AU2016203241B2

AU2016203241B2 - Immunogenic composition

Info

Publication number: AU2016203241B2
Application number: AU2016203241A
Authority: AU
Inventors: Cindy Castado
Original assignee: GlaxoSmithKline Biologicals SA
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 2011-05-27
Filing date: 2016-05-18
Publication date: 2018-05-10
Anticipated expiration: 2032-05-25
Also published as: AU2016203241A1

Abstract

The present invention relates to fusion proteins comprising fragments from toxin A and/or toxin B of Clostridium difficile, in particular the invention relates to proteins comprising a first fragment and a second fragment, wherein (v) the first fragment is a toxin A repeating domain fragment; (vi) the second fragment is a toxin B repeating domain fragment; (vii) the first fragment has a first proximal end; (viii) the second fragment has a second proximal end; and wherein the first fragment and the second fragment are adjacent to one another and wherein the polypeptide elicits antibodies that neutralize toxin A or toxin B or both. The invention further relates to compositions comprising fragments or variants of SEQ ID NO:1 0, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35.

Description

The present invention relates to fusion proteins comprising fragments from toxin A and/or toxin B of Clostridium difficile, in particular the invention relates to proteins comprising a first fragment and a second fragment, wherein (v) the first fragment is a toxin A repeating domain fragment; (vi) the second fragment is a toxin B repeating domain fragment; (vii) the first fragment has a first proximal end; (viii) the second fragment has a second proximal end; and wherein the first fragment and the second fragment are adjacent to one another and wherein the polypeptide elicits antibodies that neutralize toxin A or toxin B or both. The invention further relates to compositions comprising fragments or variants of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,

SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NQ:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35.

2016203241 16 Apr 2018

IMMUNOGENIC COMPOSITION

TECHNICAL FIELD [001] The present application is a divisional application from Australian patent application number 2012264902, the entire disclosure of which is incorporated herein by reference.

[001a] The present invention relates to antigens from Clostridium difficile. In particular the invention relates to recombinant protein antigens comprising fragments of toxin A and/or toxin B. The invention additionally relates to immunogenic compositions or vaccines comprising these antigens, and the use of the vaccines and immunogenic compositions of the invention in prophylaxis or therapy. The invention also relates to methods of immunising using the compositions of the invention, and the use of the compositions of the invention in the manufacture of a medicament.

BACKGROUND [002] C.difficile is the most important cause of nosocomial intestinal infections and is the major cause of pseudomembranous colitis in humans (Bartlett etalAm. J. Clin. Nutr. 11 suppl:2521-6 (1980)). The overall associated mortality rate for individuals infected with C.difficile was calculated to be 5.99% within 3 months of diagnosis, with higher mortality associated with advanced age, being 13.5% in patients over 80 years (karas et al Journal of

Infection 561:1-9 (2010)). The current treatment for C.difficile infection is the administration of antibiotics (metronidazole and vancomycin), however there has been evidence of strains which are resistant to these antibiotics (Shah etal., Expert Rev. Anti Infect. Ther. 8(5), 555564 (2010)). Accordingly there is a need for immunogenic compositions capable of inducing antibodies to, and /or a protective immune response to, C. difficile.

BRIEF SUMMARY [003] The enterotoxicity of C.difficile is primarily due to the action of two toxins, toxin A and toxin B. These are both potent cytotoxins (Lyerly et al Current Microbiology 21:29-32 (1990). The C-terminal domains of toxin A and toxin B comprise repeating units, for example the C30 terminal domain of toxin A is made up of contiguous repeating units (Dove et al Infect.

Immun. 58:480-499 (1990)), for this reason the C-terminal domain may be referred to as the ‘repeating domain’. These repeat portions can be separated further into short repeats (SRs) and long repeats (LRs) as described in Ho et a/(PNAS 102:18373-18378 (2005)).

[004] The structure of a 127-aa fragment from the C terminus of the toxin A repeat domain has been determined (Ho et al PNAS 102:18373-18378 (2005)). This fragment formed a βsolenoid-like fold, composed predominantly of β strands with a low proportion of a helices.

[005] It has been demonstrated that fragments of toxin A, in particular fragments of the Cterminal domain, can lead to a protective immune response in hamsters (Lyerly et al Current Microbiology 21:29-32 (1990)), WO96/12802 and WOOO/61762.

[006] There is known to be difficulty involved in designing fusion proteins which fold correctly during expression. The polypeptides of the present invention are fusion proteins in which the native β solenoid-like structure is maintained, and which are seen to provide an immune response against both toxin A and toxin B in mice.

[006a] In a first aspect of the present invention, there is provided a polypeptide comprising a first fragment and a second fragment, wherein (i) the first fragment is a C. difficile toxin A repeating domain fragment;

(ii) the second fragment is a C. difficile toxin B repeating domain fragment;

2016203241 16 Apr 2018 (iii) the first fragment has a first proximal end;

(iv) the second fragment has a second proximal end; and wherein the first proximal end is within a short repeat and the second proximal end is within a short repeat; and wherein the first fragment and the second fragment are adjacent to one another and wherein the polypeptide elicits antibodies that neutralize toxin A or toxin B or both. [007] In a first aspect of the invention there may be provided a polypeptide comprising a first fragment and a second fragment, wherein (i) the first fragment is a toxin A repeating domain fragment;

(ii) the second fragment is a toxin B repeating domain fragment;

(iii) the first fragment has a first proximal end;

(iv) the second fragment has a second proximal end; and polypeptide elicits antibodies that neutralize toxin A or toxin B or both [008] In a second aspect of the invention there may be provided a polypeptide comprising:

(i) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,

SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35;

(ii) A variant having at least 90%, 95%, 98%, 99% or 100% similarity to SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35; or (iii) A fragment of at least 250, 280, 300, 350, 380, 400, 430, 450, 480, 500, 530, 550, 580, or 600 amino acids of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35.

[009] In a third aspect of the invention there may be provided a polynucleotide encoding a polypeptide of the invention.

[010] In a fourth aspect of the invention there may be provided a vector comprising a polynucleotide of the invention linked to an inducible promoter.

[011] In a fifth aspect of the invention there may be provided a host cell comprising a vector of the invention or a polynucleotide of the invention.

[012] In a sixth aspect of the invention there may be provided an immunogenic composition comprising a polypeptide of the invention and a pharmaceutically acceptable excipient.

[013] In a seventh aspect of the invention there may be provided a vaccine comprising an immunogenic composition of the invention.

[014] In an eighth aspect of the invention there may be provided a use of an immunogenic composition of the invention or a vaccine of the invention in the treatment or prevention of C.difficile disease.

[015] In a ninth aspect of the invention there may be provided a use of an immunogenic composition of the invention or a vaccine of the invention in the preparation of a medicament for the prevention or treatment of C.difficile disease.

2016203241 16 Apr 2018 [016] In a tenth aspect of the invention there may be provided a method of preventing or treating C.difficile disease comprising administering an immunogenic composition of the invention or a vaccine of the invention to a patient.

BRIEF DESCRIPTION OF THE DRAWINGS [017] FIG. 1 - Sequence listings of polypeptides of the invention.

[018] FIG. 2 - Pictorial representation of the C-terminal domains of ToxA and ToxB, with the SR repeats depicted as white boxes and the LR boxes depicted as black boxes.

2a

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2016203241 18 May 2016 [019] FIG. 3 - Pictorial representation of a junction between the third SR VIII of ToxA and the fourth SR II of Tox B used in Fusion 1.

[020] FIG.4 - Pictorial representation of a junction between the second SR VIII of ToxA and the third SR II of Tox B used in Fusion 2.

[021] FIG.5 - Pictorial representation of a junction between LRVII of ToxA and LRII of ToxB used in Fusion 3 (containing only part of LRVII of ToxA and part of LR II of ToxB).

[022] FIG.6 - Pictorial representation of a junction between the second SR VIII of ToxA and the third SR I of ToxB used in Fusion 4.

[023] FIG.7 - Pictorial representation of a junction comprising a glycine linker between the last residue of the ToxA protein sequence and the beginning of the fourth SRII of ToxB used in Fusion

5.

[024] FIG.8 - Graphs describing the distribution of C.difficile ToxA-ToxB fusions 1-5 as determined by sedimentation velocity analytical ultracentrifugation. Panel a) describes the distribution of Fusion 1, panel b) describes the distribution of Fusion 2, panel c) describes the 5 distribution of Fusion 3, panel d) describes the distribution of Fusion 4 and panel e) describes the distribution of Fusion 5.

[025] FIG.9 - Graph describing the Far-UV spectrum of Fusions, 2, 3, 4, and 5 measured using circular dichroism. The spectrum for fusion 2 is represented by a line with the points depicted as small squares, the spectrum for fusion 3 is represented by a line with the points depicted as small diamond shapes, fusion 4 is represented by a line with the points depicted as circles, and fusion 5 is represented by a line with the points depicted as cross shapes.

[026] FIG.10 - Graph describing the near-UV spectrum of Fusions 2, 3, 4, and 5 measured using circular dichroism. The spectrum for fusion 2 is represented by a line with the points depicted as cross shapes, the spectrum for fusion 3 is represented by a line with the points depicted as circles, the spectrum for fusion 4 is represented by a line with the points depicted as triangles, and the spectrum for fusion 5 is represented by a line with the points depicted as small diamond shapes.

[027] FIG.11 - Graph showing anti-ToxA immunogenicity in mice immunised with a fragment of the C-terminus of toxin A (aa 2387-2706), a fragment of the C-terminus of toxin B (aa 1 /SOSO 2360), or fusions 1,2, 3, 4 or 5.

[028] FIG.12 - Graph showing hemagglutination inhibition in mice immunised with a fragment of the C-terminus of toxin A (aa 2387-2706), a fragment of the C-terminus of toxin B (aa 17502360), or fusions 1,2, 3, 4 or 5.

[029] FIG.13 - Graph showing anti-ToxB immunogenicity in mice immunised with a fragment 35 of the C-terminus of toxin A (aa 2387-2706), a fragment of the C-terminus of toxin B (aa 17502360), or fusions 1,2, 3, 4 or 5.

[030] FIG.14 - Cyotoxicity inhibition titres from mice immunised with a fragment of the Cterminus of toxin A (aa 2387-2706), a fragment of the C-terminus of toxin B (aa 1750-2360), or fusions 1,2, 3, 4 or 5.

[031] FIG.15 - Graphs describing the distribution of C.difficile ToxA-ToxB fusions F52New,

F54Gly, F54New and F5ToxB as determined by sedimentation velocity analytical ultracentrifugation. Panel a) describes the distribution of F52New, panel b) describes the

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2016203241 18 May 2016 distribution of F54Gly, panel c) describes the distribution of F54New and panel d) describes the distribution of F5ToxB.

[032] FIG.16 - Graph describing the Far-UV spectrum of fusions F52New, F54Gly, F54New and F5ToxB measured using circular dichroism. The spectrum for F52New is represented by a line with the points depicted as double crosses, the spectrum for F54Gly is represented by a line with the points depicted as triangles, F54New is represented by a line with the points depicted as squares, and F5ToxB is represented by a line with the points depicted as cross shapes.

[033] FIG.17 - Graph describing the Near-UV spectrum of fusions F52New, F54Gly, F54New and F5ToxB measured using circular dichroism. The spectrum for F52New is represented by a line with the points depicted as double crosses, the spectrum for F54Gly is represented by a line with the points depicted as triangles, F54New is represented by a line with the points depicted as squares, and F5ToxB is represented by a line with the points depicted as cross shapes.

[034] FIG.18 - Graph showing anti-ToxA ELISA results for mice immunised with the F2, F52New, F54Gly, G54New or F5ToxB fusions.

[035] FIG.19 - Graph showing anti-ToxB ELISA results for mice immunised with the F2, F52New, F54Gly, F54Newor F5ToxB fusions.

[036] FIG.20 - Graph showing hemagglutination inhibition in mice immunised with the F2, F52New, F54Gly, F54Newor F5ToxB fusions.

[037] FIG.21 - Graph showing cytotoxicity titres in HT29 cells from mice immunised with the F2, F52New, F54Gly, F54Newor F5ToxB fusions.

[038] FIG.22 - Graph showing cytotoxicity titres in IMR90 cells from mice immunised with the F2, F52New, F54Gly, F54Newor F5ToxB fusions.

DETAILED DESCRIPTION

POLYPEPTIDES [039] The invention relates to a polypeptide comprising a first fragment and a second fragment, wherein (i) the first fragment is a toxin A repeating domain fragment;

(ii) the second fragment is a toxin B repeating domain fragment;

(iii) the first fragment has a first proximal end;

(iv) the second fragment has a second proximal end; and wherein the first fragment and the second fragment are adjacent to one another and wherein the polypeptide elicits antibodies that neutralize toxin A or toxin B or both.

[040] The term polypeptide refers to a contiguous sequence of amino acids.

[041] The term ‘toxin A repeating domain’ refers to the C-terminal domain of the toxin A protein from C.difficile, comprising repeated sequences. This domain refers to amino acids 18322710 of toxin A from strain VPI10463 (ATCC43255) and their equivalents in a different strain, the sequence of amino acids 1832-2710 from strain VPI10463 (ATCC43255) corresponds to amino acids 1832-2710 of SEQ ID NO:1.

[042] The term ‘toxin B repeating domain’ refers to the C-terminal domain of the toxin B protein from C.difficile. This domain refers to amino acids 1834-2366 from strain VP110463

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2016203241 18 May 2016 (ATCC43255) and their equivalents in a different strain, the sequence of amino acids 1834-2366 from strain VPI10463 (ATCC43255) corresponds to amino acids 1834-2366 of SEQ ID NO:2.

[043] The C.difficile toxins A and B are conserved proteins, however the sequence differs a small amount between strains, moreover the amino acid sequence for toxins A and B in different 5 strains may differ in number of amino acids.

[044] The invention therefore the term toxin A repeating domain and/or toxin B repeating domain to refer to a sequence which is a variant with 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1832-2710 of SEQ ID NO:1 or a variant with 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1834-2366 of SEQ ID NO:2. In one embodiment a 0 ‘variant’ is a polypeptide that varies from the referent polypeptides by conservative amino acid substitutions, whereby a residue is substituted by another with the same physico-chemical properties. Typically such substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin, and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. In one embodiment a ‘fragment’ is a polypeptide 5 which comprises a contiguous portion of at least 250 amino acids of a polypeptide.

[045] Furthermore the amino acid numbering may differ between the C-terminal domains of toxin A (or toxin B) from one strain and toxin A (or toxin B) from another strain. For this reason the term ‘equivalents in a different strain’ refers to amino acids which correspond to those of a reference strain (e.g., C. difficile VPI10463), but which are found in a toxin from a different strain and which may thus be numbered differently. A region of ‘equivalent’ amino acids may be determined by aligning the sequences of the toxins from the different strains. The amino acids numbers provided throughout refer to those of strain VPI10463.

[046] The term ‘fragment’ of a polypeptide or protein refers to a contiguous portion of at least 100, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600 amino acids from that polypeptide or protein. The term ‘first fragment’ refers to a contiguous portion of at least 100,

250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600 amino acids of the toxin A repeating domain. The term ‘second fragment’ refers to a contiguous portion of at least 100, 200, 230, 250, 280, 300, 350, 400, 450 or 500 amino acids of the toxin B repeating domain.

[047] The term ‘first proximal end’ refers to the end of the first fragment (Tox A fragment) which is covalently linked to the second fragment (ToxB fragment) or covalently linked to a linker sequence between the first and second fragment and is closest to the second fragment in primary structure. The term ‘second proximal end’ refers to the end of the second fragment which is covalently linked to the first fragment (ToxA fragment) or covalently linked to a linker sequence between the first and second fragment and is closest to the first fragment in primary structure.

[048] The polypeptide may be part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.

[049] The fragments may be positioned such that the N-terminus of the first fragment is adjacent to the C-terminus of the second fragment, alternatively the C-terminus of the first fragment may be adjacent to the N-terminus of the second fragment, or the C-terminus of the first fragment may be adjacent to the C-terminus of the second fragment, or the N-terminus of the first fragment may be adjacent to the N-terminus of the second fragment.

[050] The word ‘adjacent’ means separated by less than or exactly 20, 15, 10, 8, 5, 2, 1 or 0 amino acids in the primary structure.

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2016203241 18 May 2016 [051] The polypeptide of the invention elicits antibodies that neutralise toxin A or toxin B or both. In one embodiment the polypeptide elicits antibodies that neutralise toxin A. In a further embodiment the polypeptide elicits antibodies that neutralise toxin B. In a further embodiment the polypeptide elicits antibodies that neutralise toxin A and toxin B.

[052] Whether a polypeptide elicits antibodies against a toxin can be measured by immunising mice with an immunogenic composition comprising the polypeptide, collecting sera and analysing the anti-toxin titres of the sera using by ELISA. The sera should be compared to a reference sample obtained from mice which have not been immunised. An example of this technique can be found in example 6. The polypeptide of the invention elicits antibodies that neutralise toxin A if the sera against the polypeptide gives an ELISA readout more than 10%, 20%, 30%, 50%, 70%,

80%, 90%, or 100% higher than the reference sample.

[053] In a further embodiment the polypeptide of the invention elicits a protective immune response in a mammalian host against strains of C.difficile. In one embodiment the mammalian host is selected from the group consisting of mouse, rabbit, guinea pig, non-human primate, monkey and human. In one embodiment the mammalian host is a mouse. In a further embodiment the mammalian host is a human.

[054] Whether a polypeptide elicits a protective immune response in a mammalian host against strains of C.difficile can be determined using a challenge assay. In such an assay the mammalian host is vaccinated with the polypeptide and challenged by exposure to C.difficile, the time which the mammal survives after challenge is compared with the time which a reference mammal that has not been immunised with the polypeptide survives. A polypeptide elicits a protective immune response if a mammal immunised with the polypeptide survives at least 10%, 20%, 30%, 50%, 80%, 80%, 90%, or 100% longer than a reference mammal which has not been immunised after challenge with C.difficile.

[055] The native structure of the C-terminal domains from toxins A and B consist of an extended β solenoid-like structure. This structure consists of primarily β sheet structures, with a minority of a helical structures as seen in Ho et al (PNAS 102:18373-18378 (2005)). The secondary structures present can be determined using circular dichroism. For example measuring the shape and the magnitude of the CD spectra in the far-UV region (190-250nm) and comparing the results with those of known structures. This can be carried out using an optical path of 0.01cm from 178 to 250nm, with a 1nm resolution and bandwidth on a Jasco J-720 spectropolarimeter, for example as seen in example 5 below.

[056] In one embodiment the first fragment comprises less than 25%, 23%, 20%, 18%, 15%, 10%, or 7% alpha helical secondary structure. In one embodiment the second fragment comprises less than 28%, 25%, 23%, 20%, 18%, 15%, 10%, or 7% alpha helical secondary structure. In a further embodiment both the first fragment and the second fragment comprise less than 28%, 25%, 23%, 20%, 18%, 15%, 10%, or 7% alpha helical secondary structure.

[057] In one embodiment the first fragment comprises more than 20%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, or 42% beta sheet structure. In one embodiment the second fragment comprises more than 20%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, or 42% beta sheet structure.

In a further embodiment both the first fragment and the second fragment comprises more than 20%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, or 42% beta sheet structure.

[058] Figure 2 depicts the organisation of the C-terminal domains of ToxA and ToxB. The Cterminal domain of toxin A is made up of 8 repeat portions (designated repeat portion I, repeat portion II, repeat portion III, repeat portion IV, repeat portion V, repeat portion VI, repeat portion

VII and repeat portion VIII) each of these repeat portions can be further divided into short repeats (SRs) which are depicted as white boxes in figure 2 and long repeats (LRs) which are depicted as black boxes in figure 2 (except for Tox A repeat portion VIII which does not have a long repeat). Each of the long repeats has some structural and sequence similarity to the other long repeats.

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Similarly the short repeats have some sequence and structural similarity to one another. The Cterminal domain of toxin B is made up of 5 repeat portions subdivided into SRs and LRs. Each repeat portion contains one LR and between 2 and 5 SRs (except for Tox B repeat portion V which does not have a long repeat). For the purposes of the disclosure the phrase ‘a repeat portion’ refers to one of the eight repeat portions of ToxA (designated I, II, III, IV, V, VI, VII, and

VIII ) or one of the five repeat portions of ToxB (designated I, II, III, IV or VI). As used herein the term ‘first repeat portion’ refers to a repeat portion (or partial repeat portion) from the toxin A repeating domain. The term ‘second repeat portion’ refers to a repeat portion (or partial repeat portion) from the toxin B repeating domain . For the purposes of the disclosure the term ‘long repeat’ refers to one of the LR domains depicted as black boxes in Figure 2. For the purposes of the disclosure the term ‘short repeat’ refers to one of the SR domains depicted as white boxes in Figure 2.

[059] Thus for example, repeat portion I of ToxA contains three SRs and one LR, which can be referred to as the first SRI of ToxA, the second SRI of ToxA, the third SRI of ToxA and the LRI of ToxA, respectively.

[060] The first proximal end is considered to be within a ‘repeat portion’ if the first fragment ends in an amino acid that is within that repeat portion (i.e, the first proximal end contains only part of the repeat portion sequence). Similarly the second proximal end is considered to be within a ‘repeat portion’ if the second fragment ends in an amino acid that is within that repeat portion.

For example the first proximal end is within ‘a repeat portion I of ToxA’ if the first fragment ends with any one of amino acids 1832-1924 (inclusive) of VPI10463 or their equivalent in another strain. The first proximal end is not within a short repeat-long repeat-short repeat portion if the first fragment ends with an amino acid that is not within that short-repeat-long repeat-short repeat portion.

[061] The amino acid positions of each domain has been defined for toxin A and toxin B from strain VPI10463 (ATCC43255). These are as follows

Name	Start position	End position
ToxA_l	SR1	1832	1852
	SR2	1853	1873
	SR3	1874	1893
	LR	1894	1924
ToxA_ll	SR1	1925	1944
	SR2	1945	1965
	SR3	1966	1986
	SR4	1987	2007
	SR5	2008	2027
	LR	2028	2058
ToxA_lll	SR1	2059	2078

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	SR2	2079	2099
	SR3	2100	2120
	SR4	2121	2141
	SR5	2142	2161
	LR	2162	2192
ToxA_IV	SR1	2193	2212
	SR2	2213	2233
	SR3	2234	2253
	SR4	2254	2275
	LR	2276	2306
ToxA_V	SR1	2307	2326
	SR2	2327	2347
	SR3	2348	2368
	SR4	2369	2389
	SR5	2390	2409
	LR	2410	2440
ToxA_VI	SR1	2441	2460
	SR2	2461	2481
	SR3	2482	2502
	SR4	2503	2522
	LR	2523	2553
ToxA_VII	SR1	2554	2573
	SR2	2574	2594
	SR3	2595	2613
	LR	2614	2644
ToxA_VIII	SR1	2645	2664
	SR2	2665	2686

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	SR3	2687	2710
ToxB_l	SR1	1834	1854
	SR2	1855	1876
	SR3	1877	1896
	LR	1897	1926
ToxB_ll	SR1	1927	1946
	SR2	1947	1967
	SR3	1968	1987
	SR4	1988	2007
	SR5	2008	2027
	LR	2028	2057
ToxB_lll	SR1	2058	2078
	SR2	2079	2099
	SR3	2100	2119
	SR4	2120	2139
	SR5	2140	2159
	LR	2160	2189
ToxB_IV	SR1	2190	2212
	SR2	2213	2233
	SR3	2234	2253
	SR4	2254	2273
	SR5	2274	2293
	LR	2294	2323
ToxB_V	SR1	2324	2343
	SR2	2344	2366

Table 1 [062] For this reason the term ‘repeat portion’ may refer to amino acids 1832-1924, 19252058, 2059-2192, 2193-2306, 2307-2440, 2441-2553, 2554-2644 or 2645-2710 of toxin A (SEQ

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ID NO:1), or amino acids 1834-1926, 1927-2057, 2058-2189, 2190-2323 or 2324-2366 of toxin B (SEQ ID NO:2) or their equivalents in a different strain of C.difficile.

[063] For this reason the term ‘short repeat’ may refer to amino acids 1832-1852, 1853-1873, 1874-1893, 1925-1944 1945-1965, 1966-1986, 1987-2007, 2008-2027, 2059-2078, 2079-2099,

2100-2120, 2121-2141,2142-2161, 2193-2212, 2213-2233, 2234-2253, 2254-2275, 2307-2326,

2327-2347, 2348-2368, 2369-2389, 2390-2409, 2441-2460, 2461-2481, 2482-2502, 2503-2522, 2554-2573, 2574-2594, 2595-2613, 2645-2664, 2665-2686 or 2687-2710 of toxin A (SEQ ID NO:1) or amino acids 1834-1854, 1855-1876, 1877-1896, 1927-1946, 1947-1967, 1968-1987, 1988-2007, 2008-2027, 2058-2078, 2079-2099, 2100-2119, 2120-2139, 2140-2159, 2190-2212,

2213-2233, 2234-2253, 2254-2273, 2274-2293, 2324-2343 or 2344-2366 of toxin B (SEQ ID

NO:2) or their equivalents in a different strain of C.difficile.

[064] Similarly the term ‘long repeat’ may refer to amino acids 1894-1924, 2028-2058, 21622192, 2276-2306, 2410-2440, 2523-2553 or 2614-2644 of toxin A (SEQ ID NO:1)or amino acids 1897-1926, 2028-2057, 2160-2189 or 2294-2323 of toxin B (SEQ ID NO:2) or their equivalents in 5 a different strain of C.difficile.

[065] Similarly the term ‘short repeat-long repeat-short repeat portion’ may refer to amino acids 1874-1944, 2008-2078, 2142-2212, 2254-2326, 2390-2460, 2503-2573, or 2595-2664 of toxin A (SEQ ID NO: 1) or amino acids 1877-1946, 2008-2078, 2140-2212 or 2274-2343 of toxin B (SEQ ID NO:2) or their equivalents in a different strain of C.difficile. The term ‘does not disrupt a short repeat-long repeat-short repeat portion’ means that the proximal end is in a region which does not disrupt the structure of the short-repeat-long repeat-short repeat portion, in general this means that the proximal end is not within a long repeat and not within the short repeats making up a short repeat-long repeat-short repeat portion, except the proximal end may be in the region of 1, 2, 3, 4, 5 or 6 amino acids of the short repeat which are furthest away from the long repeat in sequence. In an embodiment the term ‘does not disrupt a short-repeat-long repeat-short repeat portion’ means that the proximal end is not within the short repeat-long repeat-short repeat portion.

[066] In one embodiment the first proximal end is within a short repeat. In one embodiment the second proximal end is within a short repeat. In one embodiment the first proximal end and the second proximal end are within a short repeat. In one embodiment the first proximal end does not disrupt a short-repeat long repeat-short repeat portion. In one embodiment the second proximal end does not disrupt a short-repeat long repeat-short repeat portion. In one embodiment the first proximal end and the second proximal end do not disrupt a short-repeat long repeat-short repeat portion.

[067] In one embodiment the first proximal end is not within amino acids 1878-1940, 21462208, 2012-2074, 2258-2322, 2394-2456, 2507-2569, 2599-2660 or 2593-2660 of toxin A (SEQ ID NO:1) or their equivalents in a different strain of C.difficile. In a second embodiment the second proximal end is not within amino acids 1881-1942, 2012-2074, 2144-2208, or 2278-2339 of toxin B (SEQ ID NO:2) or their equivalents in a different strain of C.difficile. In a further embodiment the first proximal end is not within amino acids 1878-1940, 2146-2208, 2012-2074,

2258-2322, 2394-2456, 2507-2569, 2599-2660 or 2593-2660 of toxin A (SEQ ID NO:1) or their equivalents in a different strain of C.difficile and the second proximal end is not within amino acids 1881-1942, 2012-2074, 2144-2208, or 2278-2339 of toxin B (SEQ ID NO:2) or their equivalents in a different strain of C.difficile.

[068] In one embodiment the first proximal end is within repeat portion V (amino acids 23072440 of SEQ ID NO:1 or their equivalent in a different strain), VI (amino acids 2441-2553 of SEQ ID NO:1 or their equivalent in a different strain), VII (amino acids 2554-2644 of SEQ ID NO:1or their equivalent in a different strain) or VIII (amino acids 2645-2710 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A. In a further embodiment the first proximal end is within repeat portion VII (amino acids 2554-2644 of SEQ ID NO:1 or their equivalent in a different strain)

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2016203241 18 May 2016 of toxin A. In a further embodiment the first proximal end is within repeat portion VIII (amino acids 2645-2710 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A.

[069] In one embodiment the second proximal end is within repeat portion I (amino acids 1834-1926 of SEQ ID NO:2 or their equivalent in a different strain), II (amino acids 1927-2057 of 5 SEQ ID NO:2 or their equivalent in a different strain), or iii (amino acids 2058-2189 of SEQ ID

NO:2 or their equivalent in a different strain) of toxin B. In a further embodiment the second proximal end is within repeat portion II (amino acids 1927-2057 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B. In a further embodiment the second proximal end is within repeat portion I (amino acids 1834-1926 of SEQ ID NO:2 or their equivalent in a different 0 strain) of toxin B.

[070] In one embodiment the first proximal end is within repeat portion VIII (amino acids 26452710 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A and the second proximal end is within repeat portion I (amino acids 1834-1926 of SEQ ID NO:2 or their equivalent in a different strain)of toxin B. In a further embodiment the first proximal end is within repeat portion 5 VIII (amino acids 2645-2710 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A and the second proximal end is within repeat portion II (amino acids 1927-2057 of SEQ ID NO:2 or their equivalent in a different strain)of toxin B. In a further embodiment the first proximal end is within repeat portion VII (amino acids 2554-2644 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A and the second proximal end is within repeat portion I (amino acids 1834-1926 0 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B. In a further embodiment the first proximal end is within repeat portion VII (amino acids 2554-2644 of SEQ ID NO:1 or their equivalent in a different strain) of toxin A and the second proximal end is within repeat portion II (amino acids 1927-2057 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B. In a further embodiment the first proximal end is within repeat portion VI amino acids 2441-2553 of 5 SEQ ID NO:1 or their equivalent in a different strain) and the second proximal end is within repeat portion I (amino acids 1834-1926 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B. In a further embodiment the first proximal end is within repeat portion VI amino acids 24412553 of SEQ ID NO:1 or their equivalent in a different strain) and the second proximal end is within repeat portion II (amino acids 1927-2057 of SEQ ID NO:2 or their equivalent in a different 0 strain) of toxin B. In a further embodiment the first proximal end is within repeat portion V (amino acids 2307-2440 of SEQ ID NO:1 or their equivalent in a different strain) and the second proximal end is within repeat portion I (amino acids 1834-1926 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B. In a further embodiment the first proximal end is within repeat portion V (amino acids 2307-2440 of SEQ ID NO:1 or their equivalent in a different strain) and the second 5 proximal end is within repeat portion II (amino acids 1927-2057 of SEQ ID NO:2 or their equivalent in a different strain) of toxin B.

[071] In one embodiment the first proximal end is within amino acids 2690-2710, or 26952710, or 2700-2710 of SEQ ID NO:1 or their equivalent in a different strain. In a further embodiment the first proximal end is within amino acids 2670-2700, or 2675-2695, or 2680-2690 of SEQ ID NO:1 or their equivalent in a different strain. In one embodiment the second proximal end is within amino acids 1860-1878 of Toxin B or their equivalents in a different strain. In one embodiment the second proximal end is within amino acids 1950-1980, 1955-1975 or 1960-1970 of SEQ ID NO:2 or their equivalent in a different strain. In a further embodiment the second proximal end is within amino acids 1978-2008, 1983-2003 or 1988-1998 of SEQ ID NO:2 or their equivalent in a different strain. In a further embodiment the second proximal end is within amino acids 1860-1878, 1854-1876, 1857-1887, 1862-1882, or 1867-1877 ofSEQIDNO:2 or their equivalent in a different strain.

[072] In one embodiment the first fragment consists of an entire toxin A repeating domain (amino acids 1832-2710). In one embodiment the second fragment consists of an entire toxin B repeating domain (amino acids 1833-2366).

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2016203241 18 May 2016 [073] In one embodiment the first proximal end is within short repeat 3 of repeat portion VIII of toxin A (amino acids 2687-2710 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 4 of repeat portion II of toxin B (amino acids 19882007 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first 5 proximal end is within short repeat 3 of repeat portion VIII of toxin A (amino acids 2687-2710 of

SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 3 of repeat portion II of toxin B (amino acids 1968-1987 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 3 of repeat portion VIII of toxin A (amino acids 2687-2710 of SEQ ID NO:1 or their equivalents in a 0 different strain) and the second proximal end is within short repeat 2 of repeat portion II of toxin B (amino acids 1947-1967 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 3 of repeat portion VIII of toxin A (amino acids 2687-2710 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 3 of repeat portion I of toxin B (amino acids 1877-1896 of SEQ 5 ID NO:2 or their equivalents in a different strain. In one embodiment the first proximal end is within short repeat 3 of repeat portion VIII of toxin A (amino acids 2687-2710 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion I of toxin B (amino acids 1855-1876 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 3 of repeat portion 0 VIII of toxin A (amino acids 2687-2710 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 1 of repeat portion I of toxin B (amino acids 1834-1854 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VIII of toxin A (amino acids 2665-2686 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short 5 repeat 4 of repeat portion II of toxin B (amino acids 1988-2007 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VIII of toxin A (amino acids 2665-2686 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 3 of repeat portion II of toxin B (amino acids 1968-1987 of SEQ ID NO:2 or their equivalents in a different strain). In one 0 embodiment the first proximal end is within short repeat 2 of repeat portion VIII of toxin A (amino acids 2665-2686 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion II of toxin B (amino acids 1947-1967 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VIII or toxin A (amino acids 2665-2686 of SEQ ID NO:1 or 5 their equivalents in a different strain) and the second proximal end is within short repeat 3 of repeat portion I of Toxin B (amino acids 1877-1896 of toxin B or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portionVI 11 of toxin A (amino acids 2665-2686 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion I of toxin B (amino acids 1855-1876 40 of SEQ ID NO:2 or their equivalents in a different strain. In one embodiment the first proximal end is within short repeat 2 of repeat portion VIII of toxin A (amino acids 2665-2686 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 1 of repeat portion I of toxin B (amino acids 1834-1854 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion 45 vii of toxin A (amino acids 2574-2594 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 4 of repeat portion II of toxin B (amino acids 1988-2007 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion vii of toxin A (amino acids 2574-2594 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short 50 repeat 3 of repeat portion II of toxin B (amino acids 1668-1987 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion vii of toxin A (amino acids 2574-2594 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion II of toxin B (amino acids 1947-1967 of SEQ ID NO:2 or their equivalents in a different strain). In one 55 embodiment the first proximal end is within short repeat 2 of repeat portion VII of toxin A (amino acids 2574-2594 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion I of toxin B (amino acids 1855-1876 of SEQ

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ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VII of toxin A (amino acids 2574-2594 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 1 of repeat portion I of toxin B (amino acids 1834-1854 of SEQ ID NO:2 or their equivalents in a 5 different strain). In one embodiment the first proximal end is within short repeat 3 of repeat portion

VI of toxin A (amino acids 2482-2502 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 4 of repeat portion II of toxin B (amino acids 1988-2007 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 3 of repeat portion VI of toxin A (amino acids 2482-2502 of 0 SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 3 of repeat portion II of toxin B (amino acids 1968-1987 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 3 of repeat portion VI of toxin A (amino acids 2482-2502 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion II of toxin B 5 (amino acids 1947-1967 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 3 of repeat portion VI of toxin A (amino acids 2482-2502 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion I of toxin B (amino acids 1855-1876 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is 0 within short repeat 3 of repeat portion VI of toxin A (amino acids 2482-2502 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 1 of repeat portion I of toxin B (amino acids 1834-1854 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VI of toxin A (amino acids 2461-2481 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 4 of repeat portion II of toxin B (amino acids

1988-2007 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VI of toxin A (amino acids 2461-2481 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 3 of repeat portion II of toxin B (amino acids 1968-1987 of SEQ ID NO:2 or their 0 equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VI of toxin A (amino acids 2461-2481 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion II of toxin B (amino acids 1947-1967 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VI of toxin A (amino 5 acids 2461-2481 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 2 of repeat portion I of toxin B (amino acids 1855-1876 of SEQ ID NO:2 or their equivalents in a different strain). In one embodiment the first proximal end is within short repeat 2 of repeat portion VI of toxin A (amino acids 2461-2481 of SEQ ID NO:1 or their equivalents in a different strain) and the second proximal end is within short repeat 1 of 40 repeat portion I of toxin B (amino acids 1834-1854 of SEQ ID NO:2 or their equivalents in a different strain).

[074] In one embodiment the first proximal end is within amino acids 2690-2710, or 26952710, or 2700-2710 of SEQ ID NO:1 or their equivalent in a different strain and the second proximal end is within amino acids 1950-1980, 1955-1975 or 1960-1970 of SEQ ID NO:2 or their equivalent in a different strain. In one embodiment the first proximal end is within amino acids

2690-2710, or 2695-2710, or 2700-2710 of SEQ ID NO:1 or their equivalent in a different strain and the second proximal end is within amino acids 1978-2008, 1983-2003 or 1988-1998 of SEQ ID NO:2 or their equivalent in a different strain. In one embodiment the first proximal end is within amino acids 2690-2710, or 2695-2710, or 2700-2710 of SEQ ID NO:1 or their equivalent in a different strain and the second proximal end is within amino acids 1857-1887, 1862-1882, or

1867-1877 of SEQ ID NO:2 or their equivalent in a different strain. In one embodiment the first proximal end is within amino acids 2670-2700, or 2675-2695, or 2680-2690 of SEQ ID NO:1 or their equivalent in a different strain and the second proximal end is within amino acids 19501980, 1955-1975 or 1960-1970 of SEQ ID NO:2 or their equivalent in a different strain. In one embodiment the first proximal end is within amino acids 2670-2700, or 2675-2695, or 2680-2690 of SEQ ID NO:1 or their equivalent in a different strain and the second proximal end is within

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2016203241 18 May 2016 amino acids 1978-2008, 1983-2003 or 1988-1998 of SEQ ID NO:2 or their equivalent in a different strain. In one embodiment the first proximal end is within amino acids 2670-2700, or 2675-2695, or 2680-2690 of SEQ ID NO:1 or their equivalent in a different strain and the second proximal end is within amino acids 1857-1887, 1862-1882, 1860-1878 or 1867-1877 of SEQ ID 5 NO:2 or their equivalent in a different strain.

[075] In one embodiment the first fragment comprises at least 100, 200, 300, 400 or 450 amino acids. In one embodiment the second fragment comprises at least 100, 200, 300 or 400 amino acids.

[076] In one embodiment the polypeptide further comprises a linker. This linker may be 0 between the first proximal end and the second proximal end, alternatively the linker may link the distal ends of the first fragment and/or the second fragment to a further sequence of amino acids.

[077] A peptide linker sequence may be employed to separate the first fragment and second fragment. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the 5 following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first fragment and/or the second fragments; and (3) the lack of hydrophobic or charged residues that might react with the Tox A and/or ToxB functional epitopes. Peptide linker sequences may contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in 0 the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci.

USA 83:8258-8262 (1986); U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.

[078] In one embodiment the linker comprises between 1-19, 1-15, 1-10, 1-5, 1-2, 5-20, 5-15, 5-15, 10-20, or 10-15 amino acids. In one embodiment the linker is a glycine linker, the linker may comprise multiple (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 or 19) contiguous glycine residues, or alternatively the linker may comprise some glycine residues and some residues of other amino acids such as alanine. In a further embodiment the linker comprises a single glycine residue.

[079] In an embodiment the polypeptide of the invention is part of a larger fusion protein. The fusion proteins may further comprise amino acids encoding an immunogenic portion of a further protein antigen. For example the fusion protein may further comprise an immunogenic portion of a protein antigen obtained or derived from a bacterium selected from the group consisting of S.pneumoniae, H.influenzae, N.meningitidis, E.coli, M.cattarhalis, C.tentani, C.diphtheriae, B.pertussis, S.epidermidis, enterococci, S.aureus, and Pseudomonas aeruginosa. In this case the linker may be between the first fragment or the second fragment and a further immunogenic portion of a protein antigen.

[080] The term immunogenic portion thereof or ‘immunogenic fragment’ refers to a fragment of a polypeptide wherein the fragment comprises an epitope that is recognized by cytotoxic T lymphocytes, helper T lymphocytes or B cells. Suitably, the immunogenic portion will comprise at least 30%, suitably at least 50%, especially at least 75% and in particular at least 90% (e.g. 95% or 98%) of the amino acids in the reference sequence. The immunogenic portion will suitably comprise all of the epitope regions of the reference sequence.

[081] In one embodiment the polypeptide comprises an immunogenic fragment of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO27. In one embodiment the polypeptides comprises an immunogenic fragment of at least 500, 550, 600, 650, 700, 750, 780, 800, 830, 850, 880, 900,

920, or 950 amino acids of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO27. In a further embodiment the polypeptide comprises a variant of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO27, in a further embodiment the polypeptide comprises a variant having at least 80%, 85%, 90%, 92%, 95%,

98%, 99%, or 100% sequence identity to SEQ ID NO:3-SEQ ID NO:7.

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2016203241 18 May 2016 [082] In one embodiment the polypeptide comprises more than 450, 475, 500, 525, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or 850 amino acids from toxin A. In one embodiment the polypeptide comprises less than 850, 825, 800, 775, 750, 725, 700, 675, 650, 625, or 600 amino acids from toxin A. In one embodiment the polypeptide comprises more than 350, 375,

400, 425, 450, 475, 500 or 525 amino acids from toxin B. In one embodiment the polypeptide comprises less than 525, 500, 475, or 450 amino acids from toxin B.

[083] The term ‘identity’ is known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness 0 between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence 5 Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence

Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to 0 determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the Needle program BLASTP, BLASTN (Altschul, S.F. etal., J. Molec. Biol. 215: 403-410 (1990), and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST family of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul,

S„ et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S„ et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

[084] Parameters for polypeptide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,

Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 10

Gap extension penalty:0.5

A program useful with these parameters is publicly available as the ‘needle’ program from EMBOSS package (Rice P.et al, Trends in Genetics 2000 col.16(6):276-277). The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).

[085] In order to determine the identity of a reference sequence to SEQ ID NO:1, in one embodiment the sequence identity is calculated over the entire length of the reference sequence. In a further embodiment the sequence identity is calculated over the entire length of the sequence in SEQ ID NO:1. In order to determine the identity of a reference sequence to SEQ ID NO:2, in one embodiment the sequence identity is calculated over the entire length of the reference sequence. In a further embodiment the sequence identity is calculated over the entire length of the sequence in SEQ ID NO:2.

[086] In a further aspect of the invention there is provided a polypeptide comprising (i) SEQ ID

NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID

NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35, (ii) a variant having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99% or 100% identity to SEQ ID NO: 10-19; or (iii) a fragment of at least 100, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500,

530, 550, 580 or 600 amino acids of SEQ ID NQ:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID

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NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 orSEQ ID NO:35. In a further embodiment the polypeptide comprises SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,

SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29,

SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35, ii) a variant having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99% or 100% identity to SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID 0 NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ

ID NO:35; or (iii) a fragment of at least 100, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600 amino acids of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID 5 NO:33 or SEQ ID NO:34 or SEQ ID NO:35. In a further embodiment the polypeptide comprises

SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 orSEQ ID NO:35, ii) a variant having at least 80%, 85%, 88%, 90%, 92%, 95%, 98%, 99% or 100% identity 0 to SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID

NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 orSEQ ID NO:35; or (iii) a fragment of at least 100, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600 amino acids of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID 5 NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID

NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35.

[087] In one embodiment the polypeptide comprises more than 450, 475, 500, 525, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or 850 amino acids from toxin A. In one embodiment the polypeptide comprises less than 850, 825, 800, 775, 750, 725, 700, 675, 650, 625, or 600 amino acids from toxin A. In one embodiment the polypeptide comprises more than 350, 375,

[088] In a further embodiment the polypeptide elicits neutralising antibodies that neutralise toxin A or toxin B or both. In a further embodiment the polypeptide elicits antibodies that neutralise toxin A. In a further embodiment the polypeptide elicits antibodies that neutralise toxin B. In a further embodiment the polypeptide elicits antibodies that neutralise toxin A and toxin B. The polypeptide of the invention elicits antibodies that neutralise toxin A if the sera against the polypeptide gives an ELISA readout more than 10%, 20%, 30%, 50%, 70%, 80%, 90% or 100% higher than the reference sample.

[089] In a further embodiment the polypeptide of the invention elicits a protective immune response in a mammalian host against strains of C.difficile. In one embodiment the mammalian host is selected from the group consisting of mouse, rabbit, guinea pig, monkey, non-human primate and human. In one embodiment the mammalian host is a mouse. In a further embodiment the mammalian host is a human.

[090] Whether a polypeptide elicits a protective immune response in a mammalian host against strains of C.difficile can be determined using a challenge assay. In such an assay the mammalian host is vaccinated with the polypeptide and challenged by exposure to C.difficile, the time which the mammal survives after challenge is compared with the time which a reference mammal that has not been immunised with the polypeptide survives. A polypeptide elicits a protective immune response if a mammal immunised with the polypeptide survives at least 10%, 20%, 30%, 50%, 70%, 80%, 90%, or 100% longer than a reference mammal which has not been

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2016203241 18 May 2016 immunised after challenge with C.difficile. In one embodiment the polypeptide of the invention elicits a protective immune response against strains of C.difficile in a mammal selected from the group consisting of mouse, guinea pig, monkey and human. In one embodiment the mammal is a mouse, in a further embodiment the mammal is a human.

[091] The native structure ofthe C-terminal (repeat) domains from toxins A and B consist of an extended β solenoid-like structure. This structure consists of primarily β sheet structures, with a minority of a helical structures as seen in Ho et al (PNAS 102:18373-18378 (2005)). The secondary structures present can be determined using circular dichroism. For example measuring the shape and the magnitude of the CD spectra in the far-UV region (190-250nm) and comparing the results with those of known structures. This can be carried out using an optical path of 0.01cm from 178 to 250nm, with a 1nm resolution and bandwidth on a Jasco J-720 spectropolarimeter, for example as seen in example 5 below.

[092] In one embodiment the polypeptide comprises less than 25%, 23%, 20%, 28%, 15%, 10%, or 7% alpha helical secondary structure. In a further embodiment the polypeptide comprises more than 20%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, or 42% beta sheet structure.

POLYNUCLEOTIDES [093] The invention further provides a polynucleotide encoding a polypeptide of the invention. For the purposes of the invention the term ‘polynucleotide(s)’ generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified

RNA or DNA including single and double-stranded regions/forms.

[094] The term polynucleotide encoding a peptide as used herein encompasses polynucleotides that include a sequence encoding a peptide or polypeptide of the invention. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the peptide or polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.

[095] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal similarity to the nucleotide sequence of any native (i.e. naturally occuring) gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention, for example polynucleotides that are optimized for human and/or primate and/or E.coli codon selection.

[096] Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, Μ. H. et al., Nucl. Acids Res.

Symp. Ser. pp. 215-223 (1980), Horn et al., Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge et al., Science 269:202-204 (1995)) and automated synthesis may be achieved, for example, using the ASI 431 A Peptide Synthesizer (Perkin Elmer, Palo Alto, CA).

[097] Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression ofthe gene product. For example, DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new

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2016203241 18 May 2016 restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.

VECTORS [098] In a further aspect of the invention the present invention relates vector comprising a polynucleotide of the invention linked to an inducible promoter such that when the promoter is induced a polypeptide encoded by the polynucleotide is expressed.

[099] A further aspect of the invention comprises said vector wherein the inducible promoter is activated by addition of a sufficient quantity of IPTG (Isopropyl β-D-l-thiogalactopyranoside) preferably to the growth medium. Optionally this is at a concentration of between 0.1 and 10mM,

0.1 and 5mM, 0.1 and 2.5mM, 0.2 and 10mM, 0.2 and 5mM, 0.2 and 2.5mM, 0.4 and 10mM, 1 and 10mM, 1 and 5mM, 2.5 and 10mM, 2.5 and 5mM, 5 and 10mM. Alternatively the promoter may be induced by a change in temperature or pH.

HOST CELLS [0100] For recombinant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: A

LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, conjugation, transduction, scrape loading, ballistic introduction and infection.

[0101] Representative examples of appropriate hosts include gram negative bacterial cells, such as cells of, E. coli, Acinetobacter, Actinobacillus, Bordetella, Brucella, Campylobacter,

Cyanobacteria, Enterobacter, Erwinia, Franciscella, Helicobacter, hemophilus, Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Treponema, Vibrio, Yersinia. In one embodiment the host cell is an Escherichia coli cell. Alternatively gram positive bacterial cells may also be used. A great variety of expression systems can be used to produce the polypeptides of the invention. In one embodiment the vector is derived from bacterial plasmids. Generally any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in

Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

IMMUNOGENIC COMPOSITIONS AND VACCINES [0102] There is further provided an immunogenic composition comprising a polypeptide of the invention and a pharmaceutically acceptable excipient.

[0103] In one embodiment the immunogenic composition further comprises an adjuvant. The choice of a suitable adjuvant to be mixed with bacterial toxins or conjugates made using the processes of the invention is within the knowledge of the person skilled in the art. Suitable adjuvants include an aluminium salt such as aluminium hydroxide gel or aluminum phosphate or alum, but may also be other metal salts such as those of calcium, magnesium, iron or zinc, or

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2016203241 18 May 2016 may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized saccharides, or polyphosphazenes.

[0104] In one embodiment the immunogenic composition further comprises additional antigens. In one embodiment the additional antigens are antigens derived from a bacterium selected from 5 the group consisting of S.pneumoniae, H.influenzae, N.meningitidis, E.coli, M.cattarhalis, tetanus, diphtheria, pertussis, S.epidermidis, enterococci, S.aureus, and Pseudomonas aeruginosa. In a further embodiment the immunogenic composition of the invention may comprise further antigens from C.difficile for example the S-layer proteins (W001/73030).

[0105] There is further provided a vaccine comprising the immunogenic composition, this 0 vaccine may further comprise a pharmaceutically acceptable excipient.

[0106] The vaccine preparations containing immunogenic compositions of the present invention may be used to protect a mammal susceptible to C.difficile infection or treat a mammal with a C.difficile infection, by means of administering said vaccine via systemic or mucosal route. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or 5 subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts. Although the vaccine of the invention may be administered as a single dose, components thereof may also be co-administered together at the same time or at different times (for instance pneumococcal saccharide conjugates could be administered separately, at the same time or 1-2 weeks after the administration of the any bacterial protein component of the vaccine 0 for coordination of the immune responses with respect to each other). In addition to a single route of administration, 2 different routes of administration may be used. For example, saccharides or saccharide conjugates may be administered intramuscularly (IM) or intradermally (ID) and bacterial proteins may be administered intranasally (IN) or intradermally (ID). In addition, the vaccines of the invention may be administered IM for priming doses and IN for booster doses.

[0107] The content of toxins in the vaccine will typically be in the range 1-250pg, preferably 550pg, most typically in the range 5 - 25pg. Following an initial vaccination, subjects may receive one or several booster immunizations adequately spaced. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton,

US Patent 4,235,877.

[0108] In one aspect of the invention is provided a vaccine kit, comprising a vial containing an immunogenic composition of the invention, optionally in lyophilised form, and further comprising a vial containing an adjuvant as described herein. It is envisioned that in this aspect of the invention, the adjuvant will be used to reconstitute the lyophilised immunogenic composition.

[0109] A further aspect of the invention is a method of preventing or treating C.difficile infection comprising administering to the host an immunoprotective dose of the immunogenic composition or vaccine or kit of the invention. In one embodiment there is provided a method of preventing or treating primary and/or recurrence episodes of c.difficile infection comprising administering to the host an immunoprotective dose of the immunogenic composition or vaccine or kit of the invention.

[0110] A further aspect of the invention is an immunogenic composition of the invention for use in the treatment or prevention of C.difficile disease. In one embodiment there is provided an immunogenic composition of the invention for use in the treatment or prevention of primary and/or recurrence episodes of C.difficile disease.

[0111] A further aspect of the invention is use of the immunogenic composition or vaccine or kit of the invention in the manufacture of a medicament for the treatment or prevention of C.difficile disease. In one embodiment there is provided an immunogenic composition of the invention for

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2016203241 18 May 2016 use in the manufacture of a medicament for the treatment or prevention of primary and/or recurrence episodes of C.difficile disease.

[0112] Around” or “approximately” are defined as within 10% more or less of the given figure for the purposes of the invention.

[0113] The terms “comprising”, “comprise” and “comprises” herein are intended by the inventors to be optionally substitutable with the terms “consisting of’, “consist of” and “consists of”, respectively, in every instance. The term “comprises” means “includes.” Thus, unless the context requires otherwise, the word “comprises,” and variations such as “comprise” and “comprising” will be understood to imply the inclusion of a stated compound or composition (e.g., nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps, or groups thereof. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” [0114] Embodiments herein relating to “vaccine compositions” of the invention are also applicable to embodiments relating to “immunogenic compositions” of the invention, and vice versa.

[0115] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin,

Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.

(eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-5698).

[0116] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “plurality” refers to two or more. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, may be approximate.

[0117] All references or patent applications cited within this patent specification are incorporated by reference herein in their entirety.

[0118] In order that this invention may be better understood, the following examples are set 35 forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

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EXAMPLES

Example 1: Design of five C.difficile ToxA-ToxB fusions [0119] Fusion proteins containing fragments of the C-terminal repeating domains of ToxA and ToxB 5 were designed. These fusions contained a fragment of the C-terminal repeating domain of ToxA and a fragment of the C-terminal repeating domain of ToxB, and a junction between the C-terminal end of the

ToxA fragment and the N terminal end of the ToxB fragment. Two strategies were devised, in the first strategy; the fusion was designed such that the long solenoid structure was maintained at the junction between the two fragments. In the second strategy, the two fragments of the fusions are separated by a linker to allow their independent correct folding.

[0120] The C-terminal part of ToxA and B is composed of repeated sequences: short repeats (SR) and long repeats (LR) (PNAS 2005 vol 102 : 18373-18378).

[0121] The partial known 3D structure for the C-terminal domain of ToxA (PNAS 2005 Greco et al, vol 102 : 18373-18378 ; Nature Structural & Molecular biology 2006 vol 13(5) : 460-461 ; PDB codes : 2F6E,

2G7C and 2QJ6).

[0122] The inventors predicted that there are two kinds of important interactions between residues of the C-terminal part of ToxA and ToxB. The first interaction is occurring between residues contained in a LR and its preceding SR and is important to maintain the solenoid-like structure. The second type of interaction occurs between residues contained in a LR and the following SR and this interaction is mediating the carbohydrate-binding function of the toxin.

[0123] A new “structural-functional” repeat SR-LR-SR was defined. The structure of this repeat was maintained intact in our designed fusions.

[0124] Figure 2 represents the C-terminal domains of ToxA and ToxB and the defined “SR-LR-SR” box.

[0125] The positions of the short (SR) and long repeats (LR) of ToxA and ToxB repeats are presented 5 in table 1.

[0126] A list of the “SR-LR-SR” boxes contained in the C-terminal domain of ToxA and ToxB is presented in Table 2.

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Name	Start position	End position
ToxA_1	1874	1944
ToxA_2	2008	2078
ToxA_3	2142	2212
ToxA_4	2254	2326
ToxA_5	2390	2460
ToxA_6	2503	2573
ToxA_7	2595	2664
ToxB_1	1877	1946
ToxB_2	2008	2078
ToxB_3	2140	2212
ToxB_4	2274	2343

Table 2 [0127] Finally, the number of SRs between two LRs will be maintained in the designed fusions to keep the long solenoid-like structure.

[0128] Before the design of junctions for the fusions, two working hypotheses were defined: first hypothesis, the shorter the fusions, the better the probability for the fusions to be stably over expressed; second hypothesis, according to the concept of “SR-LR-SR” boxes, the start position has to be chosen in order to ensure a correct folding of the first SR of this previously defined SR-LR-SR box. Thus the fusions start at the beginning of the SR that precedes the SR-LR-SR box. Using these two hypothesis, three start positions were analysed: residue 2370, 2234 and 2121 of ToxA.

[0129] The start position 2370 was excluded. The start position 2234 was also excluded because one of the residues involved in interactions important for the protein structural stability is not conserved. So, it was decided that all the designed fusion will begin at residue 2121 of ToxA.

[0130] All fusions will end at the last residue of ToxB.

[0131] Four fusions (F1-4) were designed in order to maintain the entire fusion in a long solenoid-like structure between the two fusion fragments.

[0132] The fusions 1 (F1) and 2 (F2) were designed using the same hypothesis. All SR protein sequences of ToxA and ToxB had been compared using a multiple alignment software (ClustalW Thompson JD et al. (1994) Nucleic Acids Res., 22, 4673-4680). The more similar sequences were the third SR VIII of ToxA and the third SR II of ToxB and third SR III of ToxB. In order to make a choice between these two SR of ToxB, a structural homology modelling (using the SwissModel interface - Arnold K etal. (2006) Bioinformatics, 22, 195-201) was performed on the C-terminal part of ToxB using the

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2016203241 18 May 2016 known 3D structure of partial ToxA C-terminal domain (PDB code : 2QJ6). Using the third SR VIII of ToxA, the best local structural superposition (performed using SwissPDBViewer - Guex N et al. (1997), Electrophoresis 18, 2714-2723) was obtained with the third SR II of ToxB. So, two junctions were designed : the first one is between the third SR VIII of ToxA and the fourth SR II of ToxB (F1) and the second one is between the second SR VIII of ToxA and the third SR II of ToxB (F2). These junctions are presented in figure 3 and 4 respectively.

[0133] To design the fusion 3 (F3), a global structural superposition was performed between both the known structure of the partial C-terminal domain of ToxA and the predicted structure of C-terminal domain of ToxB (using SwissModel and SwissPDBViewer softwares). The best superposition was found between

LR VII of ToxA and LR II of ToxB. So, it was decided to make a junction in this similar LR. The junction was performed firstly in a region where the sequence is conserved between ToxA and ToxB, after that in order to keep in the ToxA part of the fusion, the residues in interaction with the preceding SR and lastly, in order to keep in the ToxB part, the residues in interaction with the following SR. This junction is shown in figure 5.

[0134] For the design of fusion 4 (F4), the C-terminal domain of ToxB was divided in 4 fragments and a more precise homology modelling (SwissModel) was performed on them. The split was realised in order to keep intact the “SR-LR-SR” boxes (each domain finishes at the end of the SR that follows a LR). A structural superposition between the predicted structures of these fragment and the known 3D structure of ToxA was made and the best structural surperposition was obtained for the third SR of ToxB (SR I) and the last SR of ToxA (third SR VIII). So, the junction was done between the second SR VIII of ToxA and the third SRIof ToxB. This design is presented in figure 6.

[0135] The last fusion (F5) was designed in order to allow an independent correct folding of the two fragments of the fusion. The linker was added between the last residue of the ToxA protein sequence and the beginning of the fourth SR II of ToxB (always taking into account the importance of an intact “SR-LR5 SR” box). Only one exogenous residue (Glycine) was added as linker and located between two existing Glycines. Thus, the linker can also be described as composed of 3 Glycines surrounding by known (for ToxA) and predicted (for ToxB) beta-strand. This last design is shown in figure 7.

Example 2: Cloning expression and purification of the fusion proteins

Expression plasmid and recombinant strain [0136] Genes encoding the fusion proteins of partial C-terminal domains of ToxA and ToxB (SEQ ID NO:3, 4, 5, 6 and 7) and a His tag were cloned into the pET24b(+) expression vector (Novagen) using the Ndel/Xhol restriction sites using standard procedures. The final construct was generated by the transformation of E. coli strain BLR (DE3) with the recombinant expression vector according to standard method with CaCI2-treated cells (Hanahan D. « Plasmid transformation by Simanis. » In Glover, D. M. (Ed), DNA cloning. IRL Press London. (1985): p. 109-135.).

Host strain:

[0137] BLR(DE3). BLR is a recA derivative of BL21. Strains having the designation (DE3) are lysogenic 40 for a λ prophage that contains an IPTG inducible T7 RNA polymerase, λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.

[0138] Genotype : E.coli BLR::DE3 strain, F ompThsdS_B(r_B m_Bj gal dcm (DE3) A(srl-recA)306::Tn70 (Tet^R)

Expression of the recombinant proteins:

[0139] An E.coli transformant was stripped from agar plate and used to inoculate 200 ml of LBT broth ± 1% (w/v) glucose + kanamycin (50 pg/ml) to obtain O.D.600nm between 0.1 -0.2. Cultures were incubated overnight at 37 °C, 250 RPM.

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2016203241 18 May 2016 [0140] This overnight culture was diluted to 1:20 in 500 ml of LBT medium containing kanamycin (50 pg/ml) and grown at 37°C at a stirring speed of 250 rpm until O.D.620 reached 0.5/0.6.

[0141] At O.D.600nm around 0.6, the culture was cooled down before inducing the expression of the 5 recombinant protein by addition of 1 mM isopropyl β-D-l-thiogalactopyranoside (IPTG; EMD Chemicals

Inc., catalogue number: 5815) and incubated overnight at 23 °C, 250 RPM.

[0142] After overnight induction (around 16 hours), O.D.₆oonm was evaluated after induction and culture was centrifuged at 14 000 RPM for 15 minutes and pellets were frozen at -20°C separately.

Purification:

[0143] The bacterial pellet was resuspended in 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl and a mixture of protease inhibitor (Complete, Roche). Bacteria were lysed using a French Press system 20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation for example at 20 OOOg for 30 min at 4°C.

[0144] The 6-His tagged-protein was purified under native conditions on IMAC. The soluble components were loaded on a GE column (15 ml for example) (Ni loaded) preequilibrated with the same buffer used to bacterial resuspension. After loading on the column, the column was washed with the same buffer. Elution was performed using a 20mM bicine buffer (pH 8.0) containing 500 mM NaCl and different concentrations of imidazole (5-600 mM). After gel analysis, more pure fractions were selected, concentrated and loaded on SEC chromatography for further purification step.

[0145] Fractions containing the fusion proteins were selected on the basis of purity by SDS-PAGE and dialyzed against bicine buffer (20mM Bicine, 150 mM NaCl,with or without 5mM EDTA pH8.0), Protein concentration was determined using DC Protein Assay of BioRad. Proteins were thus pooled, sterilefiltered on 0.22 pm, stored at -80°C.

[0146] Alternatively, IMAC purification was preceded by a DEAE purification step using 2mM bicine buffer (pH 8.0) for loading and washing, and eluted using a gradient with the same buffer but with 1M NaCl added.

Example 3 - Cloning expression and purification of the separate C.difficile Tox A and Tox B fragments

Expression plasmid and recombinant strain.

[0147] Genes encoding the protein fragments of ToxA and ToxB (SEQ ID NO:8 and SEQ ID NO:9) and a His tag were cloned into the pET24b(+) expression vector (Novagen) using the Ndel/Xhol restriction sites using standard procedures. The final construct was generated by the transformation of E. coli strain BLR (DE3) with the recombinant expression vector according to standard method with CaCI2-treated cells (Hanahan D. « Plasmid transformation by Simanis. » In Glover, D. M. (Ed), DNA cloning. IRL Press London. (1985): p. 109-135.).

Host strain:

[0148] BLR(DE3). BLR is a recA derivative of BL21. Strains having the designation (DE3) are lysogenic for a λ prophage that contains an IPTG inducible T7 RNA polymerase, λ DE3 lysogens are designed for protein expression from pET vectors This strain is also deficient in the Ion and ompT proteases.

[0149] Genotype : E.coli BLR::DE3 strain, F ompThsdS_B(r_B~ m_B) gal dcm (DE3) A(srl-recA)306::Tn70 45 (Tet^R)

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Expression of the recombinant proteins:

[0150] A E.coli transformant was stripped from agar plate and used to inoculate 200 ml of LBT broth ± 1% (w/v) glucose + kanamycin (50 pg/ml) to obtain O.D.₆₀onm between 0.1 -0.2. Cultures were incubated overnight at 37 °C, 250 RPM.

[0151] This overnight culture was diluted to 1:20 in 500 ml of LBT medium containing kanamycin (50 pg/ml) and grown at 37°C at a stirring speed of 250 rpm until O.D.₆2o reached 0.5/0.6.

[0152] At an O.D.at 600nm of around 0.6, the culture was cooled down before inducing the expression ofthe recombinant protein by addition of 1 mM isopropyl β-D-l-thiogalactopyranoside (IPTG; EMD Chemicals Inc., catalogue number: 5815) and incubated overnight at 23 °C, 250 RPM.

[0153] After the overnight induction (around 16 hours), O.D. at 600nm was evaluated after induction and culture was centrifuged at 14 000 RPM for 15 minutes and pellets were frozen at -20°C separately.

Purification:

[0154] The bacterial pellet was resuspended in 20 mM bicine buffer (pH 8.0) containing 500 mM NaCI 5 supplemented by a mixture of protease inhibitor (Complete without EDTA, Roche cat 11873580001) and benzonase. (Roche cat 1.01695.0001). Bacteria were lysed using a French Press system 2 X 20 000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation at 34 OOOg or 48 OOOg for 25-30 min at 4°C. Supernatant was harvested and filtrated on 0.22 pm filter.

[0155] The 6-His tagged-protein was purified under native conditions on IMAC. The soluble 0 components were loaded on a GE column (for example 15ml) (Ni loaded ) pre-equilibrated with the same buffer used to bacterial resuspension. After loading, the column was washed with the same buffer.

For ToxA:

[0156] Elution was performed using a 20mM bicine buffer (pH 8.0) containing 500 mM NaCI and 5 different concentrations of imidazole (5-100 mM). After gel analysis, more pure fractions were selected, concentrated and loaded on SEC chromatography (SUPERDEX™ 75) for further purification step in the same buffer without imidazole.

For ToxB:

[0157] A second wash was performed with 20mM bicine buffer (pH 8.0) containing 500 mM NaCI and

0.5 % deoxycholate or same buffer with 150 mM NaCI. Elution was performed using a 20mM bicine buffer (pH 8.0) containing 500 mM NaCI and different concentrations of imidazole (10-500 mM). After gel analysis, more pure fractions were selected, supplemented with 5 mM EDTA and loaded on SEC chromatography (SUPERDEX™ 200) for further purification step in same buffer with 5 mM EDTA.

[0158] Fractions containing ToxA or ToxB fragments were selected on the basis of purity by SDS-PAGE and dialyzed against bicine buffer (20mM Bicine, 150 mM NaCI, pH8.0), protein concentration was determined using RCDC Protein Assay of BioRad. Proteins were thus pooled, sterile-filtered on 0.22 pm, stored at -80°C.

Example 4 - Molecular weight evaluation of the five C.difficile ToxA-ToxB fusions [0159] Analytical ultracentrifugation is used to determine the homogeneity and size distribution in solution of the different species within a protein sample by measuring the rate at which molecules move in response to a centrifugal force. This is based on the calculation of the coefficients of sedimentation of the

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2016203241 18 May 2016 different species that are obtained by sedimentation velocity experiment, which depend on their molecular shape and mass.

1. Protein samples are spun in a Beckman-Coulter PROTEOMELAB™ XL-1 analytical ultracentrifuge at 42 000RPM after the AN-60Ti rotor had been equilibrated to 15°C.

a. F1 fusion protein, 500pg/ml, 20mM Bicine, 150mM NaCl, pH8,0

b. F2 fusion protein, 500pg/ml, 20mM Bicine, 150mM NaCl, pH8,0

c. F3 fusion protein, 500pg/ml, 20mM Bicine, 150mM NaCl, pH8,0

d. F4 fusion protein, 500pg/ml, 20mM Bicine, 150mM NaCl, pH8,0

e. F5 fusion protein, 500pg/ml, 20mM Bicine, 150mM NaCl, pH8,0

2. For data collection, 160 scans were recorded at 280nm every 5 minutes.

3. Data analysis was performed using the program SEDFIT for determination of the C(S) distribution. Determination of the partial specific volume of the proteins was performed with the SEDNTERP software from their amino acid sequence. SEDNTERP was also used to determine the viscosity and the density of the buffer.

4. The molecular weight of the different species was determined from the C(S) distribution plot (concentration vs sedimentation coefficient), considering that it’s a better representation of the raw data than the C(M) distribution (concentration vs molecular weight) to characterize the size distribution of a mixture.

[0160] Figure 8 describes the distribution of the ToxA-ToxB fusions as determined by sedimentation velocity analytical ultracentrifugation.

[0161] The molecular weight of the major species detected from the C(S) distribution of all five ToxAToxB fusion proteins corresponds to their monomeric form. The best fit frictional ratios determined for the five fusions are all between 2 and 2,2. This may indicate that the proteins are present in solution as an elongated form, which would be consistent with the protein structure.

Example 5- evaluation of secondary and tertiary structures of C.difficile ToxA-ToxB fusions by circular dichroism and fluorescence spectroscopy [0162] Circular dichroism is used to determine the secondary structure composition of a protein by measuring the difference in the absorption of left-handed polarized light versus right-handed polarized light which is due to structural asymmetry. The shape and the magnitude of the CD spectra in the far-UV region (190-250nm) are different whether a protein exhibits a beta-sheet, alpha-helix or random coil structure. The relative abundance of each secondary structure type in a given protein sample can be calculated by comparison to reference spectra.

[0163] The tertiary structure of a protein sample can be assessed by the evaluation of the immobilisation of the aromatic amino acids. The observation of a CD signal in the near-UV region (25050nm) may be attributable to the polarization of phenylalanine, tyrosine and tryptophane residues and is a good indication that the protein is folded into a well defined structure.

[0164] The following protocol was used:

1. Far UV spectra are measured using an optical path of 0,01cm from 178 to 250nm, with a 1nm resolution and bandwidth on a Jasco J-720 spectropolarimeter. Temperature of the cell is

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2016203241 18 May 2016 maintained at 23°C by a Peltier thermostated RTE-111 cell block. A nitrogen flow of 10L/min is maintained during the measurements.

2. Near-UV spectra are measured using an optical path of 0,01cm from 250 to 300nm, with a 1nm resolution and bandwidth on a Jasco J-720 spectropolarimeter. Temperature of the cell is maintained at 23°C by a Peltier thermostated RTE-111 cell block. A nitrogen flow of 6L/min is maintained during the measurements.

[0165] The observation of the far-UV spectra (figure 9) for all five ToxA-ToxB fusion proteins suggests a weak content of alpha helix structures and a high content of beta sheet structures. Also, all proteins exhibited a maximum at 230nm, which is unusual for soluble globular proteins. This particularity has been well characterized in the literature and is associated with a small group of proteins known for their absence of alpha helix and their high content in beta sheet and aromatic amino acids (Zsila, Analytical Biochemistry,391( 2009) 154-156). Those particularities are coherent with the structure that is expected for the ToxA-ToxB fusion proteins. Crystal structures of 13 proteins exhibiting the characteristic CD spectra with a positive signal at 230nm were compared (Protein Data Bank). The average secondary structure content of those proteins is 42% beta sheet ±9% and 7% alpha helix ±6%. This strongly indicates that the spectral signature of the ToxA-ToxB fusion proteins is diagnostic of a high beta sheet and low alpha helix containing protein.

[0166] The observation of the shape of the near-UV spectra (figure 10) for all five fusion proteins indicates that at least some of the aromatic amino acids are immobilised, which is a strong indication of a compact and specific tertiary structure. Moreover, the treatment of the protein with a denaturing concentration of urea caused the disappearance of the near-UV signal, which is an additional indication that this characteristic spectra was due to protein folding.

Example 6 - Immunisation of mice with Tox A or Tox B fragments and ToxA-ToxB fusions [0167] Balb/C mice were immunized with the constructs described in examples 2 and 3.

Mice immunization [0168] Groups of 15 female Balb/c mice were immunized IM at days 0, 14 and 28 with 3pg or10 pg of 0 the separate fragments of toxA and toxB (see example 2) as well as with ToxA-ToxB fusions proteins (see example 3) adjuvanted with AS03B. A control group of 10 mice was vaccinated with AS03B alone.

[0169] Anti-ToxA and anti-ToxB ELISA titers were determined in individual sera collected at day 42 (post III).

[0170] Hemagglutination inhibition titers were determined in pooled Post III sera.

Anti-ToxA and anti-ToxB ELISA response: Protocol [0171] Samples of the toxA or toxB fragments were coated at 1 pg/ml in phosphate buffered saline (PBS) on high-binding microtitre plates (Nunc MAXISORP™), overnight at 4° C. The plates were blocked with PBS-BSA 1% for 30 min at RT with agitation. The mice anti-sera are prediluted 1/500 in PBS40 BSA0.2%-TWEEN™ 0.05%. and then, further twofold dilutions were made in microplates and incubated at RT for 30 min with agitation. After washing, bound murine antibody was detected using Jackson ImmunoLaboratories Inc. peroxidase-conjugated affiniPure Goat Anti-Mouse IgG (H+L) (ref: 115-035-003) diluted 1:5000 in PBS-BSA0.2%-tween 0.05%. The detection antibodies were incubated for 30 min. at room temperature (RT) with agitation. The color was developed using 4 mg O-phenylenediamine (OPD) +

5 pi H₂O₂ per 10 ml pH 4.5 0.1M citrate buffer for 15 minutes in the dark at room temperature. The reaction was stopped with 50 μΙ HCI, and the optical density (OD) was read at 490 nm relative to 620 nm.

[0172] The level of anti-ToxA or anti-ToxB antibodies present in the sera was expressed in mid-point titers. A GMT was calculated for the 15 samples in each treatment group (10 for the control group).

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Hemagglutination inhibition assay: Protocol [0173] Serial twofold dilutions of mice pooled antisera (25μΙ) were performed in phosphate buffered saline (PBS) in 96-well U-bottom microplates.

[0174] 25 μΙ of native Toxin A (0,2 pg/well) were then added and the plates were incubated at room temperature for 30 minutes.

[0175] After incubation, 50 μΙ of purified rabbit erythrocytes diluted at 2% were added to each well. The plates were incubated at 37°C for 2 hours.

[0176] Plates were analysed visually, with hemagglutination presenting as diffuse red cells in the well 0 and the inhibition of hemagglutination observed as a red point settled in the well.

[0177] The inhibition titers were defined as the reciprocal of the highest dilution of the serum inhibiting hemagglutination.

Cytotoxicity assay [0178] IMR90 fibroblast cells were cultured at 37°C with 5% CO₂, in EMEM + 10% fetal bovine serum + 1% glutamine + 1% antibiotics (penicillin-streptomycin-amphotericin) and were seeded in 96-well tissue culture plates at a density of 5.10⁴ cells/well.

[0179] After 24h, the cell media was removed from the wells.

[0180] Serial twofold dilutions of mice pooled antisera (50μΙ) were performed in cell media.

[0181] 50 μΙ of native Toxin B (0.5ng/ml) is then added and the plates incubated at 37°C with 5% CO2 for 24 hours.

[0182] Cells were observed after 24 hours, and the proportion of rounded cells was determined.

[0183] The inhibition titers were defined as the reciprocal of the highest dilution of the serum inhibiting 50% cell rounding.

Results:

[0184] Elisa results, using Tox A antibodies are described in figure 11. Anti-Tox A antibodies were induced after immunization with the ToxA alone but also with each of the 5 fusions.

[0185] The functional properties of these antibodies were tested in the hemagglutination assay. This 30 assay is only adapted for Tox A evaluation as no hemagglutination is observed with ToxB.

[0186] Haemagglutination inhibition titres are described in figure 12. Haemaglutination inhibition was observed with the anti-Tox A fragment sera or sera directed against each of the ToxA-ToxB fusions.

[0187] An ELISA using ToxB antibodies was also performed; the results of this are illustrated in Figure 13. Anti-Tox B antibodies were induced after immunization with the ToxB fragment alone but also with the

F2, F3 and F4 fusions.

[0188] Cytotoxicity inhibition titres are described in figure 14. Inhibition titers obtained using sera from mice immunised with the ToxB fragment or the ToxA-ToxB fusions were greater than that obtained using control sera.

Example 7 Design, cloning, expression and purification of 4 further fusion proteins

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2016203241 18 May 2016 [0189] Four further fusion proteins were designed using the design principles described in example 1, these were named F54 Gly (SEQ ID NO:21), F54 New (SEQ ID NO:23), F5 ToxB (SEQ ID NO:25) and F52 New (SEQ ID NO:27).

[0190] These fusion proteins were expressed according to the procedure described in example 2.

Example 8 - Molecular weight evaluation of the C.difficile ToxA-ToxB fusions described in SEQ ID

NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27 [0191] The molecular weight of the fusions described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, 0 and SEQ ID NO:27 were determined as described in example 4.

[0192] Figure 15 describes the distribution of these four further fusion proteins as determined by sedimentation velocity analytical ultracentifugation.

[0193] The molecular weight of the main species determined from the C(S) distribution of all four protein fusions described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27 corresponds to their monomeric form and all proteins exhibit sedimentation properties similar to F1 to F5 fusions.

Example 9 - evaluation of secondary and tertiary structures of C.difficile ToxA-ToxB fusions described in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27 [0194] The secondary and tertiary structures of the fusions described in SEQ ID NO:21, SEQ ID NO:23,

SEQ ID NO:25, and SEQ ID NO:27 were assessed according to the method described in example 5. The far UV CD for these fusion proteins can be found in figure 16, and the near UV spectra for these fusions can be found in figure 17.

[0195] Analysis of the near and far UV CD spectra of the proteins described in SEQ ID NO:21, SEQ ID

NO:23, SEQ ID NO:25, and SEQ ID NO:27 shows that all four have the same high beta sheet structure than F1 to F5 fusions. In addition, observation of the near UV spectra show no significant difference in the position of the aromatic amino acids in the tertiary structure compared to F1 to F5 fusions.

Example 10 - immunisation of mice with Tox A-Tox B fusions [0196] Balb/c mice were immunised with the four fusion protein constructs F54 Gly (SEQ ID NO:21),

F54 New (SEQ ID NO:23), F5 ToxB (SEQ ID NO:25) and F52 New (SEQ ID NO:27) as described in example 6.

[0197] An ELISA was carried out using the anti-ToxA and anti-ToxB ELISA response:protocol described 35 in example 6 except here the samples of the toxA or toxB fragments were coated at 2pg/ml in phosphate buffered saline on high-binding microtitre plates. A hemagglutination inhibition assay was performed as described in example 6. A toxB cytotoxicity assay was performed as described in example 6. A further toxA cytotoxicity assay was performed as described below.

ToxA cytotoxicity assay

2016203241 16 Apr 2018 [0198] HT29 cells were cultured at 37°C with 5%CO2 in DMEM +10% fetal bovine serum +1% glutamine +1% antibiotics (penicillin-streptomycin-amphotericin) and were seeded in 96well tissue culture plates at a density of 5.104 cells/well.

[0199] After 24h, the cell media was removed from the wells.

[0200] Serial twofold dilutions of mice pooled antisera (50μ1) were performed in cell media.

[0201] 50μ1 of native Toxin B (0.15ng/ml) is then added and the plates incubated at 37°C with 5% CO2 for 48 hours.

[0202] Cells were observed after 48 hours and the proportion of rounded cells were determined.

[0203] The results of the anti-toxA ELISA, anti-toxB Elisa, Haemagglutination inhibition and cytotoxicity assays are described in Figures 18, 19 ,20, 21 and 22 respectively.

[0204] A reference herein to a patent document or any other matter identified as prior art, is not to be taken as an admission that the document or other matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

[0205] Where any or all of the terms comprise, comprises, comprised or comprising are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components.

2016203241 16 Apr 2018

Claims

The claims defining the invention are as follows:

1. A polypeptide comprising a first fragment and a second fragment, wherein (i) the first fragment is a C. difficile toxin A repeating domain fragment;

(ii) the second fragment is a C. difficile toxin B repeating domain fragment;

(iii) the first fragment has a first proximal end;

(iv) the second fragment has a second proximal end; and wherein the first proximal end is within a short repeat and the second proximal end is within a short repeat; and wherein the first fragment and the second fragment are adjacent to one another and wherein the polypeptide elicits antibodies that neutralize toxin A or toxin B or both.
2. The polypeptide of claim 1 wherein the polypeptide elicits a protective immune response in a mammalian host against strains of C.difficile.
3. The polypeptide of any one of claims 1 -2 wherein the first fragment and/or the second fragment comprise less than 25%, 20%, 18% or 15% alpha helical structure.
4. The polypeptide of any preceding claim wherein the first fragment and/or the second fragment comprise more than 25%, 30%, 35%, 38% or 40% beta sheet structure.
5. The polypeptide of any preceding claim wherein the first proximal end does not disrupt a short repeat-long repeat-short repeat portion.
6. The polypeptide of any preceding claim wherein the second proximal end does not disrupt a short repeat-long repeat-short repeat portion.
7. The polypeptide of any preceding claim wherein the first proximal end and the second proximal end do not disrupt short repeat-long repeat-short repeat portions.
8. The polypeptide of any preceding claim wherein the first proximal end is not within amino acids 1878-1940, 2012-2074, 2146-2208, 2258-2322, 2394-2456, 2507-2569 or 2598-2660 of toxin A.
9. The polypeptide of any preceding claim wherein the second proximal end is not within amino acids 1881-1942, 2012-2074, 2144-2208 or 2278-2339 of toxin B.
10. A polynucleotide encoding the polypeptide of any one of claims 1 -9.

30 11. An immunogenic composition comprising the polypeptide of any one of claims 1 -9 and a pharmaceutically acceptable excipient.

12. The immunogenic composition of claim 11 further comprising an adjuvant.

13. A use of the polypeptide of any one of claims 1 to 9, a polynucleotide of claim 10, or an immunogenic composition of claim 11 or 12 in the treatment or prevention of C.difficile

35 disease.

14. A method of treating or preventing C.difficile disease, said method comprising administering to a subject in need thereof a polypeptide of any one of claims 1 to 9, a polynucleotide of claim 10, or an immunogenic composition of claim 11 or 12.

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FIGURE 1

SEQ ID NO:1 - sequence of toxin A

MSLISKEELIKLAYSIRPRENEYKTILTNLDEYNKLTTNNNENKYLQLKKLNESIDVFMN KYKTSSRNRALSNLKKDILKEVILIKNSNTSPVEKNLHFVWIGGEVSDIALEYIKQWADI NAEYNIKLWYDSEAFLVNTLKKAIVESSTTEALQLLEEEIQNPQFDNMKFYKKRMEFIYD RQKRFINYYKSQINKPTVPTIDDIIKSHLVSEYNRDETVLESYRTNSLRKINSNHGIDIR ANSLFTEQELLNIYSQELLNRGNLAAASDIVRLLALKNFGGVYLDVDMLPGIHSDLFKTI SRPSSIGLDRWEMIKLEAIMKYKKYINNYTSENFDKLDQQLKDNFKLIIESKSEKSEIFS KLENLNVSDLEIKIAFALGSVINQALISKQGSYLTNLVIEQVKNRYQFLNQHLNPAIESD NNFTDTTKIFHDSLFNSATAENSMFLTKIAPYLQVGFMPEARSTISLSGPGAYASAYYDF INLQENTIEKTLKASDLIEFKFPENNLSQLTEQEINSLWSFDQASAKYQFEKYVRDYTGG SLSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDISYEATCNLF SKNPKNSIIIQRNMNESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEF NTSEFARLSVDSLSNEISSFLDTIKLDISPKNVEVNLLGCNMFSYDFNVEETYPGKLLLS IMDKITSTLPDVNKNSITIGANQYEVRINSEGRKELLAHSGKWINKEEAIMSDLSSKEYI FFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDYIYY EKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSV RFINKSNGESVYVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNT LNAAFFIQSLIDYSSNKDVLNDLSTSVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDT INVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLKKELEAKVGVLAINMSLSIAAT VASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESKKYGPLKT EDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIP SLSIYSAIGIETENLDFSKKIMMLPNAPSRVFWWETGAVPGLRSLENDGTRLLDSIRDLY PGKFYWRFYAFFDYAITTLKPVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGA GGTYSLLLSSYPISTNINLSKDDLWIFNIDNEVREISIENGTIKKGKLIKDVLSKIDINK NKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSLLLSGDKNYLISN LSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNDST LEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNES VYSSYLDFVKNSDGHHNTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVE FICDNNKNIDIYFGEWKTSSSKSTIFSGNGRNVVVEPIYNPDTGEDISTSLDFSYEPLYG IDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHKKVNINLDSSSFEYKWS TEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFKSF NSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNLVTGWQTINGK KYYFDINTGAALTSYKIINGKHFYFNNDGVMQLGVFKGPDGFEYFAPANTQNNNIEGQAI VYQSKFLTLNGKKYYFDNNSKAVTGWRIINNEKYYFNPNNAIAAVGLQVIDNNKYYFNPD TAIISKGWQTVNGSRYYFDTDTAIAFNGYKTIDGKHFYFDSDCVVKIGVFSTSNGFEYFA PANTYNNNIEGQAIVYQSKFLTLNGKKYYFDNNSKAVTGLQTIDSKKYYFNTNTAEAATG WQTIDGKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQ IGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNK KYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKG PNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNL NTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFY FNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVT GLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIM QIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDG NRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAI RYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGV DGVKAPGIYG

SEQ ID NO:2 - sequence of toxin B

MSLVNRKQLEKMANVRFRTQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYI DTYKKSGRNKALKKFKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKD VNSDYNVNVFYDSNAFLINTLKKTVVESAINDTLESFRENLNDPRFDYNKFFRKRMEIIY DKQKNFINYYKAQREENPELIIDDIVKTYLSNEYSKEIDELNTYIEESLNKITQNSGNDV RNFEEFKNGESFNLYEQELVERWNLAAASDILRISALKEIGGMYLDVDMLPGIQPDLFES

1/35

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IEKPSSVTVDFWEMTKLEAIMKYKEYIPEYTSEHFDMLDEEVQSSFESVLASKSDKSEIF SSLGDMEASPLEVKIAFNSKGIINQGLISVKDSYCSNLIVKQIENRYKILNNSLNPAISE DNDFNTTTNTFIDSIMAEANADNGRFMMELGKYLRVGFFPDVKTTINLSGPEAYAAAYQD LLMFKEGSMNIHLIEADLRNFEISKTNISQSTEQEMASLWSFDDARAKAQFEEYKRNYFE GSLGEDDNLDFSQNIVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYEAACNLFAK TPYDSVLFQKNIEDSEIAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNT DIFAGFDVDSLSTEIEAAIDLAKEDISPKSIEINLLGCNMFSYSINVEETYPGKLLLKVK DKISELMPSISQDSIIVSANQYEVRINSEGRRELLDHSGEWINKEESIIKDISSKEYISF NPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDTQIVEER IEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRF INKETGESIFVETEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLN AAFFIQSLIEYNSSKESLSNLSVAMKVQVYAQLFSTGLNTITDAAKVVELVSTALDETID LLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLRQEIEAKIGIMAVNLTTATTAIIT SSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKHVSLVETEGVFTLLD DKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHL SIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEG EFYWRYFAFIADALITTLKPRYEDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSG GTYALSLSQYNMGINIELSESDVWIIDVDNVVRDVTIESDKIKKGDLIEGILSTLSIEEN KIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLISGELKILMLNS NHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMD DSKPSFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDES GVAEILKFMNRKGNTNTSDSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQ FEFICDENDNIQPYFIKFNTLETNYTLYVGNRQNMIVEPNYDLDDSGDISSTVINFSQKY LYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYINEKINVNINDLSIRY VWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFT PSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNLITGFVTVG DDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEG EAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFN SDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFA HHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIG LSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIG VFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESD KYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIED KMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYI AATGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:3 - sequence of Fusion 1

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGFVSINDNKHYFD DSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTA VVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFY IDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFN PETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPD GFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:4 - sequence of Fusion 2

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE

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AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFD DSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTA VVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFY IDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFN PETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPD GFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:5 - sequence of Fusion 3

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAHHNEDLGNEEGEEISYSG ILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSD SGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIE TGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMF YFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPD TAQLVISE

SEQ ID NO:6 - sequence of Fusion 4

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFA PANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGV MQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGI LNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDS GIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIET GWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFY FGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDT AQLVISE

SEQ ID NO:7 - sequence of Fusion 5

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGGGFVSINDN

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KHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFD DSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNID DNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESD KYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGV FNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:8 sequence of individual toxin A fragment

MASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVT GLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFA PANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGL PQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAA GGLFEIDGVIYFFGVDGVKAP

SEQ ID NO:9 - sequence of individual toxin B fragment

MILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFTPSYYEDGLIGYDLGLVSLYNEKF YINNFGMMVSGLIYINDSLYYFKPPVNNLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGV FSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIG DYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGN EEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVT INDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDV YYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQF GYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVII DGEEYYFDPDTA

SEQ ID NO:10 - sequence of toxin A fragment from fusion 1

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAP

SEQ ID NO:11 - sequence of toxin A fragment from fusion 2

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAG

SEQ ID NO:12 - sequence of toxin A fragment from fusion 3

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN

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TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS

KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEY

SEQ ID NO:13 - sequence of toxin A fragment from fusion 4

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAG

SEQ ID NO:14 - sequence of toxin A fragment from fusion 5

MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEF LTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANN ESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAE AATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFE YFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFN TNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNS KAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQN RFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYG

SEQ ID NO:15 - sequence of toxin B fragment from fusion 1

GFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNF NNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGII ESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWI YDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGE DGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQL VISE

SEQ ID NO:16 - sequence of toxin B fragment from fusion 2

GLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHH NEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIM QVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLV RVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNE NGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAA TGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:17 - sequence of toxin B fragment from fusion 3

FAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFND DGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEY SGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNY YFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDE YIAATGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:18 - sequence of toxin B fragment from fusion 4

GETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKEL DGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQ

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IGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGL SLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANT VNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIM RTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWL DLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:19 - sequence of toxin B fragment from fusion 5

GFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNF NNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGII ESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWI YDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGE DGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQL VISE

SEQ ID NO :20 - nucleotide sequence of F54 Gly

ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGCAATTGCAAGCACCGGCTATACCATTATCAACGGCA

AACACTTTTATTTTAACACCGACGGCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCAAATACCGATGC

CAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGACCCTGAACGGCAAAAAATACTACTTTGGCAGCGATAGCAAAGCAGTT

ACCGGTTGGCGCATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTGTGCACCATTAACAACGACAAAT

ATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTACATTACCATCGAACGCAACAACTTTTATTTCGATGCCAACAACGAAAGCAAAATGGT

GACCGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATAACAACATTGAAGGTCAGGCGATCGTTTATCAG

AACAAATTCCTGACGCTGAATGGTAAGAAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGCTGGCAGACAATTGACGGGAAGAAATATT

ACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGACGGTAAAAAGTACTACTTCAACCTGAACACAGCCGAAGCAGCCAC

AGGATGGCAGACTATTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCATCTACCGGTTATACCAGCATTAACGGTAAACATTTC

TACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCAAAGGTCCAAATGGTTTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACA

TCGAGGGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGGAAAAAATATTACTTTGGTAGCGATTCTAAAGCCGTTACCGGTCT

GCGTACCATTGATGGTAAAAAATACTACTTTAATACGAATACAGCCGTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACTATTTT

AACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATTTCGGGTAAACACTTCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCT

TTAAAGGACCTGATGGGTTCGAATATTTTGCGCCTGCGAACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTATCAGAATCGCTTTCT

GTATCTGCACGACAACATCTATTATTTTGGCAACAATTCAAAAGCAGCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCG

AATACCGCAATGGGTGCAAATGGCTACAAAACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGCCGCAGATCGGGGTATTTAAAGGTA

GCAACGGCTTCGAATACTTCGCTCCAGCGAATACGGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGCT

GGGCAAAATCTACTACTTTGGCAATAACAGTAAAGCAGTTACTGGATGGCAGACAATCAATGGTAAAGTGTACTATTTTATGCCGGATACCGCC

ATGGCAGCAGCCGGTGGTCTGTTTGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCACCGGGAATATACGGTGGTACCG

GCTTTGTGACCGTGGGTGATGATAAATACTATTTCAATCCGATTAACGGTGGTGCAGCGAGCATTGGCGAAACCATCATCGATGACAAAAACTA

TTATTTCAACCAGAGCGGTGTGCTGCAGACCGGTGTGTTTAGCACCGAAGATGGCTTTAAATATTTTGCGCCAGCGAACACCCTGGATGAAAAC

CTGGAAGGCGAAGCGATTGATTTTACCGGCAAACTGATCATCGATGAAAACATCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGGA

AAGAACTGGATGGCGAAATGCATTATTTTTCTCCGGAAACCGGTAAAGCGTTTAAAGGCCTGAACCAGATCGGCGATTACAAATACTACTTCAA

CAGCGATGGCGTGATGCAGAAAGGCTTTGTGAGCATCAACGATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTGGGCTATACCGAA

ATTGATGGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGATTGGCGTGTTCAATACCGAAGATGGTTTCAAATACTTCGCGCACCATA

ACGAAGATCTGGGTAACGAAGAAGGCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCTACTACTTTGATGATAGCTTTAC

CGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAGCAAATATTATTTCGATGAAGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATTAAC

GATGGCCAGTACTATTTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAAGTGTTCTATTTCAGCGATAGCGGCATTA

TTGAAAGCGGCGTGCAGAACATTGATGATAACTACTTCTACATCGATGATAACGGCATTGTGCAGATCGGCGTTTTTGATACCAGCGATGGCTA

CAAATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAATATAGCGGTCTGGTGCGTGTGGGCGAAGATGTGTAT

TATTTCGGCGAAACCTATACCATCGAAACCGGCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTACTTTAATCCGGAAACGAAAAAAG

CGTGCAAAGGCATTAACCTGATCGATGATATCAAATACTATTTTGATGAAAAAGGCATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAA

CTATTACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATGTTCTACTTCGGCGAAGATGGTGTTATGCAGATT

GGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCATCAGAATACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATACCGGCT

GGCTGGATCTGGATGAAAAACGCTACTACTTCACCGATGAATACATTGCGGCGACCGGCAGCGTGATTATTGATGGCGAAGAATACTACTTCGA

TCCGGATACCGCGCAGCTGGTGATTAGCGAACATCATCATCATCACCAT

SEQ ID NO :21 - amino acid of F54Gly

MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLN

GKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVF

KGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYY

FNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILY

6/35

WO 2012/163817

PCT/EP2012/059805

2016203241 18 May 2016

QNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDG

IMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKT

IDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMP

DTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGGTGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFS

TEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFN

SDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGIL

NFNNKIYYFDDSFTAWGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESG

VQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESD

KYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPD

GFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISEHHHHHH

SEQ ID NO :22 - nucleotide sequence of F54 New

ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGCAATTGCAAGCACCGGCTATACCATTATCAACGGCA

AACACTTTTATTTTAACACCGACGGCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCAAATACCGATGC

CAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGACCCTGAACGGCAAAAAATACTACTTTGGCAGCGATAGCAAAGCAGTT

ACCGGTTGGCGCATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTGTGCACCATTAACAACGACAAAT

ATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTACATTACCATCGAACGCAACAACTTTTATTTCGATGCCAACAACGAAAGCAAAATGGT

GACCGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATAACAACATTGAAGGTCAGGCGATCGTTTATCAG

AACAAATTCCTGACGCTGAATGGTAAGAAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGCTGGCAGACAATTGACGGGAAGAAATATT

ACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGACGGTAAAAAGTACTACTTCAACCTGAACACAGCCGAAGCAGCCAC

AGGATGGCAGACTATTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCATCTACCGGTTATACCAGCATTAACGGTAAACATTTC

TACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCAAAGGTCCAAATGGTTTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACA

TCGAGGGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGGAAAAAATATTACTTTGGTAGCGATTCTAAAGCCGTTACCGGTCT

GCGTACCATTGATGGTAAAAAATACTACTTTAATACGAATACAGCCGTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACTATTTT

AACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATTTCGGGTAAACACTTCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCT

TTAAAGGACCTGATGGGTTCGAATATTTTGCGCCTGCGAACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTATCAGAATCGCTTTCT

GTATCTGCACGACAACATCTATTATTTTGGCAACAATTCAAAAGCAGCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCG

AATACCGCAATGGGTGCAAATGGCTACAAAACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGCCGCAGATCGGGGTATTTAAAGGTA

GCAACGGCTTCGAATACTTCGCTCCAGCGAATACGGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGCT

GGGCAAAATCTACTACTTTGGCAATAACAGTAAAGCAGTTACTGGATGGCAGACAATCAATGGTAAAGTGTACTATTTTATGCCGGATACCGCC

ATGGCAGCAGCCGGTGGTCTGTTTGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTTACCGGCTTTGTGACCGTGG

GTGATGATAAATACTATTTCAATCCGATTAACGGTGGTGCAGCGAGCATTGGCGAAACCATCATCGATGACAAAAACTATTATTTCAACCAGAG

CGGTGTGCTGCAGACCGGTGTGTTTAGCACCGAAGATGGCTTTAAATATTTTGCGCCAGCGAACACCCTGGATGAAAACCTGGAAGGCGAAGCG

ATTGATTTTACCGGCAAACTGATCATCGATGAAAACATCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGGAAAGAACTGGATGGCG

AAATGCATTATTTTTCTCCGGAAACCGGTAAAGCGTTTAAAGGCCTGAACCAGATCGGCGATTACAAATACTACTTCAACAGCGATGGCGTGAT

GCAGAAAGGCTTTGTGAGCATCAACGATAACAAACACTATTTCGATGATAGCGGTGTGATGAAAGTGGGCTATACCGAAATTGATGGCAAACAT

TTCTACTTCGCGGAAAACGGCGAAATGCAGATTGGCGTGTTCAATACCGAAGATGGTTTCAAATACTTCGCGCACCATAACGAAGATCTGGGTA

ACGAAGAAGGCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCTACTACTTTGATGATAGCTTTACCGCGGTGGTGGGCTG

GAAAGATCTGGAAGATGGCAGCAAATATTATTTCGATGAAGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATTAACGATGGCCAGTACTAT

TTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAAGTGTTCTATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGC

AGAACATTGATGATAACTACTTCTACATCGATGATAACGGCATTGTGCAGATCGGCGTTTTTGATACCAGCGATGGCTACAAATATTTCGCACC

GGCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAATATAGCGGTCTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGAAACC

TATACCATCGAAACCGGCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTACTTTAATCCGGAAACGAAAAAAGCGTGCAAAGGCATTA

ACCTGATCGATGATATCAAATACTATTTTGATGAAAAAGGCATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACTATTACTTCAACGA

AAACGGTGAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATGTTCTACTTCGGCGAAGATGGTGTTATGCAGATTGGTGTTTTTAACACC

CCGGATGGCTTCAAATACTTTGCCCATCAGAATACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATACCGGCTGGCTGGATCTGGATG

AAAAACGCTACTACTTCACCGATGAATACATTGCGGCGACCGGCAGCGTGATTATTGATGGCGAAGAATACTACTTCGATCCGGATACCGCGCA

GCTGGTGATTAGCGAACATCATCATCATCACCAT

SEQ ID NO :23 amino acid sequence of F54 New

MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN

EFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESK

MVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTI

7/35

WO 2012/163817

PCT/EP2012/059805

2016203241 18 May 2016

DGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTD ANNIE

GQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHF

YFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMG

ANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGK

VYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAVTGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVF

STEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYF

NSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGI

LNFNNKIYYFDDSFTAWGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIES

GVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENES

DKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTP

DGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISEHHHHHH

SEQ ID NO :24 nucleotide sequence of F5 ToxB

ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGCAATTGCAAGCACCGGCTATACCATTATCAACGGCA

AACACTTTTATTTTAACACCGACGGCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCAAATACCGATGC

CAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGACCCTGAACGGCAAAAAATACTACTTTGGCAGCGATAGCAAAGCAGTT

ACCGGTTGGCGCATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTGTGCACCATTAACAACGACAAAT

ATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTACATTACCATCGAACGCAACAACTTTTATTTCGATGCCAACAACGAAAGCAAAATGGT

GACCGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATAACAACATTGAAGGTCAGGCGATCGTTTATCAG

AACAAATTCCTGACGCTGAATGGTAAGAAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGCTGGCAGACAATTGACGGGAAGAAATATT

ACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGACGGTAAAAAGTACTACTTCAACCTGAACACAGCCGAAGCAGCCAC

AGGATGGCAGACTATTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCATCTACCGGTTATACCAGCATTAACGGTAAACATTTC

TACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCAAAGGTCCAAATGGTTTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACA

TCGAGGGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGGAAAAAATATTACTTTGGTAGCGATTCTAAAGCCGTTACCGGTCT

GCGTACCATTGATGGTAAAAAATACTACTTTAATACGAATACAGCCGTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACTATTTT

AACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATTTCGGGTAAACACTTCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCT

TTAAAGGACCTGATGGGTTCGAATATTTTGCGCCTGCGAACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTATCAGAATCGCTTTCT

GTATCTGCACGACAACATCTATTATTTTGGCAACAATTCAAAAGCAGCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCG

AATACCGCAATGGGTGCAAATGGCTACAAAACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGCCGCAGATCGGGGTATTTAAAGGTA

GCAACGGCTTCGAATACTTCGCTCCAGCGAATACGGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGCT

GGGCAAAATCTACTACTTTGGCAATAACAGTAAAGCAGTTACTGGATGGCAGACAATCAATGGTAAAGTGTACTATTTTATGCCGGATACCGCC

ATGGCAGCAGCCGGTGGTCTGTTTGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTGAGCGGTCTGATTTATATTA

ACGATAGCCTGTATTACTTTAAACCACCGGTGAATAACCTGATTACCGGCTTTGTGACCGTGGGTGATGATAAATACTATTTCAATCCGATTAA

CGGTGGTGCAGCGAGCATTGGCGAAACCATCATCGATGACAAAAACTATTATTTCAACCAGAGCGGTGTGCTGCAGACCGGTGTGTTTAGCACC

GAAGATGGCTTTAAATATTTTGCGCCAGCGAACACCCTGGATGAAAACCTGGAAGGCGAAGCGATTGATTTTACCGGCAAACTGATCATCGATG

AAAACATCTATTACTTCGATGATAACTATCGTGGTGCGGTGGAATGGAAAGAACTGGATGGCGAAATGCATTATTTTTCTCCGGAAACCGGTAA

AGCGTTTAAAGGCCTGAACCAGATCGGCGATTACAAATACTACTTCAACAGCGATGGCGTGATGCAGAAAGGCTTTGTGAGCATCAACGATAAC

AAACACTATTTCGATGATAGCGGTGTGATGAAAGTGGGCTATACCGAAATTGATGGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGA

TTGGCGTGTTCAATACCGAAGATGGTTTCAAATACTTCGCGCACCATAACGAAGATCTGGGTAACGAAGAAGGCGAAGAAATTAGCTATAGCGG

CATCCTGAACTTCAACAACAAAATCTACTACTTTGATGATAGCTTTACCGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAGCAAATATTAT

TTCGATGAAGATACCGCGGAAGCGTATATTGGCCTGAGCCTGATTAACGATGGCCAGTACTATTTTAACGATGATGGCATTATGCAGGTGGGTT

TCGTGACCATTAATGATAAAGTGTTCTATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGCAGAACATTGATGATAACTACTTCTACATCGA

TGATAACGGCATTGTGCAGATCGGCGTTTTTGATACCAGCGATGGCTACAAATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGC

CAGGCGGTGGAATATAGCGGTCTGGTGCGTGTGGGCGAAGATGTGTATTATTTCGGCGAAACCTATACCATCGAAACCGGCTGGATTTATGATA

TGGAAAACGAAAGCGATAAATATTACTTTAATCCGGAAACGAAAAAAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAATACTATTTTGA

TGAAAAAGGCATTATGCGTACCGGTCTGATTAGCTTCGAAAACAACAACTATTACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAAC

ATCGAAGATAAAATGTTCTACTTCGGCGAAGATGGTGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCATCAGA

ATACCCTGGATGAAAATTTCGAAGGTGAAAGCATTAACTATACCGGCTGGCTGGATCTGGATGAAAAACGCTACTACTTCACCGATGAATACAT

TGCGGCGACCGGCAGCGTGATTATTGATGGCGAAGAATACTACTTCGATCCGGATACCGCGCAGCTGGTGATTAGCGAACATCATCATCATCAC

CAT

SEQ ID NO :25 amino acid sequence of F5 ToxB

MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN

8/35

WO 2012/163817

PCT/EP2012/059805

2016203241 18 May 2016

EFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESK

MVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTI

DGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIE

GQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHF

YFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGGAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMG

ANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGK

VYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAVSGLIYINDSLYYFKPPVNNLITGFVTVGDDKYYFNPINGGAASIGE

TIIDDKNYYFNGSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYF

SPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKY

FAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAWGWKDLEDGSKYYFDEDTAEAYIGLSLINDGGYYFNDDGIMQ

VGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDV

YYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINI

EDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPD

TAQLVISEHHHHHH

SEQ ID NO :26 - nucleotide sequence of F52 new

ATGGCAACCGGTTGGCAGACCATCGATGGCAAAAAATATTATTTTAATACCAACACCGCAATTGCAAGCACCGGCTATACCATTATCAACGGCA

AACACTTTTATTTTAACACCGACGGCATTATGCAGATTGGTGTGTTTAAAGGTCCGAACGGCTTTGAATACTTTGCACCGGCAAATACCGATGC

CAATAATATTGAAGGCCAGGCCATTCTGTATCAGAATGAATTTCTGACCCTGAACGGCAAAAAATACTACTTTGGCAGCGATAGCAAAGCAGTT

ACCGGTTGGCGCATCATCAACAATAAGAAATATTACTTCAACCCGAATAATGCAATTGCAGCAATTCATCTGTGCACCATTAACAACGACAAAT

ATTATTTCAGCTATGACGGTATTCTGCAGAATGGCTACATTACCATCGAACGCAACAACTTTTATTTCGATGCCAACAACGAAAGCAAAATGGT

GACCGGTGTTTTCAAAGGCCCTAATGGTTTTGAGTATTTCGCTCCGGCAAACACCCATAATAACAACATTGAAGGTCAGGCGATCGTTTATCAG

AACAAATTCCTGACGCTGAATGGTAAGAAATACTATTTCGATAATGACAGCAAAGCCGTGACCGGCTGGCAGACAATTGACGGGAAGAAATATT

ACTTTAATCTGAATACCGCAGAAGCAGCAACCGGTTGGCAAACGATCGACGGTAAAAAGTACTACTTCAACCTGAACACAGCCGAAGCAGCCAC

AGGATGGCAGACTATTGATGGAAAAAAATACTATTTCAACACCAACACCTTTATTGCATCTACCGGTTATACCAGCATTAACGGTAAACATTTC

TACTTCAACACCGATGGTATCATGCAGATCGGCGTTTTCAAAGGTCCAAATGGTTTCGAATACTTTGCCCCTGCCAATACAGATGCAAATAACA

TCGAGGGTCAGGCAATCCTGTACCAAAACAAATTTCTGACCCTGAATGGGAAAAAATATTACTTTGGTAGCGATTCTAAAGCCGTTACCGGTCT

GCGTACCATTGATGGTAAAAAATACTACTTTAATACGAATACAGCCGTTGCGGTTACAGGCTGGCAGACCATTAACGGGAAAAAATACTATTTT

AACACAAATACCAGCATTGCCTCAACGGGTTATACCATTATTTCGGGTAAACACTTCTACTTTAATACCGATGGTATTATGCAAATCGGAGTCT

TTAAAGGACCTGATGGGTTCGAATATTTTGCGCCTGCGAACACTGATGCGAACAATATCGAAGGACAGGCAATCCGCTATCAGAATCGCTTTCT

GTATCTGCACGACAACATCTATTATTTTGGCAACAATTCAAAAGCAGCCACCGGCTGGGTTACAATTGATGGCAACCGCTACTATTTCGAACCG

AATACCGCAATGGGTGCAAATGGCTACAAAACCATCGATAATAAAAATTTCTATTTTCGCAACGGTCTGCCGCAGATCGGGGTATTTAAAGGTA

GCAACGGCTTCGAATACTTCGCTCCAGCGAATACGGACGCGAACAATATTGAGGGTCAAGCGATTCGTTATCAAAACCGTTTTCTGCATCTGCT

GGGCAAAATCTACTACTTTGGCAATAACAGTAAAGCAGTTACTGGATGGCAGACAATCAATGGTAAAGTGTACTATTTTATGCCGGATACCGCC

ATGGCAGCAGCCGGTGGTCTGTTTGAAATTGATGGCGTGATCTATTTTTTTGGTGTGGATGGTGTTAAAGCAGTGAAAGGCCTGAACCAGATCG

GCGATTACAAATACTACTTCAACAGCGATGGCGTGATGCAGAAAGGCTTTGTGAGCATCAACGATAACAAACACTATTTCGATGATAGCGGTGT

GATGAAAGTGGGCTATACCGAAATTGATGGCAAACATTTCTACTTCGCGGAAAACGGCGAAATGCAGATTGGCGTGTTCAATACCGAAGATGGT

TTCAAATACTTCGCGCACCATAACGAAGATCTGGGTAACGAAGAAGGCGAAGAAATTAGCTATAGCGGCATCCTGAACTTCAACAACAAAATCT

ACTACTTTGATGATAGCTTTACCGCGGTGGTGGGCTGGAAAGATCTGGAAGATGGCAGCAAATATTATTTCGATGAAGATACCGCGGAAGCGTA

TATTGGCCTGAGCCTGATTAACGATGGCCAGTACTATTTTAACGATGATGGCATTATGCAGGTGGGTTTCGTGACCATTAATGATAAAGTGTTC

TATTTCAGCGATAGCGGCATTATTGAAAGCGGCGTGCAGAACATTGATGATAACTACTTCTACATCGATGATAACGGCATTGTGCAGATCGGCG

TTTTTGATACCAGCGATGGCTACAAATATTTCGCACCGGCCAATACCGTGAACGATAACATTTATGGCCAGGCGGTGGAATATAGCGGTCTGGT

GCGTGTGGGCGAAGATGTGTATTATTTCGGCGAAACCTATACCATCGAAACCGGCTGGATTTATGATATGGAAAACGAAAGCGATAAATATTAC

TTTAATCCGGAAACGAAAAAAGCGTGCAAAGGCATTAACCTGATCGATGATATCAAATACTATTTTGATGAAAAAGGCATTATGCGTACCGGTC

TGATTAGCTTCGAAAACAACAACTATTACTTCAACGAAAACGGTGAAATGCAGTTCGGCTACATCAACATCGAAGATAAAATGTTCTACTTCGG

CGAAGATGGTGTTATGCAGATTGGTGTTTTTAACACCCCGGATGGCTTCAAATACTTTGCCCATCAGAATACCCTGGATGAAAATTTCGAAGGT

GAAAGCATTAACTATACCGGCTGGCTGGATCTGGATGAAAAACGCTACTACTTCACCGATGAATACATTGCGGCGACCGGCAGCGTGATTATTG

ATGGCGAAGAATACTACTTCGATCCGGATACCGCGCAGCTGGTGATTAGCGAACATCATCATCATCACCAT

SEQ ID NO :27 - amino acid sequence of F52 New

MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLN

9/35

WO 2012/163817

PCT/EP2012/059805

2016203241 18 May 2016

GKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVF

KGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYY

FNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILY

QNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDG

IMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKT

IDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMP

DTAMAAAGGLFEIDGVIYFFGVDGVKAVKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFY

FAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAWGWKDLEDGSKYYFDEDTAEAY

IGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVN

DNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISF

ENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDE

YIAATGSVIIDGEEYYFDPDTAQLVISEHHHHHH

SEQ ID NO:28 - sequence of toxin A fragment of F54 Gly

MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN EFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDA NNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNT AEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNG FEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGN NSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRY QNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYG

SEQ ID NO:29 - sequence of toxin A fragment of F54 New

MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN EFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDA NNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNT AEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNG FEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGN NSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRY QNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAV

SEQ ID NO:30 - sequence of toxin A fragment of F5 ToxB

MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN EFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDA NNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNT AEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNG FEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGN NSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRY QNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAV

SEQ ID NO:31 - sequence of toxin A fragment of F52 New

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MATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQN EFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDA NNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNT AEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNG FEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYY FNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGN NSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRY QNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAV

SEQ ID NO:32 - sequence of toxin B fragment of F54Gly

TGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGK LIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSG VMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVG WKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDD NGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPET KKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFK YFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:33 - sequence of toxin B fragment of F54 New

TGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGK LIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSG VMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVG WKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDD NGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPET KKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFK YFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE

SEQ ID NO:34 - sequence of toxin B fragment of F5 ToxB

SGLIYINDSLYYFKPPVNNLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKY FAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSD GVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYS GILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFS DSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTI ETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKM FYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDP DTAQLVISE

SEQ ID NO:35 - sequence of toxin B fragment of F52 New

KGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAH HNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGI MQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGL VRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFN ENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIA ATGSVIIDGEEYYFDPDTAQLVISE
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FIGURE 2 ] SR | LR C-terminal domain of ToxA ιιι·ιιιι·ιιιι·ιιι·ιιιι·ιιι·ιι·ιιι

I_,_I............_v._________L...........,........................A...........,....................A......................._r_________J, ,.....................I_________,..................t.J

I ? Ϊ I I I < Γ

I II III IV V VI VII VIII

C-terminal domain of ToxB ·ιι·ι·ιιι· (__L_____„__I________

Ϊ T 1 Ϊ

I II III IV V
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C-terminal domain of ToxA

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FIGURE 3

El···

II III IV V

C-terminal domain of ToxB
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FIGURE 4

ΙΙΙ·ΙΙΙΙ·ΙΙΙΙ·ΙΙΙ·ΙΙΙΙ·ΙΙΙ·ΙΙ·ΙΊ

I 1 I_, 1 , 1 , 1 t ] r ι ϊ

I II III IV V Vi VII VIII

T'lHIIIMIIIWII '—Γ —¹ 1

I ‘ II III IV V

C-terminal domain of ToxA

C-terminal domain of ToxB
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FIGURE 5

Hi·· jLJ

I II III IV V

C-terminal domain of ToxB
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FIGURE 6

! i Ϊ — ·ιιι·ιι·π 1 1 * ' +A ? 1 ? II III IV V Vl VII V |-£| u:: M 1

„-J-,ηρ

II III IV V

C-terminal domain of ToxB
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FIGURE 7 ιιι·ιιιι·ιιιι·ιιι·ιιιι·ιιι·ιι·ιι

I . I I I , 1 ί , J J , ₁ , Ϊ , , ι ii iii iv v v: vn vm ' kJ ι y , ι u ίΤΓΙ·ΙΙΙΙ·ΙΙΙΙ·ΙΙ

L

I II III IV V

C-terminal domain of ToxB
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FIGURE 8

a)

Flvar Lot P1010J jan 2010

b)

F2 var Lot BMP87 dec 2010
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c)

d)

F4 var lot BMP89 (20Apr2012)

1.60

1.20 ^υ 0.80

0.40

0.00

—zJ ---!

0 100 200 300 400 500 600 700 800 900 1,000

MW (KDa)

e)
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F5 var lot BMP90 (20Apr2012)
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FIGURE 9

Far-UV CD of ToxA-ToxB fusions —x—f5var —f2varl —·—f4var —♦—f3var
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FIGURE 10

Near-UV CD of ToxA-ToxB fusions —f2v2n —·—f3n —f4n —·—f5n
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FIGURE 11

10000

1000

100

20100673 : Mice immunization with C. difficile ToxA-Cter, ToxB-Cter and fusion proteins 2d generation formulated in AS03B ELISA a-ToxA : concentrations (pg/ml) on Post III sera

1215

1073

937 839

771 ~=Ej~ ¹⁴⁷⁵ 1160

I

I I 11III tuO <

tuO

d.

o on <

cuo

d.

o on <

cuo

d.

o on <

cuo

d.

o cuo <

tuO

d.

o cuo <

tuO

d.

o cuo <

tuO

d.

o cuo <

tuO =i m

tUD <

tuO =ί

ΓΟ

929 1061 871 θ29 1277 τ i _ A...............

φ φ

ΙΛ ΙΛ on <

cuo =i

ΓΟ on <

cuo =i

ΓΟ

ToxA (aa

23872706)

ToxB (aa

17502360)

FI

F2

F3

F4

F5

ToxA (aa

23872706)

ToxB (aa

17502360)

FI

F2

III.

tuO <

tuO =ί

ΓΟ

F3 >>>

cuo <

tuO =i

ΓΟ

F4 cuo <

tuO =ί

ΓΟ

F5 §

T?

ci m

ro o

co
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FIGURE 12

10000

1000

100

1280

GO o

Ό

CUD <

CD

Zl o

ToxA (aa

23872706) o

Ό

CD <

CD

Zl

O

ToxB (aa

17502360)

Post III

20100673 : Mice immunization with C. difficile ToxA-Cter, ToxB-Cterand fusion proteins 2d generation formulated in AS03B

Inhibition hemagglutination assay : mid-point titers on Post III sera

512Q

5120

3840

1920

1920 1280 o

Ό

CD <

CD

Zl

O o

Ό

CD <

CD

Zl o

o

Ό

CD <

CD

Zl

O o

Ό

CD <

CD

Zl o

o

T5

CD <

CD

Zl o

o

Ό

CD <

CD

Zl

CO

FI

F2

F3

F4

F5

ToxA (aa

23872706)

240 o

o

CD <

CD

Zl

CO

ToxB (aa

17502360)

7680

Page 24 of 35
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FIGURE 13

10000

20100673 : Mice immunization with C. difficile ToxA-Cter, ToxB-Cter and fusion proteins 2d generation formulated in AS03B ELISA a-ToxB : concentrations (pg/ml) on Post III sera

1000

100 o

Ό tuO <

tuO

d.

o o

Ό tuO <

tuO

d.

o o

Ό tuO <

tuO

d.

o o

Ό tuO <

tuO =ί o

o

Ό tuO <

tuO =ί

O o

Ό tuO <

tuO =ί

O o

Ό tuO <

tuO =ί o

o

Ό tuO <

tuO =ί m

o

Ό tuO <

tuO =ί m

o

Ό tuO <

tuO =ί m

o

Ό tuO <

tuO =ί m

o

Ό tuO <

tuO =ί m

o

Ό tuO <

tuO =ί m

o

Ό tuO <

tuO =ί m

ToxA ToxB (aa (aa 2387- 1750,2706),2360),

FI

F2

F3

F4

F5

ToxA ToxB (aa (aa 2387- 1750.2706).2360).

FI

F2

F3

F4

F5 ©

I

4?

o <
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FIGURE 14

10000

1000

100 o

Ό tUD <

tuo

d.

o

ToxA (aa

23872706)

20100673 : Mice immunization with C. difficile ToxA-Cter, ToxB-Cter and fusion proteins 2d generation formulated in AS03B

320

640

320

320

160 160 30

160

320 >>>

o

Ό tUD <

tuo

d.

o o

Ό tUD <

tuo

d.

o o

Ό tUD <

tuo

d.

o o

Ό tUD <

tuo =i o

o

Ό tUD <

tuo =i o

o

Ό tUD <

tuo =i o

o

Ό tUD <

tuo =i

ΓΟ o

Ό tUD <

tuo =i

ΓΟ o

Ό tUD <

tuo =i

ΓΟ o

Ό tUD <

tuo =i

ΓΟ o

Ό tUD <

tuo =i

ΓΟ o

Ό tUD <

tuo =i

ΓΟ o

Ό tUD <

tuo =i

ΓΟ

ToxB (aa

17502360)

FI

F2

F3

F4

F5

ToxA (aa

23872706)

ToxB (aa

17502360)

FI

F2

F3

F4

F5 §

=3

T?

ci m

ro o

co
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b)

c)

FIGURE 15

a)

F52 new lotBMP154 (03oct2011)

F54 Gly Lot BMP155 (23Sep2011)
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d)

F54 New Lot BMP156 (23Sep2011)

F5 ToxB Lot BMP157 (21sep2011)
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FIGURE 16

-Ω

D u

Far UV CD of new fusion proteins

2.5

1.5

0.5

-0.5

-1.5 nm

F54 new

F54gly

F5 ToxB

F52 new
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FIGURE 17

Near UV CD of new constructs

Λ

D u

nm

F54 new —F54gly —F5ToxB

F52 new
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FIGURE 18

Mice immunization with C. difficile fusion proteins formulated in AS03B Anti-ToxA ELISA: ELISA titers (pg/ml) on individual Post III sera 1nnnn ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ 1 nnn . ₇₆₄ ⁹³⁷ 841 704 761 100 - 10 - 1 ¹¹¹11 1 1 II 1 F2 F52new F54Gly F54new F5 ToxB Geomean 764 937 841 704 761
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FIGURE 19
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FIGURE 20

Mice immunization with C. difficile fusion proteins formulated in AS03B Hemagglutination inhibition assay : inhibition titers on Post III pooled sera

10000 T2560

1000

100

1280 1280 - 1280 1280 ...... 1 1 1 m m n 1 I 1

F2 2nd F52new 3d F54Gly 3d F54new 3d F5 ToxB 3d generation generation generation generation generation 1280 1280 2560 1280 1280
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FIGURE 21

Mice immunization with C. difficile fusion proteins formulated in AS03B Cytotoxicity inhibition assay on HT29 cells : inhibition titers on Post III pooled sera

10000

1000

100

3240

3240 Pools Post III

F2 2nd i F52new 3d F54Gly 3d i F54new 3d 1 F5 ToxB 3d generation i generation i generation i generation ι generation 3240 1 3240 1 3240 i 3240 3240
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FIGURE 22

Mice immunization with C. difficile fusion proteins formulated in AS03B Cytotoxicity inhibition assay on IMR90 cells (for ToxB) inhibition titers on Post III pooled sera

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VB64650 Seq List SEQUENCE LISTING <110> GlaxoSmithKline Biologicals s.a <120> Immunogenic Composition <130> VB64650 <160> 35 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 2710 <212> PRT <213> Clostridium Difficile <400> 1

Met 1 Ser Leu lie Ser 5 Lys Glu Gl u Leu lie 10 Lys Leu Al a Tyr Ser 15 lie Arg Pro Arg Glu Asn Glu Tyr Lys Thr lie Leu Thr Asn Leu Asp Glu 20 25 30 Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gin Leu 35 40 45 Lys Lys Leu Asn Glu Ser lie Asp Val Phe Met Asn Lys Tyr Lys Thr 50 55 60 Ser Ser Arg Asn Arg Al a Leu Ser Asn Leu Lys Lys Asp lie Leu Lys 65 70 75 80 Glu Val lie Leu lie Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95 Leu Hi s Phe Val T rp lie Gly Gly Glu Val Ser Asp lie Al a Leu Glu 100 105 110 Tyr lie Lys Gin T rp Al a Asp lie Asn Al a Glu Tyr Asn lie Lys Leu 115 120 125 T rp Tyr Asp Ser Glu Al a Phe Leu Val Asn Thr Leu Lys Lys Al a lie 130 135 140 Val Glu Ser Ser Thr Thr Glu Al a Leu Gin Leu Leu Glu Glu Glu lie 145 150 155 160 Gin Asn Pro Gin Phe Asp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu 165 170 175 Phe lie Tyr Asp Arg Gin Lys Arg Phe lie Asn Tyr Tyr Lys Ser Gin 180 185 190 lie Asn Lys Pro Thr Val Pro Thr lie Asp Asp lie lie Lys Ser Hi s 195 200 205 Leu Val Ser Glu Tyr Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg 210 215 220 Thr Asn Ser Leu Arg Lys lie Asn Ser Asn Hi s Gly lie Asp lie Arg 225 230 235 240 Al a Asn Ser Leu Phe Thr Glu Gin Glu Leu Leu Asn lie Tyr Ser Gin 245 250 255 Glu Leu Leu Asn Arg Gly Asn Leu Al a Al a Al a Ser Asp lie Val Arg 260 265 270 Leu Leu Al a Leu Lys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met 275 280 285 Leu Pro Gly lie Hi s Ser Asp Leu Phe Lys Thr lie Ser Arg Pro Ser 290 295 300 Ser lie Gly Leu Asp Arg T rp Glu Met lie Lys Leu Glu Al a lie Met 305 310 315 320 Lys Tyr Lys Lys Tyr lie Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335 Leu Asp Gin Gin Leu Lys Asp Asn Phe Lys Leu lie lie Glu Ser Lys 340 345 350 Ser Glu Lys Ser Glu lie Phe Ser Lys Leu Glu Asn Leu Asn Val Ser 355 360 365 Asp Leu Glu lie Lys lie Al a Phe Al a Leu Gly Ser Val lie Asn Gin 370 375 380

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VB64650 Seq Li st Al a Leu lie Ser Lys Gin Gly Ser Tyr Leu Thr Asn Leu Val lie Glu 385 390 395 400 Gin Val Lys Asn Arg Tyr Gin Phe Leu Asn Gin Hi s Leu Asn Pro Al a 405 410 415 lie Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys lie Phe Hi s Asp 420 425 430 Ser Leu Phe Asn Ser Al a Thr Al a Glu Asn Ser Met Phe Leu Thr Lys 435 440 445 lie Al a Pro Tyr Leu Gin Val Gly Phe Met Pro Glu Al a Arg Ser Thr 450 455 460 lie Ser Leu Ser Gly Pro Gly Al a Tyr Al a Ser Al a Tyr Tyr Asp Phe 465 470 475 480 lie Asn Leu Gin Glu Asn Thr lie Glu Lys Thr Leu Lys Al a Ser Asp 485 490 495 Leu lie Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gin Leu Thr Glu 500 505 510 Gin Glu lie Asn Ser Leu T rp Ser Phe Asp Gin Al a Ser Al a Lys Tyr 515 520 525 Gin Phe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu 530 535 540 Asp Asn Gly Val Asp Phe Asn Lys Asn Thr Al a Leu Asp Lys Asn Tyr 545 550 555 560 Leu Leu Asn Asn Lys lie Pro Ser Asn Asn Val Glu Glu Al a Gly Ser 565 570 575 Lys Asn Tyr Val Hi s Tyr lie lie Gin Leu Gin Gly Asp Asp lie Ser 580 585 590 Tyr Glu Al a Thr cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser lie 595 600 605 lie lie Gin Arg Asn Met Asn Glu Ser Al a Lys Ser Tyr Phe Leu Ser 610 615 620 Asp Asp Gly Glu Ser lie Leu Glu Leu Asn Lys Tyr Arg lie Pro Glu 625 630 635 640 Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe lie Gly Hi s Gly 645 650 655 Lys Asp Glu Phe Asn Thr Ser Glu Phe Al a Arg Leu Ser Val Asp Ser 660 665 670 Leu Ser Asn Glu lie Ser Ser Phe Leu Asp Thr lie Lys Leu Asp lie 675 680 685 Ser Pro Lys Asn Val Glu Val Asn Leu Leu Gly cys Asn Met Phe Ser 690 695 700 Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser 705 710 715 720 lie Met Asp Lys lie Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser 725 730 735 lie Thr lie Gly Al a Asn Gin Tyr Glu Val Arg lie Asn Ser Glu Gly 740 745 750 Arg Lys Glu Leu Leu Al a Hi s Ser Gly Lys T rp lie Asn Lys Glu Glu 755 760 765 Al a lie Met Ser Asp Leu Ser Ser Lys Glu Tyr lie Phe Phe Asp Ser 770 775 780 lie Asp Asn Lys Leu Lys Al a Lys Ser Lys Asn lie Pro Gly Leu Al a 785 790 795 800 Ser lie Ser Glu Asp lie Lys Thr Leu Leu Leu Asp Al a Ser Val Ser 805 810 815 Pro Asp Thr Lys Phe lie Leu Asn Asn Leu Lys Leu Asn lie Glu Ser 820 825 830 Ser lie Gly Asp Tyr lie Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn 835 840 845 lie lie Hi s Asn Ser lie Asp Asp Leu lie Asp Glu Phe Asn Leu Leu 850 855 860 Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu 865 870 875 880 Asp Glu Lys Tyr Leu lie Ser Phe Glu Asp lie Ser Lys Asn Asn Ser 885 890 895 Thr Tyr Ser Val Arg Phe lie Asn Lys Ser Asn Gly Glu Ser Val Tyr 900 905 910 Val Glu Thr Glu Lys Glu lie Phe Ser Lys Tyr Ser Glu Hi s lie Thr 915 920 925

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VB64650 Seq Li st Lys Gl u lie Ser Thr lie Lys Asn Ser lie lie Thr Asp Val Asn Gly 930 935 940 Asn Leu Leu Asp Asn lie Gin Leu Asp His Thr Ser Gin Val Asn Thr 945 950 955 960 Leu Asn Ala Ala Phe Phe lie Gin Ser Leu lie Asp Tyr Ser Ser Asn 965 970 975 Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gin Leu Tyr 980 985 990 Al a Gln Leu Phe Ser Thr Gly Leu Asn Thr lie Tyr Asp Ser lie Gin 995 1000 1005 Leu Val Asn Leu lie Ser Asn Ala Val Asn Asp Thr lie Asn Val Leu 1010 1015 1020 Pro Thr lie Thr Glu Gly lie Pro lie Val Ser Thr lie Leu Asp Gly 1025 1030 1035 1040 lie Asn Leu Gly Ala Ala lie Lys Glu Leu Leu Asp Glu His Asp Pro 1045 1050 1055 Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val Leu Ala lie Asn 1060 1065 1070 Met Ser Leu Ser lie Ala Ala Thr Val Ala Ser lie Val Gly lie Gly 1075 1080 1085 Al a Glu Val Thr lie Phe Leu Leu Pro lie Al a Gly lie Ser Ala Gly 1090 1095 1100 lie Pro Ser Leu Val Asn Asn Glu Leu lie Leu His Asp Lys Ala Thr 1105 1110 1115 1120 Ser Val Val Asn Tyr Phe Asn His Leu Ser Glu Ser Lys Lys Tyr Gly 1125 1130 1135 Pro Leu Lys Thr Glu Asp Asp Lys lie Leu Val Pro lie Asp Asp Leu 1140 1145 1150 Val lie Ser Glu lie Asp Phe Asn Asn Asn Ser lie Lys Leu Gly Thr 1155 1160 1165 Cys Asn lie Leu Ala Met Glu Gly Gly Ser Gly His Thr Val Thr Gly 1170 1175 1180 Asn lie Asp His Phe Phe Ser Ser Pro Ser lie Ser Ser His lie Pro 1185 1190 1195 1200 Ser Leu Ser lie Tyr Ser Ala lie Gly lie Glu Thr Glu Asn Leu Asp 1205 1210 1215 Phe Ser Lys Lys lie Met Met Leu Pro Asn Al a Pro Ser Arg Val Phe 1220 1225 1230 Trp Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp 1235 1240 1245 Gly Thr Arg Leu Leu Asp Ser lie Arg Asp Leu Tyr Pro Gly Lys Phe 1250 1255 1260 Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Al a lie Thr Thr Leu Lys 1265 1270 1275 1280 Pro Val Tyr Glu Asp Thr Asn lie Lys lie Lys Leu Asp Lys Asp Thr 1285 1290 1295 Arg Asn Phe lie Met Pro Thr lie Thr Thr Asn Glu lie Arg Asn Lys 1300 1305 1310 Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr Tyr Ser Leu Leu Leu 1315 1320 1325 Ser Ser Tyr Pro lie Ser Thr Asn lie Asn Leu Ser Lys Asp Asp Leu 1330 1335 1340 Trp lie Phe Asn lie Asp Asn Glu Val Arg Glu lie Ser lie Glu Asn 1345 1350 1355 1360 Gly Thr lie Lys Lys Gly Lys Leu lie Lys Asp Val Leu Ser Lys lie 1365 1370 1375 Asp lie Asn Lys Asn Lys Leu lie lie Gly Asn Gin Thr lie Asp Phe 1380 1385 1390 Ser Gly Asp lie Asp Asn Lys Asp Arg Tyr lie Phe Leu Thr Cys Glu 1395 1400 1405 Leu Asp Asp Lys lie Ser Leu lie lie Glu lie Asn Leu Val Ala Lys 1410 1415 1420 Ser Tyr Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr Leu lie Ser Asn 1425 1430 1435 1440 Leu Ser Asn Thr lie Glu Lys lie Asn Thr Leu Gly Leu Asp Ser Lys 1445 1450 1455 Asn lie Ala Tyr Asn Tyr Thr Asp Glu Ser Asn Asn Lys Tyr Phe Gly 1460 1465 1470

Page 3

2016203241 18 May 2016

VB64650 Seq Li st Ala lie Ser Lys Thr Ser Gin Lys Ser lie lie His Tyr Lys Lys Asp 1475 1480 1485 Ser Lys Asn lie Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe Asn 1490 1495 1500 Ser Lys Asp Phe lie Ala Glu Asp lie Asn Val Phe Met Lys Asp Asp 1505 1510 1515 1520 lie Asn Thr lie Thr Gly Lys Tyr Tyr Val Asp Asn Asn Thr Asp Lys 1525 1530 1535 Ser lie Asp Phe Ser lie Ser Leu Val Ser Lys Asn Gin Val Lys Val 1540 1545 1550 Asn Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser Ser Tyr Leu Asp Phe 1555 1560 1565 Val Lys Asn Ser Asp Gly His His Asn Thr Ser Asn Phe Met Asn Leu 1570 1575 1580 Phe Leu Asp Asn lie Ser Phe Trp Lys Leu Phe Gly Phe Glu Asn lie 1585 1590 1595 1600 Asn Phe Val lie Asp Lys Tyr Phe Thr Leu Val Gly Lys Thr Asn Leu 1605 1610 1615 Gly Tyr Val Glu Phe lie Cys Asp Asn Asn Lys Asn lie Asp lie Tyr 1620 1625 1630 Phe Gly Glu Trp Lys Thr Ser Ser Ser Lys Ser Thr lie Phe Ser Gly 1635 1640 1645 Asn Gly Arq Asn Val Val Val Glu Pro lie Tyr Asn Pro Asp Thr Gly 1650 1655 1660 Glu Asp lie Ser Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly 1665 1670 1675 1680 lie Asp Arg Tyr lie Asn Lys Val Leu lie Al a Pro Asp Leu Tyr Thr 1685 1690 1695 Ser Leu lie Asn lie Asn Thr Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro 1700 1705 1710 Glu lie lie Val Leu Asn Pro Asn Thr Phe Hi s Lys Lys Val Asn lie 1715 1720 1725 Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys T rp Ser Thr Glu Gly Ser 1730 1735 1740 Asp Phe lie Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys lie Leu 1745 1750 1755 1760 Gl n Lys lie Arq lie Lys Gly lie Leu Ser Asn Thr Gin Ser Phe Asn 1765 1770 1775 Lys Met Ser lie Asp Phe Lys Asp lie Lys Lys Leu Ser Leu Gly Tyr 1780 1785 1790 lie Met Ser Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu Asp Arg 1795 1800 1805 Asp His Leu Gly Phe Lys lie lie Asp Asn Lys Thr Tyr Tyr Tyr Asp 1810 1815 1820 Glu Asp Ser Lys Leu Val Lys Gly Leu lie Asn lie Asn Asn Ser Leu 1825 1830 1835 1840 Phe Tyr Phe Asp Pro lie Glu Phe Asn Leu Val Thr Gly Trp Gin Thr 1845 1850 1855 lie Asn Gly Lys Lys Tyr Tyr Phe Asp lie Asn Thr Gly Ala Ala Leu 1860 1865 1870 Thr Ser Tyr Lys lie lie Asn Gly Lys His Phe Tyr Phe Asn Asn Asp 1875 1880 1885 Gly Val Met Gin Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 1890 1895 1900 Phe Ala Pro Ala Asn Thr Gin Asn Asn Asn lie Glu Gly Gin Ala lie 1905 1910 1915 1920 Val Tyr Gin Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe 1925 1930 1935 Asp Asn Asn Ser Lys Ala Val Thr Gly Trp Arg lie lie Asn Asn Glu 1940 1945 1950 Lys Tyr Tyr Phe Asn Pro Asn Asn Ala lie Al a Ala Val Gly Leu Gin 1955 1960 1965 Val lie Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala lie lie 1970 1975 1980 Ser Lys Gly Trp Gin Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr 1985 1990 1995 2000 Asp Thr Ala lie Ala Phe Asn Gly Tyr Lys Thr lie Asp Gly Lys His 2005 2010 2015

Page 4

2016203241 18 May 2016

VB64650 Seq List

Phe Tyr Phe Asp Ser Asp Cys Val Val Lys lie Gly Val Phe Ser Thr 2020 2025 2030

Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Asn Asn Asn 2035 2040 2045 lie Glu Gly Gin Ala lie Val Tyr Gin Ser Lys Phe Leu Thr Leu Asn 2050 2055 2060

Gly Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Al a Val Thr Gly Leu 2065 2070 2075 2080 Gin Thr lie Asp Ser Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Al a Glu 2085 2090 2095 Ala Ala Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 2100 2105 2110 Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gin Thr lie Asp Gly Lys 2115 2120 2125 Lys Tyr Tyr Phe Asn Thr Asn Thr Ala lie Al a Ser Thr Gly Tyr Thr 2130 2135 2140 lie lie Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie Met Gin 2145 2150 2155 2160 lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a 2165 2170 2175 Asn Thr Asp Ala Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn 2180 2185 2190 Glu Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser 2195 2200 2205 Lys Ala Val Thr Gly Trp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe 2210 2215 2220 Asn Pro Asn Asn Al a lie Ala Ala lie His Leu Cys Thr lie Asn Asn 2225 2230 2235 2240 Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie 2245 2250 2255 Thr lie Glu Arg Asn Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys 2260 2265 2270 Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a 2275 2280 2285 Pro Ala Asn Thr Hi s Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr 2290 2295 2300 Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn 2305 2310 2315 2320 Asp Ser Lys Ala Val Thr Gly Trp Gin Thr lie Asp Gly Lys Lys Tyr 2325 2330 2335 Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly T rp Gin Thr lie 2340 2345 2350 Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr 2355 2 360 2365 Gly Trp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr 2370 2375 2380 Phe lie Ala Ser Thr Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr 2385 2390 2395 2400 Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asn 2405 2410 2415 Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Al a Asn Asn lie Glu 2420 2425 2430 Gly Gin Ala lie Leu Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435 2440 2445 Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr 2450 2455 2460 lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val 2465 2470 2475 2480 Thr Gly Trp Gin Thr lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 2485 2490 2495 Thr Ser lie Ala Ser Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe 2500 2505 2510 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 2515 2520 2525 Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Al a Asn Asn lie 2530 2535 2540 Glu Gly Gl n Al a lie Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp 2545 2550 2555 2560 Page 5

2016203241 18 May 2016

VB64650 Seq List

Asn lie Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val 2565 2570 2575

Thr lie Asp Gly Asn Arg Tyr Tyr 2580 Phe Glu 2585 Pro Asn Thr Ala Met 2590 Gly Al a Asn Gly Tyr Lys Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn 2595 2600 2605 Gly Leu Pro Gin lie Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr 2610 2615 2620 Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn lie Glu Gly Gl n Al a lie 2625 2630 2635 2640 Arg Tyr Gin Asn Arg Phe Leu His Leu Leu Gly Lys lie Tyr Tyr Phe 2645 2650 2655 Gly Asn Asn Ser Lys Ala Val Thr Gly Trp Gin Thr lie Asn Gly Lys 2660 2665 2670 Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Al a Al a Al a Gly Gly Leu 2675 2680 2685 Phe Glu lie Asp Gly Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys 2690 2695 2700 Al a Pro Gly lie Tyr Gly

2705 2710 <210> 2 <211> 2366 <212> PRT <213> Clostridium Difficile <400> 2

Met Ser Leu Val Asn Arg Lys Gin Leu Glu Lys Met Al a Asn Val Arg 1 5 10 15 Phe Arg Thr Gin Glu Asp Glu Tyr Val Al a lie Leu Asp Al a Leu Glu 20 25 30 Glu Tyr Hi s Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45 Leu Lys Asp lie Asn Ser Leu Thr Asp lie Tyr lie Asp Thr Tyr Lys 50 55 60 Lys Ser Gly Arg Asn Lys Al a Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80 Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95 Asn Leu Hi s Phe Val T rp lie Gly Gly Gin lie Asn Asp Thr Al a lie 100 105 110 Asn Tyr lie Asn Gin T rp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 Val Phe Tyr Asp Ser Asn Al a Phe Leu lie Asn Thr Leu Lys Lys Thr 130 135 140 Val Val Glu Ser Al a lie Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175 Glu lie lie Tyr Asp Lys Gin Lys Asn Phe lie Asn Tyr Tyr Lys Al a 180 185 190 Gin Arg Glu Glu Asn Pro Glu Leu lie lie Asp Asp lie Val Lys Thr 195 200 205 Tyr Leu Ser Asn Glu Tyr Ser Lys Glu lie Asp Glu Leu Asn Thr Tyr 210 215 220 lie Glu Glu Ser Leu Asn Lys lie Thr Gin Asn Ser Gly Asn Asp Val 225 230 235 240 Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255 Gin Glu Leu Val Glu Arg T rp Asn Leu Al a Al a Al a Ser Asp lie Leu 260 265 270 Arg lie Ser Al a Leu Lys Glu lie Gly Gly Met Tyr Leu Asp Val Asp 275 280 285 Met Leu Pro Gly lie Gin Pro Asp Leu Phe Glu Ser lie Glu Lys Pro 290 295 300 Ser Ser Val Thr Val Asp Phe T rp Glu Met Thr Lys Leu Glu Al a lie 305 310 315 320

Page 6

2016203241 18 May 2016

VB64650 Seq Li st Met Lys Tyr Lys Glu Tyr lie Pro Glu Tyr Thr Ser Glu Hi s Phe Asp 325 330 335 Met Leu Asp Glu Glu Val Gin Ser Ser Phe Glu Ser Val Leu Al a Ser 340 345 350 Lys Ser Asp Lys Ser Glu lie Phe Ser Ser Leu Gly Asp Met Glu Al a 355 360 365 Ser Pro Leu Glu Val Lys lie Al a Phe Asn Ser Lys Gly lie lie Asn 370 375 380 Gin Gly Leu lie Ser Val Lys Asp Ser Tyr cys Ser Asn Leu lie Val 385 390 395 400 Lys Gin lie Glu Asn Arg Tyr Lys lie Leu Asn Asn Ser Leu Asn Pro 405 410 415 Al a lie Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe lie 420 425 430 Asp Ser lie Met Al a Glu Al a Asn Al a Asp Asn Gly Arg Phe Met Met 435 440 445 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460 Thr lie Asn Leu Ser Gly Pro Glu Al a Tyr Al a Al a Al a Tyr Gin Asp 465 470 475 480 Leu Leu Met Phe Lys Glu Gly Ser Met Asn lie Hi s Leu lie Glu Al a 485 490 495 Asp Leu Arg Asn Phe Glu lie Ser Lys Thr Asn lie Ser Gin Ser Thr 500 505 510 Glu Gin Glu Met Al a Ser Leu T rp Ser Phe Asp Asp Al a Arg Al a Lys 515 520 525 Al a Gin Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540 Glu Asp Asp Asn Leu Asp Phe Ser Gin Asn lie Val Val Asp Lys Glu 545 550 555 560 Tyr Leu Leu Glu Lys lie Ser Ser Leu Al a Arg Ser Ser Glu Arg Gly 565 570 575 Tyr lie Hi s Tyr lie Val Gin Leu Gin Gly Asp Lys lie Ser Tyr Glu 580 585 590 Al a Al a Cys Asn Leu Phe Al a Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605 Gin Lys Asn lie Glu Asp Ser Glu lie Al a Tyr Tyr Tyr Asn Pro Gly 610 615 620 Asp Gly Glu lie Gin Glu lie Asp Lys Tyr Lys lie Pro Ser lie lie 625 630 635 640 Ser Asp Arg Pro Lys lie Lys Leu Thr Phe lie Gly Hi s Gly Lys Asp 645 650 655 Glu Phe Asn Thr Asp lie Phe Al a Gly Phe Asp Val Asp Ser Leu Ser 660 665 670 Thr Glu lie Glu Al a Al a lie Asp Leu Al a Lys Glu Asp lie Ser Pro 675 680 685 Lys Ser lie Glu lie Asn Leu Leu Gly cys Asn Met Phe Ser Tyr Ser 690 695 700 lie Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys 705 710 715 720 Asp Lys lie Ser Glu Leu Met Pro Ser lie Ser Gin Asp Ser lie lie 725 730 735 Val Ser Al a Asn Gin Tyr Glu Val Arg lie Asn Ser Glu Gly Arg Arg 740 745 750 Glu Leu Leu Asp Hi s Ser Gly Glu T rp lie Asn Lys Glu Glu Ser lie 755 760 765 lie Lys Asp lie Ser Ser Lys Glu Tyr lie Ser Phe Asn Pro Lys Glu 770 775 780 Asn Lys lie Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu 785 790 795 800 Leu Gin Glu lie Arg Asn Asn Ser Asn Ser Ser Asp lie Glu Leu Glu 805 810 815 Glu Lys Val Met Leu Thr Glu cys Glu lie Asn Val lie Ser Asn lie 820 825 830 Asp Thr Gin lie Val Glu Glu Arg lie Glu Glu Al a Lys Asn Leu Thr 835 840 845 Ser Asp Ser lie Asn Tyr lie Lys Asp Glu Phe Lys Leu lie Glu Ser 850 855 860

Page 7

2016203241 18 May 2016

VB64650 Seq Li st lie Ser Asp Ala Leu Cys Asp Leu Lys Gln Gin Asn Glu Leu Glu Asp 865 870 875 880 Ser His Phe lie Ser Phe Glu Asp lie Ser Glu Thr Asp Glu Gly Phe 885 890 895 Ser lie Arg Phe lie Asn Lys Glu Thr Gly Glu Ser lie Phe Val Glu 900 905 910 Thr Glu Lys Thr lie Phe Ser Glu Tyr Ala Asn His lie Thr Glu Glu 915 920 925 lie Ser Lys lie Lys Gly Thr lie Phe Asp Thr Val Asn Gly Lys Leu 930 935 940 Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn 945 950 955 960 Ala Ala Phe Phe lie Gin Ser Leu lie Glu Tyr Asn Ser Ser Lys Glu 965 970 975 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gin Val Tyr Ala Gin 980 985 990 Leu Phe Ser Thr Gly Leu Asn Thr lie Thr Asp Ala Ala Lys Val Val 995 1000 1005 Glu Leu Val Ser Thr Ala Leu Asp Glu Thr lie Asp Leu Leu Pro Thr 1010 1015 1020 Leu Ser Glu Gly Leu Pro lie lie Ala Thr lie lie Asp Gly Val Ser 1025 1030 1035 1040 Leu Gly Ala Ala lie Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 Arg Gin Glu lie Glu Ala Lys lie Gly lie Met Ala Val Asn Leu Thr 1060 1065 1070 Thr Ala Thr Thr Ala lie lie Thr Ser Ser Leu Gly lie Ala Ser Gly 1075 1080 1085 Phe Ser lie Leu Leu Val Pro Leu Ala Gly lie Ser Ala Gly lie Pro 1090 1095 1100 Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val 1105 1110 1115 1120 Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe 1125 1130 1135 Thr Leu Leu Asp Asp Lys lie Met Met Pro Gin Asp Asp Leu Val lie 1140 1145 1150 Ser Glu lie Asp Phe Asn Asn Asn Ser lie Val Leu Gly Lys Cys Glu 1155 1160 1165 lie Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp lie 1170 1175 1180 Asp His Phe Phe Ser Ala Pro Ser lie Thr Tyr Arq Glu Pro His Leu 1185 1190 1195 1200 Ser lie Tyr Asp Val Leu Glu Val Gl n Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215 Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230 Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245 Lys Leu Leu Asp Arg lie Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 Arg Tyr Phe Ala Phe lie Ala Asp Ala Leu lie Thr Thr Leu Lys Pro 1265 1270 1275 1280 Arg Tyr Glu Asp Thr Asn lie Arg lie Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 Ser Phe lie Val Pro lie lie Thr Thr Glu Tyr lie Arg Glu Lys Leu 1300 1305 1310 Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325 Gin Tyr Asn Met Gly lie Asn lie Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340 lie lie Asp Val Asp Asn Val Val Arg Asp Val Thr lie Glu Ser Asp 1345 1350 1355 1360 Lys lie Lys Lys Gly Asp Leu lie Glu Gly lie Leu Ser Thr Leu Ser 1365 1370 1375 lie Glu Glu Asn Lys lie lie Leu Asn Ser Hi s Glu lie Asn Phe Ser 1380 1385 1390 Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser lie

1395 1400 1405

Page 8

2016203241 18 May 2016

VB64650 Seq List

Leu Glu Gly lie Asn Ala lie lie Glu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420

Tyr Lys 1425 Leu Leu lie Ser Gly 1430 Glu Leu Lys lie Leu 1435 Met Leu Asn Ser 1440 Asn His lie Gin Gin Lys lie Asp Tyr lie Gly Phe Asn Ser Glu Leu 1445 1450 1455 Gl n Lys Asn lie Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 1470 Gly Phe lie Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485 Pro Asp Val Val Leu lie Ser Lys Val Tyr Met Asp Asp Ser Lys Pro 1490 1495 1500 Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val lie Thr 1505 1510 1515 1520 Lys Asp Asn Val Asn lie Leu Thr Gly Tyr Tyr Leu Lys Asp Asp lie 1525 1530 1535 Lys lie Ser Leu Ser Leu Thr Leu Gin Asp Gl u Lys Thr lie Lys Leu 1540 1545 1550 Asn Ser Val Hi s Leu Asp Glu Ser Gly Val Al a Glu lie Leu Lys Phe 1555 1560 1565 Met Asn Ara Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580 Leu Glu Ser Met Asn lie Lys Ser lie Phe Val Asn Phe Leu Gin Ser 1585 1590 1595 1600 Asn lie Lys Phe lie Leu Asp Al a Asn Phe lie lie Ser Gly Thr Thr 1605 1610 1615 Ser lie Gly Gin Phe Glu Phe lie cys Asp Glu Asn Asp Asn lie Gin 1620 1625 1630 Pro Tyr Phe lie Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr 1635 1640 1645 Val Gly Asn Arg Gin Asn Met lie Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 Asp Ser Gly Asp lie Ser Ser Thr Val lie Asn Phe Ser Gin Lys Tyr 1665 1670 1675 1680 Leu Tyr Gly lie Asp Ser Cys Val Asn Lys Val Val lie Ser Pro Asn 1685 1690 1695 lie Tyr Thr Asp Glu lie Asn lie Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710 Thr Tyr Pro Glu Val lie Val Leu Asp Al a Asn Tyr lie Asn Glu Lys 1715 1720 1725 lie Asn Val Asn lie Asn Asp Leu Ser lie Arg Tyr Val T rp Ser Asn 1730 1735 1740 Asp Gly Asn Asp Phe lie Leu Met Ser Thr Ser Glu Glu Asn Lys Val 1745 1750 1755 1760 Ser Gin Val Lys lie Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu 1765 1770 1775 Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro Val 1780 1785 1790 Ser Glu lie lie Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805 lie Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 lie Asn Asn Phe Gly Met Met Val Ser Gly Leu lie Tyr lie Asn Asp 1825 1830 1835 1840 Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu lie Thr Gly Phe 1845 1850 1855 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro lie Asn Gly Gly 1860 1865 1870 Ala Ala Ser lie Gly Glu Thr lie lie Asp Asp Lys Asn Tyr Tyr Phe 1875 1880 1885 Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900 Phe Lys Tyr Phe Al a Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920 Gl u Al a lie Asp Phe Thr Gly Lys Leu lie lie Asp Glu Asn lie Tyr 1925 1930 1935 Tyr Phe Asp Asp Asn Tyr Arg Gly Al a Val Glu Trp Lys Glu Leu Asp

1940 1945 1950

Page 9

2016203241 18 May 2016

VB64650 Seq Li st Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965 Leu Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 1970 1975 1980 Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn Lys His Tyr Phe Asp 1985 1990 1995 2000 Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys His 2005 2010 2015 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn Thr 2020 2025 2030 Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn 2035 2040 2045 Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn 2050 2055 2060 Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val Gly Trp Lys 2065 2070 2075 2080 Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 Ala Tyr lie Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr Phe Asn 2100 2105 2110 Asp Asp Gly lie Met Gin Val Gly Phe Val Thr lie Asn Asp Lys Val 2115 2120 2125 Phe Tyr Phe Ser Asp Ser Gly lie lie Glu Ser Gly Val Gin Asn lie 2130 2135 2140 Asp Asp Asn Tyr Phe Tyr lie Asp Asp Asn Gly lie Val Gin lie Gly 2145 2150 2155 2160 Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175 Val Asn Asp Asn lie Tyr Gly Gin Ala Val Glu Tyr Ser Gly Leu Val 2180 2185 2190 Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu 2195 2200 2205 Thr Gly Trp lie Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 2210 2215 2220 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly lie Asn Leu lie Asp Asp 2225 2230 2235 2240 lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met Arg Thr Gly Leu lie 2245 2250 2255 Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin 2260 2265 2270 Phe Gly Tyr lie Asn lie Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp 2275 2280 2285 Gly Val Met Gin lie Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr 2290 2295 2300 Phe Ala His Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser lie 2305 2310 2315 2320 Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335 Asp Glu Tyr lie Ala Ala Thr Gly Ser Val lie lie Asp Gly Glu Glu 2340 2345 2350 Tyr Tyr Phe Asp Pro Asp Thr Ala Gin Leu Val lie Ser Glu

2355 2360 2365 <210> 3 <211> 966 <212> PRT <213> Clostridium Difficile <400> 3

Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60

Page 10

2016203241 18 May 2016

VB64650 Seq Li st Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95 lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110 Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125 Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220 Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285 Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335 Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp 500 505 510 Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu 515 520 525 Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Val 530 535 540 Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 545 550 555 560 Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly Val lie 565 570 575 Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Pro Gly Phe Val Ser lie 580 585 590 Asn Asp Asn Lys Hi s Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly 595 600 605

Page 11

2016203241 18 May 2016

VB64650 Seq Li st Tyr Thr Glu lie Asp Gly Lys His Phe Tyr Phe Al a Glu Asn Gly Glu 610 615 620 Met Gin lie Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Al a 625 630 635 640 Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr 645 650 655 Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser 660 665 670 Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr 675 680 685 Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie Gly Leu Ser Leu lie 690 695 700 Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly lie Met Gin Val Gly 705 710 715 720 Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly lie 725 730 735 lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn Tyr Phe Tyr lie Asp 740 745 750 Asp Asn Gly lie Val Gin lie Gly Val Phe Asp Thr Ser Asp Gly Tyr 755 760 765 Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp Asn lie Tyr Gly Gin 770 775 780 Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr 785 790 795 800 Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp lie Tyr Asp Met Glu 805 810 815 Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Al a cys 820 825 830 Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys 835 840 845 Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr 850 855 860 Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr lie Asn lie Glu Asp 865 870 875 880 Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gin lie Gly Val Phe 885 890 895 Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s Gin Asn Thr Leu Asp 900 905 910 Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr Gly T rp Leu Asp Leu 915 920 925 Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr lie Al a Al a Thr Gly 930 935 940 Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a 945 950 955 960 Gin Leu Val lie Ser Glu

965 <210> 4 <211> 966 <212> PRT <213> Clostridium Difficile <400> 4

Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60 Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95 lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110

Page 12

2016203241 18 May 2016

VB64650 Seq Li st Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125 Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220 Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285 Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335 Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp 500 505 510 Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu 515 520 525 Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Val 530 535 540 Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 545 550 555 560 Thr Al a Met Al a Al a Al a Gly Gly Leu Asn Gin lie Gly Asp Tyr Lys 565 570 575 Tyr Tyr Phe Asn Ser Asp Gly Val Met Gin Lys Gly Phe Val Ser lie 580 585 590 Asn Asp Asn Lys Hi s Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly 595 600 605 Tyr Thr Glu lie Asp Gly Lys His Phe Tyr Phe Al a Glu Asn Gly Glu 610 615 620 Met Gin lie Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Al a 625 630 635 640 Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr 645 650 655

Page 13

2016203241 18 May 2016

VB64650 Seq Li st Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser 660 665 670 Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr 675 680 685 Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie Gly Leu Ser Leu lie 690 695 700 Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly lie Met Gin Val Gly 705 710 715 720 Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly lie 725 730 735 lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn Tyr Phe Tyr lie Asp 740 745 750 Asp Asn Gly lie Val Gin lie Gly Val Phe Asp Thr Ser Asp Gly Tyr 755 760 765 Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp Asn lie Tyr Gly Gin 770 775 780 Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr 785 790 795 800 Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp lie Tyr Asp Met Glu 805 810 815 Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Al a cys 820 825 830 Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys 835 840 845 Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr 850 855 860 Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr lie Asn lie Glu Asp 865 870 875 880 Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gin lie Gly Val Phe 885 890 895 Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s Gin Asn Thr Leu Asp 900 905 910 Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr Gly T rp Leu Asp Leu 915 920 925 Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr lie Al a Al a Thr Gly 930 935 940 Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a 945 950 955 960 Gin Leu Val lie Ser Glu

965 <210> 5 <211> 833 <212> PRT <213> Clostridium Difficile <400> 5

Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60 Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95 lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110 Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125 Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160

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2016203241 18 May 2016

VB64650 Seq Li st Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220 Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285 Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335 Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Hi s Hi s Asn Glu Asp 500 505 510 Leu Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn 515 520 525 Phe Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val 530 535 540 Gly T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp 545 550 555 560 Thr Al a Glu Al a Tyr lie Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr 565 570 575 Tyr Phe Asn Asp Asp Gly lie Met Gin Val Gly Phe Val Thr lie Asn 580 585 590 Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly lie lie Glu Ser Gly Val 595 600 605 Gin Asn lie Asp Asp Asn Tyr Phe Tyr lie Asp Asp Asn Gly lie Val 610 615 620 Gin lie Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro 625 630 635 640 Al a Asn Thr Val Asn Asp Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser 645 650 655 Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr 660 665 670 Thr lie Glu Thr Gly T rp lie Tyr Asp Met Glu Asn Glu Ser Asp Lys 675 680 685 Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Al a cys Lys Gly lie Asn Leu 690 695 700

Page 15

2016203241 18 May 2016

VB64650 Seq Li st lie Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met Arg Thr 705 710 715 720 Gly Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly 725 730 735 Glu Met Gin Phe Gly Tyr lie Asn lie Glu Asp Lys Met Phe Tyr Phe 740 745 750 Gly Glu Asp Gly Val Met Gin lie Gly Val Phe Asn Thr Pro Asp Gly 755 760 765 Phe Lys Tyr Phe Al a Hi s Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly 770 775 780 Glu Ser lie Asn Tyr Thr Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr 785 790 795 800 Tyr Phe Thr Asp Glu Tyr lie Al a Al a Thr Gly Ser Val lie lie Asp 805 810 815 Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a Gin Leu Val lie Ser 820 825 830 Glu

<210> 6 <211> 1057 <212> PRT <213> Clostridium Difficile <400> 6

Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60 Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95 lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110 Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125 Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220 Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285 Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335

Page 16

2016203241 18 May 2016

VB64650 Seq Li st Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp 500 505 510 Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu 515 520 525 Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Val 530 535 540 Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 545 550 555 560 Thr Al a Met Al a Al a Al a Gly Gly Glu Thr lie lie Asp Asp Lys Asn 565 570 575 Tyr Tyr Phe Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe Ser Thr 580 585 590 Glu Asp Gly Phe Lys Tyr Phe Al a Pro Al a Asn Thr Leu Asp Glu Asn 595 600 605 Leu Glu Gly Glu Al a lie Asp Phe Thr Gly Lys Leu lie lie Asp Glu 610 615 620 Asn lie Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Al a Val Glu T rp Lys 625 630 635 640 Glu Leu Asp Gly Glu Met Hi s Tyr Phe Ser Pro Glu Thr Gly Lys Al a 645 650 655 Phe Lys Gly Leu Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser 660 665 670 Asp Gly Val Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn Lys Hi s 675 680 685 Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu lie Asp 690 695 700 Gly Lys Hi s Phe Tyr Phe Al a Glu Asn Gly Glu Met Gin lie Gly Val 705 710 715 720 Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp 725 730 735 Leu Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn 740 745 750 Phe Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val 755 760 765 Gly T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp 770 775 780 Thr Al a Glu Al a Tyr lie Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr 785 790 795 800 Tyr Phe Asn Asp Asp Gly lie Met Gin Val Gly Phe Val Thr lie Asn 805 810 815 Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly lie lie Glu Ser Gly Val 820 825 830 Gin Asn lie Asp Asp Asn Tyr Phe Tyr lie Asp Asp Asn Gly lie Val 835 840 845 Gin lie Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro 850 855 860 Al a Asn Thr Val Asn Asp Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser 865 870 875 880 Pag le 17

2016203241 18 May 2016

VB64650 Seq Li st Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr 885 890 895 Thr lie Glu Thr Gly Trp lie Tyr Asp Met Glu Asn Glu Ser Asp Lys 900 905 910 Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala cys Lys Gly lie Asn Leu 915 920 925 lie Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met Arg Thr 930 935 940 Gly Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly 945 950 955 960 Glu Met Gin Phe Gly Tyr lie Asn lie Glu Asp Lys Met Phe Tyr Phe 965 970 975 Gly Glu Asp Gly Val Met Gin lie Gly Val Phe Asn Thr Pro Asp Gly 980 985 990 Phe Lys Tyr Phe Ala His Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly 995 1000 1005 Glu Ser lie Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr 1010 1015 1020 Tyr Phe Thr Asp Glu Tyr lie Ala Ala Thr Gly Ser Val lie lie Asp 1025 1030 1035 1040 Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a Gin Leu Val lie Ser 1045 1050 1055 Glu

<210> 7 <211> 971 <212> PRT <213> Clostridium Difficile <400> 7 Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60 Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95 lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110 Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125 Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220 Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285

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2016203241 18 May 2016

VB64650 Seq Li st Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335 Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp 500 505 510 Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu 515 520 525 Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Val 530 535 540 Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 545 550 555 560 Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly Val lie 565 570 575 Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Pro Gly lie Tyr Gly Gly 580 585 590 Gly Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr Phe Asp Asp Ser Gly 595 600 605 Val Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys Hi s Phe Tyr Phe 610 615 620 Al a Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn Thr Glu Asp Gly 625 630 635 640 Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly 645 650 655 Glu Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr 660 665 670 Tyr Phe Asp Asp Ser Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu 675 680 685 Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie 690 695 700 Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly 705 710 715 720 lie Met Gin Val Gly Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe 725 730 735 Ser Asp Ser Gly lie lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn 740 745 750 Tyr Phe Tyr lie Asp Asp Asn Gly lie Val Gin lie Gly Val Phe Asp 755 760 765 Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp 770 775 780 Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly 785 790 795 800 Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp 805 810 815 lie Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu 820 825 830

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2016203241 18 May 2016

VB64650 Seq Li st Thr Lys Lys Al a cys Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr 835 840 845 Tyr Phe Asp Glu Lys Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu 850 855 860 Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr 865 870 875 880 lie Asn lie Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met 885 890 895 Gin lie Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s 900 905 910 Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr 915 920 925 Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr 930 935 940 lie Al a Al a Thr Gly Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe 945 950 955 960 Asp Pro Asp Thr Al a Gin Leu Val lie Ser Glu

965 970 <210> 8 <211> 321 <212> PRT <213> Clostridium Difficile <400> 8

Met Al a Ser Thr Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe 1 5 10 15 Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly 20 25 30 Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly 35 40 45 Gin Al a lie Leu Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys 50 55 60 Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie 65 70 75 80 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr 85 90 95 Gly T rp Gin Thr lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr 100 105 110 Ser lie Al a Ser Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr 115 120 125 Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp 130 135 140 Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu 145 150 155 160 Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn 165 170 175 lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr 180 185 190 lie Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a 195 200 205 Asn Gly Tyr Lys Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly 210 215 220 Leu Pro Gin lie Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe 225 230 235 240 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg 245 250 255 Tyr Gin Asn Arg Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly 260 265 270 Asn Asn Ser Lys Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val 275 280 285 Tyr Tyr Phe Met Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe 290 295 300 Glu lie Asp Gly Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a 305 310 315 320

Pro

Page 20

2016203241 18 May 2016 <210> 9 <211> 612 <212> PRT <213> Clostridium Difficile

VB64650 Seq List <400> 9

Met lie Leu Met Ser Thr Ser Glu Glu Asn Lys Val Ser Gin Val Lys 1 5 10 15 lie Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu Al a Asn Lys Leu 20 25 30 Ser Phe Asn Phe Ser Asp Lys Gin Asp Val Pro Val Ser Glu lie lie 35 40 45 Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu lie Gly Tyr Asp 50 55 60 Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr lie Asn Asn Phe 65 70 75 80 Gly Met Met Val Ser Gly Leu lie Tyr lie Asn Asp Ser Leu Tyr Tyr 85 90 95 Phe Lys Pro Pro Val Asn Asn Leu lie Thr Gly Phe Val Thr Val Gly 100 105 110 Asp Asp Lys Tyr Tyr Phe Asn Pro lie Asn Gly Gly Al a Al a Ser lie 115 120 125 Gly Glu Thr lie lie Asp Asp Lys Asn Tyr Tyr Phe Asn Gin Ser Gly 130 135 140 Val Leu Gin Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe 145 150 155 160 Al a Pro Al a Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Al a lie Asp 165 170 175 Phe Thr Gly Lys Leu lie lie Asp Glu Asn lie Tyr Tyr Phe Asp Asp 180 185 190 Asn Tyr Arg Gly Al a Val Glu T rp Lys Glu Leu Asp Gly Glu Met Hi s 195 200 205 Tyr Phe Ser Pro Glu Thr Gly Lys Al a Phe Lys Gly Leu Asn Gin lie 210 215 220 Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val Met Gin Lys Gly 225 230 235 240 Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr Phe Asp Asp Ser Gly Val 245 250 255 Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys Hi s Phe Tyr Phe Al a 260 265 270 Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn Thr Glu Asp Gly Phe 275 280 285 Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu 290 295 300 Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr Tyr 305 310 315 320 Phe Asp Asp Ser Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu Asp 325 330 335 Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie Gly 340 345 350 Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly lie 355 360 365 Met Gin Val Gly Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe Ser 370 375 380 Asp Ser Gly lie lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn Tyr 385 390 395 400 Phe Tyr lie Asp Asp Asn Gly lie Val Gin lie Gly Val Phe Asp Thr 405 410 415 Ser Asp Gly Tyr Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp Asn 420 425 430 lie Tyr Gly Gin Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu 435 440 445 Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp lie 450 455 460 Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr 465 470 475 480

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2016203241 18 May 2016

VB64650 Seq Li st Lys Lys Al a cys Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr Tyr 485 490 495 Phe Asp Glu Lys Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu Asn 500 505 510 Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr lie 515 520 525 Asn lie Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gin 530 535 540 lie Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s Gin 545 550 555 560 Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr Gly 565 570 575 T rp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr lie 580 585 590 Al a Al a Thr Gly Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe Asp 595 600 605 Pro Asp Thr Al a 610

<210> 10 <211> 587 <212> PRT <213> Clostridium Difficile <400> 10

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2016203241 18 May 2016

VB64650 Seq Li st Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp 500 505 510 Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu 515 520 525 Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Val 530 535 540 Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 545 550 555 560 Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly Val lie 565 570 575 Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Pro 580 585

<210> 11 <211> 567 <212> PRT <213> Clostridium Difficile <400> 11

Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60 Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95 lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110 Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125 Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220

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2016203241 18 May 2016

VB64650 Seq Li st Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285 Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335 Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp 500 505 510 Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu 515 520 525 Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Val 530 535 540 Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 545 550 555 560 Thr Al a Met Al a Al a Al a Gly 565

<210> 12 <211> 505 <212> PRT <213> Clostridium Difficile <400> 12

Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60 Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95 lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110 Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125

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2016203241 18 May 2016

VB64650 Seq Li st Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220 Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285 Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335 Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr

500 505 <210> 13 <211> 567 <212> PRT <213> Clostridium Difficile <400> 13

Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60 Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95

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VB64650 Seq Li st lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110 Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125 Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220 Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285 Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335 Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp 500 505 510 Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu 515 520 525 Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Val 530 535 540 Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 545 550 555 560 Thr Al a Met Al a Al a Al a Gly 565

<210> 14 <211> 591 <212> PRT <213> Clostridium Difficile <400> 14

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2016203241 18 May 2016

VB64650 Seq Li st Met Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 1 5 10 15 Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys Hi s Phe 20 25 30 Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys Gly Pro 35 40 45 Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie 50 55 60 Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu Asn Gly 65 70 75 80 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly T rp Arg 85 90 95 lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a lie Al a 100 105 110 Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr 115 120 125 Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn Asn Phe 130 135 140 Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 145 150 155 160 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s Asn Asn 165 170 175 Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu Thr Leu 180 185 190 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val Thr Gly 195 200 205 T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Al a 210 215 220 Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe 225 230 235 240 Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly 245 250 255 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr Gly Tyr 260 265 270 Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met 275 280 285 Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro 290 295 300 Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu Tyr Gin 305 310 315 320 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 325 330 335 Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys Tyr Tyr 340 345 350 Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr lie Asn 355 360 365 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser Thr Gly 370 375 380 Tyr Thr lie lie Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly lie 385 390 395 400 Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Al a 405 410 415 Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr 420 425 430 Gin Asn Arg Phe Leu Tyr Leu His Asp Asn lie Tyr Tyr Phe Gly Asn 435 440 445 Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn Arg Tyr 450 455 460 Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys Thr lie 465 470 475 480 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie Gly Val 485 490 495 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp 500 505 510 Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg Phe Leu 515 520 525 Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys Al a Val 530 535 540

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2016203241 18 May 2016

VB64650 Seq Li st Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 545 550 555 560 Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly Val lie 565 570 575 Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Pro Gly lie Tyr Gly

580 585 590 <210> 15 <211> 379 <212> PRT <213> Clostridium Difficile <400> 15

Gly Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr Phe Asp Asp Ser Gly 1 5 10 15 Val Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys Hi s Phe Tyr Phe 20 25 30 Al a Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn Thr Glu Asp Gly 35 40 45 Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly 50 55 60 Glu Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr 65 70 75 80 Tyr Phe Asp Asp Ser Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu 85 90 95 Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie 100 105 110 Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly 115 120 125 lie Met Gin Val Gly Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe 130 135 140 Ser Asp Ser Gly lie lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn 145 150 155 160 Tyr Phe Tyr lie Asp Asp Asn Gly lie Val Gin lie Gly Val Phe Asp 165 170 175 Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp 180 185 190 Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly 195 200 205 Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp 210 215 220 lie Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu 225 230 235 240 Thr Lys Lys Al a cys Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr 245 250 255 Tyr Phe Asp Glu Lys Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu 260 265 270 Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr 275 280 285 lie Asn lie Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met 290 295 300 Gin lie Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s 305 310 315 320 Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr 325 330 335 Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr 340 345 350 lie Al a Al a Thr Gly Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe 355 360 365 Asp Pro Asp Thr Al a Gin Leu Val lie Ser Glu 370 375

<210> 16 <211> 399 <212> PRT <213> Clostridium Difficile

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2016203241 18 May 2016

VB64650 Seq Li st <400> 16 Gly Leu Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly 1 5 10 15 Val Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr Phe 20 25 30 Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys 35 40 45 Hi s Phe Tyr Phe Al a Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn 50 55 60 Thr Glu Asp Gly Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp Leu Gly 65 70 75 80 Asn Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn 85 90 95 Asn Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val Gly T rp 100 105 110 Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Al a 115 120 125 Glu Al a Tyr lie Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr Phe 130 135 140 Asn Asp Asp Gly lie Met Gin Val Gly Phe Val Thr lie Asn Asp Lys 145 150 155 160 Val Phe Tyr Phe Ser Asp Ser Gly lie lie Glu Ser Gly Val Gin Asn 165 170 175 lie Asp Asp Asn Tyr Phe Tyr lie Asp Asp Asn Gly lie Val Gin lie 180 185 190 Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro Al a Asn 195 200 205 Thr Val Asn Asp Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser Gly Leu 210 215 220 Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie 225 230 235 240 Glu Thr Gly T rp lie Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr 245 250 255 Phe Asn Pro Glu Thr Lys Lys Al a Cys Lys Gly lie Asn Leu lie Asp 260 265 270 Asp lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met Arg Thr Gly Leu 275 280 285 lie Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met 290 295 300 Gin Phe Gly Tyr lie Asn lie Glu Asp Lys Met Phe Tyr Phe Gly Glu 305 310 315 320 Asp Gly Val Met Gin lie Gly Val Phe Asn Thr Pro Asp Gly Phe Lys 325 330 335 Tyr Phe Al a Hi s Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser 340 345 350 lie Asn Tyr Thr Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe 355 360 365 Thr Asp Glu Tyr lie Al a Al a Thr Gly Ser Val lie lie Asp Gly Glu 370 375 380 Glu Tyr Tyr Phe Asp Pro Asp Thr Al a Gin Leu Val lie Ser Glu

385 390 395 <210> 17 <211> 328 <212> PRT <213> Clostridium Difficile <400> 17

Phe Al a Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu lie 1 5 10 15 Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr Tyr Phe Asp 20 25 30 Asp Ser Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu Asp Gly Ser 35 40 45 Lys Tyr Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie Gly Leu Ser 50 55 60

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2016203241 18 May 2016

VB64650 Seq Li st Leu lie Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly lie Met Gin 65 70 75 80 Val Gly Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser 85 90 95 Gly lie lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn Tyr Phe Tyr 100 105 110 lie Asp Asp Asn Gly lie Val Gin lie Gly Val Phe Asp Thr Ser Asp 115 120 125 Gly Tyr Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp Asn lie Tyr 130 135 140 Gly Gin Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val 145 150 155 160 Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp lie Tyr Asp 165 170 175 Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys 180 185 190 Al a cys Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr Tyr Phe Asp 195 200 205 Glu Lys Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu Asn Asn Asn 210 215 220 Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr lie Asn lie 225 230 235 240 Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gin lie Gly 245 250 255 Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s Gin Asn Thr 260 265 270 Leu Asp Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr Gly T rp Leu 275 280 285 Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr lie Al a Al a 290 295 300 Thr Gly Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp 305 310 315 320 Thr Al a Gin Leu Val lie Ser Glu

325 <210> 18 <211> 490 <212> PRT <213> Clostridium Difficile <400> 18

Gly Glu Thr lie lie Asp Asp Lys Asn Tyr Tyr Phe Asn Gin Ser Gly 1 5 10 15 Val Leu Gin Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe 20 25 30 Al a Pro Al a Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Al a lie Asp 35 40 45 Phe Thr Gly Lys Leu lie lie Asp Glu Asn lie Tyr Tyr Phe Asp Asp 50 55 60 Asn Tyr Arg Gly Al a Val Glu T rp Lys Glu Leu Asp Gly Glu Met Hi s 65 70 75 80 Tyr Phe Ser Pro Glu Thr Gly Lys Al a Phe Lys Gly Leu Asn Gin lie 85 90 95 Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val Met Gin Lys Gly 100 105 110 Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr Phe Asp Asp Ser Gly Val 115 120 125 Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys Hi s Phe Tyr Phe Al a 130 135 140 Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn Thr Glu Asp Gly Phe 145 150 155 160 Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu 165 170 175 Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr Tyr 180 185 190 Phe Asp Asp Ser Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu Asp 195 200 205

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2016203241 18 May 2016

VB64650 Seq Li st Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie Gly 210 215 220 Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly lie 225 230 235 240 Met Gin Val Gly Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe Ser 245 250 255 Asp Ser Gly lie lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn Tyr 260 265 270 Phe Tyr lie Asp Asp Asn Gly lie Val Gin lie Gly Val Phe Asp Thr 275 280 285 Ser Asp Gly Tyr Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp Asn 290 295 300 lie Tyr Gly Gin Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu 305 310 315 320 Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp lie 325 330 335 Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr 340 345 350 Lys Lys Al a cys Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr Tyr 355 360 365 Phe Asp Glu Lys Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu Asn 370 375 380 Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr lie 385 390 395 400 Asn lie Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gin 405 410 415 lie Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s Gin 420 425 430 Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr Gly 435 440 445 T rp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr lie 450 455 460 Al a Al a Thr Gly Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe Asp 465 470 475 480 Pro Asp Thr Al a Gin Leu Val lie Ser Glu

485 490 <210> 19 <211> 379 <212> PRT <213> Clostridium Difficile <400> 19

Gly Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr Phe Asp Asp Ser Gly 1 5 10 15 Val Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys Hi s Phe Tyr Phe 20 25 30 Al a Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn Thr Glu Asp Gly 35 40 45 Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly 50 55 60 Glu Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr 65 70 75 80 Tyr Phe Asp Asp Ser Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu 85 90 95 Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie 100 105 110 Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly 115 120 125 lie Met Gin Val Gly Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe 130 135 140 Ser Asp Ser Gly lie lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn 145 150 155 160 Tyr Phe Tyr lie Asp Asp Asn Gly lie Val Gin lie Gly Val Phe Asp 165 170 175 Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp 180 185 190

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VB64650 Seq Li st Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly 195 200 205 Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp 210 215 220 lie Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu 225 230 235 240 Thr Lys Lys Al a cys Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr 245 250 255 Tyr Phe Asp Glu Lys Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu 260 265 270 Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr 275 280 285 lie Asn lie Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met 290 295 300 Gin lie Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s 305 310 315 320 Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr 325 330 335 Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr 340 345 350 lie Al a Al a Thr Gly Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe 355 360 365 Asp Pro Asp Thr Al a Gin Leu Val lie Ser Glu 370 375

<210> 20 <211> 3339 <212> DNA <213> Clostridium Difficile <400> 20 atggcaaccg gttggcagac catcgatggc aaaaaatatt attttaatac caacaccgca 60 attgcaagca ccggctatac cattatcaac ggcaaacact tttattttaa caccgacggc 120 attatgcaga ttggtgtgtt taaaggtccg aacggctttg aatactttgc accggcaaat 180 accgatgcca ataatattga aggccaggcc attctgtatc agaatgaatt tctgaccctg 240 aacggcaaaa aatactactt tggcagcgat agcaaagcag ttaccggttg gcgcatcatc 300 aacaataaga aatattactt caacccgaat aatgcaattg cagcaattca tctgtgcacc 360 attaacaacg acaaatatta tttcagctat gacggtattc tgcagaatgg ctacattacc 420 atcgaacgca acaactttta tttcgatgcc aacaacgaaa gcaaaatggt gaccggtgtt 480 ttcaaaggcc ctaatggttt tgagtatttc gctccggcaa acacccataa taacaacatt 540 gaaggtcagg cgatcgttta tcagaacaaa ttcctgacgc tgaatggtaa gaaatactat 600 ttcgataatg acagcaaagc cgtgaccggc tggcagacaa ttgacgggaa gaaatattac 660 tttaatctga ataccgcaga agcagcaacc ggttggcaaa cgatcgacgg taaaaagtac 720 tacttcaacc tgaacacagc cgaagcagcc acaggatggc agactattga tggaaaaaaa 780 tactatttca acaccaacac ctttattgca tctaccggtt ataccagcat taacggtaaa 840 catttctact tcaacaccga tggtatcatg cagatcggcg ttttcaaagg tccaaatggt 900 ttcgaatact ttgcccctgc caatacagat gcaaataaca tcgagggtca ggcaatcctg 960 taccaaaaca aatttctgac cctgaatggg aaaaaatatt actttggtag cgattctaaa 1020 gccgttaccg gtctgcgtac cattgatggt aaaaaatact actttaatac gaatacagcc 1080 gttgcggtta caggctggca gaccattaac gggaaaaaat actattttaa cacaaatacc 1140 agcattgcct caacgggtta taccattatt tcgggtaaac acttctactt taataccgat 1200 ggtattatgc aaatcggagt ctttaaagga cctgatgggt tcgaatattt tgcgcctgcg 1260 aacactgatg cgaacaatat cgaaggacag gcaatccgct atcagaatcg ctttctgtat 1320 ctgcacgaca acatctatta ttttggcaac aattcaaaag cagccaccgg ctgggttaca 1380 attgatggca accgctacta tttcgaaccg aataccgcaa tgggtgcaaa tggctacaaa 1440 accatcgata ataaaaattt ctattttcgc aacggtctgc cgcagatcgg ggtatttaaa 1500 ggtagcaacg gcttcgaata cttcgctcca gcgaatacgg acgcgaacaa tattgagggt 1560 caagcgattc gttatcaaaa ccgttttctg catctgctgg gcaaaatcta ctactttggc 1620 aataacagta aagcagttac tggatggcag acaatcaatg gtaaagtgta ctattttatg 1680 ccggataccg ccatggcagc agccggtggt ctgtttgaaa ttgatggcgt gatctatttt 1740 tttggtgtgg atggtgttaa agcaccggga atatacggtg gtaccggctt tgtgaccgtg 1800 ggtgatgata aatactattt caatccgatt aacggtggtg cagcgagcat tggcgaaacc 1860 atcatcgatg acaaaaacta ttatttcaac cagagcggtg tgctgcagac cggtgtgttt 1920 agcaccgaag atggctttaa atattttgcg ccagcgaaca ccctggatga aaacctggaa 1980 ggcgaagcga ttgattttac cggcaaactg atcatcgatg aaaacatcta ttacttcgat 2040 gataactatc gtggtgcggt ggaatggaaa gaactggatg gcgaaatgca ttatttttct 2100 ccggaaaccg gtaaagcgtt taaaggcctg aaccagatcg gcgattacaa atactacttc 2160

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VB64650 Seq List aacagcgatg gcgtgatgca gaaaggcttt gtgagcatca acgataacaa acactatttc 2220 gatgatagcg gtgtgatgaa agtgggctat accgaaattg atggcaaaca tttctacttc 2280 gcggaaaacg gcgaaatgca gattggcgtg ttcaataccg aagatggttt caaatacttc 2340 gcgcaccata acgaagatct gggtaacgaa gaaggcgaag aaattagcta tagcggcatc 2400 ctgaacttca acaacaaaat ctactacttt gatgatagct ttaccgcggt ggtgggctgg 2460 aaagatctgg aagatggcag caaatattat ttcgatgaag ataccgcgga agcgtatatt 2520 ggcctgagcc tgattaacga tggccagtac tattttaacg atgatggcat tatgcaggtg 2580 ggtttcgtga ccattaatga taaagtgttc tatttcagcg atagcggcat tattgaaagc 2640 ggcgtgcaga acattgatga taactacttc tacatcgatg ataacggcat tgtgcagatc 2700 ggcgtttttg ataccagcga tggctacaaa tatttcgcac cggccaatac cgtgaacgat 2760 aacatttatg gccaggcggt ggaatatagc ggtctggtgc gtgtgggcga agatgtgtat 2820 tatttcggcg aaacctatac catcgaaacc ggctggattt atgatatgga aaacgaaagc 2880 gataaatatt actttaatcc ggaaacgaaa aaagcgtgca aaggcattaa cctgatcgat 2940 gatatcaaat actattttga tgaaaaaggc attatgcgta ccggtctgat tagcttcgaa 3000 aacaacaact attacttcaa cgaaaacggt gaaatgcagt tcggctacat caacatcgaa 3060 gataaaatgt tctacttcgg cgaagatggt gttatgcaga ttggtgtttt taacaccccg 3120 gatggcttca aatactttgc ccatcagaat accctggatg aaaatttcga aggtgaaagc 3180 attaactata ccggctggct ggatctggat gaaaaacgct actacttcac cgatgaatac 3240 attgcggcga ccggcagcgt gattattgat ggcgaagaat actacttcga tccggatacc 3300 gcgcagctgg tgattagcga acatcatcat catcaccat 3339 <210> 21 <211> 1113 <212> PRT <213> Clostridium Difficile <400> 21

Met Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10 15 Thr Asn Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys 20 25 30 Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys 35 40 45 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn 50 55 60 Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu 65 70 75 80 Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly 85 90 95 T rp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a 100 105 110 lie Al a Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe 115 120 125 Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn 130 135 140 Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val 145 150 155 160 Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s 165 170 175 Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu 180 185 190 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val 195 200 205 Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn 210 215 220 Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr 225 230 235 240 Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie 245 250 255 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr 260 265 270 Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly 275 280 285 lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe 290 295 300 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu 305 310 315 320 Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly

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325 VB64650 330 Seq Li st 335 Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys 340 345 350 Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr 355 360 365 lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser 370 375 380 Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr Phe Asn Thr Asp 385 390 395 400 Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 405 410 415 Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie 420 425 430 Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn lie Tyr Tyr Phe 435 440 445 Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn 450 455 460 Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys 465 470 475 480 Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie 485 490 495 Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn 500 505 510 Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg 515 520 525 Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys 530 535 540 Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met 545 550 555 560 Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly 565 570 575 Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Pro Gly lie Tyr 580 585 590 Gly Gly Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn 595 600 605 Pro lie Asn Gly Gly Al a Al a Ser lie Gly Glu Thr lie lie Asp Asp 610 615 620 Lys Asn Tyr Tyr Phe Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe 625 630 635 640 Ser Thr Glu Asp Gly Phe Lys Tyr Phe Al a Pro Al a Asn Thr Leu Asp 645 650 655 Glu Asn Leu Glu Gly Glu Al a lie Asp Phe Thr Gly Lys Leu lie lie 660 665 670 Asp Glu Asn lie Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Al a Val Glu 675 680 685 T rp Lys Glu Leu Asp Gly Glu Met Hi s Tyr Phe Ser Pro Glu Thr Gly 690 695 700 Lys Al a Phe Lys Gly Leu Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe 705 710 715 720 Asn Ser Asp Gly Val Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn 725 730 735 Lys Hi s Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu 740 745 750 lie Asp Gly Lys Hi s Phe Tyr Phe Al a Glu Asn Gly Glu Met Gin lie 755 760 765 Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Al a Hi s Hi s Asn 770 775 780 Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie 785 790 795 800 Leu Asn Phe Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a 805 810 815 Val Val Gly T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp 820 825 830 Glu Asp Thr Al a Glu Al a Tyr lie Gly Leu Ser Leu lie Asn Asp Gly 835 840 845 Gin Tyr Tyr Phe Asn Asp Asp Gly lie Met Gin Val Gly Phe Val Thr 850 855 860 lie Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly lie lie Glu Ser

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VB64650 Seq List

865 870 875 880

Gly Val Gin Asn lie 885 Asp Asp Asn Tyr Phe Tyr lie 890 Asp Asp Asn 895 Gly lie Val Gin lie Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe 900 905 910 Ala Pro Al a Asn Thr Val Asn Asp Asn lie Tyr Gly Gin Al a Val Glu 915 920 925 Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu 930 935 940 Thr Tyr Thr lie Glu Thr Gly Trp lie Tyr Asp Met Glu Asn Glu Ser 945 950 955 960 Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala cys Lys Gly lie 965 970 975 Asn Leu lie Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met 980 985 990 Arg Thr Gly Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu 995 1000 1005 Asn Gly Glu Met Gin Phe Gly Tyr lie Asn lie Glu Asp Lys Met Phe 1010 1015 1020 Tyr Phe Gly Glu Asp Gly Val Met Gin lie Gly Val Phe Asn Thr Pro 1025 1030 1035 1040 Asp Gly Phe Lys Tyr Phe Al a His Gin Asn Thr Leu Asp Glu Asn Phe 1045 1050 1055 Glu Gly Glu Ser lie Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys 1060 1065 1070 Arg Tyr Tyr Phe Thr Asp Glu Tyr lie Ala Ala Thr Gly Ser Val lie 1075 1080 1085 lie Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a Gin Leu Val 1090 1095 1100 lie Ser Glu Hi s Hi s Hi s His His His 1105 1110

<210> 22 <211> 3324 <212> DNA <213> Clostridium Difficile <400> 22 atggcaaccg gttggcagac catcgatggc aaaaaatatt attttaatac caacaccgca 60 attgcaagca ccggctatac cattatcaac ggcaaacact tttattttaa caccgacggc 120 attatgcaga ttggtgtgtt taaaggtccg aacggctttg aatactttgc accggcaaat 180 accgatgcca ataatattga aggccaggcc attctgtatc agaatgaatt tctgaccctg 240 aacggcaaaa aatactactt tggcagcgat agcaaagcag ttaccggttg gcgcatcatc 300 aacaataaga aatattactt caacccgaat aatgcaattg cagcaattca tctgtgcacc 360 attaacaacg acaaatatta tttcagctat gacggtattc tgcagaatgg ctacattacc 420 atcgaacgca acaactttta tttcgatgcc aacaacgaaa gcaaaatggt gaccggtgtt 480 ttcaaaggcc ctaatggttt tgagtatttc gctccggcaa acacccataa taacaacatt 540 gaaggtcagg cgatcgttta tcagaacaaa ttcctgacgc tgaatggtaa gaaatactat 600 ttcgataatg acagcaaagc cgtgaccggc tggcagacaa ttgacgggaa gaaatattac 660 tttaatctga ataccgcaga agcagcaacc ggttggcaaa cgatcgacgg taaaaagtac 720 tacttcaacc tgaacacagc cgaagcagcc acaggatggc agactattga tggaaaaaaa 780 tactatttca acaccaacac ctttattgca tctaccggtt ataccagcat taacggtaaa 840 catttctact tcaacaccga tggtatcatg cagatcggcg ttttcaaagg tccaaatggt 900 ttcgaatact ttgcccctgc caatacagat gcaaataaca tcgagggtca ggcaatcctg 960 taccaaaaca aatttctgac cctgaatggg aaaaaatatt actttggtag cgattctaaa 1020 gccgttaccg gtctgcgtac cattgatggt aaaaaatact actttaatac gaatacagcc 1080 gttgcggtta caggctggca gaccattaac gggaaaaaat actattttaa cacaaatacc 1140 agcattgcct caacgggtta taccattatt tcgggtaaac acttctactt taataccgat 1200 ggtattatgc aaatcggagt ctttaaagga cctgatgggt tcgaatattt tgcgcctgcg 1260 aacactgatg cgaacaatat cgaaggacag gcaatccgct atcagaatcg ctttctgtat 1320 ctgcacgaca acatctatta ttttggcaac aattcaaaag cagccaccgg ctgggttaca 1380 attgatggca accgctacta tttcgaaccg aataccgcaa tgggtgcaaa tggctacaaa 1440 accatcgata ataaaaattt ctattttcgc aacggtctgc cgcagatcgg ggtatttaaa 1500 ggtagcaacg gcttcgaata cttcgctcca gcgaatacgg acgcgaacaa tattgagggt 1560 caagcgattc gttatcaaaa ccgttttctg catctgctgg gcaaaatcta ctactttggc 1620 aataacagta aagcagttac tggatggcag acaatcaatg gtaaagtgta ctattttatg 1680 ccggataccg ccatggcagc agccggtggt ctgtttgaaa ttgatggcgt gatctatttt 1740

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VB64650 Seq List tttggtgtgg atggtgttaa agcagttacc ggctttgtga ccgtgggtga tgataaatac 1800 tatttcaatc cgattaacgg tggtgcagcg agcattggcg aaaccatcat cgatgacaaa 1860 aactattatt tcaaccagag cggtgtgctg cagaccggtg tgtttagcac cgaagatggc 1920 tttaaatatt ttgcgccagc gaacaccctg gatgaaaacc tggaaggcga agcgattgat 1980 tttaccggca aactgatcat cgatgaaaac atctattact tcgatgataa ctatcgtggt 2040 gcggtggaat ggaaagaact ggatggcgaa atgcattatt tttctccgga aaccggtaaa 2100 gcgtttaaag gcctgaacca gatcggcgat tacaaatact acttcaacag cgatggcgtg 2160 atgcagaaag gctttgtgag catcaacgat aacaaacact atttcgatga tagcggtgtg 2220 atgaaagtgg gctataccga aattgatggc aaacatttct acttcgcgga aaacggcgaa 2280 atgcagattg gcgtgttcaa taccgaagat ggtttcaaat acttcgcgca ccataacgaa 2340 gatctgggta acgaagaagg cgaagaaatt agctatagcg gcatcctgaa cttcaacaac 2400 aaaatctact actttgatga tagctttacc gcggtggtgg gctggaaaga tctggaagat 2460 ggcagcaaat attatttcga tgaagatacc gcggaagcgt atattggcct gagcctgatt 2520 aacgatggcc agtactattt taacgatgat ggcattatgc aggtgggttt cgtgaccatt 2580 aatgataaag tgttctattt cagcgatagc ggcattattg aaagcggcgt gcagaacatt 2640 gatgataact acttctacat cgatgataac ggcattgtgc agatcggcgt ttttgatacc 2700 agcgatggct acaaatattt cgcaccggcc aataccgtga acgataacat ttatggccag 2760 gcggtggaat atagcggtct ggtgcgtgtg ggcgaagatg tgtattattt cggcgaaacc 2820 tataccatcg aaaccggctg gatttatgat atggaaaacg aaagcgataa atattacttt 2880 aatccggaaa cgaaaaaagc gtgcaaaggc attaacctga tcgatgatat caaatactat 2940 tttgatgaaa aaggcattat gcgtaccggt ctgattagct tcgaaaacaa caactattac 3000 ttcaacgaaa acggtgaaat gcagttcggc tacatcaaca tcgaagataa aatgttctac 3060 ttcggcgaag atggtgttat gcagattggt gtttttaaca ccccggatgg cttcaaatac 3120 tttgcccatc agaataccct ggatgaaaat ttcgaaggtg aaagcattaa ctataccggc 3180 tggctggatc tggatgaaaa acgctactac ttcaccgatg aatacattgc ggcgaccggc 3240 agcgtgatta ttgatggcga agaatactac ttcgatccgg ataccgcgca gctggtgatt 3300 agcgaacatc atcatcatca ccat 3324 <210> 23 <211> 1108 <212> PRT <213> Clostridium Difficile <400> 23

Met Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10 15 Thr Asn Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys 20 25 30 Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys 35 40 45 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn 50 55 60 Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu 65 70 75 80 Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly 85 90 95 T rp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a 100 105 110 lie Al a Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe 115 120 125 Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn 130 135 140 Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val 145 150 155 160 Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s 165 170 175 Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu 180 185 190 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val 195 200 205 Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn 210 215 220 Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr 225 230 235 240 Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie 245 250 255 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr 260 265 270

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VB64650 Seq Li st Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly 275 280 285 lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe 290 295 300 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu 305 310 315 320 Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly 325 330 335 Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys 340 345 350 Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr 355 360 365 lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser 370 375 380 Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr Phe Asn Thr Asp 385 390 395 400 Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 405 410 415 Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie 420 425 430 Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn lie Tyr Tyr Phe 435 440 445 Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn 450 455 460 Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys 465 470 475 480 Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie 485 490 495 Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn 500 505 510 Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg 515 520 525 Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys 530 535 540 Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met 545 550 555 560 Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly 565 570 575 Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Val Thr Gly Phe 580 585 590 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro lie Asn Gly Gly 595 600 605 Al a Al a Ser lie Gly Glu Thr lie lie Asp Asp Lys Asn Tyr Tyr Phe 610 615 620 Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe Ser Thr Glu Asp Gly 625 630 635 640 Phe Lys Tyr Phe Al a Pro Al a Asn Thr Leu Asp Glu Asn Leu Glu Gly 645 650 655 Glu Al a lie Asp Phe Thr Gly Lys Leu lie lie Asp Glu Asn lie Tyr 660 665 670 Tyr Phe Asp Asp Asn Tyr Arg Gly Al a Val Glu T rp Lys Glu Leu Asp 675 680 685 Gly Glu Met Hi s Tyr Phe Ser Pro Glu Thr Gly Lys Al a Phe Lys Gly 690 695 700 Leu Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 705 710 715 720 Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr Phe Asp 725 730 735 Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys Hi s 740 745 750 Phe Tyr Phe Al a Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn Thr 755 760 765 Glu Asp Gly Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp Leu Gly Asn 770 775 780 Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn 785 790 795 800 Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val Gly T rp Lys 805 810 815

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2016203241 18 May 2016

VB64650 Seq Li st Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe 820 825 Asp Glu Asp Thr Ala Glu 830 Al a Tyr lie Gly Leu Ser Leu lie Asn Asp 835 840 Gly Gin Tyr Tyr Phe Asn 845 Asp Asp Gly lie Met Gin Val Gly Phe Val 850 855 Thr lie Asn Asp Lys Val 860 Phe 865 Tyr Phe Ser Asp Ser Gly lie lie Glu 870 Ser 875 Gly Val Gin Asn lie 880 Asp Asp Asn Tyr Phe Tyr lie Asp Asp Asn 885 890 Gly lie Val Gin lie Gly 895 Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr 900 905 Phe Ala Pro Ala Asn Thr 910 Val Asn Asp Asn lie Tyr Gly Gin Ala Val 915 920 Glu Tyr Ser Gly Leu Val 925 Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly 930 935 Glu Thr Tyr Thr lie Glu 940 Thr 945 Gly Trp lie Tyr Asp Met Glu Asn Glu 950 Ser 955 Asp Lys Tyr Tyr Phe 960 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly 965 970 lie Asn Leu lie Asp Asp 975 lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie 980 985 Met Arg Thr Gly Leu lie 990 Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn 995 1000 Glu Asn Gly Glu Met Gin 1005 Phe Gly Tyr lie Asn lie Glu Asp Lys Met 1010 1015 Phe Tyr Phe Gly Glu Asp 1020 Gly Val Met Gin lie Gly Val Phe Asn Thr 1025 1030 Pro Asp Gly Phe Lys Tyr 1035 1040 Phe Ala His Gin Asn Thr Leu Asp Glu Asn Phe 1045 1050 Glu Gly Glu Ser lie 1055 Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu 1060 1065 Lys Arg Tyr Tyr Phe Thr 1070 Asp Glu Tyr lie Ala Ala Thr Gly Ser Val 1075 1080 lie lie Asp Gly Glu Glu 1085 Tyr Hi s Tyr Phe Asp Pro Asp Thr Ala Gin Leu 1090 1095 His His His Val lie Ser Glu His His 1100

1105 <210> 24 <211> 3387 <212> DNA <213> Clostridium Difficile <400> 24 atggcaaccg gttggcagac catcgatggc aaaaaatatt attttaatac caacaccgca 60 attgcaagca ccggctatac cattatcaac ggcaaacact tttattttaa caccgacggc 120 attatgcaga ttggtgtgtt taaaggtccg aacggctttg aatactttgc accggcaaat 180 accgatgcca ataatattga aggccaggcc attctgtatc agaatgaatt tctgaccctg 240 aacggcaaaa aatactactt tggcagcgat agcaaagcag ttaccggttg gcgcatcatc 300 aacaataaga aatattactt caacccgaat aatgcaattg cagcaattca tctgtgcacc 360 attaacaacg acaaatatta tttcagctat gacggtattc tgcagaatgg ctacattacc 420 atcgaacgca acaactttta tttcgatgcc aacaacgaaa gcaaaatggt gaccggtgtt 480 ttcaaaggcc ctaatggttt tgagtatttc gctccggcaa acacccataa taacaacatt 540 gaaggtcagg cgatcgttta tcagaacaaa ttcctgacgc tgaatggtaa gaaatactat 600 ttcgataatg acagcaaagc cgtgaccggc tggcagacaa ttgacgggaa gaaatattac 660 tttaatctga ataccgcaga agcagcaacc ggttggcaaa cgatcgacgg taaaaagtac 720 tacttcaacc tgaacacagc cgaagcagcc acaggatggc agactattga tggaaaaaaa 780 tactatttca acaccaacac ctttattgca tctaccggtt ataccagcat taacggtaaa 840 catttctact tcaacaccga tggtatcatg cagatcggcg ttttcaaagg tccaaatggt 900 ttcgaatact ttgcccctgc caatacagat gcaaataaca tcgagggtca ggcaatcctg 960 taccaaaaca aatttctgac cctgaatggg aaaaaatatt actttggtag cgattctaaa 1020 gccgttaccg gtctgcgtac cattgatggt aaaaaatact actttaatac gaatacagcc 1080 gttgcggtta caggctggca gaccattaac gggaaaaaat actattttaa cacaaatacc 1140 agcattgcct caacgggtta taccattatt tcgggtaaac acttctactt taataccgat 1200 ggtattatgc aaatcggagt ctttaaagga cctgatgggt tcgaatattt tgcgcctgcg 1260 aacactgatg cgaacaatat cgaaggacag gcaatccgct atcagaatcg ctttctgtat 1320

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VB64650 Seq List ctgcacgaca acatctatta ttttggcaac aattcaaaag cagccaccgg ctgggttaca 1380 attgatggca accgctacta tttcgaaccg aataccgcaa tgggtgcaaa tggctacaaa 1440 accatcgata ataaaaattt ctattttcgc aacggtctgc cgcagatcgg ggtatttaaa 1500 ggtagcaacg gcttcgaata cttcgctcca gcgaatacgg acgcgaacaa tattgagggt 1560 caagcgattc gttatcaaaa ccgttttctg catctgctgg gcaaaatcta ctactttggc 1620 aataacagta aagcagttac tggatggcag acaatcaatg gtaaagtgta ctattttatg 1680 ccggataccg ccatggcagc agccggtggt ctgtttgaaa ttgatggcgt gatctatttt 1740 tttggtgtgg atggtgttaa agcagtgagc ggtctgattt atattaacga tagcctgtat 1800 tactttaaac caccggtgaa taacctgatt accggctttg tgaccgtggg tgatgataaa 1860 tactatttca atccgattaa cggtggtgca gcgagcattg gcgaaaccat catcgatgac 1920 aaaaactatt atttcaacca gagcggtgtg ctgcagaccg gtgtgtttag caccgaagat 1980 ggctttaaat attttgcgcc agcgaacacc ctggatgaaa acctggaagg cgaagcgatt 2040 gattttaccg gcaaactgat catcgatgaa aacatctatt acttcgatga taactatcgt 2100 ggtgcggtgg aatggaaaga actggatggc gaaatgcatt atttttctcc ggaaaccggt 2160 aaagcgttta aaggcctgaa ccagatcggc gattacaaat actacttcaa cagcgatggc 2220 gtgatgcaga aaggctttgt gagcatcaac gataacaaac actatttcga tgatagcggt 2280 gtgatgaaag tgggctatac cgaaattgat ggcaaacatt tctacttcgc ggaaaacggc 2340 gaaatgcaga ttggcgtgtt caataccgaa gatggtttca aatacttcgc gcaccataac 2400 gaagatctgg gtaacgaaga aggcgaagaa attagctata gcggcatcct gaacttcaac 2460 aacaaaatct actactttga tgatagcttt accgcggtgg tgggctggaa agatctggaa 2520 gatggcagca aatattattt cgatgaagat accgcggaag cgtatattgg cctgagcctg 2580 attaacgatg gccagtacta ttttaacgat gatggcatta tgcaggtggg tttcgtgacc 2640 attaatgata aagtgttcta tttcagcgat agcggcatta ttgaaagcgg cgtgcagaac 2700 attgatgata actacttcta catcgatgat aacggcattg tgcagatcgg cgtttttgat 2760 accagcgatg gctacaaata tttcgcaccg gccaataccg tgaacgataa catttatggc 2820 caggcggtgg aatatagcgg tctggtgcgt gtgggcgaag atgtgtatta tttcggcgaa 2880 acctatacca tcgaaaccgg ctggatttat gatatggaaa acgaaagcga taaatattac 2940 tttaatccgg aaacgaaaaa agcgtgcaaa ggcattaacc tgatcgatga tatcaaatac 3000 tattttgatg aaaaaggcat tatgcgtacc ggtctgatta gcttcgaaaa caacaactat 3060 tacttcaacg aaaacggtga aatgcagttc ggctacatca acatcgaaga taaaatgttc 3120 tacttcggcg aagatggtgt tatgcagatt ggtgttttta acaccccgga tggcttcaaa 3180 tactttgccc atcagaatac cctggatgaa aatttcgaag gtgaaagcat taactatacc 3240 ggctggctgg atctggatga aaaacgctac tacttcaccg atgaatacat tgcggcgacc 3300 ggcagcgtga ttattgatgg cgaagaatac tacttcgatc cggataccgc gcagctggtg 3360 attagcgaac atcatcatca tcaccat 3387 <210> 25 <211> 1129 <212> PRT <213> Clostridium Difficile <400> 25

Met Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10 15 Thr Asn Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys 20 25 30 Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys 35 40 45 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn 50 55 60 Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu 65 70 75 80 Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly 85 90 95 T rp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a 100 105 110 lie Al a Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe 115 120 125 Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn 130 135 140 Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val 145 150 155 160 Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s 165 170 175 Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu 180 185 190 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val 195 200 205

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VB64650 Seq Li st Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn 210 215 220 Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr 225 230 235 240 Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie 245 250 255 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr 260 265 270 Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly 275 280 285 lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe 290 295 300 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu 305 310 315 320 Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly 325 330 335 Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys 340 345 350 Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr 355 360 365 lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser 370 375 380 Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr Phe Asn Thr Asp 385 390 395 400 Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 405 410 415 Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie 420 425 430 Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn lie Tyr Tyr Phe 435 440 445 Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn 450 455 460 Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys 465 470 475 480 Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie 485 490 495 Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn 500 505 510 Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg 515 520 525 Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys 530 535 540 Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met 545 550 555 560 Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly 565 570 575 Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Val Ser Gly Leu 580 585 590 lie Tyr lie Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn 595 600 605 Leu lie Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn 610 615 620 Pro lie Asn Gly Gly Al a Al a Ser lie Gly Glu Thr lie lie Asp Asp 625 630 635 640 Lys Asn Tyr Tyr Phe Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe 645 650 655 Ser Thr Glu Asp Gly Phe Lys Tyr Phe Al a Pro Al a Asn Thr Leu Asp 660 665 670 Glu Asn Leu Glu Gly Glu Al a lie Asp Phe Thr Gly Lys Leu lie lie 675 680 685 Asp Glu Asn lie Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Al a Val Glu 690 695 700 T rp Lys Glu Leu Asp Gly Glu Met Hi s Tyr Phe Ser Pro Glu Thr Gly 705 710 715 720 Lys Al a Phe Lys Gly Leu Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe 725 730 735 Asn Ser Asp Gly Val Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn 740 745 750

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2016203241 18 May 2016

VB64650 Seq Li st Lys His Tyr Phe 755 Asp Asp Ser Gly Val Met 760 Lys Val Gly Tyr Thr Glu 765 lie Asp Gly Lys 770 His Phe Tyr Phe Ala Glu 775 Asn Gly Glu Met Gin lie 780 Gly Val Phe Asn 785 Thr Glu Asp Gly Phe Lys 790 Tyr 795 Phe Ala His His Asn 800 Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu 805 810 lie Ser Tyr Ser Gly lie 815 Leu Asn Phe Asn 820 Asn Lys lie Tyr Tyr Phe 825 Asp Asp Ser Phe Thr Ala 830 Val Val Gly Trp 835 Lys Asp Leu Glu Asp Gly 840 Ser Lys Tyr Tyr Phe Asp 845 Glu Asp Thr Ala 850 Glu Ala Tyr lie Gly Leu 855 Ser Leu lie Asn Asp Gly 860 Gin Tyr Tyr Phe 865 Asn Asp Asp Gly lie Met 870 Gin 875 Val Gly Phe Val Thr 880 lie Asn Asp Lys Val Phe Tyr Phe Ser Asp 885 890 Ser Gly lie lie Glu Ser 895 Gly Val Gin Asn 900 lie Asp Asp Asn Tyr Phe 905 Tyr lie Asp Asp Asn Gly 910 lie Val Gin lie 915 Gly Val Phe Asp Thr Ser 920 Asp Gly Tyr Lys Tyr Phe 925 Ala Pro Ala Asn 930 Thr Val Asn Asp Asn lie 935 Tyr Gly Gin Ala Val Glu 940 Tyr Ser Gly Leu 945 Val Arg Val Gly Glu Asp 950 Val 955 Tyr Tyr Phe Gly Glu 960 Thr Tyr Thr lie Glu Thr Gly Trp lie Tyr 965 970 Asp Met Glu Asn Glu Ser 975 Asp Lys Tyr Tyr 980 Phe Asn Pro Glu Thr Lys 985 Lys Ala Cys Lys Gly lie 990 Asn Leu lie Asp 995 Asp lie Lys Tyr Tyr Phe 1000 Asp Glu Lys Gly lie Met 1005 Arg Thr Gly Leu 1010 lie Ser Phe Glu Asn Asn 1015 Asn Tyr Tyr Phe Asn Glu 1020 Asn Gly Glu Met 1025 Gin Phe Gly Tyr lie Asn 1030 lie Glu Asp Lys Met Phe 1035 1040 Tyr Phe Gly Glu Asp Gly Val Met Gin lie Gly 1045 1050 Val Phe Asn Thr Pro 1055 Asp Gly Phe Lys Tyr Phe Ala His Gin Asn 1060 1065 Thr Leu Asp Glu Asn Phe 1070 Glu Gly Glu Ser 1075 lie Asn Tyr Thr Gly Trp 1080 Leu Asp Leu Asp Glu Lys 1085 Arg Tyr Tyr Phe 1090 Thr Asp Glu Tyr lie Ala 1095 Al a Thr Gly Ser Val lie 1100 lie Asp Gly Glu 1105 lie Ser Glu His Glu Tyr Tyr Phe Asp Pro 1110 His His His His His 1125 Asp Thr Ala Gin Leu Val 1115 1120

<210> 26 <211> 2985 <212> DNA <213> Clostridium Difficile <400> 26 atggcaaccg gttggcagac catcgatggc aaaaaatatt attttaatac caacaccgca 60 attgcaagca ccggctatac cattatcaac ggcaaacact tttattttaa caccgacggc 120 attatgcaga ttggtgtgtt taaaggtccg aacggctttg aatactttgc accggcaaat 180 accgatgcca ataatattga aggccaggcc attctgtatc agaatgaatt tctgaccctg 240 aacggcaaaa aatactactt tggcagcgat agcaaagcag ttaccggttg gcgcatcatc 300 aacaataaga aatattactt caacccgaat aatgcaattg cagcaattca tctgtgcacc 360 attaacaacg acaaatatta tttcagctat gacggtattc tgcagaatgg ctacattacc 420 atcgaacgca acaactttta tttcgatgcc aacaacgaaa gcaaaatggt gaccggtgtt 480 ttcaaaggcc ctaatggttt tgagtatttc gctccggcaa acacccataa taacaacatt 540 gaaggtcagg cgatcgttta tcagaacaaa ttcctgacgc tgaatggtaa gaaatactat 600 ttcgataatg acagcaaagc cgtgaccggc tggcagacaa ttgacgggaa gaaatattac 660 tttaatctga ataccgcaga agcagcaacc ggttggcaaa cgatcgacgg taaaaagtac 720

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2016203241 18 May 2016

VB64650 Seq List tacttcaacc tgaacacagc cgaagcagcc acaggatggc agactattga tggaaaaaaa 780 tactatttca acaccaacac ctttattgca tctaccggtt ataccagcat taacggtaaa 840 catttctact tcaacaccga tggtatcatg cagatcggcg ttttcaaagg tccaaatggt 900 ttcgaatact ttgcccctgc caatacagat gcaaataaca tcgagggtca ggcaatcctg 960 taccaaaaca aatttctgac cctgaatggg aaaaaatatt actttggtag cgattctaaa 1020 gccgttaccg gtctgcgtac cattgatggt aaaaaatact actttaatac gaatacagcc 1080 gttgcggtta caggctggca gaccattaac gggaaaaaat actattttaa cacaaatacc 1140 agcattgcct caacgggtta taccattatt tcgggtaaac acttctactt taataccgat 1200 ggtattatgc aaatcggagt ctttaaagga cctgatgggt tcgaatattt tgcgcctgcg 1260 aacactgatg cgaacaatat cgaaggacag gcaatccgct atcagaatcg ctttctgtat 1320 ctgcacgaca acatctatta ttttggcaac aattcaaaag cagccaccgg ctgggttaca 1380 attgatggca accgctacta tttcgaaccg aataccgcaa tgggtgcaaa tggctacaaa 1440 accatcgata ataaaaattt ctattttcgc aacggtctgc cgcagatcgg ggtatttaaa 1500 ggtagcaacg gcttcgaata cttcgctcca gcgaatacgg acgcgaacaa tattgagggt 1560 caagcgattc gttatcaaaa ccgttttctg catctgctgg gcaaaatcta ctactttggc 1620 aataacagta aagcagttac tggatggcag acaatcaatg gtaaagtgta ctattttatg 1680 ccggataccg ccatggcagc agccggtggt ctgtttgaaa ttgatggcgt gatctatttt 1740 tttggtgtgg atggtgttaa agcagtgaaa ggcctgaacc agatcggcga ttacaaatac 1800 tacttcaaca gcgatggcgt gatgcagaaa ggctttgtga gcatcaacga taacaaacac 1860 tatttcgatg atagcggtgt gatgaaagtg ggctataccg aaattgatgg caaacatttc 1920 tacttcgcgg aaaacggcga aatgcagatt ggcgtgttca ataccgaaga tggtttcaaa 1980 tacttcgcgc accataacga agatctgggt aacgaagaag gcgaagaaat tagctatagc 2040 ggcatcctga acttcaacaa caaaatctac tactttgatg atagctttac cgcggtggtg 2100 ggctggaaag atctggaaga tggcagcaaa tattatttcg atgaagatac cgcggaagcg 2160 tatattggcc tgagcctgat taacgatggc cagtactatt ttaacgatga tggcattatg 2220 caggtgggtt tcgtgaccat taatgataaa gtgttctatt tcagcgatag cggcattatt 2280 gaaagcggcg tgcagaacat tgatgataac tacttctaca tcgatgataa cggcattgtg 2340 cagatcggcg tttttgatac cagcgatggc tacaaatatt tcgcaccggc caataccgtg 2400 aacgataaca tttatggcca ggcggtggaa tatagcggtc tggtgcgtgt gggcgaagat 2460 gtgtattatt tcggcgaaac ctataccatc gaaaccggct ggatttatga tatggaaaac 2520 gaaagcgata aatattactt taatccggaa acgaaaaaag cgtgcaaagg cattaacctg 2580 atcgatgata tcaaatacta ttttgatgaa aaaggcatta tgcgtaccgg tctgattagc 2640 ttcgaaaaca acaactatta cttcaacgaa aacggtgaaa tgcagttcgg ctacatcaac 2700 atcgaagata aaatgttcta cttcggcgaa gatggtgtta tgcagattgg tgtttttaac 2760 accccggatg gcttcaaata ctttgcccat cagaataccc tggatgaaaa tttcgaaggt 2820 gaaagcatta actataccgg ctggctggat ctggatgaaa aacgctacta cttcaccgat 2880 gaatacattg cggcgaccgg cagcgtgatt attgatggcg aagaatacta cttcgatccg 2940 gataccgcgc agctggtgat tagcgaacat catcatcatc accat 2985 <210> 27 <211> 995 <212> PRT <213> Clostridium Difficile <400> 27

Met Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10 15 Thr Asn Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys 20 25 30 Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys 35 40 45 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn 50 55 60 Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu 65 70 75 80 Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly 85 90 95 T rp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a 100 105 110 lie Al a Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe 115 120 125 Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn 130 135 140 Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val 145 150 155 160 Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s 165 170 175 Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu

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2016203241 18 May 2016

180 VB64650 185 Seq Li st 190 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val 195 200 205 Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn 210 215 220 Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr 225 230 235 240 Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie 245 250 255 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr 260 265 270 Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly 275 280 285 lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe 290 295 300 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu 305 310 315 320 Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly 325 330 335 Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys 340 345 350 Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr 355 360 365 lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser 370 375 380 Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr Phe Asn Thr Asp 385 390 395 400 Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 405 410 415 Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie 420 425 430 Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn lie Tyr Tyr Phe 435 440 445 Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn 450 455 460 Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys 465 470 475 480 Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie 485 490 495 Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn 500 505 510 Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg 515 520 525 Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys 530 535 540 Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met 545 550 555 560 Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly 565 570 575 Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Val Lys Gly Leu 580 585 590 Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val Met 595 600 605 Gin Lys Gly Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr Phe Asp Asp 610 615 620 Ser Gly Val Met Lys Val Gly Tyr Thr Glu lie Asp Gly Lys Hi s Phe 625 630 635 640 Tyr Phe Al a Glu Asn Gly Glu Met Gin lie Gly Val Phe Asn Thr Glu 645 650 655 Asp Gly Phe Lys Tyr Phe Al a His Hi s Asn Glu Asp Leu Gly Asn Glu 660 665 670 Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn Phe Asn Asn Lys 675 680 685 lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val Gly T rp Lys Asp 690 695 700 Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Al a Glu Al a 705 710 715 720 Tyr lie Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr Phe Asn Asp

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725 VB64650 730 Seq Li st 735 Asp Gly lie Met Gin Val Gly Phe Val Thr lie Asn Asp Lys Val Phe 740 745 750 Tyr Phe Ser Asp Ser Gly lie lie Glu Ser Gly Val Gin Asn lie Asp 755 760 765 Asp Asn Tyr Phe Tyr lie Asp Asp Asn Gly lie Val Gin lie Gly Val 770 775 780 Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro Al a Asn Thr Val 785 790 795 800 Asn Asp Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser Gly Leu Val Arg 805 810 815 Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu Thr 820 825 830 Gly T rp lie Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn 835 840 845 Pro Glu Thr Lys Lys Al a cys Lys Gly lie Asn Leu lie Asp Asp lie 850 855 860 Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met Arg Thr Gly Leu lie Ser 865 870 875 880 Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin Phe 885 890 895 Gly Tyr lie Asn lie Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly 900 905 910 Val Met Gin lie Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe 915 920 925 Al a Hi s Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser lie Asn 930 935 940 Tyr Thr Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp 945 950 955 960 Glu Tyr lie Al a Al a Thr Gly Ser Val lie lie Asp Gly Glu Glu Tyr 965 970 975 Tyr Phe Asp Pro Asp Thr Al a Gin Leu Val lie Ser Glu Hi s Hi s Hi s 980 985 990 Hi s Hi s Hi s 995

<210> 28 <211> 593 <212> PRT <213> Clostridium Difficile <400> 28

Met Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10 15 Thr Asn Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys 20 25 30 Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys 35 40 45 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn 50 55 60 Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu 65 70 75 80 Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly 85 90 95 T rp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a 100 105 110 lie Al a Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe 115 120 125 Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn 130 135 140 Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val 145 150 155 160 Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s 165 170 175 Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu 180 185 190 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val

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2016203241 18 May 2016

VB64650 Seq List

195 200 205 Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn 210 215 220 Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr 225 230 235 240 Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie 245 250 255 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr 260 265 270 Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly 275 280 285 lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe 290 295 300 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu 305 310 315 320 Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly 325 330 335 Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys 340 345 350 Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr 355 360 365 lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser 370 375 380 Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr Phe Asn Thr Asp 385 390 395 400 Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 405 410 415 Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie 420 425 430 Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn lie Tyr Tyr Phe 435 440 445 Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn 450 455 460 Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys 465 470 475 480 Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie 485 490 495 Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn 500 505 510 Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg 515 520 525 Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys 530 535 540 Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met 545 550 555 560 Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly 565 570 575 Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Pro Gly lie Tyr 580 585 590 Gly

<210> 29 <211> 589 <212> PRT <213> Clostridii jm Difficile <400> 29 Met Ala Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10 15 Thr Asn Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys 20 25 30 His Phe Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys 35 40 45 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn 50 55 60 Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu

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VB64650 Seq Li st 65 70 75 80 Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly 85 90 95 T rp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a 100 105 110 lie Al a Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe 115 120 125 Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn 130 135 140 Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val 145 150 155 160 Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s 165 170 175 Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu 180 185 190 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val 195 200 205 Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn 210 215 220 Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr 225 230 235 240 Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie 245 250 255 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr 260 265 270 Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly 275 280 285 lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe 290 295 300 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu 305 310 315 320 Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly 325 330 335 Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys 340 345 350 Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr 355 360 365 lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser 370 375 380 Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr Phe Asn Thr Asp 385 390 395 400 Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 405 410 415 Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie 420 425 430 Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn lie Tyr Tyr Phe 435 440 445 Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn 450 455 460 Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys 465 470 475 480 Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie 485 490 495 Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn 500 505 510 Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg 515 520 525 Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys 530 535 540 Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met 545 550 555 560 Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly 565 570 575 Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Val 580 585

<210> 30

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2016203241 18 May 2016

VB64650 Seq List <211> 589 <212> PRT <213> Clostridium Difficile <400> 30

Met Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10 15 Thr Asn Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys 20 25 30 Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys 35 40 45 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn 50 55 60 Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu 65 70 75 80 Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly 85 90 95 T rp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a 100 105 110 lie Al a Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe 115 120 125 Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn 130 135 140 Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val 145 150 155 160 Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s 165 170 175 Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu 180 185 190 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val 195 200 205 Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn 210 215 220 Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr 225 230 235 240 Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie 245 250 255 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr 260 265 270 Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly 275 280 285 lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe 290 295 300 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu 305 310 315 320 Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly 325 330 335 Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys 340 345 350 Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr 355 360 365 lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser 370 375 380 Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr Phe Asn Thr Asp 385 390 395 400 Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 405 410 415 Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie 420 425 430 Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn lie Tyr Tyr Phe 435 440 445 Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn 450 455 460 Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys 465 470 475 480 Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie 485 490 495 Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn

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500 VB64650 505 Seq Li st 510 Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg 515 520 525 Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys 530 535 540 Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met 545 550 555 560 Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly 565 570 575 Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Val 580 585

<210> 31 <211> 589 <212> PRT <213> Clostridium Difficile <400> 31

Met Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10 15 Thr Asn Thr Al a lie Al a Ser Thr Gly Tyr Thr lie lie Asn Gly Lys 20 25 30 Hi s Phe Tyr Phe Asn Thr Asp Gly lie Met Gin lie Gly Val Phe Lys 35 40 45 Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Asp Al a Asn 50 55 60 Asn lie Glu Gly Gin Al a lie Leu Tyr Gin Asn Glu Phe Leu Thr Leu 65 70 75 80 Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Al a Val Thr Gly 85 90 95 T rp Arg lie lie Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Al a 100 105 110 lie Al a Al a lie Hi s Leu cys Thr lie Asn Asn Asp Lys Tyr Tyr Phe 115 120 125 Ser Tyr Asp Gly lie Leu Gin Asn Gly Tyr lie Thr lie Glu Arg Asn 130 135 140 Asn Phe Tyr Phe Asp Al a Asn Asn Glu Ser Lys Met Val Thr Gly Val 145 150 155 160 Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn Thr Hi s 165 170 175 Asn Asn Asn lie Glu Gly Gin Al a lie Val Tyr Gin Asn Lys Phe Leu 180 185 190 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Al a Val 195 200 205 Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn 210 215 220 Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie Asp Gly Lys Lys Tyr 225 230 235 240 Tyr Phe Asn Leu Asn Thr Al a Glu Al a Al a Thr Gly T rp Gin Thr lie 245 250 255 Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe lie Al a Ser Thr 260 265 270 Gly Tyr Thr Ser lie Asn Gly Lys Hi s Phe Tyr Phe Asn Thr Asp Gly 275 280 285 lie Met Gin lie Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe 290 295 300 Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Leu 305 310 315 320 Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly 325 330 335 Ser Asp Ser Lys Al a Val Thr Gly Leu Arg Thr lie Asp Gly Lys Lys 340 345 350 Tyr Tyr Phe Asn Thr Asn Thr Al a Val Al a Val Thr Gly T rp Gin Thr 355 360 365 lie Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser lie Al a Ser 370 375 380 Thr Gly Tyr Thr lie lie Ser Gly Lys Hi s Phe Tyr Phe Asn Thr Asp

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VB64650 Seq Li st 385 390 395 400 Gly lie Met Gin lie Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 405 410 415 Phe Al a Pro Al a Asn Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie 420 425 430 Arg Tyr Gin Asn Arg Phe Leu Tyr Leu Hi s Asp Asn lie Tyr Tyr Phe 435 440 445 Gly Asn Asn Ser Lys Al a Al a Thr Gly T rp Val Thr lie Asp Gly Asn 450 455 460 Arg Tyr Tyr Phe Glu Pro Asn Thr Al a Met Gly Al a Asn Gly Tyr Lys 465 470 475 480 Thr lie Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gin lie 485 490 495 Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Al a Pro Al a Asn 500 505 510 Thr Asp Al a Asn Asn lie Glu Gly Gin Al a lie Arg Tyr Gin Asn Arg 515 520 525 Phe Leu Hi s Leu Leu Gly Lys lie Tyr Tyr Phe Gly Asn Asn Ser Lys 530 535 540 Al a Val Thr Gly T rp Gin Thr lie Asn Gly Lys Val Tyr Tyr Phe Met 545 550 555 560 Pro Asp Thr Al a Met Al a Al a Al a Gly Gly Leu Phe Glu lie Asp Gly 565 570 575 Val lie Tyr Phe Phe Gly Val Asp Gly Val Lys Al a Val 580 585

<210> 32 <211> 513 <212> PRT <213> Clostridium Difficile <400> 32

Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro lie 1 5 10 15 Asn Gly Gly Al a Al a Ser lie Gly Glu Thr lie lie Asp Asp Lys Asn 20 25 30 Tyr Tyr Phe Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe Ser Thr 35 40 45 Glu Asp Gly Phe Lys Tyr Phe Al a Pro Al a Asn Thr Leu Asp Glu Asn 50 55 60 Leu Glu Gly Glu Al a lie Asp Phe Thr Gly Lys Leu lie lie Asp Glu 65 70 75 80 Asn lie Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Al a Val Glu T rp Lys 85 90 95 Glu Leu Asp Gly Glu Met Hi s Tyr Phe Ser Pro Glu Thr Gly Lys Al a 100 105 110 Phe Lys Gly Leu Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser 115 120 125 Asp Gly Val Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn Lys Hi s 130 135 140 Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu lie Asp 145 150 155 160 Gly Lys Hi s Phe Tyr Phe Al a Glu Asn Gly Glu Met Gin lie Gly Val 165 170 175 Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp 180 185 190 Leu Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn 195 200 205 Phe Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val 210 215 220 Gly T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp 225 230 235 240 Thr Al a Glu Al a Tyr lie Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr 245 250 255 Tyr Phe Asn Asp Asp Gly lie Met Gin Val Gly Phe Val Thr lie Asn 260 265 270 Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly lie lie Glu Ser Gly Val

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2016203241 18 May 2016

VB64650 Seq List

275 280 285 Gin Asn lie Asp Asp Asn Tyr Phe Tyr lie Asp Asp Asn Gly lie Val 290 295 300 Gin lie Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro 305 310 315 320 Al a Asn Thr Val Asn Asp Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser 325 330 335 Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr 340 345 350 Thr lie Glu Thr Gly T rp lie Tyr Asp Met Glu Asn Glu Ser Asp Lys 355 360 365 Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Al a cys Lys Gly lie Asn Leu 370 375 380 lie Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met Arg Thr 385 390 395 400 Gly Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly 405 410 415 Glu Met Gin Phe Gly Tyr lie Asn lie Glu Asp Lys Met Phe Tyr Phe 420 425 430 Gly Glu Asp Gly Val Met Gin lie Gly Val Phe Asn Thr Pro Asp Gly 435 440 445 Phe Lys Tyr Phe Al a Hi s Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly 450 455 460 Glu Ser lie Asn Tyr Thr Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr 465 470 475 480 Tyr Phe Thr Asp Glu Tyr lie Al a Al a Thr Gly Ser Val lie lie Asp 485 490 495 Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a Gin Leu Val lie Ser 500 505 510 Glu

<210> 33 <211> 513 <212> PRT <213> Clostridium Difficile <400> 33 Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro lie 1 5 10 15 Asn Gly Gly Al a Al a Ser lie Gly Glu Thr lie lie Asp Asp Lys Asn 20 25 30 Tyr Tyr Phe Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe Ser Thr 35 40 45 Glu Asp Gly Phe Lys Tyr Phe Al a Pro Al a Asn Thr Leu Asp Glu Asn 50 55 60 Leu Glu Gly Glu Al a lie Asp Phe Thr Gly Lys Leu lie lie Asp Glu 65 70 75 80 Asn lie Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Al a Val Glu T rp Lys 85 90 95 Glu Leu Asp Gly Glu Met Hi s Tyr Phe Ser Pro Glu Thr Gly Lys Al a 100 105 110 Phe Lys Gly Leu Asn Gin lie Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser 115 120 125 Asp Gly Val Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn Lys Hi s 130 135 140 Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu lie Asp 145 150 155 160 Gly Lys Hi s Phe Tyr Phe Al a Glu Asn Gly Glu Met Gin lie Gly Val 165 170 175 Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp 180 185 190 Leu Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn 195 200 205 Phe Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val 210 215 220 Gly T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp

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VB64650 Seq Li st 225 230 235 240 Thr Al a Glu Al a Tyr lie Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr 245 250 255 Tyr Phe Asn Asp Asp Gly lie Met Gin Val Gly Phe Val Thr lie Asn 260 265 270 Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly lie lie Glu Ser Gly Val 275 280 285 Gin Asn lie Asp Asp Asn Tyr Phe Tyr lie Asp Asp Asn Gly lie Val 290 295 300 Gin lie Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro 305 310 315 320 Al a Asn Thr Val Asn Asp Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser 325 330 335 Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr 340 345 350 Thr lie Glu Thr Gly T rp lie Tyr Asp Met Glu Asn Glu Ser Asp Lys 355 360 365 Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Al a cys Lys Gly lie Asn Leu 370 375 380 lie Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met Arg Thr 385 390 395 400 Gly Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly 405 410 415 Glu Met Gin Phe Gly Tyr lie Asn lie Glu Asp Lys Met Phe Tyr Phe 420 425 430 Gly Glu Asp Gly Val Met Gin lie Gly Val Phe Asn Thr Pro Asp Gly 435 440 445 Phe Lys Tyr Phe Al a Hi s Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly 450 455 460 Glu Ser lie Asn Tyr Thr Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr 465 470 475 480 Tyr Phe Thr Asp Glu Tyr lie Al a Al a Thr Gly Ser Val lie lie Asp 485 490 495 Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a Gin Leu Val lie Ser 500 505 510

Glu <210> 34 <211> 534 <212> PRT <213> Clostridium Difficile <400> 34

Ser Gly Leu lie Tyr lie Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro 1 5 10 15 Val Asn Asn Leu lie Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr 20 25 30 Tyr Phe Asn Pro lie Asn Gly Gly Al a Al a Ser lie Gly Glu Thr lie 35 40 45 lie Asp Asp Lys Asn Tyr Tyr Phe Asn Gin Ser Gly Val Leu Gin Thr 50 55 60 Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe Al a Pro Al a Asn 65 70 75 80 Thr Leu Asp Glu Asn Leu Glu Gly Glu Al a lie Asp Phe Thr Gly Lys 85 90 95 Leu lie lie Asp Glu Asn lie Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly 100 105 110 Al a Val Glu T rp Lys Glu Leu Asp Gly Glu Met Hi s Tyr Phe Ser Pro 115 120 125 Glu Thr Gly Lys Al a Phe Lys Gly Leu Asn Gin lie Gly Asp Tyr Lys 130 135 140 Tyr Tyr Phe Asn Ser Asp Gly Val Met Gin Lys Gly Phe Val Ser lie 145 150 155 160 Asn Asp Asn Lys Hi s Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly 165 170 175 Tyr Thr Glu lie Asp Gly Lys His Phe Tyr Phe Al a Glu Asn Gly Glu

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2016203241 18 May 2016

180 VB64650 185 Seq Li st 190 Met Gin lie Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Al a 195 200 205 Hi s Hi s Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr 210 215 220 Ser Gly lie Leu Asn Phe Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser 225 230 235 240 Phe Thr Al a Val Val Gly T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr 245 250 255 Tyr Phe Asp Glu Asp Thr Al a Glu Al a Tyr lie Gly Leu Ser Leu lie 260 265 270 Asn Asp Gly Gin Tyr Tyr Phe Asn Asp Asp Gly lie Met Gin Val Gly 275 280 285 Phe Val Thr lie Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly lie 290 295 300 lie Glu Ser Gly Val Gin Asn lie Asp Asp Asn Tyr Phe Tyr lie Asp 305 310 315 320 Asp Asn Gly lie Val Gin lie Gly Val Phe Asp Thr Ser Asp Gly Tyr 325 330 335 Lys Tyr Phe Al a Pro Al a Asn Thr Val Asn Asp Asn lie Tyr Gly Gin 340 345 350 Al a Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr 355 360 365 Phe Gly Glu Thr Tyr Thr lie Glu Thr Gly T rp lie Tyr Asp Met Glu 370 375 380 Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Al a cys 385 390 395 400 Lys Gly lie Asn Leu lie Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys 405 410 415 Gly lie Met Arg Thr Gly Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr 420 425 430 Phe Asn Glu Asn Gly Glu Met Gin Phe Gly Tyr lie Asn lie Glu Asp 435 440 445 Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gin lie Gly Val Phe 450 455 460 Asn Thr Pro Asp Gly Phe Lys Tyr Phe Al a Hi s Gin Asn Thr Leu Asp 465 470 475 480 Glu Asn Phe Glu Gly Glu Ser lie Asn Tyr Thr Gly T rp Leu Asp Leu 485 490 495 Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr lie Al a Al a Thr Gly 500 505 510 Ser Val lie lie Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a 515 520 525 Gin Leu Val lie Ser Glu

530 <210> 35 <211> 400 <212> PRT <213> Clostridium Difficile <400> 35

Lys Gly Leu Asn Gin lie Gly Asp Tyr Lys 10 Tyr Tyr Phe Asn Ser 15 Asp 1 5 Gly Val Met Gin Lys Gly Phe Val Ser lie Asn Asp Asn Lys Hi s Tyr 20 25 30 Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu lie Asp Gly 35 40 45 Lys Hi s Phe Tyr Phe Al a Glu Asn Gly Glu Met Gin lie Gly Val Phe 50 55 60 Asn Thr Glu Asp Gly Phe Lys Tyr Phe Al a Hi s Hi s Asn Glu Asp Leu 65 70 75 80 Gly Asn Glu Glu Gly Glu Glu lie Ser Tyr Ser Gly lie Leu Asn Phe 85 90 95 Asn Asn Lys lie Tyr Tyr Phe Asp Asp Ser Phe Thr Al a Val Val Gly 100 105 110 T rp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr

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2016203241 18 May 2016

115 120 VB64650 Seq Li st 125 Al a Glu Al a Tyr lie Gly Leu Ser Leu lie Asn Asp Gly Gin Tyr Tyr 130 135 140 Phe Asn Asp Asp Gly lie Met Gin Val Gly Phe Val Thr lie Asn Asp 145 150 155 160 Lys Val Phe Tyr Phe Ser Asp Ser Gly lie lie Glu Ser Gly Val Gin 165 170 175 Asn lie Asp Asp Asn Tyr Phe Tyr lie Asp Asp Asn Gly lie Val Gin 180 185 190 lie Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Al a Pro Al a 195 200 205 Asn Thr Val Asn Asp Asn lie Tyr Gly Gin Al a Val Glu Tyr Ser Gly 210 215 220 Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr 225 230 235 240 lie Glu Thr Gly T rp lie Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr 245 250 255 Tyr Phe Asn Pro Glu Thr Lys Lys Al a cys Lys Gly lie Asn Leu lie 260 265 270 Asp Asp lie Lys Tyr Tyr Phe Asp Glu Lys Gly lie Met Arg Thr Gly 275 280 285 Leu lie Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu 290 295 300 Met Gin Phe Gly Tyr lie Asn lie Glu Asp Lys Met Phe Tyr Phe Gly 305 310 315 320 Glu Asp Gly Val Met Gin lie Gly Val Phe Asn Thr Pro Asp Gly Phe 325 330 335 Lys Tyr Phe Al a Hi s Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu 340 345 350 Ser lie Asn Tyr Thr Gly T rp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr 355 360 365 Phe Thr Asp Glu Tyr lie Al a Al a Thr Gly Ser Val lie lie Asp Gly 370 375 380 Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Al a Gin Leu Val lie Ser Glu 385 390 395 400

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