EP4658302A1 - Immunogenic composition - Google Patents

Abstract

The present invention relates to immunogenic compositions comprising Clostridioides difficile (C. difficile) immunogens. More particularly the present invention relates to immunogenic compositions comprising a first immunogen and a second immunogen the first immunogen comprising a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment and the second immunogen comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. The present invention further relates to nucleic acids encoding C. difficile polypeptides and the use of said immunogenic compositions and nucleic acids in medicine.

Description

IMMUNOGENIC COMPOSITION TECHNICAL FIELD [0001] The present invention is in the field of vaccinology and more particularly in the field of vaccines against Clostridioides difficile (C. difficile). Particularly, the present invention relates to immunogenic compositions comprising a combination of C. difficile immunogens. More particularly, the present invention relates to nucleic acids (for example an mRNA) encoding at least one C. difficile polypeptide, for example toxin A (TcdA) or Toxin B (TcdB) of C. difficile or fragment thereof. BACKGROUND OF THE INVENTION [0002] Clostridioides difficile (C. difficile), a gram-positive anaerobic bacterium, is the most important cause of nosocomial intestinal infections and is the major cause of pseudomembranous colitis in humans (Bartlett et al Am. J. Clin. Nutr.11 suppl: 2521-6 (1980), Leffler et al N. Eng.J. Med. 372; 1539-1548 (2015)). 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 (CDI) is the administration of antibiotics (metronidazole and vancomycin and fidaxomicin), however there has been evidence of strains which are resistant to these antibiotics (Shah et al., Expert Rev. Anti Infect. Ther. 8(5), 555–564 (2010)). Another approach to treating recurrent CDI is the use of monoclonal antibodies against C. difficile toxin B (bezlotoxumab) which is licensed for use in adults in the United States and Europe (Akiyama et al J. Clin. Gastroenterol.55; 43-51 (2021). Another treatment option recently approved in the United States for cases of recurring antibiotic -resistant CDI involves fecal transplant from healthy donors. However, with the emergence of highly virulent drug-resistant strains, a preventative approach is required to combat CDI. To date no vaccine is available to prevent CDI. Accordingly, there is a need for immunogenic compositions capable of inducing antibodies to, and/or a protective immune response to, C. difficile. [0003] The enterotoxicity of C. difficile is primarily due to the action of two toxins, toxin A (TcdA) and toxin B (TcdB). The C-terminal domains of toxin A and toxin B comprise repeating units, for example the C-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 al (PNAS 102:18373-18378 (2005)). [0004] Immunogenic compositions comprising antigens from C. difficile have been described. WO96/12802 and WO00/61762 and Lyerly et al (Current Microbiology 21:29-32 (1990)) relate to fragments of toxin A, in particular fragments of the C-terminal domain, for inducing a protective immune response in hamsters. WO9920304 relates to a mixture of co- purified toxin A and toxin B inactivated by incubation in formaldehyde. WO00/61762 relates to immunogenic compositions comprising either the full-length C-terminal domain or fragments of the C-terminal domain of toxin A and toxin B of C. difficile. Further vaccine candidates for a C. difficile vaccine include detoxified full length toxin A and toxin B (WO 14/144594, WO 14/144567), genetically detoxified Toxin A and Toxin B (WO 20/201985) or fusion proteins containing portions of Toxin A and Toxin B (WO12/59805, WO12/163811, WO12/28741). A phase 3 study examining the safety, immunogenicity and efficacy of a toxoid-based vaccine led to disappointing results and termination of this vaccine development (de Bruyn et al Lancet Infect. Dis.21; 252-262 (2021). [0005] In the absence of an approved vaccine against C. difficile infection, new compositions or vaccines with improved immunogenicity are needed. SUMMARY OF THE INVENTION [0006] The inventors of the present application surprisingly discovered that immunogenic compositions comprising a combination of two C. difficile immunogens induced an improved immune response, compared to either immunogen administered alone. The inventors discovered that improved neutralizing titers were obtained when both immunogens were administered in combination, suggesting a synergistic and/or additive effect. Furthermore, when administered in combination the immunogens were discovered to demonstrate protective efficacy. The inventors have in addition established that both protein and mRNA form of bacterial toxoids, for example C. difficile toxoids, can be efficiently used to elicit an immune response, particularly a protective immune response in a subject. [0007] Accordingly, in a first aspect there is provided an immunogenic composition comprising a first immunogen and a second immunogen: the first immunogen comprising a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment; and the second immunogen comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. It is preferred that the first immunogen is a first polypeptide, and the second immunogen is a second polypeptide. [0008] It is envisaged that one or more nucleic acid polynucleotide sequences may encode bacterial toxoid antigen, for example, the first and second polypeptides of the present disclosure. [0009] Thus, in a second aspect there is provided a nucleic acid encoding the first polypeptide as referred to in the first aspect and the second polypeptide as referred to in the first aspect, wherein said nucleic acid is a single polynucleotide encoding said first and second polypeptides. [00010] In a third aspect there is provided a nucleic acid encoding the first polypeptide as referred to in the first aspect. [00011] In a fourth aspect there is provided a nucleic acid encoding the second polypeptide as referred to in the first aspect. [00012] More particularly, the combination of the nucleic acid of the third aspect and the nucleic acid of the fourth aspect is herein provided, wherein said nucleic acids are separate polynucleotides. [00013] In a fifth aspect there is provided a carrier comprising the nucleic acid of the second aspect. [00014] In a sixth aspect there is provided a carrier comprising the nucleic acid of the third aspect. [00015] In a seventh aspect there is provided a carrier comprising the nucleic acid of the fourth aspect. [00016] In an eighth aspect there is provided a pharmaceutical composition comprising the immunogenic composition of the first aspect and a pharmaceutically acceptable excipient. [00017] In a ninth aspect there is provided a pharmaceutical composition comprising the nucleic acid of the second, third or fourth aspect, or the carrier of fifth, sixth or seventh aspect and a pharmaceutically acceptable excipient. [00018] In a tenth aspect there is provided a pharmaceutical composition comprising the first polypeptide as referred to in the first aspect and either the nucleic acid of the fourth aspect or the carrier of the seventh aspect, said pharmaceutical composition further comprising a pharmaceutically acceptable excipient. [00019] In an eleventh aspect there is provided a pharmaceutical composition comprising the second polypeptide referred to in the first aspect and either the nucleic acid of the third aspect or the carrier of the sixth aspect said pharmaceutical composition further comprising a pharmaceutically acceptable excipient. [00020] In a twelfth aspect there is provided a vaccine comprising the pharmaceutical composition the eighth, ninth, tenth or eleventh aspect. [00021] In a thirteenth aspect there is provided the pharmaceutical composition of the eighth, ninth, tenth or eleventh aspect or the vaccine of the twelfth aspect for use in medicine. [00022] In a fourteenth aspect there is provided the pharmaceutical composition of the eighth, ninth, tenth or eleventh aspect or the vaccine of the twelfth aspect for use in a method of raising an immune response in a subject, optionally a protective immune response in a subject. [00023] In a fifteenth aspect there is provided the pharmaceutical composition of the eighth, ninth, tenth or eleventh aspect or the vaccine of the twelfth aspect for use in the treatment or prevention of C. difficile disease. [00024] In a sixteenth aspect there is provided the pharmaceutical composition of the eighth, ninth, tenth or eleventh aspect or the vaccine of the twelfth aspect for use in a method of vaccinating a subject against C. difficile disease, optionally wherein the vaccination is prophylactic. [00025] In a seventeenth aspect there is provided a method of inducing an immune response against C. difficile in a subject comprising administering to the subject an immunologically effective amount of the pharmaceutical composition of the eighth, ninth, tenth or eleventh aspect or the vaccine of the twelfth aspect. [00026] In an eighteenth aspect, there is provided a method of treating or preventing C. difficile disease comprising administering the pharmaceutical composition of the eighth, ninth, tenth or eleventh aspect or the vaccine of the twelfth aspect to a subject in need thereof. [00027] In a nineteenth aspect there is provided the use of the pharmaceutical composition of the eighth, ninth, tenth or eleventh aspect or the vaccine of the twelfth aspect in the manufacture of a medicament. [00028] In a twentieth aspect there is provided the use of the pharmaceutical composition of the eighth, ninth, tenth or eleventh aspect or the vaccine of the twelfth aspect in the manufacture of a medicament against C. difficile disease. [00029] In a twenty-first aspect there is provided the pharmaceutical composition or vaccine for use according to the thirteenth, fourteenth, fifteenth or sixteenth aspect or the method of seventeenth or eighteenth aspect wherein the subject is a human subject, optionally wherein the human subject is over 50 years old. BRIEF DESCRIPTION OF THE FIGURES FIG.1A: Schematic representation of the first immunogen (i.e., first polypeptide) demonstrating schematically the proximal and distal ends of the toxin A and toxin B CROP domain fragments. Figure is not drawn to scale and is provided purely for illustrative purposes. FIG.1B: Schematic representation of ToxB showing the location of TD1 (1072-1452) within the delivery and receptor binding domain (DRBD) of the toxin B. Also shown is the location of the glycosyltransferase domain (GTD), cysteine protease domain (CPD) and the combined repetitive oligopeptide (CROP) domain. FIG.2: Schematic overview of study design for Example 3 FIG.3: Graph showing change in mean weight as a percentage compared to baseline. Data is shown by group (F22µg/AS01 vs TD12µg vs AS01) over 7-days following challenge with C. difficile 6529 strain spores. FIG.4: Graph showing change in weight as a percentage compared to baseline. Data is shown by group (F20.06µg/AS01 vs TD10.06µg vs F20.06µg + TD10.06µg/AS01 vs AS01) over 7 days following challenge with C. difficile 6529 strain spores. FIG.5: Graph showing change in weight as a percentage compared to baseline. Data is shown by group (F20.06µg/AS01 vs TD12µg vs F20.06µg + TD12µg/AS01 vs AS01) over 7 days following challenge with C. difficile 6529 strain spores. FIG.6: Schematic overview of the study design for Example 4. FIG.7: Graph showing Anti-TD1 IgG specific antibody at 13PII and 14PIII in pooled serum. FIG.8A: Graph showing neutralizing responses at 14PIII against homologous r087 ToxB, against heterologous r027 and r078 ToxB (Individual) - Individual values and geometric mean with 95% CI. FIG.8B: Graph showing neutralizing responses at 13PII and 14PIII against homologous r087 ToxB, against heterologous r027 and r078 ToxB (Individual) – Pooled sera. FIG.9: Graph showing neutralizing responses against homologous r087 ToxA, at 13 PII and 14PIII. Fig.10: Schematic overview of the study design for Example 5. Fig.11: Graph showing % weight loss at day 2 post C. difficile challenge. Fig.12A, Fig.12B and Fig.12C: Graphs showing concentration of S100A8 or G-CSF or KC in caecum at day 2 post C. difficile challenge. Fig.13A, Fig.13B and Fig.13C: Graph showing the individual and median scoring of loss of goblet cells or presence of inflammatory cells, or submucosal edema in colon at day 2 post C. difficile challenge. Fig.14: Graph showing individual anti-TcdA F2 C-ter IgG antibody titers measured by ELISA (dot in the graph). The GMT of each group is represented as a horizontal line with 95% confidence intervals depicted using error bars. Fig.15: Graph showing individual anti-TcdB F2 C-ter IgG antibody titers measured by ELISA (dot in the graph). The GMT of a group is represented as a horizontal line with 95% confidence intervals depicted using error bars. Fig.16: Graph showing individual anti-TD1 IgG antibody titers measured by ELISA (dot in the graph). The GMT of a group is represented as a horizontal line with 95% confidence intervals depicted using error bars. Fig.17: Graph showing individual anti-TcdB r087 neutralization response (dot in the graph). The GMT of a group is represented as a horizontal line with 95% confidence intervals depicted using error bars. Fig 18. Graph showing individual anti-TcdB r027 neutralization response (dot in the graph). The GMT of a group is represented as a horizontal line with 95% confidence intervals depicted using error bars. Fig.19: Graph showing individual anti-GTD IgG antibody titers measured by ELISA (dot in the graph). The GMT of each group is represented as a horizontal line with 95% confidence intervals depicted using error bars. Fig.20: Graph showing individual anti-TcdB r087 neutralization response (dot in the graph). The GMT of a group is represented as a horizontal line with 95% confidence intervals depicted using error bars. Fig.21: Graph showing individual anti-TcdB r027 neutralization response (dot in the graph). The GMT of a group is represented as a horizontal line with 95% confidence intervals depicted using error bars. Fig.22: Schematic overview of the study design for Example 8. Fig.23: Weight change from baseline (expressed in %) at day 2 post 6529 C. difficile strain spore challenge. The means and their 95% of confidence interval are provided by group. Fig.24: S100A8 levels (pg/mg of protein) in caecum measured by Luminex (dot in the graph). The GMT of a group is represented as a horizontal line and the 95% confidence interval is provided by error bars. Fig.25: S100A8 levels (pg/mg of protein) in colon measured by Luminex (dot in the graph). The GMT of a group is represented as a horizontal line and the 95% confidence interval is provided by error bars. Fig.26: Schematic overview of the study design for Example 9. Fig.27: Change in weight (%) by group at day 2 post 6529 C. difficile strain spore challenge - Mean and 95% confidence intervals are provided. Fig.28: Graph showing S100A8 level (pg per mg of protein) in caecum with GMT and 95% confidence intervals indicated. Fig.29: Graph showing S100A8 level (pg per mg of protein) in colon with GMT and 95% confidence intervals indicated. DETAILED DESCRIPTION OF THE INVENTION [00030] Prior to setting forth the invention in detail, it may be helpful to the understanding of one of ordinary skill to define the following terms: [00031] Unless otherwise explained or defined herein, 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. For example, 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 Encyclopaedia 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 1-56081-569-8). [00032] All references, including publications of patent and or patent applications cited within this patent specification are incorporated by reference herein. [00033] 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. The term “at least one” refers to one or more. [00034] Unless specified otherwise, where a numerical range is provided, it is inclusive, i.e., the endpoints are included. [00035] The terms “at least”, “no more than” and other such terms preceding a list of values are applicable to all members of said list (not merely the first member thereof), unless otherwise stated. [00036] The term “comprising” encompasses “including” as well as “consisting” e.g., a composition “comprising” X may consist exclusively of X or may include something additional e.g., X + Y. [00037] The term “about” in relation to a numerical value x is optional and means, for example, x+10%. [00038] The word “substantially” does not exclude “completely” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the present disclosure. [00039] Amino acids refers to an amino acid selected from the group consisting of alanine (ala, A), arginine (arg, R), asparagine (asn, N) , aspartic acid (asp,D), cysteine (cys, C) ,glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile,I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), valine (val, V). [00040] A “subject” as used herein is an animal, such as a mammal, including humans, non-human primates and non-primate mammals such as members of the rodent genus (including but not limited to mice and rats), the Cavia genus (including but not limited to guinea pigs) and members of the order Lagomorpha (including but not limited to rabbits). As used herein, the subject is preferably a human. [00041] As used herein, the term “sequence identity” refers to the degree of sameness of two sequences, such as amino acid sequences. This may be determined by comparing the two sequences aligned in an optimum manner and in which the sequence to be compared can comprise additions or deletions with respect to the reference sequence for an optimum alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the residue is identical between the two sequences, dividing this number of identical positions by the total number of positions in the longer of the two sequences and multiplying the result obtained by 100 in order to obtain the percentage sequence identity between these two sequences. For example, it is possible to use the BLAST program, available on the website, https://blast.ncbi.nlm.nih.gov/Blast.cgi, the parameters used being those given by default; the matrix chosen for an amino acid sequence alignment being, for example, the matrix “BLOSUM 62” proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program. Immunogenic Composition [00042] The present disclosure provides an immunogenic composition comprising a first immunogen and a second immunogen. Both immunogens are obtained from the sequences of the toxin A and toxin B polypeptides of C. difficile. Most C. difficile strains produce two major toxins, i.e., Toxin A (TcdA) and Toxin B (TcdB). These toxins are encoded by the genes tcdA and tcdB within the organism’s Pathogenicity loci (PaLoc), while certain C. difficile strains may produce a binary toxin called C. difficile transferase (CDT), closely related to the Clostridium perfringens binary toxin. All these toxins are part of the large clostridial glucosylating toxin (LCGT) family, more appropriately called clostridial glucosylating toxins (Di Bella S et al. Toxins (Basel).2016 May 3;8(5):134). Toxin A may be referred to herein as ToxA. Toxin B may be referred to herein as ToxB. [00043] In a first aspect, there is provided an immunogenic composition comprising a first immunogen and a second immunogen: the first immunogen comprising a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment and the second immunogen comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. [00044] In a preferred embodiment the first immunogen is a first polypeptide, and the second immunogen is a second polypeptide. As such, reference herein to the first polypeptide and second polypeptide corresponds to references to the first immunogen and second immunogen respectively. In an embodiment there is provided an immunogenic composition comprising a first polypeptide and a second polypeptide the first polypeptide comprising a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment and the second polypeptide comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the DRBD of toxin B. [00045] As used herein the term “immunogenic composition” relates to a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental or clinical setting) that is capable of eliciting a specific immune response, e.g., against a pathogen, such as C. difficile. As such, an immunogenic composition includes one or more antigens (for example, polypeptide antigens) or antigenic epitopes. An immunogenic composition can also include one or more additional components capable of eliciting or enhancing an immune response, such as an adjuvant. In certain instances, immunogenic compositions are administered to elicit an immune response that protects the subject, wholly or partially, against symptoms or conditions induced by a pathogen. [00046] The term polypeptide refers to a contiguous sequence of amino acids. [00047] In an embodiment the immunogenic composition of the first aspect further comprises at least one GTD polypeptide such as those described below under the heading “GTD polypeptides” e.g. in an embodiment said immunogenic composition further comprises either a ToxB- GTD and/or ToxA-GTD polypeptide. First Polypeptide [00048] The immunogenic composition of the first aspect comprises a first immunogen comprising a C. difficile toxin A combined repetitive oligopeptides (CROP) domain fragment and a C. difficile toxin B CROP domain fragment. Said first immunogen is preferably a first polypeptide. C. difficile toxin A and toxin B are alternatively known as TcdA and TcdB. [00049] In an embodiment the first polypeptide comprises a C. difficile toxin A combined repetitive oligopeptides (CROP) domain fragment wherein said C. difficile toxin A CROP domain fragment is an immunogenic fragment of the toxin A CROP domain; and a C. difficile toxin B CROP domain fragment wherein said C. difficile toxin B CROP domain fragment is an immunogenic fragment of the toxin B CROP domain. [00050] An immunogenic fragment refers to a portion of a polypeptide that is capable of inducing an immune response, for example wherein said portion is not the whole polypeptide. An immunogenic fragment is a fragment of a polypeptide or protein and refers to a contiguous portion, such as at least 100, 150, 180, 200, 230, 250, 300, 350, 380, 400, 450, 480, 500, 530, 550, 580 or 600 contiguous amino acids, from that polypeptide or protein. [00051] The toxin A CROP domain refers to the toxin A repeating domain. Said toxin A CROP domain (or toxin A repeating domain) corresponds to the C-terminal domain of the toxin A protein from C. difficile that comprises repeated sequences. For example, the C- terminal domain of the toxin A protein may be amino acids 1832-2710 from strain VPI10463 (ATCC43255) and/or their equivalents in a different C. difficile strain. Amino acids 1832-2710 from strain VPI10463 (ATCC43255) corresponds to amino acids 1832-2710 of SEQ ID NO 1. [00052] The toxin B CROP domain refers to the toxin B repeating domain. Said toxin B CROP domain (or toxin B repeating domain) corresponds to the C-terminal domain of the toxin B protein from C. difficile that comprises repeated sequences. For example, the C-terminal domain of the toxin B protein may be amino acids 1834-2366 from strain VPI10463 (ATCC43255) and/or their equivalents in a different C. difficile strain. Amino acids 1834-2366 from strain VPI10463 (ATCC43255) corresponds to amino acids 1834-2366 of SEQ ID NO:2. [00053] 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 strains may differ in the number of amino acids. As a result, the sequence of the toxin A CROP domain and toxin B CROP domain may also differ. [00054] As such, in an embodiment, the toxin A CROP domain comprises at least 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1832-2710 of SEQ ID NO:1. In an embodiment, the toxin B CROP domain comprises at least 90%, 95%, 98%, 99% or 100% sequence identity to amino acids 1834-2366 of SEQ ID NO:2. [00055] Furthermore, the amino acid numbering may differ between the C-terminal domains or N-terminal domains of toxin A or toxin B from one strain and the toxin A or toxin B from another strain. For this reason, the term ‘equivalent in a different strain’ refers to amino acids which correspond 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. Determining equivalent positions of amino acids within different strains is within the expertise of the skilled person. [00056] In an embodiment the toxin A CROP domain fragment of the first polypeptide comprises a proximal end (i.e. proximal end of the toxin A CROP domain fragment) and a distal end (i.e. distal end of the toxin A CROP domain fragment); and the toxin B CROP domain fragment of the first polypeptide comprises a proximal end (i.e. proximal end of the toxin B CROP domain fragment) and a distal end (i.e. distal end of the toxin B CROP domain fragment), wherein the proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment are adjacent to each other. This is shown schematically in Fig.1A. [00057] In an embodiment, the toxin A CROP domain fragment and the toxin B CROP domain fragment are covalently linked together, optionally via a linker sequence. In an embodiment the toxin A CROP domain fragment and the toxin B CROP domain fragment are covalently linked together to form a fusion protein, optionally wherein said covalent linkage is via a linker sequence. In an embodiment the first immunogen (or first polypeptide) is a fusion protein. [00058] As explained, both the toxin A CROP domain fragment and the toxin B CROP domain fragments comprise: ^ A proximal end (i.e., the proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment); and ^ A distal end (i.e., the distal end of the toxin A CROP domain fragment and the distal end of the toxin B CROP domain fragment) [00059] The term ‘proximal end of the toxin A CROP domain fragment’ refers to the end of toxin A CROP domain fragment which is covalently linked to the toxin B CROP domain fragment or covalently linked to a linker sequence between the toxin A and toxin B CROP domain fragments. The “proximal end of the toxin A CROP domain fragment” is thus identifiable relative to the proximity to the toxin B CROP domain fragment. The term ‘proximal end of the toxin B CROP domain fragment’ refers to the end of the toxin B CROP domain fragment which is closest to the toxin A CROP domain fragment in primary structure (amino acid sequence). [00060] The proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment are adjacent to each other. The term ‘adjacent’ means separated by less than or exactly 20, 15, 10, 8, 5, 2, 1 or 0 amino acids in the primary structure. [00061] In an embodiment the distal end of the toxin A CROP domain fragment and the distal end of the toxin B CROP domain fragment are at either terminus of the first polypeptide. [00062] The toxin A CROP domain 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) and long repeats (LRs) (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. Similarly, the short repeats have some sequence and structural similarity to one another. [00063] Similarly, the toxin B CROP domain 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). [00064] For the purposes of the disclosure ‘a repeat portion’ refers to one of the eight repeat portions of the ToxA CROP domain (designated I, II, III, IV, V, VI, VII or VIII) or one of the five repeat portions of ToxB CROP domain (designated I, II, III, IV or V) or a partial repeat portion from the toxin A or toxin B repeating domain. [00065] The proximal end of the toxin A CROP domain fragment is considered to be within a ‘repeat portion’ if the toxin A CROP domain fragment ends in an amino acid that is within that repeat portion (i.e., the proximal end of the toxin A CROP domain fragment contains any part of the repeat portion sequence). Similarly, the proximal end of the toxin B CROP domain fragment is considered to be within a ‘repeat portion’ if the toxin B CROP domain fragment starts in an amino acid that is within that repeat portion. For example, the proximal end of the toxin A CROP domain fragment is within ‘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 proximal end of the toxin A CROP domain fragment is within a ‘long repeat’ or a ‘short repeat’ if the toxin A CROP domain fragment ends in an amino acid that is within a ‘long repeat’ or a ‘short repeat’, similarly the proximal end of the toxin B CROP domain fragment is within a ‘long repeat’ or a ‘short repeat’ if the second fragment ends in an amino acid that is within a ‘long repeat’ or a ‘short repeat’. [00066] The amino acid positions of each repeat portion have been defined for toxin A and toxin B from strain VPI10463 (ATCC43255). These are as follows (Table 1). Name Start position End position ToxA I SR1 1832 1852 SR2 1853 1873 SR3 1874 1893 LR 1894 1924 ToxA II SR1 1925 1944 SR2 1945 1965 SR3 1966 1986 SR4 1987 2007 SR5 2008 2027 LR 2028 2058 ToxA III SR1 2059 2078 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 SR3 2687 2710 ToxB I SR1 1834 1854 SR2 1855 1876 SR3 1877 1896 LR 1897 1926 ToxB II SR1 1927 1946 SR2 1947 1967 SR3 1968 1987 SR4 1988 2007 SR5 2008 2027 LR 2028 2057 ToxB III 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 [00067] In one embodiment, the repeat portion of toxin A refers to amino acids 1832- 1924, 1925-2058, 2059-2192, 2193-2306, 2307-2440, 2441-2553, 2554-2644 or 2645-2710 of toxin A (SEQ ID NO:1) or an equivalent in a different strain of C. difficile. [00068] In another embodiment, the repeat portion of toxin B refers to amino acids 1834- 1926, 1927-2057, 2058-2189, 2190-2323 or 2324-2366 of toxin B (SEQ ID NO:2) or an equivalent in a different strain of C. difficile. [00069] The term ‘short repeat’ may refer to amino acids 1832-1852, 1853-1873, 1874- 1893, 1925-19441945-1965, 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. [00070] Similarly, the term ‘long repeat’ may refer to amino acids 1894-1924, 2028-2058, 2162-2192, 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 a different strain of C. difficile. [00071] In an embodiment the proximal end toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment are fused together. In an embodiment said fusion of the proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment maintains a long solenoid structure across the junction between the two fragments. The secondary structures present can be determined using circular dichroism (CD). For example, the secondary structure may be determined by 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 WO2014/086787. [00072] In an embodiment, the proximal end of the toxin A CROP domain fragment is within a toxin A repeat portion (i.e., toxin A repeat portion I, II, III, IV, V, VI, VII or VIII) and the proximal end of the toxin B CROP domain fragment is within a toxin B repeat portion (i.e., toxin B repeat portion I, II, III, IV or V). In an embodiment the toxin A repeat portion and the toxin B repeat portion have high structural similarity to one another. Two sequences can be considered to have high structural similarity when their percentage identity is higher than 40%, 45%, 50% or 60% (Marti- Renom et al. Annu. Rev. Biophys. Biomol Struct.2000 vol.29:291-325). The presence of high structural similarity can be determined by comparing the two sequences using the SwissModel and SwissPDB Viewer softwares. [00073] In an embodiment the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A. In an embodiment the proximal end of the toxin A CROP domain fragment is within amino acids 2645-2710 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain. [00074] In an embodiment the proximal end of the toxin A CROP domain fragment is within amino acids 2700-2710 or 2680-2690 of toxin A. In an embodiment the proximal end of the toxin A CROP domain fragment is within amino acids 2700-2710 or 2680-2690 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain. [00075] In an embodiment the proximal end of the toxin B CROP domain fragment is within repeat portion I (amino acids 1834-1926) of toxin B. In an embodiment the proximal end of the toxin B CROP domain fragment is within amino acids 1834-1926 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00076] In an embodiment the proximal end of the toxin B CROP domain fragment is within repeat portion II (amino acids 1927-2057) of toxin B. In an embodiment the proximal end of the toxin B CROP domain fragment is within amino acids 1927-2057 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00077] In an embodiment the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A and wherein proximal end of the toxin B CROP domain fragment is within repeat portion I (amino acids 1834-1926) of toxin B. In an embodiment, the proximal end of the toxin A CROP domain fragment is within amino acids 2645-2710 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain and the proximal end of the toxin B CROP domain fragment is within amino acids 1834-1926 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00078] In an embodiment the proximal end of the toxin A CROP domain fragment is within short repeat 3 of repeat portion VIII (amino acids 2687-2710) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within short repeat 1 of repeat portion I of toxin B (amino acids 1834-1854). In an embodiment, the proximal end of the toxin A CROP domain fragment is within amino acids 2687-2710 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1834-1854 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00079] In an embodiment the proximal end of the toxin A CROP domain fragment is within amino acid 2705-2710 of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1850 and 1860 of toxin B. In an embodiment the proximal end of the toxin A CROP domain fragment is amino acid within 2705-2710 of SEQ ID NO: 1(strain VPI10463) or an equivalent position in a different strain and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1850 and 1860 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00080] In an embodiment the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within repeat portion II (amino acids 1927-2057) of toxin B. In an embodiment the proximal end of the toxin A CROP domain fragment is within amino acids 2645-2710 of SEQ ID NO :1 (strain VPI10463) or an equivalent position in a different strain and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1927-2057 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00081] In an embodiment the proximal end of the toxin A CROP domain fragment is within short repeat 2 of repeat portion VIII (amino acids 2665-2686) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within short repeat 3 of repeat portion II of toxin B (amino acids 1968-1987).In an embodiment the proximal end of the toxin A CROP domain fragment is within amino acids 2665-2686 of SEQ ID NO : 1 (strain VPI10463) or an equivalent position in a different strain and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1968-1987 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00082] In an embodiment, the proximal end of the toxin A CROP domain fragment is within amino acids 2680-2690 of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1960-1970 of toxin B. In an embodiment the proximal end of the toxin A CROP domain fragment is within amino acids 2680-2690 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1960-1970 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00083] In an embodiment, the proximal end of the toxin A CROP domain fragment is within short repeat 3 of repeat portion VIII (amino acids 2687-2710) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within short repeat 4 of repeat portion II of toxin B (amino acids 1988-2007).In an embodiment the proximal end of the toxin A CROP domain fragment is within amino acids 2687-2710 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1988-2007 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00084] In an embodiment, the proximal end of the toxin A CROP domain fragment is within amino acids 2705-2710 of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1980-1990 of toxin B. In an embodiment, the proximal end of the toxin A CROP domain fragment is within amino acids 2705-2710 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1980-1990 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00085] In an embodiment, the distal end of the toxin A CROP domain fragment is within repeat portion III (2059-2192) or repeat portion V (2307-2440) of toxin A. In an embodiment, the distal end of the toxin A CROP domain fragment is within amino acids 2059-2192 or amino acids 2307- 2440 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain. [00086] In an embodiment the distal end of the toxin B CROP domain fragment is within repeat portion V (2324-2366) of toxin B. In an embodiment the distal end of the toxin B CROP domain fragment is within amino acids 2324-2366 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. In an embodiment the distal end of the toxin B CROP domain fragment corresponds to the C-terminal amino acid of the toxin B polypeptide, optionally amino acid 2366 of toxin B. [00087] In an alternative embodiment the immunogenic composition of the first aspect comprises a first polypeptide and a second polypeptide wherein the first polypeptide comprises a C. difficile toxin A CROP domain fragment wherein said C. difficile toxin A CROP domain fragment is the entire C. difficile toxin A CROP domain (amino acids 1832-2710); and a C. difficile toxin B CROP domain fragment wherein said C. difficile toxin B CROP domain fragment is the entire C. difficile toxin B CROP domain. In an embodiment the entire C. difficile toxin A CROP domain corresponds to amino acids 1832-2710 of SEQ ID NO: 1 (strain VPI10463) or an equivalent position in a different strain and the entire C. difficile toxin B CROP domain corresponds to amino acids 1833-2366 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. [00088] In an embodiment the first polypeptide further comprises a linker. In an embodiment the linker may link the distal end of the toxin A CROP domain fragment and/or the distal end of the toxin B CROP domain fragment to a further sequence of amino acids. However, in a preferred embodiment the linker is between the proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment. In this embodiment the linker (i.e., a peptide linker sequence) is employed to separate the toxin A CROP domain fragment and the toxin B CROP domain fragment. [00089] 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 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 ToxA and/or ToxB functional epitopes. Peptide linker sequences may contain Glycine (Gly), Asparagine (Asn) and Serine (Ser) residues. Other near neutral amino acids, such as Thr and Ala may also be used in 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. [00090] In an embodiment the linker comprises 1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 10-20 or 10-15 amino acids. In an embodiment the linker comprises 1, 2 ,34, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In a preferred embodiment the linker comprises 1-5 amino acids (i.e., 1, 2, 3, 4 or 5 amino acids). [00091] In an embodiment the linker is a glycine linker. A glycine linker may comprise multiple 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. In an alternative embodiment the linker comprises or consists of SEQ ID NO: 15. [00092] In an embodiment, the first polypeptide comprises more than 350, 375, 400 or 425 amino acids from toxin A. In an embodiment, the first polypeptide comprises less than 750, 725, 700, 675, 650, 625, or 600 amino acids from toxin A. In an embodiment the first polypeptide comprises between 400 and 600 amino acids from toxin A. Said amino acids from toxin A are from the toxin A CROP domain. [00093] In an embodiment the first polypeptide comprises more than 250, 300 or 350 amino acids from toxin B. In an embodiment, the first polypeptide comprises less than 675, 650, 625, 600, 575 or 550 amino acids from toxin B. In an embodiment, the first polypeptide comprises between 370 and 520 amino acids from toxin B. Said amino acids from toxin B are from the toxin B CROP domain. [00094] In an embodiment the first polypeptide comprises: (i) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; or (ii) A variant having at least 90%, 95%, 98%, 99% or 100% similarity or identity to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; or (iii) A fragment of at least 250, 280, 300, 350, 380, 400, 430, 450, 480, 500, 530, 550, 580, or 600 contiguous amino acids of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14. [00095] In an embodiment the first polypeptide comprises a sequence at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least 97.5% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In an embodiment the first polypeptide comprises a sequence at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In an embodiment the first polypeptide comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. Second Polypeptide [00096] The immunogenic composition of the first aspect comprises a first immunogen and a second immunogen, said second immunogen comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. Preferably the second immunogen is a polypeptide (i.e., the second polypeptide). Thus, in an embodiment, the immunogenic composition of the first aspect comprises a first polypeptide and a second polypeptide, said second polypeptide comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the DRBD domain of toxin B. [00097] The delivery and receptor binding domain (DRBD) of toxin B refers to the domain of toxin B that binds to cell surface receptors, prior to the toxin entering the cell via endocytosis. Acidification in the endosome triggers conformational changes in the toxins that prompt the DRBD to form a pore and deliver the GTD and the CPD across the endosomal membrane. The DRBD also serves to protect the hydrophobic pore-forming region (residues 957–1129), which is predicted to be released upon endosome acidification in order to form a pore that delivers the GTD and the CPD to the cytosol. (Chen P et al. Nat Struct Mol Biol.2019 Aug;26(8):712-719). [00098] In an embodiment the DRBD of toxin B corresponds to amino acids 840-1833 of toxin B from strain VPI10463 or an equivalent position in a different strain. In an embodiment the DRBD of toxin B corresponds to amino acids 840-1833 of SEQ ID NO: 2 (strain VPI10463) or an equivalent position in a different strain. It will be understood by the person skilled in the art that the specific amino acid numbering denoting the DRBD will differ between strains, however, it is within the skilled persons expertise to determine equivalent positions in other strains of C. difficile, for example using sequence alignment tools. For example, the DRBD in the M68 strain corresponds to amino acids 841-1834 of toxin B (M68), i.e., amino acids 841-1834 of SEQ ID NO: 18. [00099] In an embodiment, the second polypeptide comprises at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325 or at least 350 contiguous amino acids of the DRBD of toxin B. In an embodiment the second polypeptide is between 150 and 700 amino acids in length, between 200 and 600 amino acids in length, between 250 and 550 amino acids in length, between 300 and 450 amino acids in length, between 350 and 400 amino acids in length or between 375 and 385 amino acids in length. In an embodiment the second polypeptide, consists of between 150 and 700 amino acids, between 200 and 600 amino acids, between 250 and 550 amino acids, between 300 and 450 amino acids, between 350 and 400 amino acids or between 375 and 385 amino acids. [000100] In an embodiment the second polypeptide corresponds to amino acids 1071- 1451 of SEQ ID NO: 2 (strain VPI10463) or amino acids 1072-1452 SEQ ID NO: 18 (strain M68) or equivalent positions in a different strain, for example at equivalent positions in sequences having at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 18. [000101] In an embodiment the second polypeptide comprises a sequence at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95%, at least 97.5%, at least 99% or 100% identical to SEQ ID NO: 16 or SEQ ID NO: 17. In an embodiment, the second polypeptide comprises a sequence at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16 or SEQ ID NO: 17. In an embodiment the second polypeptide comprises or consists of SEQ ID NO: 16 or SEQ ID NO: 17. [000102] The sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 17 differ by a single amino acid, more particularly in V73 in SEQ ID NO: 16 (M68 strain) is I73 in SEQ ID NO: 17 (VPI10463 strain). It will be understood by the person skilled in the art that SEQ ID NO: 16 may be obtained by mutating a closely related sequence, such as a sequence with greater than 90% sequence identity. [000103] In an embodiment the second polypeptide comprises a sequence at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least 97.5% identical to SEQ ID NO: 16. In an embodiment, the second polypeptide comprises a sequence at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16. In an embodiment the second polypeptide comprises or consists of SEQ ID NO: 16. GTD Polypeptides [000104] Further provided herein are glucosyl transferase domain (GTD) polypeptides. The GTD domain refers to the N-terminal region of the enzymatic domain of both TcdA and TcdB. [000105] In an embodiment, the immunogenic composition disclosed herein further comprises a third immunogen wherein said third immunogen is a third polypeptide. In an embodiment said third polypeptide is a glucosyl transferase domain (GTD) polypeptide such as those disclosed in this section. Said GTD polypeptide may comprise the GTD from TcdA (herein referred to as a ToxA-GTD polypeptide) or from TcdB (herein referred to as a ToxB-GTD polypeptide) [000106] In an embodiment, there is provided a ToxB-GTD polypeptide. The abbreviation "ToxB-GTD” refers to the glucosyl transferase domain of TcdB. The ToxB-GTD polypeptide (SEQ ID NO: 48 encoded by the nucleic acid sequence of SEQ ID NO: 49) is a fragment of TcdB that corresponds to amino acids 1-543 of SEQ ID NO: 2. [000107] The ToxB-GTD polypeptide may additionally be included in the compositions of the present disclosure. The ToxB-GTD polypeptide is a polypeptide that comprises or consists of an amino acid sequence: (a) having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, 99.9%, or more) to SEQ ID NO: 48; and/or (b) that is a fragment of at least "n" consecutive amino acids of SEQ ID NO: 48, or of a polypeptide having 50% or more identity to SEQ ID NO: 48, wherein "n" is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 250, 300, 400, 500, 540, or more). Preferred fragments comprise an epitope of SEQ ID NO: 48. Other preferred fragments lack one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 48 while retaining at least one epitope of SEQ ID NO: 48. Amino acid fragments of the ToxB- GTD polypeptide may thus comprise an amino acid sequence of e.g. up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 125, up to 150, up to 175, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or up to 540, consecutive amino acid residues of SEQ ID NO: 48. [000108] The ToxB-GTD polypeptide that may additionally be included in the compositions of the present disclosure may be detoxified. Detoxification may be achieved by mutating the amino acid sequence or the encoding nucleic acid sequence of the wild-type ToxB-GTD polypeptide using any appropriate method known in the art e.g. site- directed mutagenesis. Preferably, the ToxB-GTD polypeptide comprises one or more amino acid substitutions (i.e.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, or more mutations), relative to the wild- type ToxB-GTD polypeptide sequence of SEQ ID NO: 48. For example, the ToxB-GTD polypeptide comprises one or more amino acid substitutions (i.e.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more mutations), e.g. at amino acid positions 17, 102, 139, 269, 270, 273, 284, 286, 288, 384, 449, 444, 445, 448, 449, 450, 451, 452, 455, 461, 463, 472, 515, 518, and/or 520, relative to the wild-type ToxB-GTD polypeptide sequence of SEQ ID NO: 48. For example, the ToxB-GTD polypeptide may comprise substitutions at 1, 2, 3, 4 or 5 positions corresponding to amino acids 270, 273, 284, 286 and/or 288 of the ToxB-GTD polypeptide sequence of SEQ ID NO: 48. In particular, 1, 2, 3, 4 or 5 amino acids at positions corresponding to amino acids 270, 273, 284, 286 and/or 288 of the ToxB-GTD polypeptide sequence of SEQ ID NO: 48 may be substituted, preferably by alanine residues. Where amino acids 270, 273, 284, 286 and/or 288 of SEQ ID NO: 48 are substituted, the substitutions are preferably D270A, R273A, Y284A, D286A and/or D288A, most preferably D270A, R273A, Y284A, D286A and D288A. These substitutions correspond to substitutions D270A, R273A, Y284A, D286A and D288A of SEQ ID NO: 2. The amino acid sequence of a detoxified ToxB-GTD polypeptide having alanine substitutions at these positions is provided in SEQ ID NO: 50. [000109] Where the ToxB-GTD polypeptide comprises two amino acid substitutions, the substitutions are preferably not at amino acid positions 102 and 278, or amino acid positions 102 and 288, of the ToxB-GTD polypeptide sequence of SEQ ID NO: 48. The detoxified ToxB-GTD polypeptide that may additionally be included in the compositions of the disclosure may thus be a polypeptide that comprises or consists of an amino acid sequence: (a) having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, 99.9%, or more) to SEQ ID NO: 50 ; and/or (b) that is a fragment of at least "n" consecutive amino acids of SEQ ID NO: 50, or of a polypeptide having 50% or more identity to SEQ ID NO: 50, wherein "n" is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 250, 300, 400, 500, 540, or more). Amino acid fragments of detoxified ToxB-GTD polypeptide may thus comprise an amino acid sequence of e.g. up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 125, up to 150, up to 175, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or up to 540, consecutive amino acid residues of SEQ ID NO: 50. Preferred fragments comprise an epitope of SEQ ID NO: 50. Other preferred fragments lack one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 50 while retaining at least one epitope of SEQ ID NO: 50. [000110] In an alternative embodiment, the GTD polypeptide may be the glucosyl transferase domain of TcdA (as opposed to TcdB as discussed above) and is referred to herein as ToxA-GTD. The abbreviation "ToxA-GTD” thus refers to the glucosyl transferase domain of TcdA, which is located within the N-terminal region of the enzymatic domain (ED). The ToxA-GTD polypeptide (SEQ ID NO: 51, encoded by the nucleic acid sequence of SEQ ID NO: 52) is a fragment of TcdA that corresponds to amino acids 1-541 of SEQ ID NO: 1. [000111] The ToxA-GTD polypeptide may additionally be included in the compositions of the present disclosure. The ToxA-GTD polypeptide is a polypeptide that comprises or consists of an amino acid sequence: (a) having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, 99.9%, or more) to SEQ ID NO: 51; and/or (b) that is a fragment of at least "n" consecutive amino acids of SEQ ID NO: 51, or of a polypeptide having 50% or more identity to SEQ ID NO: 51, wherein "n" is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 250, 300, 400, 500, 540, or more). Preferred fragments comprise an epitope of SEQ ID NO: 51. Other preferred fragments lack one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 51 while retaining at least one epitope of SEQ ID NO: 51. [000112] Amino acid fragments of ToxA-GTD may thus comprise an amino acid sequence of e.g. up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 125, up to 150, up to 175, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or up to 540, consecutive amino acid residues of SEQ ID NO: 51. [000113] The ToxA-GTD polypeptide that may additionally be included in the compositions of the disclosure may be detoxified. Detoxification may be achieved by mutating the amino acid sequence or the encoding nucleic acid sequence of the wild-type ToxA-GTD polypeptide using any appropriate method known in the art e.g. site- directed mutagenesis. Preferably, the ToxA-GTD polypeptide comprises one or more amino acid substitutions (i.e.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more mutations), relative to the wild-type ToxA- GTD polypeptide sequence of SEQ ID NO: 51. For example, the ToxA-GTD polypeptide may comprise substitutions at 1, 2 or 3 positions corresponding to amino acids 283, 285 and 287 of the ToxA-GTD polypeptide sequence of SEQ ID NO: 51. In particular, 1, 2, or 3 amino acids at positions corresponding to amino acids 283, 285 and 287 of the ToxA-GTD polypeptide sequence of SEQ ID NO:51 may be substituted, preferably by alanine residues (i.e. Y283A, D285A, D287A). These mutations correspond to positions 283, 285 and 287 of SEQ ID NO: 1. The amino acid sequence of a detoxified ToxA-GTD polypeptide having alanine substitutions at these positions is provided in SEQ ID NO: 53. Where the ToxA-GTD polypeptide comprises one amino acid substitution, the substitution is preferably not at amino acid position 278 of the ToxA-GTD polypeptide sequence of SEQ ID NO: 51. Where the ToxA-GTD polypeptide comprises two amino acid substitutions, the substitutions are preferably not at amino acid positions 101 and 278, of the ToxA-GTD polypeptide sequence of SEQ ID NO: 51. Where the ToxA-GTD polypeptide comprises three amino acid substitutions, the substitutions are preferably not at amino acid positions 101, 278 and 519, or amino acid positions 101, 287 and 519, of the ToxA-GTD polypeptide sequence of SEQ ID NO: 51. The detoxified ToxA-GTD polypeptide that may additionally be included in the compositions of the disclosure may thus be a polypeptide that comprises or consists of an amino acid sequence: (a) having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, 99.9%, or more) to SEQ ID NO: 53; and/or (b) that is a fragment of at least "n" consecutive amino acids of SEQ ID NO: 53, or of a polypeptide having 50% or more identity to SEQ ID NO: 53, wherein "n" is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 250, 300, 400, 500, 540, or more). Preferred fragments comprise an epitope of SEQ ID NO: 53. Other preferred fragments lack one or more amino acids (e.g.1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 53 while retaining at least one epitope of SEQ ID NO: 53. Amino acid fragments of detoxified ToxA-GTD may thus comprise an amino acid sequence of e.g. up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 125, up to 150, up to 175, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or up to 540, consecutive amino acid residues of SEQ ID NO: 53. [000114] In an embodiment, the immunogenic composition of the invention comprises a third polypeptide and said third polypeptide is a GTD polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95% or at least 97.5% sequence identity to SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 53, preferably SEQ ID NO: 50 or 51. In an embodiment, the third polypeptide is a GTD polypeptide comprising or consisting of SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 53, preferably SEQ ID NO: 50 or 51. [000115] In an embodiment, the first polypeptide and/or the second polypeptide (or the third polypeptide) comprises additional amino acid sequences. 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. In an embodiment, the first polypeptide and/or second polypeptide (or the third polypeptide) comprises a poly-histidine tag. In an embodiment, the first polypeptide and/or the second polypeptide (or the third polypeptide) may additionally comprise a signal peptide. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered. [000116] In an embodiment, the immunogenic composition according to the first aspect further comprises additional antigens. For example, in an embodiment the additional antigens are antigens derived from a bacterium selected from the group consisting of Streptococcus pneumonia, Haemophilus influenzae, Neisseria meningitidis, Escherichia coli, Moraxella catarrhalis, Clostridioides tetani, Corynebacterium diptherieriae, Bordetella pertussis, Staphylococcus epidermidis, enterococci, and Staphylococcus aureus. In a particular embodiment, the immunogenic composition further comprises additional C. difficile antigens e.g., a saccharide from C. difficile. [000117] In an embodiment, the immunogenic composition according to the first aspect further comprises an adjuvant. [000118] As used herein “adjuvant” means a compound or substance (or combination of compounds or substances) that, when administered to a subject in conjunction with an antigen or antigens, for example as part of an immunogenic composition or vaccine, increases or enhances the subject’s immune response to the administered antigen or antigens, compared to the immune response obtained in the absence of adjuvant. With respect to the present disclosure, the adjuvant may additionally mean a compound or substance (or combination of compounds or substances) that, when administered to a subject in conjunction with the first immunogen and second immunogen, increases or enhances the subject’s immune response to said immunogens. [000119] In one embodiment the adjuvant comprises an immunologically active saponin fraction. In certain embodiments, the immunogenic composition is formulated with an adjuvant comprising an immunologically active saponin fraction presented in the form of a liposome. In an embodiment, the immunologically active saponin fraction is QS21. [000120] The adjuvant may further comprise a lipopolysaccharide, optionally a lipid A derivative. In one embodiment, the adjuvant system includes the lipid A derivative 3D-MPL and QS21. For example, in one embodiment, the adjuvant contains 3D-MPL and QS21 in a liposomal formulation. [000121] In an embodiment, the adjuvant further comprises a sterol, optionally wherein the adjuvant comprises cholesterol. In one specific embodiment, the adjuvant comprises QS21 and cholesterol. In an embodiment the adjuvant further comprises 1, 2-Dioleoyl-sn-Glycero-3- phosphocholine (DOPC). For example, one specific adjuvant system comprises cholesterol, DOPC, 3D-MPL and QS21. [000122] In one specific embodiment, the immunogenic composition comprises an adjuvant formulated in a dose that includes: from about 0.1 to about 0.5 mg cholesterol; from about 0.25 to about 2 mg DOPC; from about 10 µg to about 100 µg 3D-MPL; and from about 10 µg to about 100 µg QS21. In a further specific embodiment, the immunogenic composition includes, and adjuvant formulated in a dose that includes from about 0.1 to about 0.5mg cholesterol, from about 0.25 to about 2mg DOPC, from about 10μg to about 100μg 3D-MPL, and from about 10μg to about 100μg QS21. In one a further specific embodiment, the adjuvant is formulated in a single dose that contains: about 0.25 mg cholesterol; about 1.0 mg DOPC; about 50 µg 3D-MPL; and about 50 µg QS21. In other embodiments, the immunogenic composition is formulated with a fractional dose (that is a dose, which is a fraction of the preceding single dose formulations, such as one half of the preceding quantity of components (cholesterol, DOPC, 3D-MPL and QS21), ¼ of the preceding quantity of components, or another fractional dose (e.g., 1/3, 1/6, etc.) of the preceding quantity of components. [000123] In one embodiment, the immunogenic compositions comprise an adjuvant containing combinations of lipopolysaccharide and Quillaja saponins that have been disclosed previously, for example in EP0671948. This patent demonstrated a strong synergy when a lipopolysaccharide (3D-MPL) was combined with a Quillaja saponin (QS21). [000124] The adjuvant may further comprise immunostimulatory oligonucleotides (for example, CpG) or a carrier. [000125] In a specific embodiment, QS21 is provided in its less reactogenic composition where it is quenched with an exogenous sterol, such as cholesterol for example. Several particular forms of less reactogenic compositions wherein QS21 is quenched with an exogenous cholesterol exist. In a specific embodiment, the saponin /sterol is in the form of a liposome structure (WO 96/33739, Example 1). In this embodiment the liposomes suitably contain a neutral lipid, for example phosphatidylcholine, which is suitably non-crystalline at room temperature, for example eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilauryl phosphatidylcholine. The liposomes may also contain a charged lipid which increases the stability of the lipsome-QS21 structure for liposomes composed of saturated lipids. In these cases, the amount of charged lipid is suitably 1- 20% w/w, suitably 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), suitably 20-25%. [000126] Suitable sterols include ^-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In one particular embodiment, the adjuvant composition comprises cholesterol as sterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 11th Edn., page 341, as a naturally occurring sterol found in animal fat. [000127] Where the active saponin fraction is QS21, the ratio of QS21: sterol will typically be in the order of 1:100 to 1:1 (w/w), suitably between 1:10 to 1:1 (w/w) or 1:5 to 1:1 (w/w). Suitably excess sterol is present, the ratio of QS21: sterol being at least 1:2 (w/w). In one embodiment, the ratio of QS21: sterol is 1:5 (w/w). In an embodiment the sterol is cholesterol. [000128] In one embodiment, the invention provides a dose of an immunogenic composition comprising immunologically active saponin, (e.g., QS21), at a level of 60µg or less, for example between 1 and 60µg. In one embodiment, the dose of the immunogenic composition comprises QS21 at a level of approximately around 50 µg, for example between 45 and 55 µg, suitably between 46 – 54 µg or between 47 and 53 µg or between 48 and 52 µg or between 49 and 51 µg, or 50 µg per dose. [000129] In another embodiment the dose of the immunogenic composition comprises QS21 at a level of around 25 µg, for example between 20 – 30 µg, suitably between 21 – 29 µg or between 22 and 28 µg or between 23 and 27 µg or between 24 and 26 µg, or 25 µg. [000130] In another embodiment, the dose of the immunogenic composition comprises QS21 at a level of around 10 µg per, for example between 5 and 15 µg, suitably between 6 and 14 µg, for example between 7 and 13 µg or between 8 and 12 µg or between 9 and 11 µg, or 10µg. [000131] Specifically, a 0.5 ml vaccine dose volume contains 25 µg or 50 µg of QS21 per dose. Specifically, a 0.5 ml vaccine dose volume contains 50 µg of QS21 per dose. [000132] The lipopolysaccharide may be a non-toxic derivative of lipid A, particularly monophosphoryl lipid A or more particularly 3-Deacylated monophoshoryl lipid A (3D – MPL). [000133] 3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A. and is referred throughout the document as MPL or 3D-MPL. See, for example, US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL primarily promotes CD4+ T cell responses with an IFN-^ (Th1) phenotype. 3D-MPL can be produced according to the methods disclosed in GB 2220211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. In an embodiment, the compositions of the present invention use small particle 3D-MPL. Small particle 3D-MPL has a particle size such that it may be sterile-filtered through a 0.22 ^m filter. Such preparations are described in WO 94/21292. [000134] The invention therefore provides a dose of an immunogenic composition comprising lipopolysaccharide, (e.g., 3D-MPL), at a level of 75µg or less, for example between 1 and 60µg. In one embodiment the lipopolysaccharide is present at an amount of about 50μg per dose. [000135] In one embodiment, the dose of the immunogenic composition comprises 3D-MPL at a level of around 50 µg, for example between 45 – 55 µg, suitably between 46 – 54 µg or between 47 and 53 µg or between 48 and 52 µg or between 49 and 51 µg, or 50 µg. [000136] In one embodiment, the dose of the immunogenic composition comprises 3D-MPL at a level of around 25 µg, for example between 20 – 30 µg, suitably between 21 – 29 µg or between 22 and 28 µg or between 23 and 27 µg or between 24 and 26 µg, or 25 µg. [000137] In another embodiment, the dose of the immunogenic composition comprises 3D- MPL at a level of around 10 µg, for example between 5 and 15 µg, suitably between 6 and 14 µg, for example between 7 and 13 µg or between 8 and 12 µg or between 9 and 11 µg, or 10µg. [000138] In one embodiment, the volume of the dose is 0.5 ml. In a further embodiment, the immunogenic composition is in a volume suitable for a dose which volume is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In a further embodiment, the human dose is between 1 ml and 1.5 ml. [000139] Specifically, a 0.5 ml vaccine dose volume contains 25 µg or 50 µg of 3D-MPL per dose. Specifically, a 0.5 ml vaccine dose volume contains 50 µg of 3D-MPL per dose. [000140] The dose of the immunogenic composition according to any aspect of the invention suitably refers to human dose. By the term “human dose” is meant a dose which is in a volume suitable for human use. Generally, this is between 0.3 and 1.5 ml. In one embodiment, a human dose is 0.5 ml. In a further embodiment, a human dose is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In a further embodiment, a human dose is between 1 ml and 1.5 ml. [000141] Suitable compositions of the invention are those wherein liposomes are initially prepared without MPL (as described in WO 96/33739), and MPL is then added, suitably as small particles of below 100 nm particles or particles that are susceptible to sterile filtration through a 0.22 μm membrane. The MPL is therefore not contained within the vesicle membrane (known as MPL out). Compositions where the MPL is contained within the vesicle membrane (known as MPL in) also form an aspect of the invention. The polypeptide comprising a C. difficile toxin A fragment and / or a C. difficile toxin B fragment can be contained within the vesicle membrane or contained outside the vesicle membrane. [000142] In a specific embodiment, QS21 and 3D-MPL are present in the same final concentration per dose of the immunogenic composition. In one aspect of this embodiment, a dose of immunogenic composition comprises a final level of 25 µg of 3D-MPL and 25 µg of QS21 or 50 µg of 3D- MPL and 50 µg of QS21. [000143] In an alternative embodiment the adjuvant comprises an oil in water emulsion. Said oil-in-water emulsion can include an oil phase that incorporates a metabolisable oil, and an additional oil phase component, such as a tocol. The oil-in-water emulsion may also contain an aqueous component, such as a buffered saline solution (e.g., phosphate buffered saline). In addition, the oil-in- water emulsion typically contains an emulsifier. In one embodiment, the metabolizable oil is squalene. In one embodiment, the tocol is alpha-tocopherol. In one embodiment, the emulsifier is a nonionic surfactant emulsifier (such as polyoxyethethylene sorbitan monooleate, TWEEN80™). In exemplary embodiments, the oil-in-water emulsion contains squalene and alpha tocopherol in a ratio which is equal or less than 1 (w/w). Thus, in and embodiment, said oil in water emulsion comprises a metabolisable oil, a tocol and an emulsifying agent. [000144] In an embodiment the metabolisable oil in the oil-in-water emulsion may be present in an amount of 0.5-10mg. In an embodiment the metabolisable oil is squalene. In order for any oil in water composition to be suitable for human administration, the oil phase of the emulsion system has to comprise a metabolisable oil. The meaning of the term metabolisable oil is well known in the art. Metabolisable can be defined as ‘being capable of being transformed by metabolism’ (Dorland’s Illustrated Medical Dictionary, W.B. Sanders Company, 25th edition (1974)). The oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE ^ (caprylic/capric triglycerides made using glycerol from vegetable oil sources and medium- chain fatty acids (MCTs) from coconut or palm kernel oils) and others. A particularly suitable metabolisable oil is squalene. Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22- tetracosahexaene) is an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast. Squalene is a metabolisable oil by virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no.8619). [000145] Suitably the metabolisable oil is present in the adjuvant composition in an amount of 0.5-10 mg, 1-10mg, 2-10mg, 3-9mg, 4-8mg, 5-7mg or 5-6 mg (e.g., 2-3, 5-6, or 9-10mg), specifically about 5.35 mg. [000146] In an embodiment, the tocol in the oil-in-water emulsion may be present in an amount of 0.5 – 11 mg. Tocols are well known in the art and are described in EP0382271. In an embodiment the tocol is alpha-tocopherol. In an embodiment the tocol is alpha-toxopherol or a derivative thereof such as alpha-tocopherol succinate (also known as vitamin E succinate). Said tocol is suitably present in in an amount of 0.5-11 mg, 1-11mg, 2-10mg, 3-9mg, 4-8mg, 5-7mg, 5-6 mg (e.g., 10- 11, 5-6, 2.5-3.5 or 1-3 mg). In a specific embodiment the tocol is present in an amount of about 5.94 mg. [000147] In an embodiment, the emulsifying agent may be present in an amount of 0.4 -4mg. In an embodiment, the emulsifying agent is polyoxyethylene sorbitan monooleate. In an embodiment, the polyoxyethylene sorbitan monooleate is selected from the group comprising: Polysorbate® 80 or Tween® 80. Said emulsifying agent is suitably present in the adjuvant composition in an amount of 0.1- 5, 0.2-5, 0.3-4, 0.4-3 or 2-3 mg (e.g., 0.4-1.2, 2-3 or 4-5 mg) emulsifying agent. In a specific embodiment the emulsifying agent is present in an amount of about 0.97 mg or about 2.425 mg. [000148] In one embodiment, the amounts of specific components present in the composition are the amounts present in a 0.5 ml human dose. In a further embodiment, the immunogenic composition is in a volume suitable for a human dose which volume is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In a further embodiment, the human dose is between 1 ml and 1.5 ml. [000149] Where the adjuvant is in a liquid form and is to be combined with a liquid form of a polypeptide composition, the adjuvant composition in a human dose will be a fraction of the intended final volume of the human dose, for example approximately half of the intended final volume of the human dose, for example a 350 µl volume for an intended human dose of 0.7ml, or a 250 µl volume for an intended human dose of 0.5 ml. The adjuvant composition is diluted when combined with the immunogenic composition, to provide the final human dose of vaccine. The final volume of such dose will of course vary dependent on the initial volume of the adjuvant composition and the volume of the immunogenic composition added to the adjuvant composition. In an alternative embodiment, a liquid adjuvant is used to reconstitute a lyophilised immunogenic composition. In this embodiment, the human dose of the adjuvant composition is approximately equal to the final volume of the human dose. The liquid adjuvant composition is added to the vial containing the lyophilised immunogenic composition. The final human dose can vary between 0.5 and 1.5 ml. [000150] The method of producing oil-in-water emulsions is well known to the person skilled in the art. Commonly, the method comprises mixing the tocol-containing oil phase with a surfactant such as a PBS/ polyoxyethylene sorbitan monooleate solution, followed by homogenisation using a homogenizer. It would be clear to a man skilled in the art that a method comprising passing the mixture twice through a syringe needle would be suitable for homogenising small volumes of liquid. Equally, the emulsification process in microfluidiser (M110S Microfluidics machine, maximum of 50 passes, for a period of 2 minutes at maximum pressure input of 6 bar (output pressure of about 850 bar)) could be adapted by the man skilled in the art to produce smaller or larger volumes of emulsion. The adaptation could be achieved by routine experimentation comprising the measurement of the resultant emulsion until a preparation was achieved with oil droplets of the required diameter. [000151] In an oil in water emulsion, the oil and emulsifier should be in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline. [000152] In an embodiment the oil-in-water emulsion of the present invention have a small oil droplet size in the sub-micron range. Suitably the droplet sizes will be in the range 120 to 750 nm, such as from 120 to 600 nm in diameter. In a particular embodiment the oil-in water emulsion contains oil droplets of which at least 70% by intensity are less than 500 nm in diameter. In an embodiment the oil-in water emulsion contains oil droplets of which at least 80% by intensity are less than 300 nm in diameter or at least 90% by intensity are in the range of 120 to 200 nm in diameter. [000153] In an alternative embodiment the adjuvant is an aluminum-based adjuvant, optionally aluminum hydroxide, or aluminum phosphate. Suitable aluminium-based adjuvants include hydroxides, phosphates or mixtures thereof. The adjuvants can take any suitable form (e.g., gel, crystalline, amorphous etc). The first and/or second immunogens disclosed herein can be precipitated with or adsorbed onto he aluminium-based adjuvant using methods known to the skilled person. In an embodiment, the aluminium-based adjuvant is aluminium hydroxide or aluminium phosphate. In an embodiment, the adjuvant is aluminium hydroxide. In an embodiment, the adjuvant is aluminium phosphate. In an embodiment, the first and/or second immunogen are adsorbed onto aluminium hydroxide or aluminium phosphate. [000154] In an embodiment the immunogenic composition according to the first aspect elicit antibodies that neutralize toxin A or toxin B or both. In an embodiment, said immunogenic composition according to the first aspect elicits antibodies that neutralize toxin A or toxin B or both, following administration to a subject e.g., a mammal. [000155] In an embodiment the immunogenic composition of the invention comprises a first polypeptide and a second polypeptide, wherein said first polypeptide elicits antibodies that neutralize toxin A and toxin B, and wherein said second polypeptide elicits antibodies that neutralize toxin B. [000156] The phrase ‘elicits neutralising antibodies’ means that the when the immunogenic compositions are used to immunise a mammal, for example a mouse, a guinea pig or a human, the mammal generates neutralising antibodies. Whether a composition elicits neutralizing antibodies against a toxin can be measured by immunizing mice with an immunogenic composition of the present invention, collecting sera and analysing the anti-toxin titers of the sera using by enzyme-linked immunosorbent assay (ELISA). The sera could be compared to a reference sample obtained from mice which have not been immunised. An example of this technique can be found in example 4. The immunogenic composition 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. [000157] In a further embodiment the immunogenic composition of the invention elicits a protective immune response in a mammalian host against strains of C. difficile. The phrase ‘elicit a protective immune response’ means that when the immunogenic composition of the invention is used to immunise a mammal such as a mouse, guinea pig or human, the mammal generates antibodies capable of protecting the mammal from death caused by C. difficile. In one embodiment the mammalian host is selected from the group consisting of mouse, rabbit, guinea pig, monkey, non- human primate, or human. In one embodiment the mammalian host is a mouse. In a further embodiment the mammalian host is a human [000158] Whether an immunogenic composition 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 composition 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 composition survives. An immunogenic composition 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 after challenge with C. difficile than a reference mammal which has not been immunised with the composition. In one embodiment the immunogenic composition of the invention elicits a protective immune response against C. difficile in a mammal selected from the group consisting of mouse, guinea pig, monkey, or human. In one embodiment the mammal is a mouse, in a further embodiment the mammal is a human. Nucleic Acid [000159] Also provided are nucleic acids encoding at least one polypeptide comprising a bacterial toxoid or toxin or an immunogenic fragment of a bacterial toxoid or toxin. In an embodiment, the nucleic acids encode at least one polypeptide comprising a C. difficile toxoid or toxin or an immunogenic fragment of a bacterial toxoid or toxin. In an embodiment, the nucleic acid encodes a C. difficile TcdA toxoid. In an embodiment, the C. difficile TcdA toxoid contains a mutation in a glucosyltransferase domain and a cysteine protease domain. In an embodiment the mutation in the TcdA glucosyltransferase domain is a substitution at amino acid 285 and/or 287 of SEQ ID NO:29 of WO 12/143902, relative to the corresponding wild type sequence. In an embodiment, the mutation in the TcdA cysteine protease domain is a substitution at position 158 of SEQ ID NO:32 of WO 12/143902, relative to the corresponding wild type sequence. In an embodiment, the nucleic acid encodes a C. difficile TcdB toxoid. In an embodiment the mutation in the TcdB glucosyltransferase domain is a substitution at amino acid 286 and/or 288 of SEQ ID NO:31 of WO 12/143902, relative to the corresponding wild type sequence. In an embodiment, the mutation in the TcdB cysteine protease domain is a substitution at position 155 of SEQ ID NO:33 of WO 12/143902, relative to the corresponding wild type sequence. In an embodiment the nucleic acid encodes a C. difficile TcdA toxoid and a C. difficile TcdB toxoid. In an embodiment, the mutation in the TcdA glucosyltransferase domain is a substitution at amino acid 285 and/or 287 of SEQ ID NO:29 of WO 12/143902, relative to the corresponding wild type sequence and the mutation in the TcdA cysteine protease domain is a substitution at position 158 of SEQ ID NO:32 of WO 12/143902, relative to the corresponding wild type sequence and the mutation in the TcdB glucosyltransferase domain is a substitution at amino acid 286 and/or 288 of SEQ ID NO:31 of WO 12/143902, relative to the corresponding wild type sequence and the mutation in the TcdB cysteine protease domain is a substitution at position 155 of SEQ ID NO:33 of WO 12/143902, relative to the corresponding wild type sequence. In an embodiment, the bacterial toxoid is as described in WO 12/143902, WO 20/201985 or WO 21/255690. In further embodiments, the nucleic acid encodes at least one polypeptide comprising a fragment of toxin A or toxin B of C. difficile, for example the fragment of toxin A or toxin B described herein or fusion proteins comprising at least one or at least two of the fragments of toxin A and/or toxin B described herein. [000160] Also provided are nucleic acids encoding the first and/or second polypeptides referred to in respect of the first aspect above. [000161] In a second aspect there is provided a nucleic acid encoding a) the first polypeptide as referred to in the first aspect; and b) the second polypeptide as referred to in the first aspect, wherein said nucleic acid is a single polynucleotide encoding said first and second polypeptides. [000162] In a third aspect there is provided a nucleic acid encoding the first polypeptide as referred to in the first aspect. In an embodiment there is provided a nucleic acid encoding the first polypeptide as referred to the first aspect, wherein said first polypeptide comprises a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment. In an embodiment, the nucleic acid encoding the first polypeptide as referred to in the first aspect, encodes a polypeptide with a sequence that is at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least 97.5% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In an embodiment, the nucleic acid encoding the first polypeptide as referred to in the first aspect, encodes a polypeptide that comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. [000163] In a fourth aspect there is provided a nucleic acid encoding the second polypeptide as referred to in the first aspect. In an embodiment there is provided a nucleic acid encoding the second polypeptide as referred to in the first aspect, wherein said second polypeptide comprises a fragment of C. difficile toxin B, said fragment comprising at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. In an embodiment, the nucleic acid encoding the second polypeptide referred to in the first aspect, encodes a polypeptide with a sequence that is at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least 97.5% identical to SEQ ID NO: 16 or SEQ ID NO: 17. In an embodiment, the nucleic acid encoding the second polypeptide referred to in the first aspect, encodes a polypeptide that comprises or consists of SEQ ID NO: 16 or SEQ ID NO: 17. [000164] Further provided is a nucleic acid encoding the third polypeptide. In an embodiment there is provided a nucleic acid encoding the third polypeptide wherein said third polypeptide is a glucosyl transferase domain (GTD) polypeptide. Said GTD polypeptide may comprise the GTD from TcdA (herein referred to as a ToxA-GTD polypeptide) or from TcdB (herein referred to as a ToxB-GTD polypeptide). In an embodiment, the nucleic acid encoding third polypeptide encodes a polypeptide with a sequence that is at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least 97.5% identical to SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 53. [000165] Thus, the present disclosure provides nucleic acids encoding both the first polypeptide and second polypeptide (i.e., in a single polynucleotide) as well as nucleic acids encoding the first polypeptide, second and third polypeptides individually. The nucleic acid of the present disclosure may be DNA or RNA (including hybrids thereof) but is preferably RNA. Thus, in a preferred embodiment the nucleic acid is RNA. [000166] Therefore, in an embodiment there is provided an RNA (e.g. mRNA) encoding the first polypeptide as referred to in the first aspect and the second polypeptide as referred to in the first aspect wherein said RNA is a single polynucleotide encoding said first and second polypeptides. There is further provided an RNA encoding the first polypeptide as referred to the first aspect. There is further provided an RNA encoding the second polypeptide as referred to in the first aspect. There is further provided an RNA encoding the third polypeptide. [000167] Nucleic acid (i.e., DNA or RNA) analogues, such as those containing modified backbones (e.g., peptide nucleic acids (PNAs) or phosphorothioates) or modified bases, are within the scope of the present disclosure. The nucleic acid may be linear, circular and/or branched, but will generally be linear. Typically, the nucleic acid will be in recombinant form, i.e., a form which does not occur in nature. [000168] The nucleic acid of the second, third or fourth aspect may be for the expression of the first polypeptide and/or the second polypeptide of the present disclosure in vitro from a host cell (i.e., the nucleic acid is, or is part of, an expression vector). Suitable nucleic acid expression vectors (in particular, DNA expression vectors) can comprise, for example, (1) an origin of replication; (2) a selectable marker gene; (3) one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, or a terminator), and/or one or more translation signals; and (4) a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell. [000169] However, in a preferred embodiment, the nucleic acid is for the expression of the first polypeptide and/or the second polypeptide (and/or the third polypeptide) of the present disclosure in vivo in a subject (i.e., the nucleic acid is, or is part of, a nucleic acid-based vaccine). In this embodiment, in addition to a nucleic acid sequence encoding the first polypeptide and/or the second polypeptide (and/or the third polypeptide) of the present disclosure, the nucleic acid may comprise one or more heterologous sequences, such as a sequence encoding a further protein (e.g., as detailed below) and/or a control sequence, in particular a promoter or an internal ribosome entry site. [000170] Nucleic acids of the present disclosure may be codon optimised. In some embodiments, nucleic acids of the present disclosure may be codon optimised for expression in human cells. Codon optimisation refers to the use of specific codons, which, while not altering the sequence of the expressed protein (given genetic code redundancy), may increase translation efficacy and/or half- life of the nucleic acid. [000171] The nucleic acid (e.g., RNA) may encode either the first polypeptide, the second polypeptide or the third polypeptide of the present disclosure only (i.e., the nucleic acid encodes a single protein). Alternatively, the nucleic acid may encode multiple proteins, for example, the nucleic acid may encode both the first polypeptide and the second polypeptide, wherein said nucleic acid is a single polynucleotide. [000172] The nucleic acid of the present disclosure is, in particular, provided in purified or substantially purified form; that is, substantially free from other nucleic acids (e.g., free or substantially free from naturally occurring nucleic acids, such as further nucleic acids expressed by a host cell). Said nucleic acids is generally at least 50% pure (by weight), such as at least 60%, 70%, 80%, 90%, or 95% pure (by weight). [000173] The present disclosure also provides, in a further independent aspect, a vector comprising one or more nucleic acids of the present disclosure. [000174] In a preferred embodiment, the nucleic acid of the present disclosure is RNA. “RNA” refers to an artificial (or, defined differently, recombinant) ribonucleic acid encoding the first polypeptide of the present disclosure, the second polypeptide of the present disclosure or both the first and second polypeptide of the present disclosure, which may be translated in a cell (i.e., mRNA). In an embodiment, the RNA is neither, nor comprised within, a viral vector or virus-based vaccine (such as a live-attenuated virus vaccine). In a particular embodiment, the RNA is mRNA. [000175] RNA molecules can have various lengths but are typically 500-20,000 ribonucleotides long e.g., 1000-20,000, 1000-15,000, 1000-10,000, 1000-5000, 1000-3000, 1000-2500, 1000-2500 or 1000-2000 ribonucleotides long. [000176] In an embodiment, the RNA is non-self-replicating. [000177] In a separate embodiment the RNA is self-replicating. Self-replicating RNA can be produced using replication elements derived from, e.g., alphaviruses, and substituting sequences encoding the structural viral proteins with that encoding the first polypeptide and/or second polypeptide of the present disclosure. A self-replicating RNA molecule is typically a positive-strand molecule which can be directly translated after delivery to a cell, and this translation provides an RNA- dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of the encoded protein (i.e., the first polypeptide and/or second polypeptide of the present disclosure or the third polypeptide of the present disclosure); or may be transcribed to provide further transcripts with the same sense as the delivered RNA, which are translated to provide in situ expression of the encoded protein. The overall result of this sequence of transcriptions is substantial amplification in the number of the introduced RNAs, and so the encoded first polypeptide and/or second polypeptide of the present disclosure becomes a major polypeptide product of the cells. [000178] In such embodiments wherein the RNA is self-replicating, it may encode (i) an RNA- dependent RNA polymerase which can transcribe RNA from the self-replicating RNA and (ii) the first polypeptide and/or second polypeptide of the present disclosure or the third polypeptide of the present disclosure. The polymerase can be an alphavirus replicase e.g., comprising one or more of alphavirus proteins nsP1, nsP2, nsP3 and nsP4. Such alphavirus-based self-replicating RNA can use a replicase from, for example, a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus (EEEV), or a Venezuelan equine encephalitis virus (VEEV). Mutant or wild-type virus sequences can be used e.g., the attenuated TC83 mutant of VEEV has been used for self-replicating RNA (see WO 2005/113782). Thus, a self-replicating RNA encoding a first polypeptide and/or second polypeptide of the present disclosure (or the third polypeptide of the present disclosure) may have two open reading frames. The first (5') open reading frame encodes a replicase, in particular an alphavirus replicase (e.g., as detailed above); the second (3') open reading frame encodes the first polypeptide and/or the second polypeptide of the present disclosure. Further open reading frames may also be present, encoding (i) one or more further proteins; and/or (ii) accessory polypeptides. [000179] Generally, the RNA comprises a 5’ cap, such as a 7’-methylguanosine, which may be added via enzymatic means or a non-enzymatic reaction. The RNA may have the following exemplary 5’ caps: - a 7’-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotide by a triphosphate bridge (also referred to as “Cap O”); - a 7’-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotide by a triphosphate bridge, and wherein the first 5’ ribonucleotide comprises a 2’-methylated ribose (2’-O-Me) (also referred to as “Cap 1”); - a 7’-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotides by a triphosphate bridge, and wherein the first and second 5’ ribonucleotides comprise a 2’-methylated ribose (2’-O- Me) (also referred to as “Cap 2”); - or a 7’-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotides by a triphosphate bridge, and wherein the first, second and third 5’ ribonucleotides comprise a 2’-methylated ribose (2’-O-Me). [000180] In an embodiment, the 5’ cap comprises a 7’-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleoside by a triphosphate bridge, and wherein the first 5’ ribonucleoside comprises a 2’- methylated ribose (2’-O-Me), e.g., the 5’ end of the RNA has the structure m7G(5')ppp(5')(2'OMeA)pG. In an embodiment, this cap is added non-enzymatically through the use of the following reagent: [000181] Said reagent is sold as CLEANCAP Reagent AG (TRILINK BIOTECHNOLOGIES). [000182] In other embodiments, a cap may be added resulting in the 5’ end of the RNA having the structure m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. This cap may be added non-enzymatically through the use of the following reagent: [000183] Said reagent is sold as CLEANCAP Reagent AG (3’OMe) (TRILINK BIOTECHNOLOGIES). [000184] Generally, the RNA comprises a 3’ poly-adenosine (“poly-A”) tail, e.g., comprising 10-700 A ribonucleotides. The poly-A tail may comprise at least two non-contiguous stretches of A ribonucleotides (also referred to as a “split poly-A tail”), or a (in particular, only one) contiguous stretch of A ribonucleotides. The total number of A ribonucleotides (“As”) in at least two non-contiguous stretches may be, for example, 10-700, such as 10-600, 10-500, 20-500, 50-500, 70-500, 100-500, 20- 400, 30-300, 40-200, 50-150, 70-120, 100-120, or, in particular, 100-120. In an embodiment, the 3’ poly- A tail comprises a contiguous stretch of 100-500 A ribonucleotides. [000185] The total number of As in a (in particular, only one) contiguous stretch may be, for example, 10-700; such as 10-600, 20-600 or in particular 40-600 (such as 50-600, 80-600, 80-550, 100- 500; or 40-70, 50-65 or 55-65). Wherein at least two non-contiguous stretches of As are used, these may be of differing length. For example, a first stretch may be 10-150 As in length, such as 10-100, 10- 50, 15-50, 20-50, 20-40, 25-40, or, in particular 25-35 As in length. For example, a second stretch may be 10-150 As in length, such as 10-150, 20-120, 30-100, 40-90, 50-90, 60-90, 65-90, 70-90, or, in particular, 80-90 As in length. The first stretch may be located 5’ or 3’ relative to the second stretch. However, in a particular embodiment, the first stretch is located 5’ relative to the second stretch. In a further particular embodiment, the polyA tail comprises, in the 5’ to 3’ direction, a first and a second non-contiguous stretch of As, that are 25-35 and 80-90 As in length respectively. In a further particular embodiment, the polyA tail comprises, in the 5’-3’ direction, a first and a second non-contiguous stretch of As, that are 25-35 and 65-90 As in length respectively. In some embodiments, the at least two non- contiguous stretches of As is from, or is part of, the 3’ untranslated region (UTR), e.g., as detailed below. [000186] In an embodiment the RNA comprises (in addition to any 5' cap structure) modified ribonucleotides, i.e., ribonucleotides that are modified in structure relative to standard A, C, G or U ribonucleotides. In an embodiment the RNA may include one or more modified ribonucleotides. In other embodiments, the RNA does not comprise modified ribonucleotides, i.e., the RNA contains standard A, C, G or U ribonucleotides only (except for any 5’ cap structure, if present, e.g., as detailed above). [000187] In embodiments wherein one or more modified ribonucleotides are used, said one or more modified ribonucleotides may be, or may comprise, N1-methylpseudouridine (“1mΨ”); pseudouridine (“Ψ”); N1-ethylpseudouridine; 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2- methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6- glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine (m6A); N6- threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2- methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O- dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine; N6,N6- dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6- threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza- adenosine; N1-methyl-adenosine; N6,N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; .alpha.-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2- (halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP; 2'- Deoxy-2'-a-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8- (amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido- adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza- adenosine; 7-methyladenine; 1-Deazaadenosine TP; 2'Fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2- Amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-Ethynyladenosine TP; 2-aminoadenine; 2- Aminoadenosine TP; 2-Amino-ATP; 2'-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2'-b- Ethynyladenosine TP; 2-Bromoadenosine TP; 2'-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2',2'-difluoroadenosine TP; 2'-Deoxy-2'-a-mercaptoadenosine TP; 2'-Deoxy-2'-a- thiomethoxyadenosine TP; 2'-Deoxy-2'-b-aminoadenosine TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'- Deoxy-2'-b-bromoadenosine TP; 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b-fluoroadenosine TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine TP; 2'-Deoxy-2'-b- thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2- methoxy-adenine; 2-methylthio-adenine; 2-Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3- Deazaadenosine TP; 4'-Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5'- Homo-adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9- Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7- deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2- thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4- acetylcytidine; 2'-O-methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O- methylcytidine; Lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2'-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo- cytidine; .alpha.-thio-cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP; 2'-Azido-2'-deoxy-CTP; 2'- Deoxy-2'-a-aminocytidine TP; 2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2'-O- dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine: 5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6- aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1- methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine: 2-methoxy-cytidine; 2-thio-5-methyl- cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1- deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza- zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP; 2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP; 2'O-methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a- Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP; 2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'- difluorocytidine TP; 2'-Deoxy-2'-a-mercaptocytidine TP; 2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'- Deoxy-2'-b-aminocytidine TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP; 2'- Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy- 2'-b-mercaptocytidine TP; 2'-Deoxy-2'-b-thiomethoxycytidine TP; 2'-O-Methyl-5-(1-propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(1- Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5'-Homo-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2'-O-dimethylguanosine; N2-methylguanosine; Wyosine; 1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7- deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O- trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; N1-methyl-guanosine; .alpha.-thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2'-Amino-2'-deoxy-GTP; 2'-Azido-2'-deoxy- GTP; 2'-Deoxy-2'-a-aminoguanosine TP; 2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine; 6- (alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8- (alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8- (thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio- guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio- 7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio- guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl- N2-isobutyl-guanosine TP; 2'-a-Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP; 2'-b- Ethynylguanosine TP; 2'-b-Trifluoromethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'-Deoxy- 2'-a-mercaptoguanosine TP; 2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine TP; 2'-Deoxy-2'-b- chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy-2'-b- mercaptoguanosine TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP; 5'-Homo-guanosine TP; 8-bromo-guanosine TP; 9- Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine; 1,2'-O-dimethylinosine; 2'-O- methylinosine; 7-methylinosine; 2'-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy- thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5- hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine; Dihydrouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5- carboxypropyl)pseudouridine; 1-methylpseduouridine; 1-methyl-pseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-O-methyluridine; 3-(3-amino-3- carboxypropyl)uridine; 3,2'-O-dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine; 5- (carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester, 5,2'-O- dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O- methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5- carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2'-O-methyluridine; 5- carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- caboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-methoxycaeoonylmethyl-2'-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5- methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2- selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5- Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; N1- methyl-pseudo-uridine; N1-ethyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)-2-thiouridine TP; 5-(iso-Pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5- propynyl uracil; .alpha.-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouridine; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouridine; 1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouridine; 1 (aminoalkylaminocarbonylethylenyl)- pseudouridine; 1 (aminocazbonylethylenyl)-2(thio)-pseudouridine; 1 (aminocarbonylethylenyl)-2,4- (dithio)pseudouridine; 1 (aminocarbonylethylenyl)-4 (thio)pseudouridine; 1 (aminocarbonylethylenyl)- pseudouridine; 1 substituted 2(thio)-pseudouridine; 1 substituted 2,4-(dithio)pseudouridine; 1 substituted 4 (thio)pseudouridine; 1 substituted pseudouridine; 1-(aminoalkylamino- carbonylethylenyl)-2-(thio)-pseudouridine; 1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; 1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2 (thio)pseudouridine; 2' deoxy uridine; 2' fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl, 2'amino, 2'azido, 2'fluoro-guanosine; 2'-Amino-2'-deoxy-UTP; 2'-Azido-2'-deoxy-UTP; 2'-Azido-deoxyuridine TP; 2'-O- methylpseudouridine; 2' deoxy uridine; 2' fluorouridine; 2'-Deoxy-2'-a-aminouridine TP; 2'-Deoxy-2'-a- azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouridine; 4- (thio)pseudouridine; 4-(thio)uracil; 4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouridine; 5-(alkyl)-2,4 (dithio)pseudouridine; 5-(alkyl)-4 (thio)pseudouridine; 5-(alkyl)pseudouridine; 5-(alkyl)uracil; 5- (alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5- (dimethylaminoalkyl)uracil; 5-(guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazole-1-alkyl)uracil; 5- (methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5- (methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2- (thio)pseudouridine; 5-(methyl)-2,4 (dithio)pseudouridine; 5-(methyl)-4 (thio)pseudouridine; 5- (methyl)pseudouridine; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)- 2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5- aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; Pseudo-UTP-1-2-ethanoic acid; Pseudouridine; 4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine; 1-methyl-1-deaza- pseudouridine; 1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza- pseudouridine; 2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2- thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy- pseudouridine; 4-thio-1-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; (.+-.)1-(2-Hydroxypropyl)pseudouridine TP; (2R)-1-(2- Hydroxypropyl)pseudouridine TP; (2S)-1-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo-vinyl)ara- uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo- vinyl)uridine TP; 1-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; 1-(2,2-Diethoxyethyl)pseudouridine TP; 1-(2,4,6-Trimethylbenzyl)pseudouridine TP; 1-(2,4,6- Trimethyl-benzyl)pseudo-UTP; 1-(2,4,6-Trimethyl-phenyl)pseudo-UTP; 1-(2-Amino-2- carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP; 1-(2-Hydroxyethyl)pseudouridine TP; 1-(2- Methoxyethyl)pseudouridine TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; 1-(3,4- Dimethoxybenzyl)pseudouridine TP; 1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino- propyl)pseudo-UTP; 1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1-(4-Amino-4- carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP; 1-(4-Amino-butyl)pseudo-UTP; 1-(4- Amino-phenyl)pseudo-UTP; 1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP; 1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP; 1-(4- Iodobenzyl)pseudouridine TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP; 1-(4- Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo- UTP; 1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP; 1-(4- Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP; 1-(4-Nitro-phenyl)pseudo-UTP; 1-(4- Thiomethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1-(4- Trifluoromethylbenzyl)pseudouridine TP; 1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo- UTP; 1,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)- propionyl]pseudouri- dine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl} pseudouridine TP; 1- Acetylpseudouridine TP; I-Alkyl-6-(1-propynyl)-pseudo-UTP; 1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1- Alkyl-6-allyl-pseudo-UTP; 1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP; 1-Alkyl-6- vinyl-pseudo-UTP; 1-Allylpseudouridine TP; 1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2-pseudouridine TP; 1- Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl- pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP; 1-Cyclooctylmethyl-pseudo-UTP; 1- Cyclooctyl-pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1- Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo- UTP; 1-Homoallylpseudouridine TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP; 1- Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-alpha-thio-pseudo-UTP; 1- Methanesulfonylmethylpseudouridine TP; 1-Methoxymethylpseudouridine TP; 1-Methyl-6-(2,2,2- Trifluoroethyl)pseudo-UTP; 1-Methyl-6-(4-morpholino)-pseudo-UTP; 1-Methyl-6-(4-thiomorpholino)- pseudo-UTP; 1-Methyl-6-(substituted phenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP; 1-Methyl- 6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP; 1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6- chloro-pseudo-UTP; 1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP; 1- Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP; 1-Methyl-6-ethyl-pseudo- UTP; 1-Methyl-6-fluoro-pseudo-UTP; 1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino- pseudo-UTP; 1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP; 1-Methyl-6-iso-propyl- pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP; 1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6- phenyl-pseudo-UTP; 1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP; 1-Methyl-6- trifluoromethoxy-pseudo-UTP; 1-Methyl-6-trifluoromethyl-pseudo-UTP; 1- Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1- Pivaloylpseudouridine TP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP; 1-propynyl- pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1-Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl- pseudo-UTP; 1-Vinylpseudouridine TP; 2,2'-anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5- Methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP; 2'-a- Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP; 2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'- difluorouridine TP; 2'-Deoxy-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy- 2'-b-aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP; 2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b- chlorouridine TP; 2'-Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP; 2'-Deoxy-2'-b- mercaptouridine TP; 2'-Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2- methoxyuridine; 2'-O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'- Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5- Cyanouridine TP; 5-Dimethylaminouridine TP; 5'-Homo-uridine TP; 5-iodo-2'-fluoro-deoxyuridine TP; 5- Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP; 5- Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6-(4-Morpholino)-pseudo-UTP; 6-(4- Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP; 6-Azido- pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl- pseudo-UTP; 6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP; 6-Hydroxy- pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP; 6-Methylamino- pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo- UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine 1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4- methylbenzoic acid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2- [2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-{2-(2-ethoxy)- ethoxy)-ethoxy}-ethoxy]-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)- ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3- propionic acid; Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid; Pseudo-UTP-N1-6- hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid; Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP- N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1- yl: 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa)- naphthalene; 2 (amino)purine; 2,4,5-(trimethyl)phenyl; 2' methyl, 2'amino, 2'azido, 2'fluoro-cytidine; 2' methyl, 2'amino, 2'azido, 2'fluoro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluoro-uridine; 2'-amino-2'- deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'- deoxyribose; 2'-fluoro-modified bases; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo- pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl; 3- (methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4- (methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5- (methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6- chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)- phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7- (aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)- phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7- (guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl; 7-(guanidiniumalkylhydroxy)-1- (aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)- phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7- (guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3- (diaza)-2-(oxo)-phenoxazin-1-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7- (aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3- (diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho- (aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho-substituted-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; O6-substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl- pyrrolo-pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2- on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino- purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'-OH-ara-guanosine TP; 5-(2- carbomethoxyvinyl)uridine TP; or N6-(19-Amino-pentaoxanonadecyl)adenosine TP. [000188] In some embodiments, the percentage of standard As substituted with A- substitutable modified nucleotide (e.g. those above) is at least: 0.1%, 0.5%, 0.8%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In some embodiments, the percentage of standard As substituted with m⁶A may be 0.1-5%, in particular 0.5-2%, in particular 0.8-1.2%, such as about 1% (or 1%); in these embodiments the RNA may be circular RNA. Low substitution levels with m6A (e.g., 1%) have been shown in inhibit innate immune activation (Chen et al. Mol Cell. 2019 76, 1: 96-109). In some embodiments, the percentage of standard Cs substituted with cytosine-substitutable modified nucleotide (e.g., those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In some embodiments, the percentage of standard Gs substituted with G-substitutable modified nucleotide (e.g., those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or 100%. In an embodiment, the percentage of standard Us substituted with U-substitutable modified nucleotide (e.g., those above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%; more preferably with 1mΨ and/or Ψ (even more preferably 1mΨ). [000189] In an embodiment, the modified ribonucleotides (i.e., the one or more modified ribonucleotides) is, or comprise, 1mΨ and/or Ψ. In such embodiments, the RNA may comprise 1mΨ and/or Ψ, and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e., there are no standard U nucleotides, nor modified U ribonucleotides other than 1mΨ and/or Ψ, in the RNA, i.e., 100% U substitution). In particular, the RNA may comprise 1mΨ and/or Ψ, and neither standard U ribonucleotides nor other modified ribonucleotides (i.e., there are no standard U nucleotides, nor modified ribonucleotides of any type - A, C, G or U substitutable - other than 1mΨ and/or Ψ, in the RNA, i.e., 100% U substitution with no other modified nucleotides being allowed). The RNA may comprise Ψ, and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e., 100% U substitution with Ψ). In particular, the RNA may comprise Ψ, and neither standard U ribonucleotides nor other modified ribonucleotides (i.e., 100% U substitution with Ψ with no other modified nucleotides being allowed). In an embodiment, the RNA comprises 1mΨ, and neither standard U ribonucleotides nor other modified U ribonucleotides (i.e., 100% U substitution with 1mΨ). In an embodiment, the RNA comprises 1mΨ, and neither standard U ribonucleotides nor other modified ribonucleotides (i.e., 100% U substitution with 1mΨ with no other modified nucleotides being allowed). [000190] In an embodiment, the RNA is codon optimised. Codon optimisation may provide an elevated GC content, relative to non-codon optimised RNA encoding the same protein(s). The GC content (the percentage of all ribonucleotides (or, defined alternatively, all “nitrogenous bases”) in the RNA which are G or C) of the RNA may be at least 10%, such as at least 20%, 30%, 35% or at least 40%, 45%, 46%, 47%, 48%, 49%, or at least 50%. The GC content of the RNA may be 10-70%, such as 20-65%, 30-65%, 35-65%, 40-60%, 45-55%, 46-53%, 47-51%, or 48-50%. In an embodiment the RNA comprises a GC content of 40%-60%. Codon optimisation may provide an elevated C content relative to non-codon optimised RNA encoding the same protein(s). The percentage of C-optimisable codons in the RNA which have been substituted, as a result of codon optimisation, for a codon with greater C content (while encoding the same amino acid) may be least 30%, such as at least 40%, 50%, 55% or at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% or at least 72%; The percentage of C-optimisable codons in the RNA which have been substituted, as a result of codon optimisation, for a codon with greater C content (while encoding the same amino acid) may be 30-80%, such as 40-90%, 45-90%, 50-80%, 55- 80%, 60-80%, 65-75%, 66-75%, 67-75%, 68-75%, 69-75%, 70-74%, 71-74% or 72-74%. [000191] In an embodiment, the RNA comprises a 5’ and/or a 3’ untranslated region (UTR). In an embodiment, the RNA comprises both a 5’ and 3’ UTR; e.g., selected from the 5’and 3’ UTRs of RNA transcripts of the following genes (i.e., the following human genes): beta-actin, albumin, ATP synthase beta subunit, fibroblast activation protein (“FAP”), H4 clustered histone 15 (“HIST2H4A”), glyceraldehyde-3-phosphate dehydrogenase, heat shock protein family A (Hsp70) member 8 gene, interleukin-2 gene (“IL-2”), and transferrin. In some embodiments, the RNA comprises a 5’ and a 3’ UTR selected from: - SEQ ID NO: 19 and 20, respectively, - SEQ ID NO: 21 and 22, respectively, - SEQ ID NO: 23 and 24, respectively, - SEQ ID NO: 25 and 26, respectively, - SEQ ID NO: 27 and 28, respectively, and - RNA sequences at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 19, 21, 23, 25 or 27 (for the 5’ UTR) and RNA sequences at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 20, 22, 24, 26 or 28 (for the 3’ UTR) (in particular, the pairing of 5’ and 3’ UTRs having such identity to SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26, and SEQ ID NO: 27 and 28, respectively); with RNA sequences according to SEQ ID NO: 19 and 20, SEQ ID NO: 25 and 26, SEQ ID NO: 27 and 28 (and RNA sequences having such identity thereto, e.g., at least 95% or greater) being more preferred; and RNA sequences according to SEQ ID NO: 19 and 20 (and RNA sequences having such identity thereto, e.g., at least 95% or greater) being even more preferred. [000192] Both the 3’ and 5’ UTR may influence expression of the first polypeptide and/or second polypeptide of the present disclosure (or the third polypeptide of the present disclosure) through a variety of mechanisms. Without wishing to be found by this theory, the 5’ UTR may affect the expression of the encoded polypeptide e.g., via pre-initiation complex regulation, closed-loop regulation, upstream open reading frame regulations (i.e., reinitiation), provision of internal ribosome entry sites, and provision of microRNA binding sites. Without wishing to be found by this theory, the 3’ UTR may affect the expression of the encoded protein of the present disclosure e.g., via providing regulation regions that post-transcriptionally influence expression, e.g., influencing translation efficiency, localisation of the RNA, stability of the RNA, polyadenylation, and circularization of the RNA. [000193] In one specific embodiment, the RNA is circular RNA. [000194] In an embodiment, the RNA fulfils any 2, 3, 4 or 5 of the following criteria (for example, (a) (b), (d) and (f); (a), (a), (b), (c), (d) and (f); or (a), (b), (d), (e) and (f): (a) is non-self-replicating; (b) is single stranded; (c) comprises a 5’ cap, which is a 7’-methylguanosine linked 5’-to-5’ to the 5’ first ribonucleotide by a triphosphate bridge, and wherein the first 5’ ribonucleotide comprises a 2’-methylated ribose (2’-O-Me); (d) comprises a 3’poly-A tail; (e) comprises 1mΨ, and neither standard U ribonucleotides nor other modified ribonucleotides (f) comprises a 5’ and a 3’ UTR. [000195] In an embodiment, the RNA fulfils all of criteria (a) – (f), above. [000196] In an embodiment, the nucleic acid of the invention is RNA and the RNA comprises an open reading frame (ORF) encoding the first polypeptide as referred to in the first aspect of the present disclosure and/or the second polypeptide as referred to in the first aspect of the present disclosure (or the third polypeptide of the present disclosure i.e. a GTD polypeptide, optionally a ToxA- GTD polypeptide or a ToxB-GTD polypeptide). [000197] In an embodiment, the nucleic acid of the invention (e.g., RNA) comprises, in the 5’ to 3’ direction: i) a 5’ Cap, ii) a 5’ UTR, iii) an ORF encoding the first polypeptide as referred to in the first aspect of the present disclosure and/or the second polypeptide as referred to in the first aspect of the present disclosure (or the third polypeptide of the present disclosure i.e. a GTD polypeptide, optionally a ToxA-GTD polypeptide or a ToxB-GTD polypeptide).), iv) a 3’UTR, and v) a 3’ poly-A tail (in particular, the 5’ Caps; 5’ UTRs, 3’UTRs and 3’ poly-A tails as detailed above throughout this subsection). [000198] In an embodiment, the nucleic acid of the invention comprises a stop codon between the ORF and the 3’ UTR. In an embodiment, the nucleic acid is RNA, said RNA comprising a stop codon between the ORF and the 3’ UTR, optionally wherein the stop codon comprises or consists of SEQ ID NO: 57. [000199] In an embodiment, the ORF encoding the first polypeptide as referred to in the first aspect of the present disclosure (i.e. comprising a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment) comprises or consists of SEQ ID NO: 29; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 29. In an embodiment, the ORF encoding the second polypeptide as referred to in the first aspect of the present disclosure (i.e. comprising a fragment of C. difficile toxin B, said fragment comprising at least 100 contiguous amino acids of the DRBD of toxin B) comprises or consists of SEQ ID NO: 30; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 30. In an embodiment, the ORF encodes for the ToxB-GTD polypeptide as referred to above. In an embodiment, the ORF encoding the GTD polypeptide comprises or consists of SEQ ID NO: 35; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 35 [000200] In an embodiment, the RNA comprises an open reading frame comprising or consisting of SEQ ID NO: 29; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 29 (first polypeptide wherein said first polypeptide comprises a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment). [000201] In an embodiment, the RNA comprises an open reading frame comprising or consisting of SEQ ID NO: 30; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 30 (second polypeptide wherein said second polypeptide comprises a fragment of C. difficile toxin B, said fragment comprising at least 100 contiguous amino acids of the DRBD of toxin B). [000202] In an embodiment, the RNA comprises an open reading frame comprising or consisting of SEQ ID NO: 35; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 35 (ToxB-GTD polypeptide). [000203] In an embodiment, open reading frames that encode the polypeptides disclosed herein (or sequences at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical thereto) may further comprise a sequence encoding for a purification tag e.g., a poly-histidine tag. The design of RNA sequences encoding for poly-histidine tags is within the remit of the person skilled in the art. An exemplar sequence encoding for a 6-His tag is provided in SEQ ID NO: 31. [000204] In an embodiment, open reading frames that encode the polypeptides disclosed herein (or sequences at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical thereto) may further comprise a signal sequence. Signal sequences encode short peptides (for example the peptides provided herein as SEQ ID NO: 36 to 47), present either at the N- or occasionally the C- terminus, that prompt cells to translocate and secrete the protein (e.g., to the lumen of the endoplasmic reticulum). Exemplary signal sequences are for example provided in Román et al 2016; Journal of Biotechnology, vol 239, pg47-6. In an embodiment the signal sequence is a native signal sequence i.e., is the signal sequence that is natively present within the coding sequence of the polypeptide which naturally exists to direct intracellular trafficking of the protein (e.g., to the lumen of the endoplasmic reticulum). In an embodiment, the signal sequence is a non-native signal sequence, i.e., is an artificial signal sequence or is a signal sequence that has been obtained from a heterologous protein (i.e., a different protein to the protein encoded by the nucleic acid). Exemplary signal sequences are provided in SEQ ID NO: 32 (Human serum albumin), SEQ ID NO: 33 (Human IgG) and SEQ ID NO: 34 (Luc). In embodiments where the ORF further comprises a signal sequence (e.g., a non-native signal sequence), a linker sequence may additionally be present, wherein said linker is positioned between the signal sequence and the protein coding sequence. In an embodiment, said linker encodes a cleavage site. [000205] In preferred embodiments, the RNA (e.g. mRNA) comprises or consists of the sequence of: SEQ ID NO: 54; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical thereto, preferably encoding the first polypeptide of the present disclosure (i.e. wherein said first polypeptide comprises a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment), SEQ ID NO: 55; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical thereto, preferably encoding the second polypeptide of the present disclosure (i.e. wherein said second polypeptide comprises a fragment of C. difficile toxin B, said fragment comprising at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B), SEQ ID NO: 56; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical thereto, preferably encoding the third polypeptide of the present disclosure (i.e. wherein said third polypeptide is a GTD polypeptide, optionally a ToxB-GTD polypeptide). [000206] In an embodiment the RNA comprises an open reading frame comprising or consisting of positions 54-3002 of SEQ ID NO: 54; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to said positions. In an embodiment, the open reading frame comprises or consists of positions 54-2003 of SEQ ID NO: 54 wherein positions 54-107 of SEQ ID NO: 54 comprise a signal peptide. [000207] In an embodiment the RNA comprises an open reading frame comprising or consisting of positions 54-1256 of SEQ ID NO: 55; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to said positions. In an embodiment, the open reading frame comprises or consists of positions 54-1256 of SEQ ID NO: 55 wherein positions 54-110 of SEQ ID NO: 55 comprise a signal peptide. [000208] In an embodiment the RNA comprises an open reading frame comprising or consisting of positions 54-1730 of SEQ ID NO: 56; or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to said positions. In an embodiment, the open reading frame comprises or consists of positions 54-1730 of SEQ ID NO: 56 wherein positions 54-104 of SEQ ID NO: 56 comprise a signal peptide. [000209] The RNA can conveniently be prepared by in vitro transcription (IVT). IVT can use a (DNA) template created and propagated in plasmid form in bacteria or created synthetically (for example by gene synthesis and/or polymerase chain-reaction (PCR) engineering methods). For instance, a DNA-dependent RNA polymerase (such as the bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe the replicating RNA from a DNA template. Appropriate capping and poly-A addition reactions can be used as required (although the poly-A tail is usually encoded within the DNA template). [000210] Nucleic acid (especially RNA) by themselves and unprotected, may be degraded by the subject’s nucleases and may require a carrier to facilitate target cell entry. Accordingly, the present disclosure also provides a carrier comprising the nucleic acid of the second aspect (e.g., RNA), the nucleic acid of the third aspect (e.g., RNA) or the nucleic acid of the fourth aspect (e.g., RNA) of the present disclosure. Further provided is a carrier comprising the nucleic acid encoding the third polypeptide. As used herein the term “carrier” refers to a delivery vector or vehicle that functions to protect the nucleic acid from being degraded (i.e., by endogenous nucleases) prior to delivery and uptake into the target cell. [000211] The carrier may be lipid-based (e.g., a lipid nanoparticle or cationic nanoemulsion), polymer-based (e.g., comprising polyamines, dendrimers and/or copolymers), peptide or protein-based (e.g., comprising protamine, a cationic cell-penetrating peptide, and/or an anionic peptide conjugated to a positively charged polymer), cell-based (e.g., antigen presenting cells, such as dendritic cells loaded with the nucleic acid), or virus-based (e.g., viral replicon particles). In particular embodiments, the carrier is non-virion, i.e., free or substantially free of viral capsid. [000212] In particular, lipid-based carriers provide a means to protect the nucleic acid (e.g., RNA), e.g., through encapsulation, and deliver it to target cells for protein expression. In certain embodiments, the lipid-based carrier is, or comprises, a cationic nano-emulsion (“CNE”). CNEs and methods for their preparation are described in, for example, WO2012/006380. With a CNE, the nucleic acid (e.g., RNA) which encodes the RSV-F protein of the present disclosure is complexed with a CNE particle, in particular comprising an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged molecule, thereby anchoring the molecule to the emulsion particles. In a particular embodiment, a lipid-based carrier is a lipid inorganic nanoparticle (“LION”). [000213] In an embodiment, the nucleic acids (e.g., RNA) of the second, third and fourth aspect are encapsulated in a lipid nanoparticle (LNP). In an embodiment, the nucleic acid encoding the third polypeptide is encapsulated in an LNP. Thus, in an embodiment the carrier is an LNP. Thus, in an embodiment, the present disclosure also provides an LNP encapsulating the nucleic acid of the second aspect, an LNP encapsulating the nucleic acid of the third aspect or an LNP encapsulating the nucleic acid of the fourth aspect of the present disclosure. Further provided is an LNP encapsulating a nucleic acid, said nucleic acid encoding the third polypeptide of the present disclosure (i.e. a GTD polypeptide). [000214] A plurality of such LNPs will be part of a composition (e.g. a pharmaceutical composition as detailed in the section entitled Pharmaceutical compositions below) comprising free and/or encapsulated nucleic acid (e.g., RNA), and in some embodiments the LNPs encapsulate at least: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or at least 100% of the total number of nucleic acid (e.g., RNA) molecules in the composition. At least 80% of the LNPs in the composition may be 20-200 nm, 40-190 nm, 60-180 nm or, in particular, 80-160 nm in diameter. In a particular embodiment, substantially all, or all, LNPs in the composition are 20-200 nm, 40-190 nm, 60- 180 nm or, in particular, 80-160 nm in diameter. [000215] The LNP can comprise multilamellar vesicles (MLV), small uniflagellar vesicles (SUV), or large unilamellar vesicles (LUV). [000216] The amount of nucleic acid (e.g., RNA) per LNP can vary, and the number of individual nucleic acid molecules per LNP can depend on the characteristics of the particle being used. For RNA molecules, in general, an LNP may include 1-500 RNA molecules, e.g., <200, <100, <50, <20, <10, <5, or 1-4. Generally, an LNP includes fewer than 10 different species of RNA e.g., fewer than 5, 4, 3, or 2 different species. In an embodiment the LNP includes a single RNA species (i.e., all RNA molecules in the particle have the same sequence). [000217] LNPs according to the present disclosure may be formed from a single lipid (e.g., a cationic lipid) or, in particular, from a mixture of lipids. In particular, the mixture comprises various classes of lipids, such as: (a) a mixture of cationic lipids and sterols, (b) a mixture of cationic lipids and neutral lipids, (c) a mixture of cationic lipids and polymer-conjugated lipids, (d) a mixture of cationic lipids, sterols and polymer-conjugated lipids, or (e) a mixture of cationic lipids, neutral lipids and polymer-conjugated lipids; or (f) a mixture of cationic lipids, sterols and neutral lipids; or (g) a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids. [000218] Further classes of lipids, such as anionic lipids, may also be present in a mixture of lipids. In an embodiment, the lipid nanoparticle comprises a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids. [000219] The cationic lipid may have a pKa of 5.0-10.0, 5.0-9.0, 5.0-8.5, 5.0-8.0, 5.0-7.9, 5.0- 7.8, 5.0-7.7 or 5.0-7.6. In an embodiment, the cationic lipid has a pKa of 5.0-8.0; optionally 5.0-7.6. The pKa of the cationic lipid is distinct to the pKa of the LNP as a whole (sometimes called “apparent pKa”). pKa may be determined via any well-known method, such as via a toluene nitrosulphonic acid (TNS) fluorescence assay or acid base titration. [000220] In an embodiment, the cationic lipid comprises a tertiary or quaternary amine group. In a specific embodiment the cationic lipid comprises a tertiary amine group. Exemplary cationic lipids comprising tertiary amine groups include: 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy- 3dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3- (N-methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N- dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl- 4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); or MC3 (see, e.g. US20100324120). [000221] In some embodiments, the cationic lipid has the structure of lipid RV28, RV31, RV33, RV37, RV39 RV42, RV44, RV73, RV75, RV81, RV84, RV85, RV86, RV88, RV91, RV92, RV93, RV94, RV95, RV96, RV97, RV99 or RV101, as disclosed in WO2021/038508. In a further embodiment, the cationic lipid has the structure:

[000222] In an embodiment, the cationic lipid has the structure: (also referred to as lipid RV39). [000223] In another embodiment, the cationic lipid has the structure: [000224] In another embodiment, the cationic lipid has the structure:

[000225] The lipids in the LNP may comprise (in mole %) 20-80, 25-75, 30-70, or 35-65%, 30- 60, 40-55 or 40-50% cationic lipid; such as about 40% (or 40%), about 42% (or 42%), about 44% (or 44%), about 46% (or 46%) or about 48% (or 48%) cationic lipid. The lipids in the LNP may comprise (in mole %) at least 20, 25 or at least 35%, or at least 40% cationic lipid. The lipids in the LNP may comprise (in mole %) no more than 80, no more than 70, no more than 60 or no more than 50% cationic lipid. [000226] The molar ratio of protonatable nitrogen atoms in the LNP’s cationic lipids to phosphates in the RNA (a.k.a “N:P” ratio), may be in the range of (including the endpoints) 1:1-20:1, 2:1-10:1, 3:1-9:1, 4:1-8:1, 4.5:1-7.5:1, 4.5:1-6.5:1 or 5.0:1-6.5:1. [000227] In an embodiment, the polymer-conjugated lipid is a PEGylated lipid. In the LNP, the PEGs of such PEGylated lipids may have average molecular weight of 0.5-11.0 kDa, such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0, 1.0-3.5, 1.0-3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, 1.7- 2.3, 1.8-2.2, 1.9-2.1 kDa or about 2.0 (or 2.0 kDa). In an embodiment, the PEG has a molecular weight of 1-3 kDa. The average molecular weight of such PEGs may be expressed as the median molecular weight. Alternatively, in an LNP, at least 80% of the PEGs of such PEGylated lipids may have molecular weight of 0.5-11.0 kDa, such as 0.5-8.0, 0.8-8.0, 0.8-7.0, 0.8-6.0, 0.8-5.0, 0.8-4.0, 1.0-4.0, 1.0-3.5, 1.0- 3.0, 1.2-2.8, 1.4-2.6, 1.5-2.5, 1.6-2.4, 1.7-2.3, 1.8-2.2, 1.9-2.1 kDa or about 2.0 kDa (or 2.0 kDa). The PEGylated lipid may have the structure: [000228] Exemplary PEGylated lipids include 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, 1,2- dimyristoyl-sn-glycero-2-phosphoethanolamine-N-[methoxy(polyethylene glycol)] and 1,2-dimyristoyl- rac-glycerol-3-methoxypolyethylene glycol. In an embodiment, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000. [000229] The lipids in the LNP may comprise (in mole %) 0.1-8.0, 0.4-7.0, 0.6-6.0, 0.8-4.0, 0.8- 3.5% or 1.0-3.0% polymer-conjugated lipid (e.g., PEGylated lipid); such as about 1.0 (or 1.0%), about 1.5% (or 1.5%), about 2.0% (or 2.0%) or about 2.5% (or 2.5%) polymer-conjugated lipid (e.g., PEGylated lipid). The lipids in the LNP may comprise (in mole %) at least 0.1%, at least 0.5%, at least 0.8%, or at least 1% polymer-conjugated lipid (e.g., PEGylated lipid). The lipids in the LNP may comprise (in mole %) no more than 8.0, no more than 6.0, no more than 4.0 or no more than 3.0% polymer-conjugated lipid (e.g., PEGylated lipid). [000230] In an embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), although other neutral lipids available to the skilled person may also be used. [000231] The lipids in the LNP may comprise (in mole %) 0-15.0, 0.1-15.0, 2.0-14.0, 5.0-13.0, 6.0-12.0, 7.0-11.0, 8.0-11.0% or 9.0-11.0% neutral lipid, such as about 9.4% (or 9.4%), about 9.6% (or 9.6%), about 9.8% (or 9.8%) or about 10.0% (or 10%) neutral lipid. The lipids in the LNP may comprise (in mole %) at least 0.1, at least 5.0, at least 7.0, at least 8.0 or at least 9.0% neutral lipid. The lipids in the LNP may comprise (in mole %) no more than 15.0, no more than 13.0, no more than 12.0, or no more than 11.0% neutral lipid. [000232] Exemplary sterols include cholesterol, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, symosterol, lathosteriol, 14- demethyl-lanosterol, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, 14-demethyl-14- dehydrolanosterol (FF-MAS), diosgenin, dehydroepiandrosterone sulfate (DHEA sulfate), dehydroepiandrosterone, sitosterol, lanosterol-95, 4,4-dimethyl(d6)-cholest-8(9), 14-dien-3β-ol (dihydro-FF-MAS-d6), 4,4-dimethyl(d6)-cholest-8(9)-en-3β-ol (dihydro T-MAS-d6), zymostenol, sitostanol, campestanol, camperstanol, 7-dehydrodesmosterol, pregnenolone, 4,4-dimethyl-cholest- 8(9)-en-3β-ol (dihyrdro T-MAS), Δ5-avensterol, brassicasterol, dihydro FF-MAS, 24-methylene cholesterol, oxysterols, deuterated sterols, fluorinated sterols, sulfonated sterols, phosphorylated sterols, A-ring substituted sterols, cholest-5-ene-3ß,4ß-diol, 5α-cholestan-3ß-ol, 4-cholesten-3-one, cholesta-8(9),24-dien-3-one, cholesta-8(9),24-dien-3-one, 2,2,3,4,4-pentadeuterio-5a-cholestan-3ß-ol, cholesteryl phosphocholine, cholesteryl-d7 pentadecanoate, cholesteryl-d7 palmitate, B-ring substituted sterols, cholestanol, 5ß,6ß-epoxy-d7, 3ß-hydroxy-5-cholestene-7-one, 6α-hydroxy-5α- cholestane, cholestanol, 5α,6α-epoxy, cholest-5-en-3ß,7α-diol, cholest-5-en-3ß,7ß-diol, cholestanol, 5α,6α-epoxy-d7, Δ5,7-cholesterol, cholesta-5,8(9)-dien-3ß-ol, cholesta-5,8(14)-dien-3ß-ol, 7α- hydroxy-4-cholesten-3-one, zymostenol-d7, zymostenol, 7-dehydrodesmosterol, 3b,5a-dihydroxy- cholestan-6-one, D-ring substituted sterols, 3ß-hydroxy-5α-cholest-8(14)-en-15-one, 3ß-hydroxy-5α- cholestane-15-one, 5α-cholest-8(14)-ene-3ß,15α-diol, 5α-cholest-8(14)-ene-3ß,15ß,-diol, lanosterol- 95, 5α-7,24-cholestadiene, 14-dehydro zymostenol, ergosta-5,7,9(11),22-tetraen-3ß-ol, cholest-5-ene- 3ß,25-diol, cholest-(25R)-5-ene-3ß,27-diol, 24(R/S),25-epoxycholesterol, 24(S),25-epoxycholesterol, 24(R/S),25-epoxycholesterol-d6, cholest-5-ene-3ß,22(S)-diol, cholest-5-ene-3ß,22(R)-diol, cholest-5- ene-3ß,24(S)-diol, cholest-5-ene-3ß,24(R)-diol, 27-hydroxy-4-cholesten-3-one, campestanol, N,N- dimethyl-3ß-hydroxycholenamide, 25,27-dihydroxycholesterol, N,N-dimethyl-3ß- hydroxycholenamide, 25,27-dihydroxycholesterol, 5-cholestene-3β,20α-diol, 24S,25-epoxy-5α- cholest-8(9)-en-3β-ol, 24(S/R),25-epoxylanost-8(9)-en-3β-ol, 7-keto-27-hydroxycholesterol, 7α,27- dihydroxy-4-cholesten-3-one, 7α,27-dihydroxycholesterol, 7ß,27-dihydroxycholesterol, 5α,6ß- dihydroxycholestanol, 7α,25-dihydroxycholesterol, 7β,25-dihydroxycholesterol, 7α,24(S)- dihydroxycholesterol, 7α,24(S)-dihydroxy-4-cholesten-3-one, 7-keto-25-hydroxycholesterol, 7α,24S,27-trihydroxycholesterol, dihydrotestosterone, testosterone, estrone, estrogen, estradiol, corticosterone, cortisol, or 24S,27-dihydroxycholesterol. [000233] In an embodiment, the sterol is cholesterol or a cholesterol-based lipid (e.g., any of those provided in the foregoing paragraph). [000234] The lipids in the LNP may comprise (in mole %) 20-80, 25-80, 30-70, 30-60, 35-60, 40-60, 40-50 or 41-49% sterol, such as about 42% (or 42%), about 43% (or 43%), about 44% (or 44%), about 46% (or 46%), or about 48% (or 48%) sterol. The lipids in the LNP may comprise (in mole %) at least 20, at least 30, at least 35, at least 40 or at least 41% sterol. The lipids in the LNP may comprise (in mole %) no more than 80, no more than 70, no more than 60 or no more than 50% sterol. [000235] In an embodiment, the LNP may have the following mole % in combination: 30-60% cationic lipid (such as 35-55%, or 40-50%), 35-70% sterol (such as 40-55%, or 41-49%), 0.8-4.0% polymer-conjugated lipid (such as 0.8-3.5%, or 1.0-3.0%), and 0-15% neutral lipid (such as 6.0-12.0% or 8.0-11.0%). [000236] Such LNPs encapsulating nucleic acids (e.g., RNA) may be formed by admixing a first solution comprising the nucleic acids with a second solution comprising lipids which form the LNP. The admixing may be performed by any suitable means available to the skilled person, e.g., a T-mixer, microfluidics, or an impinging jet mixer. Admixing may be followed by filtration to obtain a desirable LNP size distribution (e.g., those as detailed above in this subsection). The filtration may be performed by any suitable means available to the skilled person, e.g., tangential-flow filtration or cross-flow filtration. [000237] In a further independent aspect, the present disclosure provides a method of preparing an LNP encapsulating the nucleic acid (e.g., RNA) of the second aspect, a method of preparing an LNP encapsulating the nucleic acid (e.g., RNA) of the third aspect or a method of preparing an LNP encapsulating the nucleic acid (e.g., RNA) of the fourth aspect, comprising admixing a first solution comprising the nucleic acid and a second solution comprising lipids which form the LNP (e.g. using the means as set out in the foregoing paragraph); and optionally filtering the obtained admixture (e.g. using the means as set out in the foregoing paragraph). Pharmaceutical Compositions [000238] In a further independent aspect, the present disclosure also provides a pharmaceutical composition comprising the immunogenic composition of the first aspect and a pharmaceutically acceptable excipient. Thus, there is provided a pharmaceutical composition comprising an immunogenic composition, said immunogenic composition comprising a first immunogen and a second immunogen: the first immunogen comprising a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment; and the second immunogen comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. It is preferred that the first immunogen is a first polypeptide, and the second immunogen is a second polypeptide. [000239] In a further independent aspect, the present disclosure also provides a pharmaceutical composition comprising the nucleic acid of the second aspect and a pharmaceutically acceptable excipient. Said aspect thus provides, a pharmaceutical composition comprising a nucleic acid encoding a) the first polypeptide as referred to in the first aspect; and b) the second polypeptide as referred to in the first aspect, wherein said nucleic acid is a single polynucleotide encoding said first and second polypeptides, and a pharmaceutically acceptable excipient. Further provided is a pharmaceutical composition comprising a carrier, said carrier comprising the nucleic acid of the second aspect and a pharmaceutically acceptable excipient i.e. a pharmaceutical composition comprising a carrier, said carrier comprising a nucleic acid encoding a) the first polypeptide as referred to in the first aspect; and b) the second polypeptide as referred to in the first aspect, wherein said nucleic acid is a single polynucleotide encoding said first and second polypeptides and a pharmaceutically acceptable excipient. [000240] In a further independent aspect, the present disclosure also provides a pharmaceutical composition comprising the nucleic acid of the third aspect and a pharmaceutically acceptable excipient. Said aspect thus provides, a pharmaceutical composition comprising a nucleic acid encoding the first polypeptide as referred to in the first aspect, and a pharmaceutically acceptable excipient. Further provided is a pharmaceutical composition comprising a carrier, said carrier comprising the nucleic acid of the third aspect and a pharmaceutically acceptable excipient i.e., a pharmaceutical composition comprising a carrier, said carrier comprising a nucleic acid encoding the first polypeptide as referred to in the first aspect and a pharmaceutically acceptable excipient. [000241] In a further independent aspect, the present disclose also provides a pharmaceutical composition comprising the nucleic acid of the fourth aspect and a pharmaceutically acceptable excipient. Said aspect thus provides, a pharmaceutical composition comprising a nucleic acid encoding the second polypeptide as referred to in the first aspect, and a pharmaceutically acceptable excipient. Further provided is a pharmaceutical composition comprising a carrier, said carrier comprising the nucleic acid of the third aspect and a pharmaceutically acceptable excipient i.e., a pharmaceutical composition comprising a carrier, said carrier comprising a nucleic acid encoding the first polypeptide as referred to in the first aspect and a pharmaceutically acceptable excipient. [000242] Thus, the present disclosure provides pharmaceutical compositions comprising ^ The immunogenic composition of the first aspect and a pharmaceutically acceptable excipient. ^ The nucleic acid of the second aspect (or carrier comprising the nucleic acid of the second aspect), and a pharmaceutically acceptable excipient. ^ The nucleic acid of the third aspect (or carrier comprising the nucleic acid of the third aspect) and a pharmaceutically acceptable excipient. ^ The nucleic acid of the fourth aspect (or carrier comprising the nucleic acid of the fourth aspect) and a pharmaceutically acceptable excipient. [000243] In a specific and preferred embodiment there is provided a pharmaceutical composition comprising the nucleic acid of the third aspect (or carrier comprising the nucleic acid of the third aspect) and the nucleic acid of the fourth aspect (or carrier comprising the nucleic acid of the fourth aspect) and a pharmaceutically acceptable excipient. In said embodiment, the nucleic acid of the third aspect and the nucleic acid of the fourth aspect are separate polynucleotides. In a specific and preferred embodiment there is provided a pharmaceutical composition comprising the nucleic acid of the third aspect (or carrier comprising the nucleic acid of the third aspect) and the nucleic acid of the fourth aspect (or carrier comprising the nucleic acid of the fourth aspect) and a pharmaceutically acceptable excipient, wherein the nucleic acid of the third aspect encodes the first polypeptide as referred to in the first aspect of the present disclosure and wherein the nucleic acid of the fourth aspect encodes the second polypeptide as referred to in the first aspect of the present disclosure. In said embodiment, the nucleic acid of the third aspect and the nucleic acid of the fourth aspect are separate polynucleotides. In an embodiment, said pharmaceutical composition further comprises the nucleic acid encoding the third polypeptide (i.e. GTD polypeptide) or a carrier comprising the nucleic acid encoding the third polypeptide. [000244] In a specific and preferred embodiment there is provided a pharmaceutical composition comprising the nucleic acid of the third aspect (or carrier comprising the nucleic acid of the third aspect) and the nucleic acid of the fourth aspect (or carrier comprising the nucleic acid of the fourth aspect) and a pharmaceutically acceptable excipient, wherein the nucleic acid of the third aspect is an RNA comprising an open reading frame comprising or consisting of SEQ ID NO: 29; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 29; and the nucleic acid of the fourth aspect is an RNA comprising an open reading frame comprising or consisting of SEQ ID NO: 30; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 30. In an embodiment, the pharmaceutical composition further comprises a nucleic acid encoding the third polypeptide of the present disclosure (or a carrier comprising said nucleic acid)) wherein the nucleic acid encoding the third polypeptide is an RNA comprising an open reading frame comprising or consisting of SEQ ID NO: 35; or an RNA sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 35. [000245] In a specific and preferred embodiment there is provided a pharmaceutical composition comprising an RNA of SEQ ID NO: 54 (or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical thereto) and an RNA of SEQ ID NO: 55 (or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical thereto). In an embodiment said pharmaceutical composition further comprises an RNA of SEQ ID NO: 56 (or an RNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical thereto). [000246] However, the present disclosure also contemplates pharmaceutical compositions comprising a mixture of polypeptide and nucleic acid modalities. As such there is provided a pharmaceutical composition comprising the first polypeptide as referred to in the first aspect and the nucleic acid of the fourth aspect (i.e., a nucleic acid encoding the second polypeptide as referred to in the first aspect), said pharmaceutical composition further comprising a pharmaceutically acceptable excipient. [000247] Further provided is a pharmaceutical composition comprising the second polypeptide according to the first aspect and the nucleic acid according to third aspect (i.e., a nucleic acid encoding the first polypeptide as referred to in the first aspect), said pharmaceutical composition further comprising a pharmaceutically acceptable excipient. [000248] The pharmaceutical compositions disclosed herein are generally for immunising subjects against disease, preferably against C. difficile disease. Accordingly, pharmaceutical compositions of the present disclosure are generally considered vaccine compositions. Thus, in an embodiment, a vaccine comprising any of the pharmaceutical compositions disclosed herein is provided. [000249] As used herein the term “excipient” refers to a substance that serves as a vehicle or medium for a pharmaceutical composition. Pharmaceutically acceptable excipients are well-known in the art, see, e.g., Gennaro.2000. Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [000250] Pharmaceutical compositions of the present disclosure may be in plain water (e.g., “w.f.i.”) or in a buffer e.g., a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. Buffer salts will typically be included in the 5-20mM range. [000251] Pharmaceutical compositions of the present disclosure may have a pH between 5.0 and 9.5 e.g., between 6.0 and 8.0. Pharmaceutical compositions of the present disclosure may comprise excipients present for the purposes of pH adjustment e.g., sodium hydroxide and/or hydrochloric acid. [000252] Pharmaceutical compositions of the present disclosure compositions may include sodium salts (e.g., sodium chloride) to give tonicity. A concentration of 10±2 mg/mL NaCl is typical, e.g., about 9 mg/mL (or 9 mg/mL). [000253] Pharmaceutical compositions of the present disclosure may include metal ion chelators (in particular, in embodiments wherein such compositions comprise RNA). These can prolong RNA stability by removing ions which can accelerate phosphodiester hydrolysis. Thus, such compositions may include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are typically present at between 10-500 μΜ e.g., 0.1 mM. A citrate salt, such as sodium citrate, can also act as a chelator, while advantageously also providing buffering activity. [000254] Pharmaceutical compositions of the present disclosure may have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g., between 240-360 mOsm/kg, or between 290-310 mOsm/kg. [000255] Pharmaceutical compositions of the present disclosure may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. Mercury-free compositions are preferred, and preservative-free vaccines can be prepared. [000256] Pharmaceutical compositions of the present disclosure may be aseptic or sterile. [000257] Pharmaceutical compositions of the present disclosure may be non-pyrogenic e.g., containing <1 EU (endotoxin unit, a standard measure) per dose, such as <0.1 EU per dose. [000258] Pharmaceutical compositions of the present disclosure may be gluten free. [000259] Pharmaceutical compositions or vaccines of the present disclosure may be prepared in unit dose form. In some embodiments a unit dose may have a volume of between 0.1 -1.0 mL e.g., about 0.5mL (or 0.5mL). [000260] Pharmaceutical compositions or vaccines of the present disclosure may be prepared as injectables, either as solutions or suspensions. The composition may be prepared for pulmonary administration e.g., by an inhaler, using a fine spray. The composition may be prepared for nasal, aural or ocular administration e.g., as spray or drops. Injectables for intramuscular administration are typical. [000261] Pharmaceutical compositions or vaccine of the present disclosure comprise an immunologically effective amounts of the first polypeptide and/or second polypeptide referred to in the first aspect of the present disclosure, nucleic acid (e.g., RNA) and/or carrier (e.g., lipid nanoparticle), as well as any other components, as needed. [000262] By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention, preferably prevention of C. difficile disease. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individuals to be treated (e.g., non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. In embodiments wherein pharmaceutical compositions of the present disclosure comprise RNA, the RNA content will generally be expressed in terms of the amount of RNA per dose. A preferred dose has <120µg RNA e.g., <100µg (e.g., 10-120µg or 10-100 µg, such as 10µg, 25µg, 50µg, 75µg or 100µg, or about 10µg, 25µg, 50µg, 75µg or 100µg), but expression can be seen at much lower levels e.g., <1µg/dose, <100ng/dose, <10ng/dose, <1ng/dose, etc. [000263] Pharmaceutical compositions of the present disclosure may be lyophilised. [000264] In a further independent aspect, the present disclosure also provides a delivery device (e.g., syringe, nebuliser, sprayer, inhaler, dermal patch, etc.) comprising a pharmaceutical composition of the present disclosure. This device can be used to administer the composition to a vertebrate subject. [000265] In a further independent aspect, the present disclosure also provides a method of preparing a pharmaceutical composition, comprising formulating the first polypeptide and/or second polypeptide referred to in the first aspect of the present disclosure, nucleic acid (e.g., RNA) or carrier (e.g., lipid nanoparticle) of the present disclosure with a pharmaceutically acceptable excipient, to produce said composition. In particular, said pharmaceutical composition has the features as detailed above throughout this section. In a further independent aspect, the present disclosure also provides a kit comprising the first polypeptide and/or second polypeptide referred to in the first aspect of the present disclosure, a nucleic acid, carrier or pharmaceutical composition or delivery device of the present disclosure, and instructions for use. Medical Use [000266] There is further provided a pharmaceutical composition of the disclosure or vaccine of the disclosure for use in medicine. More particularly, there is provided a pharmaceutical composition of the disclosure or vaccine of the disclosure for use in the treatment or prevention of C. difficile disease. [000267] There is further provided a pharmaceutical composition of the disclosure or vaccine of the disclosure for use in a method of raising an immune response in a subject, optionally a protective immune response in a subject. Further provided is a pharmaceutical composition of the disclosure or vaccine of the disclosure for use in a method of vaccinating a subject against C. difficile disease, optionally wherein the vaccination is prophylactic. [000268] As used herein, “C. difficile disease” refers to any infection or disease caused by toxins released by C. difficile. Examples of C. difficile disease are antibiotic-associated diarrhea (AAD), pseudomembranous colitis and toxic megacolon, which can be life-threatening. [000269] The pharmaceutical compositions of the present disclosure or vaccines of the present disclosure 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 pharmaceutical composition or vaccine via a systemic or mucosal route. These administrations may include injection via the intramuscular, intraperitoneal, intradermal, or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts. Although the pharmaceutical composition or 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. In addition to a single route of administration, 2 different routes of administration may be used. [000270] 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. [000271] There is further provided a method of inducing an immune response against C. difficile in a subject comprising administering to the subject an immunologically effective amount of the pharmaceutical composition of or the vaccine of the present disclosure. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment, protection or prevention. Administration of an immunologically effective amount elicits an immune response, including a protective immune response. This amount can vary depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (e.g., non-human primate, primate, etc.), the capacity of the individual’s immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor’s assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range. [000272] There is further provided a method of treating or preventing C. difficile disease comprising administering the pharmaceutical composition or the vaccine of the disclosure to a subject in need thereof. [000273] There is further provided the use of the pharmaceutical composition or the vaccine of the present disclosure in the manufacture of a medicament. Further provided is the use of the pharmaceutical composition or the vaccine of the present disclosure in the manufacture of a medicament against C. difficile disease. [000274] In an embodiment, said use or method of treatment may comprise administering to a subject at least one dose of the pharmaceutical composition or vaccine of the present disclosure. In an embodiment, said use or method of treatment comprises administering two doses of the pharmaceutical composition or vaccine of the present disclosure to a subject. In an embodiment, said use or method of treatment comprises administering three doses of the pharmaceutical composition or vaccine of the present disclosure to a subject. [000275] Embodiments are further described in the subsequent numbered clauses: 1. An immunogenic composition comprising a first immunogen and a second immunogen: the first immunogen comprising a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment; and the second immunogen comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. 2. The immunogenic composition of clause 1 wherein the first immunogen is a first polypeptide, and the second immunogen is a second polypeptide. 3. The immunogenic composition of clause 2 wherein the first polypeptide comprises a C. difficile toxin A CROP domain fragment wherein said C. difficile toxin A CROP domain fragment is an immunogenic fragment of the toxin A CROP domain; and a C. difficile toxin B CROP domain fragment wherein said C. difficile toxin B CROP domain fragment is an immunogenic fragment of the toxin B CROP domain. 4. The immunogenic composition of clause 2 or clause 3 wherein the toxin A CROP domain fragment of the first polypeptide comprises a proximal end (i.e., proximal end of the toxin A CROP domain fragment) and a distal end (i.e., distal end of the toxin A CROP domain fragment); and the toxin B CROP domain fragment of the first polypeptide comprises a proximal end (i.e., proximal end of the toxin B CROP domain fragment) and a distal end (i.e., distal end of the toxin B CROP domain fragment), wherein the proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment are adjacent to each other. 5. The immunogenic composition of clause 4 wherein the distal end of the toxin A CROP domain fragment and the distal end of the toxin B CROP domain fragment are at either terminus of the first polypeptide. The immunogenic composition of clause 4 wherein the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A. The immunogenic composition according to clause 6 wherein the proximal end of the toxin A CROP domain fragment is within amino acids 2700-2710 or 2680-2690 of toxin A. The immunogenic composition of clause 4 wherein the proximal end of the toxin B CROP domain fragment is within repeat portion I (amino acids 1834-1926) of toxin B. The immunogenic composition of clause 4 wherein the proximal end of the toxin B CROP domain fragment is within repeat portion II (amino acids 1927-2057) of toxin B. The immunogenic composition according to clause 4 wherein the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A and wherein proximal end of the toxin B CROP domain fragment is within repeat portion I (amino acids 1834-1926) of toxin B. An immunogenic composition according to clause 10 wherein the proximal end of the toxin A CROP domain fragment is within short repeat 3 of repeat portion VIII (amino acids 2687- 2710) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within short repeat 1 of repeat portion I of toxin B (amino acids 1834-1854). An immunogenic composition according to clause 10 or clause 11 wherein the proximal end of the toxin A CROP domain fragment is amino acid 2705-2710 of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1850 and 1860 of toxin B. The immunogenic composition according to clauses 4 wherein the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within repeat portion II (amino acids 1927-2057) of toxin B. An immunogenic composition according to clause 13 wherein the proximal end of the toxin A CROP domain fragment is within short repeat 2 of repeat portion VIII (amino acids 2665- 2686) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within short repeat 3 of repeat portion II of toxin B (amino acids 1968-1987). An immunogenic composition according to clause 13 and clause 14 wherein the proximal end of the toxin A CROP domain fragment is within amino acids 2680-2690 of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1960-1970 of toxin B. An immunogenic composition according to clause 13 wherein the proximal end of the toxin A CROP domain fragment is within short repeat 3 of repeat portion VIII (amino acids 2687- 2710) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within short repeat 4 of repeat portion II of toxin B (amino acids 1988-2007). An immunogenic composition according to clause 16 wherein the proximal end of the toxin A CROP domain fragment is within amino acids 2705-2710 of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within amino acids 1980-1990 of toxin B. The immunogenic composition of clauses 4-17 wherein the distal end of the toxin A CROP domain fragment is within repeat portion III (2059-2192) or repeat portion V (2307-2440) of toxin A. The immunogenic composition of clauses 4-17 wherein the distal end of the toxin B CROP domain fragment is within repeat portion V (2324-2366) of toxin B. The immunogenic composition of clause 19 wherein the distal end of the toxin B CROP domain fragment corresponds to the C-terminal amino acid of the toxin B polypeptide, optionally amino acid 2366 of toxin B. The immunogenic composition of clause 2 wherein the first polypeptide comprises a C. difficile toxin A CROP domain fragment wherein said C. difficile toxin A CROP domain fragment is the entire C. difficile toxin A CROP domain (amino acids 1832-2710); and a C. difficile toxin B CROP domain fragment wherein said C. difficile toxin B CROP domain fragment is the entire C. difficile toxin B CROP domain (amino acids 1833-2366) The immunogenic composition according to any of clauses 2-21 wherein the first polypeptide further comprises a linker. The immunogenic composition according to clause 22 wherein the linker comprises 1-20 amino acids. The immunogenic composition according to clause 22 or clause 23 wherein the linker comprises 1-5 amino acids. The immunogenic composition according to clauses 22-24 wherein the linker is a glycine linker. The immunogenic composition according to clauses 22-24 wherein the linker comprises or consists of SEQ ID NO: 15. The immunogenic composition according to clauses 22-26 wherein the linker is between the proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment. The immunogenic composition according to any of clauses 2-27 wherein the first polypeptide comprises more than 350, 375, 400 or 425 amino acids from toxin A. The immunogenic composition according to any of clauses 2-28 wherein the first polypeptide comprises less than 750, 725, 700, 675, 650, 625, or 600 amino acids from toxin A. The immunogenic composition according to any of clauses 2-29 wherein the first polypeptide comprises more than 250, 300 or 350 amino acids from toxin B. The immunogenic composition according to any of clauses 2-30 wherein the first polypeptide comprises less than 675, 650, 625, 600, 575 or 550 amino acids from toxin B. The immunogenic composition according to any one of clauses 2-31 wherein the first polypeptide comprises: (i) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; or (ii) A polypeptide or variant having at least 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; or (iii) A fragment of at least 250, 280, 300, 350, 380, 400, 430, 450, 480, 500, 530, 550, 580, or 600 contiguous amino acids of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14. The immunogenic composition according to any of clauses 2-32 the first polypeptide comprises a sequence at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least 97.5% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. The immunogenic composition according to any preceding clause wherein the first polypeptide comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. The immunogenic composition according to any preceding clause wherein the first polypeptide comprises or consists of SEQ ID NO: 3. The immunogenic composition of clause 2 wherein the second polypeptide comprises a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the DRBD of toxin B wherein the DRBD of toxin B corresponds to amino acids 840-1833 of SEQ ID NO: 2 (strain VPI10463 (ATCC43255), amino acids 841-1834 of SEQ ID NO: 18 (strain M68) or at equivalent positions in the toxin B of other strains of C. difficile. The immunogenic composition of clause 2 wherein the second polypeptide comprises at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325 or at least 350 contiguous amino acids of the DRBD of toxin B. The immunogenic composition of clause 2 wherein the second polypeptide is between 150 and 700 amino acids, between 200 and 600 amino acids, between 250 and 550 amino acids, between 300 and 450 amino acids, between 350 and 400 amino acids or between 375 and 385 amino acids. The immunogenic composition of clause 2 wherein the second polypeptide comprises a sequence at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least 97.5% identical to SEQ ID NO: 16 or SEQ ID NO: 17. The immunogenic composition of clause 39 wherein the second polypeptide comprises SEQ ID NO: 16 or SEQ ID NO: 17. The immunogenic composition of any preceding clause further comprising a third immunogen, said third immunogen being a third polypeptide. The immunogenic composition of clause 41 wherein said third polypeptide comprises the glucosyl transferase domain of TcdB or TcdA of C. difficile. The immunogenic composition of clause 41 or clause 42 wherein said third polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95% or at least 97.5% sequence identity to SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 53. The immunogenic composition according to any preceding clause further comprising additional antigens. The immunogenic composition of clause 44 wherein the additional antigens are antigens derived from a bacterium selected from the group consisting of Streptococcus pneumonia, Haemophilus influenzae, Neisseria meningitidis, Escherichia coli, Moraxella catarrhalis, Clostridioides tetani, Corynebacterium diptherieriae, Bordetella pertussis, Staphylococcus epidermidis, enterococci, and Staphylococcus aureus. The immunogenic composition of any of the preceding clauses further comprising a saccharide from C. difficile. The immunogenic composition according to any previous clause further comprising an adjuvant. The immunogenic composition according to clause 47 wherein the adjuvant comprises an immunologically active saponin fraction. The immunogenic composition according to clause 48 wherein the immunologically active saponin fraction is presented in the form of a liposome. The immunogenic composition according to clause 48 or clause 49 wherein the immunologically active saponin fraction is QS21 The immunogenic composition according to clauses 48-50 wherein the adjuvant further comprises a lipopolysaccharide, optionally wherein said lipopolysaccharide is a lipid A derivative, optionally wherein the lipid A derivative is 3D-MPL. The immunogenic composition according to clauses 48-51 wherein the adjuvant further comprises a sterol, optionally wherein said sterol is cholesterol. The immunogenic composition according to clause 48-52 wherein the adjuvant further comprises 1, 2-Dioleoyl-sn-Glycero-3-phosphocholine (DOPC). The immunogenic composition of any of clauses 48-53, wherein the adjuvant comprises cholesterol, DOPC, 3D-MPL and QS21. The immunogenic composition of clause 47 wherein said adjuvant comprises an oil in water emulsion, wherein said oil in water emulsion comprises a metabolisable oil, a tocol and an emulsifying agent. The immunogenic composition according to clause 55 wherein the metabolisable oil is squalene. The immunogenic composition according to clause 55 wherein the tocol is alpha-tocopherol. The immunogenic composition according to clause 55 wherein the emulsifying agent is polyoxyethylene sorbitan monooleate. The immunogenic composition according to clause 58 wherein the polyoxyethylene sorbitan monooleate is selected from the group comprising: Polysorbate® 80 or Tween® 80. The immunogenic composition according to clause 47 wherein the adjuvant is ASO1 or ASO3 The immunogenic composition according to clause 47 wherein the adjuvant is an aluminum- based adjuvant, optionally aluminum hydroxide or aluminum phosphate. The immunogenic composition according any of clauses 2-61 wherein the first polypeptide and/or second polypeptide has been chemically detoxified. The immunogenic composition according to any of clauses 1-62 wherein said immunogenic composition elicits antibodies that neutralize toxin A or toxin B or both. The immunogenic composition according to any of clauses 2-62 wherein said first polypeptide elicits antibodies that neutralize toxin A and toxin B, and wherein said second polypeptide elicits antibodies that neutralize toxin B. A nucleic acid encoding at least one polypeptide comprising a bacterial toxoid or toxin or an immunogenic fragment of a bacterial toxoid or toxin. The nucleic acid of clause 65 wherein the at least one polypeptide comprises a C. difficile toxoid or toxin or an immunogenic fragment of a C. difficile toxin. The nucleic acid of clause 65 or 66 wherein the at least one polypeptide is a fragment of toxin A or toxin B of C. difficile. 68. The nucleic acid of clause 65 wherein the at least one polypeptide is a fragment from toxin B of C. difficile. 69. The nucleic acid of clause 65 wherein the at least one polypeptide is a fragment from toxin A of C. difficile. 70. The nucleic acid of any one of clauses 65-69 wherein the nucleic acid encodes at least two polypeptides comprising a fragment of toxin A or toxin B of C. difficile. 71. The nucleic acid of clause 70 wherein the at least two polypeptides both comprise a fragment of toxin B of C. difficile. 72. The nucleic acid of any one of clauses 68-71 wherein the fragment of toxin B comprises at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. 73. The nucleic acid of any one of clauses 65-72 wherein the fragment of toxin B comprises at least part of a CROP domain. 74. The nucleic acid of any one of clauses 65 – 73 wherein the fragment of toxin B comprises at least part of the glucosyl transferase domain of C. difficile toxin B. 75. The nucleic acid of claim 74 wherein the fragment of toxin B comprises amino acids 1-543 of toxin B from C. difficile. 76. A nucleic acid encoding a) the first polypeptide as referred to in clauses 2-35 and b) the second polypeptide as referred to in clause 2 and clauses 36-40 wherein said nucleic acid is a single polynucleotide encoding said first and second polypeptides. 77. A nucleic acid encoding the first polypeptide as referred to in clauses 2-35. 78. A nucleic acid encoding the second polypeptide as referred to in clause 2 and clauses 36-40. A nucleic acid encoding the third polypeptide as referred to in clauses 41-43. The nucleic acid of clauses 65-79 wherein the nucleic acid is RNA. The nucleic acid of clause 80 wherein the RNA is non-self-replicating RNA. The nucleic acid of clause 80 wherein the RNA is self-replicating RNA (SAM). The nucleic acid of clause 65-82, comprising, in the 5’ to 3’ direction: i) a 5’ Cap, ii) a 5’ UTR, iii) an open reading frame encoding the first polypeptide as referred to in any of clauses 2-35, the second polypeptide as referred to in clause 2 and any of clauses 36-40 and/or the third polypeptide as referred to in clauses 41-43. iv) a 3’UTR, and v) a 3’ poly-A tail. The nucleic acid of clause 83, wherein the 5’ cap comprises a 7’-methylguanosine linked 5’- to-5’ to the 5’ first ribonucleoside by a triphosphate bridge, and wherein the first 5’ ribonucleoside comprises a 2’-methylated ribose (2’-O-Me). The nucleic acid of clause 83 wherein the 3’ poly-A tail comprises a contiguous stretch of 100-500 A ribonucleotides. The nucleic acid of clause 83 wherein the 3’ poly-A tail comprises at least two non- contiguous stretches of A ribonucleotides; optionally 25-35 and 65-90 ribonucleotides in length respectively; optionally orientated in the 5’ to 3’ direction. The nucleic acid according to any of clauses 65-86 comprising a modified ribonucleotide. The nucleic acid of clause 87 wherein the modified ribonucleotide is 1mΨ. The nucleic acid of clause 88 comprising 1mΨ and neither standard U ribonucleotides nor other modified U ribonucleotides; optionally wherein the RNA comprises 1mΨ and neither standard U ribonucleotides nor other modified ribonucleotides. The nucleic acid of clauses 80-89 wherein the RNA has a GC content of 40-60%. A carrier comprising the nucleic acid of clauses 65-75. A carrier comprising the nucleic acid of clause 76. A carrier comprising the nucleic acid of clause 77. A carrier comprising the nucleic acid of clause 78. A carrier comprising the nucleic acid of clause 79. The carrier according to any of clauses 91-95, wherein said carrier is a lipid nanoparticle. The carrier of clause 96 wherein the lipid nanoparticle comprises a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids. The carrier of clause 97 wherein the cationic lipid has a pKa of 5.0-8.0; optionally 5.0-7.6. The carrier of clause 97 and clause 98 wherein the cationic lipid comprises a tertiary amine group. The carrier of clause 97 wherein the polymer-conjugated lipid is a PEGylated lipid, optionally wherein the PEG has an average molecular weight of 1-3 kDa. The carrier of clause 97 wherein the sterol is cholesterol or a cholesterol-based lipid. The carrier of clauses 97-101 wherein the lipid nanoparticle comprises (in mole %) 30- 60% cationic lipid, 35-70% sterol, 0.8-4.0% polymer-conjugated lipid, and 0-15% neutral lipid; optionally 40-50% cationic lipid, 41-49% sterol, 1.0-3.0% polymer-conjugated lipid and 8.0-11.0% neutral lipid. A pharmaceutical composition comprising the immunogenic composition of clauses 1-64 and a pharmaceutically acceptable excipient. A pharmaceutical composition comprising the nucleic acid of any of clauses 65-75, clause 76, clause 77, clause 78 or clause 79 or the carrier of clause 91, clause 92, clause 93, clause 94 or clause 95 and a pharmaceutically acceptable excipient. A pharmaceutical composition comprising the carrier of clause 93 and the carrier of clause 94 and a pharmaceutically acceptable excipient, optionally wherein said pharmaceutical composition further comprises the carrier of clause 95. A pharmaceutical composition comprising the nucleic acid of clause 78 or the carrier of clause 94 and a pharmaceutically acceptable excipient. A pharmaceutical composition comprising: the first polypeptide according to clause 2-35 and the nucleic acid according to clause 78 or the carrier according to clause 94, said pharmaceutical composition further comprising a pharmaceutically acceptable excipient. A pharmaceutical composition comprising: the second polypeptide according to clause 2 and clauses 36-40 and the nucleic acid according to clause 77 or the carrier according to clause 93, said pharmaceutical composition further comprising a pharmaceutically acceptable excipient. A vaccine comprising the pharmaceutical composition of any of clauses 103-108. The pharmaceutical composition of clauses 103-108 or the vaccine of clause 109 for use in medicine. The pharmaceutical composition of clauses 103-108 or the vaccine of clause 109 for use in a method of raising an immune response in a subject, optionally a protective immune response in a subject. 112. The pharmaceutical composition of clauses 103-108 or the vaccine of clause 109 for use in the treatment or prevention of C. difficile disease. 113. The pharmaceutical composition of clauses 103-108 or the vaccine of clause 109 for use in a method of vaccinating a subject against C. difficile disease, optionally wherein the vaccination is prophylactic. 114. A method of inducing an immune response against C. difficile in a subject comprising administering to the subject an immunologically effective amount of the pharmaceutical composition of clauses 103-108 or the vaccine of clause 109. 115. A method of treating or preventing C. difficile disease comprising administering the pharmaceutical composition of clause 103-108 or the vaccine of clause 109 to a subject in need thereof. 116. Use of the pharmaceutical composition of clauses 103-108 or the vaccine of clause 109 in the manufacture of a medicament. 117. Use of the pharmaceutical composition of clauses 103-108 or the vaccine of clause 109 in the manufacture of a medicament against C. difficile disease. 118. The pharmaceutical composition or vaccine for use according to clause 110-113 or the method of clause 114 or clause 115 wherein the subject is a human subject, optionally wherein the human subject is over 50 years old. The invention is further illustrated by the following non-limiting examples. EXAMPLES Example 1: Cloning, expression, and purification of the F2 and TD1 constructs F2: Genes encoding the F2 fusion protein (SEQ ID NO: 3) and a His tag was cloned into the pET24b(+) expression vector (Novagen) using the NdeI/XhoI 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 CaCl2-treated cells (Hanahan D. In Glover, D. M. (Ed), DNA cloning. IRL Press London. (1985): p.109-135.). 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 lon and ompT proteases. Genotype : E. coli BLR::DE3 strain, F- ompT hsdS_B(r_B- m_B-) gal dcm (DE3) Δ(srl-recA)306::Tn10 (TetR) An E. coli transformant was stripped from agar plate and used to inoculate 200 ml of LBT broth ± 1% (w/v) glucose + kanamycin (50 µg/ml) to obtain O.D.600nm between 0.1 -0.2. Cultures were incubated overnight at 37 ^C, 250 RPM. This overnight culture was diluted to 1:20 in 500 ml of LBT medium containing kanamycin (50 µg/ml) and grown at 37°C at a stirring speed of 250 rpm until O.D.620 reached 0.5/0.6. At O.D.600nm around 0.6, the culture was cooled down before inducing the expression of the recombinant protein by addition of 1 mM isopropyl β-D-1- thiogalactopyranoside (IPTG) and incubated overnight at 23 ^C, 250 RPM. After overnight induction (around 16 hours), O.D.600nm was evaluated after induction and culture was centrifuged at 14000 RPM for 15 minutes and pellets were frozen at -20 ^C separately. 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 20000 PSI. Soluble (supernatant) and insoluble (pellet) components were separated by centrifugation for example at 20000g for 30 min at 4°C. 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. 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, sterile-filtered on 0.22 µm, stored at -80°C. 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. TD1: The genes encoding TD1 (residues 1072-1452 with a l0x His-tag at the C-terminus) was cloned into a modified pET 28a vector which has a 6x His/SUMO (Saccharomyces cerevisiae Smt3p) tag introduced to the N-terminus. All mutants were generated by two-step PCR and verified by DNA sequencing. TD1 was expressed in E. coli 4strain BL21-Star (DE3, Invitrogen). Bacteria were cultured at 37°C in LB medium containing kanamycin or ampicillin. The temperature was reduced to 16°C when OD₆₀₀ reached ~0.8. Expression was induced with 1 mM IPTG and continued at 16°C overnight. The cells were harvested by centrifugation and stored at -80°C until use. TD1 was purified using Ni2+affinity resins in a buffer containing 50mM Tris, pH 8.5, 500mM NaCl and 10mM imidazole. The protein was eluted with high imidazole buffer (50 mM tris, pH 8.5, 500mM NaCl and 300mM imidazole) and then dialyzed at 4°C against a buffer containing 20mM Tris, pH 8.5, 1mM TCEP and 40mM NACl. The His6-SUMO tag was cleaved by SUMO protease and further purified by Mono-Q ion exchange chromatography (GE Healthcare) in a buffer containing 20mM Tris, pH 8.5 and eluted with a NaCl gradient. Example 2: Materials and Methods Primary challenge model with 6529 strain of Clostridioides difficile The model described herein was based on the model developed by Chen et al (2008). In brief, mice received an antibiotic cocktail (kanamycin 0,4 mg/ml, gentamycin 0,035 mg/ml, colistin 850 U/ml, metronidazole 0,215 mg/ml, vancomycin 0,045 mg/ml) within the drinking water from day 6 to day 3 before challenge, followed by an IP injection of clindamycin (10 mg/kg) the day before the challenge. Challenge is performed by administering with a gastric tube 5.105 cfu of the 6529 strain (ribotype 027). Mortality (generally low), weight loss and any symptoms (like diarrhea) were daily monitored for 6 to 7 days post-challenge. Mice were daily weighed from day 0 to day 7 post- challenge. Results were expressed as the relative weight (%) compared to day 0. Serological analysis of anti-Toxin A Ct, Toxin B Ct and TD1 IgG by Enzyme Linked Immuno-Sorbent Assay (ELISA). The Toxin A F2 Ct fragment (comprising amino acids 2387 to 2706) was coated at 4 µg/ml, GTD (Toxin B fragment comprising amino acids 1 to 543) was coated at 2 µg/ml, Toxin B F2 Ct fragment (comprising amino acids 1968 to 2366) and TD1 (Toxin B fragment comprising amino acids 1072 to 1452) were coated at 1 µg/ml in phosphate buffered saline (PBS) on high-binding microtiter plates (Nunc MAXISORPTM) overnight at 4°C. The plates were blocked with PBS-Bovine Serum Albumin (BSA) 1% for 30 minutes at room temperature (RT) under shaking. Then serial two-fold dilutions of mouse sera in PBS-BSA 0.2%-TWEENTM 0.05% were incubated at RT for 30 minutes under shaking. After washing, peroxidase-conjugated anti-mouse IgG antibodies (Jackson ImmunoLaboratories) diluted 1/5000 in PBS-BSA 0.2%- TWEEN^TM 0.05% were added for 30 minutes at RT under shaking. After an additional washing step, the bound mouse antibodies were detected by adding a solution containing 4 mg O-phenylenediamine and 5 µl H2O2 per 10 ml of 0.1M citrate buffer pH 4.5 for 15 minutes in the dark at RT. The colorimetric reaction was stopped by adding HCl 1N and the optical densities (OD), which are directly proportional to the amount of antibodies presents in the serum, were measured using a spectrophotometer. The level of specific anti-Toxin A Ct or anti-Toxin B Ct or anti-TD1 IgG antibodies present in mouse sera was calculated by the four-parameter method in comparison to the curve of a calibrated standard using the SofMaxPro software. Results were expressed as µg/ml. Toxin A-mediated cytotoxicity neutralization assays Human colonic epithelial cells HT29 were cultured at 37°C with 5% CO2 in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 1% glutamine and 1% of an antibiotic cocktail containing penicillin, streptomycin and amphotericin. HT29 cells were seeded in 96-well black tissue culture plates (Greiner Bio-one) at a density of 4.10 E3 cells/well. After 24 hours, the medium was removed from the wells. Then 50 µl of three-fold serial dilutions of mouse sera followed by 50 μl of native Toxin A (Toxin A r087 at 67ng/ml - Batch GSK P054) were added into the plates and incubated at 37°C with 5% CO2 for 6 days. After removal of antisera / toxin mixture, 100µl of DNA-staining Hoechst fluorescent reagent (BD Pharmingen, Ref: 561908) diluted 1/500 in PBS were added for 2 hours in the dark at RT. After removal of Hoechst dye solution, the plate was dried and the fluorescence-covered surface of each well (corresponding to the well surface still covered by living cells, which is minimal in case of full cytotoxicity and maximal in absence of any cytotoxicity) was determined using the AxioVision microscope software. Neutralization titers were defined as the reciprocal dilution of serum inducing a 50% inhibition of the Toxin A cytotoxic effect. Toxin B-mediated cytotoxicity neutralization assays Human colonic epithelial cells HCT-116 cells were cultured at 37°C with 5% CO2 in Minimum Essential Medium (MEM) Eagle with Earle's BSS supplemented with 10% fetal bovine serum, 1% glutamine and 1% of an antibiotic cocktail containing penicillin, streptomycin and amphotericin. HCT116 cells were seeded in 96-well black tissue culture plates (Greiner Bio-one) at a density of 1.10E3 cells/well. After 24 hours, the medium was removed from the wells. Then 50 µl of three-fold serial dilutions of mouse sera followed by 50 μl of native Toxin B (Toxin B r087 from List labs at 50 ng/ml, Toxin B r078 from TGC Biomics at 1ng/ml and Toxin B r027 from TGC Biomics at 20ng/ml) were added into the plates and incubated at 37°C with 5% CO2 for 6 days. After removal of antisera / toxin mixture, 100µl of DNA-staining Hoechst fluorescent reagent (BD Pharmingen, Ref: 561908) diluted 1/500 in PBS were added for 2 hours in the dark at RT. After removal of Hoechst dye solution, the plate was dried and the fluorescence-covered surface of each well (corresponding to the well surface still covered by living cells, which is minimal in case of full cytotoxicity and maximal in absence of any cytotoxicity) was determined using the AxioVision microscope software. Neutralization titers were defined as the reciprocal dilution of serum inducing a 50% inhibition of the Toxin B cytotoxic effect. Measurement of inflammatory markers Caecum and colon sections were collected and were snap frozen on dry ice and stored at -80°C until further analysis. Samples were homogenized twice in 1 mL of PBS containing an anti-protease inhibitor cocktail (Halt Protease &Phosphatase inhibitor cocktail - Thermo Scientific) diluted at 1/100 by GentleMACS™ Dissociator (program: m_lung_02_01 C 37sec). The homogenates were cleared by centrifugation (10 min at 14000 rpm) and stored at −70°C un^l analysis. Total protein was measured using BCA assay (Thermo Scientific Pierce BCA) according to manufacturer’s instructions. Inflammatory biomarkers detection: Luminex technology was utilised according to manufacturer’s instructions (Mouse S100A8 and S100A9 from R&D System Biotechne (LXSAMSM-02), Mouse IL6, Mouse KC/Groa, Mouse IL1b from Millipore – Milliplex MCYMAG-70K). Colon Histopathology Colon samples were processed to paraffin blocks, sectioned at approximately 4µm, put on a Superfrost glass slide using standard techniques. Sections were stained with haematoxylin and eosin and the slides were cover-slipped. Slides were examined using a standard light microscope by a trained pathologist. Histopathological findings were recorded following a 0 to 4 scoring scheme (0 = none, 1 = mild, 2 = moderate, 3 = marked and 4 = severe). Statistical Analysis The distributions of Elisa and Neutralization titers are assumed to be lognormal. To evaluate the immunogenicity effect, by endpoint and by time point, the statistical method was an Analysis of Variance (ANOVA) on the log10 values with one fixed factor (group). Heterogeneity of variance (identical variances not assumed for the different levels of the factor combinations) was included in the model. These models were used to estimate geometric means and their 95% CIs as well as geometric mean ratios and their 95% CIs. Example 3: Evaluation of the protective efficacy of TD1 at two doses (0.06µg or 2µg) alone or combined with a suboptimal dose of F2 by IM route in the C. difficile C57Bl6 mouse model (strain 6529 (r027)) challenge. The primary objective was to evaluate the protective efficacy of TD1 at two doses (2µg and 0.06µg) and combined to the suboptimal dose of F2 in terms of weight loss. Female 7 week(s) old C57BL/6JOlaHsd Mouse (7 groups, 8 animal(s) per group) were randomly assigned to the study groups and immunized 2 times Intramuscular at day 0 and day 14 with 50µl of the following formulations: ^ 2µg F2 adjuvanted with AS01 ^ 0.06 µg F2 adjuvanted with AS01 ^ 2µg TD1 adjuvanted with AS01 ^ 0.06µg F2 and 2µg TD1 adjuvanted with AS01 ^ 0.06µg TD1 adjuvanted with AS01 ^ 0.06µg F2 and 0.06µg TD1 adjuvanted with AS01 ^ AS01 The AS01 adjuvant has 50 µg QS21 presented in the form of a liposome, 50 µg 3D-MPL, 0.25 mg cholesterol and 1.0 mg DOPC per 0.5ml dose. A dose of 50μl suitable for immunizing mice contains 5μg QS21, 5μg 3D-MPL, 0.025mg cholesterol and 0.1mg DOPC. The immunized C57Bl6 mice were then treated with antibiotics at day 22 to day 27 and then were challenged at day 28 with 400µl of 6529 C. difficile strain spores (ribotype 027) at 9,6210E5 spores/mouse via intra-gastric (IG) route. Sera from individual animals were collected at day 22 to measure IgG specific of ToxA Ct, ToxB Ct and TD1 and to measure also neutralizing titers anti-ToxA on HT29 cell lines (strain r087 and r027) and anti-ToxB (strain r087 and r027) on HCT116 cell lines in sera at day 22 (8PII). Feces were collected at day 28 to measure total IgG and IgG specific of ToxA Ct, ToxB Ct and TD1. The bacterial load was assessed by counting vegetative cells and spores in feces collected at day 28, day 30, day 31, day 32 and day 35. The immunized mice were monitored in terms of weight loss, mortality from day 28 to day 35. Details of the study design are provided in Figure 2. Results A mean profile of weight change from baseline (expressed in %) over time was estimated for each group after challenge – This data is reported in Figure 3. The comparison between F2 (2 µg dose) and TD1 (2 µg dose) at the peak of the disease (day 2 post-challenge) show that TD1 (2 µg dose) induced a significant (6.7 %, p=0.0021) but partial reduction of weight loss compared to the negative control AS01 while F2 (2 µg dose) was almost fully protective. F2 was significantly more protective than TD1 (15.5%, p=0.0001). This was expected because F2 targets both Toxin A and Toxin B whereas TD1 targets only Toxin B. This data suggests that, for protection against weight loss, targeting both Toxin B & Toxin A is important. However, combining 0.06 µg of F2 and 0.06 µg of TD1 significantly improved the protection against weight loss compared to 0.06 µg of F2 (4.7%, p=0.0269) or 0.06µg of TD1 (7%, p=0.0015) alone (see Figure 4). Furthermore, a significant difference (7.3%, p=0.001) was observed when combining 2 µg of TD1 and 0.06 µg of F2 as compared to F20.06µg alone (see Fig 5). The difference observed (2.9%, p=0.1694) as compared to TD12µg alone was not significant. No interference and no clear added value effect were shown by combining TD1 and F2 at 8d post II in the three serological responses (data not shown). No interference and added value were observed by combining TD1 and F2 at 8d post II on neutralizing Abs titers against Toxin B (data not shown). Summary: TD1 (2 µg dose) induced a significant but partial reduction of weight loss while F2 (2 µg), was almost fully protective. Interestingly, combining 0.06 µg F2 and 0.06 µg TD1 did significantly improve the protection against weight loss compared to 0.06 µg of F2 or 0.06 µg TD1 alone, showing that both antigens are complementary. Example 4: Immunogenicity of Clostridioides difficile TD1 antigen in C57Bl6 mouse model The primary objective of the study was to evaluate the immunogenicity of TD1 alone and the added value when TD1 is combined to F2. Female C57BL/6JOlaHsd mice (5 weeks old) were randomly assigned to the study groups. Animals were immunized intramuscularly at day 0, 14 day 28 with 50µl of the following formulations: ^ TD16µg /AS01 ^ TD12µg /AS01 ^ F22µg /AS01 ^ TD12µg + F22µg /AS01 ^ AS01 Sera were collected from individual animals at day 27 (13 Post II) and day 42 (14Post III) to measure the anti-Toxin A Ct, anti-Toxin B Ct and anti-TD1 IgG responses by an Enzyme-Linked ImmunoSorbent Assay (ELISA). In order to assess the functionality of elicited antibodies, anti- Toxin A (r087) and anti-Toxin B (r087, r027 and r078) neutralizing titers were measured in sera collected at day 27 and day 42. The study design is summarized in Fig 6. Results: ^ TD1 is highly immunogenic in mice, similar immunogenicity is observed between the two doses and at the two timepoints (13PII and 14PIII) – see Fig 7. ^ A minor negative interference on TD1 response is observed when TD1 is combined to F2 as compared to TD1 alone (Fig 7), however this interference is not observed on anti-F2 response in terms of anti-ToxA IgG and anti-ToxB IgG (data not shown). Neutralisation data can be seen in Fig 8A (individual values) and Fig 8B (pooled sera) and is summarised as follows: Neutralizing response against homologous r087 ToxB: ^ The TD1 antigen induces higher neutralizing titers than F2 at 14PIII (individual values). ^ The combo TD1-F2 induces a higher neutralization response than each single antigen (TD1 2 µg vs F22 µg alone) at 13 post II (pooled sera). ^ No interference is seen in 14 post-III when TD1 is mixed with F2. Neutralizing response against heterologous r027 ToxB: ^ TD1 antigen induces high neutralizing titers while anti-F2 Abs are ineffective in neutralizing r027 ToxB at 14PIII. ^ The combo TD12 µg - F22 µg induces a higher neutralization response than each single antigen (TD12 µg vs F22 µg alone) at 13 Post II. ^ A two-fold reduction is observed in post-III when TD1 is combined with F2 as compared to TD12 µg alone. Neutralizing response against heterologous r078 ToxB: ^ TD1 induces higher neutralizing titers than F2 ^ The combo TD1-F2 induces a higher neutralization response than each single antigen (TD1 2 µg vs F22 µg alone) at 14 Post III. Neutralizing responses against homologous r087 ToxA: As expected, TD1 does not induce any anti-ToxA neutralizing antibodies. This is not surprising considering that TD1 target only the Tox B antigen unlike F2 antigen which target the C terminus sequences of Toxin A and Toxin B (see Fig 9). However, TD1 does not impact the F2 dependent induction of anti-ToxA neutralizing antibodies when administered in combination. Conclusion The results showed that: ^ TD1 is highly immunogenic in mice at the two doses tested (2 µg and 6 µg). ^ TD1 induces more potent and cross-reactive ToxB neutralizing Abs than F2 antigen. ^ The TD1/F2 antigen combo induces higher neutralizing responses than each single antigen in the 3 Toxin B (r087, r027, r078) neutralization assays. ^ TD1 does not induce any ToxA neutralizing Abs as expected. Example 5: Assessment of the protective effect of F2 +TD1 immunization on gut damage and inflammation in the mouse C difficile (strain 6529(r027)) challenge model Objective: The purpose of this study was to assess the effect of the TD1 and F2 combination (at two doses: 6µg and 2µg) in the protection conferred to toxin-mediated gut damage and inflammation. Study Design The study design is shown schematically in Figure 10. Female 5-week-old C57BL/6JOlaHsd Mouse (5 animals per group) were randomly assigned to the study. Mice from group 1 to 6 were immunized intramuscularly (gastrocnemius m. Left) at day 0, 14 and day 28 with 50µl of the following formulations (see table 2): Table 2: Summary of Groups Group C57BL6 Immunization Challenge with r027 N=10 6529 C. diff strain? (separated into two studies of n=5) G₁ 2µg F2 adjuvanted with AS01 _{Yes G2} 2µg TD1 adjuvanted with AS01 _{Yes G3} 6µg TD1 adjuvanted with AS01 _{Yes G4} 2µg F2 and 2µg TD1 adjuvanted with AS01 _{Yes G5} 2µg F2 and 6µg TD1 adjuvanted with AS01 _{Yes G6} 4.7µg Toxoid B adjuvanted with Al(OH)3 Yes [Toxoid B used in this study was a native toxin B purified from C. difficile and inactivated by formaldehyde (obtained from QUADRATECH DIAGNOSTICS - #QTXAG-203-500)] G7 No Yes G8 No No Mice from group 1 to group 7 were treated with antibiotics at day 36 to day 41 and then were challenged at day 42 with 400µl of 6529 C. difficile strain spores (ribotype 027) at 1.510*6 Spores/400µL via the intragastric route. A negative control was included (G8), who were not immunized and not challenged. Sera were collected from individual animals at day 28 (14PII) and day 36 (8PIII) and were tested for anti-TD1, anti-ToxA Ct, anti-ToxB Ct using ELISA. To assess the functionality of elicited antibodies anti-Toxin B (087-027) neutralizing titers were measured in sera collected at day 36. Feces were collected (pool of 2 mice) at day 42 (14PIII) and then were tested for anti-TD1, anti- ToxA Ct, anti-ToxB Ct using ELISA. All mice were followed in term of weight loss, mortality at day 0 to day2 post challenge. Feces, caecum, colon and sera were collected at day 2 post challenge to measure inflammatory markers by Luminex. The colon was cut into two sections such that histopathological analysis could also be conducted. Results: Protective effect of F2+TD1 in the mouse C. difficile challenge model Weight Loss – Data shown in Fig.11 o TD1 (2 µg) induced a higher protection than previously. o Similar protection levels were observed in the various vaccinated groups Inflammatory markers – Caecum – day 2 post C. difficile challenge – Data shown in Fig 12A, Fig. 12B and Fig.12C o F2 induced a partial protection whatever the tissue and the inflammatory marker o Reduced production of S100A8 (calprotectin), G-CSF and KC in group immunized with F22 µg + TD16 µg (G5) compared to groups immunized with each antigen alone (G1 & G3); clear effect of F22 µg + TD12 µg (G4) as well but only in terms of G-CSF production o Such differences were not seen when looking at S100A9 & IL-6 in caecum and S100A8/A9, G-CSF, KC & IL-6 in colon and feces o Whatever the cytokine/chemokine measured, Toxoid B did not perform better than F2 and was less effective than F2 + TD1 (6µg). Colon Histopathology– day 2 post C. difficile challenge – Data shown in Fig.13A, Fig.13B and Fig. 13C. o F2 induced a partial protection against tissue damage, whatever the parameter o A beneficial effect on goblet cell loss (Fig 13A), inflammatory cells in colon (Fig.13B) and submucosal edema (Fig.35C) was seen in mice immunized with F22 µg + TD16 µg (G5) compared to groups immunized with each antigen alone (G1 & G3). o Such effects were not seen when looking at tissue involvement, mucosal hyperplasia (median score = 0 in non-vaccinated mice) & crypt involvement (score = 0 in all groups except non-vaccinated mice) – data not shown. o Whatever the parameter addressed, Toxoid B did not perform better than F2 and was less effective than F2 + TD1 (6µg) Conclusion A better protection against weight loss was obtained with TD1 (~ F2 and Toxoid B) in this three- immunization schedule compared to previous results obtained in a two-immunization schedule (Example 3); protection levels similar in the various vaccinated groups. F2 induced a partial protection against gut inflammation and damage whilst Toxoid B performed similarly to F2. Beneficial effect of combining F2 and TD1 (at 6 µg) was shown on several inflammatory markers, inflammatory cells, loss of goblet cells and sub-mucosal edema. Example 6: Immunogenicity of C. difficile LNP-F2 mRNA and LNP-TD1 mRNA in C57BL6 mice (20230100) Objectives: - To assess the immunogenicity of LNP-F2 mRNA or LNP-TD1 mRNA at three doses (8µg, 2µg, 0,5µg) compared to each protein (F22µg or TD12µg). - To assess the immunogenicity of the combination of LNP-F2 mRNA and LNP-TD1 mRNA at two doses (2µg and 0,5µg) compared to each LNP mRNA at the same dose. Two endpoints were assessed as follows: - anti-TcdA F2 Ct, anti-TcdB F2 Ct and anti-TD1 IgG response measured by ELISA on sera at 21PI, 21PII and 14PIII. - Neutralizing Ab response anti-TcdA (r087) and anti-TcdB (r087 and r027) evaluated on sera at day 56 (14PIII) in individual sera. Study Design: Female 5-week-old C57BL/6JOlaHsd Mouse (8 animals per group) were randomly assigned to the study. Mice from group 1 to 11 were immunized intramuscularly (gastrocnemius m. Left) at day 0, 21 and day 42 with 50µl of the following formulations: 1. LNP-F2 mRNA 8µg 2. LNP-F2 mRNA 2µg 3. LNP-F2 mRNA 0,5µg 4. LNP-TD1 mRNA 8µg 5. LNP-TD1 mRNA 2µg 6. LNP-TD1 mRNA 0,5µg 7. LNP-F2 mRNA 2µg + LNP-TD1 mRNA 2µg 8. LNP-F2 mRNA 0,5µg + LNP-TD1 mRNA 0,5µg 9. F22µg/AS01 10. TD12µg/AS01 11. F22µg + TD12µg/AS01 An additional group of naive mice was added (mice from group12 are not immunized). Sera was collected from individual animals at day 21 (21PI), day 42 (21PII) and at day 56 (14PIII) Sera from day 42 and day 56 were tested for anti-TD1, anti-TcdA F2 C-ter, anti-TcdB F2 C-ter by ELISA. To assess the functionality of elicited antibodies anti-Toxin B (087-027) neutralizing titers were measured in sera collected at day 56. Results: Anti-TcdA and Anti-TcdB (F2 C-ter) IgG by Elisa - Individual sera day 56 (14PIII) Data is shown in Figure 14 (Anti-TcdA) and Figure 15 (Anti-TcdB). For both anti-TcdA and anti-TcdB the IgG ELISA titers induced by F2 mRNA were comparable to that of 2 µg of F2 protein (at the 2 highest mRNA doses). Comparable titers were also observed for the LNP-F2 / LNP-TD1 combination at both doses tested. Anti -TD1 IgG by ELISA - Individual sera day 42 (14 PIII) Data is shown in Figure 16. TD1 mRNA induced anti-TD1 IgG titers that were comparable to that of 2 µg of TD1 protein at all doses tested. As expected, neither LNP-F2 mRNA nor F2 protein induced anti-TD1 titers at any dose. Neutralizing Ab responses against homologous r087 TcdB - Individual sera at day 56 (14PIII) Data is shown in Figure 17 Anti-F2 and anti-TD1 mAbs were effective in neutralizing r087 ToxB with comparable responses observed between mRNA and protein groups. No interference was observed when F2 and TD1 were combined, again in both mRNA and protein groups. Neutralizing Ab responses against heterologous r027 TcdB - Individual sera at day 56 (14PIII) Data is shown in Figure 18. As observed previously, anti-F2 Abs (induced by both mRNA and protein) were ineffective in neutralizing r027 ToxB. Neutralizing antibody responses to TcdB r027 were comparable in TD1 mRNA and TD1 protein groups. No impact of mixing TD1 & F2 (as mRNA & as proteins) on the neutralizing response to TcdB r027 was observed. Example 7: Immunogenicity of C. difficile LNP-ToxB-GTD mRNA in C57BL6 mice Objective: To assess the immunogenicity of LNP-ToxB-GTD mRNA (coding region of SEQ ID NO: 35) and to compare the immunogenicity to a detoxified recombinant ToxB-GTD protein (SEQ ID NO:50) adjuvanted with AS01. Study Design Female 5-week-old C57BL/6JOlaHsd Mouse (8 animals per group) were randomly assigned to the study. Mice from group 1 to 6 were immunized intramuscularly (gastrocnemius m. Left) at day 0, 21 and day 42 with 50µl of the following formulations: 1. LNP-ToxB-GTD mRNA 8µg 2. LNP-ToxB-GTD mRNA 2µg 3. LNP-ToxB-GTD mRNA 0,5µg 4. ToxB-GTD 6µg/AS01 5. ToxB-GTD 2µg/AS01 6. ToxB-GTD 0.7µg/AS01 An additional group of naive mice was included (group 7) but were not immunized. Sera was collected from individual animals at day 21 (21PI), day 42 (21PII) and at day 56 (14PIII). Sera from day 56 was tested for anti-GTD using an ELISA assay. To assess the functionality of elicited antibodies anti-Toxin B (087-027) neutralizing titers were measured in sera collected at day 42 and day 56. Results: Antibody Titers: As can be seen from Fig.19, ToxB-GTD mRNA is as immunogenic (in terms of anti-GTD IgG titers) as ToxB-GTD protein adjuvanted with AS01 in 14PIII (day 56) sera. Neutralizing antibody response (anti-TcdB): As can be seen from Fig.20 (r087 nAb) and Fig.21 (r027 nAb) higher neutralization titers were observed in groups administered with ToxB-GTD mRNA compared to groups administered ToxB-GTD protein (adjuvanted with AS01). Example 8: Protective efficacy of F2/TD1 (proteins & mRNA) and F2/TD1/GTD (proteins) combos in the mouse Clostridium difficile challenge model Please see Figure 22 for study design details. Briefly, female 5-week-old C57BL/6JOlaHsd Mouse were randomly assigned to the study. Mice from group 1 to 6 were immunized intramuscularly (gastrocnemius m. Left) at day 0, 14 and day 28 with 50µl of the following formulations (see table 3 below, column “Antigen Formulation”): Table 3 Group n Antigen Formulation Antibiotic Treatment Challenge (Yes/No) (Yes/No) 1 8 ^{F22µg / AS01} Y Y 2 8 ^{TD16µg / AS01} Y Y 3 8 ^{GTD 6µg / AS01} Y Y 4 8 ^{F22µg / TD16µg/ AS01} Y Y 5 8 ^{F22µg / TD16µg / GTD} Y Y 6µg/ AS01 6 8 ^{F2 mRNA 2µg / TD1 mRNA} Y Y 2µg 7 8 / Y Y 8 6 / Y N 9 6 / N N *NB: Due to technical limitation this study was conducted in two parallel experiments therefore n=8 refers to n=2x4 and n=6 refers to n=2x3. Following immunization with the antigen formulation, mice from group 1 to group 7 were treated with antibiotics at day 36 to day 41 and then were challenged at day 42 with 400µl of 6529 C. difficile strain spores (ribotype 027) at 1.5x106 Spores/mouse via intragastric route. Mice from group 8 only received the antibiotic treatment and were not challenged. A negative control was also included (G9), these mice were not immunized, not treated with antibiotics and not challenged. Sera were collected from individual animals at day 36 (8 days post third dose (8PIII)) for measurement of anti-TD1, anti-TcdA F2 Cter, anti-TcdB F2 Cter and anti-GTD antibodies by ELISA. To assess the functionality of elicited antibodies anti-TcdA (r087) and anti-TcdB (r087-r027) neutralizing titers were also measured. All mice were monitored in terms of weight loss and mortality from day 0 to day2 post C. difficile challenge. Furthermore, two days post-challenge a number of tissues were collected as follows: - Caecum and colon were collected to measure inflammatory markers by Luminex. Results: Weight Loss: A mean profile of weight change from baseline (expressed in %) at day 2 post 6529 C. difficile strain spore challenge was calculated for each group. The means and their 95% of confidence interval are shown in Figure 23. In this study, a trend was observed towards higher protection with the combinations (i.e. F2+TD1 protein, F2+TD1 mRNA and F2+TD1+GTD protein) compared to single proteins. Calprotectin (S100A8) level in caecum and colon at day 2 post C.difficile challenge: Individual S100A8 levels (pg/mg of protein) were measured in the caecum (Fig.24) and colon (Fig.25) via Luminex (dot in the graphs). The GMT of a group is represented as a horizontal line and the CI as the vertical interval around. - Protein data: As can be observed from the data, single proteins induced similar levels of protection. All were partially protective compared to non-immunized challenged mice. There was a trend for higher protection with the combinations F2+TD1 and more notably, F2+TD1+GTD. - mRNA data: F2/TD1 mRNA significantly more protective than F2/TD1/AS01 in both caecum and colon. Example 9: Assessment of the protective effect of mRNA combos (F2&TD1 mRNA or F2&TD1&GTD mRNA) compared to protein combos (F2&TD1 or F2&TD1&GTD) on gut damage and inflammation in the mouse C. difficile challenge model. The objective of this study was to assess the protective effect of mRNA combos (F2&TD1 mRNA or F2&TD1&GTD mRNA) at two doses (2µg and 0,5µg) in comparison to corresponding protein combos (F2&TD1 or F2&TD1&GTD), against gut inflammation and damage in the mouse C. difficile challenge model. A secondary objective of the study was to assess the added value of GTD mRNA when combined to F2&TD1 mRNA at the two doses (2µg and 0,5µg). Please see Figure 26 for study design details. Female 5-week-old C57BL/6JOlaHsd mice were randomly assigned to the study. Mice from group (G) 1 to 6 were immunized intramuscularly (gastrocnemian m. Left) at day 0, 14 and day 28 with 50µl of the following formulations: ^ G1: 2µg F2 + 6µg TD1 adjuvanted with AS01 ^ G2: 2µg F2 mRNA + 2µg TD1 mRNA ^ G3: 0.5µg F2 mRNA + 0.5µg TD1 mRNA ^ G4: 2µg F2 + 6µg TD1 + 6µg GTD adjuvanted with AS01 ^ G5: F2 mRNA 2µg / TD1 mRNA 2µg / GTD mRNA 2µg ^ G6: F2 mRNA 0.5µg / TD1 mRNA 0.5µg / GTD mRNA 0.5µg Mice from group 1 to group 7 were treated with antibiotics at day 36 to day 41 and then were challenged at day 42 with 400µl of 6529 C. difficile strain spores (ribotype 027) at 1.5 x 106 Spores/mouse via intragastric route. Mice from group 8 only received the antibiotic treatment at day 36 to day 41 and were not challenged. A summary of the study design is provided in Table 4 below. Table 4: Group N Vaccine Adjuvant antibiotic challenge treatment G1 10 ^{F22µg + TD16µg} AS01 Yes Yes G2 10 ^{F2 mRNA 2µg / TD1 mRNA 2µg} Yes Yes G3 10 ^{F2 mRNA 0.5µg / TD1 mRNA 0.5µg} Yes Yes G4 10 ^{F22µg / TD16µg / GTD 6µg} AS01 Yes Yes G5 10 ^{F2 mRNA 2µg / TD1 mRNA 2µg / GTD mRNA} Yes Yes 2µg G6 10 ^{F2 mRNA 0.5µg / TD1 mRNA 0.5µg / GTD} Yes Yes mRNA 0.5µg G7 (+ CTR) 10 / Yes Yes G8 (- CTR) 6 / Yes / *NB: Due to technical limitation this study was conducted in two parallel experiments therefore n=10 refers to n=2x5 and n=6 refers to n=2x3. Sera was collected from individual animals at day 36 (8PIII). All mice were followed in terms of weight loss and mortality from day 0 to day2 post C. difficile challenge. Caecum, colon and sera were collected at day 2 post challenge to measure inflammatory markers by Luminex. Results: - A mean profile of weight change from baseline (expressed in %) at day 2 post 6529 C. difficile strain spore challenge was calculated for each group. All the immunized groups induced protection against weight loss after C.difficile challenge (see Fig.27). - Calprotectin (S100A8) levels in caecum and colon was measured at day 2 post C.difficile challenge (see Figure 28 (caecum) and Figure 29 (colon)) with data showing strong protection across all groups, particularly with the higher dose (i.e.2 µg) F2/TD1/GTD mRNA in the colon. SEQUENCE LISTING Uracil (“u”) residues in the below RNA sequences are denoted as (“t”) in the corresponding SEQ LISTING (ST.26). Any spacing is to be ignored (sequence to be read as one continuous sequence). 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 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 2 MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEG QAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGIL QNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYY FDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTN TFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKK YYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFN TDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGN RYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFL HLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLNQIGDYKYYFNSDGVMQKGFVSIND NKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGIL NFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTIND KVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLV RVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISF ENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTG WLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO:4 – sequence of Fusion 5 MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEG QAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGIL QNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYY FDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTN TFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKK YYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFN TDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGN RYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFL HLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGGGF VSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEIS YSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGF VTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVE YSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRT GLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGES INYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO:5 – C-TAB.G5 fusion protein MVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYF NLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTD GIMQIGVFKGPNGFEYFAPANTHNNNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKY YFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAP ANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNK NFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQT INGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGRSMHNLITGFVTVGDDKYYFNPIN GGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIY YFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDS GVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYY FDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDS GIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYY FGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFN ENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKR YYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO:6 C-TAB.G5.1 fusion protein VTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFN LNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDG IMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYY FNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPA NTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKN FYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTI NGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYGRSMHNLITGFVTVGDDKYYFNPING GAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYY FDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSG VMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYF DDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSG IIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYF GETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNE NGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRY YFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO:7 – sequence of toxin A fragment from fusion 2 MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEG QAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGIL QNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYY FDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTN TFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKK YYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFN TDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGN RYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFL HLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAG SEQ ID NO:8 – sequence of toxin A fragment from fusion 5 MGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEG QAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGIL QNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYY FDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTN TFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKK YYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFN TDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGN RYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFL HLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYG SEQ ID NO: 9 seq of toxin a from CTAB G5 MVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYF NLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTD GIMQIGVFKGPNGFEYFAPANTHNNNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKY YFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAP ANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNK NFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQT INGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYG SEQ ID NO: 10 seq of toxin a from CTAB G5 VTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFN LNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDG IMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYY FNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPA NTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKN FYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTI NGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYG SEQ ID NO:11 – sequence of toxin B fragment from fusion 2 GLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTE DGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYI GLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTS DGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKA CKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFN TPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLV ISE SEQ ID NO:12– sequence of toxin B fragment from fusion 5 GFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEE ISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQV GFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQA VEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIM RTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEG ESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 13 sequence of toxin B from CTAB G5 NLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDE NLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSD GVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGN EEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDD GIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDN IYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFD EKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLD ENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 14 sequence of toxin B from CTAB G5.1 NLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDE NLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSD GVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGN EEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDD GIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDN IYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFD EKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLD ENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 15 Linker RSMH SEQ ID NO: 16 – TD1 (M68) LTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKH VSLVETEGVFTLLDDKVMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHT VTDDIDHFFSAPSITYREPHLSIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWE TGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRYEDTNI RINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVW IIDVDNVVRDVTIESDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFV SLTFSILEGINAIIEVDLLSKSYKLLISGELKILMLNSNHIQQKIDYIG SEQ ID NO: 17 TD1 (VPI10463) LTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKH VSLVETEGVFTLLDDKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHT VTDDIDHFFSAPSITYREPHLSIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWE TGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRYEDTNI RINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVW IIDVDNVVRDVTIESDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFV SLTFSILEGINAIIEVDLLSKSYKLLISGELKILMLNSNHIQQKIDYIG SEQ ID NO:18 – TcdB of C. difficile M68 strain MSLVNRKQLEKMANVRFRVQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDTYIDTYKKS GRNKALKKFKEYLVIEILELKNSNLTPVEKNLHFIWIGGQINDTAINYINQWKDVNSDYNVNVFYD SNAFLINTLKKTIIESASNDTLESFRENLNDPEFNHTAFFRKRMQIIYDKQQNFINYYKAQKEENP DLIIDDIVKTYLSNEYSKDIDELNAYIEESLNKVTENSGNDVRNFEEFKTGEVFNLYEQELVERNL AGASDILRVILKNIGGVYLDVDMLPGIHPDLFKDINKPDSVKTAVDWEEMQLEAIMKHKEYIPEYT SKHFDTLDEEVQSSFESVLASKSDKSEIFLPLGDIEVSPLEVKIAFAKGSIINQALISAKDSYCSD LLIKQIQNRYKILNDTLGPIISQGNDFNTTMNNFGESLGAIANEENISFIAKIGSYLRVGFYPEAN TTITLSGPTIYAGAYKDLLTFKEMSIDTSILSSELRNFEFPKVNISQATEQEKNSLWQFNEERAKI QFEEYKKNYFEGALGEDDNLDFSQNTVTDKEYLLEKISSSTKSSERGYVHYIVQLQGDKISYEAAC NLFAKNPYDSILFQKNIEDSEVAYYYNPTDSEIQEIDKYRIPDRISDRPKIKLTFIGHGKAEFNTD IFAGLDVDSLSSEIETAIGLAKEDISPKSIEINLLGCNMFSYSVNVEETYPGKLLLRVKDKVSELM PSMSQDSIIVSANQYEVRINSEGRRELLDHSGEWINKEESIIKDISSKEYISFNPKENKIIVKSKN LPELSTLLQEIRNNSNSSDIELEEKVMLAECEINVISNIETQVVEERIEEAKSLTSDSINYIKNEF KLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRFINKETGESIFVETEKTIFSEYANHI TEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLNAAFFIQSLIEYNSSKESLSNLSVAMKVQVYA QLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLR QEIEAKIGIMAVNLTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDY FKHVSLVETEGVFTLLDDKVMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFS APSITYREPHLSIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIR DNYEGEFYWRYFAFIADALITTLKPRYEDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSGG TYALSLSQYNMGINIELSESDVWIIDVDNVVRDVTIESDKIKKGDLIEGILSTLSIEENKIILNSH EINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLISGELKILMLNSNHIQQKIDYIGFN SELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMDDSKPSFGYYSNNLKDVKVI TKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNRKGSTNTSDSLMSFL ESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENNNIQPYFIKFNTLETNYTLYVGN RQNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEV IVLDANYINEKINVNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFN FSDKQDVPVSEIILSFTPSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPV NNLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLD ENLEGEAIDFTGKLIIDENIYYFEDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDDKYYFNS DGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLG NEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFND DGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVND NIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFDPETKKACKGINLIDDIKYYF DEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTL DENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE SEQ ID NO: 19: RNA sequence of 5’ UTR of “UTR4” (HIST2H4A 5’ UTR) AGGAGAAGCU GUCUAUCGGG CUCCAGCGGU C SEQ ID NO: 20: RNA sequence of 3’ UTR of “UTR4” (HIST2H4A 3’ UTR) GCCGCCGCUC CAGCUUUGCA CGUUUCGAUC CCAAAGGCCC UUUUUAGGGC CGACCA SEQ ID NO: 21: RNA sequence of 5’ UTR of “UTR3” (FAP 5’ UTR) AGAACGCCCC CAAAAUCUGU UUCUAAUUUU ACAGAAAUCU UUUGAAACUU GGCACGGUAU UCAAAAGUCC GUGGAAAGAA AAAAACCUUG UCCUGGCUUC AGCUUCCAAC UACAAAGACA GACUUGGUCC UUUUCAACGG UUUUCACAGA UCCAGUGACC CACGCUCUGA AGACAGAAUU AGCUAACUUU CAAAAACAUC UGGAAAA SEQ ID NO: 22: RNA sequence of 3’ UTR of “UTR3” (FAP 3’ UTR) AAACGAUGCA GAUGCAAGCC UGUAUCAGAA UCUGAAAACC UUAUAUAAAC CCCUCAGACA GUUUGCUUAU UUUAUUUUUU AUGUUGUAAA AUGCUAGUAU AAACAAACAA AUUAAUGUUG UUCUAAAGGC UGUUAAAAAA AAGAUGAGGA CUCAGAAGUU CAAGCUAAAU AUUGUUUACA UUUUCUGGUA CUCUGUGAAA GAAGAGAAAA GGGAGUCAUG CAUUUUGCUU UGGACACAGU GUUUUAUCAC CUGUUCAUUU GAAGAAAAAU AAUAAAGUCA GAAG SEQ ID NO: 23: RNA sequence of 5’ UTR of “UTR7” (IL-25’ UTR) GUUCCCUAUC ACUCUCUUUA AUCACUACUC ACAGUAACCU CAACUCCUGC CACA SEQ ID NO: 24: RNA sequence of 3’ UTR of “UTR7” (IL-23’ UTR) UUAAGUGCUU CCCACUUAAA ACAUAUCAGG CCUUCUAUUU AUUUAAAUAU UUAAAUUUUA UAUUUAUUGU UGAAUGUAUG GUUUGCUACC UAUUGUAACU AUUAUUCUUA AUCUUAAAAC UAUAAAUAUG GAUCUUUUAU GAUUCUUUUU GUAAGCCCUA GGGGCUCUAA AAUGGUUUCA CUUAUUUAUC CCAAAAU SEQ ID NO: 25: RNA sequence of possible 5’ UTR AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC SEQ ID NO: 26: RNA sequence of possible 3’ UTR GCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG CACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC SEQ ID NO: 27: RNA sequence of possible 5’ UTR GAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC SEQ ID NO: 28: RNA sequence of possible 3’ UTR CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUC CCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCC AGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAA CAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUC AAUUUCGUGCCAGCCACACCCUGGAGCUAGC SEQ ID NO: 29 RNA sequence (F2_no signal peptide_no His tag) GGCUGGCAGACAAUCGACGGCAAGAAGUACUACUUCAACACCAACACCGCCAUUGCCAGCACCGGC UACACCAUCAUCAACGGCAAGCACUUCUACUUUAAUACCGACGGCAUCAUGCAGAUUGGCGUGUUC AAGGGCCCCAACGGCUUCGAGUAUUUCGCCCCUGCCAACACCGACGCCAACAAUAUCGAAGGCCAG GCCAUCCUGUACCAGAACGAGUUUCUGACCCUGAACGGGAAGAAGUAUUAUUUCGGCAGCGACAGC AAGGCCGUGACCGGCUGGCGGAUUAUCAACAACAAGAAAUAUUACUUUAACCCGAACAACGCUAUC GCCGCCAUCCACCUGUGCACCAUCAACAAUGACAAGUACUAUUUCAGCUACGAUGGCAUCCUGCAG AACGGCUACAUCACCAUCGAGCGGAACAAUUUCUACUUCGAUGCCAACAACGAGAGCAAGAUGGUC ACCGGGGUGUUCAAAGGACCUAAUGGCUUUGAGUACUUCGCUCCCGCUAAUACCCACAACAACAAC AUCGAGGGACAAGCCAUCGUCUAUCAGAACAAGUUCCUGACGCUCAAUGGCAAAAAGUAUUACUUU GACAACGACUCCAAGGCUGUCACCGGAUGGCAGACCAUUGAUGGGAAAAAGUACUACUUUAACCUG AACACCGCCGAGGCCGCCACAGGCUGGCAAACUAUUGAUGGAAAGAAGUACUAUUUCAAUCUCAAU ACGGCCGAAGCUGCUACUGGAUGGCAAACGAUAGACGGAAAGAAGUAUUACUUUAAUACGAACACC UUUAUCGCCUCCACCGGGUACACCUCCAUUAACGGGAAACACUUUUAUUUCAACACGGACGGGAUU AUGCAAAUCGGGGUUUUCAAGGGGCCGAAUGGAUUCGAAUACUUCGCACCAGCCAAUACGGAUGCU AACAACAUUGAAGGACAGGCUAUUCUCUACCAAAACAAAUUCCUCACACUGAACGGCAAAAAGUAC UACUUUGGCUCCGAUAGCAAGGCUGUUACAGGCCUGAGAACUAUCGACGGAAAAAAGUAUUAUUUC AACACAAAUACCGCCGUGGCAGUGACAGGCUGGCAGACGAUUAACGGGAAGAAAUAUUACUUCAAU ACCAAUACCAGCAUAGCCUCCACGGGCUAUACAAUCAUCUCCGGCAAACAUUUUUACUUUAACACA GAUGGUAUAAUGCAAAUUGGAGUCUUUAAGGGACCUGACGGUUUUGAAUACUUUGCCCCAGCUAAC ACAGACGCAAACAACAUAGAAGGGCAAGCAAUCAGAUAUCAGAAUCGGUUCCUGUACCUGCACGAC AACAUCUACUACUUCGGCAACAACUCCAAAGCCGCUACCGGCUGGGUCACCAUUGACGGCAAUCGG UAUUACUUCGAGCCCAAUACCGCCAUGGGCGCCAACGGAUACAAGACCAUCGAUAACAAGAACUUU UACUUCCGGAACGGGCUGCCCCAAAUCGGAGUGUUUAAAGGCAGCAACGGAUUUGAGUAUUUUGCU CCGGCCAACACUGAUGCAAACAAUAUUGAGGGUCAAGCUAUACGGUAUCAGAACCGCUUCCUGCAU CUGCUGGGCAAAAUCUACUAUUUUGGGAACAACAGUAAAGCCGUCACUGGCUGGCAGACUAUCAAU GGCAAAGUCUACUACUUCAUGCCCGACACCGCUAUGGCUGCCGCUGGCGGACUUAACCAGAUUGGA GACUACAAGUAUUACUUCAACUCCGACGGCGUGAUGCAGAAAGGCUUCGUGUCCAUCAACGACAAC AAGCACUAUUUUGACGACAGCGGCGUCAUGAAGGUCGGAUACACCGAGAUUGACGGAAAACAUUUC UAUUUCGCCGAGAACGGGGAGAUGCAAAUCGGCGUGUUCAACACCGAGGACGGCUUCAAGUACUUU GCUCACCACAACGAGGACCUGGGCAACGAGGAAGGCGAGGAAAUCAGCUACUCCGGCAUCCUGAAC UUUAACAACAAAAUCUAUUACUUCGACGACAGCUUCACCGCCGUCGUCGGAUGGAAGGACCUGGAA GAUGGCUCUAAGUACUAUUUUGAUGAGGACACAGCCGAGGCCUACAUCGGCCUGUCUCUGAUUAAC GACGGCCAGUACUACUUCAAUGACGAUGGCAUCAUGCAAGUGGGCUUCGUCACCAUCAAUGAUAAG GUGUUCUACUUCAGCGAUAGCGGCAUCAUCGAGAGCGGCGUGCAGAACAUCGACGACAACUACUUC UACAUCGAUGAUAACGGCAUCGUCCAGAUAGGGGUUUUCGACACCUCCGACGGGUACAAAUAUUUC GCACCCGCAAACACAGUGAACGAUAACAUCUAUGGCCAGGCCGUGGAAUACUCCGGCCUCGUUAGA GUGGGCGAAGAUGUUUACUAUUUCGGGGAGACUUACACCAUCGAAACCGGCUGGAUAUACGACAUG GAAAACGAGAGCGACAAGUACUACUUCAAUCCCGAGACAAAGAAGGCCUGCAAGGGCAUCAACCUG AUUGACGACAUUAAGUACUACUUCGACGAGAAGGGCAUCAUGCGGACCGGCCUGAUUAGCUUCGAG AACAACAACUACUAUUUCAACGAGAAUGGCGAGAUGCAGUUCGGCUAUAUCAAUAUCGAGGACAAG AUGUUUUACUUCGGCGAGGAUGGCGUUAUGCAGAUAGGGGUGUUCAAUACCCCUGAUGGGUUCAAG UAUUUCGCUCAUCAGAACACCCUGGACGAGAACUUCGAGGGCGAGAGCAUCAAUUACACCGGCUGG CUGGACCUGGAUGAGAAGCGCUACUACUUUACCGACGAGUACAUUGCCGCCACUGGCUCCGUGAUU AUCGACGGCGAGGAAUAUUACUUCGACCCCGACACAGCCCAGCUGGUCAUCUCUGAA SEQ ID NO: 30: RNA Sequence (TD1_no signal peptide_no linker_no His tag) CUAACCACCGCCACCACCGCCAUAAUCACAAGCAGCCUGGGCAUCGCUAGCGGCUUCAGCAUCCUG CUGGUGCCCCUGGCCGGCAUCAGCGCCGGCAUCCCUAGCCUGGUGAACAACGAGCUGGUGCUGAGA GACAAGGCCACCAAGGUGGUGGACUACUUCAAGCACGUGAGCCUGGUGGAGACCGAGGGCGUGUUC ACCCUGCUGGACGACAAGGUGAUGAUGCCCCAAGACGACCUGGUGAUCAGCGAGAUCGACUUCAAC AACAACAGCAUCGUGCUGGGCAAGUGCGAGAUCUGGAGAAUGGAGGGCGGCAGCGGCCACACCGUG ACCGACGACAUCGACCACUUCUUCAGCGCCCCUAGCAUCACCUACAGAGAGCCCCACCUGAGCAUC UACGACGUGCUGGAGGUGCAGAAGGAGGAGCUGGACCUGAGCAAGGACCUGAUGGUGCUGCCCAAC GCCCCCAACAGAGUGUUCGCCUGGGAGACCGGCUGGACCCCCGGCCUGAGAAGCCUGGAGAACGAC GGCACCAAGCUGCUGGACAGAAUCAGAGACAACUACGAGGGCGAGUUCUACUGGAGAUACUUCGCC UUCAUCGCCGACGCCCUGAUCACCACCCUGAAGCCUAGAUACGAGGACACCAACAUCAGAAUCAAC CUGGACAGCAACACACGGAGCUUCAUCGUGCCCAUCAUCACCACCGAGUACAUCAGAGAGAAGCUG AGCUACAGCUUCUACGGCAGCGGCGGCACCUACGCCCUGAGCCUGUCUCAGUACAACAUGGGCAUC AACAUCGAGCUGAGCGAGAGCGACGUGUGGAUCAUCGACGUGGACAACGUGGUGAGAGACGUGACC AUCGAGAGCGACAAGAUCAAGAAGGGCGACCUGAUCGAGGGCAUCCUGAGCACCCUGAGCAUCGAG GAGAACAAGAUCAUCCUGAACAGCCACGAGAUCAACUUCAGCGGCGAGGUGAACGGCAGCAACGGC UUCGUGAGCCUGACCUUUAGCAUUCUGGAGGGUAUCAACGCCAUCAUCGAGGUGGACCUGCUGAGC AAGAGCUACAAGCUGCUGAUCAGCGGCGAGCUGAAGAUCCUGAUGCUGAACAGCAACCACAUUCAG CAGAAGAUCGACUACAUGGC SEQ ID NO: 31: RNA (6-His tag) CAUCACCAUCACCAUCAC SEQ ID NO: 32: RNA (ALB signal sequence) AUGAAGUGGGUCACCUUCAUCAGCCUGCUGUUUCUGUUCAGCAGCGCCUACAGC SEQ ID NO: 33: RNA (hIgG signal sequence) AUGGACUGGACCUGGCGGGUGUUUUGUCUGCUGGCCGUGACACCGGGCGCCCACCCC SEQ ID NO: 34: RNA (Luc signal sequence) AUGGGCGUGAAGGUGCUGUUCGCCCUGAUCUGCAUCGCCGUAGCCGAAGCU SEQ ID NO: 35: RNA Sequence (ToxB-GTD_no signal peptide_no linker_no His tag) AGCCUGGUGAACAGAAAGCAGCUGGAGAAGAUGGCCAACGUGAGAUUCAGAACCCAAGAGGACGAG UACGUGGCCAUCCUGGACGCCCUGGAGGAGUACCACAACAUGAGCGAGAACACGGUUGUGGAGAAG UAUCUGAAGCUGAAGGACAUCAACAGCCUGACCGACAUCUACAUCGACACCUACAAAAAGAGCGGC AGAAACAAGGCCCUGAAGAAGUUCAAGGAGUACCUGGUGACCGAGGUGCUGGAGCUGAAGAACAAC AACCUGACCCCCGUGGAGAAGAACCUGCACUUCGUGUGGAUCGGCGGGCAGAUUAACGAUACAGCC AUCAACUACAUCAAUCAGUGGAAGGACGUGAACAGCGACUACAACGUGAACGUGUUCUACGACAGC AACGCCUUCCUGAUCAACACCCUGAAGAAGACAGUGGUGGAGUCCGCCAUCAACGAUACGCUGGAG AGCUUCAGAGAGAACCUGAACGACCCUAGAUUCGACUACAACAAGUUCUUCAGAAAGAGAAUGGAG AUCAUCUACGACAAGCAGAAGAACUUCAUCAACUACUACAAGGCUCAGAGAGAGGAGAACCCCGAG CUGAUCAUCGACGACAUCGUGAAGACCUACCUGAGCAACGAGUACAGCAAGGAGAUCGACGAGCUG AACACCUACAUCGAGGAGAGCCUGAACAAGAUCACACAGAACAGCGGCAACGACGUACGCAACUUC GAGGAAUUUAAGAACGGCGAGAGCUUCAACCUGUACGAGCAAGAGCUGGUGGAGAGAUGGAACCUG GCCGCCGCUAGCGCCAUCCUGGCCAUCAGCGCCCUGAAGGAGAUCGGCGGCAUGGCCCUGGCCGUG GCCAUGCUGCCCGGCAUUCAGCCCGACCUGUUCGAGAGCAUCGAGAAGCCUAGCAGCGUGACCGUG GACUUCUGGGAGAUGACCAAGCUGGAGGCCAUCAUGAAGUACAAGGAGUACAUCCCCGAGUACACA AGCGAGCACUUCGACAUGCUGGACGAAGAGGUGCAGAGCAGCUUCGAAUCGGUGCUGGCUAGCAAG AGCGACAAGAGCGAGAUCUUCAGCAGUCUGGGUGAUAUGGAGGCUAGCCCCCUGGAGGUGAAGAUC GCCUUCAACAGCAAGGGCAUCAUCAACCAAGGCCUGAUCAGCGUGAAGGACAGCUACUGCAGCAAC CUGAUCGUGAAGCAGAUCGAGAACAGAUACAAGAUCCUGAACAACAGCCUGAACCCCGCCAUCAGC GAGGACAACGACUUCAACACCACCACCAACACCUUCAUCGACAGCAUCAUGGCCGAGGCCAACGCC GACAACGGCAGAUUCAUGAUGGAGCUGGGCAAGUACCUGAGAGUGGGCUUCUUCCCCGACGUGAAG ACCACCAUCAACCUGAGCGGCCCCGAGGCCUACGCCGCCGCCUACCAAGACCUGCUGAUGUUCAAG GAGGGCAGCAUGAACAUCCACCUGAUCGAGGCCGACCUGAGAAACUUCGAGAUCAGUAAGACCAAC AUCUCUCAGAGCACCGAGCAAGAGAUGGCUAGCCUGUGGUCCUUCGAUGAUGCUAGAGCCAAGGCU CAGUUCGAGGAGUACAAGAGAAACUACUUCGAGGGCAGCCUG SEQ ID NO:36 – signal peptide CD33mod MAPLLLLLPLLWAGALA SEQ ID NO:37 – signal peptide tPA MKRGLCCVLLLCGAVFVSPS SEQ ID NO:38 – signal peptide tPA (P/A) MKRGLCCVLLLCGAVFVSAS SEQ ID NO:39 – signal peptide HA MNTQILVFALIAIIPTNADKI SEQ ID NO:40 – signal peptide Spike MFVFLVLLPLVSS SEQ ID NO:41 – signal peptide IL2 MYRMQLLSCIALSLALVTNS SEQ ID NO:42 – signal peptide IFNA2 MALTFALLVALLVLSCKSSCSVG SEQ ID NO:43 – signal peptide Luc MGVKVLFALICIAVAEA SEQ ID NO:44 – signal peptide ALB MKWVTFISLLFLFSSAYS SEQ ID NO:45 – signal peptide hIgG MDWTWRVFCLLAVTPGAHP SEQ ID NO:46 – signal peptide ALBmod MKWVTFISLLFLFSSSSRA SEQ ID NO:47 – signal peptide Tryp2 MNLLLILTFVAAAVA SEQ ID NO: 48 – ToxB-GTD protein MSLVNRKQLEKMANVRFRTQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYIDTYKKS GRNKALKKFKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKDVNSDYNVNVFYD SNAFLINTLKKTVVESAINDTLESFRENLNDPRFDYNKFFRKRMEIIYDKQKNFINYYKAQREENP ELIIDDIVKTYLSNEYSKEIDELNTYIEESLNKITQNSGNDVRNFEEFKNGESFNLYEQELVERWN LAAASDILRISALKEIGGMYLDVDMLPGIQPDLFESIEKPSSVTVDFWEMTKLEAIMKYKEYIPEY TSEHFDMLDEEVQSSFESVLASKSDKSEIFSSLGDMEASPLEVKIAFNSKGIINQGLISVKDSYCS NLIVKQIENRYKILNNSLNPAISEDNDFNTTTNTFIDSIMAEANADNGRFMMELGKYLRVGFFPDV KTTINLSGPEAYAAAYQDLLMFKEGSMNIHLIEADLRNFEISKTNISQSTEQEMASLWSFDDARAK AQFEEYKRNYFEGSL SEQ ID NO: 49 – ToxB-GTD nucleotide ATGAGTTTAGTTAATAGAAAACAGTTAGAAAAAATGGCAAATGTAAGATTTCGTACTCAAGAAGAT GAATATGTTGCAATATTGGATGCTTTAGAAGAATATCATAATATGTCAGAGAATACTGTAGTCGAA AAATATTTAAAATTAAAAGATATAAATAGTTTAACAGATATTTATATAGATACATATAAAAAATCT GGTAGAAATAAAGCCTTAAAAAAATTTAAGGAATATCTAGTTACAGAAGTATTAGAGCTAAAGAAT AATAATTTAACTCCAGTTGAGAAAAATTTACATTTTGTTTGGATTGGAGGTCAAATAAATGACACT GCTATTAATTATATAAATCAATGGAAAGATGTAAATAGTGATTATAATGTTAATGTTTTTTATGAT AGTAATGCATTTTTGATAAACACATTGAAAAAAACTGTAGTAGAATCAGCAATAAATGATACACTT GAATCATTTAGAGAAAACTTAAATGACCCTAGATTTGACTATAATAAATTCTTCAGAAAACGTATG GAAATAATTTATGATAAACAGAAAAATTTCATAAACTACTATAAAGCTCAAAGAGAAGAAAATCCT GAACTTATAATTGATGATATTGTAAAGACATATCTTTCAAATGAGTATTCAAAGGAGATAGATGAA CTTAATACCTATATTGAAGAATCCTTAAATAAAATTACACAGAATAGTGGAAATGATGTTAGAAAC TTTGAAGAATTTAAAAATGGAGAGTCATTCAACTTATATGAACAAGAGTTGGTAGAAAGGTGGAAT TTAGCTGCTGCTTCTGACATATTAAGAATATCTGCATTAAAAGAAATTGGTGGTATGTATTTAGAT GTTGATATGTTACCAGGAATACAACCAGACTTATTTGAGTCTATAGAGAAACCTAGTTCAGTAACA GTGGATTTTTGGGAAATGACAAAGTTAGAAGCTATAATGAAATACAAAGAATATATACCAGAATAT ACCTCAGAACATTTTGACATGTTAGACGAAGAAGTTCAAAGTAGTTTTGAATCTGTTCTAGCTTCT AAGTCAGATAAATCAGAAATATTCTCATCACTTGGTGATATGGAGGCATCACCACTAGAAGTTAAA ATTGCATTTAATAGTAAGGGTATTATAAATCAAGGGCTAATTTCTGTGAAAGACTCATATTGTAGC AATTTAATAGTAAAACAAATCGAGAATAGATATAAAATATTGAATAATAGTTTAAATCCAGCTATT AGCGAGGATAATGATTTTAATACTACAACGAATACCTTTATTGATAGTATAATGGCTGAAGCTAAT GCAGATAATGGTAGATTTATGATGGAACTAGGAAAGTATTTAAGAGTTGGTTTCTTCCCAGATGTT AAAACTACTATTAACTTAAGTGGCCCTGAAGCATATGCGGCAGCTTATCAAGATTTATTAATGTTT AAAGAAGGCAGTATGAATATCCATTTGATAGAAGCTGATTTAAGAAACTTTGAAATCTCTAAAACT AATATTTCTCAATCAACTGAACAAGAAATGGCTAGCTTATGGTCATTTGACGATGCAAGAGCTAAA GCTCAATTTGAAGAATATAAAAGGAATTATTTTGAAGGTTCTCTT SEQ ID NO: 50 – ToxB-GTD detox MSLVNRKQLEKMANVRFRTQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYIDTYKKS GRNKALKKFKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKDVNSDYNVNVFYD SNAFLINTLKKTVVESAINDTLESFRENLNDPRFDYNKFFRKRMEIIYDKQKNFINYYKAQREENP ELIIDDIVKTYLSNEYSKEIDELNTYIEESLNKITQNSGNDVRNFEEFKNGESFNLYEQELVERWN LAAASAILAISALKEIGGMALAVAMLPGIQPDLFESIEKPSSVTVDFWEMTKLEAIMKYKEYIPEY TSEHFDMLDEEVQSSFESVLASKSDKSEIFSSLGDMEASPLEVKIAFNSKGIINQGLISVKDSYCS NLIVKQIENRYKILNNSLNPAISEDNDFNTTTNTFIDSIMAEANADNGRFMMELGKYLRVGFFPDV KTTINLSGPEAYAAAYQDLLMFKEGSMNIHLIEADLRNFEISKTNISQSTEQEMASLWSFDDARAK AQFEEYKRNYFEGSL SEQ ID NO: 51 – ToxA-GTD MSLISKEELIKLAYSIRPRENEYKTILTNLDEYNKLTTNNNENKYLQLKKLNESIDVFMNKYKTSS RNRALSNLKKDILKEVILIKNSNTSPVEKNLHFVWIGGEVSDIALEYIKQWADINAEYNIKLWYDS EAFLVNTLKKAIVESSTTEALQLLEEEIQNPQFDNMKFYKKRMEFIYDRQKRFINYYKSQINKPTV PTIDDIIKSHLVSEYNRDETVLESYRTNSLRKINSNHGIDIRANSLFTEQELLNIYSQELLNRGNL AAASDIVRLLALKNFGGVYLDVDMLPGIHSDLFKTISRPSSIGLDRWEMIKLEAIMKYKKYINNYT SENFDKLDQQLKDNFKLIIESKSEKSEIFSKLENLNVSDLEIKIAFALGSVINQALISKQGSYLTN LVIEQVKNRYQFLNQHLNPAIESDNNFTDTTKIFHDSLFNSATAENSMFLTKIAPYLQVGFMPEAR STISLSGPGAYASAYYDFINLQENTIEKTLKASDLIEFKFPENNLSQLTEQEINSLWSFDQASAKY QFEKYVRDYTGGS SEQ ID NO: 52 – ToxA-GTD nucleotide ATGTCTTTAATATCTAAAGAAGAGTTAATAAAACTCGCATATAGCATTAGACCAAGAGAAAATGAG TATAAAACTATACTAACTAATTTAGACGAATATAATAAGTTAACTACAAACAATAATGAAAATAAA TATTTACAATTAAAAAAACTAAATGAATCAATTGATGTTTTTATGAATAAATATAAAACTTCAAGC AGAAATAGAGCACTCTCTAATCTAAAAAAAGATATATTAAAAGAAGTAATTCTTATTAAAAATTCC AATACAAGCCCTGTAGAAAAAAATTTACATTTTGTATGGATAGGTGGAGAAGTCAGTGATATTGCT CTTGAATACATAAAACAATGGGCTGATATTAATGCAGAATATAATATTAAACTGTGGTATGATAGT GAAGCATTCTTAGTAAATACACTAAAAAAGGCTATAGTTGAATCTTCTACCACTGAAGCATTACAG CTACTAGAGGAAGAGATTCAAAATCCTCAATTTGATAATATGAAATTTTACAAAAAAAGGATGGAA TTTATATATGATAGACAAAAAAGGTTTATAAATTATTATAAATCTCAAATCAATAAACCTACAGTA CCTACAATAGATGATATTATAAAGTCTCATCTAGTATCTGAATATAATAGAGATGAAACTGTATTA GAATCATATAGAACAAATTCTTTGAGAAAAATAAATAGTAATCATGGGATAGATATCAGGGCTAAT AGTTTGTTTACAGAACAAGAGTTATTAAATATTTATAGTCAGGAGTTGTTAAATCGTGGAAATTTA GCTGCAGCATCTGACATAGTAAGATTATTAGCCCTAAAAAATTTTGGCGGAGTATATTTAGATGTT GATATGCTTCCAGGTATTCACTCTGATTTATTTAAAACAATATCTAGACCTAGCTCTATTGGACTA GACCGTTGGGAAATGATAAAATTAGAGGCTATTATGAAGTATAAAAAATATATAAATAATTATACA TCAGAAAACTTTGATAAACTTGATCAACAATTAAAAGATAATTTTAAACTCATTATAGAAAGTAAA AGTGAAAAATCTGAGATATTTTCTAAATTAGAAAATTTAAATGTATCTGATCTTGAAATTAAAATA GCTTTCGCTTTAGGCAGTGTTATAAATCAAGCCTTGATATCAAAACAAGGTTCATATCTTACTAAC CTAGTAATAGAACAAGTAAAAAATAGATATCAATTTTTAAACCAACACCTTAACCCAGCCATAGAG TCTGATAATAACTTCACAGATACTACTAAAATTTTTCATGATTCATTATTTAATTCAGCTACCGCA GAAAACTCTATGTTTTTAACAAAAATAGCACCATACTTACAAGTAGGTTTTATGCCAGAAGCTCGC TCCACAATAAGTTTAAGTGGTCCAGGAGCTTATGCGTCAGCTTACTATGATTTCATAAATTTACAA GAAAATACTATAGAAAAAACTTTAAAAGCATCAGATTTAATAGAATTTAAATTCCCAGAAAATAAT CTATCTCAATTGACAGAACAAGAAATAAATAGTCTATGGAGCTTTGATCAAGCAAGTGCAAAATAT CAATTTGAGAAATATGTAAGAGATTATACTGGTGGATCT SEQ ID NO: 53 – ToxA-GTD detox MSLISKEELIKLAYSIRPRENEYKTILTNLDEYNKLTTNNNENKYLQLKKLNESIDVFMNKYKTSS RNRALSNLKKDILKEVILIKNSNTSPVEKNLHFVWIGGEVSDIALEYIKQWADINAEYNIKLWYDS EAFLVNTLKKAIVESSTTEALQLLEEEIQNPQFDNMKFYKKRMEFIYDRQKRFINYYKSQINKPTV PTIDDIIKSHLVSEYNRDETVLESYRTNSLRKINSNHGIDIRANSLFTEQELLNIYSQELLNRGNL AAASDIVRLLALKNFGGVALAVAMLPGIHSDLFKTISRPSSIGLDRWEMIKLEAIMKYKKYINNYT SENFDKLDQQLKDNFKLIIESKSEKSEIFSKLENLNVSDLEIKIAFALGSVINQALISKQGSYLTN LVIEQVKNRYQFLNQHLNPAIESDNNFTDTTKIFHDSLFNSATAENSMFLTKIAPYLQVGFMPEAR STISLSGPGAYASAYYDFINLQENTIEKTLKASDLIEFKFPENNLSQLTEQEINSLWSFDQASAKY QFEKYVRDYTGGS SEQ ID NO: 54 – RNA sequence encoding F2 (5’ and 3’ UTRs are underlined) AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAAGUGGGUCA CCUUCAUCAGCCUGCUGUUUCUGUUCAGCAGCGCCUACAGCGGCUGGCAGACAAUCGACGGCAAGA AGUACUACUUCAACACCAACACCGCCAUUGCCAGCACCGGCUACACCAUCAUCAACGGCAAGCACU UCUACUUUAAUACCGACGGCAUCAUGCAGAUUGGCGUGUUCAAGGGCCCCAACGGCUUCGAGUAUU UCGCCCCUGCCAACACCGACGCCAACAAUAUCGAAGGCCAGGCCAUCCUGUACCAGAACGAGUUUC UGACCCUGAACGGGAAGAAGUAUUAUUUCGGCAGCGACAGCAAGGCCGUGACCGGCUGGCGGAUUA UCAACAACAAGAAAUAUUACUUUAACCCGAACAACGCUAUCGCCGCCAUCCACCUGUGCACCAUCA ACAAUGACAAGUACUAUUUCAGCUACGAUGGCAUCCUGCAGAACGGCUACAUCACCAUCGAGCGGA ACAAUUUCUACUUCGAUGCCAACAACGAGAGCAAGAUGGUCACCGGGGUGUUCAAAGGACCUAAUG GCUUUGAGUACUUCGCUCCCGCUAAUACCCACAACAACAACAUCGAGGGACAAGCCAUCGUCUAUC AGAACAAGUUCCUGACGCUCAAUGGCAAAAAGUAUUACUUUGACAACGACUCCAAGGCUGUCACCG GAUGGCAGACCAUUGAUGGGAAAAAGUACUACUUUAACCUGAACACCGCCGAGGCCGCCACAGGCU GGCAAACUAUUGAUGGAAAGAAGUACUAUUUCAAUCUCAAUACGGCCGAAGCUGCUACUGGAUGGC AAACGAUAGACGGAAAGAAGUAUUACUUUAAUACGAACACCUUUAUCGCCUCCACCGGGUACACCU CCAUUAACGGGAAACACUUUUAUUUCAACACGGACGGGAUUAUGCAAAUCGGGGUUUUCAAGGGGC CGAAUGGAUUCGAAUACUUCGCACCAGCCAAUACGGAUGCUAACAACAUUGAAGGACAGGCUAUUC UCUACCAAAACAAAUUCCUCACACUGAACGGCAAAAAGUACUACUUUGGCUCCGAUAGCAAGGCUG UUACAGGCCUGAGAACUAUCGACGGAAAAAAGUAUUAUUUCAACACAAAUACCGCCGUGGCAGUGA CAGGCUGGCAGACGAUUAACGGGAAGAAAUAUUACUUCAAUACCAAUACCAGCAUAGCCUCCACGG GCUAUACAAUCAUCUCCGGCAAACAUUUUUACUUUAACACAGAUGGUAUAAUGCAAAUUGGAGUCU UUAAGGGACCUGACGGUUUUGAAUACUUUGCCCCAGCUAACACAGACGCAAACAACAUAGAAGGGC AAGCAAUCAGAUAUCAGAAUCGGUUCCUGUACCUGCACGACAACAUCUACUACUUCGGCAACAACU CCAAAGCCGCUACCGGCUGGGUCACCAUUGACGGCAAUCGGUAUUACUUCGAGCCCAAUACCGCCA UGGGCGCCAACGGAUACAAGACCAUCGAUAACAAGAACUUUUACUUCCGGAACGGGCUGCCCCAAA UCGGAGUGUUUAAAGGCAGCAACGGAUUUGAGUAUUUUGCUCCGGCCAACACUGAUGCAAACAAUA UUGAGGGUCAAGCUAUACGGUAUCAGAACCGCUUCCUGCAUCUGCUGGGCAAAAUCUACUAUUUUG GGAACAACAGUAAAGCCGUCACUGGCUGGCAGACUAUCAAUGGCAAAGUCUACUACUUCAUGCCCG ACACCGCUAUGGCUGCCGCUGGCGGACUUAACCAGAUUGGAGACUACAAGUAUUACUUCAACUCCG ACGGCGUGAUGCAGAAAGGCUUCGUGUCCAUCAACGACAACAAGCACUAUUUUGACGACAGCGGCG UCAUGAAGGUCGGAUACACCGAGAUUGACGGAAAACAUUUCUAUUUCGCCGAGAACGGGGAGAUGC AAAUCGGCGUGUUCAACACCGAGGACGGCUUCAAGUACUUUGCUCACCACAACGAGGACCUGGGCA ACGAGGAAGGCGAGGAAAUCAGCUACUCCGGCAUCCUGAACUUUAACAACAAAAUCUAUUACUUCG ACGACAGCUUCACCGCCGUCGUCGGAUGGAAGGACCUGGAAGAUGGCUCUAAGUACUAUUUUGAUG AGGACACAGCCGAGGCCUACAUCGGCCUGUCUCUGAUUAACGACGGCCAGUACUACUUCAAUGACG AUGGCAUCAUGCAAGUGGGCUUCGUCACCAUCAAUGAUAAGGUGUUCUACUUCAGCGAUAGCGGCA UCAUCGAGAGCGGCGUGCAGAACAUCGACGACAACUACUUCUACAUCGAUGAUAACGGCAUCGUCC AGAUAGGGGUUUUCGACACCUCCGACGGGUACAAAUAUUUCGCACCCGCAAACACAGUGAACGAUA ACAUCUAUGGCCAGGCCGUGGAAUACUCCGGCCUCGUUAGAGUGGGCGAAGAUGUUUACUAUUUCG GGGAGACUUACACCAUCGAAACCGGCUGGAUAUACGACAUGGAAAACGAGAGCGACAAGUACUACU UCAAUCCCGAGACAAAGAAGGCCUGCAAGGGCAUCAACCUGAUUGACGACAUUAAGUACUACUUCG ACGAGAAGGGCAUCAUGCGGACCGGCCUGAUUAGCUUCGAGAACAACAACUACUAUUUCAACGAGA AUGGCGAGAUGCAGUUCGGCUAUAUCAAUAUCGAGGACAAGAUGUUUUACUUCGGCGAGGAUGGCG UUAUGCAGAUAGGGGUGUUCAAUACCCCUGAUGGGUUCAAGUAUUUCGCUCAUCAGAACACCCUGG ACGAGAACUUCGAGGGCGAGAGCAUCAAUUACACCGGCUGGCUGGACCUGGAUGAGAAGCGCUACU ACUUUACCGACGAGUACAUUGCCGCCACUGGCUCCGUGAUUAUCGACGGCGAGGAAUAUUACUUCG ACCCCGACACAGCCCAGCUGGUCAUCUCUGAAUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUG CUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCC CACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCA GCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAAC GAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCU AGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO: 55 – RNA sequence encoding TD1 (5’ and 3’ UTRs are underlined) AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGACUGGACCU GGCGGGUGUUUUGUCUGCUGGCCGUGACACCGGGCGCCCACCCCGCCCUAACCACCGCCACCACCG CCAUAAUCACAAGCAGCCUGGGCAUCGCUAGCGGCUUCAGCAUCCUGCUGGUGCCCCUGGCCGGCA UCAGCGCCGGCAUCCCUAGCCUGGUGAACAACGAGCUGGUGCUGAGAGACAAGGCCACCAAGGUGG UGGACUACUUCAAGCACGUGAGCCUGGUGGAGACCGAGGGCGUGUUCACCCUGCUGGACGACAAGG UGAUGAUGCCCCAAGACGACCUGGUGAUCAGCGAGAUCGACUUCAACAACAACAGCAUCGUGCUGG GCAAGUGCGAGAUCUGGAGAAUGGAGGGCGGCAGCGGCCACACCGUGACCGACGACAUCGACCACU UCUUCAGCGCCCCUAGCAUCACCUACAGAGAGCCCCACCUGAGCAUCUACGACGUGCUGGAGGUGC AGAAGGAGGAGCUGGACCUGAGCAAGGACCUGAUGGUGCUGCCCAACGCCCCCAACAGAGUGUUCG CCUGGGAGACCGGCUGGACCCCCGGCCUGAGAAGCCUGGAGAACGACGGCACCAAGCUGCUGGACA GAAUCAGAGACAACUACGAGGGCGAGUUCUACUGGAGAUACUUCGCCUUCAUCGCCGACGCCCUGA UCACCACCCUGAAGCCUAGAUACGAGGACACCAACAUCAGAAUCAACCUGGACAGCAACACACGGA GCUUCAUCGUGCCCAUCAUCACCACCGAGUACAUCAGAGAGAAGCUGAGCUACAGCUUCUACGGCA GCGGCGGCACCUACGCCCUGAGCCUGUCUCAGUACAACAUGGGCAUCAACAUCGAGCUGAGCGAGA GCGACGUGUGGAUCAUCGACGUGGACAACGUGGUGAGAGACGUGACCAUCGAGAGCGACAAGAUCA AGAAGGGCGACCUGAUCGAGGGCAUCCUGAGCACCCUGAGCAUCGAGGAGAACAAGAUCAUCCUGA ACAGCCACGAGAUCAACUUCAGCGGCGAGGUGAACGGCAGCAACGGCUUCGUGAGCCUGACCUUUA GCAUUCUGGAGGGUAUCAACGCCAUCAUCGAGGUGGACCUGCUGAGCAAGAGCUACAAGCUGCUGA UCAGCGGCGAGCUGAAGAUCCUGAUGCUGAACAGCAACCACAUUCAGCAGAAGAUCGACUACAUCG GCUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCC CGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUG CUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCC ACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAG GGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA SEQ ID NO: 56 – RNA sequence encoding ToxB-GTD (5’ and 3’ UTRs are underlined) AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGUGAAGG UGCUGUUCGCCCUGAUCUGCAUCGCCGUAGCCGAAGCUAGCCUGGUGAACAGAAAGCAGCUGGAGA AGAUGGCCAACGUGAGAUUCAGAACCCAAGAGGACGAGUACGUGGCCAUCCUGGACGCCCUGGAGG AGUACCACAACAUGAGCGAGAACACGGUUGUGGAGAAGUAUCUGAAGCUGAAGGACAUCAACAGCC UGACCGACAUCUACAUCGACACCUACAAAAAGAGCGGCAGAAACAAGGCCCUGAAGAAGUUCAAGG AGUACCUGGUGACCGAGGUGCUGGAGCUGAAGAACAACAACCUGACCCCCGUGGAGAAGAACCUGC ACUUCGUGUGGAUCGGCGGGCAGAUUAACGAUACAGCCAUCAACUACAUCAAUCAGUGGAAGGACG UGAACAGCGACUACAACGUGAACGUGUUCUACGACAGCAACGCCUUCCUGAUCAACACCCUGAAGA AGACAGUGGUGGAGUCCGCCAUCAACGAUACGCUGGAGAGCUUCAGAGAGAACCUGAACGACCCUA GAUUCGACUACAACAAGUUCUUCAGAAAGAGAAUGGAGAUCAUCUACGACAAGCAGAAGAACUUCA UCAACUACUACAAGGCUCAGAGAGAGGAGAACCCCGAGCUGAUCAUCGACGACAUCGUGAAGACCU ACCUGAGCAACGAGUACAGCAAGGAGAUCGACGAGCUGAACACCUACAUCGAGGAGAGCCUGAACA AGAUCACACAGAACAGCGGCAACGACGUACGCAACUUCGAGGAAUUUAAGAACGGCGAGAGCUUCA ACCUGUACGAGCAAGAGCUGGUGGAGAGAUGGAACCUGGCCGCCGCUAGCGCCAUCCUGGCCAUCA GCGCCCUGAAGGAGAUCGGCGGCAUGGCCCUGGCCGUGGCCAUGCUGCCCGGCAUUCAGCCCGACC UGUUCGAGAGCAUCGAGAAGCCUAGCAGCGUGACCGUGGACUUCUGGGAGAUGACCAAGCUGGAGG CCAUCAUGAAGUACAAGGAGUACAUCCCCGAGUACACAAGCGAGCACUUCGACAUGCUGGACGAAG AGGUGCAGAGCAGCUUCGAAUCGGUGCUGGCUAGCAAGAGCGACAAGAGCGAGAUCUUCAGCAGUC UGGGUGAUAUGGAGGCUAGCCCCCUGGAGGUGAAGAUCGCCUUCAACAGCAAGGGCAUCAUCAACC AAGGCCUGAUCAGCGUGAAGGACAGCUACUGCAGCAACCUGAUCGUGAAGCAGAUCGAGAACAGAU ACAAGAUCCUGAACAACAGCCUGAACCCCGCCAUCAGCGAGGACAACGACUUCAACACCACCACCA ACACCUUCAUCGACAGCAUCAUGGCCGAGGCCAACGCCGACAACGGCAGAUUCAUGAUGGAGCUGG GCAAGUACCUGAGAGUGGGCUUCUUCCCCGACGUGAAGACCACCAUCAACCUGAGCGGCCCCGAGG CCUACGCCGCCGCCUACCAAGACCUGCUGAUGUUCAAGGAGGGCAGCAUGAACAUCCACCUGAUCG AGGCCGACCUGAGAAACUUCGAGAUCAGUAAGACCAACAUCUCUCAGAGCACCGAGCAAGAGAUGG CUAGCCUGUGGUCCUUCGAUGAUGCUAGAGCCAAGGCUCAGUUCGAGGAGUACAAGAGAAACUACU UCGAGGGCAGCCUGUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCG UCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCAC UCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCU AGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUA UACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO: 57 UGAUGA

Claims

CLAIMS What is claimed is: 1. An immunogenic composition comprising a first immunogen and a second immunogen: the first immunogen comprising a C. difficile toxin A CROP domain fragment and a C. difficile toxin B CROP domain fragment; and the second immunogen comprising a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the delivery and receptor binding domain (DRBD) of toxin B. 2. The immunogenic composition of claim 1 wherein the first immunogen is a first polypeptide, and the second immunogen is a second polypeptide. 3. The immunogenic composition of claim 2 wherein the toxin A CROP domain fragment of the first polypeptide comprises a proximal end (i.e., proximal end of the toxin A CROP domain fragment) and a distal end (i.e., distal end of the toxin A CROP domain fragment); and the toxin B CROP domain fragment of the first polypeptide comprises a proximal end (i.e., proximal end of the toxin B CROP domain fragment) and a distal end (i.e., distal end of the toxin B CROP domain fragment), wherein the proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment are adjacent to each other. 4. The immunogenic composition of claim 3 wherein the distal end of the toxin A CROP domain fragment and the distal end of the toxin B CROP domain fragment are at either terminus of the first polypeptide. 5. The immunogenic composition of claim 3 wherein the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A. 6. The immunogenic composition according to claim 5 wherein the proximal end of the toxin A CROP domain fragment is within amino acids 2700-2710 or 2680-2690 of toxin A.

7. The immunogenic composition of claim 3 wherein the proximal end of the toxin B CROP domain fragment is within repeat portion I (amino acids 1834-1926) of toxin B. 8. The immunogenic composition of claim 3 wherein the proximal end of the toxin B CROP domain fragment is within repeat portion II (amino acids 1927-2057) of toxin B. 9. The immunogenic composition according to claim 3 wherein the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A and wherein proximal end of the toxin B CROP domain fragment is within repeat portion I (amino acids 1834-1926) of toxin B. 10. The immunogenic composition according to claim 3 wherein the proximal end of the toxin A CROP domain fragment is within repeat portion VIII (amino acids 2645-2710) of toxin A and wherein the proximal end of the toxin B CROP domain fragment is within repeat portion II (amino acids 1927-2057) of toxin B. 11. The immunogenic composition according to any of claims 2-10 wherein the first polypeptide further comprises a linker, optionally wherein the linker is between the proximal end of the toxin A CROP domain fragment and the proximal end of the toxin B CROP domain fragment, optionally wherein the linker comprises 1-20 amino acids. 12. The immunogenic composition according to any of claims 2-11 wherein the first polypeptide comprises a sequence at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least 97.5% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. 13. The immunogenic composition of claim 2 wherein the second polypeptide comprises a fragment of C. difficile toxin B which comprises at least 100 contiguous amino acids of the DRBD of toxin B wherein the DRBD of toxin B corresponds to amino acids 840-1833 of SEQ ID NO: 2 (strain VPI10463 (ATCC43255), amino acids 841-1834 of SEQ ID NO: 18 (strain M68) or at equivalent positions in the toxin B of other strains of C. difficile. 14. The immunogenic composition of claim 2 wherein the second polypeptide comprises a sequence at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95% or at least

97.5%, at least 99% or 100% identical to SEQ ID NO: 16 or SEQ ID NO: 17. 15. The immunogenic composition according to any previous claim further comprising a third immunogen, wherein the third immunogen is a third polypeptide and wherein said third polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95% or at least 97.5% sequence identity to SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 53. 16. The immunogenic composition according to any previous claim further comprising an adjuvant. 17. A nucleic acid encoding a) the first polypeptide as referred to in claim 2-12 and b) the second polypeptide as referred to in claim 2 and claims 13-14 wherein said nucleic acid is a single polynucleotide encoding said first and second polypeptides. 18. A nucleic acid encoding the first polypeptide as referred to in claim 2-12. 19. A nucleic acid encoding the second polypeptide as referred to in claim 2 and claims 13-14. 20. A nucleic acid encoding the third polypeptide as referred to in claim 15. 21. The nucleic acid of claims 17-21 wherein the nucleic acid is RNA. 22. A carrier comprising the nucleic acid of claim 17, the nucleic acid of claim 18, the nucleic acid of claim 19 or the nucleic acid of claim 20, optionally wherein the carrier is a lipid nanoparticle, optionally wherein the lipid nanoparticle comprises a mixture of cationic lipids, neutral lipids, sterols and polymer-conjugated lipids. 23. A pharmaceutical composition comprising the immunogenic composition of claims 1-16 and a pharmaceutically acceptable excipient. 24. A pharmaceutical composition comprising the nucleic acid of claim 17, the nucleic acid of claim 18, the nucleic acid of claim 19, the nucleic acid of claim 20 or the carrier of claim 22 and a pharmaceutically acceptable excipient. 25. A pharmaceutical composition, said pharmaceutical composition comprising, a carrier comprising the nucleic acid of claim 18, a carrier comprising the nucleic acid of claim 19 and a pharmaceutically acceptable excipient, optionally wherein the carrier is a lipid nanoparticle. 26. A pharmaceutical composition comprising either: the first polypeptide according to claim 2-12 and the nucleic acid according to claim 19 or, the second polypeptide according to claim 2 and claims 13-14 and the nucleic acid according to claim 18 said pharmaceutical composition further comprising a pharmaceutically acceptable excipient. 27. A vaccine comprising the pharmaceutical composition of any of claims 23-26. 28. The pharmaceutical composition of claims 23-26 or the vaccine of claim 27 for use in the treatment or prevention of C. difficile disease.