcdB (SEQ ID NO: 6). Part C shows cell morphology after treatment with an EDC-inactivated mutant TcdB (SEQ ID NO: 6). Part D shows cell morphology after treatment with wild-type toxin B (SEQ ID NO: 2). Part E shows cell morphology after treatment with a triple mutant TcdB (SEQ ID NO: 6). Similar results were observed with TcdA treatment.

Figure 10: Graph showing the neutralizing antibody titers described in Example 25 (study muCdiff2010-06).

Figure 11: Graph showing the neutralizing antibody titers described in Example 26 (study muCdiff2010-07).

Figure 12: Graph showing neutralizing antibody responses against toxins A and B in hamsters after 4 immunizations as described in Example 27 (Study ham C. difficile 2010-02).

Figure 13: Graph showing the neutralizing antibody response in hamsters following vaccination with chemically inactivated genetic mutant toxins and List Biological toxoids as described in Example 27 (Study ham C. difficile 2010-02).

Figure 14: Survival curves of hamsters in the three immunized groups compared to non-immunized controls, as described in Example 28 (Study ham C. difficile 2010-02, continued).

Figure 15: Graph showing the relative neutralizing antibody responses in hamsters to different formulations of C. difficile mutant toxin (study ham C. difficile 2010-03), as described in Example 29.

Figures 16A-B: Graphs showing strong relative neutralizing antibody responses in cynomolgus monkeys against chemically inactivated genetic mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively), as described in Example 30.

Figure 17: Amino acid sequences of the light chain (VL) and heavy chain (HL) variable regions of A3-25 mAb IgE. Signal peptide - highlighted; CDRs - italicized and underlined; constant region - bold and underlined (full sequence not shown).

Figure 18: Graph showing titration of individual toxin A monoclonal antibodies in a toxin neutralization assay using ATP levels (quantified by relative light units - RLU) as an indicator of cell viability. mAbs A80-29, A65-33, A60-22, and A3-25 neutralized toxin A with increasing concentration compared to the toxin control ( _4xEC50 ) (but not to the level of the positive rabbit anti-toxin A control). mAbs A50-10, A56-33, and A58-46 did not neutralize toxin A. The cell-only control was ^1-1.5x106 RLU.

Figure 19: Mapping of a panel of 8 epitopes of toxin B mAbs by BiaCore.

Figures 20A-C: Synergistic neutralizing activity of combinations of toxin A mAbs: Adding varying dilutions of neutralizing antibodies A60-22, A65-33, and A80-29 to increasing concentrations of A3-25 mAb synergistically enhanced neutralization of toxin A (regardless of dilution). The RLU for the toxin A only (4x _EC50 ) control is shown (< ^0.3x106 ), while the cell only control was ^2-2.5x106 RLU, as shown in Figures 20B and 20C.

Figure 21: Synergistic neutralization activity of combinations of toxin B mAbs: Neutralization of toxin B by mAbs 8-26, B60-2, and B59-3 is shown in Figure 21 A. Neutralization of toxin B was synergistically enhanced upon combining dilutions of B8-26 and B59-3 (Figure 21 B).

Figure 22: Western blot showing that expression of Rac1 GTPase is reduced from 24 to 96 hours in extracts of genetic mutant toxin B (SEQ ID NO: 6), but not in extracts treated with wild-type toxin B (SEQ ID NO: 2). The blot also shows that Rac1 is glucosylated in extracts treated with toxin B, but not in extracts treated with genetic mutant toxin B.

Figure 23A-K: Graphs representing the results of an in vitro cytotoxicity assay, where ATP levels (RLU) are plotted against: increasing concentrations of C. difficile culture media and hamster serum pools (■); crude toxin (culture harvest) from each strain and hamster serum pool (●); purified toxin (commercial toxin obtained from List Biologicals) and hamster serum pool (▲); crude toxin control; and purified toxin (◆), control. Toxin from each strain was added to cells at 4x _EC50 values. Figure 23 shows that an immunogenic composition comprising mutant TcdA (SEQ ID NO: 4) and mutant TcdB (SEQ ID NO: 6) (wherein the mutant toxin is inactivated by EDC, e.g., according to Example 29, Table 15 described herein) induced neutralizing antibodies that exhibited neutralizing activity against toxins of C. difficile from at least the following 16 different CDC strains (as compared to respective toxin-only controls): 2007886 (Figure 23A); 2006017 (Figure 23B); 2007070 (Figure 23C); 2007302 (Figure 23D); 2007838 (Figure 23E); 2007886 (Figure 23F); 2009292 (Figure 23G); 2004013 (Figure 23H); 2009141 (Figure 23I); 2005022 (Figure 23J); 2006376 (Figure 23K).

FIG24 : Illustrated are exemplary EDC/NHS inactivation of mutant C. difficile toxins, resulting in at least three possible types of modifications: cross-linking, glycine adducts, and β-alanine adducts. Part A illustrates cross-linking. The carboxylic acid residues of the triple mutant toxin are activated by the addition of EDC and NHS. The activated ester reacts with primary amines to form stable amide bonds, resulting in intra- and intermolecular cross-linking. Part B illustrates the formation of glycine adducts. Following inactivation, the residual activated ester is quenched by the addition of glycine to form a stable amide bond. Part C illustrates the formation of β-alanine adducts. Three moles of NHS can react with one mole of EDC to form activated β-alanine. This, in turn, reacts with primary amines to form stable amide bonds.

Figure 25: Illustrate exemplary mutant C. difficile toxin EDC/NHS inactivation resulting in at least one of the following types of modifications: (A) cross-linking, (B) glycine adducts, and (C) [beta]-alanine adducts.

Sequence Description

SEQ ID NO: 1 shows the amino acid sequence of wild-type Clostridium difficile 630 toxin A (TcdA).

SEQ ID NO: 2 shows the amino acid sequence of wild-type Clostridium difficile 630 toxin B (TcdB).

SEQ ID NO: 3 shows the amino acid sequence of a mutant TcdA having mutations at positions 285 and 287 compared to SEQ ID NO: 1.

SEQ ID NO: 4 shows the amino acid sequence of a mutant TcdA having mutations at positions 285, 287 and 700 compared to SEQ ID NO: 1.

SEQ ID NO: 5 shows the amino acid sequence of a mutant TcdB having mutations at positions 286 and 288 compared to SEQ ID NO: 2.

SEQ ID NO: 6 shows the amino acid sequence of a mutant TcdB having mutations at positions 286, 288, and 698 compared to SEQ ID NO: 2.

SEQ ID NO: 7 shows the amino acid sequence of a mutant TcdA having mutations at positions 269, 272, 285, 287, 460, 462 and 700 compared to SEQ ID NO: 1.

SEQ ID NO: 8 shows the amino acid sequence of a mutant TcdB having mutations at positions 270, 273, 286, 288, 461, 463 and 698 compared to SEQ ID NO: 2.

SEQ ID NO: 9 shows the DNA sequence encoding wild-type Clostridium difficile 630 toxin A (TcdA).

SEQ ID NO: 10 shows the DNA sequence encoding wild-type Clostridium difficile 630 toxin B (TcdB).

SEQ ID NO: 11 shows the DNA sequence encoding SEQ ID NO: 3.

SEQ ID NO: 12 shows the DNA sequence encoding SEQ ID NO: 4.

SEQ ID NO: 13 shows the DNA sequence encoding SEQ ID NO: 5.

SEQ ID NO: 14 shows the DNA sequence encoding SEQ ID NO: 6.

SEQ ID NO: 15 shows the amino acid sequence of wild-type Clostridium difficile R20291 TcdA.

SEQ ID NO: 16 shows the DNA sequence encoding SEQ ID NO: 15.

SEQ ID NO: 17 shows the amino acid sequence of wild-type C. difficile CD196TcdA.

SEQ ID NO: 18 shows the DNA sequence encoding SEQ ID NO: 17.

SEQ ID NO: 19 shows the amino acid sequence of wild-type Clostridium difficile VPI10463TcdA.

SEQ ID NO:20 shows the DNA sequence encoding SEQ ID NO:19.

SEQ ID NO: 21 shows the amino acid sequence of wild-type Clostridium difficile R20291TcdB.

SEQ ID NO: 22 shows the DNA sequence encoding SEQ ID NO: 21.

SEQ ID NO: 23 shows the amino acid sequence of wild-type Clostridium difficile CD196TcdB.

SEQ ID NO: 24 shows the DNA sequence encoding SEQ ID NO: 23.

SEQ ID NO: 25 shows the amino acid sequence of wild-type Clostridium difficile VPI10463TcdB.

SEQ ID NO: 26 shows the DNA sequence encoding SEQ ID NO: 25.

SEQ ID NO: 27 shows the DNA sequence of the pathogenicity locus of wild-type Clostridium difficile VPI10463.

SEQ ID NO:28 shows the amino acid sequence of residues 101-293 of SEQ ID NO:1.

SEQ ID NO:29 shows the amino acid sequence of residues 1-542 of SEQ ID NO:1.

SEQ ID NO:30 shows the amino acid sequence of residues 101-293 of SEQ ID NO:2.

SEQ ID NO:31 shows the amino acid sequence of residues 1-543 of SEQ ID NO:2.

SEQ ID NO:32 shows the amino acid sequence of residues 543-809 of SEQ ID NO:1.

SEQ ID NO:33 shows the amino acid sequence of residues 544-767 of SEQ ID NO:2.

SEQ ID NO: 34 shows the amino acid sequence of mutant TcdA, wherein residues 101, 269, 272, 285, 287, 460, 462, 541, 542, 543, 589, 655, and 700 can be any amino acid.

SEQ ID NO: 35 shows the amino acid sequence of mutant TcdB, wherein 102, 270, 273, 286, 288, 384, 461, 463, 520, 543, 544, 587, 600, 653, 698 and 751 can be any amino acid.

SEQ ID NO: 36 shows the amino acid sequence of the variable light chain of a neutralizing antibody against Clostridium difficile TcdA (A3-25mAb).

SEQ ID NO: 37 shows the amino acid sequence of the variable heavy chain of a neutralizing antibody against Clostridium difficile TcdA (A3-25 mAb).

SEQ ID NO: 38 shows the amino acid sequence of CDR1 of the variable light chain of a neutralizing antibody against Clostridium difficile TcdA (A3-25mAb).

SEQ ID NO: 39 shows the amino acid sequence of CDR2 of the variable light chain of a neutralizing antibody against Clostridium difficile TcdA (A3-25mAb).

SEQ ID NO: 40 shows the amino acid sequence of CDR3 of the variable light chain of a neutralizing antibody against Clostridium difficile TcdA (A3-25mAb).

SEQ ID NO: 41 shows the amino acid sequence of CDR1 of the variable heavy chain of a neutralizing antibody against Clostridium difficile TcdA (A3-25mAb).

SEQ ID NO: 42 shows the amino acid sequence of CDR2 of the variable heavy chain of a neutralizing antibody against Clostridium difficile TcdA (A3-25mAb).

SEQ ID NO: 43 shows the amino acid sequence of CDR3 of the variable heavy chain of a neutralizing antibody against Clostridium difficile TcdA (A3-25mAb).

SEQ ID NO:44 shows the DNA sequence encoding SEQ ID NO:3.

SEQ ID NO:45 shows the DNA sequence encoding SEQ ID NO:4.

SEQ ID NO:46 shows the DNA sequence encoding SEQ ID NO:5.

SEQ ID NO:47 shows the DNA sequence encoding SEQ ID NO:6.

SEQ ID NO: 48 shows the nucleotide sequence of the immunostimulatory oligonucleotide ODN CpG 24555.

SEQ ID NO: 49 shows the amino acid sequence of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 50 shows the amino acid sequence of the signal peptide of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 51 shows the amino acid sequence of CDR1 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 52 shows the amino acid sequence of CDR2 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 53 shows the amino acid sequence of CDR3 of the variable heavy chain of Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 54 shows the amino acid sequence of the constant region of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 55 shows the amino acid sequence of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 56 shows the amino acid sequence of the signal peptide of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 57 shows the amino acid sequence of CDR1 of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 58 shows the amino acid sequence of CDR2 of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 59 shows the amino acid sequence of CDR3 of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B8-26mAb).

SEQ ID NO: 60 shows the amino acid sequence of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 61 shows the amino acid sequence of the signal peptide of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 62 shows the amino acid sequence of CDR1 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 63 shows the amino acid sequence of CDR2 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 64 shows the amino acid sequence of CDR3 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 65 shows the amino acid sequence of the constant region of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 66 shows the amino acid sequence of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 67 shows the amino acid sequence of the signal peptide of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 68 shows the amino acid sequence of CDR1 of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 69 shows the amino acid sequence of CDR2 of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 70 shows the amino acid sequence of CDR3 of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B59-3mAb).

SEQ ID NO: 71 shows the amino acid sequence of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 72 shows the amino acid sequence of the signal peptide of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 73 shows the amino acid sequence of CDR1 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 74 shows the amino acid sequence of CDR2 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 75 shows the amino acid sequence of CDR3 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 76 shows the amino acid sequence of the constant region of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 77 shows the amino acid sequence of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 78 shows the amino acid sequence of the signal peptide of the variable light chain of Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 79 shows the amino acid sequence of CDR1 of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 80 shows the amino acid sequence of CDR2 of the variable light chain of the Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 81 shows the amino acid sequence of CDR3 of the variable light chain of Clostridium difficile TcdB neutralizing antibody (B9-30mAb).

SEQ ID NO: 82 shows the amino acid sequence of mutant TcdB, wherein the residues at positions 102, 270, 273, 286, 288, 384, 461, 463, 520, 543, 544, 587, 600, 653, 698 and 751 may be any amino acid.

SEQ ID NO: 83 shows the amino acid sequence of a mutant TcdA having mutations at positions 269, 272, 285, 287, 460, 462 and 700 compared to SEQ ID NO: 1, wherein the methionine at position 1 is deleted.

SEQ ID NO: 84 shows the amino acid sequence of a mutant Clostridium difficile toxin A having mutations at positions 285, 287, and 700 compared to SEQ ID NO: 1, wherein the methionine at position 1 is deleted.

SEQ ID NO: 85 shows the amino acid sequence of a mutant Clostridium difficile toxin B having mutations at positions 270, 273, 286, 288, 461, 463 and 698 compared to SEQ ID NO: 2, wherein the methionine at position 1 is deleted.

SEQ ID NO: 86 shows the amino acid sequence of a mutant Clostridium difficile toxin B having mutations at positions 286, 288, and 698 compared to SEQ ID NO: 2, wherein the methionine at position 1 is deleted.

SEQ ID NO: 87 shows the amino acid sequence of wild-type Clostridium difficile 2004013 TcdA.

SEQ ID NO: 88 shows the amino acid sequence of wild-type Clostridium difficile 2004111 TcdA.

SEQ ID NO: 89 shows the amino acid sequence of wild-type Clostridium difficile 2004118 TcdA.

SEQ ID NO: 90 shows the amino acid sequence of wild-type Clostridium difficile 2004205TcdA.

SEQ ID NO: 91 shows the amino acid sequence of wild-type Clostridium difficile 2004206TcdA.

SEQ ID NO: 92 shows the amino acid sequence of wild-type Clostridium difficile 2005022 TcdA.

SEQ ID NO: 93 shows the amino acid sequence of wild-type C. difficile 2005088TcdA.

SEQ ID NO: 94 shows the amino acid sequence of wild-type C. difficile 2005283 TcdA.

SEQ ID NO: 95 shows the amino acid sequence of wild-type Clostridium difficile 2005325TcdA.

SEQ ID NO: 96 shows the amino acid sequence of wild-type Clostridium difficile 2005359 TcdA.

SEQ ID NO: 97 shows the amino acid sequence of wild-type Clostridium difficile 2006017TcdA.

SEQ ID NO: 98 shows the amino acid sequence of wild-type Clostridium difficile 2007070TcdA.

SEQ ID NO: 99 shows the amino acid sequence of wild-type Clostridium difficile 2007217TcdA.

SEQ ID NO: 100 shows the amino acid sequence of wild-type Clostridium difficile 2007302 TcdA.

SEQ ID NO: 101 shows the amino acid sequence of wild-type Clostridium difficile 2007816TcdA.

SEQ ID NO: 102 shows the amino acid sequence of wild-type Clostridium difficile 2007838 TcdA.

SEQ ID NO: 103 shows the amino acid sequence of wild-type Clostridium difficile 2007858 TcdA.

SEQ ID NO: 104 shows the amino acid sequence of wild-type Clostridium difficile 2007886TcdA.

SEQ ID NO: 105 shows the amino acid sequence of wild-type Clostridium difficile 2008222TcdA.

SEQ ID NO: 106 shows the amino acid sequence of wild-type Clostridium difficile 2009078TcdA.

SEQ ID NO: 107 shows the amino acid sequence of wild-type Clostridium difficile 2009087TcdA.

SEQ ID NO: 108 shows the amino acid sequence of wild-type Clostridium difficile 2009141 TcdA.

SEQ ID NO: 109 shows the amino acid sequence of wild-type Clostridium difficile 2009292TcdA.

SEQ ID NO: 110 shows the amino acid sequence of wild-type Clostridium difficile 2004013TcdB.

SEQ ID NO: 111 shows the amino acid sequence of wild-type Clostridium difficile 2004111 TcdB.

SEQ ID NO: 112 shows the amino acid sequence of wild-type Clostridium difficile 2004118 TcdB.

SEQ ID NO: 113 shows the amino acid sequence of wild-type Clostridium difficile 2004205TcdB.

SEQ ID NO: 114 shows the amino acid sequence of wild-type Clostridium difficile 2004206TcdB.

SEQ ID NO: 115 shows the amino acid sequence of wild-type Clostridium difficile 2005022TcdB.

SEQ ID NO: 116 shows the amino acid sequence of wild-type Clostridium difficile 2005088TcdB.

SEQ ID NO: 117 shows the amino acid sequence of wild-type Clostridium difficile 2005283TcdB.

SEQ ID NO: 118 shows the amino acid sequence of wild-type Clostridium difficile 2005325TcdB.

SEQ ID NO: 119 shows the amino acid sequence of wild-type Clostridium difficile 2005359 TcdB.

SEQ ID NO: 120 shows the amino acid sequence of wild-type Clostridium difficile 2006017TcdB.

SEQ ID NO: 121 shows the amino acid sequence of wild-type Clostridium difficile 2006376TcdB.

SEQ ID NO: 122 shows the amino acid sequence of wild-type Clostridium difficile 2007070TcdB.

SEQ ID NO: 123 shows the amino acid sequence of wild-type Clostridium difficile 2007217TcdB.

SEQ ID NO: 124 shows the amino acid sequence of wild-type Clostridium difficile 2007302 TcdB.

SEQ ID NO: 125 shows the amino acid sequence of wild-type Clostridium difficile 2007816TcdB.

SEQ ID NO: 126 shows the amino acid sequence of wild-type Clostridium difficile 2007838TcdB.

SEQ ID NO: 127 shows the amino acid sequence of wild-type Clostridium difficile 2007858TcdB.

SEQ ID NO: 128 shows the amino acid sequence of wild-type Clostridium difficile 2007886TcdB.

SEQ ID NO: 129 shows the amino acid sequence of wild-type Clostridium difficile 2008222TcdB.

SEQ ID NO: 130 shows the amino acid sequence of wild-type Clostridium difficile 2009078TcdB.

SEQ ID NO: 131 shows the amino acid sequence of wild-type Clostridium difficile 2009087TcdB.

SEQ ID NO: 132 shows the amino acid sequence of wild-type Clostridium difficile 2009141 TcdB.

SEQ ID NO: 133 shows the amino acid sequence of wild-type Clostridium difficile 2009292TcdB.

SEQ ID NO: 134 shows the amino acid sequence of wild-type C. difficile 014TcdA.

SEQ ID NO: 135 shows the amino acid sequence of wild-type C. difficile 015TcdA.

SEQ ID NO: 136 shows the amino acid sequence of wild-type C. difficile 020TcdA.

SEQ ID NO: 137 shows the amino acid sequence of wild-type C. difficile 023TcdA.

SEQ ID NO: 138 shows the amino acid sequence of wild-type C. difficile 027TcdA.

SEQ ID NO: 139 shows the amino acid sequence of wild-type C. difficile 029 TcdA.

SEQ ID NO: 140 shows the amino acid sequence of wild-type C. difficile 046TcdA.

SEQ ID NO: 141 shows the amino acid sequence of wild-type Clostridium difficile 014TcdB.

SEQ ID NO: 142 shows the amino acid sequence of wild-type Clostridium difficile 015TcdB.

SEQ ID NO: 143 shows the amino acid sequence of wild-type Clostridium difficile 020TcdB.

SEQ ID NO: 144 shows the amino acid sequence of wild-type Clostridium difficile 023TcdB.

SEQ ID NO: 145 shows the amino acid sequence of wild-type Clostridium difficile 027TcdB.

SEQ ID NO: 146 shows the amino acid sequence of wild-type Clostridium difficile 029TcdB.

SEQ ID NO: 147 shows the amino acid sequence of wild-type Clostridium difficile 046TcdB.

SEQ ID NO: 148 shows the amino acid sequence of wild-type C. difficile 001 TcdA.

SEQ ID NO: 149 shows the amino acid sequence of wild-type C. difficile 002 TcdA.

SEQ ID NO: 150 shows the amino acid sequence of wild-type C. difficile 003 TcdA.

SEQ ID NO: 151 shows the amino acid sequence of wild-type C. difficile 004 TcdA.

SEQ ID NO: 152 shows the amino acid sequence of wild-type C. difficile 070TcdA.

SEQ ID NO: 153 shows the amino acid sequence of wild-type C. difficile 075TcdA.

SEQ ID NO: 154 shows the amino acid sequence of wild-type C. difficile 077TcdA.

SEQ ID NO: 155 shows the amino acid sequence of wild-type C. difficile 081 TcdA.

SEQ ID NO: 156 shows the amino acid sequence of wild-type C. difficile 117TcdA.

SEQ ID NO: 157 shows the amino acid sequence of wild-type C. difficile 131 TcdA.

SEQ ID NO: 158 shows the amino acid sequence of wild-type C. difficile 001 TcdB.

SEQ ID NO: 159 shows the amino acid sequence of wild-type Clostridium difficile 002 TcdB.

SEQ ID NO: 160 shows the amino acid sequence of wild-type Clostridium difficile 003TcdB.

SEQ ID NO: 161 shows the amino acid sequence of wild-type Clostridium difficile 004 TcdB.

SEQ ID NO: 162 shows the amino acid sequence of wild-type Clostridium difficile 070TcdB.

SEQ ID NO: 163 shows the amino acid sequence of wild-type Clostridium difficile 075TcdB.

SEQ ID NO: 164 shows the amino acid sequence of wild-type Clostridium difficile 077TcdB.

SEQ ID NO: 165 shows the amino acid sequence of wild-type Clostridium difficile 081TcdB.

SEQ ID NO: 166 shows the amino acid sequence of wild-type C. difficile 117TcdB.

SEQ ID NO: 167 shows the amino acid sequence of wild-type C. difficile 131 TcdB.

SEQ ID NO: 168 shows the amino acid sequence of wild-type C. difficile 053TcdA.

SEQ ID NO: 169 shows the amino acid sequence of wild-type C. difficile 078TcdA.

SEQ ID NO: 170 shows the amino acid sequence of wild-type C. difficile 087TcdA.

SEQ ID NO: 171 shows the amino acid sequence of wild-type C. difficile 095TcdA.

SEQ ID NO: 172 shows the amino acid sequence of wild-type C. difficile 126TcdA.

SEQ ID NO: 173 shows the amino acid sequence of wild-type Clostridium difficile 053TcdB.

SEQ ID NO: 174 shows the amino acid sequence of wild-type Clostridium difficile 078TcdB.

SEQ ID NO: 175 shows the amino acid sequence of wild-type Clostridium difficile 087TcdB.

SEQ ID NO: 176 shows the amino acid sequence of wild-type Clostridium difficile 095TcdB.

SEQ ID NO: 177 shows the amino acid sequence of wild-type C. difficile 126TcdB.

Detailed Description of the Invention

The present inventors have surprisingly discovered (among other things) mutant Clostridium difficile toxin A and toxin B, and methods thereof. The mutant portions are characterized in that they are immunogenic and exhibit reduced cytotoxicity compared to wild-type forms of the respective toxins. The present invention also relates to immunogenic portions thereof, biological equivalents thereof, and isolated polynucleotides comprising nucleic acid sequences encoding any of the foregoing.

The immunogenic compositions described herein unexpectedly demonstrate the ability to elicit novel neutralizing antibodies against C. difficile toxins, and may have the ability to confer active and/or passive protection against C. difficile challenge. The novel antibodies are directed against various epitopes of toxin A and toxin B. The inventors have also discovered that combinations of at least two neutralizing monoclonal antibodies can exhibit unexpected synergistic effects in the in vitro neutralization of each of toxin A and toxin B.

The compositions of the invention described herein are useful for treating, preventing, reducing the risk of, reducing the occurrence of, reducing the severity of, and/or delaying the onset of Clostridium difficile infection, Clostridium difficile associated disease (CDAD), syndrome, condition, symptom, and/or complications thereof in a mammal, compared to a mammal not administered the compositions of the invention.

Additionally, the present inventors have discovered recombinant non-sporulating C. difficile cells capable of stably expressing mutant C. difficile toxin A and toxin B, and methods for producing the same.

Immunogenic composition

In one aspect, the present invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin comprising an amino acid sequence having at least one mutation in the glucosyltransferase domain and at least one mutation in the cysteine protease domain relative to a corresponding wild-type Clostridium difficile toxin.

As used herein, the term "wild-type" refers to a form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and has not been intentionally modified by human manipulation. The present invention also relates to isolated polynucleotides comprising a nucleic acid sequence encoding any of the above-described polypeptides. In addition, the present invention relates to the use of any of the above-described compositions to treat or prevent C. difficile infection, C. difficile-associated diseases, syndromes, disease conditions, symptoms, and/or complications in mammals to reduce the risk, reduce the severity, reduce the incidence, and/or delay the onset of C. difficile infection compared to a mammal not administered the composition, as well as methods for preparing the compositions.

As used herein, an "immunogenic composition" or "immunogen" refers to a composition that elicits an immune response in a mammal to which the composition is administered.

An "immune response" refers to the development of a beneficial humoral (antibody-mediated) and/or cellular (antigen-specific T cells or their secreted products) response in a recipient patient to a C. difficile toxin. The immune response can be humoral, cellular, or both.

The immune response may be an active response induced by administering an immunogenic composition or immunogen, or a passive response induced by administering an antibody or sensitized T cells.

The presence of a humoral (antibody-mediated) immune response can be determined, for example, by cell-based assays known in the art, such as neutralizing antibody assays, ELISA, and the like.

The cellular immune response is typically caused by the presentation of polypeptide epitopes associated with class I or class II MHC molecules to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or other components of innate immunity. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4+ T cells) or CTLs (cytotoxic T lymphocytes) known in the art.

In one embodiment, the immunogenic composition is a vaccine composition. As used herein, a "vaccine composition" is a composition that elicits an immune response in a mammal to which it is administered. The vaccine composition can protect the immunized mammal against subsequent challenge with the immunizing agent or an immunologic cross-reactive agent. Protection can be complete or partial, with a reduction in symptoms or infection compared to an unimmunized mammal under identical conditions.

The immunogenic compositions described herein are cross-reactive, meaning they are capable of eliciting an effective immune response (e.g., a humoral immune response) against a toxin produced by a strain of C. difficile that is different from the strain from which the composition was derived. For example, an immunogenic composition described herein (e.g., derived from C. difficile 630) can elicit cross-reactive antibodies that bind to toxins produced by multiple strains of C. difficile (e.g., toxins produced by C. difficile R20291 and VPI10463). See, e.g., Example 37. Cross-reactivity indicates the cross-protective potential of a bacterial immunogen, and vice versa.

As used herein, the term "cross-protection" refers to the ability of an immune response induced by an immunogenic composition to prevent or attenuate infection caused by different bacterial strains or species of the same genus. For example, the immunogenic compositions described herein (e.g., derived from Clostridium difficile 630) can induce an effective immune response in a mammal to attenuate Clostridium difficile infection caused by strains other than 630 (e.g., Clostridium difficile R20291) in the mammal and/or attenuate Clostridium difficile disease caused by strains other than 630 (e.g., Clostridium difficile R20291) in the mammal.

Exemplary mammals in which the immunogenic composition or immunogen elicits an immune response include any mammal, such as a mouse, a hamster, a primate, and a human. In preferred embodiments, the immunogenic composition or immunogen elicits an immune response in a human to whom the composition is administered.

As described above, toxin A (TcdA) and toxin B (TcdB) are homologous glucosyltransferases that inactivate small GTPases of the Rho/Rac/Ras family. The effects of TcdA and TcdB on mammalian target cells depend on a multistep mechanism involving receptor-mediated endocytosis, membrane translocation, autoproteolytic processing, and monoglycosylation of the GTPase. Many of these functional activities have been attributed to separate regions in the primary sequences of the toxins, and imaging of the toxins has been performed to show that these molecules are structurally similar.

The wild-type gene for TcdA has approximately 8130 nucleotides that encode a reduced molecular weight protein of approximately 308 kDa with approximately 2710 amino acids. As used herein, wild-type C. difficile TcdA includes C. difficile TcdA from any wild-type strain of C. difficile. Wild-type C. difficile TcdA can include a wild-type C. difficile TcdA amino acid sequence that, when optimally aligned, for example, by using the programs GAP or BESTFIT with a predetermined gap weight, is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to SEQ ID NO: 1 (full length).

In a preferred embodiment, the wild-type C. difficile TcdA comprises the amino acid sequence set forth in SEQ ID NO: 1, which describes the wild-type amino acid sequence of TcdA from C. difficile strain 630 (also disclosed as GenBank Accession Nos. YP_001087137.1 and/or CAJ67494.1). C. difficile strain 630 is known in the art as the PCR-ribotype 012 strain. SEQ ID NO: 9 describes the wild-type gene for TcdA from C. difficile strain 630, which is also disclosed as GenBank Accession No. NC_009089.1.

Another example of a wild-type C. difficile TcdA includes the amino acid sequence set forth in SEQ ID NO: 15, which depicts the wild-type amino acid sequence of TcdA from C. difficile strain R20291 (also disclosed as GenBank Accession No. YP_003217088.1). C. difficile strain R20291 is known in the art as a highly virulent strain and a PCR-ribotype 027 strain. The amino acid sequence of TcdA from C. difficile strain R20291 is approximately 98% identical to SEQ ID NO: 1. SEQ ID NO: 16 depicts the wild-type gene from C. difficile strain R20291, which is also disclosed as GenBank Accession No. NC_013316.1.

Another example of a wild-type C. difficile TcdA includes the amino acid sequence set forth in SEQ ID NO: 17, which depicts the wild-type amino acid sequence of TcdA from C. difficile strain CD196 (also disclosed as GenBank Accession No. CBA61156.1). CD196 is a strain from a recent Canadian outbreak and is known in the art as the PCR-ribotype 027 strain. The amino acid sequence of TcdA from C. difficile strain CD196 is approximately 98% identical to SEQ ID NO: 1 and approximately 100% identical to TcdA from C. difficile strain R20291. SEQ ID NO: 18 depicts the wild-type gene for TcdA from C. difficile strain CD196, which is also disclosed as GenBank Accession No. FN538970.1.

Additional examples of the amino acid sequence of wild-type C. difficile TcdA include SEQ ID NO: 19, which describes the wild-type amino acid sequence of TcdA from C. difficile strain VPI10463 (also disclosed as GenBank Accession No. CAA63564.1). The amino acid sequence of TcdA from C. difficile strain VPI10463 is approximately 100% (99.8%) identical to SEQ ID NO: 1. SEQ ID NO: 20 describes the wild-type gene of TcdA from C. difficile strain VPI10463, which is also disclosed as GenBank Accession No. X92982.1.

Additional examples of wild-type C. difficile TcdA include TcdA from a wild-type strain of C. difficile, which is available from the Centers for Disease Control and Prevention (CDC, Atlanta, GA). The inventors have discovered that, when optimally aligned, the amino acid sequence of TcdA from a wild-type strain of C. difficile available from the CDC is at least about 99.3% to 100% identical to amino acid residues 1-821 of SEQ ID NO: 1 (TcdA from C. difficile 630). See Table 1.

The inventors also discovered that when optimally aligned (e.g., when optimally aligned over the entire length of the sequence), the amino acid sequence of TcdA from a wild-type Clostridium difficile strain can comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to about 100% identity to SEQ ID NO: 1.

Table 1: Percent identity of amino acid residues 1-821 of wild-type C. difficile strains obtained from CDC and TcdA from various wild-type C. difficile strains with amino acid residues 1-821 of SEQ ID NO: 1 when optimally aligned.

Thus, in one embodiment, the wild-type Clostridium difficile TcdA amino acid sequence comprises a sequence of at least about 500, 600, 700, or 800 contiguous residues that, when optimally aligned, for example, by using the programs GAP or BESTFIT with predetermined gap weights, is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to the same length of sequence between residues 1-900 of SEQ ID NO: 1. Examples include the strains described above (e.g., R20291, CD196, etc.) and those listed in Table 1.

In another embodiment, the wild-type C. difficile TcdA amino acid sequence comprises a sequence that, when optimally aligned, is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably about 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to any sequence selected from SEQ ID NOs: 87-109. See Table 1-a.

The wild-type gene for TcdB has approximately 7098 nucleotides encoding a protein of approximately 270 kDa with a reduced molecular weight of approximately 2366 amino acids. As used herein, wild-type C. difficile TcdB includes C. difficile TcdB from any wild-type strain of C. difficile. Wild-type C. difficile TcdB can comprise a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to SEQ ID NO: 2 when optimally aligned, for example, using the programs GAP or BESTFIT with predetermined gap weights. In a preferred embodiment, the wild-type C. difficile TcdB comprises the amino acid sequence set forth in SEQ ID NO: 2, which describes the wild-type amino acid sequence of TcdB from C. difficile strain 630 (also disclosed as GenBank Accession Nos. YP_001087135.1 and/or CAJ67492). SEQ ID NO: 10 describes the wild-type gene of TcdB from C. difficile strain 630, which is also disclosed as GenBank Accession No. NC_009089.1.

Another example of a wild-type C. difficile TcdB comprises the amino acid sequence set forth in SEQ ID NO: 21, which depicts the wild-type amino acid sequence of TcdB from C. difficile strain R20291 (also disclosed as GenBank Accession Nos. YP_003217086.1 and/or CBE02479.1). The amino acid sequence of TcdB from C. difficile strain R20291 is approximately 92% identical to SEQ ID NO: 2. SEQ ID NO: 22 depicts the wild-type gene of TcdB from C. difficile strain R20291, which is also disclosed as GenBank Accession No. NC_013316.1.

Another example of a wild-type C. difficile TcdB includes the amino acid sequence set forth in SEQ ID NO: 23, which depicts the wild-type amino acid sequence of TcdB from C. difficile strain CD196 (also disclosed as GenBank Accession Nos. YP_003213639.1 and/or CBA61153.1). SEQ ID NO: 24 depicts the wild-type gene of TcdB from C. difficile strain CD196, also disclosed as GenBank Accession No. NC_013315.1. The amino acid sequence of TcdB from C. difficile strain CD196 is approximately 92% identical to SEQ ID NO: 2.

Other examples of wild-type C. difficile TcdB amino acid sequences include SEQ ID NO: 25, which describes the wild-type amino acid sequence of TcdB from C. difficile strain VPI10463 (also disclosed as GenBank Accession Nos. P18177 and/or CAA37298). The amino acid sequence of TcdB from C. difficile strain VPI10463 is 100% identical to SEQ ID NO: 2. SEQ ID NO: 26 describes the wild-type gene of TcdB from C. difficile strain VPI10463, which is also disclosed as GenBank Accession No. X53138.1.

Other examples of wild-type C. difficile TcdB include TcdB from a wild-type strain of C. difficile, which is available from the Centers for Disease Control and Prevention (CDC, Atlanta, GA). The inventors discovered that, when optimally aligned, the amino acid sequence of TcdB from a wild-type strain of C. difficile available from the CDC is at least about 96%-100% identical to amino acid residues 1-821 of SEQ ID NO: 2 (TcdB from C. difficile 630). See Table 2.

Table 2: Percent identity of amino acid residues 1-821 of wild-type C. difficile strains obtained from CDC and TcdB from various wild-type C. difficile strains with amino acid residues 1-821 of SEQ ID NO: 2 when optimally aligned.

Thus, in one embodiment, the wild-type Clostridium difficile TcdB amino acid sequence comprises a sequence of at least about 500, 600, 700, or 800 contiguous residues that, when optimally aligned, for example, by using the programs GAP or BESTFIT with predetermined gap weights, is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably about 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to the same length of sequence between residues 1-900 of SEQ ID NO: 2. Examples include the strains described above (e.g., R20291, CD196, etc.) and those listed in Table 2.

In another embodiment, the wild-type Clostridium difficile TcdB amino acid sequence comprises a sequence that, when optimally aligned, is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably about 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to any sequence selected from SEQ ID NOs: 110-133. See Table 2-a.

The genes for toxins A and B (tcdA and tcdB) are part of a 19.6-kb locus (the pathogenicity locus, PaLoc) that contains three additional small open reading frames (ORFs), tcdD, tcdE, and tcdC, and can be considered to be useful for toxicity. PaLoc is known to be stable and conserved among toxigenic species. It is present at the same chromosomal integration site in all toxigenic strains analyzed so far. The pathogenicity locus (PaLoc) is not present in non-toxigenic strains. Therefore, the wild-type C. difficile strains described herein are characterized by the presence of the pathogenicity locus. Another preferred feature of the wild-type C. difficile strains described herein is the production of TcdA and TcdB.

In one embodiment, a wild-type strain of C. difficile is one having a pathogenicity locus that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to the pathogenicity locus of C. difficile 630 or VPI10463. The sequence of the total pathogenicity locus of C. difficile VPI10463 is registered in the EMBL database as sequence accession number X92982 and is also shown in SEQ ID NO: 26. Strains with a PaLoc identical to reference strain VPI10463 are preferably designated as toxinotype 0. Strains of toxinotypes I-VII, IX, XII-XV, and XVIII-XXIV produce TcdA and TcdB, regardless of variations in their toxin genes.

The glucosyltransferase domain is located at the N-terminus of the toxin. The glucosyltransferase activity of the toxin is associated with the toxin's cytotoxic function. Without being bound by any mechanism or theory, it is believed that the glucosyltransferase activity in both toxins catalyzes the monoglycosylation of small GTP-binding proteins in the Rho/Rac/Ras superfamily. Following glycosylation of these GTP-binding proteins, cellular physiology is significantly modified, resulting in loss of structural integrity and disruption of important signal transduction pathways in toxin-infected host cells. The Asp-Xaa-Asp (DXD) motif, which is associated with binding of manganese, uridine diphosphate (UDP), and glucose, is a hallmark of the glucosyltransferase domain. Without being bound by any mechanism or theory, it is believed that residues important for catalytic activity, such as the DXD motif, are unchanged in TcdB from known "historical" strains such as 630 and in TcdB from highly virulent strains such as R20291. The DXD motif is located at residues 285-287 (numbering according to SEQ ID NO: 1) of wild-type C. difficile TcdA and at residues 286-288 (numbering according to SEQ ID NO: 2) of wild-type C. difficile TcdB.

Global alignment algorithms (e.g., sequence analysis programs) are known in the art and can be used to optimally align two or more amino acid toxin sequences to determine whether the toxin contains a specific signature sequence (e.g., DXD in the glucosyltransferase domain, DHC in the cysteine protease domain, etc., as described below). The optimally aligned sequences are compared to the respective reference sequences (e.g., SEQ ID NO: 1 for TcdA or SEQ ID NO: 2 for TcdB) to determine the presence of the signature motif. "Optimal alignment" refers to the alignment that gives the highest percentage identity score. Such an alignment can be performed using known sequence analysis programs. In one embodiment, a CLUSTAL alignment (e.g., CLUSTALW) with predetermined parameters is used to identify a suitable wild-type toxin by comparing the query sequence to the reference sequence. The relative numbering of conserved amino acid residues is based on the residue numbering of the reference amino acid sequence to indicate small insertions or deletions (e.g., 5 amino acids or less) in the aligned sequences.

As used herein, the term "numbering according to" refers to the numbering of the residues of a reference sequence when comparing a given amino acid or polynucleotide sequence to the reference sequence. In other words, the numbers or residue positions of a given polymer are designated relative to the reference sequence rather than the actual numerical positions of the residues in the given amino acid or polynucleotide sequence.

For example, a given amino acid sequence, such as that of a highly virulent wild-type strain of Clostridium difficile, can be aligned with a reference sequence (e.g., the sequence of a historical wild-type strain of Clostridium difficile, such as 630) by introducing gaps, if necessary, to optimize the match between the two sequences. In these cases, despite the presence of gaps, the numbering of residues in a given amino acid or polynucleotide is assigned relative to the reference sequence to which the alignment is being made. As used herein, a "reference sequence" refers to a defined sequence used as a basis for sequence comparison.

Unless otherwise indicated, all amino acid positions of TcdA herein refer to the numbering of SEQ ID NO: 1. Unless otherwise indicated, all amino acid positions of TcdB herein refer to the numbering of SEQ ID NO: 2.

As used herein, the glucosyltransferase domain of TcdA can begin at exemplary residues 1, 101, or 102 of wild-type C. difficile TcdA, such as SEQ ID NO: 1, and can end at exemplary residues 542, 516, or 293. Any minimum residue position between residues 1 and 542 of TcdA can be combined with the maximum residue position to define the sequence of the glucosyltransferase domain, as long as the DXD motif region is included. For example, in one embodiment, the glucosyltransferase domain of TcdA comprises SEQ ID NO: 27, which is identical to residues 101-293 of SEQ ID NO: 1, and which includes the DXD motif region. In another embodiment, the glucosyltransferase domain of TcdA comprises SEQ ID NO: 28, which is identical to residues 1-542 of SEQ ID NO: 1.

As used herein, the glucosyltransferase domain of TcdB can begin at exemplary residues 1, 101, or 102 of wild-type C. difficile TcdB, such as SEQ ID NO: 2, and can end at exemplary residues 542, 516, or 293. Any minimum residue position between residues 1 and 543 of TcdB can be combined with the maximum residue position to define the sequence of the glucosyltransferase domain, as long as the DXD motif region is included. For example, in one embodiment, the glucosyltransferase domain of TcdB comprises SEQ ID NO: 29, which is identical to residues 101-293 of SEQ ID NO: 2, and which includes the DXD motif region. In another embodiment, the glucosyltransferase domain of TcdB comprises SEQ ID NO: 30, which is identical to residues 1-543 of SEQ ID NO: 1.

Without being bound by theory or mechanism, it is believed that the N-terminus of TcdA and/or TcdB is cleaved by autoproteolytic processing, allowing the glucosyltransferase domain to translocate and be released into the host cell cytoplasm, where it can interact with Rac/Ras/Rho GTPases. Wild-type C. difficile TcdA has been shown to be cleaved between L542 and S543. Wild-type C. difficile TcdB has been shown to be cleaved between L543 and G544.

The cysteine protease domain is associated with the autocatalytic proteolytic activity of the toxin. The cysteine protease domain is located downstream of the glucosyltransferase domain and is characterized by a catalytic triad of aspartic acid, histidine, and cysteine (DHC), such as D589, H655, and C700 of wild-type TcdA and D587, H653, and C698 of wild-type TcdB. Without being bound by mechanism or theory, it is believed that the catalytic triad is conserved between toxins from "historical" strains such as 630 and TcdB from highly virulent strains such as R20291.

As used herein, the cysteine protease domain of TcdA can begin at the exemplary residue 543 of wild-type TcdA, such as SEQ ID NO: 1, and can end at the exemplary residues 809, 769, 768, or 767. Any minimum residue position between residues 543 and 809 of wild-type TcdA can be combined with the maximum residue position to define the sequence of the cysteine protease domain, as long as the catalytic triad DHC motif region is included. For example, in one embodiment, the cysteine protease domain of TcdA comprises SEQ ID NO: 32, which has DHC motif regions located at residues 47, 113, and 158 of SEQ ID NO: 32, which correspond to D589, H655, and C700 of wild-type TcdA, numbered according to SEQ ID NO: 1, respectively. SEQ ID NO: 32 is identical to residues 543-809 of SEQ ID NO: 1, TcdA.

As used herein, the cysteine protease domain of TcdB can begin at the exemplary residue 544 of wild-type TcdB, such as SEQ ID NO: 2, and can end at the exemplary residues 801, 767, 755, or 700. Any minimum residue position between residues 544 and 801 of wild-type TcdB can be combined with the maximum residue position to define the sequence of the cysteine protease domain, as long as the catalytic triad DHC motif region is included. For example, in one embodiment, the cysteine protease domain of TcdB comprises SEQ ID NO: 33, which comprises the DHC motif regions at residues 44, 110, and 115 of SEQ ID NO: 33, which correspond to D587, H653, and C698 of wild-type TcdB, numbered according to SEQ ID NO: 2, respectively. SEQ ID NO: 33 is identical to residues 544-767 of SEQ ID NO: 2, TcdB. In another embodiment, the cysteine protease domain of TcdB comprises SEQ ID NO: 2, residues 544-801 of TcdB.

In the present application, the immunogenic composition comprises a mutant Clostridium difficile toxin. As used herein, the term "mutant" refers to a molecule that exhibits a structure or sequence that differs from the corresponding wild-type structure or sequence, for example by having crosslinks compared to the corresponding wild-type structure and/or by having at least one mutation compared to the corresponding wild-type sequence when optimally aligned, for example, by using the programs GAP or BESTFIT with predetermined gap weights. As used herein, the term "mutant" also encompasses molecules that exhibit functional properties that differ from the corresponding wild-type molecule (e.g., loss of glucosyltransferase and/or loss of cysteine protease activity).

C. difficile toxins from any of the above wild-type strains can be used as a source for producing mutant C. difficile toxins. Preferably, C. difficile 630 is a source for producing mutant C. difficile toxins.

The mutation may include a substitution, deletion, truncation, or modification of the wild-type amino acid residue normally located at that position. Preferably, the mutation is a non-conservative amino acid substitution. The present invention also encompasses an isolated polynucleotide comprising a nucleic acid sequence encoding any of the mutant toxins described herein.

As used herein, a "non-conservative" amino acid substitution refers to a change from one class of amino acids to another class of amino acids according to Table 3 below.

Examples of non-conservative amino acid substitutions include substitutions in which an aspartic acid residue (Asp, D) is replaced by an alanine residue (Ala, A). Other examples include replacement of an aspartic acid residue (Asp, D) with an asparagine residue (Asn, N), and replacement of an arginine (Arg, R), glutamic acid (Glu, E), lysine (Lys, K), and/or histidine (His, H) residue with an alanine residue (Ala, A).

Conservative substitutions refer to exchanging amino acids within the same class, for example according to Table 3.

The mutant toxins of the present invention can be prepared using techniques known in the art for preparing mutations, such as site-directed mutagenesis, mutagenesis using a mutagen (e.g., UV light), and the like. Site-directed mutagenesis is preferred. Alternatively, nucleic acid molecules having the target sequence can be directly synthesized. Such chemical synthesis methods are known in the art.

In the present invention, a mutant Clostridium difficile toxin comprises at least one mutation in the glucosyltransferase domain relative to the corresponding wild-type Clostridium difficile toxin. In one embodiment, the glucosyltransferase domain comprises at least two mutations. Preferably, the mutations reduce or eliminate the glucosyltransferase activity of the toxin compared to the glucosyltransferase activity of the corresponding wild-type Clostridium difficile toxin.

Exemplary amino acid residues in the glucosyltransferase domain of TcdA that can be mutated include at least one of the following or any combination thereof (numbered according to SEQ ID NO: 1, compared to wild-type Clostridium difficile TcdA): W101, D269, R272, D285, D287, E460, R462, S541 and L542.

Exemplary mutations in the glucosyltransferase domain of TcdA include at least one or any combination of the following (compared to wild-type Clostridium difficile TcdA): W101A, D269A, R272A, D285A, D287A, E460A, R462A, S541A, and L542G. In another preferred embodiment, the glucosyltransferase domain of TcdA includes (compared to wild-type Clostridium difficile TcdA) D285A and D287A mutations.

Exemplary amino acid residues in the glucosyltransferase domain of TcdB that can be mutated include at least one of the following or any combination thereof (numbered according to SEQ ID NO: 2, compared to wild-type Clostridium difficile TcdB): W102, D270, R273, D286, D288, N384, D461, K463, W520 and L543.

Exemplary mutations in the glucosyltransferase domain of TcdB include at least one of the following, or any combination thereof (compared to wild-type Clostridium difficile TcdB): W102A, D270A, D270N, R273A, D286A, D288A, N384A, D461A, D461R, K463A, K463E, W520A, and L543A. In a preferred embodiment, the glucosyltransferase domain of TcdB comprises (compared to wild-type Clostridium difficile TcdB) L543A. In another preferred embodiment, the glucosyltransferase domain of TcdB comprises (compared to wild-type Clostridium difficile TcdB) D286A and D288A mutations.

Any of the mutations described above can be combined with a mutation in the cysteine protease domain. In the present invention, the mutant C. difficile toxin comprises at least one mutation in the cysteine protease domain relative to the corresponding wild-type C. difficile toxin. Preferably, the mutation reduces or eliminates the cysteine protease activity of the toxin compared to the cysteine protease activity of the corresponding wild-type C. difficile toxin.

Exemplary amino acid residues in the cysteine protease domain of TcdA that can be mutated include at least one of the following, or any combination thereof (as compared to wild-type C. difficile TcdA, according to the numbering of SEQ ID NO: 1): S543, D589, H655, and C700. Exemplary mutations in the glucosyltransferase domain of TcdA include at least one of the following, or any combination thereof (as compared to wild-type C. difficile TcdA): S543A, D589A, D589N, H655A, C700A. In a preferred embodiment, the cysteine protease domain of TcdA comprises (as compared to wild-type C. difficile TcdA) a C700A mutation.

Exemplary amino acid residues in the cysteine protease domain of TcdB that may be mutated include at least one of the following, or any combination thereof (as compared to wild-type Clostridium difficile TcdB, numbered according to SEQ ID NO: 2): G544, D587, H653, and C698. In a preferred embodiment, the cysteine protease domain of TcdB comprises (as compared to wild-type Clostridium difficile TcdB) a C698A mutation. Other amino acid residues in the cysteine protease domain of TcdB that may be mutated include (as compared to wild-type TcdB) K600 and/or R751. Exemplary mutations include K600E and/or R751E.

Thus, a mutant C. difficile toxin of the invention has a mutated glucosyltransferase domain relative to a corresponding wild-type C. difficile toxin and has a mutated cysteine protease domain relative to a corresponding wild-type C. difficile toxin.

An exemplary mutant C. difficile TcdA comprises a glucosyltransferase domain comprising SEQ ID NO:29 having amino acid substitutions at positions 285 and 287 relative to the corresponding wild-type C. difficile toxin A; and a cysteine protease domain comprising SEQ ID NO:32 having an amino acid substitution at position 158 relative to the corresponding wild-type C. difficile toxin A. For example, such a mutant C. difficile TcdA comprises the amino acid sequence set forth in SEQ ID NO:4, wherein the initial methionine is optionally absent. In another embodiment, the mutant C. difficile toxin A comprises the amino acid sequence set forth in SEQ ID NO:84.

Other examples of mutant C. difficile toxin A include the amino acid sequence set forth in SEQ ID NO: 7, which has the same mutations as D269A, R272A, D285A, D287A, E460A, R462A, and C700A as SEQ ID NO: 1, wherein the initial methionine is optionally absent. In another embodiment, the mutant C. difficile toxin A includes the amino acid sequence set forth in SEQ ID NO: 83.

Another exemplary mutant TcdA comprises SEQ ID NO: 34, wherein the residues at positions 101, 269, 272, 285, 287, 460, 462, 541, 542, 543, 589, 655, and 700 can be any amino acid.

In some embodiments, the mutant C. difficile toxin exhibits reduced or eliminated autoproteolytic processing compared to the corresponding wild-type C. difficile toxin. For example, the mutant C. difficile TcdA can comprise a mutation at one of the following residues, or any combination thereof, compared to the corresponding wild-type C. difficile TcdA: S541, L542, and/or S543. Preferably, the mutant C. difficile TcdA comprises at least one of the following mutations, or any combination thereof, compared to the corresponding wild-type C. difficile TcdA: S541A, L542G, and S543A.

Another exemplary mutant C. difficile TcdA comprises (compared to the corresponding wild-type C. difficile TcdA) S541A, L542, S543, and C700 mutations.

An exemplary mutant C. difficile toxin B comprises a glucosyltransferase domain comprising SEQ ID NO: 31 having amino acid substitutions at positions 286 and 288 relative to the corresponding wild-type C. difficile toxin B, and a cysteine protease domain comprising SEQ ID NO: 33 having an amino acid substitution at position 155 relative to the corresponding wild-type C. difficile toxin B. For example, such a mutant C. difficile TcdB comprises the amino acid sequence set forth in SEQ ID NO: 6, wherein the initial methionine is optionally absent. In another embodiment, the mutant C. difficile toxin A comprises the amino acid sequence set forth in SEQ ID NO: 86.

Other examples of mutant C. difficile TcdB comprise the amino acid sequence set forth in SEQ ID NO: 8, which has the D270A, R273A, D286A, D288A, D461A, K463A, and C698A mutations relative to SEQ ID NO: 2, wherein the initial methionine is optionally absent. In another embodiment, the mutant C. difficile toxin A comprises the amino acid sequence set forth in SEQ ID NO: 85.

Another exemplary mutant TcdB comprises SEQ ID NO: 35, wherein the residues at positions 101, 269, 272, 285, 287, 460, 462, 541, 542, 543, 589, 655, and 700 can be any amino acid.

As another example, the mutant Clostridium difficile TcdB can comprise a mutation at position 543 and/or 544 compared to the corresponding wild-type Clostridium difficile TcdB. Preferably, the mutant Clostridium difficile TcdB comprises (compared to the corresponding wild-type Clostridium difficile TcdB) an L543 and/or G544 mutation. More preferably, the mutant Clostridium difficile TcdB comprises (compared to the corresponding wild-type Clostridium difficile TcdB) an L543G and/or G544A mutation.

Another exemplary mutant C. difficile TcdB comprises (compared to the corresponding wild-type C. difficile TcdB) L543G, G544A, and C698 mutations.

In one aspect, the present invention relates to isolated polypeptides having a mutation at any position within amino acid residues 1-1500, as numbered according to SEQ ID NO: 2, to define exemplary mutant Clostridium difficile toxin B. For example, in one embodiment, the isolated polypeptide comprises a mutation between amino acid residues 830 and 990 of SEQ ID NO: 2. Exemplary positions for mutation include positions 970 and 976, as numbered according to SEQ ID NO: 2. Preferably, the mutation between residues 830 and 990 is a substitution. In one embodiment, the mutation is a non-conservative substitution, wherein the Asp (D) and/or Glu (E) amino acid residues are not replaced with neutralizing amino acid residues, such as lysine (K), arginine (R), and histidine (H), when acidified. Exemplary mutations include E970K, E970R, E970H, E976K, E976R, and E976H of SEQ ID NO: 2 to define mutant Clostridium difficile toxin B.

In another aspect, the present invention relates to isolated polypeptides having a mutation at any position of amino acid residues 1-1500, as numbered according to SEQ ID NO: 1, to define exemplary mutant Clostridium difficile toxin A. For example, in one embodiment, the isolated polypeptide comprises a mutation between amino acid residues 832 and 992 of SEQ ID NO: 1. Exemplary positions for mutation include positions 972 and 978, as numbered according to SEQ ID NO: 1. Preferably, the mutation between residues 832 and 992 is a substitution. In one embodiment, the mutation is a non-conservative substitution, wherein the Asp (D) and/or Glu (E) amino acid residues are not replaced with neutralizing amino acid residues, such as lysine (K), arginine (R), and histidine (H), when acidified. Exemplary mutations include D972K, D972R, D972H, D978K, D978R, and D978H of SEQ ID NO: 1 to define mutant Clostridium difficile toxin A.

The polypeptides of the present invention may contain an initial methionine residue, in some cases due to host cell-mediated processes. Depending on, for example, the recombinant production method and/or the fermentation or growth conditions of the host cells, it is known in the art that the N-terminal methionine encoded by the translation initiation codon can be removed from the polypeptide after translation in the cell, or the N-terminal methionine can be retained in the isolated polypeptide.

Thus, in one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 4, wherein the initial methionine (at position 1) is optionally absent. In one embodiment, the initial methionine of SEQ ID NO: 4 is absent. In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 84, which is identical to SEQ ID NO: 4, but without the initial methionine.

In another aspect, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 6, wherein the initial methionine (at position 1) is optionally absent. In one embodiment, the initial methionine of SEQ ID NO: 6 is absent. In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 86, which is identical to SEQ ID NO: 6, but without the initial methionine.

In another aspect, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 7, wherein the initial methionine (at position 1) is optionally absent. In one embodiment, the present invention relates to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 83, which is identical to SEQ ID NO: 7, but without the initial methionine. In another aspect, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 8, wherein the initial methionine (at position 1) is optionally absent. In one embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 85, which is identical to SEQ ID NO: 8, but without the initial methionine.

In one aspect, the present invention relates to an immunogenic composition comprising SEQ ID NO: 4, wherein the initial methionine (at position 1) is optionally absent. In another aspect, the present invention relates to an immunogenic composition comprising SEQ ID NO: 6, wherein the initial methionine (at position 1) is optionally absent. In a further aspect, the present invention relates to an immunogenic composition comprising SEQ ID NO: 7, wherein the initial methionine (at position 1) is optionally absent. In another aspect, the present invention relates to an immunogenic composition comprising SEQ ID NO: 8, wherein the initial methionine (at position 1) is optionally absent.

In another aspect, the invention relates to an immunogenic composition comprising SEQ ID NO: 83. In one aspect, the invention relates to an immunogenic composition comprising SEQ ID NO: 84. In one aspect, the invention relates to an immunogenic composition comprising SEQ ID NO: 85. In another aspect, the invention relates to an immunogenic composition comprising SEQ ID NO: 86.

In addition to generating an immune response in a mammal, the immunogenic compositions described herein have reduced cytotoxicity compared to the corresponding wild-type Clostridium difficile toxin. Preferably, the immunogenic compositions are safe for administration to a mammal and have minimal (e.g., about 6-8 log ₁₀ reduction) to no cytotoxicity compared to the respective wild-type toxin.

The term cytotoxicity as used herein is a term understood in the art and refers to apoptotic cell death and/or a state in which one or more of the cells are abnormally damaged biochemically or biologically compared to the same cells under the same conditions but without the cytotoxic agent. Toxicity can be quantified, for example, as the amount of a substance required to induce 50% cell death in a cell or mammal (i.e., _EC50 or _ED50 , respectively), or by other methods known in the art.

Assays that indicate cytotoxicity are known in the art, such as the cell rounding assay (see, for example, Kuehne et al. Nature. 2010 Oct 7; 467(7316): 711-3). The action of TcdA and TcdB causes cells to round (e.g., lose morphology) and die, and such phenomena can be observed by light microscopy. See, for example, Figure 9.

Other exemplary cytotoxicity assays known in the art include glycosylation assays associated with [ ¹⁴ C]glucose-labeled Ras phosphorimaging assays (as described in Busch et al., J Biol Chem. 1998 Jul 31; 273(31): 19566-72) and preferably the cytotoxicity assays described in the Examples below, wherein EC ₅₀ can refer to the concentration of an immunogenic composition that exhibits at least about 50% of the cytopathic effect (CPE) in cells, preferably human diploid fibroblasts (e.g., IMR90 cells (ATCC CCL-186 ^™ ) as compared to the same cells under the same conditions in the absence of toxin. In vitro cytotoxicity assays can also be used to evaluate the concentration of a composition that inhibits at least about 50% of the cytopathic effect (CPE) induced by a wild-type Clostridium difficile toxin in cells, preferably human diploid fibroblasts (e.g., IMR90 cells (ATCC CCL-186 ^™ ) as compared to the same cells under the same conditions in the absence of toxin. Other exemplary cytotoxicity assays include the assays described in Doern et al. Cytotoxicity assays such as those described in [15] et al., J Clin Microbiol. 1992 Aug;30(8):2042-6. Cytotoxicity can also be determined by measuring ATP levels in cells treated with toxins. For example, luciferase substrates such as (Promega) can be used, which emit fluorescence measured as relative light units (RLU). In such assays, cell viability can be directly proportional to the amount of ATP in the cells or the RLU value.

In one embodiment, the cytotoxicity of the immunogenic composition is reduced by at least about 1000, 2000, 3000, 4000, 5000-, 6000-, 7000-, 8000-, 9000-, 10000-, 11000-, 12000-, 13000-fold, 14000-fold, 15000-fold or more compared to the corresponding wild-type C. difficile toxin. See, e.g., Table 20.

In another embodiment, the cytotoxicity of the immunogenic composition is reduced by at least about 2-log ₁₀ , more preferably about 3-log ₁₀ , and most preferably about 4-log ₁₀ or more compared to the corresponding wild-type toxin under the same conditions. For example, as measured by standard cytopathic effect (CPE), the EC ₅₀ of the mutant C. difficile TcdB can be about 10 ^-9 g/ml, compared to an exemplary wild-type C. difficile TcdB having an EC ₅₀ value of at least about 10 ^-12 g/ml. See, for example, Tables 7A, 7B, 8A, and 8B of the Examples section below.

In another embodiment, the _EC50 of the cytotoxicity of the mutant C. difficile toxin is at least about 50 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml, 1000 μg/ml or greater, as measured by, for example, an in vitro cytotoxicity assay described herein. Thus, in preferred embodiments, the immunogenic composition and mutant toxin are biologically safe for administration to a mammal.

Without being bound by mechanism or theory, TcdA with mutations D285 and D287 compared to wild-type TcdA and TcdB with mutations D286 and D288 compared to wild-type TcdB are expected to be defective in glycosyltransferase activity and therefore defective in inducing cytopathogenic effects. Additionally, toxins with mutations in the DHC motif are expected to be defective in autocatalytic processing and therefore not have any cytotoxic effects.

However, the inventors unexpectedly discovered that the exemplary mutant TcdA having SEQ ID NO:4 and the exemplary mutant TcdB having SEQ ID NO:6 unexpectedly exhibited cytotoxicity (albeit significantly reduced compared to wild-type C. difficile 630 toxin), despite exhibiting dysfunctional glucosyltransferase activity and dysfunctional cysteine protease activity. Without being bound by mechanism or theory, it is believed that the mutant toxins exert cytotoxicity through a novel mechanism. However, the exemplary mutant TcdA having SEQ ID NO:4 and the exemplary mutant TcdB having SEQ ID NO:6 were unexpectedly immunogenic. See the Examples below.

While chemical cross-linking of wild-type toxins may not inactivate the toxin, the inventors have further discovered that chemical cross-linking of at least one amino acid of a mutant toxin further reduces the cytotoxicity of the mutant toxin compared to the same mutant toxin lacking the chemical cross-link and compared to the corresponding wild-type toxin. Preferably, the mutant toxin is purified prior to contact with the chemical cross-linking agent.

Furthermore, although chemical cross-linking agents may alter useful epitopes, the present inventors have surprisingly discovered that genetically modified mutant Clostridium difficile toxins having at least one chemically cross-linked amino acid result in immunogenic compositions that elicit a variety of neutralizing antibodies or binding fragments thereof. Thus, epitopes associated with neutralizing antibody molecules are unexpectedly retained after chemical cross-linking.

Crosslinking (also referred to herein as "chemical inactivation" or "inactivation") is a method of chemically linking two or more molecules via covalent bonds. The terms "crosslinking reagent," "crosslinker," and "crosslinking substance" refer to molecules that are capable of reacting with and/or chemically linking to specific functional groups (primary amines, sulfhydryls, carboxyls, carbonyls, etc.) on peptides, polypeptides, and/or proteins. In one embodiment, the molecule can comprise two or more reactive ends capable of reacting with and/or chemically linking to specific functional groups (primary amines, sulfhydryls, carboxyls, carbonyls, etc.) on peptides, polypeptides, and/or proteins. Preferably, the chemical crosslinker is water-soluble. In another preferred embodiment, the chemical crosslinker is a heterobifunctional crosslinking substance. In another embodiment, the chemical crosslinker is not a bifunctional crosslinking substance. Chemical crosslinkers are known in the art.

In a preferred embodiment, the crosslinker is a zero-length crosslinker. "Zero-length" refers to a crosslinker that mediates or produces a direct crosslink between the functional groups of two molecules. For example, in the crosslinking of two polypeptides, a zero-length crosslinker will result in the formation of a bridge or crosslink between a carboxyl group of an amino acid side chain of one polypeptide and an amino group of another polypeptide without the introduction of exogenous substances. A zero-length crosslinker can catalyze, for example, the formation of an ester bond between a hydroxyl group and a carboxyl group and/or an amide bond between a carboxyl group and a primary amino group.

Exemplary suitable chemical cross-linking agents include formaldehyde; formalin; acetaldehyde; propionaldehyde; water-soluble carbodiimides (RN=C=NR'), including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 3-(2-morpholino-(4-ethyl)carbodiimide N-methyl-p-toluenesulfonic acid 1-cyclohexyl ester (CMC), N,N'-dicyclohexylcarbodiimide (DCC) and N,N'-diisopropylcarbodiimide (DIC) and derivatives thereof; as well as N-hydroxysuccinimide (NHS); phenylglyoxal; and/or UDP-dialdehyde.

Preferably, the cross-linking agent is EDC. When a mutant Clostridium difficile toxin polypeptide is chemically modified with EDC (e.g., by contacting the polypeptide with EDC), in one embodiment, the polypeptide includes (a) at least one cross-link between a side chain of an aspartic acid residue of the polypeptide and a side chain of a glutamic acid residue of the polypeptide. In one embodiment, the polypeptide includes (b) at least one cross-link between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide. In one embodiment, the polypeptide includes (c) at least one cross-link between a carboxyl group at the C-terminus of the polypeptide and an amino group at the N-terminus of the polypeptide. In one embodiment, the polypeptide includes (d) at least one cross-link between a carboxyl group at the C-terminus of the polypeptide and a side chain of a lysine residue of the polypeptide. In one embodiment, the polypeptide includes (e) at least one cross-link between a side chain of an aspartic acid residue of the polypeptide and a side chain of a lysine residue of a second isolated polypeptide. In one embodiment, the polypeptide includes (f) at least one cross-link between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of a second isolated polypeptide. In one embodiment, the polypeptide comprises (g) at least one crosslink between the carboxyl group at the C-terminus of the polypeptide and the amino group at the N-terminus of a second isolated polypeptide. In one embodiment, the polypeptide comprises (h) at least one crosslink between the carboxyl group at the C-terminus of the polypeptide and the side chain of a lysine residue of a second isolated polypeptide. See, e.g., Figures 24 and 25.

"Second isolated polypeptide" refers to any isolated polypeptide present during the reaction with EDC. In one embodiment, the second isolated polypeptide is a mutant Clostridium difficile toxin polypeptide having the same sequence as the first isolated polypeptide. In another embodiment, the second isolated polypeptide is a mutant Clostridium difficile toxin polypeptide having a different sequence than the first isolated polypeptide.

In one embodiment, the polypeptide comprises at least two modifications selected from (a)-(d). In an exemplary embodiment, the polypeptide comprises (a) at least one crosslink between a side chain of an aspartic acid residue in the polypeptide and a side chain of a glutamic acid residue in the polypeptide; and (b) at least one crosslink between a side chain of a glutamic acid residue in the polypeptide and a side chain of a lysine residue in the polypeptide. In another embodiment, the polypeptide comprises at least three modifications selected from (a)-(d). In another embodiment, the polypeptide comprises modifications (a), (b), (c), and (d).

When more than one mutant polypeptide is present during chemical modification by EDC, in one embodiment, the resulting composition comprises at least one of any of the modifications (a)-(h). In one embodiment, the composition comprises at least two modifications selected from the group consisting of modifications (a)-(h). In another embodiment, the composition comprises at least three modifications selected from the group consisting of modifications (a)-(h). In another embodiment, the composition comprises at least four modifications selected from the group consisting of modifications (a)-(h). In another embodiment, the composition comprises at least one of each of the modifications (a)-(h).

In an exemplary embodiment, the resulting composition comprises (a) at least one cross-link between the side chain of an aspartic acid residue of the polypeptide and the side chain of a glutamic acid residue of the polypeptide; and (b) at least one cross-link between the side chain of a glutamic acid residue of the polypeptide and the side chain of a lysine residue of the polypeptide. In one embodiment, the composition further comprises (c) at least one cross-link between the carboxyl group at the C-terminus of the polypeptide and the amino group at the N-terminus of the polypeptide; and (d) at least one cross-link between the carboxyl group at the C-terminus of the polypeptide and the side chain of a lysine residue of the polypeptide.

In further exemplary embodiments, the resulting composition comprises (e) at least one cross-link between the side chain of an aspartic acid residue of the polypeptide and the side chain of a lysine residue of a second isolated polypeptide; (f) at least one cross-link between the side chain of a glutamic acid residue of the polypeptide and the side chain of a lysine residue of the second isolated polypeptide; (g) at least one cross-link between the carboxyl group at the C-terminus of the polypeptide and the amino group at the N-terminus of the second isolated polypeptide; and (h) at least one cross-link between the carboxyl group at the C-terminus of the polypeptide and the side chain of a lysine residue of the second isolated polypeptide.

In further exemplary embodiments, the resulting composition comprises (a) at least one cross-link between a side chain of an aspartic acid residue of the polypeptide and a side chain of a glutamic acid residue of the polypeptide; (b) at least one cross-link between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide; (e) at least one cross-link between a side chain of an aspartic acid residue of the polypeptide and a side chain of a lysine residue of a second isolated polypeptide; and (f) at least one cross-link between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of a second isolated polypeptide.

In a preferred embodiment, the chemical crosslinking agent comprises formaldehyde, more preferably, a reagent comprising formaldehyde in the absence of lysine. Glycine and other suitable compounds with primary amines can be used as quenchers in the crosslinking reaction. Thus, in another preferred embodiment, the chemical reagent comprises formaldehyde and glycine is used.

In another preferred embodiment, the chemical cross-linking agent includes EDC and NHS. As is known in the art, NHS can be included in the EDC coupling method. However, the inventors surprisingly found that NHS can promote a further reduction in the cytotoxicity of mutant C. difficile toxins compared to the corresponding wild-type toxin, compared to genetically mutated toxins, and compared to genetically mutated toxins chemically cross-linked by EDC. See, for example, Example 22. Therefore, without being bound by mechanism or theory, mutant toxin polypeptides having a β-alanine moiety attached to the side chain of at least one lysine residue of the polypeptide (e.g., obtained from the reaction of a mutant toxin polypeptide, EDC, and NHS) can promote a further reduction in the cytotoxicity of the mutant toxin compared to, for example, a C. difficile toxin (wild-type or mutant) in which the β-alanine moiety is not present.

The use of EDC and/or NHS can also include the use of glycine or other suitable compounds having a primary amine as a quencher. Any compound having a primary amine can be used as a quencher, such as glycine methyl ester and alanine. In a preferred embodiment, the quencher compound is a non-polymeric hydrophilic primary amine. Examples of non-polymeric hydrophilic primary amines include, for example, amino sugars, amino alcohols, and amino polyols. Specific examples of non-polymeric hydrophilic primary amines include glycine, ethanolamine, glucosamine, amine-functionalized polyethylene glycol, and amine-functionalized ethylene glycol oligomers.

In one aspect, the present invention relates to mutant C. difficile toxin polypeptides having at least one amino acid side chain chemically modified with EDC and a non-polymeric hydrophilic primary amine, preferably glycine. The resulting glycine adduct (e.g., from a triple mutant toxin treated with EDC, NHS, and quenched with glycine) can facilitate reduced cytotoxicity of the mutant toxin compared to the corresponding wild-type toxin.

In one embodiment, when a mutant Clostridium difficile toxin polypeptide is chemically modified with EDC and glycine, the polypeptide comprises at least one modification of the polypeptide when modified with EDC (e.g., at least one of any of the modifications (a)-(h) above) and at least one of the following exemplary modifications: (i) a glycine moiety attached to the carboxyl moiety at the C-terminus of the polypeptide; (j) a glycine moiety attached to the side chain of at least one aspartic acid residue of the polypeptide; and (k) a glycine moiety attached to the side chain of at least one glutamic acid residue of the polypeptide. See, for example, Figures 24 and 25.

In one embodiment, at least one amino acid of mutant C. difficile TcdA is chemically cross-linked and/or at least one amino acid of mutant C. difficile TcdB is chemically cross-linked. In another embodiment, at least one amino acid of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and/or SEQ ID NO: 8 is chemically cross-linked. For example, at least one amino acid can be chemically cross-linked using a reagent comprising a carbodiimide, such as EDC. Carbodiimides can form covalent bonds between free carboxyl groups (e.g., side chains from aspartic acid and/or glutamic acid) and amino groups (e.g., in the side chains of lysine residues) to form stable amide bonds.

As another example, at least one amino acid can be chemically cross-linked using a reagent comprising NHS. NHS ester-activated cross-linking species can react with primary amines (e.g., at the N-terminus of each polypeptide chain and/or in the side chains of lysine residues) to produce amide bonds.

In another embodiment, at least one amino acid can be chemically cross-linked using reagents including EDC and NHS. For example, in one embodiment, the present invention relates to an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, wherein the polypeptide comprises at least one amino acid side chain chemically modified with EDC and NHS. In another embodiment, the present invention relates to an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, wherein the polypeptide comprises at least one amino acid side chain chemically modified with EDC and NHS. In another embodiment, the present invention relates to an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 7, or SEQ ID NO: 8. The polypeptide is modified by contacting the polypeptide with EDC and NHS. See, for example, Figures 24 and 25.

When a mutant Clostridium difficile toxin polypeptide is chemically modified with EDC and NHS (e.g., by contact), in one embodiment, the polypeptide comprises at least one modification (e.g., at least one of any of the above (a)-(h) modifications) when the polypeptide is modified with EDC and (l) a β-alanine moiety attached to the side chain of at least one lysine residue of the polypeptide.

In another aspect, the present invention relates to a mutant C. difficile toxin polypeptide, wherein the polypeptide comprises at least one amino acid side chain chemically modified with EDC, NHS, and a non-polymeric hydrophilic primary amine, preferably glycine. In one embodiment, the polypeptide comprises at least one modification where the polypeptide is modified with EDC (e.g., at least one of any of the modifications (a)-(h) above), at least one modification where the polypeptide is modified with glycine (e.g., at least one of the modifications (i)-(k) above), and (1) a β-alanine moiety attached to the side chain of at least one lysine residue of the polypeptide. See, e.g., Figures 24 and 25.

In one aspect, the present invention relates to a mutant Clostridium difficile toxin polypeptide, wherein the side chain of at least one lysine residue of the polypeptide is linked to a β-alanine moiety. In one embodiment, the side chain of a second lysine residue of the polypeptide is linked to the side chain of an aspartic acid residue and/or to the side chain of a glutamic acid residue. The "second" lysine residue of a polypeptide includes a lysine residue of the polypeptide that is not linked to a β-alanine moiety. The side chain of the aspartic acid and/or the side chain of the glutamic acid to which the second lysine residue is linked can be the side chain of the aspartic acid and/or the side chain of the glutamic acid of the polypeptide to form an intramolecular crosslink, or can be the side chain of the aspartic acid and/or the side chain of the glutamic acid of the second polypeptide to form an intermolecular crosslink. In another embodiment, the side chain of at least one aspartic acid residue and/or the side chain of at least one glutamic acid residue of the polypeptide is linked to a glycine moiety. The aspartic acid residue and/or the glutamic acid residue linked to the glycine moiety is not linked to a lysine residue.

As another example of chemically cross-linked mutant C. difficile toxin polypeptides, at least one amino acid can be chemically cross-linked using a reagent comprising formaldehyde. Formaldehyde can react with N-terminal amino acid residues and the side chains of arginine, cysteine, histidine, and lysine. Formaldehyde and glycine can form Schiff base adducts that can attach to the N-terminal primary amino group, arginine, and tyrosine residues, and to a lesser extent, asparagine, glutamine, histidine, and tryptophan residues.

Chemical cross-linking agents are believed to reduce the cytotoxicity of a toxin, for example, as measured by an in vitro cytotoxicity assay or by animal toxicity, if the treated toxin is less toxic (e.g., about 100%, 99%, 95%, 90%, 80%, 75%, 60%, 50%, 25%, or 10% less toxic) than an untreated toxin under the same conditions.

Preferably, the chemical cross-linking agent reduces the cytotoxicity of the mutant C. difficile toxin by at least about 2-log ₁₀ , more preferably about 3-log ₁₀ , and most preferably about 4-log ₁₀ or more relative to the mutant toxin under the same conditions but in the absence of the chemical cross-linking agent. Preferably, the chemical cross-linking agent reduces the cytotoxicity of the mutant toxin by at least about 5-log ₁₀ , about 6-log ₁₀ , about 7-log ₁₀ , about 8-log ₁₀ or more compared to the wild-type toxin.

In another preferred embodiment, the chemically inactivated mutant C. difficile toxin exhibits an EC50 value of greater than or at least about 50 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml, 1000 μg/ml or more, e.g., as measured by an in vitro _{cytotoxicity assay such} as those described herein.

The reaction conditions for contacting the mutant toxin with the chemical cross-linking agent are within the purview of those skilled in the art and may vary depending on the reagents used. However, the present inventors have surprisingly discovered optimal reaction conditions for contacting a mutant C. difficile toxin polypeptide with a chemical cross-linking agent while maintaining functional epitopes and reducing the cytotoxicity of the mutant toxin compared to the corresponding wild-type toxin.

Preferably, conditions are selected for contacting the mutant toxin with the cross-linking agent wherein the mutant toxin has a minimum concentration of about 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 mg/ml to a maximum concentration of about 3.0, 2.5, 2.0, 1.5, or 1.25 mg/ml. Any minimum value can be combined with any maximum value to define a suitable reaction concentration range for the mutant toxin. Most preferably, the reaction concentration of the mutant toxin is about 1.0-1.25 mg/ml.

In one embodiment, the minimum concentration of the reagents used in the reaction is about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, or 50 mM, and the maximum concentration is about 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, or 50 mM. Any minimum value can be combined with any maximum value to define a suitable concentration range for the chemical reagents of the reaction.

In a preferred embodiment where the reagent comprises formaldehyde, the concentration used is preferably any concentration from about 2 mM to 80 mM, most preferably about 40 mM. In another preferred embodiment where the reagent comprises EDC, the concentration used is preferably any concentration from about 1.3 mM to about 13 mM, more preferably from about 2 mM to 3 mM, and most preferably about 2.6 mM.

Exemplary reaction times for contacting the mutant toxin with the chemical cross-linking agent include a minimum of about 0.5, 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, or 60 hours, and a maximum of about 14, 12, 10, 7, 5, 3, 2, 1, or 12 hours. Any minimum value can be combined with any maximum value to define a suitable range of reaction times.

In preferred embodiments, the step of contacting the mutant toxin with a chemical cross-linking agent is performed for a time sufficient to reduce the cytotoxicity of the mutant C. difficile toxin to an _EC50 value of at least about 1000 μg/ml in suitable human cells, such as IMR-90 cells, compared to the same mutant toxin in the absence of the cross-linking agent in a standard in vitro cytotoxicity assay. More preferably, the reacting step is performed for a time that is at least 2-fold and most preferably at least 3-fold or longer than the time sufficient to reduce the cytotoxicity of the mutant toxin to an _EC50 value of at least about 1000 μg/ml in suitable human cells. In one embodiment, the reaction time does not exceed about 168 hours (or 7 days).

For example, in one embodiment where the reagent comprises formaldehyde, the mutant toxin is preferably contacted with the reagent for about 12 hours, which has been shown to be an exemplary time period sufficient to reduce the cytotoxicity of the mutant C. difficile toxin in a standard in vitro cytotoxicity assay to an EC ₅₀ value of at least about 1000 μg/ml in suitable human cells, such as IMR-90 cells, compared to the same mutant toxin in the absence of the cross-linking agent. In a more preferred embodiment, the reaction is conducted for about 48 hours, which is at least about 3 times the sufficient reaction time period. In such an embodiment, the reaction time preferably does not exceed about 72 hours.

In another embodiment in which the reagent comprises EDC, the mutant toxin is preferably contacted with the reagent for about 0.5 hours, more preferably at least about 1 hour, or most preferably about 2 hours. In such an embodiment, the reaction time preferably does not exceed about 6 hours.

Exemplary pH values for contacting the mutant toxin with the chemical cross-linking agent include a minimum of about pH 5.5, 6.0, 6.5, 7.0, or 7.5, and a maximum of about pH 8.5, 8.0, 7.5, 7.0, or 6.5. Any minimum value can be combined with any maximum value to define a suitable range of pH. Preferably, the reaction is carried out at a pH of 6.5-7.5, preferably pH 7.0.

Exemplary temperatures for contacting the mutant toxin with the chemical cross-linking agent include a minimum of about 2°C, 4°C, 10°C, 20°C, 25°C, or 37°C, and a maximum of about 40°C, 37°C, 30°C, 27°C, 25°C, or 20°C. Any minimum value can be combined with any maximum value to define a suitable range of reaction temperatures. Preferably, the reaction is carried out at about 20°C-30°C, most preferably 25°C.

The immunogenic compositions described above can comprise one mutant Clostridium difficile toxin (A or B). Thus, the immunogenic compositions can be provided in separate vials in a formulation or kit (e.g., a separate vial of a composition comprising mutant Clostridium difficile toxin A and a separate vial of a composition comprising mutant Clostridium difficile toxin B). The immunogenic compositions can be administered simultaneously, sequentially, or separately.

In another embodiment, the immunogenic composition described above can comprise two mutant C. difficile toxins (A and B). Any combination of mutant C. difficile toxin A and mutant C. difficile toxin B can be combined into an immunogenic composition. Thus, the immunogenic compositions can be combined in a single vial (e.g., a single vial containing a composition comprising mutant C. difficile TcdA and a composition comprising mutant C. difficile TcdB). Preferably, the immunogenic composition comprises mutant C. difficile TcdA and mutant C. difficile TcdB.

For example, in one embodiment, the immunogenic composition comprises SEQ ID NO: 4 and SEQ ID NO: 6, wherein at least one amino acid of each of SEQ ID NO: 4 and SEQ ID NO: 6 is chemically cross-linked. In another embodiment, the immunogenic composition comprises a mutant Clostridium difficile toxin A comprising SEQ ID NO: 4 or SEQ ID NO: 7, and comprises a mutant Clostridium difficile toxin B comprising SEQ ID NO: 6 or SEQ ID NO: 8, wherein at least one amino acid of each of said mutant Clostridium difficile toxins is chemically cross-linked.

In another embodiment, the immunogenic composition comprises any sequence selected from SEQ ID NO: 4, SEQ ID NO: 84, and SEQ ID NO: 83 and any sequence selected from SEQ ID NO: 6, SEQ ID NO: 86, and SEQ ID NO: 85. In another embodiment, the immunogenic composition comprises SEQ ID NO: 84 and an immunogenic composition comprising SEQ ID NO: 86. In another embodiment, the immunogenic composition comprises SEQ ID NO: 83 and an immunogenic composition comprising SEQ ID NO: 85. In another embodiment, the immunogenic composition comprises SEQ ID NO: 84, SEQ ID NO: 83, SEQ ID NO: 86, and SEQ ID NO: 85.

It should be understood that any composition of the present invention, for example, an immunogenic composition comprising mutant toxin A and/or mutant toxin B, can be used in different ratios or combinations of amounts for therapeutic effect. For example, mutant C. difficile TcdA and mutant C. difficile TcdB can be present in an immunogenic composition in a ratio ranging from 0.1:10 to 10:0.1, A:B. In another embodiment, for example, mutant C. difficile TcdB and mutant C. difficile TcdA can be present in an immunogenic composition in a ratio ranging from 0.1:10 to 10:0.1, B:A. In a preferred embodiment, the ratio is such that the composition contains a greater total amount of mutant TcdB than the total amount of mutant TcdA.

In one aspect, the immunogenic composition is capable of binding to a neutralizing antibody or binding fragment thereof. Preferably, the neutralizing antibody or binding fragment thereof is a neutralizing antibody or binding fragment thereof described below. In an exemplary embodiment, the immunogenic composition is capable of binding to an anti-toxin A antibody or antigen-binding fragment thereof, wherein the anti-toxin A antibody or binding fragment thereof comprises a variable light chain having the amino acid sequence of SEQ ID NO: 36 and a variable heavy chain having the amino acid sequence of SEQ ID NO: 37. For example, the immunogenic composition can comprise mutant Clostridium difficile TcdA, SEQ ID NO: 4, or SEQ ID NO: 7. As another example, the immunogenic composition can comprise SEQ ID NO: 84 or SEQ ID NO: 83.

In another exemplary embodiment, the immunogenic composition is capable of binding to an anti-toxin B antibody or antigen-binding fragment thereof, wherein the anti-toxin B antibody or antigen-binding fragment thereof comprises the variable light chain of B8-26 and the variable heavy chain of B8-26. For example, the immunogenic composition can comprise mutant Clostridium difficile TcdB, SEQ ID NO: 6 or SEQ ID NO: 8. As another example, the immunogenic composition can comprise SEQ ID NO: 86 or SEQ ID NO: 85.

recombinant cells

In another aspect, the invention relates to a recombinant cell or its progeny. In one embodiment, the cell or its progeny comprises a polynucleotide encoding a mutant Clostridium difficile TcdA and/or a mutant Clostridium difficile TcdB.

In another embodiment, the recombinant cell or its progeny comprises a nucleic acid sequence that encodes a polypeptide that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 when optimally aligned, for example, by using the programs GAP or BESTFIT with predetermined gap weights.

In another embodiment, the recombinant cell or its progeny comprises a nucleic acid sequence that encodes a polypeptide that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to any one of SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:83, or SEQ ID NO:85 when optimally aligned, for example, by using the programs GAP or BESTFIT with predetermined gap weights.

In further embodiments, the recombinant cell or its progeny comprises a nucleic acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to any one of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47 when optimally aligned, for example, by using the programs GAP or BESTFIT with predetermined gap weights.

Recombinant cells can be derived from any cell useful for recombinant production of the polypeptides of the present invention. For example, prokaryotic or eukaryotic cells. Preferably, the recombinant cell is derived from any cell suitable for expressing a heterologous nucleic acid sequence of greater than about 5,000, 6,000, preferably about 7,000, and more preferably about 8,000 nucleotides or longer. The prokaryotic host cell can be any Gram-negative or Gram-positive bacterium. In exemplary embodiments, the prokaryotic host cell lacks endogenous polynucleotides encoding toxins and/or spores.

Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. For example, recombinant cells can be derived from Pseudomonas fluorescens cells, as described in paragraphs [0201]-[0230] of U.S. Patent Application Publication No. 2010013762, which is incorporated herein by reference.

Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Preferably, the cell is derived from a Clostridium difficile cell.

The wild-type strains of C. difficile identified by the present inventors lack endogenous polynucleotides encoding C. difficile toxins. Strains lacking endogenous toxin A and B genes include the following strains available from the American Type Culture Collection (ATCC): C. difficile 1351 (ATCC 43593 ^™ ), C. difficile 3232 (ATCC BAA-1801 ^™ ), C. difficile 7322 (ATCC 43601 ^™ ), C. difficile 5036 (ATCC 43603 ^™ ), C. difficile 4811 (ATCC 43602 ^™ ), and C. difficile VPI 11186 (ATCC 700057 ^™ ).

Thus, in one embodiment, the recombinant Clostridium difficile cell is derived from a strain described herein. Preferably, the recombinant Clostridium difficile cell or its progeny is derived from a strain selected from the group consisting of Clostridium difficile 1351, Clostridium difficile 5036, and Clostridium difficile VPI 11186. More preferably, the recombinant Clostridium difficile cell or its progeny is derived from a Clostridium difficile VPI 11186 cell.

In preferred embodiments, the recombinant C. difficile cells or their progeny are inactivated for spore formation. Spores can be infectious, highly resistant, and promote the maintenance of C. difficile in an aerobic environment outside the host. Spores can also promote the survival of C. difficile in the host during antimicrobial therapy. Therefore, C. difficile cells lacking spore formation genes can be used to produce safe immunogenic compositions for administration to mammals. Furthermore, the use of such cells promotes safety during production, for example, protecting equipment, future products, and employees.

Examples of sporulation genes for targeted inactivation include spo0A, spoIIE, σ ^E , σ ^G , and σ ^K . Preferably, the spo0A gene is inactivated.

Methods for inactivating the sporulation genes of C. difficile are known in the art. For example, the sporulation genes can be inactivated by targeted insertion of a selectable marker, such as an antibiotic resistance marker. See, e.g., Heap et al., J Microbiol Methods. 2010 Jan;80(1):49-55; Heap et al., J. Microbiol. Methods, 2007 Sept;70(3):452-464; and Underwood et al., J Bacteriol. 2009 Dec;191(23):7296-305. See also, e.g., Minton et al., WO 2007/148091, entitled "DNA Molecules and Methods," which is incorporated herein by reference in its entirety at pages 33-66, or the corresponding U.S. publication US 20110124109 Al, paragraphs [00137]-[0227].

Methods for producing mutant Clostridium difficile toxins

In one aspect, the present invention relates to a method for producing a mutant Clostridium difficile toxin. In one embodiment, the method comprises culturing any of the recombinant cells described above, or a progeny thereof, under suitable conditions to express the polypeptide.

In another embodiment, the method comprises culturing a recombinant cell, or a progeny thereof, under conditions suitable for expressing a polynucleotide encoding a mutant Clostridium difficile toxin, wherein the cell comprises a polynucleotide encoding a mutant Clostridium difficile toxin, and wherein the mutant comprises (relative to a corresponding wild-type Clostridium difficile toxin) a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation. In one embodiment, the cell lacks an endogenous polynucleotide encoding a toxin.

In additional embodiments, the method comprises culturing a recombinant Clostridium difficile cell, or a progeny thereof, under suitable conditions to express a polynucleotide encoding a mutant Clostridium difficile toxin, wherein the cell comprises a polynucleotide encoding a mutant Clostridium difficile toxin and the cell lacks an endogenous polynucleotide encoding a toxin.

In another aspect, the present invention relates to a method for producing a mutant Clostridium difficile toxin. The method comprises the following steps: (a) contacting a Clostridium difficile cell with a recombinant Escherichia coli cell, wherein the Clostridium difficile cell lacks an endogenous polynucleotide encoding a Clostridium difficile toxin and the E. coli cell comprises a polynucleotide encoding a mutant Clostridium difficile toxin; (b) culturing the Clostridium difficile cell and the E. coli cell under suitable conditions to transfer the polynucleotide from the E. coli cell to the Clostridium difficile cell; (c) selecting a Clostridium difficile cell comprising a polynucleotide encoding a mutant Clostridium difficile toxin; (d) culturing the Clostridium difficile cell of step (c) under suitable conditions to express the polynucleotide; and (e) isolating the mutant Clostridium difficile toxin.

In the methods of the present invention, recombinant E. coli cells include a heterologous polynucleotide encoding a mutant Clostridium difficile toxin described herein. The polynucleotide can be DNA or RNA. In an exemplary embodiment, the polynucleotide encoding the mutant Clostridium difficile toxin is codon-optimized for E. coli codon usage. Methods for codon-optimizing polynucleotides are well known in the art.

In one embodiment, the polynucleotide comprises a nucleic acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polynucleotide encoding a mutant C. difficile TcdA described herein. Exemplary polynucleotides encoding a mutant C. difficile toxin A include SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 44, and SEQ ID NO: 45.

In another embodiment, the polynucleotide comprises a nucleic acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polynucleotide encoding a mutant C. difficile TcdB described herein. Exemplary polynucleotides encoding a mutant C. difficile toxin B include SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 46, and SEQ ID NO: 47. In another embodiment, the polynucleotide encodes SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

In one embodiment, the E. coli cell comprising the heterologous polynucleotide is an E. coli cell that stably hosts a heterologous polynucleotide encoding a mutant Clostridium difficile toxin. Exemplary E. coli cells include cells selected from the group consisting of Stbl2 ^™ E. coli competent cells (Invitrogen, Carlsbad, CA), OneStbl3 ^™ chemically competent E. coli (Invitrogen, Carlsbad, CA), ElectroMAX ^™ Stbl4 ^™ E. coli competent cells (Invitrogen), and E. coli CA434. In a preferred embodiment, the E. coli cloning host cell is not DH5α. More preferably, the E. coli cloning host cell is MAX Stbl2 ^™ E. coli competent cells.

The method of the present invention further comprises the step of culturing the Clostridium difficile cell and the E. coli cell under conditions suitable for transferring the polynucleotide from the E. coli cell to the Clostridium difficile cell, thereby producing a recombinant Clostridium difficile cell. In a preferred embodiment, the culturing conditions are suitable for transferring the polynucleotide from the E. coli cell (donor cell) to the Clostridium difficile cell (recipient cell) and for producing genetically stable inheritance.

Most preferably, the culture conditions are suitable for bacterial conjugation, which is known in the art. "Conjugation" refers to a specific process of transferring a polynucleotide, in which a polynucleotide (e.g., a bacterial plasmid) is transferred unidirectionally from one bacterial cell (i.e., the "donor") to another (i.e., the "recipient"). The conjugation process involves contact between the donor cell and the recipient cell. Preferably, the donor E. coli cell is an E. coli CA434 cell.

Exemplary suitable (conjugative) conditions for transferring a polynucleotide from an E. coli cell to a C. difficile cell include growing a liquid culture of C. difficile in brain heart infusion broth (BHI; Oxoid) or Schaedlers anaerobic broth (SAB; Oxoid). In another embodiment, a solid culture of C. difficile can be grown on fresh blood agar (FBA) or BHI agar. Preferably, C. difficile is grown in an anaerobic environment (e.g., 80% _N2 , 10% _CO2 , and 10% _H2 [vol/vol]) at 37°C. In one embodiment, the suitable conditions include growing E. coli aerobically in Luria-Bertani (LB) broth or on LB agar at 37°C. For conjugative transfer to C. difficile, exemplary suitable conditions include growing E. coli anaerobically on FBA. Antibiotics can be included in the liquid or solid culture medium, as is known in the art. Examples of such antibiotics include cycloserine (250 μg/ml), cefoxitin (8 μg/ml), chloramphenicol (12.5 μg/ml), thiamphenicol (15 μg/ml) and erythromycin (5 μg/ml).

The method of the present invention further comprises the step of selecting the resulting recombinant Clostridium difficile cell comprising a polynucleotide encoding a mutant Clostridium difficile toxin. In an exemplary embodiment, the recombinant Clostridium difficile cell is a recipient of the polynucleotide encoding a mutant Clostridium difficile toxin from a recombinant Escherichia coli cell.

The methods of the present invention include the step of culturing a recombinant cell or its progeny under conditions suitable for expression of a polynucleotide encoding a mutant C. difficile toxin, which results in the production of a mutant C. difficile toxin. Suitable conditions for expression of the polynucleotide by the recombinant cell include culture conditions suitable for growth of C. difficile cells, which are known in the art. For example, suitable conditions may include culturing C. difficile transformants in Brain Heart Infusion Broth (BHI; Oxoid) or Schaedlers Anaerobic Broth (SAB; Oxoid). In another embodiment, a solid C. difficile culture may be grown on FBA or BHI agar. Preferably, C. difficile is grown in an anaerobic environment (e.g., 80% N ₂ , 10% CO ₂ , and 10% H ₂ [vol/vol]) at 37°C.

In one embodiment, the method of the present invention comprises the step of isolating the resulting mutant C. difficile toxin. Methods for isolating proteins from C. difficile are known in the art.

In another embodiment, the method includes a step of purifying the resulting mutant C. difficile toxin. Methods for purifying polypeptides, such as chromatography, are known in the art.

In an exemplary embodiment, the method further comprises the step of contacting the isolated mutant C. difficile toxin with a chemical crosslinking agent as described above. Preferably, the crosslinking agent comprises formaldehyde, ethyl-3-(3-dimethylaminopropyl)carbodiimide, or a combination of EDC and NHS. Exemplary reaction conditions are described above and in the Examples section below.

In another aspect, the present invention relates to an immunogenic composition comprising a mutant Clostridium difficile toxin as described herein produced by any method, preferably produced by the method described above.

Antibody

Surprisingly, the immunogenic compositions of the present invention described above elicit novel antibodies in vivo, indicating that the immunogenic compositions contain native structures (e.g., retained antigenic epitopes) retained by the respective wild-type C. difficile toxins and that the immunogenic compositions contain epitopes. Antibodies raised against a toxin produced by one strain of C. difficile may be able to bind to a corresponding toxin produced by another strain of C. difficile. That is, antibodies and binding fragments thereof may be "cross-reactive," meaning the ability to react with similar antigenic sites on toxins produced by multiple strains of C. difficile. Cross-reactivity also includes the ability of an antibody to react or bind to an antigen that did not stimulate its production, i.e., a reaction between an antigen and an antibody raised against a different, but similar, antigen.

In one aspect, the present inventors surprisingly discovered monoclonal antibodies having a neutralizing effect on Clostridium difficile toxins and methods for preparing the same. The antibodies of the present invention can neutralize the cytotoxicity of Clostridium difficile toxins in vitro, inhibit the binding of Clostridium difficile toxins to mammalian cells, and/or can neutralize the enterotoxicity of Clostridium difficile toxins in vivo. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding any of the above-mentioned polypeptides. In addition, the present invention relates to the use of any of the above-mentioned compositions in treating and preventing Clostridium difficile infection, Clostridium difficile-related diseases, syndromes, disease conditions, symptoms and/or complications in mammals, reducing the risk, reducing the severity, reducing the incidence and/or delaying the occurrence thereof compared to mammals not administered the composition, as well as methods for preparing the compositions.

The present inventors have further discovered that a combination of at least two neutralizing monoclonal antibodies can exhibit unexpected synergistic effects in neutralizing TcdA or TcdB, respectively. Antitoxin antibodies or binding fragments thereof can be used to inhibit Clostridium difficile infection.

An "antibody" is a protein comprising at least one or two heavy (H) chain variable regions (abbreviated herein as VH) and at least one or two light (L) chain variable regions (abbreviated herein as VL). The VH and VL can be further subdivided into hypervariable regions, termed "complementarity determining regions" ("CDRs"), which are interspersed with more conserved regions called "framework regions" (FRs). The extent of the framework regions and CDRs has been precisely defined (see Kabat, E.A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al., J. Mol. Biol. 196: 901-917, 1987). The term "antibody" includes intact immunoglobulins of the IgA, IgG, IgE, IgD, IgM types (and subclasses thereof), wherein the light chains of said immunoglobulins may be of the kappa or lambda type.

The antibody molecule can be full-length (e.g., an IgG1 or IgG4 antibody). Antibodies can have various isotypes, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In a preferred embodiment, the antibody is of the IgG isotype, such as IgG1. In another preferred embodiment, the antibody is an IgE antibody.

In another embodiment, the antibody molecule includes an "antigen-binding fragment" or "binding fragment," as used herein, which refers to a portion of an antibody that specifically binds to a toxin of C. difficile (e.g., toxin A). A binding fragment is, for example, a molecule in which one or more immunoglobulin chains are not full-length but that specifically binds to a toxin.

Examples of binding portions encompassed by the term "binding fragment" of an antibody include (i) an aFab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment, consisting of the VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of the VH domain; and (vi) isolated complementarity determining regions (CDRs), with sufficient framework to specifically bind to the antigen-binding portion of, for example, a variable region. The binding fragment of the light chain variable region and the binding fragment of the heavy chain variable region, such as the two domains VL and VH of the Fv fragment, can be connected by a synthetic linker that makes them a single protein chain using recombinant methods, wherein VL and VH pair to form a monovalent molecule (called single-chain Fv (scFv), see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also encompassed within the term "binding fragment" of an antibody. These antibody portions are obtained using techniques known in the art and the use of the portion is screened in the same manner as for intact antibodies.

As used herein, an antibody that "specifically binds" to a particular polypeptide or an epitope on a particular polypeptide, or is "specific for" a particular polypeptide or an epitope on a particular polypeptide, is an antibody that binds to that particular polypeptide or an epitope on a particular polypeptide but does not substantially bind to any other polypeptide or polypeptide epitope. For example, when referring to a biomolecule (e.g., a protein, nucleic acid, antibody, etc.) that "specifically" binds to a target, the biomolecule binds to its target molecule in a heterogeneous population of molecules comprising the target and does not bind to other molecules in appreciable amounts, as measured under specified conditions (e.g., immunoassay conditions in the case of an antibody). Binding between the antibody and its target determines the presence of the target in the heterogeneous population of molecules. For example, "specific binding" or "specifically binds" means that the antibody or its binding fragment binds to a wild-type and/or mutant toxin of Clostridium difficile with an affinity that is at least twice as high as its affinity for a nonspecific antigen.

In an exemplary embodiment, the antibody is a chimeric antibody. Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, the gene encoding the Fc constant region of a mouse (or other species) monoclonal antibody molecule can be digested with a restriction enzyme to remove the region encoding the mouse Fc, and substituted with the equivalent portion of the gene encoding the human Fc constant region. Chimeric antibodies can also be produced by recombinant DNA techniques, wherein DNA encoding the mouse variable region can be linked to DNA encoding the human constant region.

In another exemplary embodiment, the antibody or binding fragment thereof is humanized using methods known in the art. For example, once a mouse antibody is obtained, at least a portion of the CDRs of the antibody can be replaced with human CDRs. Humanized antibodies can also be generated by replacing sequences of the mouse Fv variable region not directly involved in antigen binding with equivalent sequences of the human Fv variable region. General methods for generating humanized antibodies are known in the art.

For example, monoclonal antibodies against C. difficile TcdA or C. difficile TcdB can also be produced by annotated techniques such as the hybridoma technique (see, e.g., Kohler and Milstein, 1975, Nature, 256: 495-497). Briefly, an immortalized cell line is fused with lymphocytes from a mammal immunized with C. difficile TcdA, C. difficile TcdB, or a mutant C. difficile toxin as described herein, and the culture supernatant of the resulting hybridoma cells is screened to identify hybridomas that produce monoclonal antibodies that bind to C. difficile TcdA or C. difficile TcdB. Typically, the immortalized cell line is derived from lymphocytes of the same mammal. Hybridomas that produce the monoclonal antibodies of the invention are detected by screening the hybridoma culture supernatant for antibodies that bind to C. difficile TcdA or C. difficile TcdB using an assay such as ELISA. Human hybridomas can be prepared in a similar manner.

As an alternative to generating antibodies by immunization and selection, antibodies of the present invention can also be identified by screening recombinant combinatorial immunoglobulin libraries with C. difficile TcdA, C. difficile TcdB, or mutant C. difficile toxins described herein. The recombinant antibody library can be, for example, a scFv library or a Fab library. Furthermore, the antibodies described herein can also be used in competitive binding studies to identify additional anti-TcdA or anti-TcdB antibodies and binding fragments thereof. For example, additional anti-TcdA or anti-TcdB antibodies and binding fragments thereof can be identified by screening a human antibody library and identifying molecules in the library that compete with the antibodies described herein in a competitive binding assay.

In addition, antibodies encompassed by the present invention include recombinant antibodies that can be produced using phage display methods known in the art. In phage display methods, phage can be used to display antigen-binding domains expressed from a repertoire or antibody library (e.g., human or mouse). Phage expressing an antigen-binding domain that binds to an immunogen described herein (e.g., a mutant Clostridium difficile toxin) can be selected or identified using an antigen, such as a labeled antigen.

Antibodies and binding fragments thereof are also within the scope of the present invention in which specific amino acids have been substituted, deleted, or added. Preferably, the preferred antibodies have amino acid substitutions in the framework regions to improve binding to the antigen. For example, a small number of selected acceptor framework residues of the immunoglobulin chain can be replaced by corresponding donor amino acids. Preferred positions for substitution include amino acid residues near the CDRs or that can interact with the CDRs. Criteria for selecting amino acids from donors are described in U.S. Pat. No. 5,585,089 (e.g., columns 12-16). The acceptor framework can be a mature human antibody framework sequence or a consensus sequence thereof.

As used herein, a "neutralizing antibody or binding fragment thereof" refers to an antibody or binding fragment thereof that binds to a pathogen (e.g., C. difficile TcdA or TcdB) and reduces the infectivity and/or activity (e.g., reduces cytotoxicity) of the pathogen in a mammal and/or cell culture, as compared to the pathogen under the same conditions in the absence of the neutralizing antibody or binding fragment thereof. In one embodiment, the neutralizing antibody or binding fragment thereof is capable of neutralizing at least about 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the biological activity of the pathogen, as compared to the pathogen under the same conditions in the absence of the neutralizing antibody or binding fragment thereof.

As used herein, the term "anti-toxin antibody or binding fragment thereof" refers to an antibody or binding fragment thereof that binds to a respective C. difficile toxin (e.g., C. difficile toxin A or toxin B). For example, an anti-toxin A antibody or binding fragment thereof to an antibody or binding fragment thereof that binds to TcdA.

The antibodies or binding fragments thereof described herein can be produced in any wild-type and/or transgenic mammal, including, for example, mice, humans, rabbits, and goats.

When the immunogenic composition is administered to a population in advance, for example, for vaccination, the antibody response generated in the individual can be used to neutralize toxins from the same strain as well as toxins from strains that did not stimulate the production of the antibodies. See, for example, Example 37, which shows that the immunogenic composition generates cross-reactivity between toxins from the 630 strain and toxins from various wild-type C. difficile strains.

In one aspect, the present invention relates to antibodies or binding fragments thereof that are specific for TcdA of Clostridium difficile. Monoclonal antibodies that specifically bind to TcdA include A65-33, A60-22, A80-29 and/or preferably A3-25.

In one aspect, the invention relates to antibodies or binding fragments thereof that are specific for TcdA from any wild-type strain of C. difficile, such as those described above, for example, specific for SEQ ID NO: 1. In another aspect, the invention relates to antibodies or binding fragments thereof that are specific for the immunogenic compositions described above. For example, in one embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO: 4 or SEQ ID NO: 7. In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO: 4 or SEQ ID NO: 7, wherein at least one amino acid of SEQ ID NO: 4 or SEQ ID NO: 7 is cross-linked with formaldehyde, EDC, NHS, or a combination of EDC and NHS. In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO: 84 or SEQ ID NO: 83.

Antibodies or binding fragments thereof having variable heavy and variable light chain regions that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to the variable heavy and light chain regions of A65-33, A60-22, A80-29, and/or preferably A3-25 can also bind to TcdA.

In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the variable heavy chain region amino acid sequence of A3-25 set forth in SEQ ID NO: 37.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of the variable light chain region of A3-25 set forth in SEQ ID NO: 36.

In another aspect, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence set forth in SEQ ID NO:37; and a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence set forth in SEQ ID NO:36.

In another embodiment, antibodies or binding fragments thereof having the complementarity determining regions (CDRs) of the variable heavy chain and/or variable light chain of A65-33, A60-22, A80-29, and/or preferably A3-25 can bind to TcdA. The CDRs of the variable heavy chain region of A3-25 are shown in Table 4 below.

The CDRs of the variable light chain region of A3-25 are shown in Table 5 below.

In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequences of the heavy chain complementarity determining regions (CDRs) set forth in SEQ ID NOs: 41 (CDR H1), 42 (CDR H2) and 43 (CDR H3); and/or the amino acid sequences of the light chain CDRs set forth in SEQ ID NOs: 38 (CDRL1), 39 (CDR L2) and 40 (CDR L3).

In an exemplary embodiment, the antibody or binding fragment thereof specific for C. difficile toxin A specifically binds an epitope in the N-terminal region of TcdA, such as an epitope between amino acids 1-1256 of TcdA numbered according to SEQ ID NO:1.

In preferred embodiments, the antibody or binding fragment thereof specific for C. difficile toxin A specifically binds to an epitope in the C-terminal region of toxin A, such as between amino acids 1832-2710 of TcdA numbered according to SEQ ID NO: 1. Examples include A3-25, A65-33, A60-22, A80-29.

In another embodiment, the antibody or binding fragment thereof specific for Clostridium difficile toxin A specifically binds to an epitope in the "translocation" region of Clostridium difficile toxin A, for example, preferably an epitope comprising residues 956-1128 of TcdA numbered according to SEQ ID NO: 1, for example, an epitope between amino acids 659-1832 of TcdA numbered according to SEQ ID NO: 1.

In another aspect, the present invention relates to antibodies or binding fragments thereof that are specific for TcdB of C. difficile. For example, the antibody or binding fragment thereof can be specific for TcdB from any wild-type strain of C. difficile, such as those described above, e.g., specific for SEQ ID NO: 2. In another aspect, the present invention relates to antibodies or binding fragments thereof that are specific for the immunogenic compositions described above. For example, in one embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO: 6 or SEQ ID NO: 8.

In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO: 6 or SEQ ID NO: 8, wherein at least one amino acid of SEQ ID NO: 6 or SEQ ID NO: 8 is cross-linked with formaldehyde, EDC, NHS, or a combination of EDC and NHS. In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO: 86 or SEQ ID NO: 85.

Monoclonal antibodies that specifically bind TcdB include antibodies produced by the B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6 and/or preferably B8-26 clones described herein.

Antibodies or binding fragments thereof that can also bind to TcdB include antibodies or binding fragments thereof having variable heavy chain and variable light chain regions that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical to the variable heavy chain and light chain regions of B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6, preferably B8-26, B59-3 and/or B9-30.

In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the variable heavy chain region amino acid sequence of A3-25 set forth in SEQ ID NO: 49.

In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the variable heavy chain region amino acid sequence of A3-25 set forth in SEQ ID NO: 60.

In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the variable heavy chain region amino acid sequence of A3-25 set forth in SEQ ID NO:71.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of the variable light chain region of A3-25 set forth in SEQ ID NO: 55.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the variable light chain region amino acid sequence of A3-25 set forth in SEQ ID NO: 66.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of the variable light chain region of A3-25 set forth in SEQ ID NO: 77.

The amino acid sequence of the variable heavy chain of the neutralizing antibody against Clostridium difficile TcdB (B8-26mAb) is shown in SEQ ID NO: 49. See Table 25-a.

The amino acid sequence of the variable light chain of the neutralizing antibody against Clostridium difficile TcdB (B8-26mAb) is shown in SEQ ID NO: 55. See Table 25-b.

In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequence of the heavy chain CDRs set forth in SEQ ID NO: 51 (CDR H1), 52 (CDR H2) and 53 (CDR H3), and/or comprises the amino acid sequence of the light chain CDRs set forth in SEQ ID NO: 57 (CDR L1), 58 (CDRL2) and 59 (CDR L3).

The amino acid sequence of the variable heavy chain of the neutralizing antibody against Clostridium difficile TcdB (B59-3mAb) is shown in SEQ ID NO: 60. See Table 26-a.

The amino acid sequence of the variable light chain of the neutralizing antibody against Clostridium difficile TcdB (B59-3mAb) is shown in SEQ ID NO: 66. See Table 26-b.

In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequence of the heavy chain CDRs set forth in SEQ ID NOs: 62 (CDR H1), 63 (CDR H2) and 64 (CDR H3), and/or comprises the amino acid sequence of the light chain CDRs set forth in SEQ ID NOs: 68 (CDR L1), 69 (CDRL2) and 70 (CDR L3).

The amino acid sequence of the variable heavy chain of the neutralizing antibody against Clostridium difficile TcdB (B9-30mAb) is shown in SEQ ID NO: 71. See Table 27-a.

The amino acid sequence of the variable light chain of the neutralizing antibody against Clostridium difficile TcdB (B9-30mAb) is shown in SEQ ID NO: 77. See Table 27-b.

In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequence of the heavy chain CDRs set forth in SEQ ID NOs: 73 (CDR H1), 74 (CDR H2) and 75 (CDR H3), and/or comprises the amino acid sequence of the light chain CDRs set forth in SEQ ID NOs: 79 (CDR L1), 80 (CDRL2) and 81 (CDR L3).

In one aspect, the invention relates to antibodies or binding fragments thereof that are specific for wild-type C. difficile TcdB from any strain of C. difficile, such as those described above, for example, specific for SEQ ID NO: 2. In another aspect, the invention relates to antibodies or binding fragments thereof that are specific for an immunogenic composition as described above. For example, in one embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO: 6 or SEQ ID NO: 8. In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO: 6 or SEQ ID NO: 8, wherein at least one amino acid of SEQ ID NO: 6 or SEQ ID NO: 8 is cross-linked with formaldehyde, EDC, NHS, or a combination of EDC and NHS.

Antibodies or binding fragments thereof having variable heavy and variable light chain regions that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to the variable heavy and light chain regions of B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6 and/or preferably B8-26 can also bind to TcdB.

In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence of B8-26 (SEQ ID NO: 49).

In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence of B8-26 (SEQ ID NO: 55).

In another aspect, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence of B8-26 (SEQ ID NO: 49); and a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence of B8-26 (SEQ ID NO: 55). 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% the same.

In another embodiment, antibodies or binding fragments thereof having the CDRs of the variable heavy chain and/or variable light chain of B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6 and/or preferably B8-26 can also bind to TcdB.

In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequence of the heavy chain complementarity determining region (CDR) of B8-26, and/or comprises the amino acid sequence of the light chain CDR of B8-26.

In preferred embodiments, the antibody or binding fragment thereof specific for C. difficile toxin B specifically binds to an epitope in the N-terminal region of toxin B, such as between amino acids 1-1256 of TcdB as numbered according to SEQ ID NO: 2. Examples include B2-31, B5-40, B8-26, B70-2, B6-30, and B9-30.

In an exemplary embodiment, the antibody or binding fragment thereof specific for C. difficile toxin B specifically binds an epitope in the C-terminal region of toxin B, e.g., between amino acids 1832-2710 of TcdB numbered according to SEQ ID NO:2.

In another embodiment, the antibody or binding fragment thereof specific for C. difficile toxin B specifically binds to an epitope in the "translocation" region of C. difficile toxin B, e.g., preferably an epitope comprising residues 956-1128 of TcdB numbered according to SEQ ID NO: 2, e.g., an epitope between amino acids 659-1832 of TcdB. Examples include B59-3, B60-2, and B56-6.

Combination of antibodies

The anti-toxin antibodies or binding fragments thereof can be administered in combination with other anti-C. difficile toxin antibodies (e.g., other monoclonal antibodies, polyclonal gamma-globulins) or antigen-binding fragments thereof. Useful combinations include anti-toxin A antibodies or binding fragments thereof and anti-toxin B antibodies or antigen-binding fragments thereof.

In another embodiment, the combination comprises an anti-toxin A antibody or binding fragment thereof and another anti-toxin A antibody or antigen-binding fragment thereof. Preferably, the combination comprises a neutralizing anti-toxin A monoclonal antibody or binding fragment thereof and another neutralizing anti-toxin A monoclonal antibody or binding fragment thereof. Surprisingly, the inventors discovered a synergistic effect of such a combination in neutralizing toxin A cytotoxicity. For example, the combination comprises a combination of at least two of the following neutralizing anti-toxin A monoclonal antibodies: A3-25; A65-33; A60-22; and A80-29. More preferably, the combination comprises the A3-25 antibody and at least one of the following neutralizing anti-toxin A monoclonal antibodies: A65-33; A60-22; and A80-29. Most preferably, the combination comprises all four antibodies: A3-25; A65-33; A60-22; and A80-29.

In other embodiments, the combination comprises an anti-toxin B antibody or binding fragment thereof and another anti-toxin B antibody or antigen-binding fragment thereof. Preferably, the combination comprises a neutralizing anti-toxin B monoclonal antibody or binding fragment thereof and another neutralizing anti-toxin B monoclonal antibody or antigen-binding fragment thereof. Surprisingly, the inventors have discovered a synergistic effect of such combinations in neutralizing toxin B cytotoxicity. More preferably, the combination comprises a combination of at least two of the following neutralizing anti-toxin B monoclonal antibodies: B8-26; B9-30; and B59-3. Most preferably, the combination comprises all three antibodies: B8-26; B9-30; and B59-3.

In another embodiment, the combination includes an anti-toxin B antibody or binding fragment thereof and another anti-toxin B antibody or antigen-binding fragment thereof. As previously described, the present inventors have discovered that a combination of at least two neutralizing monoclonal antibodies can exhibit an unexpected synergistic effect in the neutralization of toxin A and toxin B, respectively.

In another embodiment, the agents of the invention can be formulated as a mixture, or chemically or genetically linked using techniques known in the art to result in a covalently linked antibody (or covalently linked antibody fragment) having binding properties against both toxin A and toxin B. Combination formulations can be guided by determining one or more parameters such as affinity, avidity, or biological potency of an agent alone or in combination with another agent.

Such combination therapies are preferably additive and/or synergistic in their therapeutic activity, e.g., in the inhibition, prevention (e.g., prevention of relapse), and/or treatment of a C. difficile-associated disease or condition. Administration of such combination therapies can reduce the dosage of the therapeutic agent (e.g., antibody or antibody fragment mixture, or cross-linked or genetically fused bispecific antibody or antibody fragment) required to achieve the desired effect.

It should be understood that any of the compositions of the present invention, e.g., anti-toxin A and/or anti-toxin B antibodies or antigen-binding fragments thereof, can be combined in various ratios or amounts for therapeutic effect. For example, anti-toxin A and anti-toxin B antibodies or their respective binding fragments can be present in the composition in a ratio ranging from 0.1:10 to 10:0.1 (A:B). In another embodiment, anti-toxin A and anti-toxin B antibodies or their respective binding fragments can be present in the composition in a ratio ranging from 0.1:10 to 10:0.1 (B:A).

In another aspect, the present invention relates to a method for producing neutralizing antibodies against TcdA of C. difficile. The method comprises administering the immunogenic composition described above to a mammal and recovering antibodies from the mammal. In a preferred embodiment, the immunogenic composition comprises a mutant C. difficile TcdA having SEQ ID NO: 4, wherein at least one amino acid of the mutant C. difficile TcdA is chemically cross-linked, preferably by methyl or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking. Exemplary neutralizing antibodies against TcdA that can be produced include A65-33; A60-22; A80-29 and/or A3-25.

In another aspect, the present invention relates to a method for producing neutralizing antibodies against TcdB of Clostridium difficile. The method comprises administering the immunogenic composition described above to a mammal and recovering antibodies from the mammal. In a preferred embodiment, the immunogenic composition comprises a mutant C. difficile TcdB having SEQ ID NO: 6, wherein at least one amino acid of the mutant C. difficile TcdB is chemically cross-linked, preferably by methyl or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking. Exemplary neutralizing antibodies against TcdB that can be produced include B2-31; B5-40, B70-2; B6-30; B9-30; B59-3; B60-2; B56-6; and/or B8-26.

preparation

The compositions of the present invention (e.g., for example, compositions comprising a mutant C. difficile toxin, an immunogenic composition, an antibody and/or an antibody-binding fragment thereof as described herein) can be in a variety of forms. These forms include, for example, semi-solid and solid dosage forms, suppositories, liquid forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and/or dried forms such as, for example, lyophilized powder forms, freeze-dried forms, spray-dried forms, and/or foam-dried forms. For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from a mixture containing the composition of the present invention. In exemplary embodiments, the composition is in the form of a solution or suspension in a liquid carrier suitable for injection prior to injection. In further exemplary embodiments, the composition is emulsified or encapsulated in liposomes or microparticles, such as a polylactic acid compound, polyglycolide, or copolymer.

In a preferred embodiment, the composition is lyophilized and reconstituted immediately before use.

In one aspect, the invention relates to pharmaceutical compositions comprising any of the compositions described herein (such as, for example, compositions comprising a mutant C. difficile toxin, an immunogenic composition, an antibody and/or an antibody-binding fragment thereof as described herein) formulated together with a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, stabilizer, diluent, and/or buffer that is physiologically suitable.

Exemplary stabilizers include carbohydrates such as sorbitol, mannitol, starch, dextran, sucrose, trehalose, lactose, and/or glucose; inert proteins such as albumin and/or casein; and/or other large, slowly metabolized macromolecules such as polysaccharides, such as chitosan, polylactic acid, polyglycolic acid and copolymers (such as latex-functionalized SEPHAROSE ^™ agarose, agarose, cellulose, etc.), amino acids, polyamino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). In addition, these carriers can be used as immunostimulatory substances (i.e., adjuvants).

Preferably, the composition includes trehalose. Preferred amounts of trehalose (weight %) range from a minimum of about 1%, 2%, 3%, or 4% to a maximum of about 10%, 9%, 8%, 7%, 6%, or 5%. Any minimum value can be combined with any maximum value to define a suitable range. In one embodiment, the composition includes approximately 3%-6% trehalose, and most preferably, 4.5% trehalose, for example, per 0.5 mL dose.

Examples of suitable diluents include distilled water, saline, physiological phosphate-buffered saline, glycerol, alcohols (e.g., ethanol), Ringer's solution, dextrose solution, Hanks' balanced salt solution, and/or lyophilization vehicles.

Exemplary buffers include phosphates (e.g., calcium phosphate, sodium phosphate); acetates (e.g., sodium acetate); succinates (e.g., sodium succinate); glycine; histidine; carbonate, Tris (trishydroxymethylaminomethane), and/or bicarbonate (e.g., ammonium bicarbonate) buffers. Preferably, the composition includes a Tris buffer. Preferred amounts of Tris buffer include a minimum of about 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM to a maximum of about 100 mM, 50 mM, 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, or 11 mM. Any minimum value can be combined with any maximum value to define a suitable range. In one embodiment, the composition includes approximately 8 mM to 12 mM Tris buffer, most preferably, 10 mM Tris buffer, e.g., in each 0.5 mL dose.

In another preferred embodiment, the composition includes a histidine buffer. Preferred amounts of histidine buffer include a minimum of about 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM to a maximum of about 100 mM, 50 mM, 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, or 11 mM. Any minimum value can be combined with any maximum value to define a suitable range. In one embodiment, the composition includes about 8 mM to 12 mM histidine buffer, most preferably 10 mM histidine buffer, for example, in each 0.5 mL dose.

In another preferred embodiment, the composition includes a phosphate buffer. Preferred amounts of phosphate buffer include from a minimum of about 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM to a maximum of about 100 mM, 50 mM, 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, or 11 mM. Any minimum value can be combined with any maximum value to define a suitable range. In one embodiment, the composition includes about 8 mM to 12 mM phosphate buffer, most preferably 10 mM phosphate buffer, for example, in each 0.5 mL dose.

The pH of the buffer is typically selected to stabilize the selected active material and can be determined by those skilled in the art using known methods. Preferably, the pH of the buffer is within the physiological pH range. Thus, the preferred pH range is from about 3 to about 8; more preferably, from about 6.0 to about 8.0; more preferably, from about 6.5 to about 7.5; and most preferably, from about 7.0 to about 7.2.

In some embodiments, the pharmaceutical composition may include a surfactant. Any surfactant is suitable, whether amphoteric, nonionic, cationic, or anionic. Exemplary surfactants include polyoxyethylene sorbitan ester surfactants (e.g., ), such as Polysorbate 20 and/or Polysorbate 80; polyoxyethylene fatty ethers derived from dodecyl, hexadecyl, octadecyl, and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monododecyl ether (Brij 30); Triton X 100, or t-octylphenyl polyvinyl chloride; and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate, and combinations thereof. Preferred surfactants include Polysorbate 80 (polyoxyethylene sorbitan monooleate).

Preferred amounts (weight %) of Polysorbate 80 include, from a minimum of about 0.001%, 0.005%, or 0.01%, to a maximum of about 0.010%, 0.015%, 0.025%, or 1.0%. Any minimum value can be combined with any maximum value to define a suitable range. In one embodiment, the composition includes about 0.005%-0.015% Polysorbate 80, most preferably, 0.01% Polysorbate 80.

In an exemplary embodiment, the immunogenic composition comprises trehalose and Phosphate 80. In another exemplary embodiment, the immunogenic composition comprises a Tris buffer and Polysorbate 80. In another exemplary embodiment, the immunogenic composition comprises a histidine buffer and Polysorbate 80. In another exemplary embodiment, the immunogenic composition comprises a phosphate buffer and Polysorbate 80.

In an exemplary embodiment, the immunogenic composition comprises trehalose, a Tris buffer, and Polysorbate 80. In another exemplary embodiment, the immunogenic composition comprises trehalose, a histidine buffer, and Polysorbate 80. In another exemplary embodiment, the immunogenic composition comprises trehalose, a phosphate buffer, and Polysorbate 80.

The compositions described herein may also include ingredients of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, and/or mineral oil.Examples include glycols such as propylene glycol or polyethylene glycol.

In some embodiments, the pharmaceutical composition further comprises formaldehyde. For example, in preferred embodiments, the pharmaceutical composition further comprising formaldehyde comprises an immunogenic composition wherein the mutant Clostridium difficile toxin of the immunogenic composition has been contacted with a chemical cross-linking agent comprising formaldehyde. The amount of formaldehyde present in the pharmaceutical composition can vary from a minimum of approximately 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.010%, 0.013%, or 0.015%, to a maximum of approximately 0.020%, 0.019%, 0.018%, 0.017%, 0.016%, 0.015%, 0.014%, 0.013%, 0.012%, 0.011%, or 0.010%. Any minimum value can be combined with any maximum value to define a suitable range. In one embodiment, the pharmaceutical composition comprises about 0.010% formaldehyde.

In other embodiments, the pharmaceutical compositions described herein do not include formaldehyde. For example, in preferred embodiments, the pharmaceutical compositions that do not include formaldehyde include immunogenic compositions in which at least one amino acid of a mutant C. difficile toxin is chemically cross-linked with a substance comprising EDC. More preferably, in such embodiments, the mutant C. difficile toxin has not been exposed to a chemical cross-linking agent comprising formaldehyde. As another exemplary embodiment, the lyophilized form of the pharmaceutical composition does not include formaldehyde.

In another embodiment, the compositions described herein may include an adjuvant as described below. Preferred adjuvants enhance the intrinsic immune response to the immunogen without causing conformational changes in the immunogen that could affect the qualitative form of the immune response.

Exemplary adjuvants include 3De-O-acylated monophosphoryl lipid A (MPL ^™ ) (see GB 2220211 (GSK)); aluminium hydroxide gels such as Alhydrogel ^™ (Brenntag Biosector, Denmark); aluminium salts (such as aluminium hydroxide, aluminium phosphate, aluminium sulfate), which may be used in the presence or absence of immunostimulants such as MPL or 3-DMP, QS-21, polymeric or monomeric amino acids such as polyglutamic acid or polylysine.

Additional exemplary adjuvants are immunostimulatory oligonucleotides such as CpG oligonucleotides (see, e.g., WO 1998/040100, WO 2010/067262), or saponins and immunostimulatory oligonucleotides, such as CpG oligonucleotides (see, e.g., WO 00/062800). In a preferred embodiment, the adjuvant is a CpG oligonucleotide, most preferably a CpG oligodeoxynucleotide (CpG ODN). Preferred CpG ODNs are of the B class that preferentially activate B cells. For the present invention, the CpG ODN has the amino acid sequence 5' T*C*G*T*C*G*T*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3' (SEQ ID NO: 48) wherein * represents a phosphorothioate bond. A CpG ODN of this sequence is known as CpG 24555, which is described in WO 2010/067262. In a preferred embodiment, CpG 24555 is used together with an aluminum hydroxide salt such as Alhydrogel.

Another exemplary class of adjuvants includes saponin adjuvants, such as Stimulon ^™ (QS-21, which is a triterpene glycoside or saponin, Aquila, Framingham, Mass.) or particles derived therefrom, such as ISCOMs (immunostimulatory complexes) and adjuvants. Thus, the compositions of the present invention can be provided in the form of ISCOMs, ISCOMS containing CTB, liposomes, or encapsulated in compounds such as acrylates or poly (DL-lactide-co-glycosides) to form microspheres of a size suitable for adsorption. Generally, the term "ISCOM" refers to an immunogenic complex formed between glycosides, such as triterpene saponins (particularly Quil A), and an antigen containing a hydrophobic region. In a preferred embodiment, the adjuvant is ISCOMATRIX adjuvant.

Other exemplary adjuvants include RC-529, GM-CSF, and complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA).

Another exemplary class of adjuvants are glycolipid analogs including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which has an amino acid substitution in the sugar residue.

Optionally, the pharmaceutical composition includes two or more different adjuvants. Preferred adjuvant combinations include, for example, any adjuvant combination containing at least two of the following adjuvants: alum, MPL, QS-21, ISCOMATRIX, CpG, and alcolhol. Exemplary adjuvant combinations include a combination of CpG and alcolhol.

Alternatively, in one embodiment, the composition is administered to a mammal without an adjuvant.

The compositions described herein can be administered by any route of administration, such as, for example, parenteral, topical, intravenous, mucosal, oral, subcutaneous, intraarterial, intracranial, intradural, intraperitoneal, intranasal, intramuscular, intradermal, infusion, rectal, and/or transdermal routes for prophylactic and/or therapeutic applications. In preferred embodiments, the route of administration of the compositions is parenteral, more preferably intramuscular. Typical intramuscular administration is performed in the muscles of the arm or leg.

The compositions described herein can be administered in combination with other therapies that are at least partially effective in preventing and/or treating C. difficile infection. For example, the compositions of the present invention can be administered prior to, concurrently with, or after: biological therapy; probiotic therapy; anal implants; immunotherapy (e.g., intravenous immunoglobulin); and/or accepted standard of care antibiotic treatment for C. difficile-associated disease (CDAD), such as metronidazole and/or vancomycin.

The compositions of the present invention involving toxins A and B can be administered to a mammal in any combination. For example, an immunogenic composition comprising mutant C. difficile TcdA can be administered before, simultaneously with, or after an immunogenic composition comprising mutant C. difficile TcdB. Conversely, an immunogenic composition comprising mutant C. difficile TcdB can be administered before, simultaneously with, or after an immunogenic composition comprising mutant C. difficile TcdA.

In another embodiment, an immunogenic composition comprising an anti-toxin A antibody or binding fragment thereof can be administered before, concurrently with, or after an immunogenic composition comprising an anti-toxin B antibody or binding fragment thereof. Conversely, an immunogenic composition comprising an anti-toxin B antibody or binding fragment thereof can be administered before, concurrently with, or after an immunogenic composition comprising an anti-toxin A antibody or binding fragment thereof.

In other embodiments, the compositions of the present invention can be administered before, simultaneously with, or after the pharmaceutically acceptable carrier. For example, an adjuvant can be administered before, simultaneously with, or after the composition comprising a mutant Clostridium difficile toxin. Thus, the compositions of the present invention and the pharmaceutically acceptable carrier can be packaged in the same bottle, or they can be packaged in separate bottles and mixed prior to use. The compositions can be formulated for single and/or multiple dose administration.

Methods for protecting against and/or treating Clostridium difficile infection in mammals

In one aspect, the present invention relates to methods for inducing an immune response against a Clostridium difficile toxin in a mammal. The methods comprise administering to the mammal an effective amount of a composition described herein. For example, the methods can comprise administering an effective amount to generate an immune response against a respective Clostridium difficile toxin in the mammal.

In an exemplary embodiment, the present invention relates to a method for inducing an immune response against C. difficile TcdA in a mammal. The method comprises administering to the mammal an effective amount of an immunogenic composition comprising mutant C. difficile TcdA. In another exemplary embodiment, the present invention relates to a method for inducing an immune response against C. difficile TcdB in a mammal. The method comprises administering to the mammal an effective amount of an immunogenic composition comprising mutant C. difficile TcdB.

In other embodiments, the method comprises administering to a mammal an effective amount of an immunogenic composition comprising mutant C. difficile TcdA and an effective amount of an immunogenic composition comprising mutant C. difficile TcdB. In other aspects, the compositions described herein can be used to treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of C. difficile infection, C. difficile associated disease (CDAD), syndrome, condition, symptom, and/or complications thereof in a mammal compared to a mammal not administered the composition. The method comprises administering to a mammal an effective amount of the composition.

Three clinical syndromes caused by C. difficile infection are recognized based on the severity of the infection. The most severe form is pseudomembranous colitis (PMC), which is characterized by profuse diarrhea, abdominal pain, signs of systemic illness, and a distinctive endoscopic appearance of the colon.

Antibiotic-associated colitis (AAC) is also characterized by vomiting, abdominal pain and tenderness, systemic signs (e.g., fever), and leukocytosis. Intestinal damage in AAC is less severe than in PMC, in which characteristic endoscopic appearance of the colon is absent, and mortality is low.

Finally, antibiotic-associated diarrhea (AAD, also known as Clostridium difficile-associated diarrhea (CDAD)) is a relatively mild syndrome characterized by mild to moderate diarrhea, the absence of large bowel inflammation (characterized, for example, by abdominal pain and tenderness), and systemic signs of infection (e.g., fever).

These three different devices typically occur with increasing frequency. That is, PMC typically occurs less frequently than AAC, while AAD is generally the most common clinical manifestation of C. difficile disease.

A common complication of C. difficile infection is recurrent or relapsing disease, which occurs in up to 20% of all patients who recover from C. difficile disease. Relapses are clinically characterized by AAD, AAC, or PMC. Patients who relapse once are more likely to relapse again.

As used herein, conditions of C. difficile infection include, for example, mild, mild to moderate, moderate, and severe C. difficile infection. Conditions of C. difficile infection can vary depending on the manifestation and/or severity of symptoms of the infection.

Symptoms of C. difficile infection can include physiological, biochemical, histological, and/or behavioral symptoms, such as, for example, diarrhea; colitis; spastic colitis, fever, fecal leukocytosis, and inflammation on colon biopsy; pseudomembranous colitis; hypoalbuminemia; generalized edema; leukocytosis; sepsis; abdominal pain; asymptomatic carriage; and/or complications and intermediate pathological phenotypes that occur during the course of infection, as well as combinations thereof. Thus, for example, administration of an effective amount of a composition described herein can, for example, treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of diarrhea; abdominal pain, cramping, fever, inflammation on colon biopsy, hypoalbuminemia, generalized edema, leukocytosis, sepsis, and/or asymptomatic carriage (compared to a mammal not administered the composition).

Risk factors for C. difficile infection may include, for example, current or imminent use of antimicrobial agents (encompassing any antimicrobial substance with an antimicrobial spectrum and/or activity against anaerobic bacteria, including, for example, vitamins that disrupt normal colonic microflora, such as clindamycin, cephalosporins, metronidazole, vancomycin, fluoroquinolones (including levofloxacin, moxifloxacin, gatifloxacin, and ciprofloxacin), linezolid, etc.); current or imminent discontinuation of prescribed metronidazole or vancomycin; current or imminent contact with a healthcare facility (e.g., hospital, long-term care facility, etc.) and healthcare staff; current or imminent treatment with proton pump inhibitors, H2 antagonists, and/or methotrexate, or a combination thereof; current or risk of gastrointestinal disease, such as inflammatory bowel disease; previous, current, or imminent gastrointestinal surgery or gastrointestinal treatment of a mammal; previous C. difficile infection and/or CDAD. Patients who have had or are currently experiencing recurrent, e.g., one or more, Clostridium difficile infections and/or CDAD; and humans who are at least about 65 years of age or older.

In the methods described herein, the mammal can be any mammal, such as, for example, a mouse, a hamster, a primate, and a human. In a preferred embodiment, the mammal is a human. According to the present invention, the human can include individuals who have previously exhibited a C. difficile infection, a C. difficile-associated disease, syndrome, condition, symptom, and/or complications thereof; individuals who are currently exhibiting a C. difficile infection, a C. difficile-associated disease, syndrome, condition, symptom, and/or complications thereof; and individuals who are at risk of a C. difficile infection, a C. difficile-associated disease, syndrome, condition, symptom, and/or complications thereof.

Examples of individuals showing symptoms of C. difficile infection include individuals who have shown or are showing the symptoms described above; individuals who have had or are currently suffering from C. difficile infection and/or C. difficile-associated disease (CDAD); and individuals who have had recurrent C. difficile infection and/or CDAD.

Examples of patients at risk for C. difficile infection include individuals at risk for planned antimicrobial use or individuals who are undergoing planned antimicrobial use; individuals at risk for discontinuation of prescribed metronidazole or vancomycin or individuals who are at risk for discontinuation of prescribed metronidazole or vancomycin; individuals at risk for planned exposure to a nursing facility (e.g., a hospital, long-term care facility, etc.) or nursing staff or individuals who are exposed to a nursing facility or nursing staff; and/or individuals at risk for planned treatment with or individuals who are undergoing treatment with: proton pump inhibitors, H2 antagonists, and/or methotrexate, or a combination thereof; individuals who have had or are undergoing gastrointestinal diseases such as inflammatory bowel disease; individuals who have undergone or are undergoing gastrointestinal surgery or gastrointestinal treatment; and individuals who have had or are experiencing a recurrence of C. difficile infection and/or CDAD, for example, patients who have had one or more C. difficile infection and/or CDAD; individuals who are approximately 65 years of age or older. Such at-risk patients may or may not currently be showing symptoms of C. difficile infection.

In asymptomatic patients, prevention and/or treatment can be initiated at any age (e.g., at about 10, 20, or 30 years of age). However, in one embodiment, treatment is not necessarily initiated until the patient reaches about 45, 55, 65, 75, or 85 years of age. For example, the compositions described herein can be administered to asymptomatic people aged 50-85 years.

In one embodiment, a method for treating, preventing, reducing the risk of, reducing the occurrence of, reducing the severity of, and/or delaying the onset of a Clostridium difficile infection, a Clostridium difficile-associated disease, syndrome, condition, symptom, and/or its complications in a mammal comprises administering to a mammal in need thereof, a mammal at risk of a Clostridium difficile infection, and/or a mammal suspected of having a Clostridium difficile infection, an effective amount of a composition described herein. An effective amount includes, for example, an amount sufficient to treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of a Clostridium difficile infection, a Clostridium difficile-associated disease, syndrome, condition, symptom, and/or its complications, as compared to a mammal not administered the composition. Administration of an effective amount of a composition of the present invention can, for example, treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of diarrhea, abdominal pain, cramping, fever, inflammation on colon biopsy, hypoalbuminemia, generalized edema, leukocytosis, sepsis, and/or asymptomatic carriers (compared to a mammal not administered the composition). In a preferred embodiment, the method comprises administering an effective amount of an immunogenic composition described herein to a mammal in need thereof, a mammal at risk for C. difficile infection, and/or a mammal suspected of having C. difficile infection.

In another embodiment, a method for treating, preventing, reducing the risk of, reducing the occurrence of, reducing the severity of, and/or delaying the onset of a C. difficile infection, a C. difficile-associated disease, a syndrome, a condition, a symptom, and/or its complications in a mammal comprises administering to a mammal suspected of having or experiencing a C. difficile infection an effective amount of a composition described herein. An effective amount includes, for example, an amount sufficient to treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of a C. difficile infection, a C. difficile-associated disease, a syndrome, a condition, a symptom, and/or its complications as compared to a mammal not administered the composition.

Administration of an effective amount of the composition can improve at least one sign or symptom of a C. difficile infection in a subject, such as those described below. Administration of an effective amount of a composition described herein can, for example, reduce the severity and/or reduce the recurrence of diarrhea; reduce the severity and/or reduce the recurrence of abdominal pain, cramping, fever, inflammation on colon biopsy, hypoalbuminemia, generalized edema, leukocytosis, sepsis, and/or asymptomatic carriers, etc., compared to a mammal not administered the composition. Optionally, the presence of symptoms, signs, and/or risk factors for infection is determined prior to treatment. In a preferred embodiment, the method comprises administering an effective amount of an antibody and/or binding fragment thereof described herein to a mammal suspected of having or experiencing a C. difficile infection.

Thus, an effective amount of a composition is an amount sufficient to achieve the desired effect (e.g., a prophylactic and/or therapeutic effect) in the methods of the present invention. For example, the amount of immunogen administered can vary from a minimum of about 1 μg, 5 μg, 25 μg, 50 μg, 75 μg, 100 μg, 200 μg, 500 μg, or 1 mg per injection to a maximum of about 2 mg, 1 mg, 500 μg, or 200 μg. Any minimum value can be combined with any maximum value to define a suitable range. Typically, about 10, 20, 50, or 100 μg of each immunogen is used per human injection.

The amount of the composition of the present invention administered to a subject may depend on the severity of the infection and/or individual characteristics, such as overall health, age, sex, weight, and tolerance to drugs. It may also depend on the extent, severity, and type of the disease. The effective amount may also vary depending on factors such as the route of administration, the target site, the patient's physiological state, the patient's age, whether the patient is human or animal, other therapies being administered, and whether the treatment is prophylactic or therapeutic. Those skilled in the art will be able to determine an appropriate amount based on these and other factors.

An effective amount may include one effective dose or multiple effective doses (e.g., 2, 3, 4 doses or more) for use in the methods herein. The effective dose may need to be increased gradually to optimize safety and efficacy.

A combination of an amount and a frequency of administration sufficient to achieve prophylactic and/or therapeutic use is defined as a prophylactically effective or therapeutically effective regimen. In a prophylactic and/or therapeutic regimen, the composition is typically administered in more than one dose until a sufficient immune response is achieved. Typically, the immune response is monitored and repeated doses are administered if the immune response diminishes.

The composition can be administered in multiple doses over time. Treatment can be monitored by measuring the antibody, or activated T-cell or B-cell response to the therapeutic agent (e.g., an immunogenic composition comprising a mutant C. difficile toxin) over time. If the response decreases, a booster dose is indicated.

Example

Example 1: Identification of Toxin-Negative Clostridium difficile Strains

To identify C. difficile strains lacking toxin (A and B) genes and expressing toxins, 13 C. difficile strains were tested. Culture media from the 13 C. difficile strains were tested for toxin A by ELISA. Seven strains expressed toxin A: C. difficile 14797-2, C. difficile 630, C. difficile BDMS, C. difficile W1194, C. difficile 870, C. difficile 1253, and C. difficile 2149. See Figure 3.

Six strains do not express toxin A and lack the entire pathogenicity locus: C. difficile 1351 (ATCC 43593 ^™ ), C. difficile 3232 (ATCC BAA-1801 ^™ ), C. difficile 7322 (ATCC 43601 ^™ ), C. difficile 5036 (ATCC 43603 ^™ ), C. difficile 4811 (ATCC 3602 ^™ ), and C. difficile VPI 11186 (ATCC 700057 ^™ ). VPI 11186 was selected based on its efficiency in taking up plasmid DNA by conjugation.

The same 13 strains were tested in a multiplex PCR assay using primers outside the pathogenicity locus (PaLoc; Braun et al., Gene. 1996 Nov 28; 181(1-2): 29-38. PCR results showed that DNA from the six toxin A-negative strains (by ELISA) did not amplify any genes from PaLoc (tcdA-tcdE). PaLoc flanking sequences (cdd3 and cdu2) were present (data not shown).

Example 2: Inactivation of the sporulation pathway in Clostridium difficile VPI 11186

Knocking out sporulation in a C. difficile production strain facilitates large-scale fermentation in a safe production environment. The ClosTron system was used to create a non-spore-forming C. difficile strain. See Heap et al., J Microbiol Methods. 2009 Jul;78(1):79-85. The ClosTron system was used to inactivate the target gene by site-directed insertional inactivation of the group II intron of the spo0A1 Clostridial gene. Using this ClosTron technology, sporulation was inactivated in the toxin-negative strain VPI11186. Erythromycin-resistant mutants were selected and the presence of the insertion cassette was confirmed by PCR (not shown). Loss of sporulation ability was confirmed in two independent clones.

Example 3: Genetic modification of toxin A and B genes to inactivate cytotoxic function

Full-length mutant toxin A and B open reading frames (ORFs) based on the strain 630Δ genomic sequence were designed for synthesis at Blue Heron Biotech. See, e.g., SEQ ID NOs: 9-14. The active site of the glucosyltransferase activity responsible for cytotoxicity was altered by two allelic substitutions: D285A/D287A for toxin A (see SEQ ID NO: 3) and D286A/D288A for toxin B (see SEQ ID NO: 5). Two nucleotides were mutated at each aspartate (D) codon to create an alanine (A) codon. See, e.g., SEQ ID NOs: 9-14. In addition, a pair of vectors expressing mutant toxins lacking cysteine residues were constructed by custom synthesis at Blue Heron Biotech. Seven cysteine residues from mutant toxin A and nine cysteine residues from mutant toxin B were replaced with alanine. This substitution included the catalytic cysteines of the A and B toxin autocatalytic proteases. Additionally, silent mutations were introduced where necessary to remove restriction enzyme sites used for vector construction.

Example 4: pMTL84121fdx expression vector

The plasmid shuttle vector used for expression of C. difficile mutant toxin antigens was selected from the pMTL8000-series regulatory system developed by the Minton lab (see Heap et al., J Microbiol Methods. 2009 Jul; 78(1): 79-85). The selected vector, pMTL84121fdx, contains the C. difficile plasmid pCD6 Gram+ replicon, the catP (chloramphenicol/thiamphenicol) selection marker, the p15a Gram-replicon and tra function, as well as the C. sporogenes ferredoxin promoter (fdx) and a distal multiple cloning site (MCS). Empirical data indicate that the low-copy number p15a replicon confers greater stability in E. coli compared to ColE1 selection. The fdx promoter was chosen because it gave higher expression compared to other promoters tested with the CAT reporter construct, such as tcdA, tcdB; or heterologous tetR or xylR (data not shown).

Example 5: Cloning of modified toxin ORF into pMTL84121fdx

The full-length mutant toxin A and B open reading frames (ORFs) based on the strain 630Δ genomic sequence were subcloned using standard molecular biology techniques using the pMTL84121fdx vector's multiple cloning NdeI and BglII sites. To facilitate cloning, the ORFs were flanked by an NdeI site proximal to the start codon and a BglII site immediately downstream of the stop codon.

Example 6: Site-directed mutagenesis of TcdA to create a triple mutant

The catalytic cysteine residues of the autocatalytic protease domain were substituted in SEQ ID NOs: 3 and 5 (i.e., in each "double mutant") (i.e., C700A for TcdA and C698A for TcdB). For mutagenesis of mutant toxin A, a 2.48 kb NdeI-HindIII fragment from the TcdA D285A/D287A expression plasmid was subcloned into pUC19 (using the same enzyme digestion) and site-directed mutagenesis was performed on this template. Once the new alleles were confirmed by DNA sequence analysis, the modified NdeI-HindIII fragment was reintroduced into the expression vector pMTL84121fdx to create the "triple mutants," i.e., SEQ ID NOs: 4 and 6.

Example 7: Site-directed mutagenesis of TcdB to create a triple mutant

For the mutagenesis of mutant toxin B, a 3.29 kb NdeI-EcoNI fragment from the mutant toxin B plasmid was modified and reintroduced. Due to the absence of an EcoNI site in available cloning vectors, a slightly larger 3.5 kb NdeI-EcoRV fragment was subcloned into pUC19 (prepared with NdeI-SmaI). Following mutagenesis, the modified internal 3.3 kb NdeI-EcoNI fragment was excised and used to replace the corresponding mutant toxin B expression vector, pMTL84121fdx. Since this targeted strategy was found to have low cloning efficiency, an alternative strategy for introducing the C698A allele (involving the replacement of the 1.5 kb DraIII) was also attempted. Both strategies independently produced the desired recombinants.

Example 8: Creation of additional mutant toxin variants by site-directed mutagenesis

At least 12 different mutant C. difficile toxin variants were constructed. Allelic substitutions were introduced into the N-terminal mutant toxin gene fragment by site-directed mutagenesis (kit). The recombinant toxin was also engineered as a reference control to evaluate the ability of this plasmid-based system to produce proteins with biological activity quantitatively equivalent to the native toxin purified from a wild-type C. difficile strain. In this case, the allelic substitution was introduced to restore the original glucosyltransferase substitution. In addition, a pair of cysteine-free mutant toxin vectors were constructed by custom synthesis at Blue Heron Biotech.

The 12 toxin variants include: (1) a mutant Clostridium difficile toxin A with a D285A/D287A mutation (SEQ ID NO: 3); (2) a mutant Clostridium difficile toxin B with a D286A/D288A mutation (SEQ ID NO: 5); (3) a mutant Clostridium difficile toxin A with a D285A/D287A C700A mutation (SEQ ID NO: 4); (4) a mutant Clostridium difficile toxin B with a D286A/D288A C698A mutation (SEQ ID NO: 6); (5) a recombinant toxin A with SEQ ID NO: 1; (6) a recombinant toxin B with SEQ ID NO: 2; (7) a mutant Clostridium difficile toxin A with a C700A mutation; (8) a mutant Clostridium difficile toxin B with a C698A mutation; (9) a mutant Clostridium difficile toxin B with a C700A mutation; difficile toxin A having C1169S, C1407S, C1623S, C2023S, and C2236S mutations; (10) a mutant C. difficile toxin B having C698A, C395S, C595S, C824S, C870S, C1167S, C1625S, C1687S, and C2232S mutations; (11) a mutant C. difficile toxin A having D285A, D287A, C700A, D269A, R272A, E460A, and R462A mutations (SEQ ID NO: 7); and (12) a mutant C. difficile toxin B having D270A, R273A, D286A, D288A, Mutant Clostridium difficile toxin B with D461A, K463A, and C698A mutations (SEQ ID NO: 8).

Example 9: Stability of transformants

The re-arranged plasmid was obtained with a general DH5 E. coli laboratory strain. On the contrary, after growing for 3 days on LB chloramphenicol (25 μg/ml) plates at 30°C, a slowly growing full-length mutant toxin recombinant was produced using the transformation of the Invitrogen Stbl2 ^™ E. coli host. Lower cloning efficiency was obtained with the relevant Stbl3 ^™ and Stbl4 ^™ E. coli strains, although it was found that these strains were stable for maintaining the plasmid. Subsequently, the transformant was selected in agar or liquid culture using the chloramphenicol at 30°C. It was also found that the use of LB (Miller's) substratum improved the recovery and growth of the transformant (compared to the substratum based on animal-free tryptone soy).

Example 10: Transformation of Clostridium difficile with pMTL84121fdx encoding wild-type or genetically mutant toxin genes

Transformation of C. difficile using E. coli conjugative transfer was performed essentially as described in Heap et al., Journal of Microbiological Methods, 2009. 78(1): p. 79-85. pMTL84121fdx encoding wild-type or variant mutant toxin genes was used to transform the E. coli host CA434. The E. coli host CA434 was an intermediate for the integration of expression plasmids into the C. difficile production strain VPI 11186spo0A1. CA434 is a derivative of E. coli HB101. This strain carries the Tra+Mob+R702 conjugative plasmid, which confers resistance to Km, Tc, Su, Sm/Spe, and Hg (due to Tn1831). Chemically competent or electrocompetent CA434 cells were prepared, and expression vector transformants were selected on Miller's LB CAM plates at 30°C. Pick the slow-growing colonies that appear after 3 days and expand them to the mid-logarithmic phase (~24h, 225rpm, orbital shaker, 30°C) in 3 mL LB chloramphenicol culture. Collect the E. coli culture by low-speed (5,000g) centrifugation to avoid destroying the pili, and gently resuspend the cell pellet in 1 mL PBS with a large-bore pipette. Concentrate the cells with low-speed centrifugation. Remove most of the PBS by pouring, transfer the dried sediment to an anaerobic box and resuspend it with 0.2 mL of Clostridium difficile culture, point it onto a BHIS agar plate and grow it for 8h or overnight. In the case of mutant toxin A transformants, overnight mating achieves better results. Scrape the cell mass into 0.5 mL PBS and plate 0.1 mL onto BHIS selection medium, which has been supplemented with 15 μg/mL thiamphenicol (a more powerful chloramphenicol analogue) and D-cycloserine/cefoxitin to kill the E. coli donor cells. The transformant that occurs after purifying 16-24h by re-streaking onto new BHIS (adding supplement) flat board, and test the expression of recombinant toxin or mutant toxin in subsequent culture. Prepare glycerol long-term preservation solution (glycerol permanent) and reserve seed from the clone that shows good expression. Also prepare a small amount of plasmid from 2mL culture, wherein used the Qiagen test kit step (wherein with lysozyme (optional) pretreatment cell) of modification. Described Clostridium difficile is prepared in a small amount DNA as template for PCR sequencing to verify clone integrity. Alternatively, prepare plasmid a large amount of DNA and order-checking from intestinal bacteria Stbl2 ^TM transformant.

Example 11: Analysis of Clostridium difficile Expression of Toxin A and B Triple Mutants (SEQ ID NOs: 4 and 6, respectively) and a Sevenfold B Mutant (SEQ ID NO: 8)

Transformants were grown in 2 mL cultures (for routine analysis) or in vented stopper flasks (for time course experiments). Samples (2 mL) were briefly centrifuged (10,000 rpm, 30 s) to concentrate the cells: the supernatant was discarded and concentrated 10x (Amicon-ultra 30k); the pellet was decanted and frozen at -80°C. The cell pellet was thawed on ice, resuspended in 1 mL of hydrolysis buffer (Tris-HCl pH 7.5; 1 mM EDTA, 15% glycerol), and sonicated (1 x 20 s with a microtip). The hydrolyzate was centrifuged at 4°C, and the supernatant was concentrated 5-fold. Samples of the supernatant and hydrolyzate were combined with sample buffer and heat-treated (10 min, 80°C), then loaded onto duplicate 3-8% Tris-acetate SDS-PAGE gels (Invitrogen). One gel was stained with Coomassie Brilliant Blue, and the second gel was electroblotted for Western analysis. Toxin A-specific and toxin B-specific rabbit antisera (Fitgerald; Biodesign) were used to detect mutant toxin A and B variants. Expression of the seven-fold mutant toxin B (SEQ ID NO: 8) was also confirmed by Western blot hybridization.

Example 12: Elimination of Mutant Toxin Glucosyltransferase Activity

Genetic double mutant (DM) toxins A and B (SEQ ID NOs: 3 and 5, respectively) and triple mutant (TM) toxins A and B (SEQ ID NOs: 4 and 6, respectively) did not transfer ¹⁴ C-glucose to ¹⁰ μg of RhoA, Rac1, and Cdc42 GTPase in an in vitro glucose mobilization assay (in the presence of UDP- 14 C-glucose [30 μM], 50 mM HEPES, pH 7.2, 100 mM KCl, 4 mM MgCl ₂ , 2 mM MnCl ₂ , 1 mM DTT, and 0.1 μg/μL BSA). However, wild-type A and B toxin controls (having SEQ ID NOs: 1 and 2, respectively) each efficiently transferred ¹⁴ C-glucose to the GTPase at low doses of 10 and 1 ng (and lower doses - data not shown) (Figures 4A and 4B), even in the presence of 100 μg of the mutant toxin (Figure 4B), representing at least a 100,000-fold reduction compared to the respective wild-type toxins. Similar results were detected for Cdc42 GTPase (data not shown).

Specifically, in Fig. 4B, wild-type toxin A and toxin B (1 ng) or triple mutant toxin A and triple mutant toxin B (100 μg) were incubated with hoA GTPase at 30°C for 2 hours in the presence of UDP- ¹⁴ C-glucose. As shown, 1 ng of wild-type TcdA and TcdB transferred ¹⁴ C-glucose to RhoA, but 100 μg of triple mutant toxin A and triple mutant toxin B did not. When 1 ng of wild-type TcdA or TcdB was added to the respective reactions containing 100 μg of triple mutant toxin A or triple mutant toxin B, RhoA glucosylation was detected, indicating the absence of a glucosylation inhibitor. The sensitivity of the glucosylation activity assay was estimated to be 1 ng of wild-type toxin in a background of 100 μg of mutant toxins (a 1:100,000 ratio). The results show that mutations in the glucosyltransferase active site of triple mutant toxin A and triple mutant toxin B reduced any measurable (less than 100,000-fold activity compared to the activity of the respective wild-type toxins) glucosyltransferase activity. A similar assay was also developed to characterize glucosyltransferase activity by TCA precipitation of glucosylated GTPases.

Example 13: Elimination of autocatalytic cysteine protease activity

When the cysteine protease cleavage site is mutated, the autocatalytic cleavage function is abolished in triple genetic mutants A and (TM) (SEQ ID NOs: 4 and 6, respectively). As shown in Figure 5, wild-type (wt) toxins A and B (SEQ ID NOs: 3 and 5, respectively) are cleaved in the presence of inositol-6-phosphate. Double mutant toxins A and B (SEQ ID NOs: 3 and 5, respectively) are also cleaved in the presence of inositol-6-phosphate (data not shown), similar to the wild-type. Toxin A (SEQ ID NO: 3) is cleaved from 308 kDa into two fragments of 245 and 60 kDa. Toxin B (SEQ ID NO: 5) is cleaved from 270 kDa into two fragments of 207 and 63 kDa. Triple genetic mutants A and B (TM) (SEQ ID NOs: 4 and 6, respectively) remain unaffected at 308 and 270 kDa, respectively, even in the presence of inositol-6-phosphate. See Figure 5. Thus, the genetic modification inactivates cysteine protease activity.

More specifically, in Figure 5 , 1 μg of triple mutant A and triple mutant B were incubated in parallel with wild-type TcdA and TcdB (available from List Biologicals) at room temperature (21 ± 5°C) for 90 minutes. Cleavage reactions were performed in a 20 μL volume of Tris-HCl, pH 7.5, 2 mM DTT in the presence or absence of inositol-6-phosphate (10 mM TcdA and 0.1 mM TcdB). The entire reaction volume was then loaded onto a 3-8% SDS/PAGE; protein bands were visualized by silver staining. As shown, in the presence of inositol-6-phosphate, wt Tcd A and cdB were cleaved into two protein bands, 245 kD and 60 kD, and 207 kD and 63 kD, respectively. The triple mutant toxin A and triple mutant toxin B were not cleaved, confirming that the C700A mutation in triple mutant toxin A and the C698A mutation in triple mutant toxin B blocked cleavage.

Example 14: Residual cytotoxicity of triple mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively)

The cytotoxicity of the genetically modified toxins was assessed in an in vitro cytotoxicity assay (IMR90 cells, a human diploid lung fibroblast cell line). These cells were sensitive to both toxins A and B. As an additional preferred embodiment, Vero normal kidney cells from African green monkeys (Cercopithecus aethiops) can be used in the cytotoxicity assay as they have been observed to be reasonably sensitive to toxins A and B. Preferably, HT-29 human colorectal adenocarcinoma cells are not used in the cytotoxicity assay as they show significantly reduced sensitivity to the toxins (compared to Ver and IMR90 cell lines). See, for example, Table 6 below.

Serial dilutions of genetic mutant toxin or wt toxin samples were added to the cell monolayer cultures grown in 96-well tissue culture plates. After 72 h of incubation at 37°C, the metabolically active cells of the plates were assessed by measuring cellular ATP levels, which were measured by adding luciferase-based reagents (Promega, Madison, WI) to produce fluorescence expressed as relative light units (RLU). High RLU showed that cells were alive, while low RLU showed that cells were metabolically inactive and fast-dying. The level of cytotoxicity (expressed as EC ₅₀ ) was defined as the amount of wt toxin or genetic mutant toxin that caused 50% RLU reduction (compared to the level in the cell culture control) (the details of this assay are described below). As shown in Figure 6, Table 7A, and Table 8A, the EC ₅₀ values for TcdA and TcdB were approximately 0.92 ng/mL and 0.009 ng/mL, respectively. _EC50 values for triple mutant toxin A and triple mutant toxin B were approximately 8600 ng/mL and 74 ng/mL, respectively. Despite an approximately 10,000-fold reduction in cytotoxicity relative to the wt toxin, both genetic mutant toxins demonstrated low levels of residual cytotoxicity. This residual cytotoxicity could be blocked by neutralizing antitoxin monoclonal antibodies, indicating that it was specific for the triple mutant toxins but was unlikely to be related to the enzymatic activity of the known wt toxin enzymes (glucosidase or autoproteolysis).

Both wt toxins exhibit potent in vitro cytotoxicity, with small amounts sufficient to cause a variety of effects on mammalian cells, such as cell rounding (cytopathic effect or CPE) and lack of metabolic activity (measured by ATP levels). Therefore, two in vitro assays (CPE or cell rounding assay and ATP assay) were developed to verify that no residual cytotoxicity remained in the mutant toxin drug substance. Results are expressed as _EC50 , which is the amount of toxin or mutant toxin that causes 1) 50% of the cells to develop CPE or 2) a 50% reduction in ATP levels (measured as relative light units).

In the CPE assay, samples of the drug substance are serially diluted and incubated with IMR90 cells to observe their potential cytopathic effects. The CPE assay is scored on a scale of 0 (normal cells) to 4 (~100% cell rounding), with a score of 2 (~50% cell rounding) defining the _EC50 value for the sample tested. This method was used to test the mutant toxin drug substance at a concentration of 1000 μg/mL, which is the maximum tolerable concentration that can be tested in this assay without matrix interference. Therefore, no detectable cytotoxicity is reported as an _EC50 > 1000 μg/ml.

The ATP assay is based on the measurement of the amount of fluorescent signal generated from ATP, which is proportional to the amount of metabolically active cells. The maximum tolerable concentration that can be tested in this assay (without assay interference) is approximately 200 μg/mL. Therefore, no detectable cytotoxicity is reported as _EC50 >200 μg/mL.

Different concentrations of mutant toxins A and B were added to cells in parallel (with toxin control). The endpoint of the assay was cell viability, as determined by cellular ATP levels (using Promega). The degree of fluorescence is proportional to the ATP level or the number of viable cells.

The in vitro cytotoxicity ( _EC50 ) of wild-type (wt) toxin A was 920 pg/mL, while that of toxin B was 9 pg/mL. The in vitro cytotoxicity ( _EC50 ) of mutant toxin A (SEQ ID NO: 4) was 8600 ng/mL, while that of mutant toxin B (SEQ ID NO: 6) was 74 ng/mL. Although these values represent 9348- and 8222-fold reductions, respectively, residual cytotoxicity was detected in both mutant toxins.

In other words, in an in vitro cytotoxicity assay in IMR-90 cells, the cytotoxicity of triple mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively) was significantly reduced relative to the cytotoxicity of wt toxins A and B (SEQ ID NOs: 1 and 2, respectively). As shown in Figure 6, although both triple mutant toxins exhibited a significant reduction in cytotoxicity relative to wt toxins ( ^104- fold), residual cytotoxicity was observed at higher concentrations for both triple mutant toxins.

Additionally, the residual cytotoxicity of each triple mutant toxin can be completely neutralized by toxin-specific antibodies (e.g., at least a 6-8 _logio reduction in toxicity relative to wild-type toxin toxicity). See Example 16, below.

Cell culture assays are more sensitive for detecting cytotoxicity than in vivo animal models. When delivered to a mouse lethal challenge model by the ip or iv route, wtTcdA has an _LD50 of ~50 ng/mouse, while wtTcdB is more potent with an _LD50 of ~5 ng/mouse. In contrast, the cell culture-based in vitro assay described above has _EC50 values of 100 pg/well (wtTcdA) and 2 pg/well (wtTcdB).

Example 15: Residual Cytotoxicity of Genetic Seven-fold Mutant Toxin B (SEQ ID NO: 8)

As shown in Figure 7, the _EC50 values of the triple mutant toxin B (SEQ ID NO: 6) (20.78 ng/mL) and the seven-fold mutant toxin B (SEQ ID NO: 8) (35.9 ng/mL) mutants were similar, indicating that the addition of four additional mutations to further modify the glucosyltransferase active site and GTPase substrate binding site did not further reduce the cytotoxicity of the genetic mutant toxins. The _EC50 value of the double mutant toxin B (SEQ ID NO: 5) was also similar to that of the triple and seven-fold mutant toxins (data not shown). This observation suggests that the cytotoxicity of the mutant toxins is surprisingly independent of the glucosyltransferase and substrate recognition mechanisms.

Example 16: Residual cytotoxicity of triple mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively)

To further assess the nature of the residual cytotoxicity, the mutant toxins (SEQ ID NOs: 4 and 6) were mixed and incubated with their respective neutralizing antibodies, and the mixture was added to IMR90 cell monolayers.

The results (Figure 8) show that the residual cytotoxicity of mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively) was completely eliminated by neutralizing antibodies specific for mutant toxins A (upper panel, Figure 8) and B (lower panel, Figure 8). Increasing concentrations of mutant toxins A (upper panel) and B (lower panel) were incubated with rabbit antitoxin monoclonal (pAb, 1:10 dilution) or mouse monoclonal antibody (1:50 dilution from a stock solution containing 3.0 mg IgG/mL) and then added to IMR90 cells. After incubation with IMR90 cells at 37°C for 72 hours, substrate was added and relative light units (RLU) were measured in a photophotometer using the fluorescence program to measure ATP levels. Lower ATP levels indicate higher toxicity. Controls included Tcd and TcdB (4 times their corresponding _EC50 values).

Published reports indicate that mutations in the glucosyltransferase or autocatalytic protease domains of the toxins result in complete inactivation of toxicity. However, our data are inconsistent with these published reports, which may be due to the fact that our studies tested highly purified mutant toxins at increasing concentrations, while published reports used crude culture hydrolysates; that cell rounding in mutant toxin-treated cells was observed for an increased time point (e.g., 24 hours, 48 hours, 72 hours, or 96 hours), while published reports observed it for less than 12 hours; that our cytotoxicity assays used cell lines that exhibit significantly higher sensitivity to the toxins compared to the HT-29 human colorectal adenocarcinoma cells used in the published cytotoxicity assays; and/or that unknown activities or processes other than glucosylation may contribute to the residual toxicity of the mutant toxins.

Example 17: Novel Mechanism of Cytotoxicity of Genetic Mutant Toxins

To investigate the mechanism of residual cytotoxicity of the mutant toxins, IMR-90 cells were treated with either wt toxin B (SEQ ID NO: 2) or mutant toxin B (SEQ ID NO: 6), and the glucosylation of Rac1 GTPase was investigated over time. Samples were collected from 24 to 96 hours and cell extracts were prepared. Glucosylated Rac1 was distinguished from non-glucosylated Rac1 by Western blotting using two Rac1 antibodies. One antibody (23A8) recognized both forms of Rac1, while the other (102) recognized only non-glucosylated Rac1. As shown in Figure 22, for toxin B, total Rac1 levels did not change over time, and the majority of Rac1 was glucosylated. On the other hand, treatment with mutant toxin B (SEQ ID NO: 6) resulted in a significant decrease in total Rac1, however, this Rac1 remained non-glucosylated at all time points. This indicates that treatment with the mutant toxins, but not the wt toxin, negatively affects Rac1 levels. As shown in Figure 22, at the indicated time points, actin levels were similar in cells treated with toxin and genetic mutant toxin B, and similar to mock-treated cells. This suggests that the genetic mutant toxin exerts cytotoxicity through a different mechanism than the wild-type toxin, which drives the glucosidation pathway.

Example 18: Chemical treatment of genetic mutant toxins

While genetically modified mutant toxins showing a 4-log reduction in cytotoxic activity are preferred, further reductions in cytotoxic activity (2-4 log) are contemplated. Two chemical inactivation strategies were evaluated.

The first method uses formaldehyde and glycine to inactivate mutant toxins. Formaldehyde inactivation occurs through the formation of a Schiff base (imine) between formaldehyde and primary amines on the protein. The Schiff base can then react with many amino acid residues (Arg, His, Trp, Tyr, Gln, Asn) to form intramolecular or intermolecular crosslinks. This crosslinking fixes the structure of the protein, leading to its inactivation. In addition, formaldehyde can react with glycine to form a Schiff base. This glycyl Schiff base can then react with amino acid residues to form intermolecular protein-glycine crosslinks. Formaldehyde reduces the cytotoxic activity of the genetic mutant toxins to below the detectable limit (triple mutant B (SEQ ID NO: 6) cytotoxicity is reduced to >8 log ₁₀ , while triple mutant A (SEQ ID NO: 4) is >6 log ₁₀ ). However, when the formaldehyde-inactivated (FI) triple mutant toxins are incubated at 25°C, recovery is observed over time. Cytotoxicity reversion can be prevented by adding a small amount of formaldehyde (0.01-0.02%) to the FI-triple mutant toxin stock solution. See Example 23.

Another approach uses 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) treatment to generate inactivated mutant toxins. In this approach, EDC/NHS reacts with carboxyl groups on proteins to form activated esters. This activated ester can then react with primary amines on the protein to form stable amide bonds. As with the reaction with formaldehyde, this reaction results in both intramolecular and intermolecular crosslinks. The amide bonds formed by EDC/NHS treatment are more stable and irreversible than the fragile imine bonds formed by formalin inactivation. In addition to the crosslinks formed by the reaction of the activated ester with primary amines on the polypeptide, glycine and β-alanine adducts can also form. Without being bound by mechanism or theory, glycine adducts are produced when glycine is added to anneal unreacted activated esters. The amine of glycine reacts with the activated ester on the polypeptide to form a stable amide bond. Without being bound by mechanism or theory, the β-alanine adduct is formed by the reaction of activated β-alanine with a primary amine on the polypeptide. This reaction results in a stable amide bond. The activated β-alanine is produced by the reaction of 3 moles of NHS with 1 mole of EDC.

The mutant toxin of chemical inactivation should have the _EC50 value of >=1000ug/mL for realizing cytotoxic activity with respect to the reduction of 2-4log of the mutant toxin of genetic modification (with respect to 6-8log of natural toxin).Outside the reduction of cytotoxic activity, it will be favourable to retain the key epitope determined by dot blot analysis.At present, identified many reaction conditions, it satisfies the condition of reducing cytotoxicity and recognition epitope.Prepare the mutant toxin of several batches of inactivation for animal research, and show in Table 7A and 7B, Table 8A and 8B from the analytical data of several representative batches.

List = List Biologicals; CPE = cytopathic effect assay; _EC50 = lowest concentration at which 50% of cells show cytotoxicity; mAb = monoclonal antibody; neut = neutralizing; ND = not tested; TM = activation site and cleavage mutant ("triple mutant")

List = List Biologicals; CPE = cytopathic effect assay; _EC50 = lowest concentration at which 50% of cells show cytotoxicity; mAb = monoclonal antibody; neut = neutralizing; ND = not tested; TM = activation site and cleavage mutant ("triple mutant")

Example 19: Purification

At the end of fermentation, the fermentor was cooled. The cell suspension was recovered by continuous centrifugation and resuspended in a suitable buffer. Cell lysis of the cell suspension was achieved by high-pressure homogenization. For mutant toxin A, the homogenate was flocculent and subjected to continuous centrifugation. This solution was filtered and subsequently transferred to further processing. For mutant toxin B, the homogenate was clarified by continuous centrifugation and subsequently transferred to further processing.

Mutant toxin A (SEQ ID NO: 4) was purified using two chromatography steps followed by a final buffer change. The clarified lysate was loaded onto a hydrophobic interaction chromatography (HIC) column and the bound mutant toxin was eluted with a sodium citrate gradient. The product pool from the HIC column was then loaded onto a cation exchange (CEX) column and the bound mutant toxin was eluted with a sodium chloride gradient. The CEX pool containing the purified mutant toxin A was exchanged to the final buffer by diafiltration. The purified mutant toxin A was exchanged to the final drug substance intermediate buffer by diafiltration. Following diafiltration, the retentate was filtered through a 0.2 micron filter and then chemically inactivated to the final drug substance. The protein concentration was targeted to be 1-3 mg/mL.

Mutant toxin B (SEQ ID NO: 6) was purified using two chromatography steps followed by a final buffer change. The clarified lysate was loaded onto an anion exchange (AEX) column and the bound mutant toxin was eluted with a sodium chloride gradient. Sodium citrate was added to the product collection from the AEX column and loaded onto a hydrophobic interaction chromatography (HIC) column. The bound mutant toxin was eluted with a sodium citrate gradient. The HIC collection containing the purified mutant toxin polypeptide (SEQ ID NO: 6) was exchanged to the final buffer by diafiltration. The purified mutant toxin B was exchanged to the final drug substance intermediate buffer by diafiltration. Following diafiltration, the retentate was filtered through a 0.2 micron filter and then chemically inactivated to the final drug substance. The protein concentration was targeted to be 1-3 mg/mL.

Example 20: Formaldehyde/Glycine Inactivation

After purification, genetic mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively) are inactivated with 40 mM (1.2 mg/ml) formaldehyde at 25°C for 48 hours. The inactivation is performed in 10 mM phosphate, 150 mM sodium chloride buffer (containing 40 mM (3 mg/ml) glycine) at pH 7.0±0.5. The inactivation time is set to more than three times the time required to reduce the EC ₅₀ to 1000 ug/mL in IMR90 cells. After 48 hours, the biological activity is reduced by 7 to 8 log ₁₀ relative to the native toxin. After 48 hours of incubation, the inactivated mutant toxin is exchanged to the drug substance buffer by diafiltration. For example, using a 100 kD regenerated cellulose acetate ultrafiltration cartridge, the inactivated toxin is concentrated to 1-2 mg/mL and the buffer is exchanged.

Example 21: N-(3-dimethylaminopropyl)-N,-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS) inactivation

After purification, the genetic mutant toxins (SEQ ID NO: 4 and SEQ ID NO: 6) were inactivated for 2 hours at 25°C with 0.5 mg of EDC and 0.5 mg of NHS per mg of purified genetic mutant toxins A and B (approximately 2.6 mM and 4.4 mM, respectively). The reaction was inactivated by adding glycine to a final concentration of 100 mM and incubated for an additional 2 hours at 25°C. The inactivation was performed in 10 mM phosphate, 150 mM sodium chloride buffer at pH 7.0 ± 0.5. The inactivation time was set to more than three times the time required to reduce the EC ₅₀ to 1000 ug/mL in IMR90 cells. After 2 hours, the biological activity was reduced by 7 to 8 log ₁₀ relative to the native toxin. After 4 hours of incubation, the inactivated mutant toxins were exchanged to drug substance buffer by diafiltration. For example, using a 100 kD regenerated cellulose acetate ultrafiltration cartridge, concentrate the inactivated toxin to 1-2 mg/mL and exchange the buffer.

Unless otherwise indicated, the following terms used in the Examples section refer to compositions produced according to Example 21 of this specification: "EDC/NHS-treated triple mutant toxin", "EDC-inactivated mutant toxin", "mutant toxin [A/B] drug substance", "EI-mutant toxin", "EDC/NHS-triple mutant toxin". For example, the following terms are synonymous: "EDC/NHS-treated triple mutant toxin A", "EDC-inactivated mutant toxin A", "mutant toxin A drug substance", "EI-mutant toxin A", "EDC/NHS-triple mutant toxin A". As another example, the following terms are synonymous: "EDC/NHS-treated triple mutant toxin B"; "EDC-inactivated mutant toxin B"; "mutant toxin B drug substance"; "EI-mutant toxin B"; "EDC/NHS-triple mutant toxin B".

The mutant toxin A drug substance and the mutant toxin B drug substance are both prepared in a batch process comprising (1) fermentation of a toxin-negative Clostridium difficile strain (VPI 11186) containing a plasmid encoding each genetic triple mutant toxin polypeptide in a medium comprising soy hydrolysate, yeast extract HY YEST ^™ 412 (Sheffield Bioscience), glucose, and thiamphenicol, (2) purification of the genetic mutant toxins (the "drug substance intermediates") from the cell-free lysate to a purity of at least greater than 95% using ion exchange and hydrophobic interaction chromatography, (3) chemical inactivation by treatment with EDC/NHS followed by a glycine annealing/blocking reaction, and (4) exchange to a final buffer matrix.

Example 22: Studies supporting inactivation and formulation conditions

To optimize the chemical inactivation of the genetic mutant toxins, a statistical design of experiments (DOE) was performed. Factors tested in the DOE included temperature, formaldehyde/glycine concentration, EDC/NHS concentration, and time (Tables 9 and 10). To monitor the loss of biological activity, _EC50 values were determined in IMR90 cells. In addition, the cell morphology of IMR-90 cells at various time points after treatment was observed. See Figure 9, which shows the morphology at 72 hours after treatment. To determine the effect on protein structure, epitope recognition was monitored using dot blot analysis, using a panel of monoclonal antibodies raised against different domains of the toxin.

In the formaldehyde/glycine inactivation of C. difficile mutant toxins, final reaction conditions were selected to achieve the desired level of reduction in cytotoxic activity (7 to 8 _logio ) while maximizing epitope recognition. See Example 20 above.

In the EDC/NHS inactivation of C. difficile mutant toxins, final reaction conditions were selected to achieve the desired level of reduction in cytotoxic activity (7 to 8 _logio ) while maximizing epitope recognition. See Example 21 above.

In another embodiment, the EDC-NHS reaction is annealed by adding sufficient alanine to anneal the reaction. The use of alanine can result in modifications on the mutant toxin protein similar to those observed when the reaction is annealed with glycine. For example, annealing with the addition of alanine can result in alanine moieties on the side chains of glutamic acid and/or aspartic acid residues in the mutant toxin. In another embodiment, the EDC-NHS reaction is annealed by adding sufficient glycine methyl ester to anneal the reaction.

Production of chemically inactivated triple mutant C. difficile toxins A and B under optimal conditions resulted in a further reduction in residual cytotoxicity to undetectable levels (>1000 μg/mL - the highest concentration tested by the CPE assay) while retaining antigenicity (measured by its reactivity with toxin-specific neutralizing antibodies). The results presented in Table 28 demonstrate a stepwise reduction in cytotoxicity from wt toxin to EDC/NHS-treated triple mutant toxin. Immunofluorescence labeling confirmed that the triple mutant toxins (SEQ ID NOs: 4 and 6) and the mutant toxin drug substances exhibited comparable binding to IMR-90 cells, indicating that the loss of cytotoxicity was not due to reduced cell binding (data not shown). Compared to mutant toxin A drug substance, mutant toxin B drug substance achieved a higher fold reduction in cytotoxicity, consistent with the observed ~600-fold greater potency of TcdB compared to TcdA.

Table 28. Cytotoxicity Summary

The results of the cytotoxicity assay were also evaluated for mutant toxin B modified with EDC alone, or with EDC and Sulfo-NHS. See Table 29.

Table 29

Conditions: Triple mutant toxin B ("TM TcdB") (SEQ ID NO: 6) was modified in a weight ratio of mutant toxin B: EDC: sulfo-NHS = 1: 0.5: 0.94. This ratio is molar equivalent (corresponding to the higher MW of sulfo-NHS) to the standard EDC/NHS reaction described in Example 21. To determine the effect of sulfo-NHS, the ratio of sulfo-NHS was varied from 0.5x to 2x the standard ratio. Duplicate reactions were performed in 1x PBS pH 7.0 at 25°C and initiated by the addition of EDC solution. After 2 hours, the reactions were annealed by the addition of 1 M glycine pH 7.0 (0.1 M final concentration) and incubated for an additional 2 hours. The annealed reactions were desalted using a Vivaspin 20 device and the mutant toxin B drug substance ("TMTcdB-EDC") was concentrated, sterile filtered into sterile vials and used for evaluation in cytotoxicity assays.

At the same molar ratio, Sulfo-NHS reduced the _EC50 to approximately 250 ug/mL (compared to >1000 ug/mL for NHS). Even at twice the molar ratio, Sulfo-NHS did not appear to be as effective as NHS in reducing cytotoxicity. See Table 30.

Table 30

To determine the number and type of modifications, peptide mapping was performed on both EDC/NHS and EDC/Sulfo-NHS inactivated triple mutant toxin B samples. Similar amounts of glycine adducts, cross-links, and dehydroalanine modifications were observed in both samples. However, no β-alanine was observed in the Sulfo-NHS sample.

Wild-type toxin B (SEQ ID NO: 2) was inactivated using a standard protocol (see Example 21); toxin B:EDC:NHS 1:0.5:0.5, at 25°C for 2 hours in 1x PBS pH 7.0, followed by annealing with 1 M glycine (0.1 M final concentration) and incubation for an additional 2 hours. The sample was desalted, concentrated, and used in a cytotoxicity assay. The _EC50 for this sample was <244 ng/mL.

Example 23: Recovery Study

To determine whether recovery occurs after inactivation of C. difficile mutant toxins with formaldehyde/glycine or EDC/NHS, samples of the inactivated mutant toxin (1 mg/mL) were incubated at 25°C for 5-6 weeks. Aliquots were removed weekly and the _EC50 value was determined in IMR90 cells. One formaldehyde/glycine inactivated sample contained no formaldehyde, while one sample contained 0.01% formaldehyde. The _EC50 was determined using a CPE assay.

At 25°C in the absence of residual formaldehyde, partial recovery was observed (Table 11). After 5 weeks, cytotoxic activity increased approximately 3-fold. Despite the increase in cytotoxic activity, there was still a 7 log ₁₀ reduction relative to native toxin after 5 weeks. Recovery was completely prevented by the inclusion of formalin at a concentration of 0.010%. No recovery was observed in the EDC/NHS-inactivated samples. Throughout the 6-week incubation, the _EC50 values for all four sets of EDC/NHS-treated triple mutant toxin A (SEQ ID NO: 4) and EDC/NHS-treated triple mutant toxin B (SEQ ID NO: 6) remained at the initial level of >1000 μg/mL. In contrast, the _EC50 values for FI-treated triple mutant toxin A (SEQ ID NO: 4) and FI-treated triple mutant toxin B (SEQ ID NO: 6) were unstable and decreased to unacceptably low _EC50 values, indicating either increased cytotoxicity or recovery from inactivation. See Table 11.

In addition to steadily reducing cytotoxicity to undetectable levels (>1000 μg/mL, as measured by the CPE assay), the EDC/NHS-inactivated mutant toxins retained an important epitope that is the target of toxin-neutralizing mAbs. See Table 31. The FI mutant toxins showed loss of the same antigenic determinant.

Table 31. EDC/NHS inactivation reduces the cytotoxicity of genetic mutant toxins while maintaining important antigenic determinants

a Cytotoxicity was measured using CPE assay on IMR90 cells

b Values determined by Biacore ^™ analysis using multiple neutralizing mAbs (directed against various non-overlapping toxin epitopes)

c is the average of two experiments

dFor the first three rows, neutralizing mAbs "1," "2," and "3" refer to mAbs A60-22, A80-29, and A65-33 against toxin A, respectively. For the last three rows, neutralizing mAbs "1," "2," and "3" refer to mAbs B8-26, B59-3, and B-56-15 against toxin B, respectively.

Example 24: Preclinical immunogenicity studies

Key preclinical objectives include testing compositions containing mutant toxins A and B of C. difficile in small animals and non-human primates (NHPs). Mice and hamsters are immunized to determine, among other things, whether the C. difficile compositions can elicit neutralizing antibodies against mutant toxins A and B. Following a series of immunizations of mice, hamsters, and cynomolgus macaques, the antigens are tested for induction of serum neutralizing antibody responses. In some embodiments, genetically and/or chemically inactivated mutant toxins are formulated in a neutral buffer, an aluminophosphate buffer, or a buffer containing ISCOMATRIX as an adjuvant. Neutralizing antibody responses are typically tested 2-4 weeks after each boost or final dose.

The toxin neutralization assay demonstrates the ability of antisera to neutralize the cytotoxic effects mediated by C. difficile TcdA or TcdB and can therefore be used to measure the functional activity of the antibodies present in a sample. The toxin neutralization assay is performed on the human lung fibroblast cell line IMR-90, which is sensitive to both TcdA and TcdB. Briefly, 96-well microtiter plates are seeded with IMR-90 cells as targets for toxin-mediated cytotoxicity. Each test serum sample is analyzed separately for its ability to neutralize TcdA and TcdB. Appropriate serial dilutions of the test antisera are mixed with a fixed concentration of TcdA or TcdB and incubated at 37°C in a humidified incubator (37°C/5% _CO2 ) for 90 minutes to allow neutralization of the toxin to occur. For quality control, all plates include reference standards and controls containing known titers of antitoxin antibodies. After 90 minutes, the toxin-antiserum mixture was added to the IMR-90 cell monolayer culture and the plate was incubated for another 72 hours. Subsequently, the substrate was added to the assay plate to determine the level of adenosine triphosphate (ATP) present in metabolically active cells, and it was measured as relative light units (RLU). High ATP levels indicate high cell viability, and the level is directly proportional to the amount of toxin neutralization by the antibodies present in the sample. For preclinical data, the RLU data were plotted against the dilution values of the test antiserum samples to generate a four-parameter logistic (4-PL) regression response fitting curve. Neutralization titers are expressed as the sample dilution value that exhibits a 50% reduction in cytotoxicity.

Example 25: Mouse immunogenicity study: mu Clostridium difficile 2010-06

The purpose of this study was to evaluate the immunogenicity of two forms of mutant C. difficile toxin B (SEQ ID NO: 6), each chemically inactivated by a different method. Untreated mutant toxin B (SEQ ID NO: 6) (genetically inactivated but not chemically inactivated) with or without adjuvant was used as a control in this study.

Groups of 10 mice were immunized intramuscularly with 10 μg of the immunogen according to Table 12.

Table 12. Testing of chemically inactivated mutant toxin B (SEQ ID NO: 6) in mice

Results: No adverse effects were observed after each administration of the vaccine candidate. As shown in Figure 10, after three doses of the respective mutant toxins, mice in each group developed significant and potent anti-toxin B neutralizing antibodies.

Based on the low levels at week 12, it appears that EDC-inactivated mutant toxin B (Group 2) and formalin-inactivated mutant toxin B (Group 1) produced potent neutralizing responses in mice.

In the absence of chemical inactivation, the genetic mutant toxin B (SEQ ID NO: 6) produced a neutralizing response after two doses (Group 3-4, Week 8), which was boosted after the third dose (Group 3-4, Week 12).

Example 26: Mouse immunogenicity study: mu Clostridium difficile 2010-07:

The purpose of this study was to evaluate the immunogenicity of chemically inactivated C. difficile mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively), alone and in combination. Immunogens for all groups were formulated with aluminum phosphate as the adjuvant.

Groups of 5 mice were immunized intramuscularly with 10 μg of the immunogen according to Table 13.

Table 13. Testing of chemically inactivated genetic A and B mutant toxins in mice (SEQ ID NOs: 4 and 6, respectively)

Results: There were no adverse effects after each administration of the vaccine candidate. As shown in Figure 11, after 2 doses of chemically inactivated genetic mutant toxins, antitoxin A neutralizing antibodies (groups 3-5) were boosted to titers between 34log ₁₀ , while antitoxin B neutralizing antibodies (groups 1-2, 5) remained low to undetectable, which is consistent with the data from the mouse study described above (Figure 10). Antitoxin B neutralizing antibodies were boosted to 2-3log ₁₀ in groups 1, 2 and 5 after the third dose (12-week titer) and reached their peak value (14-week titer) after the fourth dose. Antitoxin A neutralizing antibody titers in groups 3-5 increased slightly after the third (12-week titer) and fourth immunizations (14-week titer).

Example 27: Hamster immunogenicity study: Clostridium difficile 2010-02:

The purpose of this study was to evaluate the immunogenicity and protective potential of C. difficile triple mutants and chemically inactivated mutant toxins A and B in the Syrian golden hamster model. The Syrian golden hamster model represents the best challenge model for mimicking human CDAD. The same batches of mutant toxins A and B used in the mu C. difficile 2010-07 mouse study were also used in this study. As a control, one group was administered the mutant toxins without an aluminum-containing adjuvant.

Groups of 5 Syrian golden hamsters were immunized intramuscularly with 10 μg of the immunogen according to Table 14.

Table 14. Testing of chemically inactivated mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively) in hamsters (C. difficile 2010-02)

Results: No adverse effects were observed after immunization with the mutant toxins. As shown in Figure 12, after a single dose of the mutant toxins, the anti-toxin A neutralizing response was between 2-3 log ₁₀ (formalin-inactivated mutant toxins (Groups 1-2)) and between 3-4 log ₁₀ (EDC-inactivated mutant toxins (Group 3)). After the second dose, anti-toxin A antibodies were boosted in all three groups. After the third dose, anti-toxin A antibodies did not show an increase in all three groups. Similar results were observed after the fourth immunization, where an increase in titer was observed in the formalin-inactivated group (Group 2) that did not contain aluminum adjuvant.

After a single dose, anti-toxin B neutralizing responses were undetectable in the formalin-inactivated mutant toxin group (Groups 1-2) and only exceeded 2 log ₁₀ in the EDC-inactivated mutant toxin (Group 3). After the second dose, anti-toxin B neutralizing antibody titers increased to 3-4 log ₁₀ in both formalin-inactivated groups (Groups 1-2) and to 4-5 log ₁₀ in the EDC-inactivated group (Group 3). For all three groups, increases in anti-toxin B neutralizing antibody titers were observed after the third and/or fourth dose, and the groups reached peak titers at week 16 (after the last dose). See Figure 12.

In Figure 13, the levels of neutralizing antibody responses to the chemically inactivated genetic mutant toxin (Figure 12) are compared with the levels elicited by the toxoid from List Biological Laboratories, Inc. (Campbell, California) (also referred to herein as "List Bio" or "List Biologicals") (i.e., the wild-type toxin prepared by formalin inactivation purchased from List Biological Laboratories toxoid; the control reagent used to establish the hamster challenge model).

As used herein, "FI" in the figures and tables refers to formalin/glycine treatment of toxins for 2 days at 25°C, unless otherwise indicated. As used herein, "EI" in the figures and tables refers to EDC/NHS treatment for 4 hours at 30°C, unless otherwise indicated. In Figure 13 , five hamster animals were treated with each mutant toxin composition, while eleven hamster animals were treated with the toxoid purchased from List Biological.

The data in Figure 13 show that in hamsters administered according to Table 14, the neutralizing antibody titers against toxin A (Figure 13A) and toxin B (Figure 13B) induced by two doses of the immunogenic composition containing the EDC-inactivated mutant toxin were higher than the neutralizing antibody titers elicited by List Biologicals' toxinoid.

Example 28: Hamster Immunogenicity Study: Clostridium difficile 2010-02 (Continued)

To assess the protective efficacy of the mutant toxin, immunized hamsters, along with a control group of unimmunized animals, were first given an oral dose of clindamycin (30 mg/kg) to disrupt normal intestinal flora. Five days later, the hamsters were challenged with orally administered wild-type Clostridium difficile spores (630 strain, 100 cfu/animal). The animals were monitored daily for signs of CDAD, known as wet tail in hamsters, for 11 days after the challenge. Animals identified as having severe CDAD were euthanized using a clinical system that scores a number of different parameters, including post-stimulation activity, dehydration, excrement, temperature, and body weight, as are known in the art.

On day 11, the study was terminated and all animals were euthanized. Figure 14 shows the survival curves for each of the three immunized groups (Groups 1-3 according to Table 14) compared to the non-immunized control. As shown therein, all non-immunized animals developed severe CDAD and required euthanasia 1-3 days after challenge (0% survival). Both groups administered with the formalin-inactivated mutant toxin had a 60% survival curve, and no animals required euthanasia until day 3 (Group 1) or day 4 (Group 2). The group administered with the EDC-inactivated mutant toxin had an 80% survival curve, with one (of five) animals requiring euthanasia on day 7. Thus, hamsters can be protected from lethal challenge with C. difficile spores.

Example 29: Hamster Immunogenicity Study: Ham Clostridium difficile 2010-03: Immunogenicity of Genetically and Chemically Inactivated Toxins of C. difficile Mutants

The purpose of this study was to evaluate the immunogenicity of unadjuvanted C. difficile triple mutants and chemically inactivated mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively) in the Syrian golden hamster model. Mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively) from the same batches used in the mu C. difficile 2010-07 mouse study were used in this study. As a control, one group (Group 1) received phosphate-buffered saline as a placebo.

Groups of 5 or 10 Syrian golden hamsters were immunized with the immunogens according to Table 15. The animals were dosed three times. In addition, the animals were dosed every two weeks.

Results: See Figure 15. No anti-toxin A or B antibodies were observed in the placebo control group. After one dose, between 2-3 log ₁₀ of anti-toxin A neutralizing antibodies were observed in the formalin-inactivated group (Group 2) and the genetic mutant toxin group (Group 4), and between 3-4 log ₁₀ of anti-toxin A neutralizing antibodies were observed in the EDC-inactivated group (Group 3). After the second immunization with the relevant mutant toxin, an increase in anti-toxin A neutralizing antibodies was observed in each of these groups (2-4) (compare titers at weeks 2 and 3 in Figure 15). After the third dose of mutant toxin (given at week 4), the anti-toxin A neutralizing antibody titers in groups 2-4 were increased compared to their titers at week 4.

Anti-toxin B neutralizing antibodies were detectable after the second dose, with anti-toxin B neutralizing antibodies increasing to between 3-4 log ₁₀ in the formalin-inactivated group (Group 2) and the EDC-inactivated group (Group 3), and to between 2-3 log ₁₀ in the genetic triple mutant group (Group 4). After the third immunization (week 4), anti-toxin B neutralizing antibody titers were boosted to between 3-4 log ₁₀ in the formalin-inactivated mutant toxin group (Group 2) and the genetic mutant toxin group (Group 4), and to between 4-5 log ₁₀ in the EDC-inactivated mutant toxin group (Group 3).

For both anti-toxin A and anti-toxin B neutralizing antibodies, peak titers were observed at week 6 (after the third dose) in all vaccinated groups (Groups 2-4).

Evaluation of immunogenic compositions using Alcoll/CpG or ISCOMATRIX as adjuvants

Hamsters immunized with immunogenic compositions containing chemically inactivated mutant toxins formulated in alum, ISCOMATRIX, or alum/CPG 24555 (Alh/CpG) developed potent neutralizing antitoxin antisera. Peak antitoxin A and antitoxin B responses were observed to be 2-3 times higher and statistically significantly higher in the groups immunized with the mutant toxins formulated in Alh/CpG or ISCOMATRIX than in the groups immunized with the vaccine formulated in alum only. See Table 32, which shows 50% neutralization titers. Hamsters (n=10/group) were immunized at weeks 0, 2, and 4 with 10 μg of each mutant toxin A drug substance and mutant toxin B drug substance (formulated in 100 μg of alum, or 200 μg of CpG 24555 + 100 μg of alum, or 10 U of ISCOMATRIX). Serum was collected at each time point and analyzed for functional antitoxin activity in a toxin neutralization assay. Geometric mean titers are given in Table 32. Stars (*) indicate statistical significance (p < 0.05) when compared to the titer in the alcoll group.

The protective efficacy of immunogenic compositions containing mutant toxin drug substances formulated with these adjuvants was tested. Hamsters were immunized and orally administered clindamycin (30 mg/kg) at week 5 and challenged as described above. A group of unimmunized hamsters (n=5) served as controls. Improved efficacy was observed in hamsters immunized with the mutant toxin drug substance (adjuvanted with Alh/CpG or ISCOMATRIX) (100% survival) compared to alcolyl alone (70% survival). Thus, hamsters can be protected from lethal challenge with C. difficile spores.

Example 30: Inoculation of Cynomolgus Monkeys with Clostridium difficile

The purpose of this study was to test the immunogenicity of low and high doses of EDC-inactivated and formalin-inactivated C. difficile mutant toxins in cynomolgus monkeys. All mutant toxins were formulated with ANTIBIUM(R) as an adjuvant, except for one group which served as a non-adjuvanted control (Group 5).

Results: Figure 16 shows the anti-toxin neutralizing antibody responses in these animals at weeks 0, 2, 3, 4, 6, 8, and 12. Anti-toxin A titers in all five groups ranged from 2 to 3 log ₁₀ after a single dose (week 2 titers). These titers were boosted after each subsequent dose in each group. In these animals, titers did not decrease between weeks 3 and 4. Peak titers for all groups were between 4 and 5 log _10. The group without ISCOMATRIX adjuvant (Group 5) had the lowest titers at all time points, demonstrating the usefulness of ISCOMATRIX in boosting immune responses. The unadjuvanted control group (Group 5) peaked at week 12, as did the group immunized with a high dose of EDC-inactivated mutant toxin (Group 4); all other groups peaked at week 6, two weeks after the last dose. Titers for all groups were boosted after the second dose (week 3 time point). As with the anti-toxin A response, the anti-toxin B response did not decrease from week 3 to week 4. After the third dose (week 6), anti-toxin B neutralizing antibody titers were between 3-4 log ₁₀ in all groups, except for the low-dose formalin-inactivated group (Group 1) and the high-dose EDC-inactivated group (Group 4), both of which had titers > 4 log _10. Peak titers were observed in all groups at week 12, except for the low-dose EDC-inactivated group (Group 3), which had peak titers at week 8. All groups had peak titers > 4 log ₁₀ .

Example 31: Production of Monoclonal Antibodies

Although toxins A and B share considerable structural homology, the neutralizing activity of the antibodies was found to be toxin-specific. In the present invention, several antibodies were identified that are specific for individual toxins and directed against various epitopes and functional domains, exhibiting high affinity and potent neutralizing activity against native toxins. Antibodies were isolated from mice immunized with either commercially available formalin-inactivated (FI)-mutant toxins or recombinant fully-mutant toxins (SEQ ID NOs: 4 and 6, rendered avirulent by introduction of specific mutations in their catalytic sites) to generate mAbs against toxins A and B, respectively. Epitope mapping of the antibodies revealed that the majority of mAbs against toxin A (49 of 52) were directed against the non-catalytic C-terminal domain of the toxin.

Monoclonal antibodies against toxin B target three domains of the protein. Of the 17 toxin B-specific mAbs, six are specific for the N-terminus (e.g., amino acids 1-543 of wild-type C. difficile TcdB, such as 630), six are specific for the C-terminus (e.g., amino acids 1834-2366 of wild-type C. difficile TcdB, such as 630), and five are specific for the central translocation domain (e.g., amino acids 799-1833 of wild-type C. difficile TcdB, such as 630). Using mutant C. difficile toxins (e.g., SEQ ID NOs: 4 and 6) as immunizing antigens thus offers a key advantage over formalin inactivation methods, which can adversely affect the antigenic structure of mutant toxins, for presentation of most, if not all, antigenic epitopes.

Example 32: Characterization of mAb A3-25 against toxin A, comprising a variable light chain having the amino acid sequence of SEQ ID NO: 36 and a variable heavy chain having the amino acid sequence of SEQ ID NO: 37

mAb A3-25 is of particular interest because it defies all attempts to define its immunoglobulin (Ig) isotype (using commonly used isotyping kits for IgG, IgM, and IgA). Further analysis by Western blot using Ig H-chain-specific antiserum revealed that A3-25 is of the IgE isotype, which is unusual for mAbs produced. This was confirmed by nucleotide sequencing of mRNA isolated from the A3-25 hybridoma cells. The amino acid sequence deduced from the nucleotide sequences of the variable regions of the H and L chains of A3-25 is shown in Figure 17 .

To further evaluate A3-25 mAb in C. difficile infection and disease models, the Ig isotype of A3-25 mAb was changed to murine IgG1 by grafting the variable region of the epsilon H chain onto a murine gamma heavy chain according to published methods.

Example 33: Neutralizing activity and epitope mapping of toxin-specific antibodies

Additionally, in an attempt to identify functional/neutralizing antibodies, all monoclonal antibodies were evaluated for their ability to neutralize wild-type toxin in a standard cytopathic effect (CPE) assay or in a more rigorous and quantitative assay based on measurement of ATP as an indicator of cell viability.

Of a total of 52 toxin A-specific antibodies, four mAbs (A3-25, A65-33, A60-22, and A80-29 (Table 17 and Figure 18) exhibited varying levels of neutralizing activity. BiaCore competitive binding assays and hemagglutination inhibition (HI) assays were performed to map the antibody epitopes. The results indicated that these antibodies target different epitopes of the toxin A protein (Table 17). To further confirm the binding location on the protein, the antibodies were individually evaluated in Western blot or dot blot assays using toxin fragments of known sequence. All four neutralizing antibodies were found to be directed against the C-terminal region of the toxin.

Of the total 17 toxin B-specific antibodies, 9 were found to be neutralizing. Of these 9 neutralizing mAbs, 6 were directed against the N-terminus of toxin B, while the other three were directed against the translocation domain of toxin B (Table 18). Based on Biacore competitive binding assays, the 9 neutralizing monoclonal antibodies can be grouped into 4 epitope groups, as shown in Figure 19.

Table 17: Characterization of selected mAbs to toxin A

Table 18: Characterization of selected mAbs to toxin B

Example 34: Identification of novel toxin A antibody combinations with significantly enhanced neutralizing activity:

When tested separately in an ATP-based neutralization assay, four toxin A mAbs (A3-25, A65-33, A60-22, and A80-29) showed incomplete or partial toxin A neutralization activity. mAb A3-25 was the most potent antibody, while the other three were less neutralizing and A80-29 was barely above background ( FIG. 18 ). However, when A3-25 was combined with any of the other three mAbs, a synergistic effect of neutralization was observed in all three combinations, which was far greater than the sum of the neutralization of the individual antibodies, as shown in FIG. 20A-C . In addition, all three combinations exhibited the complete neutralization capacity typically observed with anti-toxin A polyclonal antibodies.

Example 35: Identification of novel toxin B antibody combinations showing significantly enhanced neutralizing activity:

We also observed synergistic neutralization of toxin B mAbs from different epitope groups (identified by BiaCore analysis). Toxin B mAb B8-26 (the most dominant mAb from Group 1) was combined with multiple mAbs from Group 3. These combinations were evaluated in a toxin B-specific neutralization assay, and the results are shown in Figure 21 and Table 19.

A synergistic neutralization effect was observed when B8-26 was combined with mAbs from epitope group 3 (Figure 21B), but not with any other mAbs (data not shown).

Example 36: Screening of safe and effective mutant toxin compositions by mAb in vitro:

Genetically engineered mutant toxins A and B of Clostridium difficile (e.g., SEQ ID NOs: 4 and 6) exhibit residual cytotoxicity (as measured by in vitro cytotoxicity). While we have achieved a ~4 log reduction in the cytotoxicity of C. difficile toxins for each mutant toxin (Table 20), further chemical inactivation of the mutant toxins, such as with formalin treatment, is preferred. However, chemical inactivation treatments can be harsh and may adversely affect key antigenic epitopes of the toxin or mutant toxin.

To optimize the bioprocess, a statistical design of experiments (DOE) was performed for the chemical inactivation of triple mutants Tcd A and B (1 mg/mL) using formalin and EDC/NHS treatment. To optimize the formalin inactivation of triple mutant TcdA, we varied the formalin/glycine concentration (20-40 mM), pH (6.5-7.5), and temperature (25-40° C.). For triple mutant TcdB, we varied the formalin/glycine concentration from 2 to 80 mM while the temperature and pH were 25° C. and 7.0, respectively. The incubation time for all formalin treatments was 24 hours. For formalin inactivation, the "40/40" in Tables 21 and 23 represents the formalin and glycine concentrations used in the reactions. For EDC/NHS treatment, we varied the concentration of EDC/NHS from 0.25 to 2.5 mg/mg of triple mutant TcdA and from 0.125 to 2.5 mg/mg of triple mutant TcdB and incubated at 25°C for 4 hours. At the end of the reaction, all samples were desalted in 10 mM phosphate, pH 7.0. After purification, the treated Tcds were analyzed for residual cytotoxicity and mAb recognition of epitopes by dot blot analysis. The goal was to identify treatment conditions that reduced cytotoxicity to the desired level (EC ₅₀ >1000 μg/mL) without adversely affecting the epitopes recognized by a panel of neutralizing antibodies (++++ or +++). The treatment conditions (marked with a check mark "√" in Tables 21-24) produced potentially safe and effective immunogenic compositions that retained reactivity to at least four neutralizing mAbs and exhibited a 6-8 log ₁₀ reduction in cytotoxicity (relative to the cytotoxicity of the respective wild-type toxin). The results of the selection are shown in Tables 21-24. Additional data from different treatment conditions for the triple mutant toxins, along with data from in vitro cytotoxicity and toxin neutralization assays, are shown in Tables 33 and 34. See also, for example, Examples 20 and 21 above, which provide further details on preferred cross-linking treatment conditions for the mutant toxins.

Table 33

Chemical cross-linking reaction conditions for triple mutant toxin A (SEQ ID NO: 4) samples mentioned in Table 33

Samples 1-4 were modified with EDC/NHS. Conditions: 30°C, 20 mM MES/150 mM NaCl, pH 6.5. Reactions were initiated by the addition of EDC. After a 2-hour reaction, 1 M glycine was added to samples A, B, and C to a final concentration of 50 mM. No glycine was added to sample D. Reactions were set up with varying weight ratios of mutant toxin A (SEQ ID NO: 4): EDC:NHS, as shown below.

Sample 5 L44905-160A 80 mM HCHO, 80 mM glycine, 80 mM NaPO4 pH 7, 1 mg/mL mutant toxin A (SEQ ID NO: 4) protein, reacted at 25°C for 48 hr.

Sample 6: L44166-166 EDC/NHS-modified mutant toxin A (SEQ ID NO: 4) in 20 mM MES/150 mM NaCl, pH 6.5, at 25°C. Mutant toxin A (SEQ ID NO: 4): EDC: NHS = 1:0.5:0.5. The reaction was initiated by the addition of EDC. After a 2-hour reaction, 1 M glycine was added to a final concentration of 0.1 M glycine and incubated for an additional 2 hours. After this time, the reaction buffer was exchanged for 1X PBS on Sephadex G25.

Sample 7 L44905-170A 80 mM HCHO, 80 mM glycine, 80 mM NaPO ₄ pH 7, 1 mg/mL mutant toxin A (SEQ ID NO: 4) protein, reacted at 35° C. for 48 hr. This formalin reaction is intended to produce excessive cross-linking, thereby strictly reducing antigen binding.

Sample 8 L44897-61 32 mM HCHO/80 mM glycine, reacted at 25°C for 72 hours.

Sample 9 L44897-63 80 mM HCHO/80 mM glycine, reacted at 25°C for 72 hours.

The following reactions all had a reaction time of 24 hr:

Sample 10 L44897-72 tube #1 25°C, 80 mM NaPi pH 6.5, 20 mM HCHO/20 mM glycine

Sample 11 L44897-72 tube #2 25°C, 80 mM NaPi pH 6.5, 40 mM HCHO/40 mM glycine

Sample 12 L44897-72 tube #3 32.5°C, 80 mM NaPi pH 7.0, 30 mM HCHO/30 mM glycine

Sample 13 L44897-72 tube #4 32.5°C, 80 mM NaPi pH 7.0, 30 mM HCHO/30 mM glycine

Sample 14 L44897-72 tube #5 32.5°C, 80 mM NaPi pH 7.0, 30 mM HCHO/30 mM glycine

Sample 15 L44897-75 tube #6 25°C, 80 mM NaPi pH 7.5, 20 mM HCHO/20 mM glycine

Sample 16 L44897-75 tube #7 25°C, 80 mM NaPi pH 7.5, 40 mM HCHO/40 mM glycine

Sample 17 L44897-75 tube #8 40°C, 80 mM NaPi pH 6.5, 20 mM HCHO/20 mM glycine

Sample 18 L44897-75 tube #9 40°C, 80 mM NaPi pH 6.5, 40 mM HCHO/40 mM glycine

Sample 19 L44897-75 tube #10 40°C, 80 mM NaPi pH 7.5, 20 mM HCHO/20 mM glycine

Sample 20 L44897-75 tube #11 40°C, 80 mM NaPi pH 7.5, 40 mM HCHO/40 mM glycine

The following eight samples were reacted in 80 mM NaPi pH 7.0 containing 78 mM HCHO and 76 mM glycine at 25°C for the indicated times:

Sample 21 L44897-101 (premodified) TxA control Time zero control sample, unmodified or exposed to HCHO/glycine

Sample 29 (L44980-004) is an EDC/NHS-modified mutant toxin A (SEQ ID NO: 4) (triple mutant toxin A (SEQ ID NO: 4)-EDC). Reaction conditions were: 25°C, 20 mM MES/150 mM NaCl pH 6.6 buffer. Triple mutant toxin A (SEQ ID NO: 4): EDC: NHS = 1:0.5:0.5 w:w:w. The reaction was initiated by the addition of EDC. After a 2-hour reaction, succinate was added to a final concentration of 0.1 M and the reaction was continued at 25°C for an additional 2 hours. The reaction was terminated by desalting on Sephadex G25.

The following 12 samples and 2 controls were used for recovery experiments, where the samples were incubated at 25°C and 37°C:

Reaction 1 = 25°C, 80 mM NaPi pH 7.0, 40 mM HCHO only (no glycine), 24 hour reaction.

Reaction 2 = 25°C, 80 mM NaPi pH 7.0, 40 mM HCHO/40 mM glycine, 24 hour reaction.

The following four samples were generated by performing reactions at 25°C in 80 mM NaPi pH 7.0, 40 mM HCHO/40 mM glycine for the times indicated:

Sample 48 L44897-139 was reacted at 25°C for 48 hr, 80 mM NaPi pH 7.0, 40 mM HCHO/40 mM glycine.

Sample 49: L44166-204 EDC/NHS-modified mutant toxin A (SEQ ID NO: 4). 25C, 1x PBS pH 7 buffer. Mutant toxin A (SEQ ID NO: 4): EDC: NHS = 1:0.5:0.5 w:w:w. React with EDC/NHS for 2 hours, then add 1M glycine to a final concentration of 0.1M and react for an additional 2 hours. Buffer exchange on Sephadex G25 to 20mM L-histidine/100mM NaCl pH 6.5.

Chemical cross-linking reaction conditions for mutant toxin B samples mentioned in Table 34

Triple mutant toxin B (SEQ ID NO: 6) was chemically cross-linked and tested according to the following reaction conditions. Sample L44905-86 was tested in an experiment involving three formalin reaction variations and two incubation temperatures. Six samples were taken each day, for a total of 18 samples. The first sample in the list was the untreated control (which brought the total to 19 samples). The untreated control included untreated triple mutant toxin B polypeptide (SEQ ID NO: 6).

Reaction 1 ("Rxn1") = 80 mM HCHO, 80 mM glycine, 80 mM NaPO4 pH 7, 1 mg/mL triple mutant toxin B (SEQ ID NO: 6) protein

Reaction 2 ("Rxn2") = 80 mM HCHO, no glycine, 80 mM NaPO4 pH 7, 1 mg/mL triple mutant toxin B (SEQ ID NO: 6) protein

Reaction 3 ("Rxn3") = 80 mM HCHO, no glycine, 80 mM NaPO4 pH 7, 1 mg/mL triple mutant toxin B (SEQ ID NO: 6) protein + cyanoborohydride capping. Cyanoborohydride capping involves adding 80 mM _CNBrH4 to the final reaction after desalting and incubating at 36°C for 24 hr.

For sample L34346-30A, 0.5 g EDC and NHS per gram of triple mutant toxin B (SEQ ID NO: 6) were added at 30°C for 4 hours in 20 mM MES, 150 mM NaCl, pH 6.5.

For sample L34346-30B, 0.5 g of EDC and NHS per gram of triple mutant toxin B (SEQ ID NO: 6) was incubated at 30° C. for 2 hours, followed by the addition of glycine (final concentration in μg/L) and incubation for another 2 hours at 30° C. in 20 mM MES, 150 mM NaCl, pH 6.5. The only difference between the two reactions, L34346-30A and L34346-30B, was the addition of glycine to reaction L34346-30B.

Example 37: Antibodies induced by immunogenic compositions are capable of neutralizing toxins from various strains of Clostridium difficile

To assess whether antibodies induced by immunogenic compositions containing mutant toxin drug substances are capable of neutralizing a broad spectrum of diverse toxin sequences, strains representing diverse ribotypes and toxinotypes were sequenced to identify the extent of genetic diversity among the strains compared to the mutant toxin drug substances. Culture supernatants containing toxins secreted from various strains were then tested in in vitro neutralization assays with serum from immunized hamsters to determine the coverage of the immunogenic compositions and their ability to protect against diverse toxins from prevalent clinical strains.

HT-29 cells (a colon cancer cell line) and IMR-90 cells were both used to test for neutralization of toxins expressed by CDC strains. HT-29 cells were more sensitive to TcdA; the EC ₅₀ of purified TcdA in these cells was 100 pg/mL (compared to 3.3 ng/mL for TcdB). On the other hand, IMR-90 cells were more sensitive to TcdB; the EC ₅₀ of purified TcdB in these cells ranged from 9-30 pg/mL (compared to 0.92-1.5 ng/mL for TcdA). The specificity of the assay for TcdA and TcdB in these cell lines was confirmed by using polyclonal and monoclonal toxin-specific antibodies. For normalization of the assay, culture filtrates of 24 CDC isolates were tested at 4 times the concentration of their respective EC ₅₀ values. Three of the strains had toxin levels that were too low to be used for testing in the neutralization assay.

24 strains representing diverse ribotypes/toxin types (covering more than 95% of the prevalent C. difficile strains in the United States and Canada) were obtained from the CDC. These isolates include strains representing ribotypes 027, 001, and 078, three CDAD strains prevalent in the United States, Canada, and the United Kingdom. Strains 2004013 and 2004118 represent ribotype 027; strain 2004111 represents ribotype 001, and strains 2005088, 2005325, and 2007816 represent ribotype 078. In order to identify the degree of genetic diversity between the clinical isolates that cause disease and the 630 strains, the toxin genes (TcdA and TcdB) from these clinical strains were fully sequenced. See Table 35. The amino acid sequences of the toxins were aligned using ClustalW in the Megalign ^™ program and analyzed for sequence identity. For tcdA, genomic alignment analysis revealed that all clinical isolates shared approximately 98-100% amino acid sequence identity with strain 630. The C-terminal portion of the tcdA gene was slightly more diverse. The same analysis was performed for the tcdB gene, which exhibited even greater sequence diversity. Notably, strains 2007838/NAP7/126 and 2007858/NAP1/unk5 exhibited the greatest diversity patterns with strain 630 in the N-terminal (79-100%) and C-terminal (88-100%; data not shown) domains.

Hamster serum pools (HS) were collected from Syrian golden hamsters immunized with immunogens comprising mutant TcdA (SEQ ID NO: 4) and mutant TcdB (SEQ ID NO: 6), wherein the mutant toxins were inactivated with EDC and formulated with aluminum phosphate according to, for example, Example 29, Table 15, supra. The results in Table 35 show that at least toxin B from each culture supernatant was neutralized by sera from immunized hamsters in an in vitro neutralization assay.

Table 35. Description of C. difficile strains from the CDC and the ability of immune hamster sera to neutralize various toxins

b Toxin levels too low to allow neutralization assays

Figure 23 shows the results of neutralization assays on IMR-90 using toxin preparations from various strains of C. difficile. The data show that TcdB neutralizing antibodies in hamster antisera were able to neutralize toxins from all 21 isolates tested, including highly virulent strains and TcdA-negative, TcdB-positive strains. At least 16 different strains of C. difficile were obtained from CDC (Atlanta, GA) (previously described) and cultured in C. difficile culture medium under suitable conditions (as known in the art and as described above). Culture supernatants containing secreted toxins were analyzed to determine their cytotoxicity ( _EC50 ) on IMR-90 monolayer cultures and then tested in a standard in vitro neutralization assay at 4-fold _EC50 with various dilutions of serum from hamsters immunized with mutant toxin A drug substance and mutant toxin B drug substance (formulated with aluminum phosphate). Crude toxin and purified toxin (commercially available toxin from List Biologicals) from the culture supernatant of each strain (not purified from individual supernatants) were tested for cytotoxicity against IMR-90 cells using the in vitro cytotoxicity assay described above.

In Figures 23A-K, the graphs show results from an in vitro cytotoxicity assay (described previously), where ATP levels (RLU) are plotted against increasing concentrations of: C. difficile culture and hamster serum pool (■); crude toxin and hamster serum pool (●); purified toxin and hamster serum pool (▲); crude toxin control; and purified toxin (◆), control. Toxins from each strain were added to cells at 4x EC ₅₀ values.

As shown in Figures 23A-K, hamsters that received the immunogens surprisingly developed neutralizing antibodies that exhibited neutralizing activity against toxins from at least 16 different C. difficile CDC strains (compared to controls given only each toxin): 2007886 (Figure 23A); 2006017 (Figure 23B); 2007070 (Figure 23C); 2007302 (Figure 23D); 2007838 (Figure 23E); 2007886 (Figure 23F); 2009292 (Figure 23G); 2004013 (Figure 23H); 2009141 (Figure 23I); 2005022 (Figure 23J); and 2006376 (Figure 23K). See also Table 35 for additional C. difficile strains from which toxins were tested and neutralized by immunogenic compositions containing mutant toxin A drug substance and mutant toxin B drug substance formulated in aluminum phosphate.

In another study, culture supernatants containing toxins secreted by various C. difficile strains (obtained from the CDC and Leeds Hospital, UK) were tested in an in vitro neutralization assay with serum from hamsters administered mutant toxin A and mutant toxin B drug substances (formulated in alum). The experimental design is shown in Table 36. The results are shown in Tables 37 and 38.

Example 38: Peptide mapping of the EDC/NHS triple mutant toxin

To characterize the EDC/NHS inactivated triple mutant toxins, peptide mapping experiments were performed on four sets of EDC/NHS-treated triple mutant toxin A (SEQ ID NO: 4) and four sets of EDC/NHS-treated triple mutant B (SEQ ID NO: 6). After digestion of the mutant toxins with trypsin, the resulting peptide fragments were separated by reverse phase HPLC. Mass spectrometry was used to identify modifications resulting from the inactivation process. For both mutant toxin A drug substance and mutant toxin B drug substance, greater than 95% of the theoretical tryptic peptides were identified. Cross-links and glycine adducts (glycine was used as a capping agent) were identified. β-Alanine adducts were also observed in both mutant toxin A drug substance and mutant toxin B drug substance. Without being limited by mechanism or theory, the β-Alanine adducts appear to result from the reaction of three moles of NHS with one mole of EDC, whereby NHS-activated β-Alanine is formed. This molecule can then react with lysine groups to form β-alanine adducts (+70Da). Low levels (0.07 mol/mol protein) of dehydroalanine (-34Da) were also observed in the EDC/NHS-treated triple mutant toxin B sample. Dehydroalanine is the result of desulfonation of cysteine residues. The same type and extent of modification were observed in all four batches of each mutant toxin, indicating that the method produces a consistent product. Peptide mapping (with greater than 95% sequence coverage) confirmed the presence of the modifications. A summary of the modifications is shown in Table 39. See also Figures 24-25. In addition, the size and charge heterogeneity of the triple mutant toxin A drug substance and the triple mutant toxin B drug substance were increased compared to the size and charge heterogeneity of the respective triple mutant toxin A and triple mutant toxin B in the absence of chemical inactivation. Consequently, size exclusion chromatography (SEC) and anion exchange chromatography (AEX) spectra had relatively broad peaks (data not shown).

Table 39. Summary of modifications observed in mutant toxin drug substances

The extent of modification was calculated by dividing the HPLC area of the modified peptide by the HPLC area of the native + modified peptide.

Example 39: Production of Drug Product

The Clostridium difficile immunogenic composition (drug product) contains two active pharmaceutical ingredients (mutant toxin A drug substance and mutant toxin B drug substance). An exemplary drug product is a lyophilized formulation containing 10 mM Tris buffer pH 7.4, 4.5% (w/w) trehalose dihydrate, and 0.01% (w/v) Polysorbate 80, including each of mutant toxin A drug substance and mutant toxin B drug substance. See Table 40. The immunogenic composition is prepared for injection by resuspending the lyophilized vaccine with a diluent or a diluent containing alum. The placebo will contain sterile normal saline solution for injection (0.9% sodium chloride).

Table 40

Buffer preparation

Add water for injection (WFI) to the blending tube. While mixing, add excipients and dissolve into the solution. Measure the pH. If necessary, adjust the pH to 7.4 ± 0.1 with HCl. Dilute the solution to the final weight with WFI and filter through a 0.22 μm Millipore Express SHC XL150 filter. Take a pre-filtration bioburden reduction sample before filtration. Sample the filtered buffer for osmolality and pH.

Preparation

Thawed mutant toxin drug substance was collected in formulation tubes based on pre-calculated amounts in the following order: First, 50% of the target dilution buffer volume required to achieve 0.6 mg/mL was added to the tube, followed by the addition of mutant toxin A drug substance and mixing at 100 rpm for 5 minutes. Mutant toxin B drug substance was then added to the tube and the solution was further diluted to the 0.6 mg/mL dilution point and mixed at 100 rpm for another 5 minutes. Samples were taken and tested for total mutant toxin concentration. Based on the mutant toxin concentration value being processed, the solution was diluted to 100% of the volume and mixed at 100 rpm for 15 minutes. The formulated drug product was sampled for pH and bioburden prefiltration. The formulated drug product was then filtered using a Millipore Express SHC XL150 for overnight storage or transferred to a transfer tube for sterile filtration.

The formulated aliquots were moved to the filling area, sampled for bioburden measurement, and sterile filtered using two Millipore Express SHC XL150 filters in series. The formulated aliquots were filled into pyrogen-free glass vials at a target volume of 0.73 mL. The filled vials were partially stoppered and loaded into the freeze dryer. Lyophilization cycles were performed as shown in Table 41. Upon completion of the cycle, the freeze drying chamber was backfilled with nitrogen to 0.8 atm, and the stopper was fully seated. The chamber was unloaded and the vials were capped with flip-off seals.

Table 41. Lyophilization Cycle Set Points for Clostridium difficile Drug Product

Drug product stability data are summarized in Table 42. The data demonstrate that the drug product is physically and chemically stable when stored at 2-8°C for at least 3 months or at 25°C or 40°C for at least 1 month. Impurity levels as determined by size exclusion chromatography (SEC) did not change under either storage condition, and in vitro antigenicity did not change up to the last time point tested.

Table 42: Stability of Lyophilized Drug ^Producta

aFor these tests, lyophilized DP was reconstituted with 60 mM NaCl diluent.

Example 40: Vaccine Diluent

For saline, 60 mM NaCl was used as the diluent for lyophilized non-adjuvanted drug products to ensure an isotonic solution upon reconstitution.

Alumina: Alumina "85" 2% (Brenntag) is a commercially available Good Manufacturing Practice (GMP) grade product composed of octahedral crystalline sheets of aluminum hydroxide. An exemplary alumina diluent formulation is shown in Table 43. This exemplary formulation can be used in combination with the pharmaceutical products described above.

Studies using alumgel adjuvanted formulations showed 100% binding of mutant toxin A drug substance and mutant toxin B drug substance to 1 mg Al/mL alumgel (pH 6.0 to 7.5). Maximum binding of both drug substances was observed at the highest protein concentration tested (300 μg/mL each).

Protein binding to alum was also tested using lyophilized drug product formulations containing 200 μg/mL of each drug substance and alum in the range of 0.25-1.5 mg/mL. The drug product was reconstituted with diluents containing varying concentrations of alum, and the percentage of each mutant toxin bound was measured. All tested concentrations of alum demonstrated 100% binding to the antigen.

The protein binding kinetics to alum were also evaluated at target doses of mutant toxin A drug substance and mutant toxin B drug substance (200 μg/mL each). The results showed that 100% of the mutant toxin drug substance was bound to alum throughout the 24-hour RT time course.

CpG 24555 and Alcohol: CpG 24555 is a synthetic 21-mer oligodeoxynucleotide (ODN) having the sequence 5-TCG TCG TTTTTC GGT GCT TTT-3 (SEQ ID NO: 48). An exemplary formulation of CpG 24555 in combination with an alcohol diluent is shown in Table 44. This exemplary formulation can be used in combination with the pharmaceutical products described above.

The adjuvant is a saponin-based adjuvant known in the art. An exemplary formulation of the adjuvant formulation is shown in Table 45. This exemplary formulation can be used in combination with the pharmaceutical products described above.

Example 41: Immunogenicity of a mutant toxin drug composition with alum as adjuvant in an NHP model and clinical proof of concept

The immunogenicity of mutant toxin drug substance compositions adjuvanted with alum was evaluated in an NHP model (specifically, cynomolgus macaques). NHPs immunized with 10 μg of each mutant toxin A drug substance and mutant toxin B drug substance composition (formulated with alum) at two-week intervals (weeks 0, 2, and 4) developed strong neutralizing antitoxin responses. See Table 46. After the third immunization, both antitoxin A and antitoxin B neutralizing responses reached the protective range and remained within or above the protective range until at least week 33 (the last time point studied).

Cynomolgus monkeys (n=8) were immunized IM with 10 μg of each mutant toxin A drug substance and mutant toxin B drug substance (formulated in 250 μg of alum). Serum was collected at each time point and analyzed for functional antitoxin activity in a toxin neutralization assay. GMTs are provided in Table 46. The range of protective titers provided in this table illustrates the range of neutralizing antibody titers that corresponded to a significant reduction in recurrence of C. difficile infection in the Merck monoclonal antibody treatment trial.

Correlation of human protective antibody titers from Merck mAb treatment trials with titers induced by Pfizer’s vaccine candidate in NHPs

A phase II efficacy study using Merck/Medarex mAb (Lowy et al., NEngl J Med 2010 Jan 21; 362(3): 197-205) appears to demonstrate a correlation between serum neutralizing anti-toxin mAb levels and prevention of CDAD relapse. Following administration of toxin-specific mAb to humans, serum antibody levels in human recipients in the range of 10-100 μg/mL demonstrated protection against relapse (70% reduction in CDAD relapse).

Immunogenic compositions containing mutant toxin drug substances were tested to determine whether they could induce potent neutralizing antibody responses in humans by comparing published data from the Merck/Medarex Phase 2 study with antibody levels induced by the immunogenic compositions in the NHP model. This was achieved by using previously published characterizations of Merck/Medarex mAbs to convert the range of these mAbs (10-100 μg/mL) in sera from subjects who did not show signs of relapse to 50% neutralization titers, and comparing these titers ("protective titer range") with those observed in the preclinical models described herein. As shown in Table 46, the immunogenic composition containing mutant toxin A and mutant toxin B drug substances (adjuvanted with alum) generated immune responses in NHPs that reached the "protective range" after the third dose and remained within or above this range until Week 33. The toxin neutralizing antibody levels induced in NHPs by the C. difficile immunogenic compositions of the invention were comparable to the serum antibody levels in the Merck/Medarex test subjects who showed protection against recurrence of CDAD.

Example 42: Immunogenicity of mutant toxin drug substance compositions adjuvanted with ISCOMATRIX or Alh/CpG 24555 (Alh/CpG) in the NHP model

In NHPs, both ISCOMATRIX and Alh/CpG statistically significantly enhanced neutralization titers against toxins A and B compared to vaccines administered with alcolhol alone (Table 47). Vaccines administered with Alh/CpG or ISCOMATRIX elicited above-background antitoxin responses at earlier time points (weeks 2-4) compared to alcolhol alone (weeks 4-6), which may have an important role in protecting against recurrence of CDAD in humans. Immunogenic compositions adjuvanted with Alh/CpG or ISCOMATRIX generated antitoxin neutralization titers that reached the protective range more quickly than those adjuvanted with alcolhol (see also Example 41) and remained within or above this range until week 33.

As shown in Table 47, at weeks 0, 2, and 4, cynomolgus monkeys were immunized IM with 10 μg of each mutant toxin A drug substance and mutant toxin B drug substance, wherein the drug substance was formulated with 250 μg of alum (n=8), or with 500 μg of CpG + 250 μg of alum (n=10), or with 45 U of ISCOMATRIX (n=10). Sera were collected at each time point and analyzed for functional antitoxin activity in the toxin neutralization assay described above. GMTs are listed in the table. Stars (*) indicate statistical significance (p<0.05) when compared to the titer in the alum group. The protective titer range represents the range of neutralizing antibody titers that correspond to a significant reduction in recurrence of C. difficile infection in the Merck/Medarex mAb treatment trial.

[00155] The doses of mutant toxin A and mutant toxin B drug substances administered (in the presence of ISCOMATRIX or Alh/CpG adjuvant) were also evaluated for their ability to generate neutralizing antitoxin antibody titers in NHPs. In one study, NHPs were administered either a low dose (10 μg) or a high dose (100 μg) of each mutant toxin drug substance (formulated in ISCOMATRIX). Responses were compared at each time point following immunization. As shown in Table 48, antitoxin neutralization titers were robust in both treatment groups. Antitoxin A titers were nearly equivalent between the low and high dose groups at most time points, while antitoxin B titers tended to be higher in the high dose group.

As shown in Table 48, cynomolgus macaques (n=5) were immunized IM with 10 μg or 100 μg of each mutant toxin A drug substance and mutant toxin B drug substance (formulated in 45 U of ISCOMATRIX) at weeks 0, 2, and 4. Serum was collected at each time point and analyzed for functional antitoxin activity in a toxin neutralization assay. GMTs are listed in the table. The protective titer range represents the range of neutralizing antibody titers that corresponded to a significant reduction in recurrence of C. difficile infection in the Merck/Medarex mAb treatment trial.

In an attempt to enhance the kinetics of the anti-toxin B response, NHPs were immunized with a constant dose of mutant toxin A drug substance (10 μg) mixed with increasing doses of mutant toxin B drug substance (10, 50, or 100 μg) in the presence of ISCOMATRIX or Alh/CpG adjuvant. Regardless of the adjuvant, there was a trend toward inducing higher anti-toxin B neutralizing responses in the groups receiving the higher dose of mutant toxin B drug substance (50 or 100 μg) compared to the 10 μg dose of mutant toxin B drug (Table 50, with * markings indicating statistically significant increases). This trend was observed at most time points after the final immunization. However, in some cases, the anti-toxin A neutralizing response showed a statistically significant decrease (marked with ^ in Table 49) as the amount of mutant toxin B was increased.

As shown in Tables 49 and 50, NHPs (10/group) were immunized IM at weeks 0, 2, and 4 with varying ratios of mutant toxin A drug substance to mutant toxin B drug substance (either as ISCOMATRIX (45 U per dose) or as Alh/CpG/ (250 μg/500 μg per dose)), using 10 μg of mutant toxin A drug substance plus 10, 50, or 100 μg of mutant toxin B drug substance (designated in Tables 49 and 50 as: 10A:10B, 10A:50B, and 10A:100B, respectively). Table 49 shows anti-toxin A titers. Table 50 shows anti-toxin B titers. GMTs are listed in the tables. The protective titer range represents the range of neutralizing antibody titers that corresponded to a significant reduction in recurrence of C. difficile infection in the Merck mAb therapy trial. The symbol ^ represents a statistically significant decrease in neutralizing titers compared to the 10A:10B group. The asterisk * represents a statistically significant increase in the neutralization titer compared to the 10A:10B group (p < 0.05).

Example 43: Five-week IM repeat dosing toxicity study with an immunogenic composition in cynomolgus monkeys with a 4-week recovery period

A five-week IM repeated-dose toxicity study was conducted in cynomolgus monkeys using PF-06425095 (an immunogenic composition comprising triple mutant toxin A drug substance and triple mutant toxin B drug substance in combination with the adjuvants aluminum hydroxide and CpG 24555) to evaluate the potential toxicity and immunogenicity of Clostridium difficile triple mutant toxin A drug substance and triple mutant toxin B drug substance in combination with aluminum hydroxide and CpG 24555 adjuvant (PF-06425095). Cynomolgus monkeys (6/sex/group) were administered IM as a prime dose of PF-06425095 at 0.2 or 0.4 mg/dose of triple mutant toxin A and triple mutant toxin B (low-dose and high-dose immunogenic composition groups, respectively), 0.5 mg aluminum as aluminum hydroxide, and 1 mg CpG 24555, as well as an adjuvant-only combination (aluminum hydroxide + CpG 24555; PF-06376915), followed by three booster doses (days 1, 8, 22, and 36). A separate group of animals (6/sex) received 0.9% isotonic saline (approximately pH 7.0). The immunogenic composition vehicle consisted of 10 mM Tris buffer (pH 7.4), 4.5% trehalose dihydrate, and 0.1% Polysorbate 80. The adjuvant control vehicle consisted of 10 mM histidine buffer (with 60 nM NaCl, pH 6.5). The total dosing volume was 0.5 mL per injection. All doses were administered into the left and/or right quadriceps muscles. Selected animals underwent a 4-week non-dosing observation period to assess the reversibility of any effects observed during the dosing phase of this study.

No adverse findings were observed in this study. PF-06425095 was well tolerated and produced only local inflammatory reactions with no evidence of systemic toxicity. During the dosing period, the following increases (dose-dependent) were observed in the immunogenic composition-treated groups compared to pre-test levels: fibrinogen (23.1% to 2.3x) on days 4 and 38, C-reactive protein (2.1x to 27.5x) on days 4 and 38 (2.3x to 101.5x), and globulin (11.1% to 24.1%) on days 36 and/or 38; consistent with the expected inflammatory response to administration of the adjuvanted immunogenic composition.

The increases in fibrinogen and C-reactive protein noted on day 4 were partially restored on day 8, with increases in fibrinogen (25.6% to 65.5%) and C-reactive protein (4.5x and 5.6x) only in the high-dose immunogenic composition group. Increases in interleukin (IL)-6 were observed in both the low- and high-dose immunogenic composition groups at 3 hours on day 1 (8.3x to 127.2x individual values at 0 hours on day 1, in response to dosing) and at 3 hours on day 36 (9.4x to 39.5x individual values at 0 hours on day 36). No changes were observed in other cytokines (IL-10, IL-12, interferon-induced protein (IP-10), and tumor necrosis factor alpha (TNF-α). Elevations in these acute phase proteins and cytokines are part of the expected normal physiological response to administration of foreign antigens. During the recovery period (cytokines were not assessed during the recovery period), there were no PF 06425095-related or adjuvant-related changes in these clinicopathological parameters. In addition, there were local changes at the injection site, which were of similar incidence and severity in the adjuvant control group and the low-dose and high-dose immunogenic composition groups; therefore, they were not directly related to PF-06425095. During the dosing period, changes included mild to moderate chronic active inflammation characterized by separation of muscle fibers caused by infiltration of macrophages, which often contained basophilic material (interpreted as aluminum-containing adjuvant), lymphocytes, plasma cells, neutrophils, eosinophils, and cells, necrotic debris, and edema. Extracellular basophilic material was also present in these lesions of chronic active inflammation. At the end of the recovery period, there was mild to moderate chronic inflammation and mononuclear cell infiltration, as well as mild fibrosis. These injection site findings represent a local inflammatory response to the adjuvant. Other microscopic changes included a slight to moderate increase in lymphocyte structure in the iliac (draining) lymph nodes and a slight increase in cellularity in the germinal centers of the spleen, which were observed during the dosing period in the adjuvant control group and the low-dose and high-dose immunogenic composition groups. At the end of the recovery period, these microscopic findings were of low severity. These effects represent immune responses to antigenic stimulation, as well as pharmacological responses to adjuvant or PF-06425095. There was no test article-related increase in anti-DNA antibodies.

In view of the absence of adverse findings, the no-observable-adverse-effect-level (NOAEL) in this study was 4 doses administered as two 0.5 mL injections for the high-dose immunogenic composition group (0.4 mg of triple mutant toxin A drug substance and triple mutant toxin B drug substance/dose as PF-06425095).

Example 44: Efficacy of Seropositive NHP Serum Passively Transferred to Hamsters

Groups of five Syrian golden hamsters were administered the antibiotic clindamycin (30 mg/kg) orally to disrupt normal intestinal flora. Five days later, the hamsters were challenged with orally administered wild-type Clostridium difficile spores (strain 630, 100 cfu/animal) and intraperitoneally (IP) administered NHP serum according to Table 51. Without being bound by mechanism or theory, disease symptoms following spore challenge typically begin approximately 30-48 hours after challenge.

[0146] Following three immunizations with mutant toxin A drug substance and mutant toxin B drug substance (A:B ratios of 10:10, 10:50, and 10:100, formulated in ISCOMATRIX, see Example 42, Tables 49 and 50 ), NHP serum was pooled from the NHP serum samples that exhibited the highest titers (anti-toxin A serum and anti-toxin B serum) for administration to hamsters. NHP sera were collected from time points of 5, 6, and 8 weeks (immunizations were performed at 0, 2, and 4 weeks) as described in Example 42. The results are shown in Tables 52-54 below. The symbol "+" represents the geometric mean (GM) in () excluding animal #3 non-responder. "*TB" represents the final bleed, the day the animal was euthanized, not all animals were euthanized on the same day.

In another study, Syrian golden hamsters were administered orally clindamycin antibiotics (30 mg/kg) to disrupt normal intestinal flora. Five days later, the hamsters were challenged with orally administered wild-type Clostridium difficile spores (strain 630, 100 cfu/animal) and NHP serum was administered intraperitoneally (IP) according to Table 55. Without being limited by mechanism or theory, disease symptoms following spore challenge typically begin approximately 30-48 hours after challenge.

NHP sera administered to hamsters were pooled from samples collected from NHPs following three immunizations with mutant toxin A drug substance and mutant toxin B drug substance (A:B ratios of 10:10, 10:50, and 10:100, formulated in alum gel and CpG 24555, see Example 42, Tables 49 and 50 ). NHP sera were collected from time points of 5, 6, 8, and 12 weeks, as described in Example 42 (NHP immunized at 0, 2, and 4 weeks). The results are shown in Tables 56-59 below. Sera from hamsters were further studied to determine inhibitory concentration (IC ₅₀ ) values, which were determined using the toxin neutralization assay described above. The levels of toxin-neutralizing antibodies induced in hamsters by the C. difficile immunogenic compositions of the present invention were comparable to the serum antibody levels in the Merck/Medarex test subjects (which showed protection against recurrence of CDAD).

*＝Decrease on that day

Example 45: Identification of Mutant Toxin Drug Substances

The primary structure of triple mutant toxin A is shown in SEQ ID NO: 4. The _NH2 -terminal Met residue at position 1 of SEQ ID NO: 4 is derived from the start codon of SEQ ID NO: 12 and is deleted in the isolated protein (e.g., see SEQ ID NO: 84). Therefore, in Examples 12 to 45, "SEQ ID NO: 4" refers to SEQ ID NO: 4 in which the start methionine (at position 1) is deleted. Purified triple mutant toxin A (SEQ ID NO: 4) (drug substance intermediate - Lot L44993-132) and EDC/NHS-treated triple mutant toxin A (SEQ ID NO: 4) ("mutant toxin A drug substance" - Lot L44898-012) both exhibit a single _NH2 -terminal sequence starting with SLISKEELIKLAYSI (positions 2-16 of SEQ ID NO: 4).

The primary structure of triple mutant toxin B is shown in SEQ ID NO: 6. The _NH2 -terminal Met residue at position 1 of SEQ ID NO: 6 is derived from the start codon of SEQ ID NO: 12 and is deleted in the isolated protein (e.g., see SEQ ID NO: 86). Therefore, in Examples 12 to 45, "SEQ ID NO: 6" refers to SEQ ID NO: 6 in which the start methionine (at position 1) is deleted. Both purified triple mutant toxin B (SEQ ID NO: 6) (drug substance intermediate - Lot 010) and EDC/NHS-treated triple mutant toxin B (SEQ ID NO: 6) ("Mutant Toxin B Drug Substance" - Lot L44906-153) exhibited a single _NH2 -terminal sequence starting with SLVNRKQLEKMANVR (positions 2-16 of SEQ ID NO: 6).

Circular dichroism (CD) spectroscopy was used to assess the secondary and tertiary structure of triple mutant A (SEQ ID NO: 4) and mutant toxin A drug substances. CD spectroscopy was also used to assess the secondary and tertiary structure of triple mutant B (SEQ ID NO: 6) and mutant toxin B drug substances. CD spectroscopy was also used to assess the potential effect of pH on structure. The effect of EDC treatment on triple mutant toxin A was analyzed by comparing CD data obtained from mutant toxin A drug substance with data obtained from triple mutant toxin A. The effect of EDC treatment on triple mutant toxin B (SEQ ID NO: 6) was analyzed by comparing CD data obtained from mutant toxin B drug substance with data obtained from triple mutant toxin B.

Far-UV CD data were obtained for the mutant toxin A drug substance at various pH values. Spectra recorded at pH 5.0-7.0 indicated a high proportion of α-helices in the secondary structure, suggesting that the polypeptide backbone of the protein adopts a well-defined conformation dominated by α-helices.

Near-UV CD spectra were also obtained for the mutant toxin A drug substance. The strongly negative ellipticity between 260 and 300 nm indicates that the aromatic side chains are in a unique rigid environment, i.e., the mutant toxin A drug substance has a tertiary structure. In fact, characteristic features originating from individual types of aromatic side chains can be distinguished in the spectrum: the shoulder at ~290 nm and the largest negative peak at ~283 nm are due to the absorption of polarized light by the ordered tryptophan side chains, the negative peak at 276 nm is from the tyrosine side chain, and the small shoulders at 262 and 268 nm indicate phenylalanine residues involved in three-dimensional contacts. The far- and near-UV results provide evidence that the mutant toxin A drug substance maintains a fully folded structure at physiological pH. The nearly identical far- and near-UV CD spectra observed at pH 5.0-7.0 indicate that no detectable structural changes occur within this pH range. CD data could not be collected at pH 3.0 and 4.0 because the protein is insoluble at these pH points. In comparative far- and near-UV CD spectra of mutant toxin A drug substance and triple mutant toxin A, the spectra of the two proteins were essentially identical under all experimental conditions studied, indicating that EDC treatment had no detectable effect on the secondary and tertiary structure of triple mutant toxin A. This finding is consistent with the gel filtration and analytical ultracentrifugation results, which showed no detectable changes in Stokes radius and sedimentation/friction coefficient, respectively.

The mutant toxin A drug substance (as well as triple mutant toxin A) contains 25 tryptophan residues, which are distributed throughout the primary sequence and can be used as convenient internal fluorescent probes. Fluorescence emission spectra of the mutant toxin A drug substance between 300-400 nm were obtained as a function of temperature. At 6.8°C, when excited at 280 nm, the mutant toxin A drug substance displayed a characteristic tryptophan fluorescence color spectrum. The fluorescence emission maximum was observed at ~335 nm, indicating that the tryptophan residues were in a non-polar environment, which is a typical internal environment of proteins rather than a polar water environment. The fluorescence emission spectrum results, together with the CD experimental results given in this report, confirmed that the mutant toxin A drug substance maintains a completely folded structure.

The fluorescence of the external probe 8-anilino-1-naphthalenesulfonic acid (ANS) was used to characterize possible conformational changes in mutant toxin A drug substance and triple mutant toxin A when pH was varied. As can be seen from the results, when mutant toxin A drug substance or triple mutant toxin A was titrated with this probe at pH 7.0, there was essentially no increase in the ANS fluorescence density, indicating that no hydrophobic surface was exposed on the protein under these conditions. Changing the pH to 2.6 resulted in a dramatic change in the ANS fluorescence quantum field as the probe concentration increased until the fluorescence quantum field reached apparent saturation. This increase in the ANS fluorescence quantum field indicates that at low pH (2.6), both mutant toxin A drug substance and triple mutant toxin A underwent pH-induced conformational changes that exposed the hydrophobic surface. Such conformational changes indicate that EDC-induced modification and inactivation of triple mutant toxin A does not limit the conformational plasticity of mutant toxin A drug substance (DS).

The effect of EDC treatment on the hydrodynamic properties of triple mutant toxin A was evaluated using size exclusion chromatography on a G4000SWXL column. Mutant toxin A drug substance and triple mutant toxin A were injected onto a G4000SWXL column equilibrated at pH 7.0, 6.0, and 5.0. The data indicated that no difference in Stokes radius between the mutant toxin A drug substance and triple mutant toxin A could be detected using size exclusion chromatography. Therefore, EDC treatment did not significantly affect the hydrodynamic properties of triple mutant toxin A and, consequently, its overall molecular shape.

Triple mutant toxin A and mutant toxin A drug substance were further analyzed using multi-angle laser light scattering (MALLS). Treatment of triple mutant toxin A with EDC resulted in a heterogeneous mixture composed of various multimeric and monomeric species. This heterogeneity reflects the introduction of numerous EDC-induced inter- and intramolecular covalent bonds (between carboxyl groups and primary amines of the protein).

The data obtained provide the physical and chemical characteristics of triple mutant toxin A and mutant toxin A drug substance (triple mutant toxin A treated with EDC) and describe the key features in its primary, secondary and tertiary structures. The data generated demonstrate that triple mutant toxin A treated with EDC results in covalent modification of its polypeptide chains without affecting the secondary and tertiary structures of the protein. Treatment with EDC results in intramolecular and intermolecular crosslinking. The biochemical and biophysical parameters obtained for mutant toxin A drug substance (and triple mutant toxin A) are presented in Table 60.

Far-UV CD data were obtained at various pH values for the mutant toxin B drug substance. Spectra recorded at pH 5.0-7.0 indicated a high proportion of α-helices in the secondary structure, suggesting that the polypeptide backbone of the protein adopts a well-defined conformation dominated by α-helices.

Near-UV CD spectra were also obtained for the mutant toxin B drug substance. The strongly negative ellipticity between 260 and 300 nm indicates that the aromatic side chains are in a unique rigid environment, i.e., the mutant toxin B drug substance has a tertiary structure. In fact, characteristic features originating from individual types of aromatic side chains can be distinguished in the spectrum: the shoulder at ~290 nm and the largest negative peak at ~283 nm are due to the absorption of polarized light by the ordered tryptophan side chain, the negative peak at 276 nm is from the tyrosine side chain, and the small shoulders at 262 and 268 nm indicate phenylalanine residues involved in three-dimensional contacts. The far- and near-UV results provide evidence that the mutant toxin B drug substance maintains a fully folded structure at physiological pH. The very similar far- and near-UV CD spectra observed at pH 5.0-7.0 indicate that no detectable structural changes occur within this pH range. CD data could not be collected at pH 3.0 and 4.0 because the protein is insoluble at these pH points.

In the comparative far- and near-UV CD spectra of mutant toxin B drug substance and triple mutant toxin B, the spectra of the two proteins were very similar between pH 5.0-7.0, indicating that EDC treatment had no detectable effect on the secondary and tertiary structure of the protein.

The triple mutant toxin B contains 16 tryptophan residues that are distributed throughout the primary sequence and can be used as a convenient internal fluorescent probe. The fluorescence emission spectrum of the mutant toxin B drug substance between 300-400 nm was obtained as a function of temperature. At 7°C, when excited at 280 nm, the mutant toxin B drug substance showed a characteristic tryptophan fluorescence color spectrum. The fluorescence emission maximum was observed at ~335 nm, indicating that the tryptophan residues are in a non-polar environment, which is a typical protein internal environment rather than a polar water environment. This result, together with the results of the CD experiment (see above), confirms that the mutant toxin B drug substance maintains a completely folded structure.

The fluorescence of the external probe 8-anilino-1-naphthalenesulfonic acid (ANS) was used to characterize possible conformational changes in mutant toxin B drug substance and triple mutant toxin B when pH was varied. As can be seen from the results, when mutant toxin B drug substance or triple mutant toxin B was titrated with this probe at pH 7.0, there was essentially no increase in the ANS fluorescence density, indicating that no hydrophobic surface was exposed on the protein under these conditions. Changing the pH to 2.6 resulted in a dramatic change in the ANS fluorescence quantum field (in the presence of mutant toxin B drug substance) as the probe concentration increased, until the fluorescence quantum field reached apparent saturation. This increase in the ANS fluorescence quantum field indicates that at low pH (2.6), the mutant toxin B drug substance underwent a pH-induced conformational change that exposed the hydrophobic surface. Such conformational changes indicate that EDC-induced modification and inactivation of triple mutant toxin B does not limit the conformational plasticity of mutant toxin B drug substance (DS).

The effect of EDC treatment on the hydrodynamic properties of triple mutant toxin B was evaluated using size exclusion chromatography on a G4000SWXL column. Mutant toxin B drug substance and triple mutant toxin B were injected onto a G4000SWXL column equilibrated at pH 7.0, 6.0, and 5.0. The data indicated that no differences in Stokes radius between mutant toxin B drug substance and triple mutant toxin B could be detected using size exclusion chromatography, and therefore, EDC treatment did not drastically affect the hydrodynamic properties of the protein, and consequently, its overall molecular shape.

Triple mutant toxin B and mutant toxin B drug substance were further analyzed using multi-angle laser light scattering (MALLS) technology. Treatment of triple mutant toxin B with EDC resulted in a more heterogeneous mixture composed of various multimeric and monomeric species. This heterogeneity reflects the introduction of numerous EDC-induced inter- and intramolecular covalent bonds (between carboxyl groups and primary amines of the protein).

The data obtained provide the physical and chemical characteristics of triple mutant toxin B and mutant toxin B drug substance (triple mutant toxin B treated with EDC) and describe the key features in its primary, secondary and tertiary structures. The data generated prove that triple mutant toxin B treated with EDC results in covalent modification of its polypeptide chains, but does not affect the secondary and tertiary structures of the protein. Treatment with EDC results in intramolecular and intermolecular crosslinking. The main biochemical and biophysical parameters obtained for mutant toxin B drug substance (and triple mutant toxin B) are given in Table 61.

Aspects of the Invention

The following items describe further embodiments of the present invention:

C1. An isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, and wherein the polypeptide comprises at least one amino acid side chain chemically modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

C2. An isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, and wherein the polypeptide comprises amino acid side chains chemically modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

C3. The isolated polypeptide according to item C1 or C2, wherein at least one aspartic acid residue side chain of said polypeptide or at least one glutamic acid residue side chain of said polypeptide is chemically modified with glycine.

C4. The isolated polypeptide according to any one of items C1 to C3, wherein the polypeptide comprises:

a) at least one cross-link between a side chain of an aspartic acid residue of said polypeptide and a side chain of a lysine residue of said polypeptide; and

b) at least one cross-link between a side chain of a glutamic acid residue of said polypeptide and a side chain of a lysine residue of said polypeptide.

C5. The isolated polypeptide according to any one of items C1 to C4, wherein said polypeptide comprises a β-alanine moiety attached to the side chain of at least one lysine residue of said polypeptide.

C6. The isolated polypeptide according to item C4, wherein said polypeptide comprises a glycine moiety attached to the side chain of an aspartic acid residue of said polypeptide or to the side chain of a glutamic acid residue of said polypeptide.

C7. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, and wherein the side chain of at least one lysine residue of the polypeptide is linked to a β-alanine moiety.

C8. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, and wherein the side chain of at least one lysine residue of the polypeptide is linked to a β-alanine moiety.

C9. The isolated polypeptide according to item C7 or C8, wherein the side chain of the second lysine residue of the polypeptide is linked to the side chain of an aspartic acid residue or to the side chain of a glutamic acid residue.

C10. The isolated polypeptide according to any one of items C7 to C9, wherein the side chain of an aspartic acid residue or a glutamic acid residue of the polypeptide is linked to a glycine moiety.

C11. The isolated polypeptide according to any one of items C1-C10, wherein said polypeptide has an _EC50 of at least about 100 μg/ml.

C12. An immunogenic composition comprising an isolated polypeptide having the amino acid sequence shown in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, and an isolated polypeptide having the amino acid sequence shown in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, and wherein the polypeptide has at least one amino acid side chain chemically modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

C13. The immunogenic composition according to item C12, wherein the polypeptide comprises at least one of any of the following:

a) at least one β-alanine moiety attached to a lysine side chain of the polypeptide;

b) at least one cross-link between the side chain of an aspartic acid residue and the side chain of a lysine residue of said polypeptide; and

c) at least one cross-link between a side chain of a glutamic acid residue and a side chain of a lysine residue of said polypeptide.

C14. The immunogenic composition of item C12, wherein said polypeptide has an EC ₅₀ of at least about 100 μg/ml.

C15. An immunogenic composition comprising an isolated polypeptide having an amino acid sequence as shown in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, and an isolated polypeptide having an amino acid sequence as shown in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, and

a) wherein at least one lysine residue of SEQ ID NO: 4 is side chain-linked to a β-alanine moiety, and

b) wherein at least one lysine residue of SEQ ID NO: 6 is side chain linked to a [beta]-alanine moiety.

C16. The immunogenic composition according to item C15, wherein the second lysine residue side chain of SEQ ID NO: 4 is linked to the side chain of an aspartic acid residue or to the side chain of a glutamic acid residue, and wherein the second lysine residue of SEQ ID NO: 6 is linked to the side chain of an aspartic acid residue or to the side chain of a glutamic acid residue.

C17. The immunogenic composition according to any one of items C12 to C16, wherein the side chain of the aspartic acid residue or the side chain of the glutamic acid residue of the polypeptide is linked to a glycine moiety, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent.

C18. The immunogenic composition according to any one of items C12 to C16, wherein the side chain of the aspartic acid residue or the side chain of the glutamic acid residue of the polypeptide is linked to a glycine moiety, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent.

C19. The immunogenic composition of any one of items C12-C18, wherein said polypeptide has an EC ₅₀ of at least about 100 μg/ml.

C20. An immunogenic composition comprising an isolated polypeptide having the amino acid sequence shown in SEQ ID NO: 84 and an isolated polypeptide having the amino acid sequence shown in SEQ ID NO: 86, wherein each polypeptide comprises:

a) at least one cross-link between a side chain of an aspartic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide;

b) at least one cross-link between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide;

c) a β-alanine moiety attached to the side chain of at least one lysine residue of said polypeptide; and

d) a glycine moiety attached to the side chain of at least one aspartic acid residue of the polypeptide or to the side chain of at least one glutamic acid residue of the polypeptide.

C21. An immunogenic composition comprising a mutant Clostridium difficile toxin A comprising a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation relative to corresponding wild-type Clostridium difficile toxin A.

C22. The composition of item C21, wherein the mutation is a non-conservative amino acid substitution.

C23. The composition of item C22, wherein the substitution comprises an alanine substitution.

C24. The composition of any one of items C21-C23, wherein the wild-type Clostridium difficile toxin A comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

C25. The composition of item C24, wherein the wild-type Clostridium difficile toxin A comprises a sequence that is at least 98% identical to SEQ ID NO: 1.

C26. The composition of item C25, wherein the wild-type Clostridium difficile toxin A comprises SEQ ID NO: 1.

C27. The composition of any one of items C21-C26, wherein the glucosyltransferase domain comprises at least two mutations.

C28. The composition according to item C27, wherein said at least two mutations are present at amino acid position 101, 269, 272, 285, 287, 269, 272, 460, 462, 541 or 542 according to the numbering of SEQ ID NO: 1.

C29. The composition of any one of items C21-C26, wherein said glucosyltransferase domain comprises SEQ ID NO: 29.

C30. The composition of item C29, wherein the glucosyltransferase domain comprises at least two non-conservative mutations, wherein the non-conservative mutations are present at amino acid positions 101, 269, 272, 285, 287, 269, 272, 460, 462, 541, or 542 of SEQ ID NO: 29, or any combination thereof.

C31. The composition of any one of items C21 to C26, wherein said cysteine protease domain comprises a mutation present at position 700, 589, 655, 543, or any combination thereof, according to the numbering of SEQ ID NO: 1.

C32. The composition of any one of items C21-C26, wherein said cysteine protease domain comprises SEQ ID NO: 32.

C33. The composition of item C32, wherein the cysteine protease domain comprises a non-conservative mutation, wherein the non-conservative mutation is present at position 1, 47, 113, 158 of SEQ ID NO: 32, or any combination thereof.

C34. The composition of item C21, wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 4.

C35. The composition of item C21, wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 84.

C36. The composition of item C21, wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 7.

C37. The composition of item C21, wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 83.

C38. The composition of any one of items C21-C33, wherein at least one amino acid of the mutant Clostridium difficile toxin A is chemically cross-linked.

C39. The composition of item C38, wherein the amino acids are chemically cross-linked by formaldehyde.

C40. The composition of item C38, wherein the amino acids are chemically cross-linked via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

C41. The composition of item C38 or C40, wherein the amino acids are chemically cross-linked via N-hydroxysuccinimide.

C42. The composition of any one of items C21 to C41, wherein said composition is recognized by an anti-toxin A neutralizing antibody or a binding fragment thereof.

C43. An immunogenic composition comprising a mutant Clostridium difficile toxin A, wherein, relative to the corresponding wild-type Clostridium difficile toxin A, the mutant Clostridium difficile toxin A comprises: a glucosyltransferase domain comprising SEQ ID NO: 29 having amino acid substitutions at positions 285 and 287; and a cysteine protease domain comprising SEQ ID NO: 32 having an amino acid substitution at position 158; wherein at least one amino acid of the mutant Clostridium difficile toxin A is chemically cross-linked.

C44. An immunogenic composition comprising SEQ ID NO: 4 or SEQ ID NO: 7, wherein at least one amino acid of SEQ ID NO: 4 or SEQ ID NO: 7 is chemically cross-linked.

C45. The composition of item C43 or C44, wherein at least one amino acid is cross-linked by formaldehyde.

C46. The composition of item C43 or C44, wherein the at least one amino acid is cross-linked via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

C47. The composition of item C43, C44, or C46, wherein at least one amino acid is cross-linked via N-hydroxysuccinimide.

C48. The composition of item C43 or C44, wherein said composition is recognized by an anti-toxin A neutralizing antibody or a binding fragment thereof.

C49. An immunogenic composition comprising SEQ ID NO:4.

C50. An immunogenic composition comprising SEQ ID NO:84.

C51. An immunogenic composition comprising SEQ ID NO:7.

C52. An immunogenic composition comprising SEQ ID NO: 83.

C53. The composition according to any one of items C49 to C52, wherein at least one amino acid is chemically cross-linked.

C54. The composition of any one of items C21-C51, wherein the composition exhibits reduced cytotoxicity relative to the corresponding wild-type Clostridium difficile toxin A.

C55. An isolated polypeptide comprising SEQ ID NO: 84.

C56. An isolated polypeptide comprising SEQ ID NO: 86.

C57. An isolated polypeptide comprising SEQ ID NO: 83.

C58. An isolated polypeptide comprising SEQ ID NO: 85.

C59. An immunogenic composition comprising a mutant Clostridium difficile toxin B comprising a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation relative to a corresponding wild-type Clostridium difficile toxin B.

C60. The composition of item C59, wherein the mutation is a non-conservative amino acid substitution.

C61. The composition of item C60, wherein the substitution comprises an alanine substitution.

C62. The composition of any one of items C59-C61, wherein the wild-type Clostridium difficile toxin B comprises a sequence that is at least 95% identical to SEQ ID NO: 2.

C63. The composition of item C62, wherein the wild-type Clostridium difficile toxin B comprises a sequence that is at least 98% identical to SEQ ID NO: 2.

C64. The composition of item C63, wherein the wild-type Clostridium difficile toxin B comprises SEQ ID NO: 2.

C65. The composition of any one of items C59-C64, wherein the glucosyltransferase domain comprises at least two mutations.

C66. The composition according to item C65, wherein said at least two mutations are present at amino acid position 102, 286, 288, 270, 273, 384, 461, 463, 520, or 543 according to the numbering of SEQ ID NO: 2.

C67. The composition of any one of items C59-C64, wherein said glucosyltransferase domain comprises SEQ ID NO: 31.

C68. The composition of item C67, wherein the glucosyltransferase domain comprises at least two non-conservative mutations, said non-conservative mutations being present at amino acid positions 102, 286, 288, 270, 273, 384, 461, 463, 520, or 543 of SEQ ID NO: 31.

C69. The composition according to any one of items C59 to C64, wherein said cysteine protease domain comprises mutations present at positions 698, 653, 587, 544, or any combination thereof, according to the numbering of SEQ ID NO: 2.

C70. The composition of any one of items C59-C64, wherein said cysteine protease domain comprises SEQ ID NO: 33.

C71. The composition of item C70, wherein said cysteine protease domain comprises a non-conservative mutation, said non-conservative mutation being present at position 1, 44, 110, 155 of SEQ ID NO: 33, or any combination thereof.

C72. The composition of item C59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 6.

C73. The composition of item C59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 86.

C74. The composition of item C59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 8.

C75. The composition of item C59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 85.

C76. The composition of any one of items C59-C71, wherein at least one amino acid of the mutant Clostridium difficile toxin B is chemically cross-linked.

C77. The composition of item C76, wherein the amino acids are chemically cross-linked by formaldehyde.

C78. The composition of item C76, wherein the amino acids are chemically cross-linked via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

C79. The composition of item C76 or C78, wherein said at least one amino acid is chemically cross-linked via N-hydroxysuccinimide.

C80. The composition of any one of items C59 to C79, wherein said composition is recognized by an anti-toxin B neutralizing antibody or a binding fragment thereof.

C81. An immunogenic composition comprising a mutant Clostridium difficile toxin B, wherein, relative to the corresponding wild-type Clostridium difficile toxin B, the mutant Clostridium difficile toxin B comprises: a glucosyltransferase domain comprising SEQ ID NO: 31 having amino acid substitutions at positions 286 and 288; and a cysteine protease domain comprising SEQ ID NO: 33 having an amino acid substitution at position 155; wherein at least one amino acid of the mutant Clostridium difficile toxin B is chemically cross-linked.

C82. An immunogenic composition comprising SEQ ID NO: 6 or SEQ ID NO: 8, wherein at least one amino acid of SEQ ID NO: 6 or SEQ ID NO: 8 is chemically cross-linked.

C83. The composition of item C81 or C82, wherein at least one amino acid is cross-linked by formaldehyde.

C84. The composition of item C81 or C82, wherein the at least one amino acid is cross-linked via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

C85. The composition of item C81, C82, or C84, wherein at least one amino acid is cross-linked via N-hydroxysuccinimide.

C86. The composition of item C81 or C82, wherein said composition is recognized by an anti-toxin B neutralizing antibody or a binding fragment thereof.

C87. An immunogenic composition comprising SEQ ID NO:6.

C88. An immunogenic composition comprising SEQ ID NO: 86.

C89. An immunogenic composition comprising SEQ ID NO:8.

C90. An immunogenic composition comprising SEQ ID NO:85.

C91. The composition of any one of items C59-C89, wherein the composition exhibits reduced cytotoxicity relative to the corresponding wild-type Clostridium difficile toxin B.

C92. An immunogenic composition comprising SEQ ID NO: 4 and an immunogenic composition comprising SEQ ID NO: 6, wherein at least one amino acid of each of SEQ ID NO: 4 and 6 is chemically cross-linked.

C93. An immunogenic composition comprising SEQ ID NO: 84 and an immunogenic composition comprising SEQ ID NO: 86, wherein at least one amino acid of each of SEQ ID NO: 84 and 86 is chemically cross-linked.

C94. The composition of item C92 or C93, wherein at least one amino acid is cross-linked by formaldehyde.

C95. The composition of item C92 or C93, wherein the at least one amino acid is cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

C96. The composition of item C92, C93, or C95, wherein at least one amino acid is cross-linked via N-hydroxysuccinimide.

C97. A recombinant cell or progeny thereof comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47.

C98. A recombinant cell or progeny thereof, comprising a nucleic acid sequence encoding SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

C99. A recombinant cell or progeny thereof, comprising a nucleic acid sequence encoding SEQ ID NO: 84.

C100. A recombinant cell or progeny thereof, comprising a nucleic acid sequence encoding SEQ ID NO: 86.

C101. A recombinant cell or progeny thereof, comprising a nucleic acid sequence encoding SEQ ID NO: 83.

C102. A recombinant cell or progeny thereof, comprising a nucleic acid sequence encoding SEQ ID NO: 85.

C103. The recombinant cell of item C97 or C98, wherein said cell is derived from a Gram-positive bacterial cell.

C104. The recombinant cell of item C97, C98, or C99, wherein said cell is derived from a Clostridium difficile cell.

C105. The recombinant cell of any one of items C97 to C104, wherein said cell lacks an endogenous polynucleotide encoding a toxin.

C106. The cell according to item C104 or C105, wherein the cell is derived from a Clostridium difficile cell selected from the group consisting of Clostridium difficile 1351, Clostridium difficile 3232, Clostridium difficile 7322, Clostridium difficile 5036, Clostridium difficile 4811, and Clostridium difficile VPI 11186.

C107. The cell of item C106, wherein the cell is a Clostridium difficile VPI 11186 cell.

C108. The cell of item C106 or C107, wherein the spore formation gene of the Clostridium difficile cell is inactivated.

C109. The cell of item C108, wherein the sporulation gene comprises a spo0A gene or a spoIIE gene.

C110. A method for producing a mutant Clostridium difficile toxin, comprising:

The recombinant cell or its progeny is cultured under suitable conditions to express a polynucleotide encoding a mutant Clostridium difficile toxin, wherein the cell comprises the polynucleotide encoding the mutant Clostridium difficile toxin, and wherein the mutant comprises a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation relative to a corresponding wild-type Clostridium difficile toxin.

C111. The method of item C110, wherein the cell lacks an endogenous polynucleotide encoding a toxin.

C112. The method of item C110, wherein the recombinant cell or its progeny comprises the cell of any one of items C97 to C111.

C113. The method of item C110, further comprising isolating the mutant Clostridium difficile toxin.

C114. The method of item C113, further comprising contacting the isolated mutant Clostridium difficile toxin with formaldehyde.

C115. The method of item C114, wherein said contacting occurs over a period of up to 14 days.

C116. The method of item C115, wherein said contacting occurs for a maximum of 48 hours.

C117. The method of item C114, wherein said contacting occurs at about 25°C.

C118. The method of item C113, further comprising contacting the isolated mutant Clostridium difficile toxin with ethyl-3-(3-dimethylaminopropyl)carbodiimide.

C119. The method of item C118, wherein said contacting occurs for a maximum of 24 hours.

C120. The method of item C120, wherein said contacting occurs for a maximum of 4 hours.

C121. The method of item C118, wherein said contacting occurs at about 25°C.

C122. The method of item C118, further comprising contacting the isolated mutant Clostridium difficile toxin with N-hydroxysuccinimide.

C123. The immunogenic composition produced by the method of any one of items C110-C122.

C124. A method for producing neutralizing antibodies against Clostridium difficile toxin A, comprising: administering to a mammal an immunogenic composition comprising SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, wherein at least one amino acid of SEQ ID NO: 4 is cross-linked by formaldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and recovering the antibody from the mammal.

C125. A method for producing neutralizing antibodies against Clostridium difficile toxin A, comprising: administering an immunogenic composition to a mammal, the immunogenic composition comprising SEQ ID NO: 84, wherein at least one amino acid of SEQ ID NO: 84 is cross-linked by formaldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and recovering antibodies from the mammal.

C126. A method for producing neutralizing antibodies against Clostridium difficile toxin B, comprising: administering to a mammal an immunogenic composition comprising SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, wherein at least one amino acid of SEQ ID NO: 6 is cross-linked by formaldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and recovering the antibody from the mammal.

C127. A method for producing neutralizing antibodies against Clostridium difficile toxin A, comprising: administering an immunogenic composition to a mammal, the immunogenic composition comprising SEQ ID NO: 86, wherein at least one amino acid of SEQ ID NO: 86 is cross-linked by formaldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and recovering the antibody from the mammal.

C128. An antibody or antibody binding fragment thereof specific for an immunogenic composition comprising SEQ ID NO: 4 wherein the methionine residue at position 1 is optionally absent, or SEQ ID NO: 7 wherein the methionine residue at position 1 is optionally absent.

C129. The antibody or antibody-binding fragment thereof according to item C128, wherein at least one amino acid in SEQ ID NO: 4 in which the methionine residue at position 1 is optionally absent, or at least one amino acid in SEQ ID NO: 7 in which the methionine residue at position 1 is optionally absent is cross-linked by formaldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

C130. An antibody or antibody-binding fragment thereof comprising: the amino acid sequence of the heavy chain complementarity determining region (CDR) shown in SEQ ID NO: 41 (CDR H1), SEQ ID NO: 42 (CDR H2) and SEQ ID NO: 43 (CDR H3), and the amino acid sequence of the light chain CDR shown in SEQ ID NO: 38 (CDR L1), SEQ ID NO: 39 (CDR L2) and SEQ ID NO: 40 (CDR L3).

C131. The antibody or antibody-binding fragment thereof according to item C128, C129, or C130, wherein the antibody or antibody-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 37; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 36.

C132. A composition comprising two or more antibodies or antibody-binding fragments thereof, wherein the antibodies or antibody-binding fragments thereof are selected from any of the antibodies or antibody-binding fragments thereof according to any one of items C128 to C131.

C133. An antibody or antibody binding fragment thereof specific for an immunogenic composition comprising: SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally absent, or SEQ ID NO: 8 wherein the methionine residue at position 1 is optionally absent.

C134. The antibody or antibody-binding fragment thereof according to item C133, wherein at least one amino acid in SEQ ID NO: 6 in which the methionine residue at position 1 is optionally absent, or at least one amino acid in SEQ ID NO: 8 in which the methionine residue at position 1 is optionally absent is cross-linked by formaldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

C135. An antibody or antibody-binding fragment thereof comprising: the amino acid sequence of the heavy chain complementarity determining region (CDR) shown in SEQ ID NO: 51 (CDRH1), SEQ ID NO: 52 (CDRH2) and SEQ ID NO: 53 (CDR H3), and the amino acid sequence of the light chain CDR shown in SEQ ID NO: 57 (CDR L1), SEQ ID NO: 58 (CDR L2) and SEQ ID NO: 59 (CDR L3).

C136. An antibody or antibody-binding fragment thereof, comprising: the amino acid sequences of the heavy chain complementarity determining regions (CDRs) shown in SEQ ID NO: 61 (CDR H1), SEQ ID NO: 62 (CDR H2) and SEQ ID NO: 63 (CDR H3), and the amino acid sequences of the light chain CDRs shown in SEQ ID NO: 68 (CDR L1), SEQ ID NO: 69 (CDR L2) and SEQ ID NO: 70 (CDR L3).

C137. An antibody or antibody-binding fragment thereof comprising: the amino acid sequences of the heavy chain complementarity determining regions (CDRs) shown in SEQ ID NO: 73 (CDR H1), SEQ ID NO: 74 (CDR H2) and SEQ ID NO: 75 (CDR H3), and the amino acid sequences of the light chain CDRs shown in SEQ ID NO: 79 (CDR L1), SEQ ID NO: 80 (CDR L2) and SEQ ID NO: 81 (CDR L3).

C138. A composition comprising two or more antibodies or antibody-binding fragments thereof, wherein the antibodies or antibody-binding fragments thereof are selected from any one of items C133 to C137.

C139. A method for treating a Clostridium difficile infection in a mammal, comprising: administering to the mammal an immunogenic composition comprising SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, and an immunogenic composition comprising SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, wherein each of SEQ ID NO: 4 and 6 has at least one amino acid cross-linked by formaldehyde.

C140. A method for treating a Clostridium difficile infection in a mammal, comprising: administering to the mammal an immunogenic composition comprising SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally absent, and an immunogenic composition comprising SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally absent, wherein SEQ ID NO: 4 and SEQ ID NO: 6 each have at least one amino acid cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

C141. A method for treating Clostridium difficile infection in a mammal, comprising administering to the mammal: an immunogenic composition comprising SEQ ID NO: 84, and an immunogenic composition comprising SEQ ID NO: 86, wherein SEQ ID NO: 84 and SEQ ID NO: 86 each have at least one amino acid cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

C142. A method for inducing an immune response to Clostridium difficile in a mammal, comprising administering to the mammal: an immunogenic composition comprising SEQ ID NO: 4 wherein the methionine residue at position 1 is optionally absent; and an immunogenic composition comprising SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally absent; wherein SEQ ID NO: 4 and SEQ ID NO: 6 each have at least one amino acid cross-linked by formaldehyde.

C143. A method for inducing an immune response to Clostridium difficile in a mammal, comprising administering to the mammal: an immunogenic composition comprising SEQ ID NO: 4 wherein the methionine residue at position 1 is optionally absent; and an immunogenic composition comprising SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally absent; wherein SEQ ID NO: 4 and SEQ ID NO: 6 each have at least one amino acid cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

C144. A method for inducing an immune response to Clostridium difficile in a mammal, comprising administering to the mammal: an immunogenic composition comprising SEQ ID NO: 84, and an immunogenic composition comprising SEQ ID NO: 86, wherein SEQ ID NO: 84 and SEQ ID NO: 86 each have at least one amino acid cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

C145. The method of any one of items C139 to C144, wherein the mammal is a mammal in need thereof.

C146. The method of any one of items C139-C144, wherein the mammal suffers from a recurrent Clostridium difficile infection.

C147. The method of any one of items C139-C144, wherein the composition is administered parenterally.

C148. The method of any one of items C139-C144, wherein the composition further comprises an adjuvant.

C149. The method of item C148, wherein the adjuvant comprises aluminum.

C150. The method of item C148, wherein the adjuvant comprises aluminum hydroxide gel and CpG oligonucleotides.

C151. The method of item C148, wherein the adjuvant comprises

Claims (9)

1. A method for generating an immunogenic mutant Clostridium difficile toxin, comprising: contacting a polypeptide having the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 84 and SEQ ID NO: 86 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for up to 3 hours; contacting said polypeptide with N-hydroxysuccinimide; and purifying the polypeptide contacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and contacted with N-hydroxysuccinimide, wherein said purification step includes filtration. 2. The method of claim 1, further comprising contacting the polypeptide with glycine. 3. The method of claim 1, further comprising contacting the polypeptide with alanine or glycine methyl ester. 4. The method of claim 1, wherein the contact is performed for a maximum of 2 hours. 5. The method of claim 1, wherein the polypeptide comprises at least one lysine residue. 6. The method of claim 1, wherein the polypeptide comprises at least one aspartic acid residue or at least one glutamic acid residue. 7. The method of claim 1, wherein the polypeptide contains at least one mutation relative to the corresponding wild-type Clostridium difficile toxin. 8. The method of claim 1, wherein the polypeptide consists of an amino acid sequence in which methionine at position 1 is absent, as shown in SEQ ID NO: 4. 9. The method of claim 1, wherein the polypeptide consists of an amino acid sequence in which methionine at position 1 is absent, as shown in SEQ ID NO: 6.