CLOSTRIDIUM DIFFICILE TOXINS A AND/OR B ANTIGEN AND EPITOPE ANTIBODY, AND PHARMACEUTICAL USES THEREOF

Abstract

It is described a Clostridium difficile (C-difficile) toxins A and/or B as a target for therapy, including passive immunotherapy, and particularly prevention of C-difficile intoxication in human or other animals. It is also described a polypeptide comprising a portion of C-difficile toxins A and/or B sequence being an epitope for anti-toxins A and/or B antibody. It is also disclosed a method for generating a neutralizing antibody directed against C-difficile toxins A and/or B. It is also provided a novel formulation that combines key toxins A and/or B epitope antibodies, located in three key domains of toxins A and/or B, for neutralisation of the toxins A and/or B, at any stage of toxins A and/or B intoxication related to C-difficile infection. The novel formulation of toxins A and/or B epitope antibodies are useful in immunotherapy, for therapeutic and/or prophylactic mediation of C-difficile intoxication.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional application of U.S. application Ser. No. 15/551,848 filed Aug. 17, 2017, which is a national phase application of PCT application No. PCT/CA2016/050170 filed Feb. 19, 2016, which claims the benefits of U.S. Provisional Patent Application No. 62,118,450, filed on Feb. 19, 2015. The content of these applications is incorporated herewith by reference.

FIELD OF THE INVENTION

[0002] The present invention generally relates to epitopes of toxins A and/or B produced by Clostridium difficile and antibodies that specifically binding to these epitopes. The invention is also related to pharmaceutical compositions and vaccines for the prevention or treatment of Clostridium difficile infection comprising any of the epitopes or antibodies thereof.

BACKGROUND OF THE INVENTION

[0003] Clostridium difficile is a Gram-positive, anaerobic, endospore-forming gastrointestinal pathogen responsible for C-difficile-associated disease (CDAD) in humans and animals with symptoms ranging in severity from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis (Rupnik et al, 2009; Leffler and Lamont, 2009; Songer, 2004; Kelly et al, 1994). Each year in North America, 1-3% of hospitalized patients receiving antibiotics become infected with C-difficile, leading to thousands of deaths and over $1 billion in associated costs to the health-care system (Wilkins and Lyerly, 2003; Kyne et al, 2002; Kelly et al, 1994). C-difficile produces two primary virulence factors, toxin A (TcdA) and toxin B (TcdB), which are large (308 kDa and 269 kDa, respectively), single-subunit exotoxins composed of a catalytic, a translocation and a cell-receptor binding domain (RBD) (Jank and Aktories, 2008; Jank et al, 2007). Recently it was suggested TcdB is solely responsible for C-difficile virulence (Lyras et al, 2009), although earlier studies have shown both anti-TcdA and anti-TcdB monoclonal antibodies (mAbs) were required for full protection of hamsters from CDAD (Babcock et al, 2006; Kink and Williams, 1998) and anti-TcdA mAbs were required for protection in mice (Corthier et al, 1991).

[0004] The current approach for treating most CDAD infections involves administration of antibiotics, most commonly metronidazole or vancomycin (Leffler and Lamont, 2009). Antibiotic treatment places selection pressure on the organism, can lead to antibiotic resistance, and suppresses or eliminates beneficial commensal microbes. However, there are several other emerging challenges warranting the development of novel therapeutics. First, there is no acute CDAD treatment targeting TcdA and/or B. These toxins are responsible for loss of epithelial barrier function in the colon by disrupting tight junctions and increasing membrane permeability, causing diarrhea and promoting severe inflammation (Rupnik et al, 2009; Jank and Aktories, 2008). Second, hypervirulent strains of C-difficile, such as the NAP1/027 isolate, over-express TcdA and TcdB (Warny et al, 2005) and have been associated with increased mortality rates and disease severity (O'Connor et al, 2009; Pepin et al, 2005). Third, an estimated 20-25% of patients suffering from CDAD experience symptomatic relapse after the initial infection is cleared, with 45% of these patients prone to subsequent relapses (Johnson, 2009). Taken together, there is a need for non-antibiotic based reagents targeting and inhibiting TcdA and TcdB for CDAD therapy. Individuals who are asymptomatic C-difficile carriers and patients who experience mild cases of CDAD tend to possess high anti-toxin A titers (Kyne et al, 2001; Kyne et al, 2000; Warny et al, 1994; Viscidi et al, 1983). Conversely, patients susceptible to relapsing C-difficile infection have low anti-TcdA immunoglobulin titers, specifically IgM, lgG2 and lgG3 isotypes (Katchar et al, 2007; Kyne et al, 2001). TcdA-neutralizing secretory IgA antibodies are also thought to play a role in regulating CDAD severity (Johal et al 2004; Kelly et al 1992). Therefore, the introduction of anti-toxin antibodies to patients suffering from severe C-difficile infection may be a therapeutically useful approach.

[0005] A limited number of animal and human studies have illustrated the effectiveness of anti-toxin Abs for treatment of CDAD. Babcock et al (2006) intravenously administered anti-TcdA and anti-TcdB mAbs to hamsters and found a significant reduction in hamster mortality in prophylactic, primary disease and relapse models when both anti-toxin mAbs were administered. A recently completed clinical trial involving these two humanized mAbs appears promising (Lowy et al, 2010). In another study, intravenous administration of anti-TcdA mAbs raised against the RBD followed by oral challenge with C-difficile resulted in protection of mice (Corthier et al, 1991). Elsewhere, a toxoid vaccine given by the intraperitoneal route to hamsters conferred protection against oral C-difficile challenge (Giannasca et al, 1999) and mice vaccinated with DNA encoding the TcdA RBD resulted in full protection from oral TcdA challenge (Gardiner et al, 2009). In humans, a number of uncontrolled studies have reported intravenous immunoglobulin (IVIG) therapy to be successful for the treatment of severe CDAD (Juang et al, 2007; Hassoun and Ibrahim, 2007; McPherson et al, 2006; Wilcox, 2004; Salcedo et al, 1997; Leung et al, 1991). IVIG involves administration of high concentrations (150-400 mg/kg) of human immunoglobulins from healthy donors which are thought to contain neutralizing anti-toxin antibodies as an estimated 60% of healthy adults have detectable TcdA- and TcdB-specific serum IgG antibodies (Viscidi et al, 983). Given that C-difficile toxins rely on attachment to epithelial cells for entry (Jank and Aktories, 2008; Jank et al, 2007), neutralizing the toxins within the lower gastrointestinal tract with antibodies may block the first step in CDAD pathogenesis. In animals, orally administered bovine immunoglobulin concentrate (BIC) containing TcdA and TcdB neutralizing IgGs were able to prevent hamster mortality when used as a propholyactic (Lyerly et al, 1991) and protected rats from the enterotoxic effects of TcdA in vivo (Kelly et al, 1996). Chicken IgY antibodies specific for toxin RBDs were shown to reduce hamster mortality when administered orally to infected animals (Kink and Williams, 1998). In humans, there have been limited reports on CDAD therapy with orally delivered Abs. Tjellstrom et al (1993) reported the successful treatment of a 31/2 year old boy suffering from severe CDAD with IgA antibody orally. Warny et al (1999) and Kelly et al (1997) examined the passage of anti-toxin bovine IgG through the human gastrointestinal tract and found a significant reduction in IgG activity, likely due to proteolytic degradation within the upper gastrointestinal tract. The limited success of both oral and systemic anti-toxin immunotherapy in clinical settings has likely been hampered by the high immunoglobulin dose requirements (150-400 mg/kg), the associated costs of these doses, and a lack of published clinical data showing the effectiveness of these treatments.

[0006] Despite such advances, there remains a need in the art for a safe and effective therapeutic for treating C-difficile-associated disease as well as for sensitive and effective reagents for the detection of toxins A and B, the factors responsible for C-difficile-associated disease.

SUMMARY OF THE INVENTION

[0007] The aforesaid and other objectives of the present invention are realized by generally providing specific Clostridium difficile (C-difficile) toxins A and/or B as a target for therapy, including passive immunotherapy, and particularly prevention of toxins A and/or B intoxication in human or other animals.

[0008] One aspect of the present invention is to provide an isolated polypeptide comprising a portion of Clostridium difficile toxins A and/or B sequence, the portion of toxins A and/or B sequence being an epitope for anti-toxins A and/or B antibody, the portion comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0009] Preferably, the portion of toxins A and/or B sequence may comprise a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0010] The aforesaid isolated peptides may be used for immunizing an animal against Clostridium difficile infection.

[0011] Another aspect of the present invention is to provide a pharmaceutical composition for generating a neutralizing antibody directed against Clostridium difficile toxins A and/or B and comprising at least two different polypeptides, each polypeptide comprising a portion of Clostridium difficile toxins A and/or B sequence, the portion of toxins A and/or B sequence being an epitope for anti-toxins A and/or B antibody, and the portion comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0012] Preferably, the pharmaceutical composition may comprise four different polypeptides, wherein the portion of toxins A and/or B sequence comprises a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0013] The aforesaid pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier.

[0014] The pharmaceutical compositions may be used for immunizing an animal against Clostridium difficile infection.

[0015] Another aspect of the present invention is to provide a vaccine composition for prevention or treatment of Clostridium difficile infection comprising at least one polypeptide which comprises a portion of Clostridium difficile toxins A and/or B sequence, the portion of toxins A and/or B sequence being an epitope for anti-toxins A and/or B antibody, and the portion comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0016] Preferably, the aforesaid vaccine composition may comprise the portion of toxins A and/or B sequence comprising a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0017] The aforesaid vaccine compositions may further comprise a pharmaceutically acceptable adjuvant.

[0018] Another aspect of the present invention is to provide a nucleic acid vaccine or DNA vaccine composition for prevention or treatment of Clostridium difficile infection comprising nucleic acids encoding at least one polypeptide which comprises a portion of Clostridium difficile toxins A and/or B sequence, the portion of toxins A and/or B sequence being an epitope for anti-toxins A and/or B antibody; the portion comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0019] Preferably, the aforesaid nucleic acid vaccine composition may comprise the portion of toxins A and/or B sequence comprising a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0020] The aforesaid nucleic acid vaccine compositions may further comprise a pharmaceutically acceptable adjuvant.

[0021] Another aspect of the present invention is to provide a method of generating a neutralizing antibody directed against Clostridium difficile toxins A and/or B. The method comprises a first step of administrating to a host an isolated polypeptide comprising a portion of the Clostridium difficile toxins A and/or B sequence, the portion comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof, for generating antibodies in the host. The method also comprises a second step of obtaining the antibodies from the host. The host may be a mammal or a bird, including bird eggs, such as but not limited to chicken eggs.

[0022] Preferably, in the first step of the method, the portion of the Clostridium difficile toxins A and/or B sequence comprises a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0023] Another aspect of the present invention is to provide a purified antibody adapted for binding to a toxins A and/or B peptide, the peptide comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0024] The aforesaid antibody may also be an active fragment thereof, a chimeric antibody, a veneered antibody, a humanized antibody or a single chain recombinant antibody based thereon as well as a bird polyclonal antibody or a bird humanized recombinant antibody.

[0025] The aforesaid antibody may be used for detecting pure toxins A and/or B or a presence of toxins A and/or B produced by Clostridium difficile in cell culture.

[0026] Another aspect of the present invention is to provide an antibody composition for prevention or treatment of Clostridium difficile infection, comprising different antibodies adapted for binding to at least two epitopes of toxins A and/or B, the at least two epitopes comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0027] Preferably, the aforesaid antibody composition may comprises different antibodies adapted for binding to four different epitopes, the epitopes comprising a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0028] Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or the treatment of Clostridium difficile toxins A and/or B intoxication, the composition comprising at least one antibody adapted for binding to at least one epitope of toxins A and/or B, the at least one epitope comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0029] Preferably, the aforesaid pharmaceutical composition may comprises at least one epitope comprising a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0030] The aforesaid pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier.

[0031] The aforesaid pharmaceutical compositions may be used for making a medicament for preventing or treating Clostridium difficile infection.

[0032] The aforesaid pharmaceutical compositions may also be used for the capture and neutralisation of Clostridium difficile toxins A and/or B, allowing a passive immunotherapy of mammals, such as humans.

[0033] The features of the present invention which are believed to be novel are set forth with particularity in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and other objects, features and advantages of the invention will become more readily apparent from the following description, reference being made to the accompanying drawings in which:

[0035] FIGS. 1A and B are schematic representation of the targeted portion of toxins A and/or B epitope antibodies.

[0036] FIGS. 2A and B illustrates the targeted sequence portion of toxins A and/or B epitope antibodies.

[0037] FIG. 3 is a photograph of a western blot showing recognition of toxins A and/or B by toxins A and/or B epitope antibody IBSCD1, according to a preferred embodiment of the present invention.

[0038] FIG. 4 is a photograph of a western blot showing recognition of toxins A and/or B by toxins A and/or B epitope antibody IBSCD2, according to a preferred embodiment of the present invention.

[0039] FIG. 5 is a photograph of a western blot showing recognition of toxins A and/or B by toxins A and/or B epitope antibody IBSCD3, according to a preferred embodiment of the present invention.

[0040] FIG. 6 is a photograph of a western blot showing recognition of toxins A and/or B by toxins A and/or B epitope antibody IBSCD4, according to a preferred embodiment of the present invention.

[0041] FIGS. 7A-D is a set of microscope photographs showing the neutralisation effect on the cell rounding and dead of Caco-2 intestinal cell line by toxins A and/or B epitope antibodies formulation in presence of the supernatant of C-difficile NAP1/027 hypervirulent strain, according to a preferred embodiment of the present invention.

[0042] FIGS. 8A-F is a set of microscope photographs showing the neutralisation effect on the cell rounding and dead of Caco-2 intestinal cell line by toxins A and/or B epitope antibodies formulation in presence of the supernatant of C-difficile NAP1/027 hypervirulent strain and purified toxin A and toxin B, according to a preferred embodiment of the present invention.

[0043] FIGS. 9A-F is a set of epifluorescence microscope photographs showing improvement of Caco-2 cell viability by the blocking antibodies against C-difficile toxin A and/or B, according to a preferred embodiment of the present invention.

[0044] FIGS. 10A-C is a set of graphs showing the protection of the Caco-2 monolayer integrity by the blocking antibodies against C-difficile toxin A and/or B, according to a preferred embodiment of the present invention.

[0045] FIGS. 11A and B is a set of graphs showing the reduction of Clostridium difficile infection in vivo by the blocking antibodies against C-difficile toxin A and/or B, according to a preferred embodiment of the present invention.

[0046] FIGS. 12A-C is a set of microscope photographs showing the reduction of mucosal damage in murine colon by the blocking antibodies against C-difficile toxin A and/or B, according to a preferred embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0047] A novel composition of anti-toxins A and/or B antibodies and toxins A and/or B epitopes will be described hereinafter. Although the invention is described in terms of specific illustrative embodiment(s), it is to be understood that the embodiment(s) described herein are by way of example only and that the scope of the invention is not intended to be limited thereby.

[0048] A broad aspect, the present invention provides a method that uses a combination of toxins A and/or B epitope antibodies, including but not limited to bird's epitope antibodies, for the capture and neutralisation of C-difficile toxins A and/or B, which permit a passive immunotherapy of humans or others mammals. The method of neutralisation aims three different key portions of toxins A and/or B among the four domains of C-difficile toxins, which are the combined repetitive oligopeptides c-terminal domain (CROPs) 10, the delivery pore forming domain 20, the auto-protease domain 30, and the N-terminal glucosyltransferase domain 40, as illustrated in FIGS. 1 and 2. These different portions of toxins A and/or B includes four different epitopes adapted for generating toxins A and/or B epitope antibodies in mammals or birds. Furthermore, FIG. 2 shows the location of the four epitopes within the toxins A and B amino acid sequences as part of the present invention.

[0049] According to a first embodiment of the present invention, it is provided an isolated peptide having the amino acid sequence: DSKKYYFNTNTAEAA (SEQ ID NO: 1). This particular peptide is a toxins A and/or B epitope peptide encompassing amino acids 2084-2098 of toxins A and/or B, which has been identified as a shared toxins A and/or B epitope using the chicken polyclonal antibodies IBSCD1 (see FIG. 3).

[0050] According to a second embodiment of the present invention, it is provided an isolated peptide having the amino acid sequence: ANQYEVRINSEGR (SEQ ID NO: 2). This particular peptide is a toxins A and/or B epitope peptide encompassing amino acids 739-751 of toxins A and/or B, which has been identified as a shared toxins A and/or B epitope using the chicken polyclonal antibodies IBSCD2 (see FIG. 4).

[0051] According to a third embodiment of the present invention, it is provided an isolated peptide having the amino acid sequence GHGKDEFNTDIFAG (SEQ ID NO: 3). This particular peptide is a toxins A and/or B epitope peptide encompassing amino acids 652-665 of toxins A and/or B, which has been identified as a shared toxins A and/or B epitope using the chicken polyclonal antibodies IBSCD3 (see FIG. 5).

[0052] According to a forth embodiment of the present invention, it is provided an isolated peptide having the amino acid sequence DEYNKLTTNNNENKYL (SEQ ID NO: 4). This particular peptide is a toxins A and/or B epitope peptide encompassing amino acids 31-46 of toxins A and/or B, which has been identified as a shared toxins A and/or B epitope using the chicken polyclonal antibodies IBSCD4 (see FIG. 6).

[0053] In accordance with another embodiment of the present invention, the isolated peptides, including combinations of one or more thereof, are adapted for generating antibodies which recognize toxins A and/or B and have a neutralising therapeutic or prophylactic activity in immunizing animals, particularly mammals, most particularly humans, who have a C-difficile intoxication.

[0054] In accordance with a preferred embodiment of the present invention, it is provided a combination of polypeptides, which comprises all the afore-mentioned amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or any combination thereof.

[0055] The afore-mentioned polypeptides may include an immunogenic peptide, particularly comprising amino acid sequence of any of SEQ ID NOS: 1-4, or an immunogenic fragment thereof. These polypeptides may also include immunogenic receptor of peptides, wherein such polypeptides comprise a combination of at least one immunogenic receptor peptide comprising amino acid sequence of any of SEQ ID NOS: 1-4, or immunogenic peptide fragment thereof.

[0056] In accordance with another embodiment of the present invention, it is provided a method for immunizing an animals, particularly mammals or birds comprising administering a toxins A and/or B epitope peptide or an immunogenic fragment thereof, whereby the animal produces antibodies that are immunoreactive with the epitope peptide exposed on partial or full length toxins A and/or B produced by C-difficile bacteria. The method for immunizing mammals or birds may comprise administering a toxins A and/or B peptide comprising amino acid sequence of any of SEQ ID NOS: 1-4 or an immunogenic fragment thereof, whereby the animal produces antibodies that are immunoreactive to full length toxins A and/or B produced by C-difficile bacteria. According to a preferred embodiment, the aforesaid method comprises the use of the four different toxins A and/or B peptides with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0057] In accordance with another embodiment of the present invention, it is provided a pharmaceutical composition for generating a neutralizing antibody directed against Clostridium difficile toxins A and/or B, the pharmaceutical composition comprising a toxins A and/or B peptide, particularly with amino acid sequence of any of SEQ ID NOS: 1-4, and a pharmaceutically acceptable carrier. According to a preferred embodiment, the pharmaceutical composition comprises the four different toxins A and/or B peptides with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and a pharmaceutically acceptable carrier.

[0058] In accordance with another embodiment of the present invention, it is provided a vaccines or immunogenic compositions that comprise one or more toxins A and/or B peptide, particularly with amino acid sequence of any of SEQ ID NOS: 1-4, and a pharmaceutically acceptable adjuvant. According to a preferred embodiment, the vaccine composition comprises the four different toxins A and/or B peptides with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and a pharmaceutically acceptable adjuvant.

[0059] The vaccine may also be used for treatment of a subject, such as a mammal, particularly a human subject, suffering from a C-difficile intoxication. Such vaccine may comprise an immunogenic amount of one or more toxins A and/or B peptides, particularly with amino acid sequence of any of SEQ ID NOS: 1-4 or immunogenic fragment thereof, and a pharmaceutically acceptable adjuvant. The aforesaid toxins A and/or B peptides may be conjugated to a carrier.

[0060] In accordance with another embodiment of the present invention, it is provided a nucleic acid vaccines or DNA vaccines comprising nucleic acids encoding immunogenic toxins A and/or B peptides, particularly with amino acid sequence of any of SEQ ID NOS: 1-4, and a pharmaceutically acceptable adjuvant. According to a preferred embodiment, the nuclei acid vaccine composition comprises the nucleic acids encoding the four different toxins A and/or B peptides with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and a pharmaceutically acceptable adjuvant. The afore-mentioned nucleic acid vaccine may further comprises at least one other polypeptide, particularly an immunomodulatory molecule peptide derived from C-difficile.

[0061] In accordance with another embodiment of the present invention, it is provided a method for diagnosis of Clostridium difficile infection comprising the steps of contacting a biological sample of a subject with at least one peptide fragment of toxins A and/or B and detecting antigen-antibody complex formation. Such at least one peptide fragment may comprises an epitope for anti-toxins A and/or B antibody with an amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0062] In accordance with another embodiment of the present invention, it is provided a kit for the diagnosis or detection of Clostridium difficile infection, the kit comprising at least one peptide fragment of toxins A and/or B and directions for diagnosing or detecting anti-toxin A and/or B antibody. Such at least one peptide fragment may comprise an epitope for anti-toxin A and/or B antibody with an amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0063] As previously mentioned, toxins A and/or B polypeptides with the amino acid sequence of any of SEQ ID NOS: 1-4 are adapted for generating toxins A and/or B epitope antibodies in mammals or birds. Such antibodies recognize toxins A and/or B and have neutralising effect on the activity of these toxins, so that the antibodies may be used for a therapeutic or prophylactic treatment against C-difficile intoxication in humans and animals.

[0064] In accordance with another embodiment of the present invention, it is provided a method of generating a neutralizing antibody directed against Clostridium difficile toxins A and/or B. The method comprises a first step of administrating to a host an isolated polypeptide comprising a portion of the Clostridium difficile toxins A and/or B sequence. The portion comprising a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof, for generating antibodies in the host. The method comprises a second step of obtaining the antibodies from the host. The host may be a mammal or a bird, including the bird eggs, such as but not limited to chicken eggs.

[0065] The aforesaid method may also comprise a first step, wherein the portion of the Clostridium difficile toxins A and/or B sequence comprises a sequence being SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0066] Referring to FIG. 3, an antibody, called IBSCD1, is adapted for binding to a key toxins A and/or B epitope, encompassing amino acids 2084-2098 of toxin A and/or B located in combined repetitive oligopeptides c-terminal domain (CROPs) 10 of toxins A and/or B. This domain plays a role in the binding to human or animal cells, as illustrated in FIG. 1, step 1.

[0067] Referring to FIGS. 4 and 5, two antibodies, called IBSCD2 and IBSCD3, are adapted for binding to toxins A and/or B epitopes, encompassing respectively amino acids 739-751 and 652-665 of toxins A and/or B located in the delivery pore forming domain 20. This domain plays a role in the endosome pore formation of the infected cell, as illustrated in FIG. 1, step 3.

[0068] Referring to FIG. 6, another antibody, called IBSCD4, bind the toxins A and/or B epitope encompassing amino acids 31-46 of toxins A and/or B located in the glucosyltransferase domain 40. This domain plays a role in the inactivation of small GTPases in the affected cells, as illustrated in FIG. 1, step 4.

[0069] In accordance with a preferred embodiment of the present invention, it is provided a novel formulation that combines key toxins A and/or B epitope antibodies, located in three key domains of toxins A and/or B, for neutralisation of the toxins A and/or B, at any stage of toxins A and/or B intoxication related to C-difficile infection. Therefore, the novel formulation of toxins A and/or B epitope antibodies may be used in immunotherapy, for therapeutic and/or prophylactic mediation of C-difficile intoxication.

[0070] In accordance with another embodiment, it is provided a purified antibody to a toxin A and/or B peptide comprising the amino acid sequence of any of SEQ ID NOS: 1-4. The above-described antibodies may specifically detect pure toxins A and/or B or the presence of these toxins produced by C-difficile in culture.

[0071] The antibody may be selected from antibodies IBSCD1, IBSCD2, IBSCD3, IBSCD4 or active fragments thereof. The antibody may include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific antibodies, and antibodies including other functionalities suiting them for diagnostic or therapeutic use. The antibody may consist of bird polyclonal or bird humanized recombinant antibodies. Furthermore, the antibody may include, but not limited to, naturally raised and recombinant prepared antibodies or fragments thereof, including single chain variants and Fv. The antibodies may also include chimereic antibodies, veneered antibodies, humanized antibodies, chicken polyclonal antibodies, chicken recombinant humanized antibodies, domain antibodies, calemized antibodies and single chain recombinant antibodies. Such antibodies can be used for passive immunization to reduce C-difficile intoxication, particularly in humans.

[0072] In accordance with another embodiment of the present invention, it is provided a pharmaceutical composition for preventing or treating C-difficile toxins A and/or B intoxication. The pharmaceutical composition may comprise at least one antibody adapted for binding to at least one epitope of toxins A and/or B, and a pharmaceutically acceptable carrier. Such epitope may comprise a amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof. According to a preferred embodiment, the pharmaceutical composition comprises the antibodies adapted for binding to the four different toxins A and/or B peptides with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and a pharmaceutically acceptable carrier.

[0073] In accordance with a another embodiment of the present invention, it is provided therapeutic methods based upon the activity of an antibody, or active fragments thereof, adapted for binding to a toxins A and/or B peptide comprising amino acid sequence of any of SEQ ID NOS: 1-4. In particular, the method may comprise antibodies, or active fragments thereof, and chimeric or synthetic antibodies derived therefrom, and can be prepared in pharmaceutical compositions, including a suitable vehicle, carrier or diluent, for administration in instances wherein therapy is appropriate, such as to treat C-difficile infection. Such methods may include oral formulations of avian of mammal anti-toxins A and/or B for prevention of toxins A and/or B intoxication. Such methods may also include modulating the half-life of the binding members, antibodies or fragments by methods known in the art such as pegylation. Such methods may further comprise additional antibodies or therapeutic agents.

[0074] In accordance with a another embodiment of the present invention, it is provided a method for diagnosis of Clostridium difficile infection comprising the steps of contacting a biological sample of a subject with at least one anti-toxins A and/or B antibody adapted for binding to at least one epitope of toxins A and/or B, and detecting antigen-antibody complex formation. Such at least one epitope may comprise an amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

[0075] In accordance with a another embodiment of the present invention, it is provided a kit for the diagnosis or detection of Clostridium difficile infection, the kit comprising at least one anti-toxins A and/or B antibody adapted for binding to at least one epitope of toxins A and/or B, and directions for diagnosing or detecting anti-toxin A and/or B antibody. Such at least one epitope may an amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof.

EXAMPLES

[0076] FIGS. 7A-D the neutralisation effect on the cell rounding and dead of Caco-2 intestinal cell line by toxins A and/or B epitope antibodies formulation in presence of the supernatant of C-difficile NAP1/027 hypervirulent strain. FIG. 7A shows intact monolayer of Caco-2 cells after incubation with 25 .mu.1 of water (negative control). FIG. 7B shows disturbed and destroyed monolayer of Caco-2 cells, with a lot of cell rounding and dead cells after incubation with 25 .mu.l of undiluted NAP1/027 strain C-difficile toxin A/B supernatant. FIG. 7C shows disturbed monolayer of Caco-2 cells, with cell rounding (arrows) and dead cells after incubation with 25 .mu.l of 1:1000 dilution of NAP1/027 strain C-difficile toxin A/B supernatant. FIG. 7D shows preserved monolayer of Caco-2 cells after incubation with 25 .mu.l of 1:1000 dilution of NAP1/027 strain C-difficile toxin A/B supernatant in the presence of a formulation containing 63 .mu.g/ml of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A/B epitope antibodies.

[0077] FIGS. 8A-F show the neutralisation effect on the cell rounding and dead of Caco-2 intestinal cell line by adding toxins A and/or B epitope antibodies formulation when in presence of the supernatant of C-difficile NAP1/027 hypervirulent strain and purified toxin A and toxin B. FIG. 8A shows complete rounding of cells, lost of adhesion and cells death after incubation with 400 ng/mL of purified toxin A. FIG. 8B shows reduction of cytotoxic effect, with decrease in rounding and cell death, after incubation with 400 ng/mL toxin A, preincubated with 63 .mu.g/mL of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies. FIG. 8C shows disturbed monolayer of Caco-2 cells, with cell rounding and dead cells and without dome formation, after incubation with 25 .mu.l of 1:100 dilution of NAP1/027 strain C-difficile toxin A/B supernatant. FIG. 8D shows preserved monolayer of Caco-2 cells after incubation with 25 .mu.l of 1:100 dilution of NAP1/027 strain C-difficile toxin A and/or B supernatant in the presence of a formulation containing 125 .mu.g/ml of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies. FIG. 8E shows complete rounding of cells, lost of adhesion and cell death after incubation with 10 ng/mL of purified toxin B. FIG. 8F shows reduction of cytotoxic effect, with decrease in rounding and cell death after incubation with 40 ng/mL toxin B, preincubated with 125 .mu.g/mL of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies.

[0078] FIGS. 9A-F show cell death induced by C. difficile toxin A and B in Caco-2 cells and the improvement of cell viability by the blocking antibodies. Caco-2 cells were seeded in chamber slides in 200 .mu.L of culture medium and incubated overnight at 37.degree. C. in CO2 incubator. The following day, cell death was induced with toxin A and B and cells were incubated for 48 h. Cells were washed with PBS and incubated with Annexin V and PI (1:10) for 15 min. Coverslips were mounted with mounting medium and DAPI. Images were taken by epifluorescence microscopy. FIG. 9A shows little or no cell death in cells treated with water. FIG. 9B shows little or no cell death due to a normal process of cell death in the presence of 125 .mu.g/mL of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 Toxins A and/or B epitope antibodies. FIG. 9C shows increased in number of green (or white), Annexin V labelled cells representing apoptotic cells, in the presence of 400 ng/mL toxin A. FIG. 9D shows a decrease in cell death with 400 ng/mL toxin A preincubated with 125 .mu.g/mL of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies. FIG. 9E shows an increase in cell death with 40 ng/mL toxin B. FIG. 9F shows a decrease in cell death with 40 ng/mL toxin B preincubated with 125 .mu.g/mL of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A/B epitope antibodies. C. difficile toxins A and B induced programmed cell death and even necrosis (represented in white), while preincubation of toxins with IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A/B epitope antibodies deceased number of apoptotic cells in all conditions. Bleu, DAPI labelled cells represent live cells.

[0079] FIGS. 10A-C show the protection of the integrity of Caco-2 monolayer by the blocking antibodies against C. difficile toxin A and B. FIG. 10A shows 7-10 days post-confluent Caco-2 monolayer that was grown on porous 4 .mu.m inserts. Only polarized monolayers of cell were used. At 6 h, the transepithelial electric resistance (TEER) of the Caco-2 monolayer rapidly decreased down to 67% when cells were stimulated with 400 ng/ml toxin A. Cells treated with toxin A preincubated with blocking antibodies IBSCD1, IBSCD2, IBSCD3 and IBSCD4 increased TEER up to 86% compared to unstimulated cells, which were reported to one. FIG. 10B shows the rapid decrease of the transepithelial electric resistance (TEER) of the Caco-2 monolayer down to 77% when cells were stimulated with 40 ng/ml toxin B compared to unstimulated cells. Use of 125 .mu.g/m of blocking antibodies restored epithelial integrity up to 93%. FIG. 10C shows that when cells were stimulates with supernatant of C. difficile NAP1/027 strain (1:10), TEER decreased to 61% compared to unstimulated after 6 h. Preincubation of 125 .mu.g/ml IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies with supernatant restored epithelial integrity up to 84%. Results represent the mean SEM of three independent experiments performed in duplicate. Statistical analysis was done with 2 way ANOVA.

[0080] FIGS. 11A-B show the reduction of Clostridium difficile infection in vivo by the blocking antibodies against C-difficile toxin A and/or B. 6-8 weeks old female mice were housed in groups of 4 in sterile cages equipped with HEPA filters and containing sterile bedding. They had access to sterile food and water ad libitum. Mice are intrinsically resistant to CDI, so they were be pre-conditioned for three days with 250 mg/L clindamycin and 400 mg/L streptomycin in the drinking water, followed by an intraperitoneal (i.p.) injection of 1 mg clindamycin/mouse to disrupt their intestinal microbiota and make them susceptible to CDI. Twenty-four hours later, mice received by gavage 10e+5 spores of the epidemic CD strain R20291 to initiate the infection. Mice were gaved twice a day with 1mg of a preparation of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies in 0.1 M carbonate buffer (pH 9.2) to neutralize the gastric acidity for 5 days. Control groups consist of uninfected mice, and infected but untreated mice. FIG. 11A shows the results from mice that were observed for a total of 7-10 days and clinical symptoms were monitored daily (diarrhea, weight loss, lethargy, etc.). FIG. 11B shows results from fresh fecal samples that were collected daily and homogenized in pre-reduced PBS, and CD were enumerated on agar plates. Clinical score end bacterial counts were the higher in untreated mice in comparison with uninfected mice. Mice receiving IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies had lower CDI score then untreated mice and lower bacterial counts.

[0081] FIGS. 12A-C show the reduction of mucosal damage in murine colon by the blocking antibodies against C-difficile toxin A and/or B. 6-8 weeks old female mice were housed in groups of 4 in sterile cages equipped with HEPA filters and containing sterile bedding. They had access to sterile food and water ad libitum. Mice are intrinsically resistant to CDI, so they were be pre-conditioned for three days with 250 mg/L clindamycin and 400 mg/L streptomycin in the drinking water, followed by an intraperitoneal (i.p.) injection of 1 mg clindamycin/mouse to disrupt their intestinal microbiota and make them susceptible to CDI. Twenty-four hours later, mice received by gavage 10e+5 spores of the epidemic CD strain R20291 to initiate the infection. Mice were gaved twice a day with 1 mg of a preparation of IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies in 0.1 M carbonate buffer (pH 9.2) to neutralize the gastric acidity for 5 days. Control groups consist of uninfected mice, and infected but untreated mice. At day ten, colons were extracted and embedded in paraffin. Hematoxylin & Eosin (H&E) staining was performed. FIG. 12A shows normal H&E staining of uninfected mice. FIG. 12B shows H&E staining of infected but untreated mice, which presented signs of mild inflammation. FIG. 12C shows H&E staining of infected mice treated with IBSCD1, IBSCD2, IBSCD3 and IBSCD4 toxins A and/or B epitope antibodies, which presented less severe mucosal damage then untreated mice. Black arrows indicate thickness of submucosal layer, immune cell infiltration and lost of epithelial layer.

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[0118] While illustrative and presently preferred embodiment(s) of the invention have been described in detail hereinabove, it is to be understood that the inventive concepts may be otherwise variously embodied and employed and that the appended claims are intended to be construed to include such variations except insofar as limited by the prior art.

Sequence CWU 1

1

6115PRTClostridium difficile 1Asp Ser Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala Ala1 5 10 15213PRTClostridium difficile 2Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg1 5 10314PRTClostridium difficile 3Gly His Gly Lys Asp Glu Phe Asn Thr Asp Ile Phe Ala Gly1 5 10416PRTClostridium difficile 4Asp Glu Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu1 5 10 1552710PRTClostridium difficile 5Met Ser Leu Ile Ser Lys Glu Glu Leu Ile Lys Leu Ala Tyr Ser Ile1 5 10 15Arg Pro Arg Glu Asn Glu Tyr Lys Thr Ile Leu Thr Asn Leu Asp Glu 20 25 30Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gln Leu 35 40 45Lys Lys Leu Asn Glu Ser Ile Asp Val Phe Met Asn Lys Tyr Lys Thr 50 55 60Ser Ser Arg Asn Arg Ala Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys65 70 75 80Glu Val Ile Leu Ile Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95Leu His Phe Val Trp Ile Gly Gly Glu Val Ser Asp Ile Ala Leu Glu 100 105 110Tyr Ile Lys Gln Trp Ala Asp Ile Asn Ala Glu Tyr Asn Ile Lys Leu 115 120 125Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala Ile 130 135 140Val Glu Ser Ser Thr Thr Glu Ala Leu Gln Leu Leu Glu Glu Glu Ile145 150 155 160Gln Asn Pro Gln Phe Asp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu 165 170 175Phe Ile Tyr Asp Arg Gln Lys Arg Phe Ile Asn Tyr Tyr Lys Ser Gln 180 185 190Ile Asn Lys Pro Thr Val Pro Thr Ile Asp Asp Ile Ile Lys Ser His 195 200 205Leu Val Ser Glu Tyr Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg 210 215 220Thr Asn Ser Leu Arg Lys Ile Asn Ser Asn His Gly Ile Asp Ile Arg225 230 235 240Ala Asn Ser Leu Phe Thr Glu Gln Glu Leu Leu Asn Ile Tyr Ser Gln 245 250 255Glu Leu Leu Asn Arg Gly Asn Leu Ala Ala Ala Ser Asp Ile Val Arg 260 265 270Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met 275 280 285Leu Pro Gly Ile His Ser Asp Leu Phe Lys Thr Ile Ser Arg Pro Ser 290 295 300Ser Ile Gly Leu Asp Arg Trp Glu Met Ile Lys Leu Glu Ala Ile Met305 310 315 320Lys Tyr Lys Lys Tyr Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335Leu Asp Gln Gln Leu Lys Asp Asn Phe Lys Leu Ile Ile Glu Ser Lys 340 345 350Ser Glu Lys Ser Glu Ile Phe Ser Lys Leu Glu Asn Leu Asn Val Ser 355 360 365Asp Leu Glu Ile Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln 370 375 380Ala Leu Ile Ser Lys Gln Gly Ser Tyr Leu Thr Asn Leu Val Ile Glu385 390 395 400Gln Val Lys Asn Arg Tyr Gln Phe Leu Asn Gln His Leu Asn Pro Ala 405 410 415Ile Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys Ile Phe His Asp 420 425 430Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn Ser Met Phe Leu Thr Lys 435 440 445Ile Ala Pro Tyr Leu Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr 450 455 460Ile Ser Leu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe465 470 475 480Ile Asn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu Lys Ala Ser Asp 485 490 495Leu Ile Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu Thr Glu 500 505 510Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala Ser Ala Lys Tyr 515 520 525Gln Phe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu 530 535 540Asp Asn Gly Val Asp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr545 550 555 560Leu Leu Asn Asn Lys Ile Pro Ser Asn Asn Val Glu Glu Ala Gly Ser 565 570 575Lys Asn Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser 580 585 590Tyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser Ile 595 600 605Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser 610 615 620Asp Asp Gly Glu Ser Ile Leu Glu Leu Asn Lys Tyr Arg Ile Pro Glu625 630 635 640Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe Ile Gly His Gly 645 650 655Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu Ser Val Asp Ser 660 665 670Leu Ser Asn Glu Ile Ser Ser Phe Leu Asp Thr Ile Lys Leu Asp Ile 675 680 685Ser Pro Lys Asn Val Glu Val Asn Leu Leu Gly Cys Asn Met Phe Ser 690 695 700Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser705 710 715 720Ile Met Asp Lys Ile Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser 725 730 735Ile Thr Ile Gly Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly 740 745 750Arg Lys Glu Leu Leu Ala His Ser Gly Lys Trp Ile Asn Lys Glu Glu 755 760 765Ala Ile Met Ser Asp Leu Ser Ser Lys Glu Tyr Ile Phe Phe Asp Ser 770 775 780Ile Asp Asn Lys Leu Lys Ala Lys Ser Lys Asn Ile Pro Gly Leu Ala785 790 795 800Ser Ile Ser Glu Asp Ile Lys Thr Leu Leu Leu Asp Ala Ser Val Ser 805 810 815Pro Asp Thr Lys Phe Ile Leu Asn Asn Leu Lys Leu Asn Ile Glu Ser 820 825 830Ser Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn 835 840 845Ile Ile His Asn Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn Leu Leu 850 855 860Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu865 870 875 880Asp Glu Lys Tyr Leu Ile Ser Phe Glu Asp Ile Ser Lys Asn Asn Ser 885 890 895Thr Tyr Ser Val Arg Phe Ile Asn Lys Ser Asn Gly Glu Ser Val Tyr 900 905 910Val Glu Thr Glu Lys Glu Ile Phe Ser Lys Tyr Ser Glu His Ile Thr 915 920 925Lys Glu Ile Ser Thr Ile Lys Asn Ser Ile Ile Thr Asp Val Asn Gly 930 935 940Asn Leu Leu Asp Asn Ile Gln Leu Asp His Thr Ser Gln Val Asn Thr945 950 955 960Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Asp Tyr Ser Ser Asn 965 970 975Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gln Leu Tyr 980 985 990Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Tyr Asp Ser Ile Gln 995 1000 1005Leu Val Asn Leu Ile Ser Asn Ala Val Asn Asp Thr Ile Asn Val 1010 1015 1020Leu Pro Thr Ile Thr Glu Gly Ile Pro Ile Val Ser Thr Ile Leu 1025 1030 1035Asp Gly Ile Asn Leu Gly Ala Ala Ile Lys Glu Leu Leu Asp Glu 1040 1045 1050His Asp Pro Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val 1055 1060 1065Leu Ala Ile Asn Met Ser Leu Ser Ile Ala Ala Thr Val Ala Ser 1070 1075 1080Ile Val Gly Ile Gly Ala Glu Val Thr Ile Phe Leu Leu Pro Ile 1085 1090 1095Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu 1100 1105 1110Ile Leu His Asp Lys Ala Thr Ser Val Val Asn Tyr Phe Asn His 1115 1120 1125Leu Ser Glu Ser Lys Lys Tyr Gly Pro Leu Lys Thr Glu Asp Asp 1130 1135 1140Lys Ile Leu Val Pro Ile Asp Asp Leu Val Ile Ser Glu Ile Asp 1145 1150 1155Phe Asn Asn Asn Ser Ile Lys Leu Gly Thr Cys Asn Ile Leu Ala 1160 1165 1170Met Glu Gly Gly Ser Gly His Thr Val Thr Gly Asn Ile Asp His 1175 1180 1185Phe Phe Ser Ser Pro Ser Ile Ser Ser His Ile Pro Ser Leu Ser 1190 1195 1200Ile Tyr Ser Ala Ile Gly Ile Glu Thr Glu Asn Leu Asp Phe Ser 1205 1210 1215Lys Lys Ile Met Met Leu Pro Asn Ala Pro Ser Arg Val Phe Trp 1220 1225 1230Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp 1235 1240 1245Gly Thr Arg Leu Leu Asp Ser Ile Arg Asp Leu Tyr Pro Gly Lys 1250 1255 1260Phe Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Ala Ile Thr Thr 1265 1270 1275Leu Lys Pro Val Tyr Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp 1280 1285 1290Lys Asp Thr Arg Asn Phe Ile Met Pro Thr Ile Thr Thr Asn Glu 1295 1300 1305Ile Arg Asn Lys Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr 1310 1315 1320Tyr Ser Leu Leu Leu Ser Ser Tyr Pro Ile Ser Thr Asn Ile Asn 1325 1330 1335Leu Ser Lys Asp Asp Leu Trp Ile Phe Asn Ile Asp Asn Glu Val 1340 1345 1350Arg Glu Ile Ser Ile Glu Asn Gly Thr Ile Lys Lys Gly Lys Leu 1355 1360 1365Ile Lys Asp Val Leu Ser Lys Ile Asp Ile Asn Lys Asn Lys Leu 1370 1375 1380Ile Ile Gly Asn Gln Thr Ile Asp Phe Ser Gly Asp Ile Asp Asn 1385 1390 1395Lys Asp Arg Tyr Ile Phe Leu Thr Cys Glu Leu Asp Asp Lys Ile 1400 1405 1410Ser Leu Ile Ile Glu Ile Asn Leu Val Ala Lys Ser Tyr Ser Leu 1415 1420 1425Leu Leu Ser Gly Asp Lys Asn Tyr Leu Ile Ser Asn Leu Ser Asn 1430 1435 1440Thr Ile Glu Lys Ile Asn Thr Leu Gly Leu Asp Ser Lys Asn Ile 1445 1450 1455Ala Tyr Asn Tyr Thr Asp Glu Ser Asn Asn Lys Tyr Phe Gly Ala 1460 1465 1470Ile Ser Lys Thr Ser Gln Lys Ser Ile Ile His Tyr Lys Lys Asp 1475 1480 1485Ser Lys Asn Ile Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe 1490 1495 1500Asn Ser Lys Asp Phe Ile Ala Glu Asp Ile Asn Val Phe Met Lys 1505 1510 1515Asp Asp Ile Asn Thr Ile Thr Gly Lys Tyr Tyr Val Asp Asn Asn 1520 1525 1530Thr Asp Lys Ser Ile Asp Phe Ser Ile Ser Leu Val Ser Lys Asn 1535 1540 1545Gln Val Lys Val Asn Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser 1550 1555 1560Ser Tyr Leu Asp Phe Val Lys Asn Ser Asp Gly His His Asn Thr 1565 1570 1575Ser Asn Phe Met Asn Leu Phe Leu Asp Asn Ile Ser Phe Trp Lys 1580 1585 1590Leu Phe Gly Phe Glu Asn Ile Asn Phe Val Ile Asp Lys Tyr Phe 1595 1600 1605Thr Leu Val Gly Lys Thr Asn Leu Gly Tyr Val Glu Phe Ile Cys 1610 1615 1620Asp Asn Asn Lys Asn Ile Asp Ile Tyr Phe Gly Glu Trp Lys Thr 1625 1630 1635Ser Ser Ser Lys Ser Thr Ile Phe Ser Gly Asn Gly Arg Asn Val 1640 1645 1650Val Val Glu Pro Ile Tyr Asn Pro Asp Thr Gly Glu Asp Ile Ser 1655 1660 1665Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly Ile Asp Arg 1670 1675 1680Tyr Ile Asn Lys Val Leu Ile Ala Pro Asp Leu Tyr Thr Ser Leu 1685 1690 1695Ile Asn Ile Asn Thr Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro Glu 1700 1705 1710Ile Ile Val Leu Asn Pro Asn Thr Phe His Lys Lys Val Asn Ile 1715 1720 1725Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr Glu Gly 1730 1735 1740Ser Asp Phe Ile Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys 1745 1750 1755Ile Leu Gln Lys Ile Arg Ile Lys Gly Ile Leu Ser Asn Thr Gln 1760 1765 1770Ser Phe Asn Lys Met Ser Ile Asp Phe Lys Asp Ile Lys Lys Leu 1775 1780 1785Ser Leu Gly Tyr Ile Met Ser Asn Phe Lys Ser Phe Asn Ser Glu 1790 1795 1800Asn Glu Leu Asp Arg Asp His Leu Gly Phe Lys Ile Ile Asp Asn 1805 1810 1815Lys Thr Tyr Tyr Tyr Asp Glu Asp Ser Lys Leu Val Lys Gly Leu 1820 1825 1830Ile Asn Ile Asn Asn Ser Leu Phe Tyr Phe Asp Pro Ile Glu Phe 1835 1840 1845Asn Leu Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr 1850 1855 1860Phe Asp Ile Asn Thr Gly Ala Ala Leu Thr Ser Tyr Lys Ile Ile 1865 1870 1875Asn Gly Lys His Phe Tyr Phe Asn Asn Asp Gly Val Met Gln Leu 1880 1885 1890Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala Pro Ala 1895 1900 1905Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln 1910 1915 1920Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn 1925 1930 1935Asn Ser Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Glu Lys 1940 1945 1950Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala Ala Val Gly Leu Gln 1955 1960 1965Val Ile Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile 1970 1975 1980Ile Ser Lys Gly Trp Gln Thr Val Asn Gly Ser Arg Tyr Tyr Phe 1985 1990 1995Asp Thr Asp Thr Ala Ile Ala Phe Asn Gly Tyr Lys Thr Ile Asp 2000 2005 2010Gly Lys His Phe Tyr Phe Asp Ser Asp Cys Val Val Lys Ile Gly 2015 2020 2025Val Phe Ser Thr Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn 2030 2035 2040Thr Tyr Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Ser 2045 2050 2055Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asn 2060 2065 2070Ser Lys Ala Val Thr Gly Leu Gln Thr Ile Asp Ser Lys Lys Tyr 2075 2080 2085Tyr Phe Asn Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr 2090 2095 2100Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala 2105 2110 2115Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn 2120 2125 2130Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly 2135 2140 2145Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val 2150 2155 2160Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu 2180 2185 2190Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser 2195 2200 2205Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr 2210 2215 2220Phe Asn Pro Asn Asn Ala Ile Ala Ala Ile His Leu Cys Thr Ile 2225 2230 2235Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly Ile Leu Gln Asn 2240 2245 2250Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe Tyr Phe Asp Ala Asn 2255 2260 2265Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly 2270 2275 2280Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn Asn Ile Glu 2285 2290 2295Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly 2300 2305 2310Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly Trp 2315 2320 2325Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala 2330 2335 2340Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr 2345 2350 2355Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile 2360 2365 2370Asp Gly Lys Lys Tyr

Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser 2375 2380 2385Thr Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr 2390 2395 2400Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe 2405 2410 2415Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly 2420 2425 2430Gln Ala Ile Leu Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435 2440 2445Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg 2450 2455 2460Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val 2465 2470 2475Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe 2480 2485 2490Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser 2495 2500 2505Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly 2510 2515 2520Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn 2525 2530 2535Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn 2540 2545 2550Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn Asn 2555 2560 2565Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr 2570 2575 2580Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr 2585 2590 2595Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile 2600 2605 2610Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala 2615 2620 2625Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln 2630 2635 2640Asn Arg Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn 2645 2650 2655Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val 2660 2665 2670Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu 2675 2680 2685Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly Val 2690 2695 2700Lys Ala Pro Gly Ile Tyr Gly 2705 271062366PRTClostridium difficile 6Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg1 5 10 15Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val65 70 75 80Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140Val Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn145 150 155 160Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val225 230 235 240Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile305 310 315 320Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp 325 330 335Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345 350Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val385 390 395 400Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420 425 430Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp465 470 475 480Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu545 550 555 560Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu 580 585 590Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile625 630 635 640Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser 660 665 670Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro 675 680 685Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys705 710 715 720Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg 740 745 750Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775 780Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu785 790 795 800Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu 805 810 815Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn Val Ile Ser Asn Ile 820 825 830Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr 835 840 845Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser 850 855 860Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp865 870 875 880Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe 885 890 895Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu 915 920 925Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn945 950 955 960Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu 965 970 975Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000 1005Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro 1010 1015 1020Thr Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly 1025 1030 1035Val Ser Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp 1040 1045 1050Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala 1055 1060 1065Val Asn Leu Thr Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu 1070 1075 1080Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala Gly 1085 1090 1095Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Val Leu 1100 1105 1110Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Lys His Val Ser 1115 1120 1125Leu Val Glu Thr Glu Gly Val Phe Thr Leu Leu Asp Asp Lys Ile 1130 1135 1140Met Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn 1145 1150 1155Asn Asn Ser Ile Val Leu Gly Lys Cys Glu Ile Trp Arg Met Glu 1160 1165 1170Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe 1175 1180 1185Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu Ser Ile Tyr 1190 1195 1200Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser Lys Asp 1205 1210 1215Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp Glu 1220 1225 1230Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr 1250 1255 1260Trp Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu 1265 1270 1275Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser 1280 1285 1290Asn Thr Arg Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile 1295 1300 1305Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr 1310 1315 1320Ala Leu Ser Leu Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu 1325 1330 1335Ser Glu Ser Asp Val Trp Ile Ile Asp Val Asp Asn Val Val Arg 1340 1345 1350Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile 1355 1360 1365Glu Gly Ile Leu Ser Thr Leu Ser Ile Glu Glu Asn Lys Ile Ile 1370 1375 1380Leu Asn Ser His Glu Ile Asn Phe Ser Gly Glu Val Asn Gly Ser 1385 1390 1395Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn 1400 1405 1410Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Leu Leu 1415 1420 1425Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser Asn His Ile 1430 1435 1440Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu Gln Lys 1445 1450 1455Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn Gly 1460 1465 1470Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys 1490 1495 1500Pro Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val 1505 1510 1515Ile Thr Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys 1520 1525 1530Asp Asp Ile Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys 1535 1540 1545Thr Ile Lys Leu Asn Ser Val His Leu Asp Glu Ser Gly Val Ala 1550 1555 1560Glu Ile Leu Lys Phe Met Asn Arg Lys Gly Asn Thr Asn Thr Ser 1565 1570 1575Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser Ile 1580 1585 1590Phe Val Asn Phe Leu Gln Ser Asn Ile Lys Phe Ile Leu Asp Ala 1595 1600 1605Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu Phe 1610 1615 1620Ile Cys Asp Glu Asn Asp Asn Ile Gln Pro Tyr Phe Ile Lys Phe 1625 1630 1635Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr Val Gly Asn Arg Gln 1640 1645 1650Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly Asp 1655 1660 1665Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly 1670 1675 1680Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn Ile Tyr 1685 1690 1695Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn Thr 1700 1705 1710Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 1715 1720 1725Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser 1730 1735 1740Asn Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn 1745 1750 1755Lys Val Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp 1760 1765 1770Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln 1775 1780 1785Asp Val Pro Val Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr 1790 1795 1800Tyr Glu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu 1805 1810 1815Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser 1820 1825 1830Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro 1835 1840 1845Val Asn Asn Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys 1850 1855 1860Tyr Tyr Phe Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu 1865 1870 1875Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val 1880 1885 1890Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe 1895 1900 1905Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile 1910 1915 1920Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe 1925 1930 1935Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp Gly 1940 1945 1950Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly 1970 1975 1980Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr 1985 1990 1995Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp 2000 2005 2010Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly 2015 2020 2025Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn 2030 2035 2040Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly 2045 2050 2055Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe 2060 2065 2070Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr 2075 2080 2085Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu 2090 2095 2100Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln

2105 2110 2115Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp 2120 2125 2130Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr 2135 2140 2145Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp 2150 2155 2160Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn 2165 2170 2175Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg 2180 2185 2190Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr 2210 2215 2220Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile 2225 2230 2235Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr 2240 2245 2250Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn 2255 2260 2265Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe 2270 2275 2280Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr 2285 2290 2295Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu 2300 2305 2310Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu 2315 2320 2325Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr 2330 2335 2340Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp 2345 2350 2355Thr Ala Gln Leu Val Ile Ser Glu 2360 2365

Claims

1) An antibody that recognizes Clostridium difficile toxins A and B, wherein said antibody specifically binds to the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.

2) The antibody of claim 1, wherein said antibody specifically binds to the sequence set forth in SEQ ID NO: 1.

3) The antibody of claim 1, wherein said antibody specifically binds to the sequence set forth in SEQ ID NO: 2.

4) The antibody of claim 1, wherein said antibody is a polyclonal antibody.

5) The antibody of claim 1, wherein said antibody is a bird antibody.

6) The antibody of claim 5, wherein said bird antibody is a chicken antibody.

7) The antibody of claim 1, wherein said antibody is in a composition that further comprises a carrier.

8) The antibody of claim 7, wherein said composition is a pharmaceutical composition and said carrier is a pharmaceutically acceptable carrier.

9) A method for immunizing a subject against Clostridium difficile infection or treating Clostridium difficile infection in a subject comprising administrating an effective amount of the antibody of claim 1 to a subject in need thereof.

10) A method for immunizing a subject against Clostridium difficile infection or treating Clostridium difficile infection in a subject comprising administrating an effective amount of the antibody of claim 4 to a subject in need thereof.

11) A method for immunizing a subject against Clostridium difficile infection or treating Clostridium difficile infection in a subject comprising administrating an effective amount of the antibody of claim 6 to a subject in need thereof.

12) The method of claim 9, wherein said subject is a human subject.

13) The method of claim 10, wherein said subject is a human subject.

14) The method of claim 11, wherein said subject is a human subject.