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

BACKGROUND

Clostridium difficile (C. difficile) is a Gram-positive anaerobic bacterium that is associated with gastrointestinal disease in humans. Colonization of C. difficile usually occurs in the colon if the natural gut flora is diminished by treatment with antibiotics. An infection can lead to antibiotic-associated diarrhea and sometimes pseudomembranous colitis through the secretion of the glucosylating toxins, toxin A and toxin B (308 and 270 kDa, respectively), which are the primary virulence factors of C. difficile.

Toxin A and toxin B are encoded within the 19 kb pathogenicity lozzs (PaLoc) by the genes tcdA and tcdB, respectively. Nonpathogenic strains of C. difficile have this locus replaced by an alternative 1 15 base pair sequence.

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

In the last decade, the numbers and severity of C. difficile outbreaks in hospitals, nursing homes, and other long-term care facilities increased dramatically. Key factors in this escalation include emergence of hypervirulent pathogenic strains, increased use of antibiotics, improved detection methods, and increased exposure to airborne spores in health care facilities.

Metronidazole and vancomycin represent the currently accepted standard of care for the antibiotic treatment of C. difficile associated disease (CDAD). However, about first episode of CDI, and up to about 50% of those patients suffer from additional recurrences. Treatment of recurrences represents a very significant challenge, and the majority of recurrences usually occur within one month of the preceding episode.

Accordingly, there is a need for immunogenic and/or therapeutic compositions and methods thereof directed to C. difficile.

SUMMARY OF THE INVENTION

These and other objectives are provided by the invention herein.

In one aspect, the invention relates to an immunogenic composition that includes a mutant C. difficile toxin A. The mutant C. difficile toxin A includes a

glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to the corresponding wild-type C. difficile toxin A. In one embodiment, at least one amino acid of the mutant C. difficile toxin A is chemically crosslinked.

In one aspect, the invention relates to an isolated polypeptide including the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, and wherein the polypeptide includes at least one amino acid side chain chemically modified by 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 crosslinked.

In one embodiment, the at least one amino acid amino acid is chemically crosslinked by 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 a respective anti-toxin neutralizing antibody or binding fragment thereof.

In one embodiment, the immunogenic composition exhibits decreased

cytotoxicity, relative to the corresponding wild-type C. difficile toxin.

In another aspect, the invention relates to an immunogenic composition that includes a mutant C. difficile toxin A, which includes a glucosyltransferase domain having SEQ ID NO: 29, which has an amino acid substitution at positions 285 and 287, and a cysteine protease domain having SEQ ID NO: 32, which has an amino acid substitution at position 158, relative to the corresponding wild-type C. difficile toxin A, wherein at least one amino acid of the mutant C. difficile toxin A is chemically

crosslinked.

In a further aspect, the invention relates to an immunogenic composition that includes a mutant C. difficile toxin A, which includes SEQ ID NO: 4, wherein at least one amino acid of the mutant C. difficile toxin A is chemically crosslinked.

In yet another aspect, the invention relates to an immunogenic composition that includes 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 that includes a mutant C. difficile toxin B. The mutant C. difficile toxin B includes a

glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to the corresponding wild-type C. difficile toxin B.

In another aspect, the invention relates to an isolated polypeptide including the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, and wherein the polypeptide includes an amino acid side chain chemically modified by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and N-Hydroxysuccinimide (NHS).

In another aspect, the invention relates to an immunogenic composition that includes a mutant C. difficile toxin B, which includes a glucosyltransferase domain having SEQ ID NO: 31 , which has an amino acid substitution at positions 286 and 288, and a cysteine protease domain having SEQ ID NO: 33, which has an amino acid substitution at position 155, relative to the corresponding wild-type C. difficile toxin B, wherein at least one amino acid of the mutant C. difficile toxin B is chemically

crosslinked.

In a further aspect, the invention relates to an immunogenic composition that includes a mutant C. difficile toxin B, which includes SEQ ID NO: 6, wherein at least one amino acid of the mutant C. difficile toxin B is chemically crosslinked.

In one aspect, the invention relates to an immunogenic composition that includes a mutant C. difficile toxin A, which includes SEQ ID NO: 4, and a mutant C. difficile toxin

B, which includes SEQ ID NO: 6, wherein at least one amino acid of each of the mutant

C. difficile toxins is chemically crosslinked.

In further aspects, the invention relates to a recombinant cell or progeny thereof, that includes a polynucleotide encoding any of the foregoing mutant C. difficile toxins, wherein the cell lacks an endogenous polynucleotide encoding a toxin.

In another aspect, the invention relates to an antibody or antibody binding fragment thereof specific to an immunogenic composition that includes a mutant C. difficile toxin.

In one aspect, the invention relates to a method of treating a C. difficile infection in a mammal. The method includes administering to the mammal an immunogenic composition that includes a mutant C. difficile toxin A, which includes SEQ ID NO: 4, and a mutant C. difficile toxin B, which includes SEQ ID NO: 6, wherein at least one amino acid of each of the mutant C. difficile toxins is crosslinked by formaldehyde.

In another aspect, the method of treating a C. difficile infection in a mammal includes administering to the mammal an immunogenic composition that includes a mutant C. difficile toxin A, which includes SEQ ID NO: 4, and a mutant C. difficile toxin

B, which includes SEQ ID NO: 6, wherein at least one amino acid of each of the mutant

C. difficile toxins is crosslinked by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide and/or N-Hydroxysuccinimide (NHS).

In one aspect, the invention relates to a method of inducing an immune response to a C. difficile infection in a mammal. The method includes administering to the mammal an immunogenic composition that includes a mutant C. difficile toxin A, which includes SEQ ID NO: 4, and a mutant C. difficile toxin B, which includes SEQ ID NO: 6, wherein at least one amino acid of each of the mutant C. difficile toxins is crosslinked by formaldehyde.

In another aspect, the method of inducing an immune response to a C. difficile infection in a mammal includes administering to the mammal an immunogenic composition that includes a mutant C. difficile toxin A, which includes SEQ ID NO: 4, and a mutant C. difficile toxin B, which includes SEQ ID NO: 6, wherein at least one amino acid of each of the mutant C. difficile toxins is crosslinked by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide and/or N-Hydroxysuccinimide (NHS).

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

In one embodiment, the methods of treating or the methods of inducing an immune response includes a mammal that has had a recurring C. difficile infection.

In one embodiment, the methods of treating or the methods of inducing an immune response includes parenterally administering the composition.

In one embodiment, the methods of treating or the methods of inducing an immune response includes an immunogenic composition that further includes an adjuvant.

In one embodiment, the adjuvant includes aluminum hydroxide gel and a CpG oligonucleotide. In another embodiment, the adjuvant includes ISCOMATRIX.

In one embodiment, the isolated polypeptide includes at least one side chain of an aspartic acid residue of the polypeptide or at least one side chain of a glutamic acid residue of the polypeptide is chemically modified by glycine.

In one embodiment, the isolated polypeptide includes at least one crosslink 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 crosslink 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 includes a beta-alanine moiety linked to a side chain of at least one lysine residue of the polypeptide.

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

In one embodiment, the isolated polypeptide includes the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, and wherein a side chain of at least one lysine residue of the polypeptide is linked to a beta-alanine moiety.

In one embodiment, the isolated polypeptide includes the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, and wherein a side chain of at least one lysine residue of the polypeptide is linked to a beta-alanine moiety.

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

In one embodiment, the isolated polypeptide includes a side chain of an aspartic acid residue or a side chain of a glutamic acid residue of the polypeptide is 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 includes an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, and an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, and wherein the polypeptides have at least one amino acid side chain chemically modified by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and N-Hydroxysuccinimide (NHS).

In one embodiment, the polypeptide includes at least one of any of: a) a) at least one beta-alanine moiety linked to a side chain of a lysine residue of the

polypeptide; b) at least one crosslink between a side chain of a lysine residue of the polypeptide and a side chain of an aspartic acid residue; and c) at least one crosslink between a side chain of a lysine residue of the polypeptide and a side chain of a glutamic acid residue.

In one embodiment, the isolated polypeptide has an EC50 of at least about 100 μg/ml.

In one aspect, the immunogenic composition includes an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, and an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, and a) wherein a side chain of at least one lysine residue of SEQ ID NO: 4 is linked to a beta-alanine moiety, and b) wherein a side chain of at least one lysine residue of SEQ ID NO: 6 is linked to a beta-alanine moiety.

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

In one embodiment, the immunogenic composition includes a side chain of an aspartic acid residue or a side chain of a glutamic acid residue of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, is linked to a glycine moiety.

In one embodiment, the immunogenic composition includes a side chain of an aspartic acid residue or a side chain of a glutamic acid residue of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, is 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 includes an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 84 and an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 86, wherein each polypeptide includes a) at least one crosslink 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 crosslink 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 beta-alanine moiety linked to a side chain of at least one lysine residue of the polypeptide; and d) a glycine moiety linked to a side chain of at least one aspartic acid residue of the polypeptide or to a side chain of at least one glutamic acid residue of the polypeptide.

BRIEF DESCRIPTION OF DRAWINGS

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

CLUSTALW alignment, default parameters.

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

CLUSTALW alignment, default parameters.

Figure 3: Graph showing identification of wild-type toxin-negative C. difficile strains. Culture media of 13 C. difficile strains were tested by ELISA for toxin A. As illustrated, seven strains expressed toxin A and 6 strains did not (strains 1351 , 3232, 7322, 5036, 481 1 and VPI 1 1 186).

Figure 4 A and B: SDS-PAGE results illustrating that triple mutant A (SEQ ID NO: 4), double mutant B (SEQ ID NO: 5), and triple mutant B (SEQ ID NO: 6) do not glucosylate Rac1 or RhoA GTPases in an in vitro glucosylation assays with UDP-14C-glucose; whereas 10 pg to 1 ng of wild type toxin B does glucosylate Rac1 .

Figure 5: Western blot indicating abrogation of cysteine protease activity in mutant toxins A and B (SEQ ID NOs: 4 and 6, respectively), as compared to

observation of cleaved fragments of wild-type toxins A and B (SEQ ID NOs: 1 and 2, respectively). See Example 13.

Figure 6: Graphs 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., about 100 μg/ml) by in vitro cytotoxicity assay in IMR-90 cells.

Figure 7: Graph showing that EC50 values are similar for the triple mutant toxin B (SEQ ID NO: 6) and hepta mutant toxin B (SEQ ID NO: 8).

Figure 8: Graph representing results from in vitro cytotoxicity tests in which the ATP levels (RLUs) are plotted against increasing concentrations of the triple mutant TcdA (SEQ ID NO: 4)(top panel) and triple mutant TcdB (SEQ ID NO: 6)(bottom panel). Residual cytotoxicity of mutant toxin A and B can be completely abrogated with neutralizing antibodies specific for mutant toxin A (top panel-pAb A and mAbs A3-25 + A60-22) and mutant toxin B (bottom panel-pAb B).

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

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

Figure 1 1 : Graph showing neutralizing antibody titers as described in Example 26 (study muCdiff2010-07).

Figure 12: Graph showing neutralizing antibody responses against toxins A and B in hamsters after four immunizations as described in Example 27 (study hamC.

difficult 2010-02)

Figure 13: Graph showing neutralizing antibody responses in hamsters after vaccination with chemically inactivated genetic mutant toxins and List Biological toxoids, as described in Example 27 (study hamC. difficile 2010-02).

Figure 14: Survival curves for three immunized groups of hamsters as compared to the non-immunized controls, described in Example 28 (study hamC. difficile2010-02, continued).

Figure 15: Graph showing relative neutralizing antibody response against different formulations of C. difficile mutant toxins in hamsters (study hamC. difficile 2010-03), as described in Example 29.

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

Figure 17: Amino acid sequences of variable regions of light (VL) and heavy (HL) chains of A3-25 mAb IgE. Signal peptide - highlighted; CDRs - italicized and

underlined; Constant region - bolded and underlined (complete sequence not shown).

Figure 18: Graph showing titration of individual toxin A monoclonal antibodies in the toxin neutralization assay using ATP levels (quantified by relative light units- RLU) as an indicator of cell viability. In comparison to the toxin (4xEC50) control, mAbs A80-29, A65-33, A60-22 and A3-25 had increasing neutralizing effects on toxin A with concentration 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 RLUs.

Figure 19: Mapping of 8 epitope groups of toxin B mAbs by BiaCore Figure 20A-C: Synergistic neutralizing activities of combinations of toxin A mAbs: Adding different dilutions of neutralizing antibodies A60-22, A65-33, and A80-29 to increasing concentrations of A3-25 mAb synergistically increased the neutralization of toxin A regardless of the dilution. The RLUs of the toxin A only (4x EC50) control is illustrated (<0.3x106) and cell only controls were 2-2.5 x106 RLUs as depicted in graphs shown in Figure 20B and Figure 20C.

Figure 21 : Synergistic neutralizing activities of toxin B mAbs: Neutralization of toxin B by mAbs 8-26, B60-2 and B59-3 is illustrated in Figure 21A. Neutralization of toxin B is synergistically increased after combining B8-26 with dilutions of B59-3 (Figure 21 B)

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

Figure 23A-K: Graph representing results from in vitro cytotoxicity tests in which the ATP levels (RLUs) are plotted against increasing concentrations of C. difficile culture media and the hamster serum pool (■);crude toxin (culture harvest) from the respective strain and the hamster serum pool (●); purified toxin (commercial toxin obtained from List Biologicals) and the hamster serum pool (▲); crude toxin (τ), control; and purified toxin (♦), control. The toxins from the respective strains were added to the cells at 4xEC50 values. Figure 23 shows that an immunogenic composition including mutant TcdA (SEQ ID NO: 4) and mutant TcdB (SEQ ID NO: 6), wherein the mutant toxins were inactivated with EDC, according to, for example, Example 29, Table

15, described herein, induced neutralizing antibodies that exhibited neutralizing activity against toxins from at least the following 16 different CDC strains of C. difficile, in comparison to the respective toxin only control: 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).

Figure 24: Illustration of an exemplary EDC/NHS inactivation of mutant C. difficile toxins, resulting in at least three possible types of modifications: crosslinks, glycine adducts, and beta-alanine adducts. Panel A illustrates crosslinking. Carboxylic

residues of triple mutant toxins are activated by the addition of EDC and NHS. The activated esters react with primary amines to form stable amide bonds, resulting in intra-and intermolecular crosslinks. Panel B illustrates formation of glycine adducts. After inactivation, residual activated esters are quenched by the addition of glycine to form stable amide bonds. Panel C illustrates formation of beta-alanine adducts. Three moles of NHS can react with one mole of EDC to form activated beta-alanine. This then reacts with primary amines to form stable amide bonds.

Figure 25: Illustration of an exemplary EDC/NHS inactivation of mutant C. difficile toxins, resulting in at least one of the following types of modifications: (A) crosslinks, (B) glycine adducts, and (C) beta-alanine adducts.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 sets forth the amino acid sequence for wild-type C. difficile 630 toxin A (TcdA).

SEQ ID NO: 2 sets forth the amino acid sequence for wild-type C. difficile 630 toxin B (TcdB).

SEQ ID NO: 3 sets forth the amino acid sequence for a mutant TcdA having a mutation at positions 285 and 287, as compared to SEQ ID NO: 1 .

SEQ ID NO: 4 sets forth the amino acid sequence for a mutant TcdA having a mutation at positions 285, 287, and 700, as compared to SEQ ID NO: 1 .

SEQ ID NO: 5 sets forth the amino acid sequence for a mutant TcdB having a mutation at positions 286 and 288, as compared to SEQ ID NO: 2.

SEQ ID NO: 6 sets forth the amino acid sequence for a mutant TcdB having a mutation at positions 286, 288, and 698, as compared to SEQ ID NO: 2.

SEQ ID NO: 7 sets forth the amino acid sequence for a mutant TcdA having a mutation at positions 269, 272, 285, 287, 460, 462, and 700, as compared to SEQ ID NO: 1 SEQ ID NO: 8 sets forth the amino acid sequence for a mutant TcdB having a mutation at positions 270, 273, 286, 288, 461 463, and 698, as compared to SEQ ID NO: 2 SEQ ID NO: 9 sets forth a DNA sequence encoding a wild-type C. difficile 630 toxin A (TcdA).

SEQ ID NO: 10 sets forth a DNA sequence encoding a wild-type C. difficile 630 toxin B (TcdB).

SEQ ID NO: 1 1 sets forth a DNA sequence encoding SEQ ID NO: 3

SEQ ID NO: 12 sets forth a DNA sequence encoding SEQ ID NO: 4

SEQ ID NO: 13 sets forth a DNA sequence encoding SEQ ID NO: 5

SEQ ID NO: 14 sets forth a DNA sequence encoding SEQ ID NO: 6

SEQ ID NO: 15 sets forth the amino acid sequence for wild-type C. difficile R20291

TcdA.

SEQ ID NO: 16 sets forth a DNA sequence encoding SEQ ID NO: 15.

SEQ ID NO: 17 sets forth the amino acid sequence for wild-type C. difficile CD196

TcdA.

SEQ ID NO: 18 sets forth a DNA sequence encoding SEQ ID NO: 17.

SEQ ID NO: 19 sets forth the amino acid sequence for wild-type C. difficile VPI10463

TcdA.

SEQ ID NO: 20 sets forth a DNA sequence encoding SEQ ID NO: 19.

SEQ ID NO: 21 sets forth the amino acid sequence for wild-type C. difficile R20291

TcdB.

SEQ ID NO: 22 sets forth a DNA sequence encoding SEQ ID NO: 21 .

SEQ ID NO: 23 sets forth the amino acid sequence for wild-type C. difficile CD196

TcdB.

SEQ ID NO: 24 sets forth a DNA sequence encoding SEQ ID NO: 23.

SEQ ID NO: 25 sets forth the amino acid sequence for wild-type C. difficile VPI10463

TcdB.

SEQ ID NO: 26 sets forth a DNA sequence encoding SEQ ID NO: 25.

SEQ ID NO: 27 sets forth a DNA sequence of a pathogenicity locus of wild-type C.

VPI10463 difficult.

SEQ ID NO: 28 sets forth the amino acid sequence for residues 101 to 293 of SEQ ID NO: 1 .

SEQ ID NO: 29 sets forth the amino acid sequence for residues 1 to 542 of SEQ ID NO: 1 .

SEQ ID NO: 30 sets forth the amino acid sequence for residues 101 to 293 of SEQ ID NO: 2.

SEQ ID NO: 31 sets forth the amino acid sequence for residues 1 to 543 of SEQ ID NO: 2.

SEQ ID NO: 32 sets forth the amino acid sequence for residues 543 to 809 of SEQ ID NO: 1 .

SEQ ID NO: 33 sets forth the amino acid sequence for residues 544 to 767 of SEQ ID NO: 2.

SEQ ID NO: 34 sets forth the amino acid sequence for a mutant TcdA, wherein residues 101 , 269, 272, 285, 287, 460, 462, 541 , 542, 543, 589, 655, and 700 may be any amino acid.

SEQ ID NO: 35 sets forth the amino acid sequence for a mutant TcdB, wherein 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: 36 sets forth the amino acid sequence for the variable light chain of a neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 37 sets forth the amino acid sequence for the variable heavy chain of a neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 38 sets forth the amino acid sequence for CDR1 of the variable light chain of neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 39 sets forth the amino acid sequence for CDR2 of the variable light chain of neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 40 sets forth the amino acid sequence for CDR3 of the variable light chain of neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 41 sets forth the amino acid sequence for CDR1 of the variable heavy chain of neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 42 sets forth the amino acid sequence for CDR2 of the variable heavy chain of neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 43 sets forth the amino acid sequence for CDR3 of the variable heavy chain of neutralizing antibody of C. difficile TcdA (A3-25 mAb).

SEQ ID NO: 44 sets forth a DNA sequence encoding SEQ ID NO: 3.

SEQ ID NO: 45 sets forth a DNA sequence encoding SEQ ID NO: 4.

SEQ ID NO: 46 sets forth a DNA sequence encoding SEQ ID NO: 5.

SEQ ID NO: 47 sets forth a DNA sequence encoding SEQ ID NO: 6.

SEQ ID NO: 48 sets forth the nucleotide sequence of immunostimulatory

oligonucleotide ODN CpG 24555.

SEQ ID NO: 49 sets forth the amino acid sequence for the variable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 50 sets forth the amino acid sequence for the signal peptide of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 51 sets forth the amino acid sequence for CDR1 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 52 sets forth the amino acid sequence for CDR2 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 53 sets forth the amino acid sequence for CDR3 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 54 sets forth the amino acid sequence for the constant region of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 55 sets forth the amino acid sequence for the variable light chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 56 sets forth the amino acid sequence for the signal peptide of the variable light chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 57 sets forth the amino acid sequence for CDR1 of the variable light chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 58 sets forth the amino acid sequence for CDR2 of the variable light chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 59 sets forth the amino acid sequence for CDR3 of the variable light chain of a C. difficile TcdB neutralizing antibody (B8-26 mAb).

SEQ ID NO: 60 sets forth the amino acid sequence for the variable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 61 sets forth the amino acid sequence for the signal peptide of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 62 sets forth the amino acid sequence for CDR1 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 63 sets forth the amino acid sequence for CDR2 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 64 sets forth the amino acid sequence for CDR3 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 65 sets forth the amino acid sequence for the constant region of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 66 sets forth the amino acid sequence for the variable light chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 67 sets forth the amino acid sequence for the signal peptide of the variable light chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 68 sets forth the amino acid sequence for CDR1 of the variable light chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 69 sets forth the amino acid sequence for CDR2 of the variable light chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 70 sets forth the amino acid sequence for CDR3 of the variable light chain of a C. difficile TcdB neutralizing antibody (B59-3 mAb).

SEQ ID NO: 71 sets forth the amino acid sequence for the variable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 72 sets forth the amino acid sequence for the signal peptide of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 73 sets forth the amino acid sequence for CDR1 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 74 sets forth the amino acid sequence for CDR2 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 75 sets forth the amino acid sequence for CDR3 of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 76 sets forth the amino acid sequence for the constant region of the variable heavy chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 77 sets forth the amino acid sequence for the variable light chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 78 sets forth the amino acid sequence for the signal peptide of the variable light chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 79 sets forth the amino acid sequence for CDR1 of the variable light chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 80 sets forth the amino acid sequence for CDR2 of the variable light chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 81 sets forth the amino acid sequence for CDR3 of the variable light chain of a C. difficile TcdB neutralizing antibody (B9-30 mAb).

SEQ ID NO: 82 sets forth the amino acid sequence for a mutant TcdB, wherein a residue 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 sets forth the amino acid sequence for a mutant TcdA having a mutation at positions 269, 272, 285, 287, 460, 462, and 700, as compared to SEQ ID NO: 1 , wherein the methionine at position 1 is absent.

SEQ ID NO: 84 sets forth the amino acid sequence for a mutant C. difficile toxin A having a mutation at positions 285, 287, and 700, as compared to SEQ ID NO: 1 , wherein the methionine at position 1 is absent.

SEQ ID NO: 85 sets forth the amino acid sequence for a mutant C. difficile toxin B having a mutation at positions 270, 273, 286, 288, 461 , 463, and 698, as compared to SEQ ID NO: 2, wherein the methionine at position 1 is absent.

SEQ ID NO: 86 sets forth the amino acid sequence for a mutant C. difficile toxin B having a mutation at positions 286, 288, and 698, as compared to SEQ ID NO: 2, wherein the methionine at position 1 is absent.

SEQ ID NO: 87 sets forth the amino acid sequence for wild-type C. difficile 2004013 TcdA.

SEQ ID NO: 88 sets forth the amino acid sequence for wild-type C. difficile 20041 1 1 TcdA.

SEQ ID NO: 89 sets forth the amino acid sequence for wild-type C. difficile 20041 18 TcdA.

SEQ ID NO: 90 sets forth the amino acid sequence for wild-type C. difficile 2004205 TcdA.

SEQ ID NO: 91 sets forth the amino acid sequence for wild-type C. difficile 2004206 TcdA.

SEQ ID NO: 92 sets forth the amino acid sequence for wild-type C. difficile 2005022 TcdA.

SEQ ID NO: 93 sets forth the amino acid sequence for wild-type C. difficile 2005088 TcdA.

SEQ ID NO: 94 sets forth the amino acid sequence for wild-type C. difficile 2005283 TcdA.

SEQ ID NO: 95 sets forth the amino acid sequence for wild-type C. difficile 2005325 TcdA.

SEQ ID NO: 96 sets forth the amino acid sequence for wild-type C. difficile 2005359 TcdA.

SEQ ID NO: 97 sets forth the amino acid sequence for wild-type C. difficile 2006017 TcdA.

SEQ ID NO: 98 sets forth the amino acid sequence for wild-type C. difficile 2007070 TcdA.

SEQ ID NO: 99 sets forth the amino acid sequence for wild-type C. difficile 2007217 TcdA.

SEQ ID NO: 100 sets forth the amino acid sequence for wild-type C. difficile 2007302 TcdA.

SEQ ID NO: 101 sets forth the amino acid sequence for wild-type C. difficile 2007816 TcdA.

SEQ ID NO: 102 sets forth the amino acid sequence for wild-type C. difficile 2007838 TcdA.

SEQ ID NO: 103 sets forth the amino acid sequence for wild-type C. difficile 2007858 TcdA.

SEQ ID NO: 104 sets forth the amino acid sequence for wild-type C. difficile 2007886 TcdA.

SEQ ID NO: 105 sets forth the amino acid sequence for wild-type C. difficile 2008222 TcdA.

SEQ ID NO: 106 sets forth the amino acid sequence for wild-type C. difficile 2009078 TcdA.

SEQ ID NO: 107 sets forth the amino acid sequence for wild-type C. difficile 2009087 TcdA.

SEQ ID NO: 108 sets forth the amino acid sequence for wild-type C. difficile 2009141 TcdA.

SEQ ID NO: 109 sets forth the amino acid sequence for wild-type C. difficile 2009292 TcdA.

SEQ ID NO: 1 10 sets forth the amino acid sequence for wild-type C. difficile 2004013 TcdB.

SEQ ID NO: 1 1 1 sets forth the amino acid sequence for wild-type C. difficile 20041 1 1 TcdB.

SEQ ID NO: 1 12 sets forth the amino acid sequence for wild-type C. difficile 20041 18 TcdB.

SEQ ID NO: 1 13 sets forth the amino acid sequence for wild-type C. difficile 2004205 TcdB.

SEQ ID NO: 1 14 sets forth the amino acid sequence for wild-type C. difficile 2004206 TcdB.

SEQ ID NO: 1 15 sets forth the amino acid sequence for wild-type C. difficile 2005022 TcdB.

SEQ ID NO: 1 16 sets forth the amino acid sequence for wild-type C. difficile 2005088 TcdB.

SEQ ID NO: 1 17 sets forth the amino acid sequence for wild-type C. difficile 2005283 TcdB.

SEQ ID NO: 1 18 sets forth the amino acid sequence for wild-type C. difficile 2005325 TcdB.

SEQ ID NO: 1 19 sets forth the amino acid sequence for wild-type C. difficile 2005359 TcdB.

SEQ ID NO: 120 sets forth the amino acid sequence for wild-type C. difficile 2006017 TcdB.

SEQ ID NO: 121 sets forth the amino acid sequence for wild-type C. difficile 2006376 TcdB.

SEQ ID NO: 122 sets forth the amino acid sequence for wild-type C. difficile 2007070 TcdB.

SEQ ID NO: 123 sets forth the amino acid sequence for wild-type C. difficile 2007217 TcdB.

SEQ ID NO: 124 sets forth the amino acid sequence for wild-type C. difficile 2007302 TcdB.

SEQ ID NO: 125 sets forth the amino acid sequence for wild-type C. difficile 2007816 TcdB.

SEQ ID NO: 126 sets forth the amino acid sequence for wild-type C. difficile 2007838 TcdB.

SEQ ID NO: 127 sets forth the amino acid sequence for wild-type C. difficile 2007858 TcdB.

SEQ ID NO: 128 sets forth the amino acid sequence for wild-type C. difficile 2007886 TcdB.

SEQ ID NO: 129 sets forth the amino acid sequence for wiId-type C. difficile 2008222

TcdB.

SEQ ID NO: 130 sets forth the amino acid sequence for wi Id -type C. difficile 2009078

TcdB.

SEQ ID NO: 131 sets forth the amino acid sequence for wi Id -type C. difficile 2009087

TcdB.

SEQ ID NO: 132 sets forth the amino acid sequence for wi Id -type C. difficile 2009141

TcdB.

SEQ ID NO: 133 sets forth the amino acid sequence for wi Id -type C. difficile 2009292

TcdB.

SEQ ID NO: 134 sets forth the amino acid sequence for wi Id -type C. difficile 014 TcdA.

SEQ ID NO: 135 sets forth the amino acid sequence for wi Id -type C. difficile 015 TcdA.

SEQ ID NO: 136 sets forth the amino acid sequence for wi Id -type C. difficile 020 TcdA.

SEQ ID NO: 137 sets forth the amino acid sequence for wi Id -type C. difficile 023 TcdA.

SEQ ID NO: 138 sets forth the amino acid sequence for wi Id -type C. difficile 027 TcdA.

SEQ ID NO: 139 sets forth the amino acid sequence for wi Id -type C. difficile 029 TcdA.

SEQ ID NO: 140 sets forth the amino acid sequence for wi Id -type C. difficile 046 TcdA.

SEQ ID NO: 141 sets forth the amino acid sequence for wi Id -type C. difficile 014 TcdB.

SEQ ID NO: 142 sets forth the amino acid sequence for wi Id -type C. difficile 015 TcdB.

SEQ ID NO: 143 sets forth the amino acid sequence for wi Id -type C. difficile 020 TcdB.

SEQ ID NO: 144 sets forth the amino acid sequence for wi Id -type C. difficile 023 TcdB.

SEQ ID NO: 145 sets forth the amino acid sequence for wi Id - ype C. difficile 027 TcdB.

SEQ ID NO: 146 sets forth the amino acid sequence for wi Id -type C. difficile 029 TcdB.

SEQ ID NO: 147 sets forth the amino acid sequence for wi Id -type C. difficile 046 TcdB.

SEQ ID NO: 148 sets forth the amino acid sequence for wi Id -type C. difficile 001 TcdA.

SEQ ID NO: 149 sets forth the amino acid sequence for wi Id -type C. difficile 002 TcdA.

SEQ ID NO: 150 sets forth the amino acid sequence for wi Id -type C. difficile 003 TcdA.

SEQ ID NO: 151 sets forth the amino acid sequence for wi Id -type C. difficile 004 TcdA.

SEQ ID NO: 152 sets forth the amino acid sequence for wi Id -type C. difficile 070 TcdA.

SEQ ID NO: 153 sets forth the amino acid sequence for wi Id -type C. difficile 075 TcdA.

SEQ ID NO: 154 sets forth the amino acid sequence for wi Id -type C. difficile 077 TcdA.

SEQ ID NO: 155 sets forth the amino acid sequence for wi Id -type C. difficile 081 TcdA.

SEQ ID NO: 156 sets forth the amino acid sequence for wi Id -type C. difficile 117 TcdA.

SEQ ID NO: 157 sets forth the amino acid sequence for wild-type C. difficile 131 TcdA

SEQ ID NO: 158 sets forth the amino acid sequence for wild -type C. difficile 001 TcdB

SEQ ID NO: 159 sets forth the amino acid sequence for wild -type C. difficile 002 TcdB

SEQ ID NO: 160 sets forth the amino acid sequence for wild -type C. difficile 003 TcdB

SEQ ID NO: 161 sets forth the amino acid sequence for wild -type C. difficile 004 TcdB

SEQ ID NO: 162 sets forth the amino acid sequence for wild -type C. difficile 070 TcdB

SEQ ID NO: 163 sets forth the amino acid sequence for wild -type C. difficile 075 TcdB

SEQ ID NO: 164 sets forth the amino acid sequence for wild -type C. difficile 077 TcdB

SEQ ID NO: 165 sets forth the amino acid sequence for wild -type C. difficile 081 TcdB

SEQ ID NO: 166 sets forth the amino acid sequence for wild -type C. difficile 1 17 TcdB

SEQ ID NO: 167 sets forth the amino acid sequence for wild -type C. difficile 131 TcdB

SEQ ID NO: 168 sets forth the amino acid sequence for wild -type C. difficile 053 TcdA

SEQ ID NO: 169 sets forth the amino acid sequence for wild -type C. difficile 078 TcdA

SEQ ID NO: 170 sets forth the amino acid sequence for wild -type C. difficile 087 TcdA

SEQ ID NO: 171 sets forth the amino acid sequence for wild -type C. difficile 095 TcdA

SEQ ID NO: 172 sets forth the amino acid sequence for wild -type C. difficile 126 TcdA

SEQ ID NO: 173 sets forth the amino acid sequence for wild -type C. difficile 053 TcdB

SEQ ID NO: 174 sets forth the amino acid sequence for wild -type C. difficile 078 TcdB

SEQ ID NO: 175 sets forth the amino acid sequence for wild -type C. difficile 087 TcdB

SEQ ID NO: 176 sets forth the amino acid sequence for wild -type C. difficile 095 TcdB

SEQ ID NO: 177 sets forth the amino acid sequence for wild -type C. difficile 126 TcdB

DETAILED DESCRIPTION

The inventors surprisingly discovered, among other things, a mutant C. difficile toxin A and toxin B, and methods thereof. The mutants are characterized, in part, by being immunogenic and exhibiting reduced cytotoxicity compared to a wild-type form of the respective toxin. The present invention also relates to immunogenic portions thereof, biological equivalents thereof, and isolated polynucleotides that include nucleic acid sequences encoding any of the foregoing.

CLAIMS:

An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, and wherein the polypeptide comprises at least one amino acid side chain chemically modified by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and N- Hydroxysuccinimide (NHS).

2. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, and wherein the polypeptide comprises an amino acid side chain chemically modified by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and N- Hydroxysuccinimide (NHS).

3. The isolated polypeptide according to claims 1 or 2, wherein at least one side chain of an aspartic acid residue of the polypeptide or at least one side chain of a glutamic acid residue of the polypeptide is chemically modified by glycine.

4. The isolated polypeptide according to any of claims 1 -3, wherein the polypeptide comprises:

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

b) at least one crosslink between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide.

5. The isolated polypeptide according to any of claims 1 -4, wherein the polypeptide comprises a beta-alanine moiety linked to a side chain of at least one lysine residue of the polypeptide.

6. The isolated polypeptide according to claim 4, wherein the polypeptide comprises a glycine moiety linked to a side chain of an aspartic acid residue of the polypeptide or to a side chain of a glutamic acid residue of the polypeptide.

7. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID

NO: 4, wherein the methionine residue at position 1 is optionally not present, and wherein a side chain of at least one lysine residue of the polypeptide is linked to a beta-alanine moiety.

8. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, and wherein a side chain of at least one lysine residue of the polypeptide is linked to a beta-alanine moiety.

9. The isolated polypeptide according to claims 7 or 8, wherein a side chain of a second lysine residue of the polypeptide is linked to a side chain of an aspartic acid residue or to a side chain of a glutamic acid residue.

10. The isolated polypeptide according to any of claims 7-9, wherein a side chain of an aspartic acid residue or a side chain of a glutamic acid residue of the polypeptide is linked to a glycine moiety.

1 1 . The isolated polypeptide as in any of claims 1 -10, wherein the polypeptide has an EC50 of at least about 100 μg/ml.

12. An immunogenic composition comprising an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, and an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, and wherein the polypeptides have at least one amino acid side chain chemically modified by 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide) (EDC) and N-Hydroxysuccinimide (NHS).

13. The immunogenic composition according to claim 12, wherein the polypeptide comprises at least one of any of:

a) at least one beta-alanine moiety linked to a side chain of a lysine residue of the polypeptide;

b) at least one crosslink between a side chain of a lysine residue of the

polypeptide and a side chain of an aspartic acid residue; and

c) at least one crosslink between a side chain of a lysine residue of the polypeptide and a side chain of a glutamic acid residue.

14. The immunogenic composition according to claim 12, wherein the polypeptides have an EC50 of at least about 100 μg/ml.

15. An immunogenic composition comprising an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, and an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, and

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

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

16. The immunogenic composition according to claim 15, wherein a side chain of a second lysine residue of SEQ ID NO: 4 is linked to a side chain of an aspartic acid residue or to a side chain of a glutamic acid residue, and wherein a second lysine residue of SEQ ID NO: 6 is linked to a side chain of an aspartic acid residue or to a side chain of a glutamic acid residue.

17. The immunogenic composition according to any of claims 12-16, wherein a side chain of an aspartic acid residue or a side chain of a glutamic acid residue of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, is linked to a glycine moiety.

18. The immunogenic composition according to any of claims 12-16, wherein a side chain of an aspartic acid residue or a side chain of a glutamic acid residue of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, is linked to a glycine moiety.

19. The immunogenic composition according to any of claims 12-18, wherein the polypeptide has an EC50 of at least about 100 pg/ml.

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

a) at least one crosslink 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 crosslink 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 beta-alanine moiety linked to a side chain of at least one lysine residue of the polypeptide; and

d) a glycine moiety linked to a side chain of at least one aspartic acid residue of the polypeptide or to a side chain of at least one glutamic acid residue of the polypeptide.

21 .An immunogenic composition comprising a mutant Clostridium difficile toxin A, which 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.

22. The composition according to claim 21 , wherein the mutation is a non-conservative amino acid substitution.

23. The composition according to claim 22, wherein the substitution comprises an

alanine substitution.

24. The composition according to any of claims 21 -23, wherein the wild-type Clostridium difficile toxin A comprises a sequence having at least 95% identity to SEQ ID NO: 1 .

25. The composition according to claim 24, wherein the wild-type Clostridium difficile toxin A comprises a sequence having at least 98% identity to SEQ ID NO: 1 .

26. The composition according to claim 25, wherein the wild-type Clostridium difficile toxin A comprises SEQ ID NO: 1 .

27. The composition according to any of claims 21 -26, wherein the glucosyltransferase domain comprises at least two mutations.

28. The composition according to claim 27, wherein the at least two mutations are

present at amino acid positions 101 , 269, 272, 285, 287, 269, 272, 460, 462, 541 , or 542, according to the numbering of SEQ ID NO: 1 .

29. The composition according to any of claims 21 -26, wherein the glucosyltransferase domain comprises SEQ ID NO: 29.

30. The composition according to claim 29, wherein the glucosyltransferase domain comprises at least two non-conservative mutations present at amino acid positions 101 , 269, 272, 285, 287, 269, 272, 460, 462, 541 , or 542, or any combination thereof, of SEQ ID NO: 29.

31 . The composition according to any of claims 21 -26, wherein the cysteine protease domain comprises a mutation present at positions 700, 589, 655, 543, or any combinations thereof, according to the numbering of SEQ ID NO: 1 .

32. The composition according to any of claims 21 -26, wherein the cysteine protease domain comprises SEQ ID NO: 32.

33. The composition according to claim 32, wherein the cysteine protease domain

comprises a non-conservative mutation present at positions 1 , 47, 1 13, 158, or any combinations thereof, of SEQ ID NO: 32.

34. The composition according to claim 21 , wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 4.

35. The composition according to claim 21 , wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 84.

36. The composition according to claim 21 , wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 7.

37. The composition according to claim 21 , wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 83.

38. The composition according to any of claims 21 -33, wherein at least one amino acid of the mutant Clostridium difficile toxin A is chemically crosslinked.

39. The composition according to claim 38, wherein the amino acid is chemically

crosslinked by formaldehyde.

40. The composition according to claim 38, wherein the amino acid is chemically

crosslinked by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide.

41 .The composition according to claim 38 or 40, wherein the amino acid is chemically crosslinked by N-hydroxysuccinimide.

42. The composition according to any of claims 21 -41 , wherein the composition is

recognized by an anti-toxin A neutralizing antibody or binding fragment thereof.

43. An immunogenic composition comprising a mutant Clostridium difficile toxin A, which comprises a glucosyltransferase domain comprising SEQ ID NO: 29 having an amino acid substitution at positions 285 and 287, and a cysteine protease domain comprising 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 crosslinked.

44. 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 crosslinked.

45. The composition according to claim 43 or 44, wherein the at least one amino acid is crosslinked by formaldehyde.

46. The composition according to claim 43 or 44, wherein the at least one amino acid is crosslinked by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide.

47. The composition according to claim 43, 44, or 46, wherein the at least one amino acid is crosslinked by N-hydroxysuccinimide.

48. The composition according to claim 43 or 44, wherein the composition is recognized by an anti-toxin A neutralizing antibody or binding fragment thereof.

49. An immunogenic composition comprising SEQ ID NO: 4.

50. An immunogenic composition comprising SEQ ID NO: 84.

51 . An immunogenic composition comprising SEQ ID NO: 7.

52. An immunogenic composition comprising SEQ ID NO: 83.

53. The composition according to any of claims 49-52, wherein at least one amino acid is chemically crosslinked.

54. The composition according to any of claims 21 -51 , wherein the composition exhibits decreased cytotoxicity, relative to the corresponding wild-type Clostridium difficile toxin A.

55. An isolated polypeptide comprising SEQ ID NO: 84.

56. An isolated polypeptide comprising SEQ ID NO: 86.

57. An isolated polypeptide comprising SEQ ID NO: 83.

58. An isolated polypeptide comprising SEQ ID NO: 85.

59. An immunogenic composition comprising a mutant Clostridium difficile toxin B, which 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 B.

60. The composition according to claim 59, wherein the mutation is a non-conservative amino acid substitution.

61 .The composition according to claim 60, wherein the substitution comprises an

alanine substitution.

62. The composition according to any of claims 59-61 , wherein the wild-type Clostridium difficile toxin B comprises a sequence having at least 95% identity to SEQ ID NO: 2.

63. The composition according to claim 62, wherein the wild-type Clostridium difficile toxin B comprises a sequence having at least 98% identity to SEQ ID NO: 2.

64. The composition according to claim 63, wherein the wild-type Clostridium difficile toxin B comprises SEQ ID NO: 2.

65. The composition according to any of claims 59-64, wherein the glucosyltransferase domain comprises at least two mutations.

66. The composition according to claim 65, wherein the at least two mutations are

present at amino acid positions 102, 286, 288, 270, 273, 384, 461 , 463, 520, or 543, according to the numbering of SEQ ID NO: 2.

67. The composition according to any of claims 59-64, wherein the glucosyltransferase domain comprises SEQ ID NO: 31 .

68. The composition according to claim 67, wherein the glucosyltransferase domain comprises at least two non-conservative mutations present at amino acid positions 102, 286, 288, 270, 273, 384, 461 , 463, 520, or 543 of SEQ ID NO: 31 .

69. The composition according to any of claims 59-64, wherein the cysteine protease domain comprises a mutation present at positions 698, 653, 587, 544, or any combinations thereof, according to the numbering of SEQ ID NO: 2.

70. The composition according to any of claims 59-64, wherein the cysteine protease domain comprises SEQ ID NO: 33.

71 .The composition according to claim 70, wherein the cysteine protease domain

comprises a non-conservative mutation present at positions 1 , 44, 1 10, 155, or any combinations thereof, of SEQ ID NO: 33.

72. The composition according to claim 59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 6.

73. The composition according to claim 59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 86.

74. The composition according to claim 59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 8.

75. The composition according to claim 59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 85.

76. The composition according to any of claims 59-71 , wherein at least one amino acid of the mutant Clostridium difficile toxin B is chemically crosslinked.

77. The composition according to claim 76, wherein the amino acid is chemically

crosslinked by formaldehyde.

78. The composition according to claim 76, wherein the amino acid is chemically

crosslinked by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide.

79. The composition according to claims 76 or 78, wherein the at least one amino acid is crosslinked by N-hydroxysuccinimide.

80. The composition according to any of claims 59-79, wherein the composition is

recognized by an anti-toxin B neutralizing antibody or binding fragment thereof.

81 .An immunogenic composition comprising a mutant Clostridium difficile toxin B, which comprises a glucosyltransferase domain comprising SEQ ID NO: 31 having an amino acid substitution at positions 286 and 288, and a cysteine protease domain comprising 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 crosslinked.

82. 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 crosslinked.

83. The composition according to claim 81 or 82, wherein the at least one amino acid is crosslinked by formaldehyde.

84. The composition according to claim 81 or 82, wherein the at least one amino acid is crosslinked by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide.

85. The composition according to claim 81 , 82, or 84, wherein the at least one amino acid is crosslinked by N-hydroxysuccinimide.

86. The composition according to claim 81 or 82, wherein the composition is recognized by an anti-toxin B neutralizing antibody or binding fragment thereof.

87. An immunogenic composition comprising SEQ ID NO: 6.

88. An immunogenic composition comprising SEQ ID NO: 86.

89. An immunogenic composition comprising SEQ ID NO: 8.

90. An immunogenic composition comprising SEQ ID NO: 85.

91 .The composition according to any of claims 59-89, wherein the composition exhibits decreased cytotoxicity, relative to the corresponding wild-type Clostridium difficile toxin B.

92. 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 NOs: 4 and 6 is chemically crosslinked.

93. 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 NOs: 84 and 86 is chemically crosslinked.

94. The composition according to claim 92 or 93, wherein the at least one amino acid is crosslinked by formaldehyde.

95. The composition according to claim 92 or 93, wherein the at least one amino acid is crosslinked by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide.

96. The composition according to claims 92, 93, or 95, wherein the at least one amino acid is crosslinked by N-hydroxysuccinimide.

97. A recombinant cell or progeny thereof, comprising SEQ ID NO: 1 1 , 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.

98. A recombinant cell or progeny thereof, comprising a nucleic acid sequence that encodes SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

99. A recombinant cell or progeny thereof, comprising a nucleic acid sequence that encodes SEQ ID NO: 84.

100. A recombinant cell or progeny thereof, comprising a nucleic acid sequence that encodes SEQ ID NO: 86.

101 . A recombinant cell or progeny thereof, comprising a nucleic acid sequence that encodes SEQ ID NO: 83.

102. A recombinant cell or progeny thereof, comprising a nucleic acid sequence that encodes SEQ ID NO: 85.

103. The recombinant cell of claim 97 or 98, wherein said cell is derived from a Gram positive bacterium cell.

104. The recombinant cell of claims 97, 98, or 99, wherein the cell is derived from a Clostridium difficile cell.

105. The recombinant cell of any of claims 97- 104, wherein the cell lacks an

endogenous polynucleotide encoding a toxin.

106. The cell according to any of claims 104, or 105 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 481 1 , and Clostridium difficile VPI 1 1 186.

107. The cell according to claim 106, wherein the cell is a Clostridium difficile VPI 1 1 186 cell.

108. The cell according to claim 106, or 107, wherein a sporulation gene of the

Clostridium difficile cell is inactivated.

109. The cell according to claim 108, wherein the sporulation gene comprises an spo0A gene or an spollE gene.

1 10. A method of producing a mutant Clostridium difficile toxin, comprising

culturing a recombinant cell or progeny thereof 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 the corresponding wild-type Clostridium difficile toxin.

1 1 1 . The method according to claim 1 10, wherein the cell lacks an endogenous

polynucleotide encoding a toxin.

1 12. The method according to claim 1 10, wherein the recombinant cell or progeny thereof comprises a cell according to any of claims 97-1 1 1 .

1 13. The method according to claim 1 10, further comprising isolating the mutant Clostridium difficile toxin.

1 14. The method according to claim 1 13, further comprising contacting the isolated mutant Clostridium difficile toxin with formaldehyde.

1 15. The method according to claim 1 14, wherein the contacting occurs for at most 14 days.

1 16. The method according to claim 1 15, wherein the contacting occurs for at most 48 hours.

1 17. The method according to claim 1 14, wherein the contacting occurs at about

25°C.

1 18. The method according to claim 1 13, further comprising contacting the isolated mutant Clostridium difficile toxin with ethyl- 3-(3-dimethylaminopropyl) carbodiimide.

1 19. The method according to claim 1 18, wherein the contacting occurs for at most 24 hours.

120. The method according to claim 120, wherein the contacting occurs for at most 4 hours.

121 . The method according to claim 1 18, wherein the contacting occurs at about

25°C.

122. The method according to claim 1 18, further comprising contacting the isolated mutant Clostridium difficile toxin with N-hydroxysuccinimide.

123. An immunogenic composition produced by the method according to any of claims 1 10- 122.

124. A method of producing a neutralizing antibody against a Clostridium difficile toxin A, comprising administering an immunogenic composition to a mammal, said immunogenic composition comprising SEQ ID NO: 4, wherein the methionine residue at position 1 is optionally not present, wherein at least one amino acid of SEQ ID NO: 4 is crosslinked 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.

125. A method of producing a neutralizing antibody against a Clostridium difficile toxin

A, comprising administering an immunogenic composition to a mammal, said immunogenic composition comprising SEQ ID NO: 84, wherein at least one amino acid of SEQ ID NO: 84 is crosslinked 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.

126. A method of producing a neutralizing antibody against a Clostridium difficile toxin

B, comprising administering an immunogenic composition to a mammal, said immunogenic composition comprising SEQ ID NO: 6, wherein the methionine residue at position 1 is optionally not present, wherein at least one amino acid of SEQ ID NO: 6 is crosslinked 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.

127. A method of producing a neutralizing antibody against a Clostridium difficile toxin A, comprising administering an immunogenic composition to a mammal, said immunogenic composition comprising SEQ ID NO: 86, wherein at least one amino acid of SEQ ID NO: 86 is crosslinked 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.

128. An antibody or antibody binding fragment thereof specific to an immunogenic composition, said immunogenic composition comprising SEQ ID NO: 4 wherein the methionine residue at position 1 is optionally not present, or SEQ ID NO: 7 wherein the methionine residue at position 1 is optionally not present,.

129. The antibody or antibody binding fragment thereof according to claim 128,

wherein at least one amino acid of SEQ ID NO: 4 wherein the methionine residue at position 1 is optionally not present, or SEQ ID NO: 7 wherein the methionine residue at position 1 is optionally not present, is crosslinked by formaldehyde, 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1 - ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

130. An antibody or antibody binding fragment thereof comprising the amino acid

sequences of the heavy chain complementarity determining regions (CDRs) set forth in SEQ ID NO: 41 (CDR H1 ), SEQ ID NO: 42 (CDR H2) and SEQ ID NO: 43 (CDR H3), and the amino acid sequences of the light chain CDRs as shown in SEQ ID NO: 38 (CDR L1 ), SEQ ID NO: 39 (CDR L2) and SEQ ID NO: 40 (CDR L3).

131 . The antibody or antibody binding fragment thereof according to claims 128, 129, or 130, wherein the antibody or antibody binding fragment thereof comprises a heavy chain, which comprises the amino acid sequence shown in SEQ ID NO: 37, and a light chain, which comprises the amino acid sequence shown in SEQ ID NO: 36.

132. A composition comprising a combination of two or more antibodies or antibody binding fragments thereof selected from any according to any of claims 128-131 .

133. An antibody or antibody binding fragment thereof specific to an immunogenic composition, said immunogenic composition comprising SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally not present, or SEQ ID NO: 8 wherein the methionine residue at position 1 is optionally not present,.

134. The antibody or antibody binding fragment thereof according to claim 133,

wherein at least one amino acid of SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally not present, or SEQ ID NO: 8 wherein the methionine residue at position 1 is optionally not present, is crosslinked by formaldehyde, 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1 - ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

135. An antibody or antibody binding fragment thereof comprising the amino acid

sequences of the heavy chain complementarity determining regions (CDRs) set forth in SEQ ID NO: 51 (CDR H1 ), SEQ ID NO: 52 (CDR H2) and SEQ ID NO: 53 (CDR H3), and the amino acid sequences of the light chain CDRs as shown in SEQ ID NO: 57 (CDR L1 ), SEQ ID NO: 58 (CDR L2) and SEQ ID NO: 59 (CDR L3).

136. An antibody or antibody binding fragment thereof comprising the amino acid

sequences of the heavy chain complementarity determining regions (CDRs) set forth 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 as shown in SEQ ID NO: 68 (CDR L1 ), SEQ ID NO: 69 (CDR L2) and SEQ ID NO: 70 (CDR L3).

137. An antibody or antibody binding fragment thereof comprising the amino acid

sequences of the heavy chain complementarity determining regions (CDRs) set forth 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 as shown in SEQ ID NO: 79 (CDR L1 ), SEQ ID NO: 80 (CDR L2) and SEQ ID NO: 81 (CDR L3).

138. A composition comprising a combination of two or more antibodies or antibody binding fragments thereof selected from any of claims 133-137.

139. A method of 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 not present, and an immunogenic composition comprising SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally not present, wherein at least one amino acid of each of SEQ ID NOs: 4 and 6 is crosslinked by formaldehyde.

140. A method of 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 not present, and an immunogenic composition comprising SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally not present, wherein at least one amino acid of each of SEQ ID NO: 4 and SEQ ID NO: 6 is crosslinked by 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1 - ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

141 .. A method of treating a 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 at least one amino acid of each of SEQ ID NO: 84 and SEQ ID NO: 86 is crosslinked by 1 - ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

142. A method of 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 not present, and an immunogenic composition comprising SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally not present, wherein at least one amino acid of each of SEQ ID NO: 4 and SEQ ID NO: 6 is crosslinked by formaldehyde.

143. A method of 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 not present, and an immunogenic composition comprising SEQ ID NO: 6 wherein the methionine residue at position 1 is optionally not present, wherein at least one amino acid of each of SEQ ID NO: 4 and SEQ ID NO: 6 is crosslinked by 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1 - ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

144. A method of 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 at least one amino acid of each of SEQ ID NO: 84 and SEQ ID NO: 86 is crosslinked by 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide and N- hydroxysuccinimide.

145. The method according to any of claims 139-144, wherein the mammal is a

mammal in need thereof.

146. The method according to any of claims 139-144, wherein the mammal has a recurring Clostridium difficile infection.

147. The method according to any of claims 139-144, wherein the composition is

administered parenterally.

148. The method according to any of claims 139-144, wherein the composition further comprises an adjuvant.

149. The method according to claim 148, wherein the adjuvant comprises aluminum.

150. The method according to claim 148, wherein the adjuvant comprises aluminum hydroxide gel and a CpG oligonucleotide.

151 . The method according to claim 148, wherein the adjuvant comprises

ISCOMATRIX®.