Abstract:
The present invention relates to recombinant  Clostridium difficile  antigens based on a polypeptide, consisting of or comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence consisting of residues 1500-700 of a  C. difficile  Toxin A sequence or a  C. difficile  Toxin B sequence; though with the proviso that the polypeptide does not include one or more Repeat Unit (RU) located between amino acid residues 1851-2710 of  C. difficile  Toxin A and/or residues 1853-2366 of a  C. difficile  Toxin B protein that consists of or comprises a first amino acid sequence and a second amino acid. Also provided is the use of said antigens for the prevention/treatment/suppression of  Clostridium difficile  infection (CDI), together with methods for generating said antigens, methods for generating antibodies that bind to said antigens, and the use of said antibodies for the prevention/treatment/suppression of CDI.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is the National Stage of International Application No. PCT/GB2013/050886, filed Apr. 4, 2013, the disclosure of which is incorporated by reference herein. 
     STATEMENT REGARDING SEQUENCE LISTING 
     The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is SQL ST25.txt. The text file is 243 KB; was created on Sep. 26, 2014 and is being submitted via EFS-Web with the filing of the specification. 
     BACKGROUND 
     The present invention relates to antigens for the prevention/treatment/suppression of  Clostridium difficile  infection (CDI). Also provided are methods for generating said antigens, methods for generating antibodies that bind to said antigens, and the use of said antibodies for the prevention/treatment/suppression of CDI. 
       Clostridium difficile  infection (CDI) is now a major problem in hospitals worldwide. The bacterium causes nosocomial, antibiotic-associated disease which manifests itself in several forms ranging from mild self-limiting diarrhoea to potentially life-threatening, severe colitis. Elderly patients are most at risk from these potentially life-threatening diseases and incidents of CDI have increased dramatically over the last 10 years. In 2010 in the UK there were over 21,000 cases of CDI with over 2,700 associated deaths. CDI costs the UK National Health Service in excess of £500M per annum. 
     The various strains of  C. difficile  may be classified by a number of methods. One of the most commonly used is polymerase chain reaction (PCR) ribotyping in which PCR is used to amplify the 16S-23S rRNA gene intergenic spacer region of  C. difficile. Reaction products from this provide characteristic band patterns identifying the bacterial ribotype of isolates. Toxinotyping is another typing method in which the restriction patterns derived from DNA coding for the C. difficile  toxins are used to identify strain toxinotype. The differences in restriction patterns observed between toxin genes of different strains are also indicative of sequence variation within the  C. difficile  toxin family. For example, there is an approximate 13% sequence difference with the C-terminal 60 kDa region of toxinotype 0 Toxin B compared to the same region in toxinotype III Toxin B. 
     Strains of  C. difficile  produce a variety of virulence factors, notable among which are several protein toxins: Toxin A, Toxin B and, in some strains, a binary toxin which is similar to  Clostridium perfringens  iota toxin. Toxin A is a large protein cytotoxin/enterotoxin which plays a role in the pathology of infection and may influence the gut colonisation process. Outbreaks of CDI have been reported with Toxin A-negative/Toxin B-positive strains, which indicates that Toxin B is also capable of playing a key role in the disease pathology. 
     The genetic sequences encoding Toxin A and Toxin B (Mw 308k and Mw 269k, respectively) are known—see, for example, Moncrief et al. (1997) Infect. Immun. 63:1105-1108. The two toxins have high sequence homology and are believed to have arisen from gene duplication. The toxins also share a common structure (see  FIG. 1 ), namely an N-terminal glucosyl transferase domain, a central hydrophobic region, four conserved cysteines, and a long series of C-terminal repeating units (RUs). 
     Both Toxins A and B exert their mechanisms of action via multi-step mechanisms, which include binding to receptors on the cell surface, internalisation followed by translocation and release of the effector domain into the cell cytosol, and finally intracellular action. Said mechanism of action involves the inactivation of small GTPases of the Rho family. In this regard, the toxins catalyse the transfer of a glucose moiety (from UDP-glucose) onto an amino residue of the Rho protein. Toxins A and B also contain a second enzyme activity in the form of a cysteine protease, which appears to play a role in the release of the effector domain into the cytosol after translocation. The  C. difficile  binary toxin modifies cell actin by a mechanism which involves the transfer of an ADP-ribose moiety from NAD onto its target protein. 
     Current therapies for the treatment of  C. difficile  infection rely on the use of antibiotics, notably metronidazole and vancomycin. However, these antibiotics are not effective in all cases and 20-30% of patients suffer relapse of the disease. Of major concern is the appearance in the UK of more virulent strains, which were first identified in Canada in 2002. These strains, which include those belonging to PCR ribotype 027 and toxinotype III, cause CDI with a directly attributable mortality more than 3-fold that observed previously. 
     New therapeutics are therefore required especially urgently since the efficacy of current antibiotics appears to be decreasing. 
     One approach is the use of antibodies which bind to and neutralise the activity of Toxin A and/or Toxin B. This is based on the knowledge that strains of  C. difficile  that do not release these toxins, so called non-toxigenic strains, do not cause CDI. By way of example, animals can be immunised, their sera collected and the antibodies purified for administration to patients—this is defined as passive immunisation. In another approach patients with CDI or subjects at risk of developing such infections can be immunised with antigens which result in an increase in circulating and mucosal antibodies directed against Toxin A and/or Toxin B—this is defined as active immunisation. 
     A critical requirement for both active and passive immunisation is the availability of suitable antigens with which to immunise the patient or animal respectively. These can comprise the natural toxins which can be purified from the media in which suitable toxigenic strains of  C. difficile  have been cultured. There are several disadvantages to this approach. Both Toxin A and Toxin B are present in culture medium in only small amounts and are difficult to purify without incurring significant losses. Thus, it is both costly and difficult to obtain the amounts necessary to meet world-wide needs. In addition, the natural toxins are unstable and toxic. 
     The above mentioned problems have resulted in there being few available  C. difficile  vaccine candidates. To date, the only CDI vaccine in late-stage development is based on a mixture of native (i.e. naturally occurring) Toxins A and B, which have been extensively inactivated by chemical modification (Salnikova et al. 2008, J. Pharm. Sci. 97:3735-3752). 
     One alternative to the use of natural toxins (and their toxoids) involves the design, development and use of recombinant fragments derived from Toxins A and B. Examples of existing antigens intended for use in treating/preventing a  C. difficile  infection include peptides based on the C-terminal repeating units (RUs) of Toxin A or Toxin B—see, for example, WO 00/61762. A problem with such antigens, however, is that they are either poorly immunogenic (i.e. the antigens produce poor antibody titres), or, where higher antibody titres are produced, the antibodies demonstrate poor neutralising efficacy against  C. difficile  cytotoxic activity (i.e. insufficient neutralising antibodies are produced). 
     There is therefore a need in the art for new vaccines/therapies/therapeutics capable of specifically addressing  C. difficile  infection (CDI). This need is addressed by the present invention, which solves one or more of the above-mentioned problems. 
     SUMMARY OF THE INVENTION 
     In summary, the present invention provides antigens that are able to induce a potent toxin-neutralising response against  C. difficile  Toxin A and/or Toxin B. The invention also provides methods for preparing recombinant antigens, and the use thereof as immunogens to enable the large-scale preparation of therapeutic antibodies. Said antibodies are able to induce a potent toxin-neutralising response against  C. difficile  Toxin A and/or Toxin B and therefore have prophylactic and/or therapeutic applications. 
     As mentioned above (see, for example, WO 00/61762), previous studies describe vaccine preparations based on the C-terminal, repeating units (RUs) of Toxin A and/or Toxin B. Said RU fragments have a poor toxin-neutralising effect, and/or are difficult to manufacture in large quantities. 
     In contrast, the present invention provides a  C. difficile  polypeptide antigen based on a Toxin A and/or a Toxin B that does not contain or include one or more (e.g. all) of the repeating units (RUs) of Toxin A and/or Toxin B. The polypeptide antigens of the invention consist of, or comprise one or more domains from the central region of the toxins. Said antigens of the invention demonstrate good toxin-neutralising immune responses and/or are readily manufactured in large quantities. 
     The present inventors have surprisingly identified that  C. difficile  antigen polypeptides which consist of or comprise one or more domains from the central region between the effector domain and the region of RUs (see  FIG. 1 ) provide a protective (toxin-neutralising) immune response that was greatly enhanced as compared to corresponding  C. difficile  toxin fragments comprising one or more of the RUs (see Tables 1 and 2). 
     Comparison of the data in Tables 1 and 2 confirms that the polypeptide antigens of the present invention elicit a considerably more potent toxin-neutralising immune response than that of a corresponding polypeptide based that includes one or more of the C-terminal repeating units of  C. difficile  toxin (exemplified by the polypeptide designated TxB2). In more detail, after a 18-week immunisation period, the toxin-neutralising immune response provided by polypeptides of the present invention was more than 60-fold higher than that provided by a corresponding RU-containing polypeptide. Thus, polypeptides of the present invention induce a potent toxin-neutralising immune response. 
     These findings are surprising for a number of reasons. Most importantly, a previous study in which animals were separately immunised with central domain fragments of  C. difficile  toxin (a fragment consisting of residues 510-1530, and a fragment consisting of residues 1530-1750) reported that these fragments failed to elicit the production of toxin-neutralising antibodies (Kink and Williams (1993) Infect. Immun. 66:2018-2025). This study therefore suggests that domains within residues 510-1530 contribute no significant antibody-binding structural determinants. In addition, a further study has showed that antibodies raised against a whole  C. difficile , while recognising a fragment consisting of the entire RU region alone, failed to recognise a fragment consisting of a central toxin region based on residues 901-1750 of the  C. difficile  same toxin (Genth et al., (2000) Infect. Immun., 68:1094-1101). This study therefore suggests that domains within residues 901-1750 contribute no significant antibody-binding structural determinants. Furthermore, while antibodies to the effector domain (residues 1-543) of  C. difficile  toxin have been shown to elicit a potent immune response (measured by simple enzyme immunoassay), said antibodies have no toxin-neutralising activity showing that antibody binding to the toxin does not correspond to toxin neutralisation (Roberts et al. (2012) Infect. Immun., 80:875-882). Collectively, it is therefore extremely surprising that recombinant immunogens based on the central domains of the  C. difficile  toxins located between the effector domain and repeat regions are capable of inducing such a potent toxin-neutralising immune response. 
     One aspect of the present invention provides a polypeptide containing, consisting of, or comprising an amino acid sequence that has at least 80% sequence identity with an amino acid sequence consisting of residues 1500-1800 (e.g. 1450-1750, or 1400-1800) of a  C. difficile  Toxin A sequence with the proviso that the polypeptide is not a polypeptide comprising one or more of (e.g. all of) the RU units between amino acid residues 1851-2710 of  C. difficile  Toxin A and/or residues 1853-2366 of a  C. difficile  Toxin B. In one embodiment, said polypeptide lacks the sequence of amino acid residues 1851-2710 of  C. difficile  Toxin A and/or residues 1853-2366 of a  C. difficile  Toxin B. 
     Another aspect of the present invention provides a polypeptide containing, consisting of or comprising an amino acid sequence that has at least 80% sequence identity with an amino acid sequence consisting of residues 542-1850 of a  C. difficile  Toxin A sequence with the proviso that the polypeptide does not comprise any of the RU units between residues 1851-2710 of  C. difficile  Toxin A or residues 1853-2366 of a  C. difficile  Toxin B. In one embodiment, said polypeptide lacks the sequence of amino acid residues 1851-2710 of  C. difficile  Toxin A and/or residues 1853-2366 of a  C. difficile  Toxin B. 
     Reference to a  C. difficile  Toxin A sequence means the amino acid sequence of a naturally-occurring  C. difficile  Toxin A (also referred to as a  C. difficile  Toxin A reference sequence). Examples of such sequences are readily understood by a skilled person, and simply for illustrative purposes, some of the more common naturally-occurring Toxin A sequences are identified in the present specification (see, for example, SEQ ID NOs: 1 and 3) as well as throughout the literature. 
     Reference to ‘at least 80% sequence identity’ throughout this specification is considered synonymous with the phrase ‘based on’ and may embrace one or more of at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 99%, and 100% sequence identity. When assessing sequence identity, a reference sequence having a defined number of contiguous amino acid residues is aligned with an amino acid sequence (having the same number of contiguous amino acid residues) from the corresponding portion of a polypeptide of the present invention. 
     In one embodiment, the polypeptide amino acid sequence is based on (i.e. has at least 80% sequence identity with) amino acid residues 1500-1700 or amino acid residues 1450-1750, or amino acid residues 1400-1800 of a  C. difficile  Toxin A. In another embodiment, the polypeptide amino acid sequence is based on amino acid residues 544-1850 of a  C. difficile  Toxin A, such as amino acid residues 564-1850, amino acid residues 584-1850, amino acid residues 594-1850, amino acid residues 614-1850, amino acid residues 634-1850, amino acid residues 654-1850, amino acid residues 674-1850, amino acid residues 694-1850, amino acid residues 714-1850, amino acid residues 734-1850, amino acid residues 754-1850, amino acid residues 767-1850, amino acid residues 770-1850, amino acid residues 774-1850, amino acid residues 794-1850, amino acid residues 814-1850, amino acid residues 834-1850, amino acid residues 854-1850, amino acid residues 874-1850, amino acid residues 894-1850, amino acid residues 914-1850, amino acid residues 934-1850, amino acid residues 954-1850, amino acid residues 974-1850, amino acid residues 994-1850, amino acid residues 1014-1850, amino acid residues 1034-1850, amino acid residues 1054-1850, amino acid residues 1074-1850, amino acid residues 1094-1850, amino acid residues 1104-1850, amino acid residues 1124-1850, amino acid residues, amino acid residues 1131-1850, amino acid residues 1144-1850, amino acid residues 1164-1850, amino acid residues 1184-1850, amino acid residues 1204-1850, amino acid residues 1224-1850, amino acid residues 1244-1850, amino acid residues 1264-1850, amino acid residues 1284-1850, amino acid residues 1304-1850, amino acid residues 1324-1850, amino acid residues 1344-1850, amino acid residues 1450-1750 or amino acid residues 1550-1850; though with the proviso that the polypeptide does not include one or more of (e.g. all of) the RU units between residues 1851-2710 of  C. difficile  Toxin A or residues 1853-2366 of a  C. difficile  Toxin B. In one embodiment, said polypeptide lacks the sequence of amino acid residues 1851-2710 of  C. difficile  Toxin A and/or residues 1853-2366 of a  C. difficile  Toxin B. By way of example only, the above amino acid position numbering may refer to the  C. difficile  Toxin A sequences identified as SEQ ID NOs: 1 and/or 3. 
     In one embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 542-1850 of a Toxin A sequence (or a portion thereof). Examples are identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NOs: 5 and 6. 
     In another embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 542-1850 of a Toxin A sequence (or a portion thereof) that substantially lacks cysteine protease activity. An example is identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 7. 
     In another embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 770-1850 of a Toxin A sequence (or a portion thereof). An example is identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 8. 
     In another embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 1130-1850 of a Toxin A sequence (or a portion thereof). An example is identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 9. 
     A related aspect of the present invention provides a polypeptide containing, consisting of, or comprising an amino acid sequence that has at least 80% sequence identity with an amino acid sequence consisting of residues 1500-1700 (e.g. 1450-1750, or 1400-1800) of a  C. difficile  Toxin B sequence with the proviso that the polypeptide does comprise one or more of (e.g. any of) the RU units between residues 1851-2710 of  C. difficile  Toxin A or residues 1853-2366 of a  C. difficile  Toxin B. In one embodiment, said polypeptide lacks the sequence of amino acid residues 1851-2710 of  C. difficile Toxin A and/or residues  1853-2366 of a  C. difficile  Toxin B. 
     In another aspect of the present invention provides a polypeptide containing, consisting of, or comprising an amino acid sequence that has at least 80% sequence identity with an amino acid sequence consisting of residues 543-1852 of a  C. difficile  Toxin B sequence with the proviso that the polypeptide does not comprise one or more of (e.g. any of) the RU units between residues 1851-2710 of  C. difficile  Toxin A or residues 1853-2366 of a  C. difficile  Toxin B. In one embodiment, said polypeptide lacks the sequence of amino acid residues 1851-2710 of  C. difficile  Toxin A and/or residues 1853-2366 of a  C. difficile  Toxin B. 
     Reference to a  C. difficile  Toxin B sequence means the amino acid sequence of a naturally-occurring  C. difficile  Toxin B (also referred to as a  C. difficile  Toxin B reference sequence). Examples of such sequences are readily understood by a skilled person, and simply for illustrative purposes some of the more common naturally-occurring Toxin B sequences are identified in the present specification (see, for example, SEQ ID NOs: 2 and 4) as well as throughout the literature. 
     In one embodiment, the polypeptide amino acid sequence is based on (i.e. has at least 80% sequence identity with) amino acid residues 1500-1700 or amino acid residues 1450-1750, or amino acid residues 1400-1800 of a  C. difficile  Toxin B. In another embodiment, the polypeptide amino acid sequence is based on amino acid residues 544-1852 of a  C. difficile  Toxin B, such as amino acid residues 564-1852, amino acid residues 584-1852, amino acid residues 594-1852, amino acid residues 614-1852, amino acid residues 634-1852, amino acid residues 654-1852, amino acid residues 674-1852, amino acid residues 694-1852, amino acid residues 714-1852, amino acid residues 734-1852, amino acid residues 754-1852, amino acid residues 767-1852, amino acid residues 770-1852, amino acid residues 774-1852, amino acid residues 794-1852, amino acid residues 814-1852, amino acid residues 834-1852, amino acid residues 854-1852, amino acid residues 874-1852, amino acid residues 894-1852, amino acid residues 914-1852, amino acid residues 934-1852, amino acid residues 954-1852, amino acid residues 974-1852, amino acid residues 994-1852, amino acid residues 1014-1852, amino acid residues 1034-1852, amino acid residues 1054-1852, amino acid residues 1074-1852, amino acid residues 1094-1852, amino acid residues 1104-1852, amino acid residues 1124-1852, amino acid residues 1131-1852, amino acid residues 1144-1852, amino acid residues 1164-1852, amino acid residues 1184-1852, amino acid residues 1204-1852, amino acid residues 1224-1852, amino acid residues 1244-1852, amino acid residues 1264-1852, amino acid residues 1284-1852, amino acid residues 1304-1852, amino acid residues 1324-1852, amino acid residues 1344-1852, amino acid residues 1450-1750 or amino acid residues 1550-1800, amino acid residues 1450-1750 or amino acid residues 1550-1850; though with the proviso that the polypeptide does not include one or more of (e.g. all of) the RU units between residues 1851-2710 of  C. difficile  Toxin A or residues 1853-2366 of a  C. difficile  Toxin B. In one embodiment, said polypeptide lacks the sequence of amino acid residues 1851-2710 of  C. difficile  Toxin A and/or residues 1853-2366 of a  C. difficile  Toxin B. By way of example only, the above amino acid position numbering may refer to the  C. difficile  Toxin B sequences identified as SEQ ID NOs: 2 and/or 4. 
     In one embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 543-1852 of a Toxin B sequence (or a portion thereof). Examples are identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NOs: 10 or 11. 
     In another embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 543-1852 of a Toxin B sequence (or a portion thereof) that substantially lacks cysteine protease activity. An example is identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 12. 
     In another embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 767-1852 of a Toxin B sequence (or a portion thereof). An example is identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 13. 
     In another embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 1145-1852 of a Toxin B sequence (or a portion thereof). An example is identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 14. 
     In another embodiment a polypeptide is provided, which comprises or consists of a sequence based on amino acid residues 1350-1852 of a Toxin B sequence (or a portion thereof). An example is identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 15. 
     The antigen polypeptides of the invention may substantially lack cysteine protease activity. In another (or the same) embodiment, antigens substantially lack glucosyl transferase activity. For example, amino acid sequence(s) providing said activity (activities) may be absent (e.g. deleted) from the antigens of the present invention. Alternatively, key amino acid residues essential for providing such activities may be either modified or deleted. Examples of amino acid modifications to substantially reduce the cysteine protease activity of Toxin A are cysteine 700 to alanine, histidine 655 to alanine, aspartic acid 589 to asparagine or a combination of more than one of these mutations. Examples of amino acid modifications to substantially reduce the cysteine protease activity of Toxin B are cysteine 698 to alanine, histidine 653 to alanine, aspartic acid 587 to asparagine or a combination of more than one of these mutations. Examples of amino acid modifications to substantially reduce the glucosyl transferase activity of Toxin A are aspartic acid 285 to alanine, aspartic acid 287 to alanine or a combination of both mutations. Examples of amino acid modifications to substantially reduce the glucosyl transferase activity of Toxin B are aspartic acid 286 to alanine, aspartic acid 288 to alanine or a combination of both mutations. These enzymatic activities are present in native Toxin A and/or Toxin B, and are associated with N-terminal domains of said Toxins (see  FIG. 1 , and/or SEQ ID NOs: 1, 2, 3 and 4). 
     The antigen polypeptides of the invention may substantially lack the glucosyl transferase domain (amino acid residues 1-542 Toxin A; amino acid residues 1-543 Toxin B) of a native  C. difficile  Toxin. In another (or the same) embodiment, the antigen substantially lacks the cysteine protease domain (amino acid residues 543-770 Toxin A; 544-767 Toxin B) of a native  C. difficile  Toxin. Said amino acid residue numbering refers to any Toxin A or Toxin B toxinotype, for example any one or more of the reference Toxin A and/or Toxin B toxinotype SEQ ID NOs recited in the present specification. Accordingly, said amino acid residue numbering may refer to any specific Toxin A and/or Toxin B reference SEQ ID NO recited in the present specification including an amino acid sequence variant having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% thereto. 
     Antigen polypeptides of the invention include chimeras in which one portion of the antigen is derived from the central domain(s) of Toxin A and/or Toxin B and a second portion of the antigen is based on a domain of a bacterial surface layer protein component (SLP) of  C. difficile  (or a fragment thereof). Inclusion of said domain has been identified by the present inventors to confer several advantages. Such SLP domains facilitate the soluble expression of antigen polypeptides of the invention. In addition, since these SLP domains are of  C. difficile  origin, it is unnecessary to cleave and remove them from constructs prior to immunisation. Indeed, the present inventors believe that antibodies to such domains recognise the intact  C. difficile  bacterium and afford additional therapeutic benefits by preventing or slowing the process of bacterial colonisation. An example of such a  C. difficile  SLP domain is based on a polypeptide comprising or consisting of the polypeptide product from  C. difficile  gene CD2767 (or a fragment thereof). By way of specific example, reference is made to a polypeptide fragment based on amino acid residues 27-401 of the polypeptide product of  C. difficile  gene CD2767 (or a portion thereof)—see, for example, a polypeptide consisting of or comprising the amino acid sequence SEQ ID NO: 16. Such a domain may be, for example, positioned at the N-terminus and/or C-terminus of a polypeptide of the invention. 
     For example, in one embodiment of the invention, a polypeptide antigen is provided which consists of or comprises a CD2767 polypeptide (e.g. based on residues 27-401 thereof) and a  C. difficile  toxin polypeptide (e.g. a central domain) as hereinbefore defined. 
     In another embodiment a polypeptide antigen is provided which consists of or comprises a CD2767 polypeptide (e.g. based on residues 27-401 thereof) and a  C. difficile  toxin polypeptide based on an amino acid sequence consisting of or comprising amino acid residues 770-1850 of a Toxin A sequence (or a portion thereof). 
     In another embodiment a polypeptide antigen is provided which consists of or comprises a CD2767 polypeptide (e.g. based on residues 27-401 thereof) and a  C. difficile  toxin polypeptide based on an amino acid sequence consisting of or comprising amino acid residues 542-1850 of a Toxin A sequence (or a portion thereof). An example is identified as a polypeptide comprising or consisting of the amino acid sequence SEQ ID NO: 17. 
     In a related embodiment of the invention and antigen is provided which consists of a chimera of CD2767 polypeptide residues 27-401 (or a portion thereof) with amino acid residues 767-1852 of a Toxin B sequence (or a portion thereof). See SEQ ID NO: 18. 
     In another embodiment of the invention a polypeptide antigen is provided which consists of or comprises a CD2767 polypeptide (e.g. based on residues 27-401 thereof) and a  C. difficile  toxin polypeptide based on an amino acid sequence consisting of or comprising amino acid residues 543-1852 of a Toxin B sequence (or a portion thereof). 
     Antigen polypeptides of the invention may additionally (or alternatively) to an SPL comprise other fusion protein partners to facilitate soluble expression. Fusion protein partners may be attached at the N- or C-terminus of the antigen construct but are usually placed at the N-terminal end. Examples of fusion partners are: NusA, thioredoxin, maltose-binding protein, small ubiquitin-like molecules (Sumo-tag). To facilitate removal of the fusion protein partner during purification, a unique protease site may be inserted between the fusion protein partner and the fusion protein per se. Such protease sites may include those for thrombin, factor Xa, enterokinase, PreScission™, Sumo™. Alternatively, removal of the fusion protein partner may be achieved via inclusion of an intein sequence between the fusion protein partner and the fusion protein per se. Inteins are self cleaving proteins and in response to a stimulus (e.g. lowered pH) are capable of self splicing at the junction between the intein and the antigen construct thus eliminating the need for the addition of specific proteases. Examples of inteins include domains derived from  Mycobacterium tuberculosis  (RecA), and  Pyrococcus horikoshii  (RadA) (Fong et al. (2010) Trends Biotechnol. 28:272-279). 
     To facilitate purification, antigens of the invention may include one or more purification tags to enable specific chromatography steps (e.g. metal ion chelating, affinity chromatography) to be included in the purification processes. Such purification tags may, for example, include: repeat histidine residues (e.g. 6-10 histidine residues), maltose binding protein, glutathione S-transferase; and streptavidin. These tags may be attached at the N- and/or C-terminus of the polypeptide antigens of the invention. To facilitate removal of such tags during purification, protease sites and/or inteins (examples above) may be inserted between the polypeptide and the purification tag(s). Examples of expression constructs for Toxin A and Toxin B derived antigens of the invention are shown in SEQ ID NOs: 17, 18, 19 and 20. 
     Thus, a typical antigen construct of the invention (starting from the N-terminus) may comprise:
         a first purification tag   an optional fusion protein partner (to facilitate expression) and/or an optional SLP   a first (preferably specific) protease sequence or intein sequence   the Toxin A and/or Toxin B antigen sequence   an optional second (preferably specific) protease sequence or intein sequence   an optional second purification tag       

     The first and second purification tags may be the same or different. Similarly, the first and second protease/intein sequence may be the same or different. The first and second options are preferably different to enable selective and controllable cleavage/purification. 
     In one embodiment, the antigen of the invention is a chimera and consists of a portion or domain of a  C. difficile  surface protein in conjunction with a toxin antigen sequence based on the central domain(s) Toxins A and/or Toxin B. 
     Accordingly, in one embodiment, a polypeptide of the invention may comprise (starting from the N-terminus):
         a first purification tag   a first (preferably specific) protease sequence or intein sequence   an antigen sequence which is a chimera of Toxin A or Toxin B and an SLP   an optional second (preferably specific) protease sequence or intein sequence   an optional second purification tag       

     Spacers may be introduced to distance the purification tag from the polypeptide—this may help to increase binding efficiency to affinity purification column media. The spacer may be placed (immediately) after the purification tag or between the fusion protein partner component and the remainder of the polypeptide per se. Similarly, spacers may be employed to distance the fusion protein partner and/or SLP from the  C. difficile  toxin component. Typical spacer sequences may consist of between 10-40 amino acid residues to give either a linear or alpha-helical structure. 
     Accordingly, in one embodiment, a polypeptide of the invention may comprise (starting from the N-terminus):
         a first purification tag   an optional first spacer sequence   a fusion protein partner (to facilitate expression) and/or an SLP   an optional second spacer sequence   a (preferably specific) protease sequence or intein sequence   the Toxin A and/or Toxin B derived antigen sequence   an optional second (preferably specific) protease sequence or intein sequence   an optional third spacer sequence   an optional second purification tag       

     Genes encoding the constructs of the invention may be generated by PCR from  C. difficile  genomic DNA and sequenced by standard methods to ensure integrity. Alternatively and preferably genes may be synthesised providing the optimal codon bias for the expression host (e.g.  E. coli, Bacillus megaterium ). Thus, the present invention provides corresponding nucleic acid sequences that encode the aforementioned polypeptides of the present invention. 
     Accordingly, a second aspect of the present invention provides a method for expressing one or more of the aforementioned polypeptide antigens of the invention, said method comprising:
         1) providing a nucleic acid sequence that encodes one or more of said polypeptide antigens in a host cell, wherein said nucleic acid sequence is operably linked to a promoter; and   2) expressing said nucleic acid sequence in the host cell.       

     Antigen polypeptides of the invention may be formulated as vaccines for human or animal use in a number of ways. For example, formulation may include treatment with an agent to introduce intra-molecular cross-links. One example of such an agent is formaldehyde, which may be incubated, for example, with antigen polypeptides of the invention for between 1-24 hours. Alternatively, longer incubation times of, for example, up to 2, 4, 6, 8 or 10 days may be employed. Following treatment with such an agent, antigens of the invention may be combined with a suitable adjuvant, which may differ depending on whether the antigen is intended for human or animal use. 
     A human or animal vaccine formulation may contain polypeptides of the present invention. Thus, in one embodiment, a vaccine formulation procedure of the present invention comprises the following steps:
         providing a recombinant polypeptide of the invention in suitable buffer system   optionally (preferably) treating said mixture with a toxoiding component such as formaldehyde   optionally transferring the polypeptide to a new buffer system   combining the polypeptide with one or more suitable adjuvants and optionally other excipients.       

     Accordingly, a third aspect of the present invention provides one or more of the aforementioned polypeptides of the invention, for use in the generation of antibodies that bind to  C. difficile  Toxin A and/or Toxin B. In one embodiment, said antibodies bind to and neutralise  C. difficile  Toxin A and/or Toxin B. 
     For immunisation of animals, the  C. difficile  recombinant antigen polypeptides of the invention may be used as immunogens separately or in combination, either concurrently or sequentially, in order to produce antibodies specific for individual  C. difficile  toxins or combinations. For example, two or more recombinant antigens may be mixed together and used as a single immunogen. Alternatively a  C. difficile  toxin antigen (e.g. Toxin A-derived) may be used separately as a first immunogen on a first animal group, and another  C. difficile  toxin antigen (e.g. Toxin B-derived) may be used separately on a second animal group. The antibodies produced by separate immunisation may be combined to yield an antibody composition directed against  C. difficile  toxins. Non-limiting examples of suitable adjuvants for animal/veterinary use include Freund&#39;s (complete and incomplete forms), alum (aluminium phosphate or aluminium hydroxide), saponin and its purified component Quil A. 
     A fourth (vaccine) aspect of the present invention provides one or more of the aforementioned polypeptide antigens of the invention, for use in the prevention, treatment or suppression of CDI (e.g. in a mammal such as man). Put another way, the present invention provides a method for the prevention, treatment or suppression of CDI (e.g. in a mammal such as man), said method comprising administration of a therapeutically effective amount of one or more of the aforementioned polypeptides of the invention to a subject (e.g. a mammal such as man). 
     By way of example, a Toxin A-based antigen (any A toxinotype) may be employed alone or in combination with a Toxin B-based antigen (any B toxinotype). Similarly, a Toxin B-based antigen (any B toxinotype) may be employed alone or in combination with a Toxin A-based antigen (any A toxinotype). Said antigens may be administered in a sequential or simultaneous manner. Vaccine applications of the present invention may further include the combined use (e.g. prior, sequential or subsequent administration) of one or more antigens such as a  C. difficile  antigen (e.g. a non-Toxin antigen; or a  C. difficile  bacterium such as one that has been inactivated or attenuated), and optionally one or more nosocomial infection antigens (e.g. an antigen, notably a surface antigen, from a bacterium that causes nosocomial infection; and/or a bacterium that causes a nosocomial infection such as one that has been inactivated or attenuated). Examples of bacteria that cause nosocomial infection include one or more of:  E. coli, Klebsiella pneumonae, Staphylococcus aureus  such as MRSA,  Legionella, Pseudomonas aeruginosa, Serratia marcescens, Enterobacter  spp,  Citrobacter  spp,  Stenotrophomonas maltophilia, Acinetobacter  spp such as  Acinetobacter baumannii, Burkholderia cepacia , and  Enterococcus  such as vancomycin-resistant  Enterococcus  (VRE). 
     In one embodiment, said vaccine application may be employed prophylactically, for example to treat a patient before said patient enters a hospital (or similar treatment facility) to help prevent hospital-acquired infection. Alternatively, said vaccine application may be administered to vulnerable patients as a matter of routine. 
     A related vaccine aspect of the invention provides one or more antibodies (comprising or consisting whole IgG and/or Fab and/or F(ab′) 2  fragments) that binds to the one or more aforementioned polypeptides of the invention, for use in the prevention, treatment or suppression of CDI (eg. in a mammal such as man). Put another way, the present invention provides a method for the prevention, treatment or suppression of CDI (e.g. in a mammal such as man), said method comprising administration of a therapeutically effective amount of said antibody (or antibodies) to a subject (e.g. a mammal such as man). 
     By way of example, an anti-Toxin A-based antigen (any A toxinotype) antibody may be employed alone or in combination with an anti-Toxin B-based antigen (any B toxinotype) antibody. Similarly, an anti-Toxin B-based antigen (any B toxinotype) antibody may be employed alone or in combination with an anti-Toxin A-based antigen (any A toxinotype) antibody. Said antibodies may be administered in a sequential or simultaneous manner. Vaccine applications of the present invention may further include the combined use (e.g. prior, sequential or subsequent administration) of one or more antibodies that bind to antigens such as a  C. difficile  antigen (e.g. a non-Toxin antigen; or a  C. difficile  bacterium), and optionally one or more antibodies that bind to one or more nosocomial infection antigens (e.g. an antigen, notably a surface antigen, from a bacterium that causes nosocomial infection; and/or a bacterium that causes a nosocomial infection). Examples of bacteria that cause nosocomial infection include one or more of:  E. coli, Klebsiella pneumonae, Staphylococcus aureus  such as MRSA,  Legionella, Pseudomonas aeruginosa, Serratia marcescens, Enterobacter  spp,  Citrobacter  spp,  Stenotrophomonas maltophilia, Acinetobacter  spp such as  Acinetobacter baumannii, Burkholderia cepacia , and  Enterococcus  such as vancomycin-resistant  Enterococcus  (VRE). 
     In one embodiment, said vaccine application may be employed prophylactically, for example once a patient has entered hospital (or similar treatment facility). Alternatively, said vaccine application may be administered to patients in combination with one or more antibiotics. 
     In one embodiment, said antibodies have been generated by immunisation of an animal (e.g. a mammal such as man, or a non-human animal such as goat or sheep) with one or more of the aforementioned antigens of the present invention. 
     In one embodiment, the antibodies of the present invention do not (substantially) bind to the repeat regions of  C. difficile  Toxin A and/or Toxin B. 
     For the preparation of vaccines for human (or non-human animal) use, the active immunogenic ingredients (whether these be antigenics of the present invention and/or corresponding antibodies of the invention that bind thereto) may be mixed with carriers or excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable carriers and excipients include, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. 
     The vaccine may further comprise one or more adjuvants. One non-limiting example of an adjuvant with the scope of the invention is aluminium hydroxide. Other non-limiting examples of adjuvants include but are not limited to: N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. 
     Typically, the vaccines are prepared as injectables, either as liquid solutions or suspensions. Of course, solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules. 
     Vaccine administration is generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes. The administration may be by parenteral injection, for example, a subcutaneous or intramuscular injection. 
     The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject&#39;s immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be particular to each subject. 
     The vaccine may be given in a single dose schedule, or optionally in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-6 separate doses, followed by other doses given at subsequent time intervals required to maintain and/or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner. 
     In one embodiment, a volume X (e.g. 1-6 ml) of buffer solution containing 10-500 μg of a polypeptide of the invention is mixed with an equivalent volume X (i.e. 1-6 ml) of adjuvant (e.g. Freund&#39;s complete adjuvant) to form an emulsion. Mixing with the adjuvant is carried out for several minutes to ensure a stable emulsion. A primary immunisation is then performed (e.g. i.m. injection) with said emulsion (e.g. 1-10 ml). In parallel, a volume X (e.g. 1-6 ml) of buffer solution containing 10-500 μg of a polypeptide of the invention is mixed with an equivalent volume X (i.e. 1-6 ml) of adjuvant (e.g. Freund&#39;s incomplete adjuvant) to form an emulsion. Mixing with the adjuvant is carried out for several minutes to ensure a stable emulsion. Subsequent immunisations (e.g. 2, 3, 4, 5 or 6) are then performed (e.g. i.m. injection) with said emulsion (e.g. 1-10 ml) on a monthly basis. Antibody titre is typically tested by sampling at a time period of approximately 2 weeks after each of said monthly immunisations, and antibody harvesting is performed when optimal antibody titre has been achieved. 
     In addition, the vaccine containing the immunogenic antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12), and/or cytokines (e.g., IFN gamma) 
     Additional formulations suitable for use with the present invention include microcapsules, suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to 10%, including for instance, about 1%-2%. 
     Antigens of the invention may also have uses as ligands for use in affinity chromatography procedures. In such procedures, antigens of the invention may be covalently immobilised onto a matrix, such as Sepharose, e.g. using cyanogen bromide-activated Sepharose. Such affinity columns may then be used to purify antibody from antisera or partially purified solutions of immunoglobulins by passing them through the column and then eluting the bound IgG fraction (e.g. by low pH). Almost all of the antibody in the eluted fraction will be directed against the antigen of the invention, with non-specific antibodies and other proteins having been removed. These affinity purified IgG fractions have applications both as immunotherapeutics and as reagents in diagnostics. For immunotherapeutics, affinity purified antibodies enable a lower dose to be administered making adverse side effects less likely. For diagnostics, affinity purified agents often give improved specificity and fewer false positive results. 
     DEFINITIONS 
       Clostridium difficile  is a species of Gram-positive bacterium of the genus  Clostridium.    
       Clostridium difficile  infection (CDI) means a bacterial infection which affects humans and animals and which results in a range of symptoms from mild self-limiting diarrhoea to life-threatening conditions such as pseudomembranous colitis and cytotoxic megacolon. In this disease,  C. difficile  replaces some of the normal gut flora and starts to produce cytotoxins which attack and damage the gut epithelium. Primary risk factors for human CDI include: receiving broad-spectrum antibiotics, being over 65 years old and being hospitalised. 
       Clostridium difficile  Toxin A is a family of protein cytotoxins/enterotoxins of approximately 300 kDa in size. Toxin A has an enzyme activity within the N-terminal region which acts to disrupt the cytoskeleton of the mammalian cell causing cell death. There a number of naturally occurring variants of Toxin A within the strains of  Clostridium difficile  which are called ‘toxinotypes’. The various toxinotypes of Toxin A have variations within their primary sequence of usually &lt;10% overall. Examples of suitable Toxin A sequences include SEQ ID NOs: 1 and 3. 
       Clostridium difficile  Toxin B is a family of protein cytotoxins of approximately 270 kDa in size which are similar to Toxin A but significantly more cytotoxic. Like Toxin A, Toxin B has an enzyme activity within the N-terminal region which acts to disrupt the cytoskeleton of the mammalian cell causing cell death. There are a number of naturally occurring variants of Toxin B within the strains of  C. difficile  which are called ‘toxinotypes’. The various toxinotypes of Toxin B have variations within their primary sequence of up to 15% overall. Examples of suitable Toxin B sequences include SEQ ID NOs: 2 and 4. 
       C. difficile  repeat units are regions within the C-terminus of Toxin A and Toxin B that contain repeating motifs which were first identified by von Eichel-Streiber and Sauerborn (1990; Gene 30:107-113). In the case of Toxin A there are 31 short repeats and 7 long repeats with each repeat consisting of a β-hairpin followed by a loop. Toxin B consists of a similar structure but with fewer repeats. The repeat units of Toxin A are contained within residues 1850-2710 and those for Toxin B within residues 1852-2366. The repeat regions play a role in receptor binding. The receptor binding regions (i.e. that define the toxin&#39;s structural binding pockets) appear to be clustered around the long repeat regions to form ‘binding modules.’ 
     Central domains of Toxin A and B are believed to play a role in translocation of the toxins into mammalian cells. The central domains of Toxin A are based on residues 542-1849 and those for Toxin B are based on residues 543-1851. Of the central domain regions of Toxin A and Toxin B, the first domain is a cysteine protease, which plays a role in the internalisation of the toxin&#39;s effector domain (which contains the glucosyl transferase activity). 
     Toxinotypes are often used to classify strains of  C. difficile . Toxinotyping is based on a method which characterises the restriction patterns obtained with the toxin genes. Toxinotypes of Toxin A and Toxin B represent variants, by primary amino acid sequence, of these protein toxins. In one embodiment, the  C. difficile  toxin is selected from one of toxinotypes 0 to XV. Preferred Toxinotypes (plus example Ribotypes and Strains) are listed in the Table immediately below. The listed Toxinotypes are purely illustrative and are not intended to be limiting to the present invention. 
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Toxinotype 
                 Example Ribotypes 
                 Example Strains 
                 Reference 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0 
                 001, 106 
                 VPI10463 
                 Rupnik et al. 
               
               
                 1 
                 003, 012, 102 
                 EX623 
                 (1998) 
               
               
                 2 
                 103 
                 AC008 
                 J. Clinical 
               
               
                 3 
                 027, 034, 075, 080 
                 R20291, QCD- 
                 Microbiol. 
               
               
                   
                   
                 32g58 
                 36: 2240-2247 
               
               
                 4 
                 023, 034, 075, 080 
                 55767 
               
               
                 5 
                 066, 078 
                 SE881 
               
               
                 6 
                 045, 063, 066 
                 51377 
               
               
                 7 
                 063 
                 57267 
               
               
                 8 
                 017, 047 
                  1470 
               
               
                 9 
                 019 
                 51680 
               
               
                 10 
                 036 
                  8864 
               
               
                 11 
                 033 
                 IS58, R11402 
                 Rupnik et al. 
               
               
                 12 
                 056 
                 IS25 
                 (2001) 
               
               
                 13 
                 070 
                 R9367 
                 Microbiology 
               
               
                 14 
                 111 
                 R10870 
                 147: 439-447 
               
               
                 15 
                 122 
                 R9385 
               
               
                   
               
             
          
         
       
     
     An “antibody” is used in the broadest sense and specifically covers polyclonal antibodies and antibody fragments so long as they exhibit the desired biological activity. For example, an antibody is a protein including at least one or two, heavy (H) chain variable regions (abbreviated herein as VHC), and at least one or two light (L) chain variable regions (abbreviated herein as VLC). The VHC and VLC regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al, J. Mol. Biol. 196:901-917, 1987, which are incorporated herein by reference). Preferably, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. 
     The VHC or VLC chain of the antibody can further include all or part of a heavy or light chain constant region. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region includes three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system. The term “antibody” includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda. 
     The term antibody, as used herein, also refers to a portion of an antibody that binds to a toxin of  C. difficile  (e.g. Toxin A or Toxin B), e.g., a molecule in which one or more immunoglobulin chains is not full length, but which binds to a toxin. Examples of binding portions encompassed within the term antibody include (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains; (ii) a F(ab′) 2  fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fc fragment consisting of the VHC and CH1 domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to bind, e.g. an antigen binding portion of a variable region. An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 1Al-ATi-Alβ; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies (as well as camelids) are also encompassed within the term antibody. These are obtained using conventional techniques known to those with skill in the art, and the portions are screened for utility in the same manner as are intact antibodies. 
     The term “fragment” means a peptide typically having at least 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290, 310, 330, 350 contiguous amino acid residues of (based on) the corresponding reference sequence. 
     The term “variant” means a peptide or peptide fragment having at least eighty, preferably at least eighty five, more preferably at least ninety percent amino acid sequence homology with a reference polypeptide sequence (e.g., a  C. difficile  toxin polypeptide amino acid sequence, and/or a fusion protein partner amino acid sequence, and/or an SLP amino acid reference sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences may be compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. 
     Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position—Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M—A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004). 
     Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes). 
     Alignment Scores for Determining Sequence Identity 
     
       
         
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 A 
                 R 
                 N 
                 D 
                 C 
                 Q 
                 E 
                 G 
                 H 
                 I 
                 L 
                 K 
                 M 
                 F 
                 P 
                 S 
                 T 
                 W 
                 Y 
                 V 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 A 
                 4 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 R 
                 −1 
                 5 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 N 
                 −2 
                 0 
                 6 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 D 
                 −2 
                 −2 
                 1 
                 6 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 C 
                 0 
                 −3 
                 −3 
                 −3 
                 9 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Q 
                 −1 
                 1 
                 0 
                 0 
                 −3 
                 5 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 E 
                 −1 
                 0 
                 0 
                 2 
                 −4 
                 2 
                 5 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 G 
                 0 
                 −2 
                 0 
                 −1 
                 −3 
                 −2 
                 −2 
                 6 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 H 
                 −2 
                 0 
                 1 
                 −1 
                 −3 
                 0 
                 0 
                 −2 
                 8 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 I 
                 −1 
                 −3 
                 −3 
                 −3 
                 −1 
                 −3 
                 −3 
                 −4 
                 −3 
                 4 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 L 
                 −1 
                 −2 
                 −3 
                 −4 
                 −1 
                 −2 
                 −3 
                 −4 
                 −3 
                 2 
                 4 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 K 
                 −1 
                 2 
                 0 
                 −1 
                 −3 
                 1 
                 1 
                 −2 
                 −1 
                 −3 
                 −2 
                 5 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 M 
                 −1 
                 −1 
                 −2 
                 −3 
                 −1 
                 0 
                 −2 
                 −3 
                 −2 
                 1 
                 2 
                 −1 
                 5 
                   
                   
                   
                   
                   
                   
                   
               
               
                 F 
                 −2 
                 −3 
                 −3 
                 −3 
                 −2 
                 −3 
                 −3 
                 −3 
                 −1 
                 0 
                 0 
                 −3 
                 0 
                 6 
                   
                   
                   
                   
                   
                   
               
               
                 P 
                 −1 
                 −2 
                 −2 
                 −1 
                 −3 
                 −1 
                 −1 
                 −2 
                 −2 
                 −3 
                 −3 
                 −1 
                 −2 
                 −4 
                 7 
                   
                   
                   
                   
                   
               
               
                 S 
                 1 
                 −1 
                 1 
                 0 
                 −1 
                 0 
                 0 
                 0 
                 −1 
                 −2 
                 −2 
                 0 
                 −1 
                 −2 
                 −1 
                 4 
                   
                   
                   
                   
               
               
                 T 
                 0 
                 −1 
                 0 
                 −1 
                 −1 
                 −1 
                 −1 
                 −2 
                 −2 
                 −1 
                 −1 
                 −1 
                 −1 
                 −2 
                 −1 
                 1 
                 5 
                   
                   
                   
               
               
                 W 
                 −3 
                 −3 
                 −4 
                 −4 
                 −2 
                 −2 
                 −3 
                 −2 
                 −2 
                 −3 
                 −2 
                 −3 
                 −1 
                 1 
                 −4 
                 −3 
                 −2 
                 11 
                   
                   
               
               
                 Y 
                 −2 
                 −2 
                 −2 
                 −3 
                 −2 
                 −1 
                 −2 
                 −3 
                 2 
                 −1 
                 −1 
                 −2 
                 −1 
                 3 
                 −3 
                 −2 
                 −2 
                 2 
                 7 
                   
               
               
                 V 
                 0 
                 −3 
                 −3 
                 −3 
                 −1 
                 −2 
                 −2 
                 −3 
                 −3 
                 3 
                 1 
                 −2 
                 1 
                 −1 
                 −2 
                 −2 
                 0 
                 −3 
                 −1 
                 4 
               
               
                   
               
             
          
         
       
     
     The percent identity is then calculated as: 
     
       
         
           
             
               
                 Total 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 number 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 of 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 idential 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 matches 
               
               
                 
                   
                     
                       [ 
                       
                         length 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         of 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         the 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         longer 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         sequence 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         plus 
                         ⁢ 
                         
                             
                         
                         ⁢ 
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                       ⁢ 
                       
                           
                       
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                         sequence 
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                         order 
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             × 
             100 
           
         
       
     
     Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. 
     
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 Conservative amino acid substitutions 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Basic: 
                 arginine 
               
               
                   
                   
                 lysine 
               
               
                   
                   
                 histidine 
               
               
                   
                 Acidic: 
                 glutamic acid 
               
               
                   
                   
                 aspartic acid 
               
               
                   
                 Polar: 
                 glutamine 
               
               
                   
                   
                 asparagine 
               
               
                   
                 Hydrophobic: 
                 leucine 
               
               
                   
                   
                 isoleucine 
               
               
                   
                   
                 valine 
               
               
                   
                 Aromatic: 
                 phenylalanine 
               
               
                   
                   
                 tryptophan 
               
               
                   
                   
                 tyrosine 
               
               
                   
                 Small: 
                 glycine 
               
               
                   
                   
                 alanine 
               
               
                   
                   
                 serine 
               
               
                   
                   
                 threonine 
               
               
                   
                   
                 methionine 
               
               
                   
                   
               
             
          
         
       
     
     In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and α-methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for clostridial polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues. 
     Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an  E. coli  S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in  Xenopus  oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method,  E. coli  cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochemistry 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993). 
     A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention. 
     Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention. 
     Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenised polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988). 
     Toxin-neutralising means the capacity of a substance to prevent the cytotoxic action of either Toxin A or Toxin B on a mammalian cell. In assays for toxin-neutralising activity, a fixed amount of toxin is mixed with various concentrations of a neutralising substance (e.g. an antibody) and the mixture applied to and incubated with a mammalian cell line (e.g. Vero cells) for a fixed time. The neutralising titre may be measured by several methods: 
     (a) The dilution of the substance (serum, antibody, purified IgG) that completely protects the cells from the cytotoxic effects of either Toxin A or Toxin B. These cytotoxic effects are evident by cell rounding and endpoint may be quantified by microscopy methods. 
     (b) The dilution of the substance (serum, antibody, purified IgG) that protects 50% of the cells (ED 50  titre) from the cytotoxic effects of either Toxin A or Toxin B. The ED 50  titre may be assessed by the use of dyes (e.g. crystal violet) which give a measure of cell integrity. Fitting titration data to either 4- or 5-parameter logistic curves provides an accurate estimation of the ED 50  titre. ED 50  estimations, which are generally more accurate than microscopy based methods, provide a quantitative estimation of the toxin-neutralising capacity of serum and purified antibodies. 
     Toxin-neutralising titres are measured in the presence of a fixed concentration of Toxin A or Toxin B which is set at a multiple of that required to induce cell death over a 24 hour incubation period. Typically, final concentrations of Toxin A may be set at 50 ng/ml and Toxin B between 0.5-2 ng/ml. The difference in the concentrations between Toxin A and Toxin B reflect the significantly higher specific cytotoxic activity of Toxin B. Thus, an ED 50  titre for an antibody of serum solution of 1000 units/ml indicates that at a 1000-fold dilution, the antibody solution is capable of neutralising 50% of the Toxin A or Toxin B cytotoxic activity. With respect to titres in serum, a toxin-neutralising titre ≧1000 unit/ml may be regarded as potent neutralising activity. 
     For highly purified IgG solutions, neutralising activity may also be expressed as the concentration of IgG (μg/ml) required to neutralise 50% of the Toxin A or Toxin B cytotoxic activity. In this case, a titre value ≦10 μg/ml IgG may be regarded as potent neutralising activity. 
       C. difficile  surface proteins (SLPs) means those proteins that are associated with the bacterial cell wall. Examples of 29  C. difficile  surface (cell wall proteins) are given in Table 1 of Fagan et al. (2011) J. Medical Microbiol. 60:1225-1228, which is hereby incorporated in its entirety by reference thereto. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  illustrates the structures of  C. difficile  Toxin A and Toxin B showing amino acid residues at the various domain boundaries. 
         FIG. 2  illustrates the purification of Toxin B recombinant fragment residues 767-1852 as a fusion protein with thioredoxin. The left-hand Figure shows a 4-12% SDS-PAGE analysis of TxB3. Columns C2-C8 show lanes with partially purified fragment. 
         FIG. 3  illustrates the purification of Toxin A recombinant fragment residues 542-1850 as a fusion protein with thioredoxin. 
         FIG. 4  depicts the expression and purification of CD2767 (residues 27-401). Purification of CD2767 (residues 27-401) by immobilised metal ion affinity chromatography. Key: column load, L; flow through fraction, FT; eluted purified CD2767 polypeptide, El. The intense band of the CD2767 polypeptide is illustrative of its high solubility. 
         FIG. 5  depicts the expression and purification of a fusion protein consisting of CD2767 (residues 27-401) with Toxin A (residues 543-1851). The fusion protein was expressed as a soluble polypeptide (top band in lanes C1 and C2) at approximately 2% of the total protein. C1 and C2 represent duplicates of the soluble fraction of the total expressed protein. 
         FIG. 6  demonstrates the antibody-mediated neutralisation of Toxin A and Toxin B as measured by the Vero cell ED 50  assay. The capacity of various dilutions of antiserum to Toxin A (residues 543-1851) (●) to prevent the cytotoxic effects of purified Toxin A (50 ng/ml) was assessed using crystal violet staining to measure cell viability. Reduced absorbance indicates a lack of cell integrity. Neutralisation by Toxin B (residues 767-1852) antiserum (▾) was assessed using Toxin B at 2 ng/ml. Each antiserum was a pool obtained from the immunisation of 3 sheep. 
     
    
    
     DETAILED DESCRIPTION 
     Examples 
     Example 1 
     Expression and Purification of Toxin B Fragment Recombinant Fragment Residues 767-1852 as a Fusion Protein with Thioredoxin (His 6 TrxTxBcentral) 
     Expression 
     L-broth (100 ml) supplemented with 200 μg/ml ampicillin and 0.4% glucose was inoculated with a scrape from a glycerol freeze (BL21 (DE3)  E. coli  harbouring plasmid pET59His 6 TrxTxBcentral) and maintained overnight at 30° C. and 180 rpm. The overnight culture was used as a 2.5% inoculum for Terrific Broth (4×1 L in 2.5 L unbaffled flasks) supplemented with 200 μg/ml ampicillin and 0.2% glucose. Cultures were maintained at 37° C. with orbital shaking (180 rpm) to an absorbance at 600 nm of 0.6. The temperature of the cultures was reduced to 16° C. and protein expression induced with the addition of 1 mM IPTG. The culture was maintained overnight at 16° C. with orbital shaking as before. Cell paste (60 g) was harvested by centrifugation (Sorvall RC3BP centrifuge, H6000A rotor, 4000 g for 20 minutes). 
     Immobilised Nickel Affinity Purification of His 6 TrxTxBcentral 
     Cells (60 g) were resuspended in buffer (pH 8, 20 mM Tris, 50 mM NaCl) and subjected to lysis using sonication. The lysate was cleared by centrifugation (Sorvall RC5C centrifuge, SS-34 rotor, 20,000 g, 20 minutes) and made up to 1 M ammonium sulphate with a saturated solution. The solution was stored on ice for 1 hour and the resultant precipitate collected by centrifugation (Heraeus Multifuge X3R centrifuge, 4000 g, 4° C.). The precipitate was resuspended in 250 ml of low imidazole buffer (pH 7.5, 50 mM Hepes, 0.5 M NaCl, 20 mM imidazole) and applied to a 30 ml nickel column (Ø26 mm) at a flow rate of 3 ml/min. The column was washed with low imidazole buffer and bound protein eluted using a gradient from 0-100% high imidazole buffer (pH 7.5, 50 mM Hepes, 0.5 M NaCl, 0.5 M imidazole). Fractions were analysed on 4-12% NuPAGE Bis-Tris polyacrylamide gels with coomassie staining. SDS PAGE of partially purified fractions are shown in  FIG. 2 . 
     Example 2 
     Expression and Purification of Toxin A Fragment Recombinant Fragment Residues 542-1850 (TxACPD) 
     L-broth (100 ml) supplemented with 100 μg/ml ampicillin and 0.2% glucose was inoculated with a glycerol freeze (BL21 (DE3)  E. coli  harbouring plasmid pET59TxACPDcentral). The culture was maintained (37° C., 180 rpm) to an absorbance at 600 nm of 0.6. The 100 ml culture was used as a 2% inoculum for Terrific Broth (4×0.75 L) supplemented with 200 μg/ml ampicillin and 0.2% glucose. Cultures were maintained at 37° C. with orbital shaking (180 rpm) to an absorbance at 600 nm of 0.6. The temperature of the cultures was reduced to 16° C. and protein expression induced 1 hour later with the addition of 1 mM IPTG. The culture was maintained overnight at 16° C. with orbital shaking as before. Cell paste (37 g) was harvested by centrifugation (Sorvall RC3BP centrifuge, H6000A rotor, 4000 g, 20 minutes) and stored at −80° C. 
     Cells (37 g) were resuspended with 260 ml buffer (20 mM Tris, 50 mM NaCl, pH 8) and subjected to lysis using sonication. The lysate was cleared by centrifugation (30,000 g, 20 minutes) and the clarified lysate stirred gently over ice whilst 130 ml of saturated ammonium sulphate solution (pH 8) was added to bring the mixture to 33% saturation. The mixture was left on ice for 15-20 minutes to allow a cloudy white precipitate to form. The precipitate was harvested by centrifugation (30,000 g, 15 minutes) and resuspended in 70 ml ‘low imidazole’ buffer (pH7.5, 50 mM Hepes, 0.5 M NaCl, 20 mM imidazole, 5% glycerol). The solution was applied to a 30 ml nickel column (Ø26 mm) at a flow rate of 2 ml/min. The column was washed at 2 ml/min until the UV absorbance of the flow through returned to near baseline levels. Bound material was eluted from the column with a 160 ml gradient (2 ml/min) to 100% ‘high imidazole’ buffer (pH7.5, 50 mM Hepes, 0.5 M NaCl, 0.5 M imidazole, 15% glycerol). Fractions were analysed on 4-12% NuPAGE Bis-Tris polyacrylamide gels and those containing the highest amount of the expression construct pooled. 
     For cleavage of the His 6 Thioredoxin tag, protein solution containing the constructs (18 ml, 1.5 mg/ml) from the first immobilised nickel column was thawed at room temperature and restriction grade thrombin (30 U) was added and the mixture was incubated at room temperature ˜20° C. for 16-18 hours. 
     The protein mix was loaded at 1 ml/min onto the column consisting of 30 ml (Ø26 mm) nickel charged chelating Sepharose column equilibrated with buffer A (50 mM HEPES, 0.5 M NaCl, 20% glycerol, pH 7.5). The column was washed with 30 ml buffer A. Bound protein was then eluted using 4% (50 ml), 8% (50 ml) and finally a gradient (120 ml) to 100% buffer B (50 mM HEPES, 0.5 M NaCl, 0.5 M imidazole, 20% glycerol, pH 7.5). The fraction containing the purified fractions were analysed by SDS PAGE and the fractions containing the purest construct pooled and dialysed into storage buffer (pH 7.5, 50 mM Hepes, 0.5 M NaCl, 20% glycerol). 
     SDS PAGE Analysis and Western Blot Analysis of Purified TxACPD Constructs 
     Purified protein solution was mixed 1:1 with 4×SDS-PAGE loading buffer supplemented with 5 mM DTT. The sample was heated at 95° C. for five minutes and loaded in duplicate (5 and 10 μl) onto a 4-12% NuPAGE Bis-Tris polyacrylamide gel. The gel was run in MES running buffer at 200 V for 45 minutes. One part of the gel was subjected to coomassie staining and the other blotted onto a nitrocellulose membrane at 40 V for 1 hour in transfer buffer. The membrane was blocked with 5% skimmed milk in tris buffered saline supplemented with 0.1% Tween 20 (TBST) for 40 minutes. The membrane was incubated for 40 minutes with sheep anti-Toxin A antibody diluted 1:25,000 in 1% skimmed milk TBST. The stock antibody concentration was 50 mg/ml. The membrane was washed for 4×15 minutes in TBST. A donkey anti-sheep antibody alkaline phosphatase conjugate was applied to the membrane at a dilution of 1:10,000 in 1% skimmed milk TBST. The solution was left on the membrane for 40 minutes with gentle agitation as before. The membrane was washed as before with TBST and the blot developed using NBT/BCIP one step reagent. SDS PAGE and Western blot are shown in  FIG. 3 . 
     Example 3 
     Expression and Purification of Residues 27-401 of  C. difficile  Protein CD2767 
     A synthetic gene which encodes residues 27-401 of  C. difficile  protein CD2767 was synthesised commercially with its codon bias optimised for expression in a host such as  E. coli . The gene was inserted into a pET28a expression vector and transformed into a BL21  E. coli  expression strain using standard molecular biology procedures. The  E. coli  expression strain was grown and protein expression induced with IPTG essentially as described in Example 1 except kanamycin was used in place of ampicillin. Cell pellets were either used directly or frozen at −20° C. 
     For protein extraction, cells were thawed and resuspended in 50 mM Tris HCl pH 8.0 buffer containing 0.5 M NaCl and 20 mM imidazole, sonicated (6×30 sec with 30 sec cooling after each) and then centrifuged at 47000×g for 20 min. The His6-tagged residue 27-401 CD2767 polypeptide was then purified from the supernatant fluid by using immobilised metal ion (Ni) affinity chromatography. Application of the sample to the column and washing was in the above Tris/NaCl/imidazole buffer. The purified construct was then eluted with a gradient to 0.5 M imidazole in the same buffer. 
     The CD2767 (residues 27-401) polypeptide was obtained as &gt;90% pure protein by the single purification step and appeared as an intense band of approx. 47 kDa on SDS PAGE ( FIG. 4 ). The protein fragment could be concentrated to &gt;120 mg/ml as measured by absorbance at 280 nm or to &gt;167 mg/ml as measured by the Bradford protein assay (bovine serum albumin as a standard). Both these assays illustrate the extremely high solubility of the CD2767 (residues 27-401) polypeptide and its potential usefulness as a solubility enhancing component within recombinant fusion proteins. 
     Example 4 
     Expression and Purification of Either Toxin a or Toxin B Recombinant Fragments as a Fusion Protein with Residues 27-401 of  C. difficile  Protein CD2767 
     A synthetic gene which encodes a fusion protein in which the N-terminus consists of residues 27-401 of  C. difficile  protein CD2767 and the C-terminus consists of Toxin B fragment recombinant fragment residues 767-1852 may be synthesised commercially with its codon bias optimised for expression in a host such as  E. coli . A synthetic gene which encodes a fusion protein in which the N-terminus consists of residues 27-401 of  C. difficile  protein CD2767 and the C-terminus consists of Toxin A fragment recombinant fragment residues 770-1850 may be similarly obtained. These and other fusion proteins may be incorporated with expression vectors with various purification tags (6 histidine) incorporated to facilitate purification. An example of such an expression construct is shown in SEQ ID NO: 19 which consists of CD2767 (residues 27-401) and Toxin A (residues 542-1850). 
     Expression and purification of the above constructs may be undertaken by similar methods as those outlined in Examples 1 and 2 and expression of a construct consisting of CD2767 (residues 27-401) as an N-terminal fusion to Toxin A (residues 543-1851) is shown in  FIG. 5 . Here, addition of the CD2767 domain to the Toxin A fragments renders it soluble and expressible as 2% of the total soluble protein. After expression in  E. coli , purification of the construct is effected by immobilised metal ion affinity chromatography and other chromatography methods such as ion exchange chromatography. 
     Example 5 
     Formulation of Antigens of the Invention for Immunisation of Animals 
     Purified  C. difficile  antigens at a concentration of between 0.5-2 mg/ml (nominally 1 mg/ml) were dialysed against a suitable buffer (e.g. 10 mM Hepes buffer pH 7.4 containing 150 mM NaCl) and then formaldehyde added to a final concentration of 0.2% and incubated for up to 7 days at 35° C. After incubation, the formaldehyde may optionally be removed by dialysis against a suitable buffer, e.g. phosphate buffered saline. 
     For sheep, 2 ml of buffer solution containing between 10 and 500 μg of the above  C. difficile  antigen is mixed with 2.6 ml of Freund&#39;s adjuvant to form an emulsion. Mixing with the adjuvant is carried out for several minutes to ensure a stable emulsion. The complete form of the adjuvant is used for the primary immunisation and incomplete Freund&#39;s adjuvant for all subsequent boosts. 
     Example 6 
     Generation of Antibodies to Antigens of the Invention 
     A number of conventional factors are taken into consideration during the preparation of antiserum in order to achieve the optimal humoral antibody response. These include: breed of animal; choice of adjuvant; number and location of immunisation sites; quantity of immunogen; and number of and interval between doses. Conventional optimisation of these parameters is routine to obtain specific antibody levels in excess of 6 g/liter of serum. 
     For sheep, an emulsion of the antigen with Freund&#39;s adjuvant was prepared as described in Example 5. The complete form of the adjuvant is used for the primary immunisation and incomplete Freund&#39;s adjuvant for all subsequent boosts. About 4.2 ml of the antigen/adjuvant mixture was used to immunise each sheep by i.m. injection and spread across 6 sites including the neck and all the upper limbs. This was repeated every 28 days. Blood samples were taken 14 days after each immunisation. 
     For comparison of the toxin-neutralising immune response to the different antigens, 3 sheep were used per antigen. They were immunised as above using an identical protocol and the same protein dose per immunisation. 
     Example 7 
     Assessment of the Neutralising Efficacy of Antisera to Toxins Using the In Vitro Cell Assay 
     The toxin neutralizing activity of the antisera against  C. difficile  Toxins was measured by cytotoxicity assays using Vero cells. A fixed amount of either purified  C. difficile  Toxin A or Toxin B was mixed with various dilutions of the antibodies, incubated for 30 min at 37° C. and then applied to Vero cells growing on 96-well tissue culture plates. Both Toxin A and Toxin B possess cytotoxic activity which results in a characteristic rounding of the Vero cells over a period of 24-72 h. In the presence of neutralising antibodies this activity is inhibited and the neutralising strength of an antibody preparation may be assessed by the dilution required to neutralise the effect of a designated quantity of either Toxin A or Toxin B. 
     Data demonstrating the neutralising activity of ovine antibody to various recombinant  C. difficile  Toxin B antigens are shown in Table 1 and Table 2. In these experiments, various dilutions of ovine antibody were mixed with Toxin B at a final concentration of 0.5 ng/ml and incubated for 30 min at 37° C. and then applied to Vero cells as above and incubated at 37° C. and monitored over a period of 24-72 h. The antibody dilutions which completely protect the cells against the cytotoxic effects of the Toxin B were calculated. 
     Table 1 shows the neutralising titres of an antigen of the invention and Table 2 shows the titres obtained using an antigen which consists of just the repeat regions. Collectively, the data in Tables 1 and 2 show the superior capacity of antigens of the invention to elicit a toxin-neutralising immune response compared to fragments containing just the repeat domains. 
     Antibody toxin neutralisation titres were also estimated by colorimetric assays based on cell staining with crystal violet (Rothman (1986) J. Clin. Pathol. 39:672-676). Vero cells were grown to confluence in 96-well cell culture plates. These assays were performed as described above using final concentrations of Toxin A and Toxin B in antibody mixtures of 50 ng/ml and 2 ng/ml, respectively. After overnight incubation, cells were washed gently with 200 μl of Dulbecco&#39;s-PBS (Sigma) which was carefully removed before the cells were fixed with 70 μl ice cold ethanol for 2 min. The ethanol was then removed and 70 μl crystal violet (1% w/v in ethanol; Pro-Lab) was added to the fixed cells and incubated for 30 minutes at 22° C. Plates were then washed carefully by immersion in deionized water to remove excess dye, dried at 37° C. and then 200 μl of 50% (v/v) ethanol added. Plates were then incubated at 37° C. in a shaker incubator (300 rpm) for 2 h before being read at 492 nm. ED 50  values were derived from the resulting toxin neutralisation curves using 4- or 5-pI nonlinear regression models ( FIG. 4 ). Thus, the ED 50  titre is the dilution of the serum or antibody required to achieve the 50% toxin-neutralising endpoint in the assay. If antibody solutions of known IgG concentrations are used, the titres may also be expressed as the concentration of IgG required to achieve the 50% toxin-neutralising endpoint. 
     Table 3 shows the toxin-neutralising ED 50  titres obtained using the crystal violet method for the serum generated using the central domains of both Toxin A and Toxin B. For both fragments, toxin-neutralising ED 50  titres in excess of 1000 unit/ml were obtained for their respective sera (see also  FIG. 6 ). 
     Toxin-neutralizing ED 50  titre values obtained for a sheep anti Toxin B (residues 767-1852) IgG solution are shown in Table 4. The neutralising titres against various toxinotypes of Toxin B were obtained for this fragment antiserum in order to assess its cross neutralising efficacy. Each purified toxinotype of Toxin B was normalised for toxicity in the assay and held at a fixed concentration of 16× the minimum toxin concentration which causes cell death in a 24 hr incubation period. Neutralising potencies are expressed in μg/ml IgG required for 50% neutralisation of the above Toxin B concentration. Less than a 4-fold difference in neutralising titres was observed which is indicative of good cross-neutralising efficacy. 
     Example 8 
     Assessment of the In Vivo Efficacy of Antiserum Generated Using Recombinant Antigens of the Invention for Treating CDI 
     To demonstrate the efficacy of the antisera generated, using recombinant antigens, to treat CDI in vivo, Syrian hamsters are passively immunised with antibodies which have neutralising activity against one or more of the toxins of  C. difficile . For assessing the efficacy of a treatment formulation, hamsters will be given antibody either intravenously or by the intraperitoneal route at various times from 6 hours post-challenge to 240 hours post challenge with  C. difficile.    
     Prior to passive immunisation hamsters are administered a broad spectrum antibiotic (e.g. clindamycin) and 12-72 h later challenged with  C. difficile  spores by mouth. Animals are then monitored for up to 15 days for symptoms of  C. difficile -associated disease. Control, non-immunised animals develop signs of the disease (e.g. diarrhoea, swollen abdomen, lethargy, ruffled fur) while those treated with ovine antibody appear normal or show statistically significant reduced incidence of disease. 
     Example 9 
     Vaccination by Peptide/Peptide Fragments of the Invention 
     A vaccine, represented by a peptide/peptide fragment of the invention is prepared by current Good Manufacturing Practice. Using such practices, peptides/peptide fragments of the invention may be bound to an adjuvant of aluminium hydroxide which is commercially available (e.g. Alhydrogel). The vaccine would normally contain a combination of antigens of the invention derived from Toxin A and Toxin B but could also contain either Toxin A or Toxin B antigens. The vaccine may also contain Toxin A and Toxin B antigens in combination with other antigens of bacterial or viral origin. 
     Purified  C. difficile  Toxin A and/or Toxin B antigen of the invention may be treated with formaldehyde at a final concentration of 0.2% and incubated for up to 24 hours at 35° C. (as described in Example 5). 
     In addition to the antigens of the invention, a typical vaccine composition comprises: 
     A) A buffer (e.g., Hepes buffer between 5 and 20 mM and pH between 7.0 and 7.5; 
     B) A salt component to make the vaccine physiologically isotonic (e.g., between 100 and 150 mM NaCl); 
     C) An adjuvant (e.g., aluminium hydroxide at a final aluminium concentration of between 100 and 700 μg per vaccine dose); and 
     D) A preservative (e.g., Thiomersal at 0.01% or formaldehyde at 0.01%). 
     Such vaccine compositions are administered to humans by a variety of different immunisation regimens, such as: 
     1. A single dose (e.g., 20 μg adsorbed fragment of the invention) in 0.5 ml administered sub-cutaneously. 
     2. Two doses (e.g., of 10 μg adsorbed fragment of the invention) in 0.5 mls administered at 0 and 4 weeks. 
     3. Three doses (e.g., of 10 μg adsorbed fragment of the invention) in 0.5 mls administered at 0, 2 and 12 weeks. 
     These vaccination regimens confer levels of protection against exposure to the homologous serotypes of  C. difficile  toxins 
     Example 10 
     Clinical Use of Antibodies Produced Using Antigens of the Invention 
     Three examples serve to illustrate the therapeutic value of the systemic ovine antibody products, produced using antigens of the invention, in patients with differing degrees of seventy in their CDI. 
     Mild CDI 
     A 67 year old male is admitted to a coronary care unit following a severe myocardial infarction. Whilst making an uneventful recovery he develops a mild diarrhoea without any other signs or symptoms. Because there have been recent episodes of CDI in the hospital, a faecal sample is sent immediately for testing and found to contain both Toxin A and Toxin B. After isolation to a single room with its own toilet he receives 250 mg of the ovine F(ab′) 2  intravenously followed by a second injection two days later. His diarrhoea stops quickly and he makes a full recovery without the need of either metranidazole or vancomycin. 
     Severe CDI with Risk of Relapse 
     A female aged 81 falls in her home and sustained a fractured left hip. She is immediately admitted to hospital and the hip is pinned successfully. Her frail condition prevented early discharge and, a few days later, she develops a productive cough for which she was given a wide spectrum antibiotic. After a further eight days she develops profuse diarrhoea with abdominal pain and tenderness and CDI is diagnosed by the appropriate faecal tests. At the time there is also evidence of systemic manifestations of the infection including a markedly raised white blood cell count, and of significant fluid loss with dehydration. The patient is started immediately on oral vancomycin and, at the same time, receives the first of five daily injections of 250 mg of the ovine F(ab′) 2 -based product intravenously. There is a rapid resolution of the signs and symptoms and of the laboratory manifestations of CDI. However, in order to avoid the risk of relapse of her CDI following stopping vancomycin, she continues to be treated for a further two weeks on an oral form of the antibody therapy. She experiences no relapse. 
     Severe CDI with Complications 
     An 87 year old female develops bronchopneumonia while resident in long-stay care facilities. The local general practitioner starts her on a course of antibiotic therapy with immediate benefit. However, eight days after stopping the antibiotic she experiences severe diarrhoea. Her condition starts to deteriorate necessitating admission to hospital where Toxin A is detected in her faeces by an ELISA test. By this time she is extremely ill with evidence of circulatory failure and her diarrhoea has stopped. The latter is found to be due a combination of paralytic ileus and toxic megacolon and an emergency total colectomy is considered essential. Since such surgery is associated with a mortality in excess of 60% she receives intravenous replacement therapy together with the contents of two ampoules (500 mg) of antibody product. By the time she is taken to the operating theatre four hours later, her general condition had improved significantly and she survives surgery. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Neutralisation titres obtained by immunisation of 
               
               
                 sheep with a recombinant Toxin B-derived antigen 
               
               
                 (TxBcentral; residues 767-1852) of the invention 
               
             
          
           
               
                   
                   
                 Immunisation 
                 Neutralisation titre 
               
               
                   
                 No of 
                 period 
                 against Toxin B 
               
               
                 Antigen 
                 Doses 
                 (weeks) 
                 (0.5 ng/ml) 
               
               
                   
               
             
          
           
               
                 Recombinant Toxin B 
                 2 
                 6 
                 480 
               
               
                 (residues 767-1852) 
                 3 
                 10 
                 5,120 
               
               
                 at 100 μg/dose 
                 4 
                 14 
                 5,120 
               
               
                   
                 5 
                 18 
                 5,120 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Neutralisation titres obtained by immunisation of sheep 
               
               
                 with a recombinant Toxin B-derived antigen (TxB2, residues 
               
               
                 1756-2366) representing the repeat regions 
               
             
          
           
               
                   
                   
                 Immunisation 
                 Neutralisation titre 
               
               
                   
                 No of 
                 period 
                 against Toxin B 
               
               
                 Antigen 
                 Doses 
                 (weeks) 
                 (0.5 ng/ml) 
               
               
                   
               
             
          
           
               
                 Recombinant Toxin B 
                 2 
                 6 
                 &lt;10 
               
               
                 (residues 1756-2366) 
                 3 
                 10 
                 10 
               
               
                 at 100 μg/dose 
                 4 
                 14 
                 10 
               
               
                   
                 5 
                 18 
                 80 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Neutralisation ED 50  titres obtained by immunisation 
               
               
                 of sheep with a recombinant antigensToxin B (residues 
               
               
                 767-1852) and Toxin A (residues 543-1851) 
               
             
          
           
               
                   
                 Neutralising titre ED 50   
               
             
          
           
               
                 Antigen 
                 ELISA Titre 
                 1 
                 2 
                 Mean 
               
               
                   
               
               
                 Toxin B (767-1852) 
                 1 × 10 5   
                 7946 ± 1027 
                 8225 ± 418 
                 8086 
               
               
                 Toxin A (542-1850) 
                 2 × 10 5   
                 2472 ± 199  
                 2096 ± 222 
                 2284 
               
               
                   
               
             
          
         
       
     
     For each antigen, 5 doses of 100 μg were given monthly to each of 3 sheep and the serum analysed at 18 weeks. ELISA titres, derived from 14 week samples, represent serum dilutions (pool from 3 animals) which gave a signal of 0.5 A 450  above background and are the mean of duplicate determinations. For the crystal violet ED 50  assay, Toxin B was used at a fixed concentration of 2 ng/ml and Toxin A at 50 ng/ml. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Neutralisation ED 50  titres against various Toxin 
               
               
                 B toxinotypes using serum antiserum generated 
               
               
                 to recombinant Toxin B (residues 767-1852) 
               
             
          
           
               
                 Immunising 
                 Assay 
                 Neutralising Potency ED 50  (μg/ml IgG) 
               
             
          
           
               
                 Antigen 
                 Toxinotype 
                 1 
                 2 
                 Mean 
               
               
                   
               
               
                 Toxin B 
                 Toxin B (0) 
                 2.5 ± 0.22 
                 2.1 ± 0.20 
                 2.3 
               
               
                 (residues 
                 Toxin B (3) 
                 7.9 ± 0.64 
                 7.5 ± 1.20 
                 7.7 
               
               
                 767-1852) 
                 Toxin B (5) 
                 7.7 ± 0.73 
                 8.1 ± 0.71 
                 7.9 
               
               
                   
                 Toxin B (10) 
                 7.2 ± 0.67 
                 9.0 ± 0.75 
                 8.1 
               
               
                   
               
             
          
         
       
     
     Antibodies to Toxin B (residues 767-1852) (toxinotype 0 sequence) were assessed for their capacity to neutralise other Toxin B toxinotypes. Purified Toxin B toxinotypes (0, 3, 5 and 10) were each titrated in the cell assay and used at a fixed concentration of 16× the minimum toxin concentration which causes cell death in a 24 hr incubation period. Neutralising potencies are expressed in μg/ml IgG required for 50% neutralisation of the above Toxin B concentration. 
     
       
         
               
             
               
             
           
               
                   
               
               
                 SED ID NOs 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   Clostridium difficile  Toxin A (Toxinotype 0) 
               
               
                 SEQ ID NO: 1 
               
               
                 MSLISKEELIKLAYSIRPRENEYKTILTNLDEYNKLTTNNNENKYLQLKKLNESIDVFMNKYKTSSRNRALSNL 
               
               
                 KKDILKEVILIKNSNTSPVEKNLHFVVVIGGEVSDIALEYIKQWADINAEYNIKLWYDSEAFLVNTLKKAIVES 
               
               
                 STTEALQLLEEEIQNPQFDNMKFYKKRMEFIYDRQKRFINYYKSQINKPTVPTIDDIIKSHLVSEYNRDETVLE 
               
               
                 SYRTNSLRKINSNHGIDIRANSLFTEQELLNIYSQELLNRGNLAAASDIVRLLALKNFGGVYLDVDMLPGIHSD 
               
               
                 LFKTISRPSSIGLDRWEMIKLEAIMKYKKYINNYTSENFDKLDQQLKDNFKLIIESKSEKSEIFSKLENLNVSD 
               
               
                 LEIKIAFALGSVINQALISKQGSYLTNLVIEQVKNRYQFLNQHLNPAIESDNNFTDTTKIFHDSLFNSATAENS 
               
               
                 MFLTKIAPYLQVGFMPEARSTISLSGPGAYASAYYDFINLQENTIEKTLKASDLIEFKFPENNLSQLTEQEINS 
               
               
                 LWSFDQASAKYQFEKYVRDYTGGSLSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDI 
               
               
                 SYEATCNLFSKNPKNSIIIQRNMNESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEFNTSEF 
               
               
                 ARLSVDSLSNEISSFLDTIKLDISPKNVEVNLLGCNMFSYDFNVEETYPGKLLLSIMDKITSTLPDVNKNSITI 
               
               
                 GANQYEVRINSEGRKELLAHSGKWINKEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDA 
               
               
                 SVSPDTKFILNNLKLNIESSIGDYIYYEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLIS 
               
               
                 FEDISKNNSTYSVRFINKSNGESVYVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTL 
               
               
                 NAAFFIQSLIDYSSNKDVLNDLSTSVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVS 
               
               
                 TILDGINLGAAIKELLDEHDPLLKKELEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLV 
               
               
                 NNELILHDKATSVVNYFNHLSESKKYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVT 
               
               
                 GNIDHFFSSPSISSHIPSLSIYSAIGIETENLDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLENDGTRLLDSI 
               
               
                 RDLYPGKFYWRFYAFFDYAITTLKPVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGAGGTYSLLLSS 
               
               
                 YPISTNINLSKDDLWIFNIDNEVREISIENGTIKKGKLIKDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIF 
               
               
                 LTCELDDKISLIIEINLVAKSYSLLLSGDKNYLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTS 
               
               
                 QKSIIHYKKDSKNILEFYNDSTLEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKV 
               
               
                 NGLYLNESVYSSYLDFVKNSDGHHNTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNN 
               
               
                 KNIDIYFGEWKTSSSKSTIFSGNGRNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLIN 
               
               
                 INTNYYSNEYYPEIIVLNPNTFHKKVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQS 
               
               
                 FNKMSIDFKDIKKLSLGYIMSNFKSFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEF 
               
               
                 NLVTGWQTINGKKYYFDINTGAALTSYKIINGKHFYFNNDGVMQLGVFKGPDGFEYFAPANTQNNNIEGQAIVY 
               
               
                 QSKFLTLNGKKYYFDNNSKAVTGWRIINNEKYYFNPNNAIAAVGLQVIDNNKYYFNPDTAIISKGWQTVNGSRY 
               
               
                 YFDTDTAIAFNGYKTIDGKHFYFDSDCVVKIGVFSTSNGFEYFAPANTYNNNIEGQAIVYQSKFLTLNGKKYYF 
               
               
                 DNNSKAVTGLQTIDSKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAIASTGYT 
               
               
                 IINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIIN 
               
               
                 NKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANT 
               
               
                 HNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEA 
               
               
                 ATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN 
               
               
                 KFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYF 
               
               
                 NTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPN 
               
               
                 TAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKA 
               
               
                 VTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYG 
               
               
                   
               
               
                   C. difficile  Toxin B (Toxinotype 0) 
               
               
                 SEQ ID NO: 2 
               
               
                 MSLVNRKQLEKMANVRFRTQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYIDTYKKSGRNKALKK 
               
               
                 FKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKDVNSDYNVNVFYDSNAFLINTLKKTVVES 
               
               
                 AINDTLESFRENLNDPRFDYNKFFRKRMEIIYDKQKNFINYYKAQREENPELIIDDIVKTYLSNEYSKEIDELN 
               
               
                 TYIEESLNKITQNSGNDVRNFEEFKNGESFNLYEQELVERWNLAAASDILRISALKEIGGMYLDVDMLPGIQPD 
               
               
                 LFESIEKPSSVTVDFWEMTKLEAIMKYKEYIPEYTSEHFDMLDEEVQSSFESVLASKSDKSEIFSSLGDMEASP 
               
               
                 LEVKIAFNSKGIINQGLISVKDSYCSNLIVKQIENRYKILNNSLNPAISEDNDFNTTTNTFIDSIMAEANADNG 
               
               
                 RFMMELGKYLRVGFFPDVKTTINLSGPEAYAAAYQDLLMFKEGSMNIHLIEADLRNFEISKTNISQSTEQEMAS 
               
               
                 LWSFDDARAKAQFEEYKRNYFEGSLGEDDNLDFSQNIVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYE 
               
               
                 AACNLFAKTPYDSVLFQKNIEDSEIAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNTDIFAGF 
               
               
                 DVDSLSTEIEAAIDLAKEDISPKSIEINLLGCNMFSYSINVEETYPGKLLLKVKDKISELMPSISQDSIIVSAN 
               
               
                 QYEVRINSEGRRELLDHSGEWINKEESIIKDISSKEYISFNPKENKITVKSKNLPELSTLLQEIRNNSNSSDIE 
               
               
                 LEEKVMLTECEINVISNIDTQIVEERIEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFED 
               
               
                 ISETDEGFSIRFINKETGESIFVETEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLNAA 
               
               
                 FFIQSLIEYNSSKESLSNLSVAMKVQVYAQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATII 
               
               
                 DGVSLGAAIKELSETSDPLLRQEIEAKIGIMAVNLTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNE 
               
               
                 LVLRDKATKVVDYFKHVSLVETEGVFTLLDDKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDI 
               
               
                 DHFFSAPSITYREPHLSIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNY 
               
               
                 EGEFYWRYFAFIADALITTLKPRYEDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNM 
               
               
                 GINIELSESDVWIIDVDNVVRDVTIESDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTF 
               
               
                 SILEGINAIIEVDLLSKSYKLLISGELKILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTK 
               
               
                 EGLFVSELPDVVLISKVYMDDSKPSFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNS 
               
               
                 VHLDESGVAEILKFMNRKGNTNTSDSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDEN 
               
               
                 DNIQPYFIKFNTLETNYTLYVGNRQNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEI 
               
               
                 NITPVYETNNTYPEVIVLDANYINEKINVNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTL 
               
               
                 ANKLSFNFSDKQDVPVSEIILSFTPSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVN 
               
               
                 NLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAID 
               
               
                 FTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHY 
               
               
                 FDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDS 
               
               
                 FTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDD 
               
               
                 NYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESD 
               
               
                 KYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIG 
               
               
                 VFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE 
               
               
                   
               
               
                   C. difficile  Toxin A (Toxinotype 3) 
               
               
                 SEQ ID NO: 3 
               
               
                 MSLISKEELIKLAYSIRPRENEYKTILTNLDEYNKLTTNNNENKYLQLKKLNESIDVFMNKYKNSSRNRALSNL 
               
               
                 KKDILKEVILIKNSNTSPVEKNLHFVWIGGEVSDIALEYIKQWADINAEYNIKLWYDSEAFLVNTLKKAIVESS 
               
               
                 TTEALQLLEEEIQNPQFDNMKFYKKRMEFIYDRQKRFINYYKSQINKPTVPTIDDIIKSHLVSEYNRDETLLES 
               
               
                 YRTNSLRKINSNHGIDIRANSLFTEQELLNIYSQELLNRGNLAAASDIVRLLALKNFGGVYLDVDMLPGIHSDL 
               
               
                 FKTIPRPSSIGLDRWEMIKLEAIMKYKKYINNYTSENFDKLDQQLKDNFKLIIESKSEKSEIFSKLENLNVSDL 
               
               
                 EIKIAFALGSVINQALISKQGSYLTNLVIEQVKNRYQFLNQHLNPAIESDNNFTDTTKIFHDSLFNSATAENSM 
               
               
                 FLTKIAPYLQVGFMPEARSTISLSGPGAYASAYYDFINLQENTIEKTLKASDLIEFKFPENNLSQLTEQEINSL 
               
               
                 WSFDQASAKYQFEKYVRDYTGGSLSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDIS 
               
               
                 YEATCNLFSKNPKNSIIIQRNMNESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEFNTSEFA 
               
               
                 RLSVDSLSNEISSFLDTIKLDISPKNVEVNLLGCNMFSYDFNVEETYPGKLLLSIMDKITSTLPDVNKDSITIG 
               
               
                 ANQYEVRINSEGRKELLAHSGKWINKEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDAS 
               
               
                 VSPDTKFILNNLKLNIESSIGDYIYYEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISF 
               
               
                 EDISKNNSTYSVRFINKSNGESVYVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTLN 
               
               
                 AAFFIQSLIDYSSNKDVLNDLSTSVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVST 
               
               
                 ILDGINLGAAIKELLDEHDPLLKKELEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLVN 
               
               
                 NELILHDKATSVVNYFNHLSESKEYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTG 
               
               
                 NIDHFFSSPYISSHIPSLSVYSAIGIKTENLDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLENNGTKLLDSIR 
               
               
                 DLYPGKFYWRFYAFFDYAITTLKPVYEDTNTKIKLDKDTRNFIMPTITTDEIRNKLSYSFDGAGGTYSLLLSSY 
               
               
                 PISMNINLSKDDLWIFNIDNEVREISIENGTIKKGNLIEDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFL 
               
               
                 TCELDDKISLIIEINLVAKSYSLLLSGDKNYLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQ 
               
               
                 KSIIHYKKDSKNILEFYNGSTLEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVN 
               
               
                 GLYLNESVYSSYLDFVKNSDGHHNTSNFMNLFLNNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNK 
               
               
                 NIDIYFGEWKTSSSKSTIFSGNGRNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLINI 
               
               
                 NTNYYSNEYYPEIIVLNPNTFHKKVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSF 
               
               
                 NKMSIDFKDIKKLSLGYIMSNFKSFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIESN 
               
               
                 LVTGWQTINGKKYYFDINTGAASTSYKIINGKHFYFNNNGVMQLGVFKGPDGFEYFAPANTQNNNIEGQAIVYQ 
               
               
                 SKFLTLNGKKYYFDNDSKAVTGWRIINNEKYYFNPNNAIAAVGLQVIDNNKYYFNPDTAIISKGWQTVNGSRYY 
               
               
                 FDTDTAIAFNGYKTIDGKHFYFDSDCVVKIGVFSGSNGFEYFAPANTYNNNIEGQAIVYQSKFLTLNGKKYYFD 
               
               
                 NNSKAVTGWQTIDSKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAEAATGWQTIDGKKYYFNTNTSIASTGYTI 
               
               
                 INGKYFYFNTDGIMQIGVFKVPNGFEYFAPANTHNNNIEGQAILYQNKFLTLNGKKYYFGSDSKAITGWQTIDG 
               
               
                 KKYYFNPNNAIAATHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTH 
               
               
                 NNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDSKKYYFNLNTAVAVTGWQTIDGEKYYFNLNTAEAA 
               
               
                 TGWQTIDGKRYYFNTNTYIASTGYTIINGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTHNNNIEGQAILYQNK 
               
               
                 FLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTYIASTGYTIISGKHFYFN 
               
               
                 TDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNDSKAATGWATIDGNRYYFEPNT 
               
               
                 AMGANGYKTIDNKNFYFRNGLPQIGVFKGPNGFEYFAPANTDANNIDGQAIRYQNRFLHLLGKIYYFGNNSKAV 
               
               
                 TGWQTINSKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYG 
               
               
                   
               
               
                   C. difficile  Toxin B (Toxinotype 3) 
               
               
                 SEQ ID NO: 4 
               
               
                 MSLVNRKQLEKMANVRFRVQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYIDTYKKSGRNKALKK 
               
               
                 FKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKDVNSDYNVNVFYDSNAFLINTLKKTIVES 
               
               
                 ATNDTLESFRENLNDPRFDYNKFYRKRMEIIYDKQKNFINYYKTQREENPDLIIDDIVKIYLSNEYSKDIDELN 
               
               
                 SYIEESLNKVTENSGNDVRNFEEFKGGESFKLYEQELVERWNLAAASDILRISALKEVGGVYLDVDMLPGIQPD 
               
               
                 LFESIEKPSSVTVDFWEMVKLEAIMKYKEYIPGYTSEHFDMLDEEVQSSFESVLASKSDKSEIFSSLGDMEASP 
               
               
                 LEVKIAFNSKGIINQGLISVKDSYCSNLIVKQIENRYKILNNSLNPAISEDNDFNTTTNAFIDSIMAEANADNG 
               
               
                 RFMMELGKYLRVGFFPDVKTTINLSGPEAYAAAYQDLLMFKEGSMNIHLIEADLRNFEISKTNISQSTEQEMAS 
               
               
                 LWSFDDARAKAQFEEYKKNYFEGSLGEDDNLDFSQNTVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYE 
               
               
                 AACNLFAKTPYDSVLFQKNIEDSEIAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNTDIFAGL 
               
               
                 DVDSLSTEIETAIDLAKEDISPKSIEINLLGCNMFSYSVNVEETYPGKLLLRVKDKVSELMPSISQDSIIVSAN 
               
               
                 QYEVRINSEGRRELLDHSGEWINKEESIIKDISSKEYISFNPKENKIIVKSKNLPELSTLLQEIRNNSNSSDIE 
               
               
                 LEEKVMLAECEINVISNIDTQVVEGRIEEAKSLTSDSINYIKNEFKLIESISDALYDLKQQNELEESHFISFED 
               
               
                 ILETDEGFSIRFIDKETGESIFVETEKAIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDATHEVNTLNAA 
               
               
                 FFIQSLIEYNSSKESLSNLSVAMKVQVYAQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPVIATII 
               
               
                 DGVSLGAAIKELSETSDPLLRQEIEAKIGIMAVNLTAATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNE 
               
               
                 LILRDKATKVVDYFSHISLAESEGAFTSLDDKIMMPQDDLVISEIDFNNNSITLGKCEIWRMEGGSGHTVTDDI 
               
               
                 DHFFSAPSITYREPHLSIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNY 
               
               
                 EGEFYWRYFAFIADALITTLKPRYEDTNIRINLDSNTRSFIVPVITTEYIREKLSYSFYGSGGTYALSLSQYNM 
               
               
                 NINIELNENDTWVIDVDNVVRDVTIESDKIKKGDLIENILSKLSIEDNKIILDNHEINFSGTLNGGNGFVSLTF 
               
               
                 SILEGINAVIEVDLLSKSYKVLISGELKTLMANSNSVQQKIDYIGLNSELQKNIPYSFMDDKGKENGFINCSTK 
               
               
                 EGLFVSELSDVVLISKVYMDNSKPLFGYCSNDLKDVKVITKDDVIILTGYYLKDDIKISLSFTIQDENTIKLNG 
               
               
                 VYLDENGVAEILKFMNKKGSTNTSDSLMSFLESMNIKSIFINSLQSNTKLILDTNFIISGTTSIGQFEFICDKD 
               
               
                 NNIQPYFIKFNTLETKYTLYVGNRQNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVIISPNIYTDEI 
               
               
                 NITPIYEANNTYPEVIVLDTNYISEKINININDLSIRYVWSNDGSDFILMSTDEENKVSQVKIRFTNVFKGNTI 
               
               
                 SDKISFNFSDKQDVSINKVISTFTPSYYVEGLLNYDLGLISLYNEKFYINNFGMMVSGLVYINDSLYYFKPPIK 
               
               
                 NLITGFTTIGDDKYYFNPDNGGAASVGETIIDGKNYYFSQNGVLQTGVFSTEDGFKYFAPADTLDENLEGEAID 
               
               
                 FTGKLTIDENVYYFGDNYRAAIEWQTLDDEVYYFSTDTGRAFKGLNQIGDDKFYFNSDGIMQKGFVNINDKTFY 
               
               
                 FDDSGVMKSGYTEIDGKYFYFAENGEMQIGVFNTADGFKYFAHHDEDLGNEEGEALSYSGILNFNNKIYYFDDS 
               
               
                 FTAVVGWKDLEDGSKYYFDEDTAEAYIGISIINDGKYYFNDSGIMQIGFVTINNEVFYFSDSGIVESGMQNIDD 
               
               
                 NYFYIDENGLVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESD 
               
               
                 KYYFDPETKKAYKGINVIDDIKYYFDENGIMRTGLITFEDNHYYFNEDGIMQYGYLNIEDKTFYFSEDGIMQIG 
               
               
                 VFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE 
               
               
                   
               
               
                   C. difficile  Toxin A 542-1850 (toxinotype 0) 
               
               
                 SEQ ID NO: 5 
               
               
                 LSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDISYEATCNLFSKNPKNSIIIQRNMN 
               
               
                 ESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEFNTSEFARLSVDSLSNEISSFLDTIKLDIS 
               
               
                 PKNVEVNLLGCNMFSYDFNVEETYPGKLLLSIMDKITSTLPDVNKNSITIGANQYEVRINSEGRKELLAHSGKW 
               
               
                 INKEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDY 
               
               
                 IYYEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSVRFINKSNGESV 
               
               
                 YVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTLNAAFFIQSLIDYSSNKDVLNDLST 
               
               
                 SVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLK 
               
               
                 KELEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESK 
               
               
                 KYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIPSLSIYSA 
               
               
                 IGIETENLDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLENDGTRLLDSIRDLYPGKFYWRFYAFFDYAITTLK 
               
               
                 PVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGAGGTYSLLLSSYPISTNINLSKDDLWIFNIDNEVR 
               
               
                 EISIENGTIKKGKLIKDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSL 
               
               
                 LLSGDKNYLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNDSTLE 
               
               
                 FNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNESVYSSYLDFVKNSDGHH 
               
               
                 NTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNKNIDIYFGEWKTSSSKSTIFSGNG 
               
               
                 RNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHK 
               
               
                 KVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFK 
               
               
                 SFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNL 
               
               
                   
               
               
                   C. difficile  Toxin A 542-1850 (toxinotype 3) 
               
               
                 SEQ ID NO: 6 
               
               
                 LSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDISYEATCNLFSKNPKNSIIIQRNMN 
               
               
                 ESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEFNTSEFARLSVDSLSNEISSFLDTIKLDIS 
               
               
                 PKNVEVNLLGCNMFSYDFNVEETYPGKLLLSIMDKITSTLPDVNKDSITIGANQYEVRINSEGRKELLAHSGKW 
               
               
                 INKEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDY 
               
               
                 IYYEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSVRFINKSNGESV 
               
               
                 YVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTLNAAFFIQSLIDYSSNKDVLNDLST 
               
               
                 SVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLK 
               
               
                 KELEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESK 
               
               
                 EYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPYISSHIPSLSVYSA 
               
               
                 IGIKTENLDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLENNGTKLLDSIRDLYPGKFYWRFYAFFDYAITTLK 
               
               
                 PVYEDTNTKIKLDKDTRNFIMPTITTDEIRNKLSYSFDGAGGTYSLLLSSYPISMNINLSKDDLWIFNIDNEVR 
               
               
                 EISIENGTIKKGNLIEDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSL 
               
               
                 LLSGDKNYLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNGSTLE 
               
               
                 FNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNESVYSSYLDFVKNSDGHH 
               
               
                 NTSNFMNLFLNNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNKNIDIYFGEWKTSSSKSTIFSGNG 
               
               
                 RNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHK 
               
               
                 KVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFK 
               
               
                 SFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIESNL 
               
               
                   
               
               
                   C. difficile  Toxin A 542-1850 (toxinotype 0) Cysteine protease negative 
               
               
                 SEQ ID NO: 7 
               
               
                 LSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDISYEATCNLFSKNPKNSIIIQRNMN 
               
               
                 ESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEFNTSEFARLSVDSLSNEISSFLDTIKLDIS 
               
               
                 PKNVEVNLLGANMFSYDFNVEETYPGKLLLSIMDKITSTLPDVNKNSITIGANQYEVRINSEGRKELLAHSGKW 
               
               
                 INKEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDY 
               
               
                 IYYEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSVRFINKSNGESV 
               
               
                 YVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTLNAAFFIQSLIDYSSNKDVLNDLST 
               
               
                 SVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLK 
               
               
                 KELEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESK 
               
               
                 KYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIPSLSIYSA 
               
               
                 IGIETENLDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLENDGTRLLDSIRDLYPGKFYWRFYAFFDYAITTLK 
               
               
                 PVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGAGGTYSLLLSSYPISTNINLSKDDLWIFNIDNEVR 
               
               
                 EISIENGTIKKGKLIKDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSL 
               
               
                 LLSGDKNYLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNDSTLE 
               
               
                 FNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNESVYSSYLDFVKNSDGHH 
               
               
                 NTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNKNIDIYFGEWKTSSSKSTIFSGNG 
               
               
                 RNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHK 
               
               
                 KVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFK 
               
               
                 SFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNL 
               
               
                   
               
               
                   C. difficile  Toxin A 770-1850 
               
               
                 SEQ ID NO: 8 
               
               
                 MSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDYIYYEKLE 
               
               
                 PVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSVRFINKSNGESVYVETEKE 
               
               
                 IFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTLNAAFFIQSLIDYSSNKDVLNDLSTSVKVQLY 
               
               
                 AQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLKKELEAKV 
               
               
                 GVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESKKYGPLKT 
               
               
                 EDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIPSLSIYSAIGIETEN 
               
               
                 LDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLENDGTRLLDSIRDLYPGKFYWRFYAFFDYAITTLKPVYEDTN 
               
               
                 IKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGAGGTYSLLLSSYPISTNINLSKDDLWIFNIDNEVREISIENG 
               
               
                 TIKKGKLIKDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSLLLSGDKN 
               
               
                 YLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNDSTLEFNSKDFI 
               
               
                 AEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNESVYSSYLDFVKNSDGHHNTSNFMN 
               
               
                 LFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNKNIDIYFGEWKTSSSKSTIFSGNGRNVVVEP 
               
               
                 IYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHKKVNINLD 
               
               
                 SSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFKSFNSENE 
               
               
                 LDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNL 
               
               
                   
               
               
                   C. difficile  Toxin A 1130-1850 
               
               
                 SEQ ID NO: 9 
               
               
                 SESKKYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIPSLS 
               
               
                 IYSAIGIETENLDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLENDGTRLLDSIRDLYPGKFYWRFYAFFDYAI 
               
               
                 TTLKPVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGAGGTYSLLLSSYPISTNINLSKDDLWIFNID 
               
               
                 NEVREISIENGTIKKGKLIKDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAK 
               
               
                 SYSLLLSGDKNYLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYND 
               
               
                 STLEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNESVYSSYLDFVKNS 
               
               
                 DGHHNTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNKNIDIYFGEWKTSSSKSTIF 
               
               
                 SGNGRNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPN 
               
               
                 TFHKKVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIM 
               
               
                 SNFKSFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNL 
               
               
                   
               
               
                   C. difficile  Toxin B (toxinotype 0) 543-1852 
               
               
                 SEQ ID NO: 10 
               
               
                 LGEDDNLDFSQNIVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYEAACNLFAKTPYDSVLFQKNIEDSE 
               
               
                 IAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNTDIFAGFDVDSLSTEIEAAIDLAKEDISPKS 
               
               
                 IEINLLGCNMFSYSINVEETYPGKLLLKVKDKISELMPSISQDSIIVSANQYEVRINSEGRRELLDHSGEWINK 
               
               
                 EESIIKDISSKEYISFNPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDTQIVE 
               
               
                 ERIEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRFINKETGESIFVE 
               
               
                 TEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLNAAFFIQSLIEYNSSKESLSNLSVAMK 
               
               
                 VQVYAQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLRQEI 
               
               
                 EAKIGIMAVNLTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKHVSLVETEG 
               
               
                 VFTLLDDKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYDVLEV 
               
               
                 QKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRY 
               
               
                 EDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVWIIDVDNVVRDVT 
               
               
                 IESDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLIS 
               
               
                 GELKILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMDDSKP 
               
               
                 SFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNRKGNTNTS 
               
               
                 DSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNYTLYVGNR 
               
               
                 QNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYIN 
               
               
                 EKINVNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFT 
               
               
                 PSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNL 
               
               
                   
               
               
                   C. difficile  Toxin B (toxinotype 3) 543-1852 
               
               
                 SEQ ID NO: 11 
               
               
                 LGEDDNLDFSQNTVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYEAACNLFAKTPYDSVLFQKNIEDSE 
               
               
                 IAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNTDIFAGLDVDSLSTEIETAIDLAKEDISPKS 
               
               
                 IEINLLGCNMFSYSVNVEETYPGKLLLRVKDKVSELMPSISQDSIIVSANQYEVRINSEGRRELLDHSGEWINK 
               
               
                 EESIIKDISSKEYISFNPKENKIIVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLAECEINVISNIDTQVVE 
               
               
                 GRIEEAKSLTSDSINYIKNEFKLIESISDALYDLKQQNELEESHFISFEDILETDEGFSIRFIDKETGESIFVE 
               
               
                 TEKAIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDATHEVNTLNAAFFIQSLIEYNSSKESLSNLSVAMK 
               
               
                 VQVYAQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPVIATIIDGVSLGAAIKELSETSDPLLRQEI 
               
               
                 EAKIGIMAVNLTAATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELILRDKATKVVDYFSHISLAESEG 
               
               
                 AFTSLDDKIMMPQDDLVISEIDFNNNSITLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYDVLEV 
               
               
                 QKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRY 
               
               
                 EDTNIRINLDSNTRSFIVPVITTEYIREKLSYSFYGSGGTYALSLSQYNMNINIELNENDTWVIDVDNVVRDVT 
               
               
                 IESDKIKKGDLIENILSKLSIEDNKIILDNHEINFSGTLNGGNGFVSLTFSILEGINAVIEVDLLSKSYKVLIS 
               
               
                 GELKTLMANSNSVQQKIDYIGLNSELQKNIPYSFMDDKGKENGFINCSTKEGLFVSELSDVVLISKVYMDNSKP 
               
               
                 LFGYCSNDLKDVKVITKDDVIILTGYYLKDDIKISLSFTIQDENTIKLNGVYLDENGVAEILKFMNKKGSTNTS 
               
               
                 DSLMSFLESMNIKSIFINSLQSNTKLILDTNFIISGTTSIGQFEFICDKDNNIQPYFIKFNTLETKYTLYVGNR 
               
               
                 QNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVIISPNIYTDEINITPIYEANNTYPEVIVLDTNYIS 
               
               
                 EKINININDLSIRYVWSNDGSDFILMSTDEENKVSQVKIRFTNVFKGNTISDKISFNFSDKQDVSINKVISTFT 
               
               
                 PSYYVEGLLNYDLGLISLYNEKFYINNFGMMVSGLVYINDSLYYFKPPIKNL 
               
               
                   
               
               
                   C. difficile  Toxin B 543-1852 Cysteine protease negative 
               
               
                 SEQ ID NO: 12 
               
               
                 LGEDDNLDFSQNIVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYEAACNLFAKTPYDSVLFQKNIEDSE 
               
               
                 IAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNTDIFAGFDVDSLSTEIEAAIDLAKEDISPKS 
               
               
                 IEINLLGANMFSYSINVEETYPGKLLLKVKDKISELMPSISQDSIIVSANQYEVRINSEGRRELLDHSGEWINK 
               
               
                 EESIIKDISSKEYISFNPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDTQIVE 
               
               
                 ERIEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRFINKETGESIFVE 
               
               
                 TEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLNAAFFIQSLIEYNSSKESLSNLSVAMK 
               
               
                 VQVYAQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLRQEI 
               
               
                 EAKIGIMAVNLTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKHVSLVETEG 
               
               
                 VFTLLDDKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYDVLEV 
               
               
                 QKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRY 
               
               
                 EDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVWIIDVDNVVRDVT 
               
               
                 IESDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLIS 
               
               
                 GELKILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMDDSKP 
               
               
                 SFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNRKGNTNTS 
               
               
                 DSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNYTLYVGNR 
               
               
                 QNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYIN 
               
               
                 EKINVNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFT 
               
               
                 PSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNL 
               
               
                   
               
               
                   C. difficile  Toxin B 767-1852 
               
               
                 SEQ ID NO: 13 
               
               
                 SIIKDISSKEYISFNPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDTQIVEER 
               
               
                 IEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRFINKETGESIFVETE 
               
               
                 KTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLNAAFFIQSLIEYNSSKESLSNLSVAMKVQ 
               
               
                 VYAQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLRQEIEA 
               
               
                 KIGIMAVNLTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKHVSLVETEGVF 
               
               
                 TLLDDKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYDVLEVQK 
               
               
                 EELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRYED 
               
               
                 TNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVWIIDVDNVVRDVTIE 
               
               
                 SDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLISGE 
               
               
                 LKILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMDDSKPSF 
               
               
                 GYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNRKGNTNTSDS 
               
               
                 LMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNYTLYVGNRQN 
               
               
                 MIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYINEK 
               
               
                 INVNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFTPS 
               
               
                 YYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNL 
               
               
                   
               
               
                   C. difficile  Toxin B 1145-1852 
               
               
                 SEQ ID NO: 14 
               
               
                 MPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYDVLEVQKEELDLSKD 
               
               
                 LMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRYEDTNIRINLD 
               
               
                 SNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVWIIDVDNVVRDVTIESDKIKKGD 
               
               
                 LIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLISGELKILMLNS 
               
               
                 NHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMDDSKPSFGYYSNNLK 
               
               
                 DVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNRKGNTNTSDSLMSFLESM 
               
               
                 NIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNYTLYVGNRQNMIVEPNYD 
               
               
                 LDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYINEKINVNINDL 
               
               
                 SIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFTPSYYEDGLIG 
               
               
                 YDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNL 
               
               
                   
               
               
                   C. difficile  Toxin B 1350-1852 
               
               
                 SEQ ID NO: 15 
               
               
                 NVVRDVTIESDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSK 
               
               
                 SYKLLISGELKILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKV 
               
               
                 YMDDSKPSFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNR 
               
               
                 KGNTNTSDSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNY 
               
               
                 TLYVGNRQNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIV 
               
               
                 LDANYINEKINVNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVS 
               
               
                 EIILSFTPSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNL 
               
               
                   
               
               
                 Protein domain from amino acid residues 27-401 of  C. difficile  CD2767 
               
               
                 SEQ ID NO: 16 
               
               
                 SNDKEMRAAWISTVYNLDWPKTKNNEAKQKKEYTDLLDKLKSVGINTAVVQVRPKSDALYKSNINPWSEYLTGT 
               
               
                 QGKDPGYDPLPFLIEEAHKRGMEFHAWFNPYRITMADESIDKLPANHPAKKNPSWVVKHGNKYYYDPGLPEVRK 
               
               
                 YIVDSIAEVVQNYDIDGVHFDDYFYPGVSFNDTATYQKYGKGQNKDDWRRENVNTLLRDVKASIKSIKPNVVFG 
               
               
                 VSPAGIWRNKSSDPTGSDTSGNESYVGTYADTRAWIKQGLIDYVVPQLYVVPIGLKAADYSKLVAVWVANEVKG 
               
               
                 TNVDLYIGQGIYKQGQSSYGGQNIAKEIVQQVTLNRKYSEIKGSMYFSAKDIANSTSIQKDLKSLYSSSEEPVT 
               
               
                 PPSNVKV 
               
               
                   
               
               
                 CD2767 (27-401) Toxin A (542-1850)-fusion protein in a expression contruct 
               
               
                 SEQ ID NO: 17 
               
               
                 MGSSHHHHHHSSGLVPRGSHMSNDKEMRAAWISTVYNLDWPKTKNNEAKQKKEYTDLLDKLKSVGINTAVVQVR 
               
               
                 PKSDALYKSNINPWSEYLTGTQGKDPGYDPLPFLIEEAHKRGMEFHAWFNPYRITMADESIDKLPANHPAKKNP 
               
               
                 SWVVKHGNKYYYDPGLPEVRKYIVDSIAEVVQNYDIDGVHFDDYFYPGVSFNDTATYQKYGKGQNKDNWRRENV 
               
               
                 NTLLRDVKASIKSIKPNVVFGVSPAGIWRNKSSDPTGSDTSGNESYVGTYADTRAWIKQGLIDYVVPQLYWPIG 
               
               
                 LKAADYSKLVAVWVANEVKGTNVDLYIGQGIYKQGQSSYGGQNIAKEIVQQVTLNRKYSEIKGSMYFSAKDIAN 
               
               
                 STSIQKDLKSLYSSSEEPVTPPSNVKVAAAPFTLSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHY 
               
               
                 IIQLQGDDISYEATCNLFSKNPKNSIIIQRNMNESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHG 
               
               
                 KDEFNTSEFARLSVDSLSNEISSFLDTIKLDISPKNVEVNLLGSNMFSYDFNVEETYPGKLLLSIMDKITSTLP 
               
               
                 DVNKNSITIGANQYEVRINSEGRKELLAHSGKWINKEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISE 
               
               
                 DIKTLLLDASVSPDTKFILNNLKLNIESSIGDYIYYEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLN 
               
               
                 NLDEKYLISFEDISKNNSTYSVRFINKSNGESVYVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQL 
               
               
                 DHTSQVNTLNAAFFIQSLIDYSSNKDVLNDLSTSVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPT 
               
               
                 ITEGIPIVSTILDGINLGAAIKELLDEHDPLLKKELEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAG 
               
               
                 ISAGIPSLVNNELILHDKATSVVNYFNHLSESKKYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAM 
               
               
                 EGGSGHTVTGNIDHFFSSPSISSHIPSLSIYSAIGIETENLDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLEN 
               
               
                 DGTRLLDSIRDLYPGKFYWRFYAFFDYAITTLKPVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGAG 
               
               
                 GTYSLLLSSYPISTNINLSKDDLWIFNIDNEVREISIENGTIKKGKLIKDVLSKIDINKNKLIIGNQTIDFSGD 
               
               
                 IDNKDRYIFLTCELDDKISLIIEINLVAKSYSLLLSGDKNYLISNLSNIIEKINTLGLDSKNIAYNYTDESNNK 
               
               
                 YFGAISKTSQKSIIHYKKDSKNILEFYNDSTLEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSIS 
               
               
                 LVSKNQVKVNGLYLNESVYSSYLDFVKNSDGHHNTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLG 
               
               
                 YVEFICDNNKNIDIYFGEWKTSSSKSTIFSGNGRNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIA 
               
               
                 PDLYTSLININTNYYSNEYYPEIIVLNPNTFHKKVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRI 
               
               
                 KGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFKSFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNS 
               
               
                 LFYFDPIEFNL 
               
               
                   
               
               
                 CD2767 (27-401) Toxin 6 (767-1852)-fusion protein in a expression contruct 
               
               
                 SEQ ID 18 
               
               
                 SNDKEMRAAWISTVYNLDWPKTKNNEAKQKKEYTDLLDKLKSVGINTAVVQVRPKSDALYKSNINPWSEYLTGT 
               
               
                 QGKDPGYDPLPFLIEEAHKRGMEFHAWFNPYRITMADESIDKLPANHPAKKNPSWVVKHGNKYYYDPGLPEVRK 
               
               
                 YIVDSIAEVVQNYDIDGVHFDDYFYPGVSFNDTATYQKYGKGQNKDDWRRENVNTLLRDVKASIKSIKPNVVFG 
               
               
                 VSPAGIWRNKSSDPTGSDTSGNESYVGTYADTRAWIKQGLIDYVVPQLYWPIGLKAADYSKLVAVWVANEVKGT 
               
               
                 NVDLYIGQGIYKQGQSSYGGQNIAKEIVQQVTLNRKYSEIKGSMYFSAKDIANSTSIQKDLKSLYSSSEEPVTP 
               
               
                 PSNVKVSIIKDISSKEYISFNPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDT 
               
               
                 QIVEERIEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRFINKETGES 
               
               
                 IFVETEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLNAAFFIQSLIEYNSSKESLSNLS 
               
               
                 VAMKVQVYAQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLL 
               
               
                 RQEIEAKIGIMAVNLTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKHVSLV 
               
               
                 ETEGVFTLLDDKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYD 
               
               
                 VLEVQKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTL 
               
               
                 KPRYEDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVWIIDVDNVV 
               
               
                 RDVTIESDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYK 
               
               
                 LLISGELKILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMD 
               
               
                 DSKPSFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNRKGN 
               
               
                 TNTSDSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNYTLY 
               
               
                 VGNRQNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDA 
               
               
                 NYINEKINVNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEII 
               
               
                 LSFTPSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNL 
               
               
                   
               
               
                 Toxin A (residues 542-1850; TxACPD) within the construct: 
               
               
                 6His-Thioredoxin-TxACPD 
               
               
                 SEQ ID 19 
               
               
                 MGSSHHHHHHSHMASDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNI 
               
               
                 DQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLARALVPRGSVTSLYKKAGSAAAPFTLS 
               
               
                 EDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDISYEATCNLFSKNPKNSIIIQRNMNES 
               
               
                 AKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEFNTSEFARLSVDSLSNEISSFLDTIKLDISPK 
               
               
                 NVEVNLLGSNMFSYDFNVEETYPGKLLLSIMDKITSTLPDVNKNSITIGANQYEVRINSEGRKELLAHSGKWIN 
               
               
                 KEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDYIY 
               
               
                 YEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSVRFINKSNGESVYV 
               
               
                 ETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTLNAAFFIQSLIDYSSNKDVLNDLSTSV 
               
               
                 KVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLKKE 
               
               
                 LEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESKKY 
               
               
                 GPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIPSLSIYSAIG 
               
               
                 IETENLDFSKKIMMLPNAPSRVFVWVETGAVPGLRSLENDGTRLLDSIRDLYPGKFYWRFYAFFDYAITTLKPV 
               
               
                 YEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGAGGTYSLLLSSYPISTNINLSKDDLWIFNIDNEVREI 
               
               
                 SIENGTIKKGKLIKDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSLLL 
               
               
                 SGDKNYLISNLSNIIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNDSTLEFN 
               
               
                 SKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNESVYSSYLDFVKNSDGHHNT 
               
               
                 SNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNKNIDIYFGEWKTSSSKSTIFSGNGRN 
               
               
                 VVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHKKV 
               
               
                 NINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFKSF 
               
               
                 NSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNL 
               
               
                   
               
               
                 Toxin B (residues 767-1852; TxBc) within the construct: 
               
               
                 6His-Thioredoxin-TxBc 
               
               
                 SEQ ID 20 
               
               
                 MGSSHHHHHHSHMASDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNI 
               
               
                 DQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLARALVPRGSVTSLYKKAGSAAAPFTSI 
               
               
                 IKDISSKEYISFNPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDTQIVEERIE 
               
               
                 EAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRFINKETGESIFVETEKT 
               
               
                 IFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLNAAFFIQSLIEYNSSKESLSNLSVAMKVQVY 
               
               
                 AQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLRQEIEAKI 
               
               
                 GIMAVNLTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKHVSLVETEGVFTL 
               
               
                 LDDKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYDVLEVQKEE 
               
               
                 LDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRYEDTN 
               
               
                 IRINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVWIIDVDNVVRDVTIESD 
               
               
                 KIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLISGELK 
               
               
                 ILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMDDSKPSFGY 
               
               
                 YSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNRKGNTNTSDSLM 
               
               
                 SFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNYTLYVGNRQNMI 
               
               
                 VEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYINEKIN 
               
               
                 VNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFTPSYY 
               
               
                 EDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNLKGGRADPAFLYKVVSAWSHPQFEK