COMPOSITIONS RELATING TO A MUTANT CLOSTRIDIUM DIFFICILE TOXIN AND
METHODS THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Patent Application
61/478,474, filed on April 22, 201 1, and U.S. Provisional Patent Application 61/478,899,
filed April 25, 201 1. The entire contents of the aforementioned applications are herein
incorporated by reference in their entireties.
FIELD
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 locus (PaLoc) by
the genes tcdA and tcdB, respectively. Nonpathogenic strains of C. difficile have this
locus replaced by an alternative 115 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
20% of patients receiving such treatment experience a recurrence of infection after a
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), Nhydroxysuccinate,
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
crossl inked.
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: 3 1, 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
g/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
g/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
g/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,
VPI1 0463, R20291 , CD1 96, 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,
VPI1 0463, R20291 , CD1 96, 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 11186).
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- 4Cglucose;
whereas 10 g 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 pg/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 muCdiff201 0-06).
Figure 11: Graph showing neutralizing antibody titers as described in Example 26
(study muCdiff201 0-07).
Figure 12 : Graph showing neutralizing antibody responses against toxins A and
B in hamsters after four immunizations as described in Example 27 (study hamC.
c//' 'c/Ve201 0-02)
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. /7 /'c/Ve201 0-02).
Figure 14: Survival curves for three immunized groups of hamsters as compared
to the non-immunized controls, described in Example 28 (study hamC. difficile20 10-02,
continued).
Figure 15 : Graph showing relative neutralizing antibody response against
different formulations of C. difficile mutant toxins in hamsters (study hamC. /7 c//e20 10-
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 (4xEC 50) 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 .5x1 06
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 (<0.3x1 06) and cell only controls were 2-2.5 x 106 RLUs as depicted in graphs
shown in Figure 20B and Figure 20C.
Figure 2 1: 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
2 1B)
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 (t ) ,
control; and purified toxin (¨) , control. The toxins from the respective strains were
added to the cells at 4xECso 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); 200401 3 (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 intraand
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: 11 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: 5 .
SEQ ID NO: 7 sets forth the amino acid sequence for wild-type C. difficile CD1 96
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 VPI1 0463
TcdA.
SEQ ID NO: 20 sets forth a DNA sequence encoding SEQ ID NO: 19 .
SEQ ID NO: 2 1 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: 2 1.
SEQ ID NO: 23 sets forth the amino acid sequence for wild-type C. difficile CD1 96
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 VPI1 0463
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.
SEQ ID NO: 28 sets forth the amino acid sequence for residues 10 1 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 10 1 to 293 of SEQ ID
NO: 2.
SEQ ID NO: 3 1 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
10 1, 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: 4 1 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: 5 1 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: 6 1 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: 7 1 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: 8 1 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 200401 3
TcdA.
SEQ ID NO: 88 sets forth the amino acid sequence for wild-type C. difficile 20041 11
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: 9 1 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 200601 7
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 200721 7
TcdA.
SEQ ID NO: 100 sets forth the amino acid sequence for wild-type C. difficile 2007302
TcdA.
SEQ ID NO: 10 1 sets forth the amino acid sequence for wild-type C. difficile 200781 6
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: 110 sets forth the amino acid sequence for wild-type C. difficile 200401 3
TcdB.
SEQ ID NO: 111 sets forth the amino acid sequence for wild-type C. difficile 20041 11
TcdB.
SEQ ID NO: 112 sets forth the amino acid sequence for wild-type C. difficile 20041 18
TcdB.
SEQ ID NO: 113 sets forth the amino acid sequence for wild-type C. difficile 2004205
TcdB.
SEQ ID NO: 114 sets forth the amino acid sequence for wild-type C. difficile 2004206
TcdB.
SEQ ID NO: 115 sets forth the amino acid sequence for wild-type C. difficile 2005022
TcdB.
SEQ ID NO: 116 sets forth the amino acid sequence for wild-type C. difficile 2005088
TcdB.
SEQ ID NO: 117 sets forth the amino acid sequence for wild-type C. difficile 2005283
TcdB.
SEQ ID NO: 118 sets forth the amino acid sequence for wild-type C. difficile 2005325
TcdB.
SEQ ID NO: 119 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 200601 7
TcdB.
SEQ ID NO: 12 1 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 200721 7
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 200781 6
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: 29 sets forth the amino acid sequence for wiId -type C. difficile 2008222
TcdB.
SEQ ID NO: 130 sets forth the amino acid sequence for wiId -type C. difficile 2009078
TcdB.
SEQ ID NO: 13 1 sets forth the amino acid sequence for wiId -type C. difficile 2009087
TcdB.
SEQ ID NO: 132 sets forth the amino acid sequence for wiId -type C. difficile 2009141
TcdB.
SEQ ID NO: 133 sets forth the amino acid sequence for wiId -type C. difficile 2009292
TcdB.
SEQ ID NO: 134 sets forth the amino acid sequence for wiId -type C. difficile 014 TcdA.
SEQ ID NO: 135 sets forth the amino acid sequence for wiId -type C. difficile 0 15 TcdA.
SEQ ID NO: 136 sets forth the amino acid sequence for wiId -type C. difficile 020 TcdA.
SEQ ID NO: 137 sets forth the amino acid sequence for wiId -type C. difficile 023 TcdA.
SEQ ID NO: 138 sets forth the amino acid sequence for wiId -type C. difficile 027 TcdA.
SEQ ID NO: 139 sets forth the amino acid sequence for wiId -type C. difficile 029 TcdA.
SEQ ID NO: 140 sets forth the amino acid sequence for wiId -type C. difficile 046 TcdA.
SEQ ID NO: 141 sets forth the amino acid sequence for wiId -type C. difficile 014 TcdB.
SEQ ID NO: 142 sets forth the amino acid sequence for wiId -type C. difficile 0 15 TcdB.
SEQ ID NO: 143 sets forth the amino acid sequence for wiId -type C. difficile 020 TcdB.
SEQ ID NO: 144 sets forth the amino acid sequence for wiId -type c . difficile 023 TcdB.
SEQ ID NO: 145 sets forth the amino acid sequence for wiId -type c . difficile 027 TcdB.
SEQ ID NO: 146 sets forth the amino acid sequence for wiId -type c . difficile 029 TcdB.
SEQ ID NO: 147 sets forth the amino acid sequence for wiId -type c . difficile 046 TcdB.
SEQ ID NO: 148 sets forth the amino acid sequence for wiId -type c . difficile 001 TcdA.
SEQ ID NO: 149 sets forth the amino acid sequence for wiId -type c . difficile 002 TcdA.
SEQ ID NO: 150 sets forth the amino acid sequence for wiId -type c . difficile 003 TcdA.
SEQ ID NO: 15 1 sets forth the amino acid sequence for wiId -type c . difficile 004 TcdA.
SEQ ID NO: 152 sets forth the amino acid sequence for wiId -type c . difficile 070 TcdA.
SEQ ID NO: 153 sets forth the amino acid sequence for wiId -type c . difficile 075 TcdA.
SEQ ID NO: 154 sets forth the amino acid sequence for wiId -type c . difficile 077 TcdA.
SEQ ID NO: 155 sets forth the amino acid sequence for wiId -type c . difficile 081 TcdA.
SEQ ID NO: 156 sets forth the amino acid sequence for wiId -type c . difficile 117 TcdA.
SEQ ID NO: 57 sets forth the amino acid sequence for wi d-type C. difficile 13 1 TcdA
SEQ ID NO: 158 sets forth the amino acid sequence for wi d-type C. difficile 001 TcdB
SEQ ID NO: 159 sets forth the amino acid sequence for wi d-type C. difficile 002 TcdB
SEQ ID NO: 160 sets forth the amino acid sequence for wi d-type C. difficile 003 TcdB
SEQ ID NO: 16 1 sets forth the amino acid sequence for wi d-type C. difficile 004 TcdB
SEQ ID NO: 162 sets forth the amino acid sequence for wi d-type C. difficile 070 TcdB
SEQ ID NO: 163 sets forth the amino acid sequence for wi d-type C. difficile 075 TcdB
SEQ ID NO: 164 sets forth the amino acid sequence for wi d-type C. difficile 077 TcdB
SEQ ID NO: 165 sets forth the amino acid sequence for wi d-type C. difficile 081 TcdB
SEQ ID NO: 166 sets forth the amino acid sequence for wi d-type C. difficile 117 TcdB
SEQ ID NO: 167 sets forth the amino acid sequence for wi d-type C. difficile 131 TcdB
SEQ ID NO: 168 sets forth the amino acid sequence for wi d-type C. difficile 053 TcdA
SEQ ID NO: 169 sets forth the amino acid sequence for wi d-type C. difficile 078 TcdA
SEQ ID NO: 170 sets forth the amino acid sequence for wi d-type C. difficile 087 TcdA
SEQ ID NO: 17 1 sets forth the amino acid sequence for wi d-type C. difficile 095 TcdA
SEQ ID NO: 172 sets forth the amino acid sequence for wi d-type C. difficile 126 TcdA
SEQ ID NO: 173 sets forth the amino acid sequence for wi d-type C. difficile 053 TcdB
SEQ ID NO: 174 sets forth the amino acid sequence for wi d-type C. difficile 078 TcdB
SEQ ID NO: 175 sets forth the amino acid sequence for wi d-type C. difficile 087 TcdB
SEQ ID NO: 176 sets forth the amino acid sequence for wi d-type c . difficile 095 TcdB
SEQ ID NO: 177 sets forth the amino acid sequence for wi d-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.
The immunogenic compositions described herein unexpectedly demonstrated the
ability to elicit novel neutralizing antibodies against C. difficile toxins and they may have
the ability to confer active and/or passive protection against a C. difficile challenge. The
novel antibodies are directed against various epitopes of toxin A and toxin B. The
inventors further discovered that a combination of at least two of the neutralizing
monoclonal antibodies can exhibit an unexpectedly synergistic effect in respective in
vitro neutralization of toxin A and toxin B.
The inventive compositions described herein may be used to treat, prevent,
decrease the risk of, decrease occurrences of, decrease severity of, and/or delay the
outset of a C. difficile infection, C. difficile associated disease (CDAD), syndrome,
condition, symptom, and/or complication thereof in a mammal, as compared to a
mammal to which the composition was not administered.
Moreover, the inventors discovered a recombinant asporogenic C. difficile cell
that can stably express the mutant C. difficile toxin A and toxin B, and novel methods for
producing the same.
Immunogenic Compositions
In one aspect, the invention relates to an immunogenic composition that includes
a mutant C. difficile toxin. The mutant C. difficile toxin includes an amino acid sequence
having at least one mutation in a glucosyltransferase domain and at least one mutation
in a cysteine protease domain, relative to the corresponding wild-type C. difficile toxin.
The term "wild-type," as used herein, refers to the 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 which has not been
intentionally modified by human manipulation. The present invention also relates to
isolated polynucleotides that include nucleic acid sequences encoding any of the
foregoing. In addition, the present invention relates to use of any of the foregoing
compositions to treat, prevent, decrease the risk of, decrease severity of, decrease
occurrences of, and/or delay the outset of a C. difficile infection, C. difficile associated
disease, syndrome, condition, symptom, and/or complication thereof in a mammal, as
compared to a mammal to which the composition is not administered, as well as
methods for preparing said 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 a cellular (mediated by antigen-specific T cells or their
secretion products) response directed against a C. difficile toxin in a recipient patient.
The immune response may be humoral, cellular, or both.
The immune response can be an active response induced by administration of
an immunogenic composition, an immunogen. Alternatively, the immune response can
be a passive response induced by administration of antibody or primed 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 a neutralizing
antibody assay, ELISA, etc.
A cellular immune response is typically elicited by the presentation of polypeptide
epitopes in association with Class I or Class I I MHC molecules to activate antigenspecific
CD4 + T helper cells and/or CD8 + cytotoxic T cells. The response may also
involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells,
astrocytes, microglia cells, eosinophils or other components of innate immunity. The
presence of a cell-mediated immunological response can be determined by proliferation
assays (CD4 + T cells) or CTL (cytotoxic T lymphocyte) assays known in the art.
In one embodiment, an 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 the composition is administered. The vaccine composition may
protect the immunized mammal against subsequent challenge by an immunizing agent
or an immunologically cross-reactive agent. Protection can be complete or partial with
regard to reduction in symptoms or infection as compared to a non-vaccinated mammal
under the same conditions.
The immunogenic compositions described herein are cross-reactive, which refers
to having a characteristic of being able to elicit an effective immune response (e.g.,
humoral immune response) against a toxin produced by another C. difficile strain that is
different from the strain from which the composition is derived. For example, the
immunogenic compositions (e.g., derived from C. difficile 630) described herein may
elicit cross-reactive antibodies that can bind to toxins produced by multiple strains of C.
difficile (e.g., toxins produced by C. difficile R20291 and VPI1 0463). See, for example,
Example 37. Cross-reactivity is indicative of the cross-protection potential of the
bacterial immunogen, and vice versa.
The term "cross-protective" as used herein refers to the ability of the immune
response induced by an immunogenic composition to prevent or attenuate infection by a
different bacterial strain or species of the same genus. For example, an immunogenic
composition (e.g., derived from C. difficile 630) described herein may induce an
effective immune response in a mammal to attenuate a C. difficile infection and/or to
attenuate a C. difficile disease caused by a strain other than 630 (e.g., C. difficile
R20291 ) in the mammal.
Exemplary mammals in which the immunogenic composition or immunogen
elicits an immune response include any mammals, such as, for example, mice,
hamsters, primates, and humans. In a preferred embodiment, the immunogenic
composition or immunogen elicits an immune response in a human to which 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
action of TcdA and TcdB on mammalian target cells depends on a multistep mechanism
of receptor-mediated endocytosis, membrane translocation, autoproteolytic processing,
and monoglucosylation of GTPases. Many of these functional activities have been
ascribed to discrete regions within the primary sequence of the toxins, and the toxins
have been imaged to show that these molecules are similar in structure.
The wild-type gene for TcdA has about 8130 nucleotides that encode a protein
having a deduced molecular weight of about 308-kDa, having about 2710 amino acids.
As used herein, a wild-type C. difficile TcdA includes a C. difficile TcdA from any wildtype
C. difficile strain. A wild-type C. difficile TcdA may include a wild-type C. difficile
TcdA amino acid sequence having at least about 90%, 9 1%, 92%, 93%, 94%, 95%,
96%, 97%, preferably about 98%, more preferably about 99% or most preferably about
100% identity to SEQ ID NO: 1 (full length) when optimally aligned, such as by the
programs GAP or BESTFIT using default gap weights.
In a preferred embodiment, the wild-type C. difficile TcdA includes an amino acid
sequence set forth in SEQ ID NO: 1, which describes the wild-type amino acid
sequence for TcdA from C. difficile strain 630 (also disclosed in GenBank accession
number YP_001 0871 37.1 and/or CAJ67494.1 ) . C. difficile strain 630 is known in the
art as being a PCR-ribotype 012 strain. SEQ ID NO: 9 describes the wild-type gene for
TcdA from C. difficile strain 630, which is also disclosed in GenBank accession number
NC_009089.1 .
Another example of a wild-type C. difficile TcdA includes an amino acid
sequence set forth in SEQ ID NO: 15, which describes the wild-type amino acid
sequence for TcdA from C. difficile strain R20291 (also disclosed in GenBank accession
number YP_00321 7088.1 ) . C. difficile strain R20291 is known in the art as being a
hypervirulent strain and a PCR-ribotype 027 strain. The amino acid sequence for TcdA
from C. difficile strain R20291 has about 98% identity to SEQ ID NO:1 . SEQ ID NO: 16
describes the wild-type gene for TcdA from C. difficile strain R20291 , which is also
disclosed in GenBank accession number NC_01 3316.1 .
An additional example of a wild-type C. difficile TcdA includes an amino acid
sequence set forth in SEQ ID NO: 17, which describes the wild-type amino acid
sequence for TcdA from C. difficile strain CD196 (also disclosed in GenBank accession
number CBA61 156.1 ) . CD1 96 is a strain from a recent Canadian outbreak, and it is
known in the art as a PCR-ribotype 027 strain. The amino acid sequence for TcdA from
C. difficile strain CD196 has about 98% identity to SEQ ID NO: 1, and has about 100%
identity to TcdA from C. difficile strain R20291 . SEQ ID NO: 18 describes the wild-type
gene for TcdA from C. difficile strain CD196, which is also disclosed in GenBank
accession number FN538970.1 .
Further examples of an amino acid sequence for a wild-type C. difficile TcdA
include SEQ ID NO: 19, which describes the wild-type amino acid sequence for TcdA
from C. difficile strain VPI10463 (also disclosed in GenBank accession number
CAA63564.1 ) . The amino acid sequence for TcdA from C. difficile strain VPI1 0463 has
about 100% (99.8%) identity to SEQ ID NO: 1. SEQ ID NO: 20 describes the wild-type
gene for TcdA from C. difficile strain VPI1 0463, which is also disclosed in GenBank
accession number X92982.1 .
Additional examples of a wild-type C. difficile TcdA include TcdA from wild-type
C. difficile strains obtainable from the Centers for Disease Control and Prevention
(CDC, Atlanta, GA). The inventors discovered that the amino acid sequence of TcdA
from wild-type C. difficile strains obtainable from the CDC include at least about 99.3%
to 100% identity, when optimally aligned, to amino acid residues 1 to 821 of SEQ ID
NO: 1 (TcdA from C. difficile 630). See Table 1.
The inventors also discovered that the amino acid sequence of TcdA from wildtype
C. difficile strains may include at least about 90%, 9 1%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, to about 100% identity, when optimally aligned (e.g., when full,
length sequences are optimally aligned) to SEQ ID NO: 1.
Table 1: wild-type C. difficile strains obtained from CDC and the percent identity
of amino acid residues 1-821 of TcdA from the respective wild-type C. difficile strain to
amino acid residues 1-821 of SEQ ID NO: 1, when optimally aligned.
Table 1: Wild-type C. difficile Strains from CDC
C. difficile Strain Approximate % Amino Acid Identity to Residues 1-821 of SEQ ID
ID NO: 1
20041 100
20041 18 99.6
2004205 100
2004206 100
2005325 99.3
2005359 99.6
200601 7 100
2007070 100
2007302 100
200781 6 99.3
2007838 99.6
2007886 99.6
2008222 100
2009078 100
2009087 100
2009141 100
2009292 99.6
Accordingly, in one embodiment, the wild-type C. difficile TcdA amino acid
sequence includes a sequence of at least about 500, 600, 700, or 800 contiguous
residues, which has at least about 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%,
preferably about 98%, more preferably about 99%, or most preferably about 100%
identity to a sequence of equal length between residues 1 to 900 of SEQ ID NO: 1 when
optimally aligned, such as by the programs GAP or BESTFIT using default gap weights.
Examples include strains described above (e.g., R20291 , CD1 96, etc) and those listed
in Table 1.
In another embodiment, the wild-type C. difficile TcdA amino acid sequence
includes a sequence having at least about 90%, 9 1%, 92%, 93%, 94%, 95%, 96%,
preferably about 97%, preferably about 98%, more preferably about 99% or most
preferably about 100% identity to any sequence selected from SEQ ID NOs: 87-1 09
when optimally aligned. See Table 1-a.
012 (004) SEQ ID NO: 15 1
014 SEQ ID NO: 134
015 SEQ ID NO: 135
017
020 SEQ ID NO: 136
023 SEQ ID NO: 137
027 SEQ ID NO: 138
029 SEQ ID NO: 139
046 SEQ ID NO: 140
053 SEQ ID NO: 168
059
070 SEQ ID NO: 152
075 SEQ ID NO: 153
077 SEQ ID NO: 154
078 SEQ ID NO: 169
081 SEQ ID NO: 155
087 SEQ ID NO: 170
095 SEQ ID NO: 17 1
106
117 SEQ ID NO: 156
126 SEQ ID NO: 172
131 SEQ ID NO: 157
The wild-type gene for TcdB has about 7098 nucleotides that encode a protein
with a deduced molecular weight of about 270 kDa, having about 2366 amino acids. As
used herein, a wild-type C. difficile TcdB includes a C. difficile TcdB from any wild-type
C. difficile strain. A wild-type C. difficile TcdB may include a wild-type amino acid
sequence having at least about 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%,
preferably about 98%, more preferably about 99% or most preferably about 100%
identity to SEQ ID NO: 2 when optimally aligned, such as by the programs GAP or
BESTFIT using default gap weights. In a preferred embodiment, the wild-type C.
difficile TcdB includes an amino acid sequence set forth in SEQ ID NO: 2, which
describes the wild-type amino acid sequence for TcdB from C. difficile strain 630 (also
disclosed in GenBank accession number YP_001 0871 35.1 and/or CAJ67492). SEQ ID
NO: 10 describes the wild-type gene for TcdB from C. difficile strain 630, which is also
disclosed in GenBank accession number NC_009089.1 .
Another example of a wild-type C. difficile TcdB includes an amino acid
sequence set forth in SEQ ID NO: 2 1, which describes the wild-type amino acid
sequence for TcdB from C. difficile strain R20291 (also disclosed in GenBank accession
number YP_00321 7086.1 and/or CBE02479.1 ) . The amino acid sequence for TcdB
from C. difficile strain R20291 has about 92% identity to SEQ ID NO: 2 . SEQ ID NO: 22
describes the wild-type gene for TcdB from C. difficile strain R20291 , which is also
disclosed in GenBank accession number NC_01 3316.1 .
An additional example of a wild-type C. difficile TcdB includes an amino acid
sequence set forth in SEQ ID NO: 23, which describes the wild-type amino acid
sequence for TcdB from C. difficile strain CD196 (also disclosed in GenBank accession
number YP_00321 3639.1 and/or CBA61 153.1 ) . SEQ ID NO: 24 describes the wildtype
gene for TcdB from C. difficile strain CD1 96, which is also disclosed in GenBank
accession number NC_01 3315.1 . The amino acid sequence for TcdB from C. difficile
strain CD1 96 has about 92% identity to SEQ ID NO: 2 .
Further examples of an amino acid sequence for a wild-type C. difficile TcdB
include SEQ ID NO: 25, which describes the wild-type amino acid sequence for TcdB
from C. difficile strain VPI10463 (also disclosed in GenBank accession number P 18 177
and/or CAA37298). The amino acid sequence for TcdB from C. difficile strain VPI1 0463
has 100% identity to SEQ ID NO: 2. SEQ ID NO: 26 describes the wild-type gene for
TcdB from C. difficile strain VPI1 0463, which is also disclosed in GenBank accession
number X531 38.1 .
Additional examples of a wild-type C. difficile TcdB include TcdB from wild-type
C. difficile strains obtainable from the Centers for Disease Control and Prevention
(CDC, Atlanta, GA). The inventors discovered that the amino acid sequence of TcdB
from wild-type C. difficile strains obtainable from the CDC include at least about 96% to
100% identity, when optimally aligned, to amino acid residue 1 to 821 of SEQ ID NO: 2
(TcdB from C. difficile 630). See Table 2 .
Table 2: wild-type C. difficile strains obtained from CDC and the % identity of
amino acid residues 1-821 of TcdB from the respective wild-type C. difficile strain to
amino acid residues 1-821 of SEQ ID NO: 2, when optimally aligned.
Accordingly, in one embodiment, a wild-type C. difficile TcdB amino acid
sequence includes a sequence of at least about 500, 600, 700, or 800 contiguous
residues, which has at least about 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, preferably
about 97%, preferably about 98%, more preferably about 99% or most preferably about
100% identity to a sequence of equal length between residues 1 to 900 of SEQ ID NO:
2 when optimally aligned, such as by the programs GAP or BESTFIT using default gap
weights. Examples include strains described above (e.g., R20291 , CD1 96, etc) and
those listed in Table 2 .
In another embodiment, the wild-type C. difficile TcdB amino acid sequence
includes a sequence having at least about 90%, 9 1%, 92%, 93%, 94%, 95%, 96%,
preferably about 97%, preferably about 98%, more preferably about 99% or most
preferably about 100% identity to any sequence selected from SEQ ID NOs: 110-1 33
when optimally aligned. See Table 2-a.
Table 2-a Wild-type C. difficile Strains
C. difficile Strain ID Toxin B, SEQ ID NO:
2004013 SEQ ID NO: 110
20041 SEQ ID NO: 111
20041 18 SEQ ID NO: 112
2004205 SEQ ID NO: 113
2004206 SEQ ID NO: 114
2005022 SEQ ID NO: 115
2005088 SEQ ID NO: 116
2005283 SEQ ID NO: 117
2005325 SEQ ID NO: 118
2005359 SEQ ID NO: 119
2006017 SEQ ID NO: 120
2006376 SEQ ID NO: 12 1
2007070 SEQ ID NO: 122
2007217 SEQ ID NO: 123
2007302 SEQ ID NO: 124
2007816 SEQ ID NO: 125
2007838 SEQ ID NO: 126
2007858 SEQ ID NO: 127
2007886 SEQ ID NO: 128
2008222 SEQ ID NO: 129
2009078 SEQ ID NO: 130
2009087 SEQ ID NO: 13 1
2009141 SEQ ID NO: 132
2009292 SEQ ID NO: 133
001 SEQ ID NO: 158
002 SEQ ID NO: 159
003 SEQ ID NO: 160
0 12 (004) SEQ ID NO: 16 1
014 SEQ ID NO: 141
0 15 SEQ ID NO: 142
0 17
020 SEQ ID NO: 143
023 SEQ ID NO: 144
027 SEQ ID NO: 145
029 SEQ ID NO: 146
046 SEQ ID NO: 147
053 SEQ ID NO: 173
059
070 SEQ ID NO: 162
075 SEQ ID NO: 163
077 SEQ ID NO: 164
078 SEQ ID NO: 174
081 SEQ ID NO: 165
087 SEQ ID NO: 175
095 SEQ ID NO: 176
106
117 SEQ ID NO: 166
126 SEQ ID NO: 177
13 1 SEQ ID NO: 167
The genes for toxins A and B (tcdA and tcdB) are part of a 19.6-kb genetic locus
(the pathogenicity locus, PaLoc) that includes 3 additional small open-reading frames
(ORFs), tcdD, tcdE, and tcdC, and may be considered useful for virulence. The PaLoc
is known to be stable and conserved in toxigenic strains. It is present at the same
chromosomal integration site in all toxigenic strains that have been analyzed to date. In
nontoxigenic strains, the pathogenicity locus (PaLoc) is not present. Accordingly, a
characteristic of the wild-type C. difficile strains described herein is the presence of a
pathogenicity locus. Another preferred characteristic of the wild-type C. difficile strains
described herein is the production of both TcdA and TcdB.
In one embodiment, the wild-type C. difficile strain is a strain having a
pathogenicity locus that is at least about 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%,
preferably about 98%, more preferably about 99% or most preferably about 100%
identical to that of C. difficile 630 or VPI10463. The total pathogenicity locus sequence
of C. difficile VPI1 0463, is registered at the EMBL database with the sequence
accession number X92982, also shown in SEQ ID NO: 26. Strains in which the PaLoc
is identical to that of the reference strain VPI1 0463 are referred to as toxinotype 0 .
Strains of toxinotypes l-VII, IX, XII-XV, and XVIII-XXIV produce both TcdA and TcdB
despite variations in their toxin genes.
At the N-terminus of the toxins, the glucosyltransferase domain is located. The
glucosyltransferase activity of the toxins is associated with the cytotoxic function of the
toxins. Without being bound by mechanism or theory, the glucosyltransferase activity in
both toxins is believed to catalyze the monoglucosylation of small GTP-binding proteins
in the Rho/Rac/Ras superfamily. After glucosylation of these GTP binding proteins,
cellular physiology is modified dramatically, resulting in a loss of structural integrity and
disruption of essential signaling pathways of the host cells infected by the toxins. The
Asp-Xaa-Asp (DXD) motif, which is involved with manganese, uridine diphosphate
(UDP), and glucose binding, is a typical characteristic for the glucosyltransferase
domain. Without being bound by mechanism or theory, it is believed that residues
critical for catalytic activity, such as the DXD motif, do not vary between a TcdB from a
known "historical" strain, such as 630, and a TcdB from a hypervirulent strain, such as
R20291 . The DXD motif is located at residues 285 to 287 of a wild-type C. difficile
TcdA, according to the numbering of SEQ ID NO: 1, and at residues 286 to 288 of a
wild-type C. difficile TcdB, according to the numbering of SEQ ID NO: 2 .
Global alignment algorithms (e.g., sequence analysis programs) are known in the
art and may be used to optimally align two or more amino acid toxin sequences to
determine if the toxin includes a particular signature motif (e.g., DXD in the
glucosyltransferase domain, DHC in the cysteine protease domain described below,
etc.). The optimally aligned sequence(s) are compared to a respective reference
sequence (e.g., SEQ ID NO:1 for TcdA or SEQ ID NO: 2 for TcdB) to determine the
existence of the signature motif. Optimal alignment" refers to an alignment giving the
highest percent identity score. Such alignment can be performed using known
sequence analysis programs. In one embodiment, a CLUSTAL alignment (such as
CLUSTALW) under default parameters is used to identify suitable wild-type toxins by
comparing the query sequence against the reference sequence. The relative numbering
of the conserved amino acid residues is based on the residue numbering of the
reference amino acid sequence to account for small insertions or deletions (for
example, five amino acids of less) within the aligned sequence.
As used herein, the term "according to the numbering of refers to the numbering
of the residues of a reference sequence when the given amino acid or polynucleotide
sequence is compared to the reference sequence. In other words, the number or
residue position of a given polymer is designated with respect to the reference
sequence rather than by the actual numerical position of the residue within the given
amino acid or polynucleotide sequence.
For example, a given amino acid sequence, such as that of a hypervirulent wildtype
C. difficile strain, can be aligned to a reference sequence (e.g., such as that of a
historical wild-type C. difficile strain, e.g., 630) by introducing gaps, if necessary, to
optimize residue matches between the two sequences. In these cases, although the
gaps are present, the numbering of the residue in the given amino acid or
polynucleotide sequence is made with respect to the reference sequence to which it has
been aligned. As used herein, a "reference sequence" refers to a defined sequence
used as a basis for a sequence comparison.
Unless stated otherwise, all references herein to amino acid positions of a TcdA
refer to the numbering of SEQ ID NO: 1. Unless stated otherwise, all references herein
to amino acid positions of a TcdB refer to the numbering of SEQ ID NO: 2 .
The glucosyltransferase domain of TcdA, as used herein, may begin at
exemplary residue 1, 10 1, or 102, and may end at exemplary residue 542, 5 16, or 293
of a wild-type C. difficile TcdA, e.g., SEQ ID NO: 1. Any minimum residue position may
be combined with a maximum residue position between residues 1 and 542 of TcdA to
define a sequence for the glucosyltransferase domain as long as the DXD motif region
is included. For example, in one embodiment, the glucosyltransferase domain of TcdA
includes SEQ ID NO: 27, which is identical to residues 10 1-293 of SEQ ID NO: 1, and it
includes the DXD motif region. In another embodiment, the glucosyltransferase domain
of TcdA includes SEQ ID NO: 28, which is identical to residues 1-542 of SEQ ID NO: 1.
The glucosyltransferase domain of TcdB, as used herein, may begin at
exemplary residue 1, 10 1, or 102, and may end at exemplary residue 543, 5 16, or 293
of a wild-type C. difficile TcdB, e.g., SEQ ID NO: 2 . Any minimum residue position may
be combined with a maximum residue position between residues 1 and 543 of TcdB to
define a sequence for the glucosyltransferase domain as long as the DXD motif region
is included. For example, in one embodiment, the glucosyltransferase domain of TcdB
includes SEQ ID NO: 29, which is identical to residues 101-293 of SEQ ID NO: 2, and it
includes the DXD motif region. In another embodiment, the glucosyltransferase domain
of TcdB includes SEQ ID NO: 30, which is identical to residues 1-543 of SEQ ID NO: 2 .
Without being bound to theory or mechanism, it is believed that the N-terminus of
TcdA and/or TcdB is cleaved by an autoproteolytic process for the glucosyltransferase
domain to be translocated and released into the host cell cytosol, 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 may be characterized by the catalytic triad aspartate,
histidine, and cysteine (DHC), e.g., D589, H655, and C700 of a wild-type TcdA, and
D587, H653, and C698 of a wild-type TcdB. Without being bound by mechanism or
theory, it is believed that the catalytic triad is conserved between a toxin from a
"historical" strain, such as 630, and a TcdB from a hypervirulent strain, such as R20291 .
The cysteine protease domain of TcdA, as used herein, may begin at exemplary
residue 543, and may end at exemplary residue 809 769, 768, or 767 of a wild-type
TcdA, e.g., SEQ ID NO: 1. Any minimum residue position may be combined with a
maximum residue position between 543 and 809 of a wild-type TcdA to define a
sequence for 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 includes SEQ ID NO: 32, which has the DHC motif region located at residues 47,
113, and 158 of SEQ ID NO: 32, which respectively correspond to D589, H655, and
C700 of a wild-type TcdA according to the numbering of SEQ ID NO: 1. SEQ ID NO:
32 is identical to residues 543 to 809 of SEQ ID NO: 1, TcdA.
The cysteine protease domain of TcdB, as used herein, may begin at exemplary
residue 544, and may end at exemplary residue 801 , 767, 755, or 700 of a wild-type
TcdB, e.g., SEQ ID NO: 2 . Any minimum residue position may be combined with a
maximum residue position between 544 and 801 of a wild-type TcdB to define a
sequence for 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 includes SEQ ID NO: 33, which includes the DHC motif region located at residues
44, 110, and 115 of SEQ ID NO: 33, which respectively correspond to D587, H653, and
C698 of a wild-type TcdB according to the numbering of SEQ ID NO: 2 . SEQ ID NO: 33
is identical to residues 544 to 767 of SEQ ID NO: 2, TcdB. In another embodiment, the
cysteine protease domain of TcdB includes residues 544-801 of SEQ ID NO: 2, TcdB.
In the present invention, the immunogenic composition includes a mutant C.
difficile toxin. The term "mutant," as used herein, refers to a molecule that exhibits a
structure or sequence that differs from the corresponding wild-type structure or
sequence, e.g., by having crosslinks as compared to the corresponding wild-type
structure and/or by having at least one mutation, as compared to the corresponding
wild-type sequence when optimally aligned, such as by the programs GAP or BESTFIT
using default gap weights. The term "mutant" as used herein further includes a
molecule that exhibits a functional property (e.g., abrogated glucosyltransferase and/or
abrogated cysteine protease activity) that differs from the corresponding wild-type
molecule.
A C. difficile toxin from any of the wild-type strains described above may be used
as a source from which a mutant C. difficile toxin is produced. Preferably, C. difficile
630 is the source from which a mutant C. difficile toxin is produced.

CLAIMS:
1. 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 NHydroxysuccinimide
(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 NHydroxysuccinimide
(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.
11. The isolated polypeptide as in any of claims 1- 10, wherein the polypeptide has
an EC5oof at least about 100 g/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 EC5oof at least about 100 g/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.
6 . 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 12-1 6, 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 12-1 6, 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 12-1 8, wherein the
polypeptide has an ECso of at least about 100 g/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.
2 1.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 wildtype
Clostridium difficile toxin A .
22. The composition according to claim 2 1, 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 2 1-23, 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 2 1-26, 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 10 1, 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 2 1-26, 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
10 1, 269, 272, 285, 287, 269, 272, 460, 462, 541 , or 542, or any combination
thereof, of SEQ ID NO: 29.
3 1.The composition according to any of claims 2 1-26, 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 2 1-26, 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, 113, 158, or any
combinations thereof, of SEQ ID NO: 32.
34. The composition according to claim 2 1, wherein the mutant Clostridium difficile toxin
A comprises SEQ ID NO: 4 .
35. The composition according to claim 2 1, wherein the mutant Clostridium difficile toxin
A comprises SEQ ID NO: 84.
36. The composition according to claim 2 1, wherein the mutant Clostridium difficile toxin
A comprises SEQ ID NO: 7 .
37. The composition according to claim 2 1, wherein the mutant Clostridium difficile toxin
A comprises SEQ ID NO: 83.
38. The composition according to any of claims 2 1-33, 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.
4 1.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 2 1-41 , 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.
5 1.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 2 1-51 , 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 wildtype
Clostridium difficile toxin B.
60. The composition according to claim 59, wherein the mutation is a non-conservative
amino acid substitution.
6 1.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: 3 1 .
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: 3 1.
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.
7 1.The composition according to claim 70, wherein the cysteine protease domain
comprises a non-conservative mutation present at positions 1, 44, 110, 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.
8 1.An immunogenic composition comprising a mutant Clostridium difficile toxin B, which
comprises a glucosyltransferase domain comprising SEQ ID NO: 3 1 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 8 1 or 82, wherein the at least one amino acid is
crosslinked by formaldehyde.
84. The composition according to claim 8 1 or 82, wherein the at least one amino acid is
crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
85. The composition according to claim 8 1, 82, or 84, wherein the at least one amino
acid is crosslinked by N-hydroxysuccinimide.
86. The composition according to claim 8 1 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.
9 1.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: 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.
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.
10 1. 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 11186.
107. The cell according to claim 106, wherein the cell is a Clostridium difficile VPI
11186 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
spoOA gene or an spollE gene.
110 . 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.
111. The method according to claim 110, wherein the cell lacks an endogenous
polynucleotide encoding a toxin.
112 . The method according to claim 110, wherein the recombinant cell or progeny
thereof comprises a cell according to any of claims 97-1 11.
113 . The method according to claim 110, further comprising isolating the mutant
Clostridium difficile toxin.
114. The method according to claim 113, further comprising contacting the isolated
mutant Clostridium difficile toxin with formaldehyde.
115 . The method according to claim 114, wherein the contacting occurs for at most 14
days.
116 . The method according to claim 115, wherein the contacting occurs for at most 48
hours.
117 . The method according to claim 114, wherein the contacting occurs at about
25°C.
118 . The method according to claim 113, further comprising contacting the isolated
mutant Clostridium difficile toxin with ethyl- 3-(3-dimethylaminopropyl) carbodiimide.
119 . The method according to claim 118, 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.
12 1. The method according to claim 118, wherein the contacting occurs at about
25°C.
122. The method according to claim 118, 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
110- 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: 4 1 (CDR H 1) , 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 L 1) , SEQ ID NO: 39 (CDR L2) and SEQ ID NO: 40 (CDR L3).
13 1. 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 128-1 3 1.
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: 5 1 (CDR H 1) , 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 L 1) , 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: 6 1 (CDR H 1) , 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 L 1) , 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 H 1) , 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 L 1) , SEQ ID NO: 80 (CDR L2) and SEQ ID NO: 8 1 (CDR L3).
138. A composition comprising a combination of two or more antibodies or antibody
binding fragments thereof selected from any of claims 133-1 37.
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 Nhydroxysuccinimide.
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 139-1 44, 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.
15 1. The method according to claim 148, wherein the adjuvant comprises
ISCOMATRIX®.