WO2023247936A1 - Compositions and methods - Google Patents

Compositions and methods Download PDF

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WO2023247936A1
WO2023247936A1 PCT/GB2023/051599 GB2023051599W WO2023247936A1 WO 2023247936 A1 WO2023247936 A1 WO 2023247936A1 GB 2023051599 W GB2023051599 W GB 2023051599W WO 2023247936 A1 WO2023247936 A1 WO 2023247936A1
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antigen
bacterium
seq
amino acid
acid sequence
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PCT/GB2023/051599
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French (fr)
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Edward Green
Benjamin Michael BRADLEY
Kathryn Naylor
Zak BEAN
Richard Mark Edwards
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Chain Biotechnology Limited
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
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Definitions

  • the invention relates to bacterial vaccines, particularly live bacterial vaccines suitable for oral administration and for stimulating humoral and/or cellular immunity.
  • Background Vaccines play a leading role in disease prevention, particularly of infectious diseases, and show promise in therapy of existing infections and chronic diseases.
  • Oral vaccines address some of the disadvantages of traditional injection-based formulations, providing improved safety and compliance and easier administration.
  • Oral vaccines may stimulate humoral and cellular responses at both systemic and mucosal sites, but there are significant challenges in their development posed by the gastrointestinal (GI) tract, as reviewed in Vela Ramirez, J. E., Sharpe, L. A., & Peppas, N. A. (2017). Current state and challenges in developing oral vaccines.
  • GI gastrointestinal
  • M cells epithelial cells
  • GALT gut-associated lymphoid tissue
  • DCs dendritic cells
  • Typical strategies in oral vaccines under development have relied on high antigen doses and potent adjuvants in order to trigger an immune response (Ramirez et al, supra).
  • Some strategies make use of Gram-negative bacterial lipopolysaccharide, Salmonella lipid A derivatives or cholera toxin that may elicit adjuvant effects, but there is a trade- off in terms of toxicity.
  • Bacterial vaccines offer promise, and live-attenuated vaccines for Vibrio cholera or Salmonella typhi vaccines have been licensed.
  • Gram-positive bacteria such as Lactococcus, which avoid LPS and may be better tolerated, have been suggested as a potential vaccine platform (Bahey-El-Din, M and Gahan, CGM (2010) Lactococcus lactis based vaccines: ‘Current status and future perspectives’, Human Vaccines, 7:1, 106- 109, DOI:10.4161/hv.7.1.13631).
  • An oral recombinant Lactobacillus vaccine is disclosed in WO 2001/021200 A1.
  • Bacterial vaccines have to date been used to target the small intestine, where the mucosal immune system has been well studied.
  • An attenuated Clostridium perfringens engineered to express high levels of antigen in inclusion bodies during sporulation has been proposed in Chen Y et al (2004) Use of a Clostridium perfringens vector to express high levels of SIV p27 protein for the development of an oral SIV vaccine, Virology 329: 226-233, ISSN 0042-6822, https://doi.org/10.1016/j.virol.2004.08.018.
  • US 5,800,821 discloses strategies to promote germination of Bacillus subtilis in the upper GI tract, namely pre-germinating spores before administration, and co-expression of invasin, which bind M cells and may promote colonization.
  • a cell wall binding system cwbA specific for Bacillus is disclosed.
  • An Example in US 5,800,821 discloses C. perfringens engineered to express is Shiga-like toxin B under control of the cpe promoter.
  • the cpe promoter is sporulation specific in Clostridium, and would not be active during vegetative growth (see Melville SB, Labbe R, Sonenshein AL. Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis. Infect Immun. 1994;62(12):5550-5558).
  • Oral vaccines licensed to date are typically intended for prevention of infection rather than as therapeutic vaccines.
  • Antibodies produced by B cells are the predominant correlate of protection for current vaccines, but cell-mediated immune functions are critical in protection against intracellular infections, and in almost all diseases, CD4 + cells are necessary to help B cell development (Stanley A.
  • the proteins are phagocytosed or endocytosed into endosomes and lysosomes by antigen presentation cells (APCs), whereby lysosomes degrade the protein into smaller peptides, some of which can (CD4 epitopes) bind to MHC class II molecules on lysosomal membranes and are presented to the cell surface to stimulate CD4 + T-cells, which in turn are required for B cells to produce antibodies (T cell help). Therefore, protein antigens have been mainly used to stimulate the body to produce antibodies.
  • APCs antigen presentation cells
  • the main pathway for the presentation of antigenic peptides on MHC Class I molecules relies on antigen that is expressed within the APC, such as following viral infection.
  • APC can also internalise antigens and present them on MHC Class I molecules to stimulate cytotoxic T lymphocytes (CTL) by a process called antigen cross presentation, which is typically an inefficient process.
  • CTL cytotoxic T lymphocytes
  • the delivery of exogenous peptides or proteins to the MHC class I pathway has been partially successful through use of chemical adjuvants such as Freund's adjuvant, and mixtures of squalene and detergents (Hilgers et al. (1999) VACCINE 17:219-228).
  • EP3235831 (Oxford Vacmedix UK Ltd) demonstrates that an artificial multi-epitope fusion protein known as a recombinant overlapping peptide (ROP) is capable of simultaneously stimulating CD4 + and CD8 + T- cell responses.
  • ROPs are made up of overlapping peptides linked by the cathepsin cleavage site target sequence and are more efficient in priming protective immunity than the whole protein from which the peptides are derived.
  • Subcutaneous immunisation with ROPs has been shown to have protective effects in a viral model and a tumour model (Zhang H et al (2009) J. Biol. Chem.
  • WO 2018/055388 discloses Clostridium engineered to express (R)-3-hydroxybutyrate (R-3-HB) as an anti-inflammatory agent, including in a simulated colon environment.
  • WO 2019/180441 discloses in vivo and pharmacokinetic profiling of R-3-HB engineered Clostridium butyricum.
  • the engineered strain could be isolated from colon samples of mice that had been dosed orally with bacterial spores.
  • the present inventors sought to exploit the ability of Clostridium to grow in anaerobic conditions to target the lower anaerobic regions of the GI tract, such as the large intestine, in order to develop a platform vaccine technology.
  • the present invention is based on the surprising discovery that Clostridium engineered for expression of antigen during anaerobic cell growth can stimulate antigen-specific immune responses.
  • the amino acid sequence and nucleic acid sequence of the OVA ROP are provided in SEQ ID NOs: 108 and 109 respectively.
  • Nucleic acid cassettes including antigen genes were prepared using an appropriate nucleic acid sequence for either ORF under the control of the fdx promoter, and the antigen gene integrated into the bacterial chromosome using techniques as described in the present application. Intracellular expression of either antigen was detected in an amount of ⁇ 80 ng/mg dry cell weight. Mice were dosed orally with 10 8 spores/mouse at days 0, 14 and 28 and sacrificed at day 42.
  • mice orally immunised with the strain expressing ROP-HPV developed both CD4 + and CD8 + T-cell response, while mice immunised with the strain expressing ROP-OVA developed CD4 + T-cell response specific to the respective antigen, according to IFN- ⁇ ELISPOT evaluation of T-cells isolated from spleens and re- stimulated with antigen.
  • the immunised mice did not develop a T-cell immune response against the C. butyricum strain itself.
  • Clostridium engineered to express a heterologous antigen in the intracellular compartment during anaerobic cell growth is capable of inducing an antigen-specific immune response in a host following oral immunisation.
  • the present disclosure provides Clostridium which expresses surface-tethered antigen or secretes antigen during anaerobic cell growth; or which expresses SARS-CoV-2 antigens in the intracellular compartment during anaerobic cell growth.
  • Clostridium expressing surface-tethered antigen can be expected to be internalised by the same cells that internalise Clostridium expressing an antigen in the intracellular compartment.
  • secreted antigen may accumulate in the vicinity of such cells and be internalised by endocytosis; or may even be secreted into cells that have already internalised Clostridium.
  • intracellular, secreted or surface-tethered forms of antigen may become suitably located for capture by B cells and generation of B cell antibody responses.
  • a first aspect of the present invention is a bacterium of the class Clostridia comprising a heterologous nucleic acid molecule encoding at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region.
  • the promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; and the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or is capable of secreting the antigen as a secreted antigen.
  • the at least one antigen is an infectious agent antigen or a tumour antigen.
  • a second aspect of the present invention is a bacterium of the class Clostridia comprising a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2.
  • the infectious agent antigen or tumour antigen is heterologous to the bacterium.
  • capable of causing expression of we mean that the promoter is capable of causing transcription and typically also translation in the bacterium, which results in the expression of the antigen.
  • the expression of antigen by the bacterium occurs during anaerobic cell growth.
  • Bacteria of the class Clostridia are obligately anaerobic bacteria, the majority of which have the ability to form spores (i.e., are spore-forming bacteria). Such bacteria may be in the form of a spore or in a vegetative form; in the latter form, the bacteria are metabolically active and typically growing.
  • Clostridia By targeting expression of the antigen to metabolically active forms of the Clostridia, it is possible to use the Clostridia as a vehicle to target antigen to the anaerobic portions of the gut. By administering the bacteria orally as spores, the bacteria remain dormant and viable during transit through the gastrointestinal tract, until they reach the anaerobic portions where they germinate and multiply.
  • Antigens By “antigen”, we mean a molecule that binds specifically to an antibody or a T-cell receptor (TCR). Antigens that bind to antibodies are called B cell antigens.
  • Suitable types of molecule include peptides, polypeptides, glycoproteins, polysaccharides, gangliosides, lipids, phospholipids, DNA, RNA, fragments thereof, portions thereof and combinations thereof.
  • Peptide and polypeptide antigens including glycoproteins, are preferred.
  • TCRs bind only peptide fragments complexed with MHC molecules.
  • the portions of an antigen that are recognised are termed “epitopes”.
  • a B cell epitope is a peptide or polypeptide, it typically comprises 3 or more amino acids, generally at least 5 and more usually at least 8 to 10 amino acids.
  • the amino acids may be adjacent amino acid residues in the primary structure of the polypeptide or may become spatially juxtaposed in the folded protein.
  • T cell epitopes are normally short primary sequences from antigens. They may bind to MHC Class I or MHC Class II molecules. Typically, MHC Class I-binding T-cell epitopes are 8 to 11 amino acids long. Class II molecules bind peptides that may be 10 to 30 residues long or longer, the optimal length being 12 to 16 residues. Peptides that bind to a particular allelic form of an MHC molecule contain amino acid residues that allow complementary interactions between the peptide and the allelic MHC molecule. The ability of a putative T-cell epitope to bind to an MHC molecule can be predicted and confirmed experimentally (Peters et al. (2020) T Cell Epitope Predictions, Annual Reviews of Immunology, Vol.
  • the antigen expressed by the bacterium of the class Clostridia is an infectious agent antigen or a tumour antigen.
  • infectious agent antigen we mean that the antigen derives from an infectious agent that is capable of infecting a susceptible host, such as a human, typically resulting in a pathology.
  • recipient from we include that the infectious agent antigen is encoded in the genome of an infectious agent, or is a variant of such an encoded antigen.
  • tumor antigen we mean that the antigen derives from an antigen that is expressed predominantly, such as almost exclusively or exclusively by tumour cells, or acts as a marker that is used in the art to distinguish a tumour cell from a healthy cell.
  • the tumour antigen is encoded in the genome of a cancer cell, or is a variant of such an encoded antigen.
  • the antigen may be an infectious agent antigen that is associated with a risk of cancer.
  • An antigen may be a fragment or portion of a complete protein, which fragment includes an epitope.
  • An “antigen segment” is a portion of an antigen, which antigen comprises an epitope.
  • a “fragment” typically comprises at least 5 amino acid residues, typically at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid residues of the antigen from which it is derived. Such fragment lengths may be sufficient to provide an epitope of the antigen.
  • An antigen or fragment may comprise at least or up to 20 amino acid residues, at least or up to 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900 or 1,000 amino acid residues. Any range involving these values is envisaged, such as between 10 and 200 amino acid resides, between 20 and 100 etc. Antigens and fragments of up to about 100, 150, 200, 250, 300, 350, 400, 500 or 550 amino acid residues are typical.
  • Typical fragments of an antigen will have an amino acid sequence comprising at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% of the corresponding native antigen or variant thereof.
  • a fragment may also be referred to as an “antigenic portion”, in other words, a portion of antigen which comprises an epitope.
  • the term “antigenic portion” is also intended to include an entire antigen, since the entire antigen is rendered antigenic by virtue of the antigenic portion.
  • a “variant” refers to a protein or peptide wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non- conservative.
  • conservative substitutions are intended combinations such as Val, Ile, Leu, Ala, Met; Asp, Glu; Asn, Gln; Ser, Thr, Gly, Ala; Lys, Arg, His; and Phe, Tyr, Trp.
  • Preferred conservative substitutions include Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • Typical variants of the antigen or portion thereof will have an amino acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% identical to the corresponding native antigen or portion thereof.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program (Thompson et al., (1994) Nucleic Acids Res., 22(22), 4673-80).
  • the parameters used may be as follows: x Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. x Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. x Scoring matrix: BLOSUM.
  • a “variant” may also refer to the nucleic acid molecule that encodes a variant antigen.
  • the at least one antigen comprises the complete amino acid sequence of a mature polypeptide which is an infectious agent antigen or a tumour antigen or variant thereof; or a fragment thereof.
  • mature polypeptide is intended to exclude a signal peptide present in the precursor of the mature polypeptide.
  • the at least one antigen comprises one or more T cell antigen segments and/or one or more B cell antigen segments.
  • An antigen segment is a portion of an antigen, which antigen comprises an epitope. Typically, an antigen segment comprises an epitope.
  • T cell antigen segments may be CD4 + T cell antigen segments or CD8 + T cell antigen segments.
  • a CD4 + T cell antigen segment is an antigen or portion thereof comprising an epitope which is capable of being presented to a CD4 + T cell in the context of MHC II.
  • a CD8 + T cell antigen segment is an antigen or portion thereof comprising an epitope which is capable of being presented to a CD8 + T cell in the context of MHC I.
  • Different antigen segments can be provided in different antigens or the same antigen. Multiple antigens or portions/fragments thereof may be used.
  • an antigen segment is in the form of a fragment of an antigen, such as a fragment comprising or consisting of a B or T cell epitope.
  • CD8 + T cell epitopes may be provided as antigen fragments of less than about 70 amino acids, such as less than 60, less than 50, less than 40.
  • the antigen is a multi-antigen fusion polypeptide comprising two or more antigen segments, such as ⁇ 3, ⁇ 4 ⁇ 5 or more or 10 or more antigen segments, optionally with an upper limit of ⁇ 30, ⁇ 20 or ⁇ 15.
  • multi-antigen fusion polypeptide we mean a polypeptide comprising antigen segments such as epitopes which are linked together, either directly or separated by appropriate linking sequences, to form an artificial polypeptide; this may be referred to as a polyepitope, artificial polyepitope, or mosaic polyepitope. Intervening sequences that occur between antigen segments in an antigen may thereby be avoided in a multi-antigen fusion polypeptide.
  • Each antigen segment may be from the same or different antigen.
  • Suitable linking sequences may be included to facilitate cleavage of antigen segments or epitopes, particularly CD8 + T cell antigen segments or epitopes, from the multi- antigen fusion polypeptide, as described in the Examples, or in EP3235831.
  • the multi-antigen fusion polypeptide comprises at least one CD4 + T cell antigen segment and at least one CD8 + T cell antigen segment.
  • the antigen segments in a multi-antigen fusion polypeptide may suitably be derived from polypeptide sequences that partially overlap in the antigen from which they are derived.
  • the first will have an N- terminal sequence that is not shared by the second, and the second will have a C- terminal sequence that is not shared by the first, and the two antigen segments will share a common sequence.
  • one antigen may be split into overlapping peptides that altogether contain the entire sequence of said antigen. In cases where there are multiple antigens, each may be present as overlapping peptides.
  • overlapping peptides encompasses recombinant overlapping peptides (ROPs), such as those described in EP3235831.
  • overlapping peptides and “ROP”, we mean that the antigen is a multi-antigen fusion polypeptide as defined above (also referred to herein as multi-antigen fusion protein) comprising two or more antigen segment sequences, i.e. peptide sequences, which partially overlap.
  • the antigen segments in a multi-antigen fusion protein are partially overlapping, and in combination encompass ⁇ 40%, ⁇ 50, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, more preferably 100% of the amino acid sequence of the antigen from which they are derived.
  • a first polypeptide may partially overlap with a second polypeptide
  • the second polypeptide may partially overlap with the third polypeptide, etc.
  • the multi-antigen fusion protein comprising overlapping peptides may comprise ⁇ 2, ⁇ 3, ⁇ 4, preferably ⁇ 5, more preferably ⁇ 10 antigen segments; optionally with an upper limit of ⁇ 30, 20 or 15 antigen segments.
  • a ROP may comprise 10 antigen segments, wherein all segments combined comprise 100% of the amino acid sequence for the whole antigen. It will be understood that not every antigen segment in a multi-antigen fusion protein necessarily contains an epitope.
  • each antigen segment comprises at least one (preferably at least 2) CD8 + epitope; at least one (preferably at least 2) CD4 + epitope; and/or at least one (such as at least 2) B cell epitope.
  • each antigen segment comprises at least one (preferably at least 2) amino acid sequence simultaneously serving as a CD8 + epitope and a CD4 + epitope. In some embodiments, each antigen segment comprises 8-50 amino acids, preferably 10-40 amino acids, more preferably 15-35 amino acids in length. In some embodiments, each antigen segment may comprise sequences of an exogenous protease cleavage site located between antigen segments. The exogenous protease is present in the host and may act extracellularly, or more typically intracellularly. For example, the sequence of a cleavage site may comprise a cleavage site of a cathepsin.
  • the cleavage site is selected from the group consisting of a cleavage site of cathepsin S (as described further in Lutzner and Kalbacher, 2008, J. Biol. Chem., 283(52):36185-36194) (e.g., Leu-Arg-Met-Lys (SEQ ID NO: 32) or a similar cleavage site), a cleavage site of cathepsin B (e.g., Met-Lys-Arg-Leu (SEQ ID NO: 33) or a similar cleavage site), a cleavage site of cathepsin K (e.g., His-Pro-Gly- Gly (SEQ ID NO: 34) or a similar restriction site), or combinations thereof.
  • a cleavage site of cathepsin S as described further in Lützner and Kalbacher, 2008, J. Biol. Chem., 283(52):36185-36194)
  • the cleavage site of cathepsin S is selected from a group consisting of X-Val/Met-X ⁇ Val/Leu-X-Hydrophobic amino acid, Arg-Cys-Gly ⁇ , -Leu, Thr-Val-Gly ⁇ , -Leu, Thr-Val-Gln ⁇ , -Leu, X-Asn-Leu-Arg ⁇ (SEQ ID NO: 35), X-Pro-Leu-Arg ⁇ (SEQ ID NO: 36), X-Ile-Val-Gln ⁇ (SEQ ID NO: 37) and X-Arg-Met-Lys ⁇ (SEQ ID NO: 38); wherein each X is independently any natural amino acid, and ⁇ represents cleavage position.
  • the cleavage site of cathepsin S is Thr-Val-Lys-Leu- Arg-Gln (SEQ ID NO: 39).
  • each antigen segment is directly connected in the artificial multi-antigen fusion protein via said sequence of cleavage site.
  • the sequence of cleavage site used to connect each antigen segment is the same or different.
  • the sequence of cleavage site is not contained in each antigen segment; or the sequence of cleavage site is contained in the antigen segment, while at least one cleavage product (or some or all of the cleavage products) formed after the antigen segment is digested is still a CD8 + epitope or CD4 + epitope.
  • the bacterium of the class Clostridia is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen or is capable of secreting the antigen as a secreted antigen.
  • exporting it is meant that a substrate – i.e. the antigen – is translocated across the bacterial plasma membrane out of the bacterial cytoplasm into the periplasmic space or extracellular milieu.
  • Suitable mechanisms of export by bacteria are known to the person skilled in the art and include in Gram-positive bacteria export by the Sec- dependent secretion pathway; the Tat-dependent secretion pathway; and Wss/Esx pathway.
  • Bacterial secretion systems and export pathways are reviewed in Green and Mecsas, 2006, Microbiol. Spectr., 4(1), doi: 10.1128/microbiolspec.VMBF-0012-2015.
  • secretion it is meant the translocation or export of the antigen from the cytoplasm of the bacterial cell across the plasma membrane into the extracellular milieu.
  • Exemplary methods of verifying antigen secretion by Clostridia are disclosed in Kovács et al., 2013, Biotechnol.
  • Biofuels, 6:117 (DOI: 10.1186/1754-6834-6-117).
  • Secreted peptides are harvested by centrifugation to separate supernatant and subsequently concentrated through Trichloroacetic acid precipitation. Following this, detection of secreted proteins is performed by the use of SDS-PAGE and western blotting.
  • tethered to the surface of the bacterium it is meant that the antigen is bound covalently or non-covalently to components of the surface of the bacterium, typically the bacterial cell wall or capsule; and hence is displayed on the surface of the bacterium.
  • An antigen may suitably be tethered to peptidoglycan comprised within the bacterial cell wall via a peptidoglycan anchoring domain or via enzymatic linkage.
  • Antigens that are tethered to the surface of the bacterium may be cleaved or degraded, thereby releasing the antigen or a fragment thereof into the extracellular milieu. The released antigen or fragment thereof is typically soluble.
  • the antigen In order to become tethered to the surface of the bacterium, the antigen must first be exported before becoming tethered.
  • an antigen is cleaved or degraded after being exported, it may not become tethered or may be tethered only transiently, even though it originally included a domain or motif required for tethering.
  • Exemplary methods of determining whether an antigen is cell-wall anchored in Clostridia are disclosed in Willson et al., 2016, Biotechnol. Biofuels, 9:109 (DOI: 10.1186/s13068-016-0526-x). Cell wall attachment is confirmed by cell fractionation isolating the supernatant through centrifugation, the cell wall and cytosolic fractions through lysozyme digestion of the cell wall and separation of protoplasts from cell wall components by centrifugation.
  • secreted antigen it is meant that the antigen has been secreted by the bacterium and is present in the extracellular milieu in a form that is not surface-tethered, and is typically soluble.
  • the Examples demonstrate successful secretion and/or surface tethering of polypeptides having a size range of between 4 kDa and 60 kDa.
  • surface- tethered or secreted antigens have a size range of up to 200 kDa, such as up to 150 kD, 100 kDa, 90 kDa, 80 kD, 70 kD or up to 60 kDa.
  • a typical lower limit may be 1 kDa, 2 kDa, 3kDa or 4kDa.
  • a LysM tethering domain is included, this would add 23 kDa to the size of the antigen.
  • a lower limit may be 24, 25, 26 or 27 kDa.
  • An average 80-90 amino acid residue polypeptide may have a molecular weight of about 10kDa.
  • the surface-tethered antigen or secreted antigen does not comprise disulphide bonds. In some embodiments, the surface-tethered antigen or secreted antigen does not comprise cysteine residues.
  • nucleic acid molecules comprising repetitive regions may not be efficiently expressed. Smaller polypeptides may be produced more efficiently than larger ones. Due to the redundancy of the genetic code, nucleic acid molecules encoding polypeptides may be re-designed such that the nucleic acid sequence is less repetitive; or may be codon optimised.
  • Suitable alternative signal sequences and/or motifs or domains that are known to be suitable for effecting secretion and/or surface tethering may be incorporated into the antigen to address the absence of secretion of the antigen by the bacteria. Cysteine residues, if present, may be removed or substituted by re- designing the coding sequence.
  • a suitable alternative promoter may be incorporated into the nucleic acid encoding the antigen, to optimise expression of the antigen by the bacteria.
  • the antigen is typically expressed as a precursor comprising an N-terminal signal peptide; and the surface-tethered antigen typically comprises a domain or motif required for tethering to peptidoglycan, optionally wherein the domain or motif is (a) a peptidoglycan anchoring domain, suitably comprising at least one LysM motif, such as a LysM domain; or (b) a sequence required for enzymatic linkage to peptidoglycan.
  • the antigen is a secreted antigen
  • the antigen is typically expressed as a precursor comprising an N-terminal signal peptide.
  • precursor is meant a polypeptide or portion thereof comprising an N-terminal signal peptide.
  • the signal peptide is present during translation of the nascent polypeptide, and is typically cleaved off such that the mature polypeptide formed following completion of translation lacks the signal peptide.
  • the signal peptide may typically comprise or be followed by a cleavage sequence at its C-terminal end to facilitate cleavage.
  • the signal peptide is capable of directing secretion of the antigen via the Sec-dependent secretion system.
  • Exemplary methods of predicting signal peptides that are capable of directing secretion of an antigen via the Sec secretion pathway are disclosed in Desvaux et al., 2005, Biochim. Biophys. Acta, 1745(2):223-253 (DOI: 10.1016/j.bbamcr.2005.04.006). Detection of signal peptides would utilise tools such as SignalP to identify proteins comprising N-terminal regions containing positively charged amino acid residues, followed by hydrophobic amino acid residues, and a carboxylic tail region containing polar amino acid residues; and comparing to a prediction of protein localisation utilising a predictive tool such as PSORT, or comparing to an appropriate mass spectrometry analysed secretome of the organism.
  • a predictive tool such as PSORT
  • Suitable signal peptides include the Usp45 signal peptide MKKKIISAILMSTILSAAAP (SEQ ID NO: 41), the Licheninase signal peptide MNKKKLKIMTFAMLVSTFLVGGLMQVPASA (SEQ ID NO: 144) or the LysM signal peptide MKKYYYLFTALISILLLSC (SEQ ID NO: 107).
  • the antigen comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 144, and/or SEQ ID NO: 107.
  • a suitable cleavage sequence to be included directly after the Usp45 signal peptide is LSVGYA (SEQ ID NO: 63), and the cleavage site is predicted to be directly after the “A”.
  • a suitable cleavage sequence to be included directly after the LysM signal peptide is “Q”, and the cleavage site is predicted to be between the final “C” of the LysM signal peptide and the “Q”.
  • a suitable cleavage sequence to be included directly after the Licheninase signal peptide is “LTT”, and the cleavage site is predicted to be directly between the Licheninase signal peptide and the “LTT”.
  • Suitable peptidoglycan anchoring domains include domains which anchor proteins to the cell surface through non-covalent attachment to the peptidoglycans found in the cell wall.
  • Non-covalent peptidoglycan anchoring domains include the LysM domain and fragments thereof.
  • the peptidoglycan anchoring domain comprises at least one LysM motif, suitably a LysM domain.
  • LysM Lysin Motif
  • LysM is a widely distributed protein motif for binding to peptidoglycans and its function is reviewed in Buist et al., 2008, Mol. Microbiol., 68(4):838-847 (doi:10.1111/j.1365- 2958.2008.06211.x).
  • Naturally occurring LysM domains typically comprise multiple copies of the LysM motif separated by spacer sequences.
  • Suitable LysM motifs may be identified from Gram positive bacteria, including Clostridia, suitably C. butyricum or C. perfringens.
  • a suitable peptidoglycan anchoring domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 LysM motifs.
  • the peptidoglycan anchoring domain comprises 2 LysM motifs, suitably from C. butyricum LysM.
  • the LysM domain comprises an amino acid sequence of SEQ ID NO: 42; or amino acids 22 to 200 of SEQ ID NO: 42; or a variant comprising at least 90%, at least 95%, at least 99% or at least 99.5% sequence identity to either sequence; or fragment comprising at least 90% of either sequence or variant.
  • the first 21 amino acid residues of the corresponding native LysM domain of SEQ ID NO: 42 i.e. MKKYYYLFLFTALISILLLSC (SEQ ID NO: 107) constitute the predicted signal peptide.
  • the LysM signal peptide and the LysM domain may conveniently be provided together, such that the precursor of the polypeptide comprises an N-terminal LysM signal peptide, and the mature polypeptide comprises the LysM domain.
  • the LysM domain may be comprised at the N-terminus or C-terminus of the antigen; or may be comprised within the antigen sequence itself. Where the LysM domain is located other than at the N-terminus, the signal peptide found in the native LysM domain is typically omitted.
  • the surface-tethered antigen may comprise a sequence required for enzymatic linkage to peptidoglycan.
  • the antigen may be anchored to the cell surface via covalent attachment to the peptidoglycans found in the cell wall.
  • the antigen may comprise a motif comprising a C-terminal Cell Wall Sortase signal (CWSS), and become anchored to the cell surface by the Sortase system, which covalently links peptides to peptidoglycan.
  • CWSS C-terminal Cell Wall Sortase signal
  • the CWSS is typically followed (from N- to C-terminus) by a segment of hydrophobic amino acids and a tail composed primarily of positively charged amino acids.
  • the Sortase system is reviewed in Dramsi et al., 2008, FEMS Microbio. Rev. 32(2):307-320 and Clancy et al., 2010, Biopolymers, 94(4):385-396.
  • Class A, Class B, Class C, Class D, Class E, and Class F are found in Gram positive bacteria: Class A, Class B, Class C, Class D, Class E, and Class F.
  • Class A, C, D, E, and F Sortase system CWSSs have the consensus amino acid sequence LPXTG (SEQ ID NO: 43; where X is any amino acid), optionally LP[A/N/K]TG (SEQ ID NO: 112).
  • the Class B Sortase system CWSS has the consensus amino acid sequence: NP(Q/K)(T/S)(N/G/S)(D/A) (SEQ ID NO: 111).
  • Other exemplary CWSS include: SPXTG (SEQ ID NO: 44) and PPXTG (SEQ ID NO: 45).
  • the antigen further comprises a cell wall sorting signal (CWSS); and optionally comprises a cell wall sorting signal comprising an amino acid sequence that is encoded by SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 111 or SEQ ID NO: 112.
  • the antigen comprising a CWSS further comprises a C-terminal segment of hydrophobic amino acids, and a tail region composed primarily of positively charged residues. Exemplary tail region sequences are disclosed in Willson et al., 2016, Biotech.
  • the cell wall sorting signal comprises an amino acid sequence of SEQ ID NO: 145 or SEQ ID NO: 146. It will be appreciated that, in order for the antigen to be covalently attached to peptidoglycan by the Sortase system, the antigen must first be exported into the periplasmic space.
  • periplasmic space it is meant — in Gram-positive bacteria – the space between the cell membrane and cell wall.
  • the precursor antigen comprises an N-terminal signal sequence – for example a Sec secretion system signal sequence – and a C-terminal CWSS.
  • the precursor antigen comprising an N-terminal signal sequence and a C-terminal CWSS further comprises a C-terminal segment of hydrophobic amino acids, and a tail composed primarily of positively charged residues.
  • the bacterium is capable of expressing the antigen as an intracellular antigen. Intracellular antigens may typically be designed without any special motifs or domains intended for interaction with the bacterial sorting systems.
  • antigens intended for expression in the intracellular compartment of the bacterium are typically not provided with N-terminal signal peptides.
  • An intracellular antigen is typically expressed as a soluble polypeptide or inclusion body in the bacterial cytoplasm, or as a combination of either form.
  • the antigen may further comprise amino acid sequences that are suitable for use as purification tags and/or tags used for immunostaining.
  • the antigen comprises a FLAG tag, a His tag, a Strep tag, an HA tag, c- Myc.
  • the antigen comprises a tag comprising an amino acid sequence that is encoded by SEQ ID NO: 46 and/or SEQ ID NO: 47.
  • the antigen comprises linkers disposed between the elements comprised by said antigen – for example, an antigen may comprise a linker disposed between a LysM domain and the antigen.
  • Suitable linkers include, for example, [Gly] 4 Ser linkers (SEQ ID NO: 48).
  • Infectious agent antigens or cancer antigens Suitable infectious agent antigens may include a viral antigen, a bacterial antigen (including a chlamydial antigen or a mycoplasma antigen), a parasite antigen, a protozoan antigen, a helminth antigen, a nematode antigen, a fungal antigen, a prion, or any combination thereof.
  • the antigen selected provides cross- immunity (also referred to as cross-protection) in that a single antigen or multiple antigens combined may confer immunity or protection against related infectious agents.
  • Cross-immunity may occur where an antigen is conserved (i.e. shared or homologous) in multiple strains or species of infectious agents. Accordingly, it may be desirable to use antigens (either single antigens or multiple combined antigens) that provide such cross-immunity.
  • the infectious agent antigen is from an infectious agent which is capable of causing an infection at a mucosal site in a susceptible host (i.e. is a mucosal infectious agent).
  • Mucosal infections may involve the following pathogens: Vibrio cholerae, coronaviruses e.g. SARS-CoV-2, influenza type A and B virus, poliovirus, rotavirus, Salmonella typhimurium, Salmonella sp. (including but not limited to S. enteria and subspecies including S. e. enterica, S. e. salamae, S. e. arizonae, S. e. diarizonae, S. e. houtenae, S. e. indica, S. enterica serovar Typhi, S. enterica serovar Typhimurium, S. enterica serovar Paratyphi; and S.
  • Vibrio cholerae coronaviruses e.g. SARS-CoV-2, influenza type A and B virus, poliovirus, rotavirus, Salmonella typhimurium, Salmonella sp.
  • S. enteria and subspecies including S.
  • adenovirus including S. sonnei, S. flexneri, and S.
  • infectious disease associated with the infectious agent may be categorised based on the location.
  • the infectious agent may be SARS- CoV-2, seasonal influenza, respiratory syncytial virus (RSV) e.g.
  • RSV-ALRI Streptococcus pneumoniae or Mycobacterium tuberculosis, which are associated with the respiratory tract
  • rotavirus Helicobacter pylori, enterotoxigenic Escherichia coli (ETEC), Salmonella, Shigella or Clostridium (difficile or perfringens), which are associated with the GI tract
  • ETEC enterotoxigenic Escherichia coli
  • Salmonella Shigella or Clostridium (difficile or perfringens)
  • syphilis gonorrhoea, herpes simplex virus 2, HPV, hepatitis B, hepatitis C or HIV, which are associated with the urogenital tract.
  • the infectious agent antigen is from an infectious agent which is capable of causing a respiratory tract infection in a susceptible host, optionally a virus such as a coronavirus, such as SARS-Cov-2; or optionally respiratory syncytial virus (RSV).
  • the infectious agent antigen is from an infectious agent which is capable of causing infection in the GI tract of susceptible host, such as gastroenteritis, optionally a virus such as a Human Rotavirus; a bacterium such as Vibrio cholerae, Campylobacter jejuni, Escherichia coli (including but not limited to ETEC, EHEC, EIEC, EPEC, EAEC, and AIEC), Shigella sp. (including S.
  • viral antigens examples include human papilloma virus (HPV) antigens; coronavirus antigens, such as SARS-CoV-2 coronavirus antigens, such as SARS-CoV-2 spike protein (for example, the coronavirus antigen may be an antigen or multiple combined antigens that confer cross-immunity to 229E, NL63, OC43 and HKU1 coronavirus strains, each of which are relevant for SARS-CoV2); human immunodeficiency virus (HIV) antigens such as products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis, e.g., hepatitis A, B, and C, hepatitis viral antigens such as the S, M, and L proteins of hepatitis, the pre-S antigen of hepatitis B virus; influenza viral antigens hemagglutinin and neuraminidase and
  • coxsackievirus A16 coxsackievirus A16 antigens.
  • Antigens of viruses that cause chronic persistent infection may be preferred, such as human papillomavirus (HPV); hepatitis C; hepatitis B; human immunodeficiency virus (HIV); herpesviruses including herpes simplex virus 1, herpes simplex virus 2 and varicella zoster virus.
  • the at least one antigen is an HPV antigen corresponding to an E1, E2, E4, E5, E6 and/or E7 protein, preferably an E1, E2, E4, E5, E6 and/or E7 protein that is conserved across one or more high-risk HPV genotypes, such as described in WO 2019/034887.
  • the HPV antigen comprises the amino acid sequence of amino acids 2 to 152 of SEQ ID NO: 1, or amino acids 2 to 140 of SEQ ID NO: 1, such as wherein the HPV antigen is encoded by nucleotides 19 to 477 of the nucleic acid sequence of SEQ ID NO: 2.
  • bacterial antigens include clostridium bacterial antigens such as Clostridium difficile (renamed Clostridioides difficile) toxin A and B; pertussis bacterial antigens such as pertussis toxin; diptheria bacterial antigens such as diptheria toxin or toxoid erythematosis, and other diptheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other bacterial antigen components; streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; Gram-negative bacilli bacterial antigens, Mycobacterium tuberculosis bacterial antigens such as heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A and other mycobacterial antigen components; Vibrio cholerae bacterial antigens such as the Cholera toxin B
  • bacterial antigens described herein are any other bacterial mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.
  • Antigens of bacteria which cause chronic persistent infection may be preferred, such as those of Mycobacterium tuberculosis, Borrelia species such as B. burgdorferi, Corynebacterium diphtheriae, Chlamydia, Vibrio cholerae, Salmonella enterica serovar Typhi; mycoplasma.
  • Fungal antigens which can be used include but are not limited to Candida fungal antigen components; Histoplasma fungal antigens, Coccidiodes fungal antigens such as spherule antigens and other Coccidiodes antigens; cryptococcal fungal antigens and other fungal antigens.
  • protozoal and other parasitic antigens include but are not limited to antigens from Plasmodium species which cause malaria, such as P.
  • tumour antigens may be used, which may be categorised as tumour-associated antigens (e.g. overexpressed proteins, differentiation antigens or cancer/testis antigens), or as tumour-specific antigens (e.g. oncoviral antigens, shared neoantigens or private neoantigens).
  • tumour-associated antigens e.g. overexpressed proteins, differentiation antigens or cancer/testis antigens
  • tumour-specific antigens e.g. oncoviral antigens, shared neoantigens or private neoantigens.
  • cancer/testis antigens are normally expressed only in immune privileged germline cells (e.g. MAGE-A1, MAGE-A3, and NY-ESO-1); differentiation antigens refers to cell lineage differentiation antigens that are not normally expressed in adult tissue (e.g. tyrosinase, gp100, MART-1, prostate specific antigen (PSA)); and overexpressed antigens simply refer to antigens that are expressed in cancer cells above healthy or normal levels (e.g. hTERT, HER2, mesothelin, and MUC-1) (Hollingsworth & Jansen (2019), npj Vaccines, 4(7)).
  • immune privileged germline cells e.g. MAGE-A1, MAGE-A3, and NY-ESO-1
  • differentiation antigens refers to cell lineage differentiation antigens that are not normally expressed in adult tissue (e.g. tyrosinase, gp100, MART-1, prostate specific antigen (PSA)); and
  • cancer antigens or tumour antigens may include, but are not limited to, K-Ras, survivin, dystroglycan, KS [1/4] pan-carcinoma antigen, ovarian carcinoma antigen (CA125), prostatic acid phosphate, PSA, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumour-associated antigens such as: CEA, TAG-72, CO17-1A; GICA 19-9, CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19, human B-lymphoma antigen-CD20, CD33, melanoma specific antigens such as ganglioside GD2, ganglioside
  • the cancer antigen or tumour antigen is a multi-antigen fusion polypeptide or recombinant overlapping peptide (ROP) for K-Ras, PSA or survivin.
  • the bacterial antigen is a Vibrio cholerae antigen.
  • V. cholerae is a diarrhoeagenic intestinal pathogenic bacterium and is the etiological agent of Cholera. Suitable V. cholerae antigens include peptides or proteins associated with or secreted by the V. cholerae bacterium. During V.
  • the bacterium secretes the cholera toxin, a heteropolymeric holotoxin consisting of one copy of the A subunit, CtxA P01555 (UniProtKB); and five copies of the B subunit, CtxB P01556 (UniProtKB).
  • the CtxA subunit catalyzes the ADP-ribosylation of Gs alpha, a GTP- binding regulatory protein, to activate the adenylate cyclase. This leads to an overproduction of cAMP and eventually to a hypersecretion of chloride and bicarbonate followed by water, resulting in the characteristic cholera stool.
  • cholerae antigens include CtxB P01556 and TcpA Q60153.
  • An exemplary CtxB antigen is VEVPGSQHIDSQKKAIERMKDTLRIA (SEQ ID NO: 149), which is derived from the P01556 native sequence.
  • An exemplary CtxB antigen is VEVPGSQHIDSQKKAIERMKNTLRIA (SEQ ID NO: 110), which has a D21N substitution compared to the native P01556 sequence.
  • a further exemplary CtxB antigen is ASLAGKREMAIITFKNGAIFQV (SEQ ID NO: 147), as disclosed in Guyon-Gruaz et al., 1986, Eur. J.
  • the antigen comprises a V. cholerae antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of CtxB, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 110, 147 or 149.
  • the antigenic portion comprising SEQ ID NO: 147 or 149 may comprise a greater portion of the complete CtxB polypeptide as provided in SEQ ID NO: 29, such as the complete CtxB.
  • the antigenic portion comprising SEQ ID NO: 110 may comprise a greater portion of the complete CtxB polypeptide as provided in SEQ ID NO: 150, such as the complete CtxB.
  • the CtxB- protein sequence (SEQ ID NO: 29) was determined from the UniProtKB submission P01556 with removal of the signal sequence (MIKLKFGVFFTVLLSSAYAHG (SEQ ID NO: 148)).
  • the V. cholerae antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of any antigenic portion of CtxB comprising SEQ ID NO: 110, 147 or 149, such as SEQ ID NO: 29 or 150.
  • a secreted antigen comprising a V. cholerae antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 27 to 129 of SEQ ID NO: 205.
  • the first 26 amino acid residues of SEQ ID NO: 205 is predicted to be the signal peptide (amino acids 1 – 20; SEQ ID NO: 41) and cleavage site (amino acids 21 – 26; SEQ ID NO: 63); and thus the precursor of the secreted antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 129 of SEQ ID NO: 205.
  • a surface-tethered antigen comprising a V.
  • cholerae antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 22 to 231 of SEQ ID NO: 30.
  • the first 21 amino acid residues of SEQ ID NO: 30 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 231 of SEQ ID NO: 30.
  • Human Rotavirus In some embodiments, the infectious agent antigen is a human rotavirus (HRV) antigen.
  • Human Rotavirus is the most common cause of diarrhoeal disease among infants and young children, causing severe gastroenteritis and contributes to a significant number of infant deaths across the world, with higher prevalence in low- and middle-income countries.
  • the HRV genome codes for six structural proteins (VP1- VP4, VP6 and VP7) and six non-structural proteins (NSP1-NSP6).
  • the infectious virion particle is formed from 3 layers of protein, whereby the outer later (outer capsid) consists of the surface proteins, VP7 and VP4, which contribute to the classification of serotypes.
  • the glycoprotein Viral Protein 7 (VP7) defines the G serotype and the protease sensitive VP4 defines the P serotype.
  • the VP4 protein protrudes on the cell surface of the virion as a spike and is involved in binding the human cell receptors including sialoglycans (such as Gangliosides GM1 and GD1a) and histo-blood group antigens (HBGAs), driving entry into the cell.
  • the VP4 is cleaved by trypsin (found in the human gut) into VP5 and VP8 to allow the virus particle to become infectious.
  • trypsin found in the human gut
  • the majority of the HRV virion capsid is made up of VP7 and VP4. Accordingly, these proteins are important in rotavirus immunity, containing epitopes for T-cell and B-cell activation as well as antigenic sites responsible for neutralisation of the virus.
  • An exemplary VP8 antigen is SEQ ID NO: 78: LDGPYQPTTFTPPNDYWILINSNTNGVVYESTNNSDFWTAVVAIEPHVNPVDRQYTIFGE SKQFNVSNDSNKWKFLEMFRSSSQNEFYNRRTLTSDTRFVGILKYGGRVWTFHGETPRAT TDSSSTANLNNISITIHSEFYIIPRSQESKCNEYINNGLPPG
  • An alternative VP8 antigen is SEQ ID NO: 155, identified in Mohanty et al., J.
  • the VP8 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of any antigenic portion of VP8 comprising SEQ ID NO: 25.
  • the antigen according to these embodiments may be secreted or surface-tethered.
  • the precursor of a secreted antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 188 of SEQ ID NO: 26.
  • the first 20 amino acids of this sequence are predicted to be the signal peptide, although an alternative signal peptide may be used.
  • the sequence was also modified by the addition of a 6 amino acid sequence (LSGVYA (SEQ ID NO:63)) at the N-terminus and a C-terminal FLAG tag (DYKDDDDK (SEQ ID NO: 46)).
  • LSGVYA 6 amino acid sequence
  • DYKDDDDK SEQ ID NO: 46
  • a sequence coding for the usp45 signal peptide MKKKIISAILMSTILSAAAP (SEQ ID NO: 41) was added at the N-terminus contiguous with the sequence encoding the 6 amino acid sequence.
  • the precursor of a secreted antigen may or may not comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 276 of SEQ ID NO: 24.
  • the first 20 amino acids of this sequence are predicted to be the signal peptide, although an alternative signal peptide may be used.
  • the secreted antigen would be based on the sequence of amino acid residues 21 to 276 of SEQ ID NO: 24.
  • the VP7 antigen may or may not be encoded by the nucleic acid sequence of SEQ ID NO: 27.
  • the HRV antigen is a multi-antigen fusion polypeptide, also referred to as a polyepitope.
  • the HRV antigen comprises one, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the epitopes of SEQ ID NOs: 67-71 and 156-161, such as one, 2, 3, 4 or 5 of the epitopes of SEQ ID NOs: 67-71; or variants or fragments comprising at least 90% sequence identity thereto.
  • the HRV antigen comprises a linker between each epitope, such as a flexible [Gly]4Ser linker (GGGGS; SEQ ID NO: 48).
  • a protease cleavage site may be included between each epitope, such as a cathepsin S cleavage site or any other suitable protease cleavage site as provided herein.
  • the epitopes may be provided in any arrangement in the antigen. Multiple copies of any one or more of the antigens may be provided, such as 2, 3 or 4 copies.
  • the antigen according to these embodiments may be secreted or surface-tethered.
  • a VP7 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 21 to 314 of SEQ ID NO: 77.
  • the first 20 amino acid residues of SEQ ID NO: 77 are predicted to be the signal peptide.
  • the precursor of a secreted antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 314 of SEQ ID NO: 77.
  • the first 20 amino acids of this sequence are predicted to be the signal peptide, although an alternative signal peptide may be used.
  • the secreted antigen would be based on the sequence of amino acid residues 21 to 314 of SEQ ID NO: 77.
  • the precursor or surface-tethered antigen may comprise a LysM domain, such as a LysM domain comprising an amino acid sequence of SEQ ID NO: 42; or amino acids 22 to 200 of SEQ ID NO: 42 or variants or fragments thereof as provided above.
  • the infectious agent antigen is a coronavirus antigen.
  • Coronavirus infection can result in the development of pathologies such as severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19).
  • the viral antigen is a SARS-CoV-2 antigen.
  • SARS-CoV-2 contains four structural proteins, including Spike (S), Envelope (E), membrane (M) and nucleocapsid (N) and at least 6 other non-structural open reading frames (ORFs) including Orf1ab.
  • S protein mediates viral attachment, fusion and entry into the host cell.
  • the SARS-CoV-2 antigen is derived from any one or more strains selected from the Wuhan strain (GenBank: NC_045512.2); SARS-CoV-2 variant strains including but not limited to the alpha variant (GenBank: MZ344997.1), the beta variant (GenBank: MW598419.1), the gamma variant (GenBank: MW642250.1), the delta variant (GenBank: MZ009823.1), the epsilon variant (GenBank: MW453103.1), the zeta variant (GenBank: MW523796.1), the eta variant (GenBank: MW560924.1), the theta variant, the iota variant (GenBank: MW643362.1), the kappa variant (GenBank: MW966601.1), the lambda variant (GenBank: MW850639.1), the mu variant, and the omicron variant (GenBank: OL672836.1); and variants thereof.
  • Wuhan strain Genetic
  • SARS-CoV-2 variant strain it is meant a SARS-CoV-2 strain comprising a genome wherein at one or more positions there have been nucleotide insertions, deletions, or substitutions; and includes transitions and transversions.
  • the insertion, deletion, or substitution may be in a nucleotide sequence that encodes a polypeptide or may be in a non-coding sequence. Where the insertion, deletion, or substitution is in a nucleotide sequence that encodes a polypeptide, the mutation may be conservative or non-conservative.
  • VOC Variants Of Concern
  • Suitable SARS-CoV-2 antigens are derived from any one or more of S, E, M, or N protein or Orf1ab polyprotein.
  • the complete amino acid sequence of the S polypeptide of the Wuhan strain is SEQ ID NO: 3.
  • the amino acid sequence of the S1 subunit of said strain is SEQ ID NO: 4 and the amino acid sequence of the S2 subunit is SEQ ID NO: 5.
  • the amino acid sequences of the E polypeptide of said strain is SEQ ID NO: 15; of the M polypeptide of said strain is SEQ ID NO: 14; of the N polypeptide of said strain is SEQ ID NO: 12; and of the Orf1ab polypeptide of said strain is SEQ ID NO: 16.
  • a suitable antigen may be derived from the receptor binding domain (RBD) of the S1 subunit, such as SEQ ID NO: 6. It may for example include the epitopes TEIYQAGSTPCNGVEGFN (SEQ ID NO: 162) and/ or NLDSKVGGNYNYLYRLFRKSN (SEQ ID NO:178) disclosed in Tai et al., 2020, Cell Mol. Immunol., 16:613-620, DOI: 10.1038/s41423-020-0400-4, or the antigenic fragment SEQ ID NO: 163 disclosed therein.
  • a suitable antigen may be derived from the S1 subunit, such as SEQ ID NO: 8, or derived from the S2 subunit, such as SEQ ID NO: 10.
  • the antigen comprises a SARS-CoV-2 antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of an antigenic portion of S, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 6, 8, 10, 162, 178 or 163.
  • the antigenic portion comprising SEQ ID NO: 6, 8, 162, 178 or 163 may comprise a greater portion of the S polypeptide as provided in SEQ ID NO: 4 or 3, such as the complete sequence of SEQ ID NO: 4 or 3.
  • the antigenic portion comprising SEQ ID NO: 10 may comprise a greater portion of the S polypeptide as provided in SEQ ID NO: 5 or 3, such as the complete sequence of SEQ ID NO: 5 or 3.
  • the SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of any antigenic portion of S comprising SEQ ID NO: 6, 8, 10 or 163.
  • the antigen according to these embodiments may be secreted or surface-tethered.
  • a surface-tethered antigen comprising a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 22 to 401 of SEQ ID NO: 7, or amino acid residues 22 to 339 of SEQ ID NO: 9, or amino acid residues 22 to 273 of SEQ ID NO: 11.
  • the first 21 amino acid residues of SEQ ID NOs: 7 and 9 are predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 401 of SEQ ID NO: 7, or 1 to 339 of SEQ ID NO: 9.
  • the SARS-CoV-2 antigen is a multi-antigen fusion polypeptide, also referred to as a polyepitope.
  • Suitable epitopes derived from the nucleoprotein are: NTASWFTALTQHGKED (SEQ ID NO: 81), DDQIGYYRRATRRIRGGDG (SEQ ID NO: 82), KMKDLSPRWYFYYLGTGPEA (SEQ ID NO: 83), NKDGIIWVATEGALNTPK (SEQ ID NO: 84), LPKGFYAEGSRGGSQASSRSSSRSRNS (SEQ ID NO: 85), AALALLLLDRLNQLESKMSGKGQQQQG (SEQ ID NO: 86), ATKAYNVTQAFGRRGPE (SEQ ID NO: 87), KHWPQIAQFAPSASAFFGMSRI (SEQ ID NO: 88), LTYTGAIKLDDKDPNF (SEQ ID NO: 89), KDQVILLNKHIDAYKTFPPTEPKKD (SEQ ID NO: 90), and FSKQLQQSMSSADSTQ (SEQ ID NO: 91).
  • SARS-CoV-2 epitopes include SQSIIAYTMSLGAEN (SEQ ID NO: 93), NPTTFHLDGEVITFD (SEQ ID NO: 94), IINLVQMAPISAMVR (SEQ ID NO: 95), SYGFQPTNGVGYQPY (SEQ ID NO: 96), APHGVVFLHVTYVPA (SEQ ID NO: 97), DGEVITFDNLKTLLS (SEQ ID NO: 98), VKPSFYVYSRVKNLN (SEQ ID NO: 99), IPTNFTISVTTEILP (SEQ ID NO: 100), VAAIFYLITPVHVMS (SEQ ID NO: 101), ATKAYNVTQAFGRRG (SEQ ID NO: 102), EVRTIKVFTTVDNIN (SEQ ID NO: 103), IASFRLFARTRSMWS (SEQ ID NO: 104), and AAAYYVGYLQPRTFL (SEQ ID NO: 105).
  • the SARS-CoV-2 antigen comprises one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the epitopes of SEQ ID NOs: 81-91, 93 to 105; or variants or fragments comprising at least 90% sequence identity thereto.
  • the SARS-CoV-2 antigen comprises a protease cleavage site between each epitope, such as a cathepsin S cleavage site such as TVKLRQ (SEQ ID NO: 39) or any other suitable protease cleavage site as provided herein.
  • the epitopes may be provided in any arrangement in the antigen.
  • a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 80.
  • a surface-tethered antigen comprising a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 22 to 496 of SEQ ID NO: 13.
  • the first 21 amino acid residues of SEQ ID NO: 13 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 496 of SEQ ID NO: 13.
  • a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 92.
  • a surface-tethered antigen comprising a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 474 of SEQ ID NO: 17.
  • the first 21 amino acid residues of SEQ ID NO: 17 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 474 of SEQ ID NO: 17.
  • a SARS-CoV-2 antigen may comprise a recombinant overlapping peptide, as disclosed in WO 2022/090679.
  • a suitable antigen disclosed therein as SEQ ID NO: 21 and provided herein as SEQ ID NO: 106 comprises 9 overlapping peptides from the S1 receptor binding domain, and 3 peptides from the C-terminal end of S2 HR2 region and proximal region of S2. Each peptide may be regarded as an antigenic portion and is linked to the next by a LRMK sequence. Immunisation studies in mice with an N- terminally His tagged version of the ROP showed high antibody responses.
  • a SARS-CoV-2 antigen may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the antigenic portions of SARS-CoV-2 present in SEQ ID NO: 106, or fragments or variants comprising at least 90% sequence identify thereto, and a protease cleavage site between each antigenic portion, such as a cathepsin S cleavage site, such as LRMK.
  • the SARS-CoV-2 antigen is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 106.
  • Secreted or surface-tethered forms may be prepared with appropriate signal peptide and surface-tethering domains or motifs, as provided herein.
  • Intracellular SARS-CoV-2 antigens are derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or E, M, N, or Orf1ab protein.
  • the S1 and S2 subunits are regarded separately, such that an antigen spanning S1 and S2 subunits (i.e. comprising a S1 sequence which is contiguous with a S2 sequence in the S protein) is excluded.
  • a SARS-CoV-2 antigen comprises only one epitope, it is typically an epitope which has not been disclosed in Fast et al.
  • SARS-CoV-2 antigens comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more antigen portions or epitopes may or may not comprise one or more epitopes disclosed in Li et al. or Fast et al., for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more epitopes.
  • the intracellular SARS- CoV-2 antigen comprises or consists of a sequence X or fragment or variant thereof having at least 90% sequence identity thereto, and either document discloses a sequence Y falling within that scope, it may be excluded that the antigen is Y. This may be the case for each such epitope in a polyepitope antigen.
  • SARS-CoV- 2 antigen comprises an antigen portion or epitope not disclosed in either document, it may comprise one or more further epitopes disclosed in either document.
  • SARS-CoV-2 antigen comprises an antigen portion or epitope disclosed in one document, it may or may not comprise one or more further epitopes disclosed in the other document.
  • KAYNVTQAF (SEQ ID NO: 164), SIIAYTMSL (SEQ ID NO: 165), HLDGEVITF (SEQ ID NO: 166), VQMAPISAM (SEQ ID NO: 167), YGFQPTNGV (SEQ ID NO: 168), VVFLHVTY (SEQ ID NO: 169), ITFDNLKTL (SEQ ID NO: 170), YVYSRVKNL (SEQ ID NO: 171), FTISVTTEI (SEQ ID NO: 172), YLITPVHVM (SEQ ID NO: 173), RTIKVFTTV (SEQ ID NO: 175), RLFARTRSM (SEQ ID NO: 176), VGYLQPRTF (SEQ ID NO: 177), ATKAYNVTQAFGRRG (SEQ ID NO: 102), SKQLQQSMSSADSTQ (SEQ ID NO: 186), TASWFTALTQ
  • Suitable intracellular SARS-CoV-2 antigen amino acid sequences are provided in SEQ ID NOs: 129 (RBD), 131 (nuc), 133 (polyepitope), 135 (SB1) and 137 (SB2). Corresponding coding sequences are provided in SEQ ID NOs: 128, 130, 132, 134 and 136.
  • the intracellular antigen comprises a SARS-CoV-2 antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of an antigenic portion of S1, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 6, 8, 162, 178 or 163.
  • the antigenic portion comprising SEQ ID NO: 6, 8, 162, 178 or 163 may comprise a greater portion of the S1 polypeptide as provided in SEQ ID NO: 4, such as the complete sequence of SEQ ID NO: 4.
  • the intracellular antigen comprises a SARS-CoV- 2 antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of an antigenic portion of S2, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 10.
  • the antigenic portion comprising SEQ ID NO: 10 may comprise a greater portion of the S2 polypeptide as provided in SEQ ID NO: 5, such as the complete sequence of SEQ ID NO: 5.
  • An intracellular antigen comprising a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of SEQ ID NO: 129, or amino acid residues 4 to 197 of SEQ ID NO: 129, or SEQ ID NO: 134, or amino acid residues 4 to 135 of SEQ ID NO: 134, or SEQ ID NO: 137 or amino acid residues 4 to 69 of SEQ ID NO: 137.
  • the intracellular SARS-CoV-2 antigen is a multi-antigen fusion polypeptide, also referred to as a polyepitope.
  • Suitable epitopes derived from the nucleoprotein are: NTASWFTALTQHGKED (SEQ ID NO: 81), DDQIGYYRRATRRIRGGDG (SEQ ID NO: 82), KMKDLSPRWYFYYLGTGPEA (SEQ ID NO: 83), NKDGIIWVATEGALNTPK (SEQ ID NO: 84), LPKGFYAEGSRGGSQASSRSSSRSRNS (SEQ ID NO: 85), AALALLLLDRLNQLESKMSGKGQQQQG (SEQ ID NO: 86), ATKAYNVTQAFGRRGPE (SEQ ID NO: 87), KHWPQIAQFAPSASAFFGMSRI (SEQ ID NO: 88), LTYTGAIKLDDKDPNF (SEQ ID NO: 89), KDQVILLNKHIDAYKTFPPTEPKKD (SEQ ID NO: 90), and FSKQLQQSMSSADSTQ (SEQ ID NO: 91).
  • SARS-CoV-2 epitopes include SQSIIAYTMSLGAEN (SEQ ID NO: 93), NPTTFHLDGEVITFD (SEQ ID NO: 94), IINLVQMAPISAMVR (SEQ ID NO: 95), SYGFQPTNGVGYQPY (SEQ ID NO: 96), APHGVVFLHVTYVPA (SEQ ID NO: 97), DGEVITFDNLKTLLS (SEQ ID NO: 98), VKPSFYVYSRVKNLN (SEQ ID NO: 99), IPTNFTISVTTEILP (SEQ ID NO: 100), VAAIFYLITPVHVMS (SEQ ID NO: 101), ATKAYNVTQAFGRRG (SEQ ID NO: 102), EVRTIKVFTTVDNIN (SEQ ID NO: 103), IASFRLFARTRSMWS (SEQ ID NO: 104), and AAAYYVGYLQPRTFL (SEQ ID NO: 105).
  • the intracellular SARS-CoV-2 antigen comprises one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the epitopes of SEQ ID NOs: 81-91, 93 to 105; or variants or fragments comprising at least 90% sequence identity thereto.
  • the SARS-CoV-2 antigen comprises a protease cleavage site between each epitope, such as a cathepsin S cleavage site such as TVKLRQ (SEQ ID NO: 39) or any other suitable protease cleavage site as provided herein.
  • the epitopes may be provided in any arrangement in the antigen.
  • An intracellular SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 80, SEQ ID NO: 92, SEQ ID NO: 131, amino acid residues 4 to 292 of SEQ ID NO: 131, SEQ ID NO: 133 or amino acid residues 4 to 270 of SEQ ID NO: 133.
  • An intracellular SARS-CoV-2 antigen may comprise a recombinant overlapping peptide, as disclosed in WO 2022/090679, suitably SEQ ID NO: 106.
  • An intracellular SARS- CoV-2 antigen may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the antigenic portions of SARS-CoV-2 present in SEQ ID NO: 106, or fragments or variants comprising at least 90% sequence identify thereto, and a protease cleavage site between each antigenic portion, such as a cathepsin S cleavage site, such as LRMK.
  • the SARS-CoV-2 antigen is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 106.
  • Campylobacter In some embodiments, the infectious agent antigen is a Campylobacter jejuni antigen.
  • the infectious agent antigen is a Campylobacter coli antigen.
  • C. jejuni and C. coli are diarrhoeagenic intestinal pathogenic bacteria.
  • Suitable C. jejuni antigens include peptides or proteins associated with or secreted by the C. jejuni or C. coli bacterium.
  • the bacteria express flagella, which are complexes comprising a basal membrane-bound secretion body and motor; and an extracellular flagellum comprising the major flagellin, FlaA, the flagella cap, FliD, the hook protein, FlgE, and the hook-filament protein, FlgK.
  • flagella which are complexes comprising a basal membrane-bound secretion body and motor
  • an extracellular flagellum comprising the major flagellin, FlaA, the flagella cap, FliD, the hook protein, FlgE, and the hook-filament protein, FlgK.
  • MOMP major outer membrane protein
  • CadF is a fibronectin-binding protein, portions of which are displayed on the bacterial cell surface, that promotes binding of C. jejuni and C. coli bacteria to the surface of cells in the gastrointestinal tract.
  • FliD contains several immunodominant epitopes, which sera from broilers react to, suggesting application in vaccines (Hung-Yueh Yeh et al Comparative Immunology, Microbiology and Infectious Diseases, Volume 49, 2016, Pages 76-81, ISSN 0147- 9571, https://doi.org/10.1016/j.cimid.2016.10.003).
  • FlaA, MOMP, PEB3, CadF peptides have been determined via in silico predictions to generate potent B cell and T cell epitopes (Yasmin et al., 2016 In Silico Pharmacol., 4:5, DOI: 10.1186/s40203- 016-0020-y).
  • FlgK, FlgE flagellar antigens with extracellular locations which produce antibody reactions in chicken sera and reduced bacterial load in chickens immunised by the recombinant proteins (Yeh et al., 2015, Arch. Microbiol., 197:353-358, DOI: 10.1007/s00203-014-1062-3).
  • Flagellin used as a vaccine candidate can produce antigen specific antibody responses (IgY, IgM and IgA) as well as reduce bacterial loads in challenge models (Lee et al., 1999, Infect. Immun., 67(11):5799-5805, DOI: 10.1128/iai.67.11.5799-5805.1999; Chintoan-Uta et al., 2016, Vaccine, 34(15):1734- 1743, DOI: 10.1016/j.vaccine.2016.02.052).
  • Exemplary antigens have been designed taking into account these disclosures. Accordingly, in some embodiments, the C.
  • jejuni antigen is selected from: FlicC, FlaA, FliD, FlgE, FlgK, MOMP, and CadF.
  • the C. jejuni antigen is FlaA or FliD.
  • the C. jejuni antigen is derived from a strain selected from: C. jejuni NCTC11168 (GenBank: NZ_CP046317), C. jejuni 81-176 (GenBank: AY681239 to AY681296), C. jejuni ST50, C. jejuni ST-257, and C. jejuni ST51.
  • the C. coli antigen is derived from a strain selected from C. coli (GenBank: NZ_CP046317). Suitable C.
  • jejuni antigens include FlaA (WP_011812789.1; SEQ ID NO: 151) and FliD (WP_002864504; SEQ ID NO: 152).
  • An exemplary FlaA antigen is SEQ ID NO: 113: GFRINTNVAALNAKANSDLNAKSLDASLSRLSSGLRINSAADDASGMAIADSLRSQANTLGQAISNGNDALG ILQTADKAMDEQLKILDTIKTKATQAAQDGQSLKTRTMLQADINKLMEELDNIANTTSFNGKQLLSGNFTNQ EFQIGASSNQTVKATIGATQSSKIGVTRFETGAQSFTSGVVGLTIKNYNGIEDFKFDNVVISTSVGTGLGAL AEEINKSADKTGVRATYDVKTTGVYAIKEGTTSQEFAINGVTIGKIEYKDGDGNGSLISAINAVKDTTGVQA SKDENGKLVLTSADGRGIKITGDIGVGSGILANQKENYGRLSLVKNDGRDINIS
  • jejuni antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of FlaA, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 113.
  • the antigen according to these embodiments may be secreted or surface-tethered.
  • a surface-tethered antigen comprising a C. jejuni antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 780 of SEQ ID NO: 114.
  • the first 21 amino acid residues of SEQ ID NO: 114 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 780 of SEQ ID NO: 114.
  • FliD antigen is SEQ ID NO: 115: AFGSLSSLGFGSGVLTQDTIDKLKEAEQKARIDPYTKKIEENTTKQKDLTEIKTKLLSFQTAVSSLADATVF AKRKVVGSISDNPPASLTVNSGVALQSMNINVTQLAQKDVYQSKGLANDGGFVNAQLNGTADLTFFSNGKEY TVTVDKNTTYRDLADKINEASGGEIVAKIVNTGEKGTPYRLTLTSKETGEDSAISFYAGKKDSNGKYQKDIN AEKIFDDLGWGLDVSASIDPDKDKKGYGIKDASLHIQTAQNAEFTLDGIKMFRSSNTVTDLGVGMTLTLNKT 5 GEINFDVQQDFEGVTKAMQDLVDAYNDLVTNLNAATDYNSETGTKGTLQGISEVNSIRSSILADLFDSQVVD GTTEDANGNKVNTKVMLSMQDFGLSLNDAGTLSFDSSKFEQKVKE
  • jejuni antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of FliD, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 115.
  • the antigen according to these embodiments may be secreted or surface-tethered.
  • a surface-tethered antigen comprising a C. jejuni antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 846 of SEQ ID NO: 116.
  • the first 21 amino acid residues of SEQ ID NO: 116 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 846 of SEQ ID NO: 116.
  • the C. jejuni antigen is a multi-antigen fusion polypeptide, also referred to as a polyepitope.
  • Suitable epitopes of FlaA are: LQTADKAMDEQLKILDTIKTKATQAAQDGQSLKTRTMLQADIN (SEQ ID NO: 117), VVISTSVGTGLGALA (SEQ ID NO: 118), ENYGRLSLVKNDGRDIN (SEQ ID NO: 119), and AGVTTLKGAMAVMDIAETAITNLDQIRADIGS (SEQ ID NO: 120).
  • Suitable epitopes of FliD are: NSGVALQSMNINVTQ (SEQ ID NO: 121), DLGVGMTLTLNKTGE (SEQ ID NO: 122), DGTTEDANGNKVNTK (SEQ ID NO: 123), EDANGNKVNTKVMLS (SEQ ID NO: 124), and ESILNKLNQQLNTVT (SEQ ID NO: 125).
  • the C. jejuni antigen comprises one, 2, 3, or all 4 of the epitopes of SEQ ID NOs: 117-120; or variants or fragments comprising at least 90% sequence identity thereto.
  • the C. jejuni antigen comprises one, 2, 3, or all 4 of the epitopes of SEQ ID NOs: 117-120; or variants or fragments comprising at least 90% sequence identity thereto.
  • the C. jejuni antigen comprises one, 2, 3, or all 4 of the epitopes of SEQ ID NOs: 117-120; or variants or fragments comprising at least 90%
  • jejuni antigen comprises one, 2, 3, 4, or all 5 of the epitopes of SEQ ID NOs: 121-125; or variants or fragments comprising at least 90% sequence identity thereto.
  • the C. jejuni antigen comprises one, 2, 3, or all 4 of the epitopes of SEQ ID NOs: 117-120; or variants or fragments comprising at least 90% sequence identity thereto, and one, 2, 3, 4, or all 5 of the epitopes of SEQ ID NOs: 121-125; or variants or fragments comprising at least 90% sequence identity thereto.
  • the C. jejuni antigen comprises one, 2, 3, 4, or all 5 of the epitopes of SEQ ID NOs: 121-125; or variants or fragments comprising at least 90% sequence identity thereto.
  • jejuni antigen comprises a linker between each epitope, such as a flexible [Gly]4Ser linker (GGGGS; SEQ ID NO: 48).
  • a protease cleavage site may be included between each epitope, such as a cathepsin S cleavage site or any other suitable protease cleavage site as provided herein.
  • the epitopes may be provided in any arrangement in the antigen. Multiple copies of any one or more of the antigens may be provided, such as 2, 3 or 4 copies.
  • the antigen according to these embodiments may be secreted or surface-tethered.
  • a FlaA antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 142.
  • a surface tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 454 of SEQ ID NO: 126.
  • the first 21 amino acids of this sequence are predicted to be the signal peptide; and thus the precursor of the surface- tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 454 of SEQ ID NO: 126.
  • a FliD antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 143.
  • a surface tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 400 of SEQ ID NO: 127.
  • the first 21 amino acids of this sequence are predicted to be the signal peptide; and thus the precursor of the surface- tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 400 of SEQ ID NO: 127.
  • Clostridia includes the orders Clostridiales, Halanaerobiales and Thermoanaerobacteriales.
  • the order Clostridiales includes the family Clostridiaceae, which includes the genus Clostridium. Clostridium is one of the largest bacterial genera. The genus is defined by rod-shaped, Gram-positive bacteria that are obligate anaerobes and capable of producing spores.
  • the Clostridial bacterium or Clostridium species is capable of forming spores.
  • Clostridium species are known to be responsible for human diseases due to the formation of toxins, https://doi.org/10.1533/9781845696337.2.820. These include C. difficile, C. botulinum, C. novyi and C. perfringens. C. difficile is included in Clostridium cluster XIa.
  • the species is not a pathogenic Clostridium species. It may or may not be an attenuated strain from such a pathogenic species, such a natural or engineered strain which does not produce toxins. Typically, attenuated strains from pathogenic species may undesirably colonise the GI tract, and may therefore preferably be avoided.
  • Several Clostridium species are found in the human lower gastrointestinal tract.
  • Clostridium cluster XIVa also known as the Clostridium Coccoides group
  • Clostridium cluster IV also known as the Clostridium leptum group
  • the Clostridium cluster XIVa includes species belonging to the Clostridium, Eubacterium, Ruminococcus, Coprococcus, Dorea, Lachnospira, Roseburia and Butyrivibrio genera.
  • Clostridium cluster IV is composed by the Clostridium, Eubacterium, Ruminococcus and Anaerofilum genera.
  • the Clostridial bacterium is a Cluster I Clostridium.
  • Pathogenic Cluster I species include C. botulinum, C.
  • Clostridium cluster I species found in the human gut are associated with disease whilst the majority are generally considered to contribute to health and wellbeing.
  • the bacteria selected from Cluster I are species associated with health benefits. These species include C. sporogenes and C. butyricum.
  • the bacterium is from cluster I, IV and/or XIVa of Clostridia, typically a non- pathogenic species.
  • the bacterium is detectable in the lower gastrointestinal tract, for example of a human, but not considered to permanently colonise or form part of the resident microbiota in the lower GI tract, for example of a human.
  • the bacterium does not colonise the GI tract. It has been observed that Clostridium that are capable of colonising the GI tract – for example, C. perfringens – are also capable of binding and/or degrading mucins (Deplancke et al., 2002, Am. J. Clin. Nutr., 76(5):1117-1125; Li and McClane, 2018, Infect. Immun.
  • the bacterium is not capable of binding and/or degrading mucins.
  • Exemplary methods of determining mucin binding and/or degradation are disclosed in Deplancke et al. (2002), Li and McClane (2016), and Low et al, 2021.
  • a suitable non- colonising species is C. butyricum.
  • C. butyricum is capable of binding mucin despite not colonising the GI tract.
  • the bacterium is capable of growing in an anaerobic section of the lower gastrointestinal tract and/or the bacterium is saccharolytic and can utilise di- and tri- saccharides in the colon.
  • the ability to grow in an anaerobic section of the lower GI tract, such as colon or terminal ileum, may be related to saccharolytic metabolism.
  • a suitable saccharolytic species is C. butyricum, which can utilise di- and tri-saccharides found in the colon.
  • Further saccharolytic species include C. acetobutylicum, C. rouseum, C. sacchrolyticum, C. saccharoperbutylacetonicum, C. pasteurianum, C. aurantybutylicum, and C.
  • C. acetobutylicum, C. rouseum, C. sacchrolyticum, C. saccharoperbutylacetonicum, C. pasteurianum, C. aurantybutylicum, and C. beijerinckii are not usually found in the human gut.
  • C. perfringens is proteolytic and may rely more on amino acids which perhaps are more readily available in the terminal small intestine. Saccharolytic Clostridia are capable of fermenting organic sugars to acids and/or solvents, and can further be distinguished from proteolytic strains by lack of ability to generate ATP via Stickland reactions, as described in in Mitchell, 1992, Res.
  • the bacterium expresses Tat-dependent secretion pathway, comprising TatA, TatB, and TatC.
  • the bacterium may comprise accessory factors allowing for such tethering. These may include a secretion system or accessory factors allowing for secretion of the antigen as provided above.
  • the bacterium expresses a Sortase system, comprising SrtA, SrtB, SrtC, and/or SrtD.
  • Sortase system is reviewed in Dramsi et al., 2008, FEMS Microbio. Rev.32(2):307- 320.
  • Clostridia sp. carry Class A (StrA), Class B (StrB), Class C (StrC), and Class D (StrD) Sortase systems; however, Sortase systems may be species-specific.
  • Clostidium difficile has a single sortase, StrB (van Leeuwen et al., 2014, FEBS Lett., 588(23):4325-33, DOI: 10.1016/j.febslet.2014.09.041; Donahue et al., 2014, BMC Microbiol., 14:219, doi: 10.1186/s12866-014-0219-1); whereas Clostridium perfringens carries both Class A and Class B Sortase systems (Tami et al., 2021, Biochem. Biophys.
  • Butyrate production is widely distributed among anaerobic bacteria belonging to the Clostridial sub-phylum and in particular, to the Clostridial clusters XIVa and IV. Butyrate-producing species are found within two predominant families of commensal human colonic Clostridia, Ruminococcaceae and Lachnospiraceae. https://doi.org/10.1111/1462-2920.13589.
  • Lachnospiraceae Within the Lachnospiraceae are included: Eubacterium rectale, Roseburia inulinivorans, Roseburia intestinalis, Dorea longicatena, Eubacterium hallii, Anaerostipes hadrus, Ruminococcus torques, Coprococcus eutactus, Blautia obeum, Dorea formicigenerans, Coprococcus catus, Within the Ruminococcaceae are included: Faecalibacterium prausnitzii, Subdoligranulum variabile, Ruminococcus bromii, Eubacterium siraeum.
  • the bacterial species produces butyric acid.
  • Butyrate-producing species include Clostridium butyricum.
  • the species is amenable to genetic engineering techniques such as transformation by electroporation or conjugation, and is typically a non-pathogenic strain.
  • Known transformable strains include industrial solvent strains including C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and C. saccharolyticum and pathogenic species including C. difficile.
  • the species is C. butyricum.
  • Suitable strains include the ‘Rowett’ strain, also referred to as (DSM10702/ATCC19398/NCTC 7423).
  • SCFAs short-chain fatty acids
  • heterologous nucleic acid molecule we mean that the nucleic acid molecule comprises one or more non-native sequences such as in the open reading frame (ORF) encoding an antigen, although it is alternatively envisaged that a native antigen coding-sequence could be provided under the control of non-native control sequences, such as to facilitate an increased level of gene expression of a native antigen during anaerobic cell growth.
  • the heterologous nucleic acid molecule comprises a gene, i.e. an ORF operatively linked to a promoter, which drives transcription of the gene.
  • Other control sequences may also be present, as known in the art (Minton et al.
  • the heterologous nucleic acid molecule may comprise a non- native gene.
  • non-native gene refers to a gene that is not in its natural environment and includes a gene from one species of a microorganism that is introduced into another species of the same genus.
  • cassette includes any heterologous nucleic acid molecule as described herein, optionally where the heterologous nucleic acid molecule comprises one or more non-native sequences including but not limited to an ORF encoding an antigen; an ORF operatively linked to a promoter; other control sequences; a non-native gene; or any combination thereof.
  • Anaerobic vegetative cell metabolism may be assessed by production of SCFAs, such as butyrate, acetate, lactate or combinations thereof from an available carbohydrate source.
  • SCFAs such as butyrate, acetate, lactate or combinations thereof from an available carbohydrate source.
  • a fermentable substrate such as a carbohydrate substrate like glucose
  • SCFAs such as butyrate, acetate, lactate or combinations thereof
  • the expressions “anaerobic cell growth” and “anaerobic vegetative cell metabolism” may be used interchangeably.
  • the promoter is selected to be active in metabolically active or growing cells. Suitable promoters are active during cell growth and may be constitutive promoters. Promoters of genes that are essential to primary metabolism may be suitable constitutive promoters.
  • the expression level of the antigen can be optimised by controlling gene expression using a promoter having a selected strength, such as a strong promoter.
  • Promoter activity can be assessed using a gene expression reporter system, such as described in Tummala et al (1999) App. Environ. Microbiol. 65: 3793- 3799.
  • a native Clostridia promoter is used.
  • Suitable promoters include the p0957 promoter of C. acetobutylicum described in the Examples which is based on SEQ ID NO: 181, and which further includes the ribosome binding site (RBS) from fdx as provided in SEQ ID NO: 182.
  • acetobutylicum promoters hbd, crt, etfA, etfB amd bcd (Alsaker and Papoutsakis (2005) J Bacteriol 187:7103-7118); and the fdx promoter from C. sporogeneses (NCIMB 10696), which can be obtained from the pMTL80000 modular shuttle plasmid (Heap et al. (2009) A modular system for Clostridium shuttle plasmids, Journal of Microbiological Methods, 78:79-85).
  • the heterologous nucleic acid molecule can be introduced into Clostridia using methods known in the art.
  • Plasmid transformation is typically achieved in Clostridia by conjugation or transformation. Methods of transformation and conjugation in Clostridia are provided in Davis, I, Carter, G, Young, M and Minton, NP (2005) “Gene Cloning in Clostridia”, In: Handbook on Clostridia (Durre P, ed) pages 37-52, CRC Press, Boca Raton, USA.
  • the heterologous nucleic acid molecule may be integrated into the genome, typically the chromosome of Clostridia, using gene integration technology, such as by Allele Coupled Exchange (ACE) as described in WO 2010/084349 and Minton et al (2016) Anaerobe 41: 104-112; or CRISPR gene editing (Atmadjaja et al. (2019) CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT), FEMS Microbiol. Lett. 366(6)).
  • ACE Allele Coupled Exchange
  • the initial creation of the pyrE deletion may be performed by a special form of ACE, as described in Minton et al, supra, or by means of retargeting mobile group II introns as described in WO 2007/148091.
  • CRISPR gene editing also has wide application in Clostridia for integration of large DNA fragments and has been successfully applied in a number of Clostridial strains, including C. acetobutylicum (Li et al. (2016) CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Biotechnol J. 11:961–72), C. beijerinckii (Li et al. (2016) and Wang et al.
  • the amount of antigen expressed will typically be lower than if the heterologous nucleic acid molecule is present on a high copy number plasmid.
  • the inventors have found in PCT Patent Application No. PCT/GB2021/053264 that an antigen-specific immune response can be effectively stimulated even when the heterologous nucleic acid molecule is integrated into the genome as a single copy.
  • the amount of antigen expressed per cell weight of clostridial cells undergoing anaerobic cell growth may typically be in the range of up to 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 ng/mg, 1 ⁇ g/ mg, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10 or 20 ⁇ g/ mg dry cell weight (but greater than 0 ng/ mg dry cell weight, typically greater than 10 ng/mg, 20 ng/mg or 40 ng/mg). Any range between any two of these values is envisaged.
  • the amount of antigen expressed per cell weight of clostridial cells undergoing anaerobic cell growth may be from 10 to 400 ng/ mg dry cell weight; 20 to 200 ng/ mg dry cell weight; 40 to 100 ng/ mg dry cell weight; 100 ng to 5 ⁇ g/ mg dry cell weight; 200 ng to 2.5 ⁇ g/ mg dry cell weight; 400-1500 ng/ mg dry cell weight; or about 800 ng/ mg dry cell weight; or it may be between 1 and 5 ⁇ g/ mg dry cell weight, or 2 and 4 ⁇ g/ mg dry cell weight, such as about 3 ⁇ g/ mg dry cell weight; or any other combination.
  • the amount can be determined by culturing clostridial cells, obtaining antigen typically comprising a detection tag such as FLAG, and quantifying the antigen by detection means such as ELISA or western blotting.
  • Antigen may be obtained by extraction from bacterial cells, or recovery from the culture medium as appropriate. Protein standards, such as FLAG- tag standards available from Sigma, may be used in such assays to construct a standard curve.
  • the amount of antigen is determined for a specific volume of cells cultured to a specific density e.g. OD1.0. The amount of antigen per dry weight of bacteria in that amount of culture is then estimated assuming the cell density in OD1.0 is 0.3 g/L.
  • the amount of antigen produced may be varied depending on the strength of the promoter, the number of copies of the heterologous nucleic acid molecule per cell etc.
  • the bacterial cell may comprise a further heterologous nucleic acid molecule encoding an immunostimulatory agent or adjuvant, which is capable of being co-expressed with the antigen.
  • Typical immunostimulatory agents may be polypeptides, such as cytokines, such as IL-12, IL-18 or GM-CSF, IFN- ⁇ , IL-2, IL-15.
  • cytokines such as IL-12, IL-18 or GM-CSF, IFN- ⁇ , IL-2, IL-15.
  • HPV16 and HPV18 E6/E7 antigens have been combined with IL- 12 in clinical trials (Hasan et al.
  • a corresponding aspect of the invention provides a method for preparing a bacterium according to the first aspect and the second aspect comprising introducing at least one heterologous nucleic acid molecule into the bacterium. It will be appreciated that where accessory factors such as sortase systems etc are not already present, these may also be introduced by means of at least one heterologous nucleic acid molecule.
  • a related aspect of the invention provides a nucleic acid molecule suitable for propagation in a bacterium of the class Clostridia comprising an antigen gene comprising a region encoding at least one surface-tethered antigen precursor and a promoter operably linked to said region, which promoter is capable of causing expression of the surface-tethered antigen precursor by a bacterium of the class Clostridia; wherein the antigen precursor comprises an N-terminal signal peptide and a domain or motif required for tethering of the antigen to peptidoglycan in a bacterium of the class Clostridia.
  • the domain or motif is a peptidoglycan anchoring domain or a sequence required for enzymatic linkage to peptidoglycan, such as wherein the peptidoglycan anchoring domain comprises at least one LysM motif, such as a LysM domain.
  • the promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth, such as wherein the promoter is the p0957 promoter of C. acetobutylicum, for example selected from SEQ ID NO: 181 and SEQ ID NO: 182, fdx promoter of C.
  • a third aspect of the invention is a pharmaceutical composition comprising a bacterium according to the first aspect or comprising the bacterium according to the second aspect.
  • a corresponding aspect of the invention provides a method for preparing a pharmaceutical composition according to the third aspect comprising formulating the bacteria with one or more pharmaceutically acceptable diluents or excipients.
  • the present invention includes pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier, excipient or further component such as therapeutic and/or prophylactic ingredient (such as adjuvant).
  • a “pharmaceutically acceptable carrier” as referred to herein is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • the carrier may include one or more excipients or diluents.
  • the Clostridia can be prepared by fermentation carried out under suitable conditions for growth of the bacteria. After fermentation, the bacteria can be purified using centrifugation and prepared to preserve activity. The bacteria in the composition are provided as viable organisms.
  • the composition can comprise bacterial spores and/or vegetative cells.
  • the bacteria can be dried to preserve the activity of the bacteria. Suitable drying methods include freeze drying, spray-drying, heat drying, and combinations thereof.
  • the obtained powder can then be mixed with one or more pharmaceutically acceptable excipients as described herein.
  • the spores and/or vegetative bacteria may be formulated with the usual excipients and components for oral administration, as described herein.
  • fatty and/or aqueous components, humectants, thickeners, preservatives, texturing agents, flavour enhancers and/or coating agents, antioxidants, preservatives and/or dyes that are customary in the pharmaceutical and food supplement industry.
  • Suitable pharmaceutically acceptable carriers include microcrystalline cellulose, cellobiose, mannitol, glucose, sucrose, lactose, polyvinylpyrrolidone, magnesium silicate, magnesium stearate and starch, or a combination thereof.
  • the bacteria can then be formed into a suitable orally ingestible forms, as described herein.
  • Suitable orally ingestible forms of probiotic bacteria can be prepared by methods well known in the pharmaceutical industry.
  • Suitable pharmaceutical carriers, excipients and formulations are described in Remington: The Science and Practice of Pharmacy 22nd Edition, The Pharmaceutical Press, London, Philadelphia, 2013.
  • Pharmaceutical compositions of the invention can be placed into dosage forms, such as in the form of unit dosages. Pharmaceutical compositions include those suitable for oral or rectal administration.
  • compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen.
  • administration is oral using a convenient dosage regimen.
  • suitable oral dosage forms include tablet, capsule, powder (e.g. a powder in sachet) and liquid.
  • the bacterium is for administering orally, it is suitably provided in the form of a spore; or in the form of a vegetative cell in a delayed release pharmaceutical composition.
  • Pharmaceutical compositions of the invention can also be formulated for rectal administration including suppositories and enema formulations.
  • a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized moulds, allowed to cool, and to solidify.
  • Enema formulations can be semi-solid including gels or ointments or in liquid form including suspensions, aqueous solutions or foams, which are known to those skilled in the art.
  • the pharmaceutical compositions of the invention are administered such that an effective amount of bacterium is delivered to an anaerobic section of the gut.
  • an amount of bacterium we include the meaning that the bacterium results in the delivery of an amount of antigen effective to induce a suitable immune response to said antigen; or to prevent, ameliorate or treat a disease.
  • the antigen will be in an amount effective to induce a CD8 + CTL response against that antigen.
  • the bacteria may be present in the pharmaceutical composition in an amount equivalent to between 1x10 5 to 1x10 11 colony forming units/g (CFU/g) of dry composition.
  • the bacteria may be present in an amount of 1x10 6 to 1x10 10 CFU per unit dosage form, preferably from about 1x10 7 to 1x10 9 CFU per unit dosage form, such as about 1x10 8 CFU per unit dosage form.
  • Pharmaceutical compositions may include adjuvants or immunostimulatory molecules, particularly pharmaceutical compositions that are formulated for delayed release. However, it is envisaged that an adjuvant may not be necessary, or may be necessary only in a quantity that is lower than would be required if the antigen were provided in a conventional polypeptide antigen vaccine formulation, or that a less toxic adjuvant only may be required.
  • compositions which lack an adjuvant are envisaged, as are those which contain only an adjuvant which is appropriate for human use, such as alum.
  • adjuvants are any substance whose admixture into the pharmaceutical composition increases or otherwise modifies the immune response to an antigen.
  • Adjuvants can include but are not limited to AlK(SO4)2, AlNa(SO4)2, AlNH(SO4)4, silica, alum, AI(OH)3, Ca3(PO4)2, kaolin, carbon, aluminium hydroxide, muramyl dipeptides, N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D- isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-s-n-glycero-3-hydroxphosphoryloxy)- ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2%
  • TLRs Toll-like receptors
  • LPS Bacterial lipopolysaccharide
  • MPL mono-phosphoryl lipid A
  • TLR5 is expressed on monocytes and DCs and responds to flagellin whereas TLR9 recognizes bacterial DNA containing CpG motifs.
  • Oligonucleotides (OLGs) containing CpG motifs are potent ligands for, and agonists of, TLR9 and have been intensively investigated for their adjuvant properties.
  • Other agents that stimulate the immune response can included, such as cytokines that are useful as a result of their lymphocyte regulatory properties.
  • Suitable cytokines may include interleukin-12 (IL-12), GM-CSF or IL-18.
  • Pharmaceutical compositions of the invention can be formulated as capsules comprising viable cells, such as vegetative cells or spores, wherein the capsules comprise a delayed-release layer or coating that allows for the release of the viable cells, typically vegetative cells in an anaerobic section of the lower GI tract following oral administration.
  • compositions of the invention are formulated to deliver the bacterium according to the first aspect or the second aspect to the GI tract, preferably by oral administration.
  • the human GI tract consists of digestive structures stretching from the mouth to the anus, including the oesophagus, stomach, and intestines.
  • the GI tract does not include the accessory glandular organs such as the liver, biliary tract or pancreas.
  • the intestines include the small intestine and large intestine.
  • the small intestine includes the duodenum, jejunum and ileum.
  • the large intestine includes the cecum, colon, rectum and anus.
  • the upper GI tract includes the buccal cavity, pharynx, oesophagus, stomach, and duodenum.
  • the lower GI tract includes the small intestine (below the duodenum) and the large intestine.
  • the pharmaceutical compositions of the invention deliver the bacterium according to the first aspect or the second aspect to the lumen or mucosal surface of the GI tract, more preferably the lumen or mucosal surface of the large intestine, and more preferably the lumen or mucosal surface of the colon.
  • the pharmaceutical compositions of the invention deliver bacterium according to the first aspect or the second aspect to anaerobic sections of the lower GI tract, preferably the colon and/or terminal small intestine (ileum, also referred to as the “terminal ileum”).
  • ileum also referred to as the “terminal ileum”.
  • a steep oxygen gradient exists within the human intestinal tract as reviewed in Zheng, Kelly and Colgan, American Journal of Physiology-Cell Physiology 2015 309:6, C350- C360. Breathable air at sea level has a Po 2 of ⁇ 145 mmHg ( ⁇ 21% O 2 ). Measurements of the healthy lung alveolus have revealed a Po2 of 100–110 mmHg.
  • the most luminal aspect of the healthy colon exists at a Po2 below 10 mmHg (1.4% O2).
  • the Po 2 drops precipitously along the radial axis from the intestinal submucosa to the lumen, which is home to trillions of anaerobic microbes.
  • the bacterium is delivered orally as a spore, it will transit through the GI tract until it reaches the anaerobic portions, where it will germinate and grow.
  • Anaerobic sections of the lower GI tract include the terminal ileum and colon.
  • the colon may have a lower PO2 than the terminal ileum, in view of Zheng, supra, and bacterial growth may therefore be more efficient in the colon.
  • P O2 required to trigger spore germination and anaerobic metabolism or growth may be in the range of 0 to 2%.
  • the human colon volume (sum of ascending/descending and transverse) is around 600ml (Pritchard, S. E. et al. (2-14) Neurogastroenterol. Motil. 26, 124-130) whereas the entire intestine of a mouse is around 1 ml in volume (McConnell, E. L., Basit, A. W. & Murdan, S. (2008) J. Pharm. Pharmacol. 60, 63-70).
  • the approximate total GI transit time is around 5-6 hours in a mouse (Padmanabhan, P., et al. (2013) EJNMMI Res. 3, 60 and Kashyap, P. C. et al. (2013) Gastroenterology 144, 967-977) and the colon transit times have been estimated to be between 23 and 40 hours in humans (Degen, L. P. & Phillips, S. F. (1996) Gut 39, 299-305 and Wagener, S., et al (2004) J. Pediatr. Surg. 39, 166-169-169). Since transit time in the human gut is five times longer than in mouse, fewer spores are needed (e.g.
  • the lab fermentation based doubling time of the bacterial strain CHN0 is similar to that for E. coli and E. coli have a gut doubling time of about 3 hours (Myhrvold, C., et al (2015) Nat. Commun.6, 10039). CHN0 may undergo 10 doublings of cells during gut transit, equating to a three order of magnitude increase in cell numbers.
  • a pharmaceutical composition taken on an empty stomach is likely to arrive in the ascending colon about 5 hours after dosing, with the actual arrival dependent largely on the rate of gastric emptying.
  • Drug delivery within the colon is greatly influenced by the rate of transit through this region.
  • capsules pass through the colon in 20-30 hours on average. Solutions and particles usually spread extensively within the proximal colon and often disperse throughout the entire large intestine.
  • the pharmaceutical compositions are for administration between meals or with food.
  • Growth of the bacterium according to the first aspect of the invention or the second aspect of the invention upon arrival in the anaerobic portion of the gut can be verified by culture, including stool culture.
  • bacteria may be cultured from portions of the GI tract obtained from the experimental animal.
  • the genetically engineered anaerobic bacteria that produce antigen can also be incorporated as part of a food product, i.e. in yoghurt, milk or soy milk, or as a food supplement; or as part of a beverage.
  • a food product i.e. in yoghurt, milk or soy milk
  • Such food products, food supplements and beverages can be prepared by methods well known in the food, supplement and beverage industry.
  • the compositions can be incorporated into animal feed products as a feed additive.
  • the growth and degree of colonisation in the gut of the genetically engineered bacteria can be controlled by the species and strain choice and/or by providing specific substrates that the bacteria thrive on as a prebiotic, either within the same dose that contains the probiotic or as a separately ingested composition.
  • the composition may also further comprise or be for administering with a prebiotic to enhance the growth of the administered probiotic.
  • the prebiotic may be administered sequentially, simultaneously or separately with a bacterium as described herein.
  • the prebiotic and bacterium can be formulated together into the same composition for simultaneous administration. Alternatively, the bacteria and prebiotic can be formulated separately for simultaneous or sequential administration.
  • Prebiotics are substances that promote the growth of probiotics in the intestines.
  • a prebiotic provides a medium that can promote the growth of the probiotic strains in the intestines.
  • One or more monosaccharides, oligosaccharides, polysaccharides, or other prebiotics that enhances the growth of the bacteria may be used.
  • the prebiotic may be selected from the group comprising of oligosaccharides, optionally containing fructose, galactose, mannose; dietary fibres, in particular soluble fibres, soy fibres; inulin; or combinations thereof.
  • Preferred prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides, xylo-oligosaccharides, oligosaccharides of soy, glycosylsucrose (GS), lactosucrose (LS), lactulose (LA), palatinose-oligosaccharides (PAO), malto-oligosaccharides, pectins, hydrolysates thereof or combinations thereof.
  • Medical uses A fourth aspect of the invention provides the bacterium of the first aspect or the second aspect; or the pharmaceutical composition of the third aspect for use in medicine.
  • a fifth aspect of the invention provides a bacterium of the class Clostridia for use in generating an antigen-specific response in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen.
  • a corresponding aspect provides a method of generating an antigen-specific immune response in a subject, comprising administering a bacterium of the class Clostridia, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen.
  • a sixth aspect of the invention provides a bacterium of the class Clostridia for use in generating an antigen-specific immune response in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2.
  • a corresponding aspect provides a method of generating an antigen-specific immune response in a subject, comprising administering a bacterium of the class Clostridia, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2.
  • a seventh aspect of the invention provides a bacterium of the class Clostridia for use in the preventive or therapeutic treatment of a disease in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen; wherein the antigen is an infectious agent antigen and the disease is the infectious disease, or the antigen is a tumour antigen and the disease is cancer.
  • a corresponding aspect provides a method of preventing, ameliorating or treating a disease in a subject, comprising administering a bacterium of the class Clostridia, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen; wherein the antigen is an infectious agent antigen and the disease is the infectious disease, or the antigen is a tumour antigen and the disease is cancer.
  • An eighth aspect of the invention provides a bacterium of the class Clostridia for use in the preventive or therapeutic treatment of COVID-19 in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2.
  • a corresponding aspect provides a method of preventing, ameliorating or treating COVID-19 in a subject, comprising administering a bacterium of the class Clostridia, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2.
  • the subject is a mammal or bird, typically a mammal, preferably a human.
  • Suitable mammals for veterinary vaccination include agricultural animals, such as ungulates, including cows, sheep or goats; or horses; or pigs; or domestic animals such as cats or dogs.
  • Suitable birds include chickens or turkeys.
  • the antigen is an infectious agent antigen
  • the subject is of a species which is susceptible to a disease caused by the infectious agent.
  • the antigen is a tumour antigen
  • the subject is of a species for which the tumour antigen is characteristic of a tumour.
  • Vaccines From concept to clinic, Paoletti and McInnes, eds, CRC Press, 1999.
  • vaccination may be effective after a single dose, or one to three inoculations may be provided about 2 weeks to six months apart.
  • the vaccination may be provided in a vaccination regimen with a different vaccine, such as a prime – boost regimen in which the vaccine of the invention is either the prime or booster vaccine, and the other of those is a different vaccine.
  • a prime – boost regimen in which the vaccine of the invention is either the prime or booster vaccine, and the other of those is a different vaccine.
  • booster There may be more than one booster.
  • such regimens will be directed at the same infectious agent or the same cancer.
  • the antigen may be any antigen as defined herein, not limited to tumour antigen or infectious agent antigen.
  • the antigen may include an artificial sequence (i.e., artificially designed sequence, which is not present in nature).
  • the antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2. Suitable antigens are as defined herein.
  • antigen-specific immune response we include any cellular or humoral immune response that is antigen-specific, i.e.
  • T cell responses such as CD4 + , CD8 + T-cell responses, or B cell (antibody) responses.
  • antigen is delivered to antigen presenting cells (APCs), especially dendritic cells (DC), which then stimulate and elicit antigen specific cytotoxic CD8 + (CTL) and/or helper CD4 + T lymphocytes.
  • APCs antigen presenting cells
  • DCs sample antigens in the microenvironment and process them intracellularly (for example, following the antigen being phagocytosed).
  • DC activation e.g. due to an inflammatory signal
  • they migrate to the lymph nodes whereby they can activate the adaptive immune response.
  • bacteria of the class Clostridia may be internalised by APCs, particularly DCs in the intestine, such as mucosal DCs.
  • APCs particularly DCs in the intestine
  • a DC that has taken up (e.g. phagocytosed) an antigen by virtue of having internalised a bacterium of the class Clostridia and/or secreted antigen may become activated, and may migrate to the lymph node and activate T-cells that have specificity to said antigen, and thence B cells.
  • the APC may be exposed to a further activating signal in addition to the bacterium of the class Clostridia, such as provided by an adjuvant, lipopolysaccharide (LPS), or inflammatory cytokine.
  • T cells express a T-cell receptor that recognises antigenic peptides that are presented by major histocompatibility complex (MHC), referred to as human leukocyte antigen (HLA) in humans.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • Helper CD4 + T-cells can effectively stimulate and amplify cytotoxic CD8 + T-cells and help B cells to produce antibodies.
  • a CD4 + response can be categorised by the type of CD4 + T-cell that is induced/activated.
  • a CD4 + response may be that of a T helper (Th) 1, Th2, and/or Th17.
  • Th1, Th2 and Th17 cells can be categorised by markers (e.g. cell surface markers), cytokine secretion and/or functional assays that are known to the skilled person.
  • markers e.g. cell surface markers
  • cytokine secretion e.g. IL-4 + IL-4 + IL-12
  • the type of CD4 + response (or combination thereof) achieved may depend on the antigen being used and/or adjuvants or other immunomodulatory molecules, which may be selected depending on the desired outcome.
  • Th2 responses are more suitable than Th1 responses for protecting against helminth infection.
  • Th1 responses which are often associated with IFN- ⁇ production, are more suitable than Th2 responses for protecting against intracellular parasites.
  • CD8 + T-cells can specifically recognise and induce apoptosis of target cells containing target antigens. Activation of specific CD8 + T-cells depends on the antigen being efficiently presented to MHC class I molecule (HLA-I antigen in humans).
  • CD8 + cytotoxic T lymphocytes are the main cell type targeted by prophylactic and therapeutic cellular immune vaccines because they can directly recognise and destroy tumour cells or cells infected by intracellular infectious agents, such as viruses.
  • CD8 + response for the purposes of targeting tumour antigens and antigens of intracellular infectious agents such as viruses, it can be advantageous to mount a CD8 + response as these cells are capable of directly recognising these antigens presented on MHC class I molecules on the cell surface.
  • CTLs are also associated with anti-tumour responses.
  • a combination of CD4 + and CD8 + responses may be beneficial, as subsets of CD4 + cells may support and/or enhance the activity of CD8 + cells by releasing cytokines into the local microenvironment. Accordingly, in some embodiments, a combination of T-cell responses is induced by the antigen.
  • MHC/HLA haplotypes differ between subjects, with each haplotype of MHC/HLA being capable of binding and thereby presenting particular types of peptide fragments.
  • the peptide fragments presented by the MHC/HLA of a first subject may differ in sequence to those presented by the MHC/HLA of a second subject.
  • MHC/HLA subtypes may differ in their ability to induce an immune response, resulting in differences within populations for responsiveness to a particular antigen.
  • B cells are able to internalise components, such as polypeptides, and present fragments of polypeptide molecules on the cell surface in complex with MHC class I or II molecules. B cells may also express on their cell surface antigen specific B cell receptors (BCR). Unlike T-cells and the TCR, which rely upon antigen being presented by MHC, the BCR can recognise antigenic epitopes without them being presented by MHC (i.e. BCR can also recognise soluble antigen). Antigen activates B cells bearing appropriate surface immunoglobulin directly to produce IgM. In some instances, B cells rely upon T-cells for activation by presenting antigen loaded to MHC class II.
  • BCR cell surface antigen specific B cell receptors
  • CD4 + T cells having responded to processed Ag, may induce immunoglobulin class-switching from IgM to IgG.
  • some antigens are able to activate B cells in a T-cell independent manner. Therefore, in some embodiments, the induction of a B cell response may be in conjunction with the induction of a T-cell response (CD4 + and/or CD8 + ).
  • Suitable antibody responses may include different isotypes, such as IgA and/or IgG isotypes. The type of antibody response achieved may depend on the antigen being used and/or adjuvants or other immunomodulatory molecules, which may be selected depending on the desired outcome.
  • IgA also referred to as sIgA in its secretory form is an antibody that plays a crucial role in the immune function of mucous membranes.
  • the amount of IgA produced in association with mucosal membranes is greater than all other types of antibody combined. In absolute terms, between three and five grams are secreted into the intestinal lumen each day. This represents up to 15% of total immunoglobulins produced throughout the body.
  • IgA has two subclasses (IgA1 and IgA2) and can be produced as a monomeric as well as a dimeric form. The IgA dimeric form is the most prevalent and is also called secretory IgA (sIgA).
  • sIgA is the main immunoglobulin found in mucous secretions, including tears, saliva, sweat, colostrum and secretions from the genitourinary tract, gastrointestinal tract, prostate and respiratory epithelium. It is also found in small amounts in blood.
  • the secretory component of sIgA protects the immunoglobulin from being degraded by proteolytic enzymes; thus, sIgA can survive in the harsh gastrointestinal tract environment and provide protection against microbes that multiply in body secretions.
  • sIgA can also inhibit inflammatory effects of other immunoglobulins.
  • IgA is a poor activator of the complement system and opsonizes only weakly. There are several subtypes of IgG.
  • the antigen-specific immune response induced by the antigen is a B-cell response.
  • the antigen-specific immune response includes the generation of antigen-specific antibodies, i.e., the antigen induces the production of antigen-specific antibodies that are specific for (i.e., bind to) said antigen.
  • the antigen-specific antibody belongs to an antibody serotype selected from the group comprising or consisting of: IgA, IgM, IgG, or any combination thereof.
  • the antigen-specific antibody is a secreted antibody, for example secretory IgA (sIgA), secretory IgM, or secretory IgG.
  • the antigen induces the production of antigen-specific IgA, antigen-specific IgM, antigen-specific IgG, or any combination thereof.
  • a bacterium comprising antigen, as described herein, can be tested for capability for inducing an antigen-specific immune response, such as following oral immunisation in a mouse model.
  • the bacterial spores (e.g. C. butyricum) encoding an antigen of interest can be administered to a group of mice by oral gavage.
  • a negative control of spores from the same bacterium but without antigen may be administered to a separate group of mice.
  • a comparison of the bacterium with antigen and such a negative control will attribute any differences as being antigen specific.
  • a suitable positive control for this experiment includes the parenteral administration by subcutaneous injection of the antigen, which will be taken up by DCs resulting in activation of an immune response to said antigen. Therefore, a comparison with this positive control will give an indication as to whether the immune response induced by the bacterium comprising antigen is equivalent to the administration of the antigen itself.
  • mice may be immunised 3 times at fortnightly intervals. Following the immunisation regimen, for example after 42 days of the regimen, the mice are sacrificed.
  • immunisation regimens may suitably be employed.
  • mice may be immunised over consecutive days with intervals between immunisations.
  • Various numbers of consecutive days of immunisation, the duration of intervals between immunisation doses, and number of times the mice are immunised may be varied. For example, the mice may be immunised for 2 consecutive days, 3 consecutive days, 4 consecutive days, or 5 consecutive days.
  • the interval between immunisation doses may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days.
  • the interval between immunisation doses may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more weeks.
  • the mice may be immunised 1 time, 2 times, 3 times, 4 times, 5 times or 6 times.
  • An immune response can be detected in a number of ways known to the skilled person. For example, splenocytes from homogenised spleens and peripheral blood mononuclear cells (PBMCs) from blood samples can collected and mononuclear cell isolates obtained using standard methods.
  • PBMCs peripheral blood mononuclear cells
  • T cells CD4 + and CD8 +
  • T cell specific marker e.g. CD8a for CD8 + T cells
  • B cells e.g. CD8a for CD8 + T cells
  • IFN- ⁇ and/or TNF ⁇ are characterized by the expression of markers (e.g. cell surface markers and/or intracellular markers) indicative of an activated cellular phenotype.
  • markers e.g. cell surface markers and/or intracellular markers
  • Cytokines such as IFN- ⁇ and/or TNF ⁇
  • ELISPOT culturing T cells in plates in the presence of anti-IFN- ⁇ or anti-TNF ⁇ antibodies, respectively, and re-stimulating the cells with wildtype antigen protein, with vegetative bacterial cells that comprise the antigen, or with antigen presenting cells that have previously been exposed to the antigen. If T cells are present that have specificity for the antigen of interest, then re- stimulation with that antigen will induce the T cells to secrete IFN- ⁇ and/or TNF ⁇ .
  • vegetative bacterial cells that do not comprise antigen is a negative control, which can be compared with the test condition to identify to what extent the IFN- ⁇ and/or TNF ⁇ secretion is antigen specific.
  • IFN- ⁇ and/or TNF ⁇ standards i.e. aliquots of these cytokines at varying concentrations
  • ELISPOT spot forming units (SPU) can be assessed, wherein an SPU for a test condition (e.g. a vaccinated group) that is at least two standard deviations higher than the average of a control group would indicate a positive result for the test condition.
  • an immune response can be tested by intracellular cytokine staining, such as that described in Zhang et al., 2009.
  • splenocytes obtained from the mice subjected to the above-described immunisation regimen can be cultured with the antigen and same negative and positive controls as the ELISPOT.
  • the cells can then be labelled with antibodies (e.g. phycoerythrin-conjugated monoclonal rat anti-mouse CD8 or CD4 antibody) or an immunoglobulin isotype control.
  • Splenocytes can then be fixed and permeabilised using a fix/perm protocol (e.g.
  • a detection antibody for intracellular antigen e.g. fluorescein isothiocyanate-conjugated anti-IFN- D ⁇ antibody.
  • Samples can then be assessed by flow cytometry, with fluorescence above that of the isotype control indicative of the antigen specific activation of the cells.
  • the co-staining with CD8 or CD4 and the IFN- ⁇ will attribute the antigen specific expression of IFN- ⁇ to either CD8 + or CD4 + T cells.
  • an immune response can be tested by detecting the expression of T cell-surface receptors or receptor ligands, typically after re-stimulation of T cells with APCs.
  • cell surface CD40 ligand expression can be assessed on CD4 + T cells, as described in Hegazy et al (2017) Gastroenterology 153: 1320- 1337.
  • the % of CD4 + T-cells expressing CD40L (CD154) following defined antigen stimulation may be determined, and non-parametric analyses performed between experimental and control groups to identify any difference in the population average antigen-specific T-cell percentage.
  • a positive result for the test condition would be indicated by a higher percentage antigen-specific (i.e., CD40L upregulated) CD4 + T- cells versus negative control group, for example at least 1% higher, at least 2% higher, at least 5% higher and/or up to 10% higher or more.
  • Cytotoxicity of CTL responses may be assessed using a chromium-51 ( 51 Cr) release assay (see B. Paige Lawrence, 2004, Current Protocols in Toxicology, 22(1):18.6.1- 18.6.27).
  • target cells expressing an antigen of interest for CTLs e.g. cancer cells expressing a tumour antigen
  • 51 Cr chromium-51
  • target cells expressing an antigen of interest for CTLs e.g. cancer cells expressing a tumour antigen
  • 51 Cr chromium-51 release assay
  • An increase in 51 Cr detection for CTLs derived from a vaccinated group would indicate a positive result for inducing an antigen specific response.
  • a positive result for a test group is indicated where the mean is at least two standard deviations higher than the mean for a control group.
  • Another suitable assay for assessing cytotoxicity is the CyQUANT LDH Cytotoxicity Assay. Lactase dehydrogenase (LDH) is a cytosolic enzyme that is released upon damage to the plasma membrane. Accordingly, LDH levels can be tested in a coculture of CTLs and target cells, using the same conditions as described for the 51 Cr release assay, to identify whether the vaccinated group has higher LDH indicative of increased cytotoxicity compared with the control group.
  • LDH Lactase dehydrogenase
  • T cell proliferation can be tested using 3 H thymidine.
  • 3 H is incorporated into new strands of chromosomal DNA during mitotic cell division, and so accumulates intracellularly as cells divide.
  • T cells (or subsets of T cells) isolated from vaccinated subjects may be compared with T cells (or subsets of T cells) isolated from control, na ⁇ ve subjects.
  • Isolated T cells can be cocultured with PBMCs or activated DCs loaded with antigen in a mixed lymphocyte reaction (MLR), and their proliferation assessed over time.
  • MLR mixed lymphocyte reaction
  • CFSE carboxyfluorescein succinimidyl ester
  • B cell responses may be assessed by quantifying the levels of antibodies in sera or other appropriate samples collected during the immunisation regimen or following termination.
  • An antibody titre is a measurement of how much antibody an organism has produced that recognizes a particular epitope, expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result.
  • Antibody titre may be tested using ELISA. Therefore, sera obtained from mice subjected to the above-described immunisation regimen can be assessed for antibody titre and compared with the same controls. A higher antibody titre, such as at least two standard deviations higher compared with the negative control would be indicative of B cell activation in an antigen specific manner.
  • vaccination may result in incremental increases, such as 2-fold to 200-fold (e.g., 20- or 200-fold), in antigen-specific antibodies, relative to an unimmunised control.
  • immunisation strategies against viruses such as HRV or SARS-CoV-2
  • neutralizing antibody responses are preferred.
  • Neutralizing antibodies are those antibodies which can inhibit or block a key component of the viral replication cycle. Viral replication may thereby be lessened and/or prevented.
  • a neutralization titer may typically be expressed as the highest serum dilution required to achieve a 50% reduction in the number of virus plaque forming unit, pfu.
  • vaccination may result in incremental increases, such as 2-fold to 200-fold (e.g., 20- or 200-fold), in neutralizing antibodies against the virus, relative to an unimmunised control.
  • the increase in neutralizing antibody titre compared to an unimmunised control may be between 10-fold and 200-fold, such as about 50-fold, about 100-fold or about 200-fold.
  • Exemplary assays for detecting and/or quantifying neutralising HRV or SARS-CoV-2 antibodies are disclosed in Morita et al., 1988, J Gen.
  • the IgA antibody titre is indicative of mucosal immunity, and so the levels of antigen- specific IgA may specifically be tested to assess the induction of mucosal immunity.
  • Suitable samples for testing for IgA include sera, faeces, contents of the colon or gut, or ileal wall extract.
  • the antigen-specific IgG titre which is indicative of systemic immunity and/or antigen-specific IgM may be tested.
  • samples of faeces or sera will be tested.
  • the ratio of antigen-specific to total IgA may be measured, and may be indicative of a B cell response. For example, total IgA and antigen-specific IgA may be determined by ELISA.
  • Non-parametric analyses may be performed between experimental and control groups to look for a difference in the population average antigen-specific IgA/IgA ratio. A positive result would be the identification of a statistically significant difference in the average antigen-specific IgA/IgA ratio between experimental and control groups.
  • Generally accepted animal models (such as those described in Ireson et al. (2019) British J Cancer 121: 101-108) can be used for testing of immunisation against cancer using a tumour or cancer antigen.
  • cancer cells human or murine
  • a bacterium comprising a tumour antigen as described herein may be delivered to a subject harbouring a tumour associated with said antigen.
  • Cancer cells can be introduced by subcutaneous injection to form a xenograft or syngeneic tumour associated with an antigen of interest.
  • the effect on the cancer cells e.g., reduction of tumour size or reduction in tumour progression (i.e., the rate at which a tumour continues to grow), which can be measured using callipers) can be assessed as a measure of the effectiveness of the immunisation.
  • More complex models include the use of patient-derived xenograft (PDX) models, in which an antigen associated with the cancer of said patient is implanted into mice (e.g. humanised mice) that have undergone an immunisation regimen as described herein.
  • PDX patient-derived xenograft
  • Suitable mouse models are disclosed, for example, in Zottnick et al., "Inducing Immunity Where It Matters: Orthotopic HPV Tumor Models and Therapeutic Vaccinations", Front. Immunol., 11:1750.
  • the levels and activity of anti-tumour CTLs may be tested, for example taking a tumour biopsy and testing the levels of CTLs (including tumour antigen specific CTLs) in the tumour microenvironment.
  • Antigen specific CTLs may be identified using MHC tetramers specific to the MHC-loaded tumour antigen, and CTLs in the tumour microenvironment can then be quantified, for example by flow cytometry.
  • a biopsy may also be tested for cytokines, by measuring those associated with an inflammatory response and T cell activation (e.g. IL-2, IFN- ⁇ , GM-CSF).
  • T cell activation e.g. IL-2, IFN- ⁇ , GM-CSF.
  • the tests also can be performed in humans, where the end point is to test for the presence of enhanced levels of circulating cytotoxic T lymphocytes against cells bearing the antigen, to test for levels of circulating antibodies against the antigen, to test for the presence of cells expressing the antigen and so forth.
  • a suitable test is described in Cai et al., 2017, which demonstrated that immunisation with ROP-survivin or ROP-HPV-E7 generated specific cellular immune responses and protected mice from inoculation with melanoma B16 cells expressing survivin or HPV- E7 proteins.
  • mice were primed subcutaneously with ROP-antigen (ROP-survivin or ROP-HPV-E7), which was compared with the wildtype antigens as a positive control, both conditions having the antigen emulsified in monophosphoryl lipid A (MPL).
  • ROP-antigen ROP-survivin or ROP-HPV-E7
  • MPL monophosphoryl lipid A
  • Immunisation was boosted subcutaneously twice at 3- week intervals with the same vaccine emulsified with MPL.
  • mice were challenged with B16-E7 or B16-survivin and subsequently assessed in ELISPOT assays.
  • ELISPOT assays were performed on PBMCs and splenocytes, as described above, with re-stimulation performed with ROP-HPV or ROP- survivin in anti-IFN- ⁇ -Ab precoated plates.
  • the data in Cai et al., 2017 demonstrate a mouse system where ROP-antigen and wildtype antigen can be used to immunise mice for anti-tumour immune responses. Therefore, the immunisation strategy of Cai et al., 2017 can be deployed as a positive control.
  • an SFU spot Forming Unit
  • a vaccinated group that is at least two standard deviations higher than the average of a control group would indicate a positive result for the test condition.
  • the skilled person can readily assess whether a bacterium of the class Clostridia encoding an antigen, such as an infectious agent antigen and/or a tumour antigen induces an immune response to said antigen.
  • an infectious agent antigen such as an infectious agent antigen and/or a tumour antigen induces an immune response to said antigen.
  • tumour antigen induces an immune response to said antigen.
  • Therapeutic or preventive treatment of an infectious disease or cancer in a subject the antigen is an infectious agent antigen and the disease is the disease caused by the infectious agent, or the antigen is a tumour antigen and the disease is cancer.
  • Suitable antigens are as described in relation to the first aspect of the invention.
  • the antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2; and the disease is COVID-19 disease.
  • Suitable antigens are as described in relation to the second aspect of the invention.
  • ameliorating or “treating” a disease, particularly cancer, we mean slowing, arresting or reducing the development of the disease or at least one of the clinical symptoms thereof; alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient; modulating the disease, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both; or preventing or delaying the onset or development or progression of the disease or disorder or a clinical symptom thereof.
  • Ameliorating or “treating” may be interpreted accordingly and may also include reducing the burden of viable infectious agent in the subject, or preventing or reducing the recurrence of dormant infectious agents into actively growing forms.
  • “Therapeutic treatment” is to be interpreted accordingly.
  • preventing we include that the agents described herein are prophylactic.
  • a preventative or prophylactic use or treatment includes a use or treatment that reduces or removes the risk of a subject contracting a disease or being infected by an infectious agent, or which reduces the risk of the subject contracting a severe form of the disease, or which reduces the severity of the disease in the event that the subject does contract the disease, for example by vaccination.
  • Treatment is to be interpreted accordingly.
  • a subject is in need of a treatment if the subject would benefit biologically, medically or in quality of life from such treatment. Treatment will typically be carried out by a physician or a veterinary surgeon who will administer a therapeutically effective amount of the bacterium or composition.
  • a therapeutically effective amount of bacterium according to the first aspect or second aspect; or composition according to the third aspect refers to an amount that will be effective for the treatments described herein, for example slowing, arresting, reducing or preventing the disease or symptom thereof.
  • the therapeutically effective amount may depend on the antigen (e.g.
  • a subject in need of therapeutic treatment is presenting symptoms of the disease.
  • a subject may be susceptible to the disease or has been tested positive for the disease but has not yet shown symptoms or is asymptomatic.
  • a subject in need of preventive treatment does not have the disease but may be at risk of developing it.
  • Preventive treatment is particularly appropriate for infectious disease.
  • the infectious disease to be treated is suitably one which may respond to an antigen specific immune response directed at the infectious agent.
  • the infectious disease, disorder or condition can be selected from those associated with the infectious agent antigens listed herein.
  • the cancer to be treated can be any cancer associated with a tumour antigen, such as those tumour antigens listed herein, particularly a cancer that has been shown to respond to immunotherapy utilising the tumour antigen.
  • Preventive treatment is particularly advantageous in relation to infectious agents which cause acute disease, although it may also be used in relation to infectious agents which cause chronic diseases.
  • Acute infections may include gastroenteritis, such as caused by a virus such as a Human Rotavirus; a bacterium such as Vibrio cholerae, Campylobacter jejuni, Escherichia coli (including but not limited to ETEC, EHEC, EIEC, EPEC, EAEC, and AIEC), Salmonella sp. (including but not limited to S. enteria and subspecies including S. e. enterica, S. e. salamae, S. e. arizonae, S. e. diarizonae, S. e. houtenae, S. e. indica, S. enterica serovar Typhi, S. enterica serovar Typhimurium, S.
  • gastroenteritis such as caused by a virus such as a Human Rotavirus
  • a bacterium such as Vibrio cholerae, Campylobacter jejuni, Escherichia coli (including but not limited to ETEC, E
  • Acute infections may include respiratory tract infections, such as caused by a virus such as a coronavirus, such as SARS-CoV-2; or respiratory syncytial virus (RSV), influenza A or B, or human parainfluenza virus (HPIV).
  • a virus such as a coronavirus, such as SARS-CoV-2; or respiratory syncytial virus (RSV), influenza A or B, or human parainfluenza virus (HPIV).
  • SARS-CoV-2 causes COVID-19 disease, which may be classified as asymptomatic or presymptomatic infection (individuals who test positive for SARS-CoV-2 using a virologic test but who have no symptoms that are consistent with COVID-19); mild illness (individuals who have any of the various signs and symptoms of COVID-19 e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell, but who do not have shortness of breath, dyspnea, or abnormal chest imaging); moderate illness (individuals who show evidence of lower respiratory disease during clinical assessment or imaging and who have an oxygen saturation (SpO2) ⁇ 94% on room air at sea level); severe illness (individuals who have SpO2 ⁇ 94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) ⁇ 300 mm Hg, a respiratory rate >30 breaths/min, or lung infiltrates >50%); or critical illness (individuals who
  • Preventive treatment of COVID-19 disease is intended to cover reduction in the severity of COVID-19 illness, such as a lower proportion of infected individuals experiencing severe illness or death; or reduction in the proportion of individuals who go on to develop symptomatic or asymptomatic infection.
  • preventive treatment of COVID-19 disease is also intended to mean preventive treatment of SARS-CoV-2 infection.
  • Therapeutic treatment is particularly advantageous in relation to cancer, or chronic infectious diseases. Chronic infectious diseases include those that are perpetuated for months or years by the infectious agent, or which exhibit periods of active growth of the infectious agent and/or symptoms, and periods of dormancy.
  • Chronic persistent infection may be caused by viruses including human papillomavirus (HPV); hepatitis C; hepatitis B; human immunodeficiency virus (HIV); herpesviruses including herpes simplex virus 1, herpes simplex virus 2 and varicella zoster virus; flavivirus associated with Yellow fever; West Nile virus; dengue virus; Zika virus; Japanese encephalitis virus; African swine fever virus; Porcine Reproductive and Respiratory Syndrome (PRRS) virus and foot-and-mouth disease virus (e.g. coxsackievirus A16).
  • SARS-CoV- 2 may cause persistent infection, such as in immunocompromised subjects (Nakajima Y et al.
  • Prolonged viral shedding of SARS-CoV-2 in an immunocompromised patient J Infect Chemother. 2021 Feb;27(2):387-389. doi: 10.1016/j.jiac.2020.12.001).
  • the duration of viral shedding of SARS-CoV-2 in acute COVID-19 disease is usually less than 10 days.
  • Therapeutic treatment of COVID-19 disease is intended to encompass treatment of patients who have already contracted SARS-CoV-2 infection, and continue to shed SARS-CoV-2 for longer than is typical in acute SARS-CoV-2 infection, e.g. longer than 10 days, or are at risk of such prolonged infection, e.g. by virtue of being immunocompromised.
  • Chronic persistent infection may be also caused by bacteria, including Mycobacterium tuberculosis, Mycobacterium bovis, Brucella, Borrelia species such as B. burgdorferi, Corynebacterium diphtheriae, Chlamydia, Vibrio cholerae, Salmonella enterica serovar Typhi; mycoplasma; fungi including Candida albicans; and various parasites including helminths and protozoa.
  • bacteria including Mycobacterium tuberculosis, Mycobacterium bovis, Brucella, Borrelia species such as B. burgdorferi, Corynebacterium diphtheriae, Chlamydia, Vibrio cholerae, Salmonella enterica serovar Typhi; mycoplasma; fungi including Candida albicans; and various parasites including helminths and protozoa.
  • Suitable cancers to be treated include melanoma and renal cell carcinoma, which are considered to be two of the most immunogenic solid tumours and have been studied extensively in vaccine development or cancers of the colon, lung, cervix, pancreas, stomach, liver, intestine, bladder, ovary, prostate, bone, brain, or head and neck.
  • Preventive treatment typically requires the establishment of immunological memory, such that the immunised subject is protected or partially protected from subsequent challenge, typically with the infectious agent antigen.
  • Immunological memory is an important consequence of adaptive immunity, as it enables a more rapid immune response to be mounted to pathogens that have been previously encountered to prevent them from contracting a disease. Immunological memory may also be important in therapeutic treatments.
  • Immunological memory in T cells can be tested using MHC tetramers that identify whether memory T cells exist for a particular antigen.
  • MHC tetramers have specificity to MHC-loaded antigen, and so an MHC tetramer can be used that is specific to an antigen of interest. These can be used on samples isolated from a subject (e.g. a blood sample or splenocytes) to measure the frequency of antigen specific T cells.
  • MHC tetramers are available for MHC class I and II, meaning that both CD4 + and CD8 + cells can be measured using MHC tetramers.
  • the MHC tetramers can be used in conjunction with fluorescent antibodies for other T cell markers to assess the proportion of antigen specific T cell subsets (e.g.
  • antigen specific Th1, Th2 and/or Th17 cells The proportion of antigen specific T cells can be assessed by flow cytometry, comparing immunised and non-immunised subjects. For example, samples obtained from mice that have undergone the immunisation regimen described herein may have blood samples and/or splenocytes assessed for MHC tetramer binding and a panel of fluorescent markers for T cell subsets. Compared with non-immunised mice, the immunised mice should have a higher proportion of cells that can be bound with an MHC tetramer, and thus identified as antigen-specific T cells, which can be further assessed by T cell subset to identify the type of T cell response induced.
  • a positive result for a test condition may be indicated by a higher percentage antigen-specific (i.e., CD40L upregulated) CD4+ T-cells versus negative control group, or the higher presence of the MHC tetramer reagent on the cell surface compared to negative control groups.
  • Suitable MHC tetramers may be generated using the methods disclosed in Ramachandiran et al., 2007, J. Immunol. Methods, 319:1-2):13-20 (DOI: 10.1016/j.jim.2006.08.014).
  • Exemplary MHC tetramers for detecting SARS-CoV-2- specific T cells are disclosed in Poluektov et al., 2021, Vaccine, 39(15):2110-2116 (DOI: 10.1016/j.vaccine.2021.03.008).
  • Immunological memory in B cells can be tested in vitro by isolating B cells from immunised and non-immunised mice (e.g. as per the immunisation regimen described herein) and re-stimulating the B cells in the presence of helper T cells specific for the same antigen.
  • B cells from immunised mice respond both quantitatively and qualitatively better, the former of which can be assessed by comparing the frequency of B cells (i.e.
  • the memory B cell antibodies produced would also have a higher affinity compared with na ⁇ ve B cells from non-immunised mice, which can be tested by purifying the produced antibodies (from immunised and non- immunised mice) and comparing their affinity for the antigen (or epitope thereof). If the antibodies produced from the immunised mice have a higher affinity, then a B cell memory response has been established that may indicate protective immunity. Corresponding in vivo studies would use such mice and challenge them with the pathogen from which the antigen is derived (e.g.
  • HPV if the antigen is an HPV antigen, for example, as described in Longet et al, 2011, Journal of Virology, 85:13253-13259; infection with HRV if the antigen is an HRV antigen such as VP8, for example as described in Lappalainen et al., 2015, Arch.
  • Virol., 160(8):2075-2078 (DOI: 10.1007/s00705-015-2461-8); and infection with SARS-CoV-2 if the antigen is a SARS-CoV-2 antigen, for example as described in Amanat et al., 2021, PLoS Biology, 19(1):e3001384 (DOI: 10.1371/journal.pbio.3001384)) to assess infection burden compared with mice challenged for the first time with the pathogen.
  • Suitable challenge models also include, for example, the Syrian Hamster challenge model disclosed in Johnson et al., 2022, J. Infect. Dis., 225(1):34-41 (DOI: 10.1093/infdis/jiab561).
  • mice are challenged with the pathogen associated with the antigen. Due to the existence of T and/or B cell immunity, immunised mice should have reduced infection burden, such as increased rates of partial or complete protection from infection compared with na ⁇ ve mice. Accordingly, a suitable in vivo system would include a challenge regimen following the immunisation regimen to assess infection burden. Suitable animal models are described in Bakaletz (2004) Developing animal models for polymicrobial diseases, Nature Reviews Microbiology, 2:552-568).
  • Immunological memory may also be tested in in vivo tumour models, including tumour challenge models, such as described in Cai et al., 2017, supra and Ireson et al., 2019, supra as described in relation to the fifth aspect of the invention.
  • tumour challenge models such as described in Cai et al., 2017, supra and Ireson et al., 2019, supra as described in relation to the fifth aspect of the invention.
  • tumour challenge models such as described in Cai et al., 2017, supra and Ireson et al., 2019, supra as described in relation to the fifth aspect of the invention.
  • LysM-CtxB 50-75 alignment (b), single amino acid exchange noted by # Figure 2: LysM-CtxB 50-75 nucleic acid sequence information showing 5’ upstream LysM domain including signal peptide, 3’ downstream FLAG tag and stop codon (SEQ ID NO: 31). LysM-CtxB 50-75- amino acid sequence is shown as SEQ ID NO: 30 and the LysM amino acid sequence as SEQ ID NO: 42. NdeI (5’) and NheI (3’) restriction sites flank the coding region.
  • Figure 3 Western blot detection of FLAG-tag linked to Usp45-CtxB (12.7kDa).
  • the blot shows plasmid based Usp45-CtxB-FLAG expression (denoted “F” or “CtxB-Full”) at OD1.0 and OD 4.0 time points.
  • CHN-0 wild-type (WT) was used as the negative control.
  • Bands demonstrating expressed Usp45-CtxB-FLAG can be seen in both whole cell lysate (WCL) and supernatant (S/N) samples, for both time points (arrow).
  • WCL whole cell lysate
  • S/N supernatant
  • Figure 4 Western blot detection of FLAG-tag linked to LysM-CtxB 50-75- (27 kDa) expressed by C. butyricum engineered to express antigens associated with the cell wall. CHN-0 wild-type (WT) was used as the negative control.
  • (A) Whole cell lysates of CADD + pMTL82151-pfdx-LysM-CtxB 50-75 -FLAG, CADD + pMTL82151-pfdx-LysM-FLAG and WT CHN-0. Samples were removed from the growth culture at the OD 600 time points indicated on the blot.
  • (B) Whole cell lysates from samples of triplicate CADD + pMTL82151-pfdx-LysM-CtxB 50-75- -FLAG grown to OD 600 1.0 and assessed against FLAG- BAPTM standards (1 ⁇ g – 50 ng).
  • (C) Whole cell lysates of CADD + LysM-CtxB 50-75- FLAG (integrated), CADD + LysM-FLAG (integrated) and WT CHN-0 as a control. Samples were removed from the growth culture at the OD 600 time points indicated on the blot.
  • Figure 6 High resolution fluorescence microscopy to demonstrate extracellular location of the CtxB50-75-FLAG antigen.
  • Brightfield (bottom) images were taken to identify bacteria, before fluorescently labelled FLAG images (top) were taken to show the LysM-CtxB 50-75- -FLAG antigen on the cell surface of the CHN-0 cells.
  • the –ve control shows bacteria that have not been exposed to the anti-FLAG antibody to detect any background staining or auto-fluorescence.
  • CHN-0 wild-type was also used as a control to demonstrate any non-specific binding of the anti-FLAG antibody.
  • An increased level of puncta is observed in the LysM-CtxB 50-75- -FLAG image which demonstrates localisation of the antigen to the cell surface.
  • FIG. 7 Nucleic and amino acid sequences of Usp45-VP8.
  • the Usp45 amino acid sequence is shown (SEQ ID NO: 41).
  • Usp45-VP8 nucleic acid sequence (SEQ ID NO: 26) consists of the Usp45 secretion signal followed by VP8 sequence, followed by a FLAG tag and stop codon. NdeI (5’) and NheI (3’) restriction sites flank the coding region.
  • Figure 8 Nucleic and amino acid sequences of Usp45-VP7 polyepitope. The nucleic acid sequence is shown as SEQ ID NO: 76 and consists of Usp45 secretion signal followed by VP7 polyepitope, followed by FLAG tag and a stop codon.
  • NdeI (5’) and NheI (3’) restriction sites flank the coding region.
  • Usp45-VP7 polyepitopes are separated by flexible [Gly]4Ser linkers, shown in SEQ ID NO: 77.
  • Figure 9 Western blot detection of FLAG-tag linked to the HRV-VP8 (20 kDa) antigen secreted by engineered C. butyricum. CHN-0 wild-type was used as the control.
  • FLAG- BAPTM standards used at a known concentration, with test samples of HRV-VP8 antigen TCA precipitated from the equivalent of 10 mL culture supernatant, at OD 600 1.0, 2.0 and 4.0 in the growth phase.
  • FIG. 10 A 10 mL sample of wild-type CHN-0 grown to OD 600 2.0 was used as a control. 40 ⁇ l protein loaded, blots blocked 5% milk, anti-FLAG (A9469) 1:5000 in TBS-T 2 h, developed using SIGMAFAST BCIP/NBT 4.5 - 5 min.
  • Figure 10 Western blot detection of FLAG tag linked to HPV-VP8 in C. butyricum engineered to secrete HRV proteins either from an integrated single gene copy (sample lanes 1-3) or from a multicopy plasmid (sample lanes 4-6) indicated by the arrow. CHN-0 wild-type was used as the control (sample lanes 7-9).
  • nucleic acid sequences in this figure consist of the LysM peptidoglycan association domain, followed by a flexible [Gly]4Ser linker, followed by the LysM-CoV-2-XX sequence, followed by a 6X His tag and stop codon. All sequences in this figure are flanked by 5’ NdeI and 3’ NheI restriction sites. Amino acid sequences (SEQ ID NOs: 7, 9, 11, 13, 17) are shown with LysM peptidoglycan association domain highlighted in bold.
  • LysM-CoV-2-Nuc amino acid sequence SEQ ID NO: 13
  • LysM-CoV-2-Comb amino acid sequence SEQ ID NO: 17
  • Figure 12 Nucleic and amino acid sequences of intracellular CoV-2-XX antigens. All compositions are identical to the LysM-CoV-2-XX antigens shown in Figure 11, but do not contain a LysM peptidoglycan anchoring domain or adjacent [Gly]4Ser linker.
  • LysM-CoV- 2-Nuc and LysM-CoV-2-Comb amino acid sequences of CoV-2-Nuc (SEQ ID NO: 131) and CoV-2-Comb (SEQ ID NO: 133) contain multiple epitopes with predicted antigenicity separated by cathepsin cleavage sites (underlined).
  • Figure 13 Western blot detection of 6XHis tag linked to LysM-SARS-CoV-2 antigens.
  • butyricum engineered to express the antigens located extracellularly via association with the peptidoglycan cell wall CHNR-0 wild-type was used as the control. Either whole cell lysates (2 mL of culture at OD6001.0 pelleted) or proteins extracted from 10 mL culture at OD6001.0 using TCA precipitation were assessed. Gels had 40 ⁇ l protein loaded, blots blocked 5% milk, anti-6XHis (1:1000) in TBS-T 1 h, anti-mouse IgG (1:5000) in TBS-T 1h, developed using SIGMAFAST BCIP/NBT for 6 min.
  • LysM-FlaA recombinant SEQ ID NO: 114
  • LysM-FliD recombinant SEQ ID NO: 116
  • LysM-FlaA polyepitope SEQ ID NO: 126
  • LysM-FliD polyepitope SEQ ID NO: 127) contain multiple epitopes with predicted antigenicity, also downstream of the LysM protein separated by a flexible [Gly]4Ser linker.
  • nucleic acid sequences in this figure consist of the LysM peptidoglycan association domain (in bold), followed by a flexible [Gly]4Ser linker, followed by the LysM-C. jejuni antigenic sequence, followed by a 6X His tag and stop codon (underlined). All sequences in this figure are flanked by 5’ NdeI and 3’ NheI restriction sites (in bold underlined italics).
  • CtxB Cholera enterotoxin subunit B
  • the Cholera enterotoxin subunit B is a 13 kDa subunit protein that makes up the pentameric ring of the Cholera enterotoxin of Vibrio cholerae. Together with the A subunit, it forms the holotoxin (choleragen).
  • the holotoxin consists of a pentameric ring of B subunits whose central pore is occupied by the A subunit.
  • the A subunit contains two chains, A1 and A2, linked by a disulfide bridge.
  • the B subunit pentameric ring directs the A subunit to its target by binding to the GM1 gangliosides present on the surface of the intestinal epithelial cells. It can bind five GM1 gangliosides. It has no toxic activity by itself.
  • Secreted expression of the CtxB protein involves expression of a truncated CtxB protein coupled to the Usp45 secretion signal.
  • the CtxB protein has been shown to provide adjuvant activities in many vaccines - Stratmann, T. Cholera Toxin Subunit B as Adjuvant–An Accelerator in Protective Immunity and a Break in Autoimmunity. Vaccines 2015, 3,579-596.
  • Extracellular expression of the CtxB protein involves the expression of a shorter 25 amino acid peptide (4 kDa) found within the CtxB protein, coupled to cell wall- associated moiety known as LysM.
  • LysM cell wall-associated moiety
  • the immunogenicity of the shorter 25 amino acid peptide (4 kDa) from CtxB has previously been demonstrated by Guyon-Gruaz et al., 1986, Eur. J. Biochem., 159:525-528.
  • the shorter 25 amino acid peptide (4 kDa) also has an almost 100% homology (one amino acid substitution of N>D) to an Enterotoxigenic Escherichia coli toxin, the heat-labile (LT) toxin, and hence may provide cross protection against V. cholerae infection and ETEC infection.
  • the LysM protein domain is a ubiquitous protein domain found in a wide variety of extracellular proteins and receptors, and anchors proteins to the cell surface through non-covalent attachment to the peptidoglycans found in the cell wall.
  • NdeI cleavage site incorporating the nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA (i.e. in the order 5’- FLAG-TAA-NheI-3’).
  • the Usp45-CtxB-FLAG construct was codon optimised for genetic engineering into C.
  • Plasmids were isolated as before and sequenced using GeneWiz sequencing services using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers (Table 1) to confirm the correct insertion of cassettes.
  • the nucleic acid sequence of the p0957-Usp45-CtxB-FLAG construct from the p0957 promoter to the stop codon is SEQ ID NO: 206.
  • the polypeptide sequence is SEQ ID NO: 205.
  • E. coli DH5 ⁇ cells were grown overnight in LB supplemented with 12.5 ⁇ g/mL chloramphenicol and stored at -80°C as 15% glycerol stocks.
  • LysM SEQ ID NO: 42
  • NdeI cleavage site incorporating the nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA (i.e. in the order 5’-FLAG-TAA-NheI-3’).
  • the LysM- CtxB 50-75 -FLAG construct was codon optimised for genetic engineering into C. butyricum and synthesised as a GeneArt String from ThermoFisher Scientific.
  • the GeneArt String was cloned into a pCRTMBlunt II-TOPOTM vector using the Zero BluntTM TOPOTM PCR Cloning Kit (ThermoFisher Scientific) as per the manufacturer’s instructions.
  • the pCRTMBlunt II-TOPOTM vector containing the LysM-CtxB 50-75 --FLAG construct was first transformed into NEB® 10-beta Competent E. coli cells (New England Biolabs) according to the manufacturer’s instructions and colonies were selected using LB Agar plates containing 50 ⁇ g/mL kanamycin.
  • Colonies positive for the plasmid as determined by kanamycin resistance were selected and the plasmid was isolated using Wizard Plus SV Miniprep DNA Purification kit (Promega) following the manufacturer’s instructions and confirmed for correct insertion using primers CH478 (SEQ ID NO: 49) and CH479 (SEQ ID NO: 50) denoted in Table 1.
  • the LysM-CtxB 50-75 -- FLAG construct was excised from the pCRTMBlunt II-TOPOTM vector using restriction endonucleases NdeI and NheI in CutSmart® buffer (all New England Biolabs Inc) according to the manufacturer’s instructions.
  • the isolated cassette was introduced into pMTL82151 (pBP1 Gram+ replicon, catP antibiotic marker, ColE1 Gram- replicon, traJ conjugal transfer function, and multiple cloning site (MCS)) already containing a pfdx (C. sporogenes ferrodoxin) promoter (SEQ ID NO: 183).
  • the plasmid was transformed into NEB® 10-beta Competent E. coli for propagation and storage, with colonies positive for plasmid uptake determined using LB agar + 25 ⁇ g/mL chloramphenicol for selection.
  • Plasmids were isolated as before and sequenced using GeneWiz sequencing services using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers (Table 1) to confirm the correct insertion of cassettes. Following sequence confirmation in E. coli NEB® 10-beta, cells were grown overnight in LB supplemented with 12.5 ⁇ g/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Sequence confirmed plasmid pMTL82151_pfdx-LysM-CtxB 50-75- -FLAG was then transformed into E. coli CA434 conjugation donor strain. Following sequence confirmation as above, E.
  • LysM-CtxB 50-75- -FLAG were grown overnight in LB supplemented with 50 ⁇ g/mL kanamycin and 12.5 ⁇ g/mL chloramphenicol and stored at -80°C as 15% glycerol stocks.
  • the LysM-CtxB 50-75- -FLAG construct was excised from the pMTL82151 vector using restriction endonucleases NotI and NheI in CutSmart® buffer (all New England Biolabs Inc) according to the manufacturer’s instructions.
  • the isolated cassette + promoter was introduced into pMTL83151 (pCB102 Gram+ replicon, catP antibiotic marker, ColE1 + tra Gram- replicon, traJ conjugal transfer function, and multiple cloning site (MCS)) already containing homologous arm sequences for the repair of the pyrE gene which is the site that is targeted for genetic insertion on the genome of the CHN-0.1 strain ( ⁇ pyrE derivative of WT CHN-0). Plasmids were transformed into E. coli NEB® 10-beta for propagation.
  • Plasmids were isolated as before and sequenced using GeneWiz sequencing services using CH22 (SEQ ID NO: 51) and CH648 (SEQ ID NO: 53) primers (Table 1) to confirm the correct insertion of cassettes. Sequence confirmed plasmids pMTL83151_pyrE repair_pfdx_LysM-CtxB 50-75- -FLAG were transformed into E. coli CA434 conjugation donors. Following sequence confirmation as above, E. coli CA434 were grown over night in LB supplemented with 50 ⁇ g/mL kanamycin and 12.5 ⁇ g/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Genetic engineering of C.
  • coli CA434 and C. butyricum CHN-0 such treated were transferred into the anoxic workstation and mixed at a ratio of 5:1 (OD 600 :OD 600 ), usually 1 mL E. coli to 0.2 mL C. butyricum.
  • the conjugation mixture was spotted onto pre-reduced non- selective RCM agar plates and incubated upright overnight. Following incubation, the mixture was harvested into 500 ⁇ L fresh pre-reduced RCM broth and spread in 100 ⁇ L volume onto fresh pre-reduced RCM agar plates supplemented with 250 ⁇ g/mL D- cycloserine and 15 ⁇ g/mL thiamphenicol. To select for C.
  • Table 1 Primers used for sequence confirmation for LysM-CtxB 50-75 -FLAG incorporation into pCRTMBlunt II-TOPOTM vectors, and pMTL82151_pfdx-LysM-CtxB 50-75- -FLAG, pMTL82121-p0957-Usp45-CtxB-FLAG, and pMTL83151_pyrE repair_pfdx_LysM- CtxB50-75-FLAG plasmid generation.
  • CH478 (SEQ ID F GTAAAACGACGGCCAG M13F NO: 49)
  • the introduction of the pMTL82151_pfdx-LysM-CtxB 50-75 -FLAG plasmid into C. butyricum CHN-0 leads to a high expression of the CtxB 50-75 -FLAG peptide by C. butyricum from a multicopy plasmid.
  • the introduction of the pMTL82121- p0957-Usp45-CtxB-FLAG plasmid into C. butyricum CHN-0 leads to a high expression and secretion of the CtxB-FLAG protein by C.
  • butyricum from a multicopy plasmid into the culture supernatant was carried out as above for pMTL82151_pfdx-LysM-CtxB 50-75 -FLAG plasmid introduction, except the base strain used was CHN-0.1 ( ⁇ pyrE derivative of WT CHN-0), the plasmid was pMTL83151_pyrE repair_pfdx_LysM-CtxB 50-75- -FLAG and additional steps were included after selection on RCM agar plates supplemented with 250 ⁇ g/mL D-cycloserine and 15 ⁇ g/mL thiamphenicol.
  • the additional steps include: colonies were then patch plated reiteratively onto Clostridium Basal Medium (CBM) agar plates and cross-checked for plasmid loss on thiamphenicol-containing selective RCM agar plates to select for mutants with restored uracil prototrophy.
  • Genomic DNA of prototroph colonies that had lost the plasmid was isolated using the GenEluteTM Bacterial Genomic DNA kit (SIGMA-Aldrich) as per the manufacturer’s instructions and used for sequencing to confirm presence of the LysM-CtxB 50-75- -FLAG cassette in the chromosome of C. butyricum using primers spanning the integration region, the promoter and respective gene construct sequence (Table 2).
  • butyricum CHN-0 + pMTL82121_p0957Usp45-CtxB-FLAG were used to inoculate fresh pre-reduced supplemented RCM broth + 15 ⁇ g/mL thiamphenicol in serial dilution and grown overnight.
  • the most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth + 15 ⁇ g/mL thiamphenicol at a starting OD 600 of 0.05.
  • OD 600 of 10 When cultures were grown to an OD 600 of 1.0 and 4.0 post initial inoculation (as indicated in Figure 3), the equivalent of OD 600 of 10 was centrifuged at 9500 ⁇ g for 6 min.
  • Dried supernatant protein pellets were then resuspended in 20 ⁇ L 5 ⁇ SDS loading dye (20% (V/V) 0.5 Tris hydrochloride pH 6.8, 23% (V/V) Glycerol, 40% (V/V) of a 10% (w/V) Sodium dodecyl sulphate (SDS) solution, 10% (V/V) 2-Mercaptoethanol, 10 mL dH2O, Bromophenol blue) + 1-5 ⁇ L 1 M Tris/HCl, pH 7.4 buffer until the protein pellets turned blue, and heat treated at 98°C for 15 minutes.
  • SDS loading dye 20% (V/V) 0.5 Tris hydrochloride pH 6.8, 23% (V/V) Glycerol, 40% (V/V) of a 10% (w/V) Sodium dodecyl sulphate (SDS) solution, 10% (V/V) 2-Mercaptoethanol, 10 mL dH2O, Bromophenol blue) + 1
  • PVDF membranes were first incubated in TBS-T blocking buffer (50 mM Tris hydrochloride, 150 mM Sodium chloride, 0.1% Tween20, pH 7.4, 5% (w/V) milk powder) for 1 h at room temperature on a shaking platform. The membrane was then washed once for 5 min in TBS-T buffer (50 mM Tris hydrochloride, 150 mM Sodium chloride, 0.1% Tween20, pH 7.4), before TBS-T containing Anti-FLAG tag® antibody Alkaline phosphatase conjugate (1:5,000; Sigma) was added for incubation at room temperature for 2 h on a shaking platform.
  • TBS-T blocking buffer 50 mM Tris hydrochloride, 150 mM Sodium chloride, 0.1% Tween20, pH 7.4
  • TBS-T buffer 50 mM Tris hydrochloride, 150 mM Sodium chloride, 0.1% Tween20, pH 7.4
  • the membrane was washed twice for 5 min at room temperature in TBS-T buffer and once for 5 min at room temperature in TBS buffer (50mM Tris hydrochloride, 150 mM Sodium chloride, pH 7.4). Alkaline phosphatase detection was performed using SIGMAFAST BCIP®/NBT substrate (SIGMA Aldrich) as per the manufacturer’s instructions. Expression can be seen in Figure 3. For plasmid-based expression, the secreted CtxB-FLAG protein was detectable to high levels on a Western blot, with a reduction of protein observed at the later time point. Confirmation of expression of LysM-CtxB 50-75 -FLAG in C. butyricum Fresh colonies of revived C.
  • butyricum CHN-0 + pMTL82151_pfdx-LysM-CtxB 50-75- -FLAG were used to inoculate fresh pre-reduced supplemented RCM broth + 15 ⁇ g/mL thiamphenicol in serial dilution and grown overnight.
  • the most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth + 15 ⁇ g/mL thiamphenicol at a starting OD 600 of 0.05.
  • the pellets were re-suspended in 40 ⁇ L 5 ⁇ SDS loading dye (20% (V/V) 0.5 Tris hydrochloride pH 6.8, 23% (V/V) Glycerol, 40% (V/V) of a 10% (w/V) Sodium dodecylsulphate (SDS) solution, 10% (V/V) 2-Mercaptoethanol, 10mL dH2O, Bromophenol blue) and heat treated at 98°C for 15 minutes.
  • SDS loading dye 20% (V/V) 0.5 Tris hydrochloride pH 6.8, 23% (V/V) Glycerol, 40% (V/V) of a 10% (w/V) Sodium dodecylsulphate (SDS) solution, 10% (V/V) 2-Mercaptoethanol, 10mL dH2O, Bromophenol blue
  • coli Positive Control Whole cell lysate ab5395 (abcam) was used as FLAG tag positive control in a 1:5 dilution or Carboxy-terminal FLAG-BAPTM Fusion Protein (FLAG-BAPTM, Sigma Aldrich) standards were used, diluted to a range of concentrations from 1000 ng – 50 ng. Separated protein were blotted onto PVDF membranes using the Tran-Blot® Turbo TM blotting system (BioRad) with the Trans-Blot® Turbo TM packs as per the manufacturer’s instructions.
  • FLAG-BAPTM Carboxy-terminal FLAG-BAPTM Fusion Protein
  • PVDF membranes were first incubated in TBS-T blocking buffer (50 mM Tris hydrochloride, 150 mM Sodium chloride, 0.1% Tween20, pH7.4, 5% (w/V) milk powder) for 1 h at room temperature on a shaking platform.
  • the blocking buffer was then replaced by TBS-T buffer (50mM Tris hydrochloride, 150mM Sodium chloride, 0.1% Tween20, pH7.4) containing Anti-FLAG tag® antibody Alkaline phosphatase conjugate (1:5,000; Sigma) for incubation at room temperature for 2 h on a shaking platform.
  • the membrane was washed twice for 5 min at room temperature in TBS-T buffer and once for 5 min at room temperature in TBS buffer (50mM Tris hydrochloride, 150 mM Sodium chloride, pH7.4). Alkaline phosphatase detection was performed using SIGMAFAST BCIP®/NBT substrate (SIGMA Aldrich) as per the manufacturer’s instructions. Expression can be seen in Figure 4A-C.
  • the LysM-CtxB 50-75- -FLAG protein was detectable to high levels on a Western blot, corresponding to ⁇ 270 ng in a specific volume of cells cultured to OD1.0.
  • CGM Clostridial Growth Medium
  • yeast extract 1 g/L sodium chloride (NaCl)
  • K 2 HPO 4 0.75 g/L potassium phosphate dibasic
  • potassium phosphate monobasic 0.75 g/L potassium phosphate monobasic
  • magnesium sulphate heptahydrate Mg 2 SO 4 .7H 2 O
  • iron (II) sulphate heptahydrate FeSO 4 .7H 2 O
  • MnSO4.4H2O 0.01 g/L ammonium sulphate
  • the most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented CGM broth + 2% glucose, 1% CaCO 3 and 0.4% glycine at a starting OD 600 of 0.05.
  • OD 600 0.7-0.9
  • the equivalent of OD 600 of 5/mL was centrifuged at 5,000 ⁇ g for 10 min. The supernatant as discarded and the pellet was washed once with 5 mL pre-reduced filter- sterilised lysis buffer (50 mM TBS with 25 mM CaCl 2 , 25 mM MgCl 2 , and 0.3 M sucrose).
  • the pellet was then resuspended in 5 mL of the pre-reduced filter sterilised lysis buffer supplemented with 3 mg/mL lysozyme (Lysozyme from chicken egg white, Sigma Aldrich) and incubated at 37°C for 3 h in an anoxic environment.
  • the digested cell suspensions were then centrifuged for 3 minutes at 5,000xg (4°C) to pellet out any undigested cells, and then the supernatant centrifuged at 9,000xg for 20 minutes to separate the cell wall fraction (supernatant) from the protoplast (pellet).
  • TCA Trichloroacetic acid
  • Dried cell wall protein pellets were then resuspended in 100 ⁇ L 5 ⁇ SDS loading dye + 10 ⁇ L 1 M Tris/HCl, pH7.4 buffer until the protein pellets turned blue. The samples were then heat treated at 98°C for 15 minutes. A maximum of 30 ⁇ L/well of the re-suspended pellets was loaded onto a Novex TM WedgewellTM 14% Tris Glycine 1.0 mm mini gel (ThermoFisher Scientific) and run in 1 ⁇ NovexTM Tris Glycine SDS Running Buffer (ThermoFisher Scientific) using 180V at room temperature.
  • PageRulerTM Plus Prestained Protein Ladder (ThermoFisher Scientific) was loaded at 10 ⁇ L/well as a marker.
  • the E. coli Positive Control Whole cell lysate ab5395 (abcam) was used as FLAG-tag positive control in a 1:5 dilution and then subject to Western blotting as described for LysM-CtxB 50-75 -FLAG whole cell lysate detection as above.
  • Cell wall fractions containing the LysM-CtxB 50-75- -FLAG can be seen in Figure 5, demonstrating the extracellular location of the CtxB50-75 peptide.
  • Pellets were washed three times before incubating with a fluorescence- conjugated secondary antibody, goat anti-mouse-Alexa-Fluor® 647 (Cell Signalling) at 1:1000 dilution (in PBS + 1% BSA) overnight without light at 4°C. Pellets were then washed five times in PBS before 10 ⁇ L of the fluorescently-labelled cells were mounted onto coverslips using Vectashield H-1700 supplemented with 10 mM MEA (Cysteamine Hydrochloride, Sigma) and left to dry before imaging.
  • mice will be administered an immunisation regimen as described herein.
  • the mice may be administered 1 x 10 8 CFU/dose orally in 3 doses, 2 weeks apart.
  • mice may be administered the dose orally in two or more immunisations comprising two or more consecutive days, with intervals of one or more days in between immunisations.
  • mice will be administered individual strains selected from a wild-type CHN- 0 strain (negative control) or the CHN-0 vaccine strain expressing the antigens from the pMTL82121-P0957-Usp45-CtxB-FLAG, pMTL82151-pfdx-LysM-CtxB 50-75- -FLAG plasmid and/or the integrated LysM-CtxB 50-75- -FLAG constructs.
  • Two final groups will be administered a commercially obtained cholera holotoxin (C-TX) as a positive control in either oral gavage or intraperitoneal (i.p.) injection.
  • C-TX cholera holotoxin
  • CD4 + T cell response will also be analysed in gut-specific tissues (small intestine and colon), where the tissue will be extracted, treated with mucolytic enzymes + EDTA and digested to a single cell suspension, as described in Di Luccia et al (2020) Cell Host & Microbe 27: 899-908. Isolated CD4 + T cells from this suspension will be re-stimulated with antigen presenting cells (APCs, previously exposed to a commercial C-TX antigen) and the change in CD40 ligand expression on the cell surface will be assessed via Flow Cytometry.
  • APCs antigen presenting cells
  • Faecal pellets will be assessed for the antigen-specific humoral response via ELISA assays to determine CtxB-specific secretory IgA (sIgA) production as a percentage of the total IgA, as described in Di Luccia et al (2020) Cell Host & Microbe 27: 899-908.
  • Materials and Methods Isolation of mononuclear cells Harvested spleens at termination will be homogenised and splenocytes isolated, whilst PBMCs will be isolated from the terminal whole blood samples. Isolation of cells will be performed using Ficoll-Paque 1.084 density gradient (GE healthcare) according to manufacturer’s instructions.
  • Cell suspension or whole blood will be layered on Ficoll- Paque media and centrifuged at 400 ⁇ g for 20-30min at RT. The mononuclear cells isolates will then be washed in balanced salt solution to remove residual contaminants.
  • mononuclear cell isolates will be purified using CD8a (Ly-a) MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.
  • a volume of 90 ⁇ L of MACS buffer PBS, 0.5% bovine serum albumin, 2mM EDTA, pH 7.2
  • MACS buffer PBS, 0.5% bovine serum albumin, 2mM EDTA, pH 7.2
  • CD8 + T-cells will then be eluted subsequently by application of buffer without magnetic field. Both CD4 + and CD8 + T-cells will be resuspended in RPMI medium before use in ELISPOT experiments.
  • IFN- ⁇ T-cell ELISPOT The Mouse IFN- ⁇ T-cell ELISPOT kit (U-CyTech Bioscience) will be used for detection of IFN- ⁇ release according to manufacturer’s instructions.
  • a total of 2.5 ⁇ 10 5 T-cells in 100 ⁇ L RPMI/well will be added to plates precoated with Anti-IFN- ⁇ antibodies and re-stimulated with C-TX holotoxin (each at 5 ⁇ g/well; SIGMA) or CHN-0 vegetative cells at 0.5 ⁇ 10 5 CFU/well.
  • Concanavalin A (Sigma Aldrich) will be added as positive control at a concentration of 5mg/mL. Plates will then be incubated overnight at 37°C and 5% CO2 before addition of biotinylated detection antibody followed by incubation with GABA conjugate and incubation with Activator I/II solution to allow for spot formation.
  • mice will then be scanned using a Celigo Image Cytometer and quantified using ImageJ software.
  • ELISPOT assays of CD4 + /CD8 + T-cells show mice CtxB-antigen-specific T-cell response, with a stronger emphasis on the CD4 + response.
  • mice immunised with the CHN-0 wild type strain developing a T-cell response.
  • a detectable CD4 + T-cell response would likely indicate a stronger, more long-lasting, and more specific vaccine response in account of the T-cell help to antibody production and recall responses.
  • enumeration of antigen-specific T cells will be undertaken using flow cytometry with two separate approaches i) detecting recall response based on CD40L upregulation following stimulation with soluble antigen (presented on pulsed antigen presenting cells), ii) Class 2 MHC tetramer (reagent in development) response.
  • a positive result for the test condition would be indicated by a higher percentage antigen-specific (i.e., CD40L upregulated) CD4 + T-cells versus negative control group, or the higher presence of the MHC tetramer reagent on the cell surface compared to negative control groups.
  • the sIgA antibody response is also known to be important in protective immunity against infections by V. cholerae, and therefore we also seek to determine the humoral response for mucosal immunity via assessment of the production of CtxB-specific secretory IgA (sIgA).
  • Example 2 Immunisation of mice using secreted Human Rotavirus antigens, VP7 and VP8, expressed by Clostridium Human Rotavirus is the most common cause of diarrhoeal disease among infants and young children, causing severe gastroenteritis and contributes to a significant number of infant deaths across the world, with higher prevalence in low- and middle-income countries. Humans are primarily infected by the species rotavirus A within which several different serotypes exist.
  • the HRV genome codes for six structural proteins (VP1-VP4, VP6 and VP7) and six non-structural proteins (NSP1-NSP6).
  • the infectious virion particle is formed from 3 layers of protein, whereby the outer later (outer capsid) consists of the surface proteins, VP7 and VP4, which contribute to the classification of serotypes.
  • the glycoprotein Viral Protein 7 (VP7) defines the G serotype and the protease sensitive VP4 defines the P serotype.
  • the VP4 protein protrudes on the cell surface of the virion as a spike and is involved in binding the human cell receptors including sialoglycans (such as Gangliosides GM1 and GD1a) and histo-blood group antigens (HBGAs), driving entry into the cell.
  • sialoglycans such as Gangliosides GM1 and GD1a
  • HBGAs histo-blood group antigens
  • the VP4 is cleaved by trypsin (found in the human gut) into VP5 and VP8 to allow the virus particle to become infectious.
  • VP7 and VP4 are important in rotavirus immunity, containing epitopes for T-cell and B-cell activation as well as antigenic sites responsible for neutralisation of the virus (specifically in the VP8 fragment of VP4) and as such, were chosen as target immunogens for the oral CHN-0 vaccine against Human Rotavirus.
  • the HRV-VP7 and -VP8 protein expression is coupled to a Sec-dependent secretion system signal peptide known as Usp45, which is a native secretion signal peptide found within C. butyricum.
  • Usp45 Sec-dependent secretion system signal peptide
  • Antigenic targets were selected for their external location on the virion outer capsid and therefore increased likelihood of generating an antigenic immune response.
  • the VP7 antigen was derived from in silico prediction for surface accessible conserved region in the VP7 protein (Ghosh et al, 2012, PLoS One, 7(7):e40749, DOI: 10.1371/journal.pone.0040749) and the regions determined were then applied to the epitope prediction servers Immune Epitope Database and Analysis Resource (IEDB; Vita et al., 2010, Nucleic Acids Res., 38:D854-862, DOI: 10.1093/nar/gkp1004) and ABCpred (Saha & Raghava, 2006, Proteins, 65:40-48, DOI: 10.1002/prot.21078) for their epitopic properties towards possible T-cell and B-cell activation.
  • IEDB Immune Epitope Database and Analysis Resource
  • the VP8 protein (as part of the VP4 protein) is another surface exposed antigen, which has previously been shown to be suitable for inducing immune responses (Xue et al., 2016, Hum. Vaccin. Immunother., 12(11):22959-2968, DOI: 10.1080/21645515.2016.1204501; Wen et al., 2012, Vaccine, 30(43):6121-6126, DOI: 10.1016/j.vaccine.2012.07.078).
  • the sequence was determined from the UniProtKB submission P11193 for the outer capsid protein VP4, whereby amino acids 1-225 (SEQ ID NO: 25) relate to the VP8 domain.
  • the first 63 amino acids including the signal sequence (MASLIYRQLLTNSYSVDLHDEIEQIGSEKTQNV TINPSPFAQTRYAPVNWGHGEINDSTTVEPI (SEQ ID NO: 57)) were removed, a 6 amino acid sequence (LSGVYA (SEQ ID NO: 63)) was added at the N-terminus and a C- terminal FLAG tag (DYKDDDDK (SEQ ID NO: 46)) was also added.
  • NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA.
  • the HRV-VP8-FLAG construct was codon optimised for genetic engineering into C.
  • plasmids were received and sequences confirmed via PCR using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers detailed in Table 1, cloning was undertaken to introduce a signal peptide (MKKKIISAILMSTILSAAAP, known as Usp45 (SEQ ID NO: 41)) between the p0957 promoter (SEQ ID NO: 182 - from C. acetobutylicum ATCC824, p0957 is the promoter for the locus tag Ca_c0957) and the HRV-VP8 open reading frame.
  • MKKKIISAILMSTILSAAAP known as Usp45 (SEQ ID NO: 41)
  • the usp45 nucleic acid sequence was amplified using primers CH754 and CH758 (see Table 4), which were designed to overlap with the RBS region of the pMTL82121_p0957 vector digested with NdeI at the 5’ and a VP8 fragment at the 3’ utilising a design that preserved the NdeI site.
  • the FLAG-tagged VP8 fragment was then amplified using primers CH758 (SEQ ID NO: 58) and CH757 (SEQ ID NO: 59) (Table 4), from the Genewiz-generated pMTL82121-p0957-HRV-VP8-FLAG plasmid, to create a fragment which contained an overlap at the 5’ to the usp45 sequence and at the 3’ to a pMTL82121-p0957 vector digested with NheI utilising a design that preserves the NheI site.
  • Hi-Fi assembly was used to assemble the entire pMTL82121-p0957-Usp45- HRV-VP8-FLAG construct from the templates Usp45 fragment and the HRV-VP8-FLAG fragment generated via PCR described above, plus an NdeI and NheI digested pMTL82121-p0957 vector.
  • the resulting constructs transformed into NEB® Stable Competent E. coli (New England Biolabs) for propagation and storage, with colonies positive for plasmid uptake determined using LB agar + 25 ⁇ g/mL chloramphenicol for selection. Plasmids were isolated as before and sequenced using GeneWiz sequencing services to confirm the correct insertion of cassettes. Following sequence confirmation in NEB® Stable Competent E.
  • the p0957-Usp45-HRV- VP8 construct was excised from the pMTL82151_p0957-Usp45-HRV-VP8-FLAG plasmid using restriction endonucleases NotI and NheI in CutSmart® buffer (all New England Biolabs Inc) according to the manufacturer’s instructions.
  • the isolated cassette + promoter was introduced into the pMTL83151_pyrE repair vector. Plasmids were transformed into NEB® Stable Competent E. coli for propagation. Plasmids were isolated as before and sequenced using GeneWiz sequencing services to confirm the correct insertion of cassettes using primers CH22 (SEQ ID NO: 51) and CH648 (SEQ ID NO: 60).
  • a polyepitope antigenic protein For the secreted HRV-VP7 protein, a polyepitope antigenic protein has been designed for expression and secretion (Figure 8). This polyepitope consists of 5 identified immunogenic epitopes, repeated 3 times and separated by flexible linkers and has been constructed as below for future incorporation into the CHN-0 strain. Table 4: Primers used in Usp45 cloning introduction to HRV-VP8 construct.
  • the epitopes were repeated 3 times within the construct to maximise antigen presentation, with the Usp45 secretion signal (MKKKIISAILMSTILSAAAP (SEQ ID NO: 41)) and a 6 amino acid sequence (LSGVYA (SEQ ID NO: 63)) added 5’ to the polyepitope construct, and also a C-terminal FLAG tag (DYKDDDDK (SEQ ID NO: 46)) was added.
  • Further modifications included for genetic engineering include an NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA.
  • the HRV-VP7-FLAG polyepitope construct was codon optimised for genetic engineering into C. butyricum, synthesised by GeneWiz and cloned into a pMTL82121_p0957 vector submitted to GeneWiz for subcloning. Once plasmids are received, the plasmids will be cloned into NEB® Stable Competent E. coli for propagation and storage, and then sequences will be confirmed via PCR using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers detailed previously in Table 1.
  • the CHN-0 strain carrying this antigen will be subject to the same in vivo analysis as detailed below for HRV-VP8-FLAG carrying CHN-0 strains.
  • Genetic engineering of C. butyricum Correct plasmids for pMTL82121-p0957-Usp45-HRV-VP8-FLAG were transformed into E. coli CA434 conjugation donor strain and conjugated into C. butyricum CHN-0 for plasmid-based expression as described previously for pMTL82151-pfdx-LysM-CtxB50- 75–FLAG constructs in Example 1.
  • Primers used to determine correct integration of the p0957-Usp45-HRV-VP8-FLAG construct into the genome are listed in Table 5 below.
  • Table 5 Primers used for confirmation of integration of Usp45-HRV-VP8 into the CHN- 0 genome.
  • the introduction of the pMTL82151_p0957-Usp45-HRV-VP8 plasmid into C. butyricum CHN-0 leads to a high expression of the secreted VP8 protein by C. butyricum from a multicopy plasmid, with a lower expression of HRV-VP8-FLAG when integrated into the genome as it is single copy. Confirmation of expression and secretion of the HRV-VP8 candidate in C.
  • a maximum of 40 ⁇ L/well of the re-suspended pellets was loaded onto a Novex TM WedgewellTM 14% Tris Glycine 1.0 mm mini gel (ThermoFisher Scientific) and run in 1 ⁇ NovexTM Tris Glycine SDS Running Buffer (ThermoFisher Scientific) using 180V at room temperature. PageRulerTM Plus Prestained Protein Ladder (ThermoFisher Scientific) was loaded at 10 ⁇ L/well as marker. Carboxy-terminal FLAG-BAPTM Fusion Protein (FLAG-BAPTM, Sigma Aldrich) standards were used, diluted to a range of concentrations from 2 ⁇ g – 50 ng.
  • the separated proteins were subject to Western blotting as described for LysM-CtxB 50- 75-FLAG in Example 1 and shown in Figure 9.
  • the secreted HRV-VP8 protein was detectable to high levels on a Western blot, corresponding to 2500 ng in a specific volume of supernatant culture (10 mL) when cells reach OD1.0 in their growth phase. Assuming the cell density in OD1.0 is 0.3 g/L, it is estimated that the secreted protein is therefore ⁇ 0. 825 ⁇ g/mg cell weight.
  • Expression of integrated Usp45-HRV-VP8-FLAG was also assessed similar to plasmid- based expression above. Fresh colonies of revived C.
  • butyricum CHN-0 + Usp45-HRV- VP8-FLAG were used to inoculate fresh pre-reduced supplemented FMC broth (no antibiotics) at a starting OD 600 of 0.05.
  • 10mL of culture was harvested before centrifugation at 13,000 ⁇ g for 10 min at 4°C and the supernatants kept for protein assessment. Proteins were extracted from supernatants using TCA precipitation and pellets prepared as described for plasmid- based expression of HRV-VP7 and HRV-VP8, before SDS-PAGE analysis and Western blotting as described above.
  • mice will be administered an immunisation regimen as described herein.
  • the mice may be administered 1 x 10 8 CFU/dose orally in 3 doses, 2 weeks apart.
  • mice may be administered the dose orally in two or more immunisations comprising two or more consecutive days, with intervals of one or more days in between immunisations.
  • mice will be administered individual strains selected from a wild-type CHN-0 strain (negative control), the CHN-0 oral vaccine strain expressing the antigens from the pMTL82151-p0957-Usp45-HRV-VP7- polyepitope-FLAG plasmid (once constructed and analysed for expression), the pMTL82151-p0957-Usp45-HRV-VP8-FLAG and/or the integrated CADD+Usp45-HRV- VP8-FLAG strain.
  • a final group will be administered the recombinant HRV-VP7 or HRV- VP8 via subcutaneous or i.p. injection (+adjuvant) as a positive control. Clinical observations will be taken throughout to determine tolerability of the test articles (weight changes and physical appearances such as hunching or coat piloerection). Table 6 — example dosing groups for CHN-0-based oral vaccines against Human Rotavirus
  • spleens will be harvested and processed to a single cell suspension and CD4 + and CD8 + cells purified individually to determine CD4 + /CD8 + -specific T cell response via IFN- ⁇ release in ELISPOT assays following antigen restimulation using either the control HRV-VP7/8 protein or CHN-0 vegetative cells and detected using the Mouse IFN- ⁇ T-cell ELISPOT kit (U-CyTech Bioscience) .
  • CD4 + T cell response will also be analysed in gut-specific tissues (small intestine and colon), where the tissue will be extracted, treated with mucolytic enzymes + EDTA and digested to a single cell suspension, as described in Di Luccia et al (2020) Cell Host & Microbe 27: 899-908. Isolated CD4 + T cells from this suspension will be re-stimulated with antigen presenting cells (APCs, previously exposed to a recombinant HRV-VP7/8 antigen) and the change in CD40 ligand expression on the cell surface will be assessed via Flow Cytometry.
  • APCs antigen presenting cells
  • Faecal pellets will be assessed for the antigen-specific humoral response will be assessed via ELISA assays to determine HRV antigen-specific secretory IgA (sIgA) production as a percentage of the total IgA, as described in Di Luccia et al (2020) Cell Host & Microbe 27: 899-908.
  • SIgA HRV antigen-specific secretory IgA
  • antigen-specific T cells will be undertaken using flow cytometry with two separate approaches i) detecting recall response based on CD40L upregulation following stimulation with soluble antigen (presented on pulsed antigen presenting cells), ii) Class 2 MHC tetramer (reagent in development) response.
  • a positive result for the test condition would be indicated by a higher percentage antigen-specific (i.e., CD40L upregulated) CD4 + T-cells versus negative control group, or the higher presence of the MHC tetramer reagent on the cell surface compared to negative control groups.
  • the sIgA antibody response is also known to be important in protective immunity against mucosal infections, and therefore we also seek to determine the humoral response for mucosal immunity via assessment of the production of HRV antigen- specific secretory IgA (sIgA).
  • sIgA HRV antigen-specific secretory IgA
  • ELISAs we expect to see an increase in antigen- specific sIgA in response to administration of the CHN-0-HRV oral vaccines, compared to the wild-type CHN-0 platform alone.
  • Example 3 Immunisation of mice using extracellular and intracellular SARS- CoV-2 peptide antigens expressed by Clostridium COVID-19 is caused by the severe acute respiratory syndrome coronavirus (SARS- CoV-2).
  • SARS-CoV-2 contains four structural proteins, including Spike (S), Envelope (E), membrane (M) and nucleocapsid (N) and at least 6 other open reading frames (ORFs).
  • S protein plays the most important role in viral attachment, fusion and entry into the host cell and thereby serves as a strong target for vaccine development.
  • the S protein contains 2 major subunits, the S1 subunit which contains the receptor binding domain (RBD) which binds to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell, and the S2 subunit which allows fusion between the virus and the host cell membrane.
  • SARS-CoV-2 epitopes were selected based on the methods described in Fast et al.
  • Li et al used peptide microarrays where 200+ peptides in the S protein were synthesised and screened for IgG and IgM responses in convalescent COVID-19 patient sera, followed by neutralisation assays.
  • Selected epitopes were identified from the Spike protein (including in the RBD) which had good predicted antigen presentation scores for MHC I and MHC II, and also linear epitopes that show stimulation of neutralising antibodies. Other epitopes were identified within the other structural proteins (M, N, E) and in a non-structural protein, Orf1ab, which have the potential to elicit a strong T cell response. Chosen epitopes were linked with a cathepsin S cleavage site for higher probability of epitope presentation via the MHCs, and as with the CtxB 50-75 -FLAG antigen in Example 1, these polyepitopes were linked to the LysM peptidoglycan-anchoring domain for extracellular expression.
  • LysM-CoV-2-RBD immunogenic epitopes determined in the RBD protein sequence of the S1 subunit of the spike protein, SEQ ID NO: 7
  • LysM-CoV-2-Nuc immunogenic epitopes determined in the nucleocapsid protein sequence, SEQ ID NO: 13
  • LysM-CoV-2-Comb immunogenic epitopes determined from the nucleocapsid, membrane, envelope, and spike protein sequences, as well as immunogenic epitopes determined in the Orf1ab non-structural protein, SEQ ID NO: 17
  • LysM-CoV-2-SB1 neutralising (B cell) linear epitopes determined in the spike protein, SEQ ID NO: 9
  • the LysM peptidoglycan-anchoring domain sequence (SEQ ID NO: 42) was also added upstream of all SARS-CoV-2 polyepitopes for extracellular localisation and the LysM domain was separated from the SARS-CoV-2 polyepitope by a flexible [Gly] 4 Ser linker.
  • Further modifications included for genetic engineering include two NdeI cleavage sites (CATATG) either side of the LysM encoding domain incorporating the nucleotide signal for aa methionine (M, ATG) and allowing removal of the LysM domain, and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the His-tag by the stop codon TAA.
  • LysM- SARS-CoV-2-His antigenic constructs were codon optimised for genetic engineering into C. butyricum and synthesised behind the p0957 promoter by GeneWiz and cloned into pMTL82121 vector submitted to GeneWiz for subcloning. Once plasmids were received, they were transformed into NEB® Stable Competent E. coli (New England Biolabs) for propagation and storage, with colonies positive for plasmid uptake determined using LB agar + 25 ⁇ g/mL chloramphenicol for selection.
  • Plasmids were isolated as before and sequenced using GeneWiz sequencing services and using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 42) primers spanning the MCS (Table 1) to confirm the correct sequence of constructs. Following sequence confirmation in NEB® Stable Competent E. coli, cells were grown overnight in LB supplemented with 12.5 ⁇ g/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Cloning of intracellular expressed SARS-COV-2-X-His antigens was performed through NdeI digestion of sequence confirmed pMTL82121_p0957-LysM-SARS-COV-2-X-His constructs to excise the LysM domain ( Figure 12).
  • coli CA434 conjugation donors and positive colonies were isolated for their resistance to 50 ⁇ g/mL kanamycin and 25 ⁇ g/mL chloramphenicol on LB agar plates. Following plasmid isolation and sequence confirmation as above, E. coli CA434 were grown over night in LB supplemented with 50 ⁇ g/mL kanamycin and 12.5 ⁇ g/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Genetic engineering of C.
  • Table 7 Primers for LysM-SARS-CoV-2-X-His construct sequencing in pMTL82121 vectors. Confirmation of expression of SARS-CoV-2 candidates in C. butyricum Fresh colonies of revived C. butyricum CHNR-0 + pMTL82121_p0957-LysM-SARS-CoV- 2-X-His or intracellular pMTL82121_p0957-SARS-CoV-2-X-His were used to inoculate fresh pre-reduced supplemented RCM broth + 15 ⁇ g/mL thiamphenicol in serial dilution and grown overnight.
  • the most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth + 15 ⁇ g/mL thiamphenicol at a starting OD 600 of 0.05.
  • OD 600 1.0, 2.0 and 4.0
  • the equivalent of OD 600 of 2/mL was centrifuged at 13,000 ⁇ g for 2 min. Supernatants were discarded and the pellets prepared for SDS-PAGE analysis as below. This process was identical for intracellular expression plasmids pMTL82121_p0957-SARS-CoV-2-X-His.
  • the pellets were re-suspended in 40 ⁇ L 5 ⁇ SDS Loading dye and heat treated at 98°C for 15 minutes.
  • the separated proteins were subject to Western blotting as described for LysM-CtxB 50- 75-FLAG in Example 1, except different antibodies for detection were used.
  • An anti- 6XHis primary antibody (1:1000 incubated for 1 h in TBS-T, Abcam) and an HRP- conjugated anti-mouse IgG secondary antibody (1:5000 incubated for 1 h in TST-T, Sigma Aldrich) were used instead of the anti-FLAG-HRP conjugated antibody.
  • Resultant blots are shown in Figure 13.
  • butyricum CHNR-0 leads to varied levels of expression of the peptidoglycan-associated SARS-CoV-2-X-His antigenic proteins by C. butyricum from multicopy plasmids.
  • a level of cleavage presumed to be between the LysM and the SARS-CoV-2 antigen is observed in most antigens, whereby a higher band at the correct expected MW is observed and a lower band at the expected size of the antigenic peptide without a LysM domain is also observed.
  • Figure 13B In the SARS-CoV-2-LysM-Nuc-His antigenic peptide expression ( Figure 13B), a ladder of bands appeared on the Western blot and therefore assessment of the supernatant of this strain was carried out.
  • the separated proteins were subject to Western blotting as described preciously and the anti-6XHis primary antibody (1:1000 incubated for 1 h in TBS-T, Abcam) and an HRP-conjugated anti-mouse IgG secondary antibody (1:5000 incubated for 1 h in TST-T, Sigma Aldrich) were used.
  • the secreted protein profile can be seen compared to CHNR-0 wild type in Figure 14.
  • the further cleavage of the peptidoglycan-associated nucleocapsid polyepitope into several peptides that are then released into the extracellular environment may be due to the pH basic nature of the protein, and the exposure of multiple epitopes from the CoV-2-Nuc-His antigen may increase the immunogenicity of the candidate leading to a better immune response.
  • Immunogenicity testing In vivo immunisation experiments will be performed to assess delivery of antigen and induction of immune responses, with a focus on cellular responses, using oral delivery of spores of engineered C. butyricum to express cell wall-associated SARS-CoV-2-X- His antigens (Table 8).
  • mice will be administered an immunisation regimen as described herein.
  • the mice may be administered 1 x 10 8 CFU/dose orally in 3 doses, 2 weeks apart.
  • mice may be administered the dose orally in two or more immunisations comprising two or more consecutive days, with intervals of one or more days in between immunisations.
  • Groups of mice will be administered individual strains selected from a wild-type CHNR-0 strain (negative control) or the CHNR-0 vaccine strain expressing the antigens from the pMTL82121-p0957-LysM-SARS-CoV-2-X-His antigen series.
  • a positive control group will be administered recombinant spike protein, nucleocapsid protein or a polyepitope protein of membrane, spike, envelope, nucleocapsid and Orf1ab protein antigens via subcutaneous or i.p. injection (+ adjuvant) as a positive control.
  • Clinical observations will be taken throughout to determine tolerability of the test articles (weight changes and physical appearances such as hunching or coat piloerection).
  • Table 8 Dosing groups for CHN-based oral vaccines against SARS-CoV-2 At sacrifice, spleens will be harvested and processed to a single cell suspension and CD4 + and CD8 + cells purified individually to determine CD4 + /CD8 + -specific T cell response via IFN- ⁇ release in ELISPOT assays following antigen restimulation using the appropriate control protein or CHNR-0 vegetative cells and detected using the Mouse IFN- ⁇ T-cell ELISPOT kit (U-CyTech Bioscience).
  • FlaA is the major component of flagellin, and FliD is the flagellar cap protein. Both FlaA and FliD are readily accessible on the surface of Campylobacter bacteria; and have previously been shown to comprise T-cell and/or B-cell epitopes (Yasmin et al, 2016, In silico pharmacol., 4:5 (DOI: 10.1186/s40203-016-0020-y); Lee et al., 1999, Infect.
  • LysM-FlaA-recombinaint denoted LysM-FlaA-recombinaint (recombinant FlaA, SEQ ID NO: 114), LysM-FlaA-polyepitope (immunogenic epitopes determined in the FlaA protein sequence, SEQ ID NO: 126), LysM-FliD-recombinant (recombinant FliD, SEQ ID NO: 115), and LysM-FliD- polyepitope (Immunogenic epitopes determined in the FliD sequence, SEQ ID NO: 127).
  • Chosen epitopes were modified by connection of the individual epitopes in some cases via a Cathepsin S cleavage sequence (TVKLRQ (SEQ ID NO: 39) (in LysM-FlaA- polyepitope, LysM-FliD-polyepitope) and all antigens had a C-terminal 6xHis tag (HHHHHH (SEQ ID NO: 47)) added for expression identification and purification.
  • TVKLRQ SEQ ID NO: 39
  • HHHHHHHH SEQ ID NO: 47
  • the LysM peptidoglycan-anchoring domain sequence (SEQ ID NO: 42) was also added upstream of all Campylobacter antigens for extracellular localisation and the LysM domain was separated from the Campylobacter antigens by a flexible [Gly]4Ser linker. Further modifications included for genetic engineering include two NdeI cleavage sites (CATATG) either side of the LysM encoding domain incorporating the nucleotide signal for aa methionine (M, ATG) and allowing removal of the LysM domain, and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the His-tag by the stop codon TAA.
  • CAATG NdeI cleavage sites
  • GCTAGC nucleotide sequence for a NheI cleavage site
  • LysM-FlaA-His and LysM-FliD-His antigenic constructs will be codon optimised for genetic engineering into C. butyricum and synthesised behind the p0957 promoter by GeneWiz and cloned into pMTL82121 vector submitted to GeneWiz for subcloning. Plasmids will be transformed into NEB® Stable Competent E. coli (New England Biolabs) for propagation and storage as set out in Examples 1-3. Sequence confirmed pMTL82121_p0957-LysM-FlaA-His and pMTL82121_p0957-LysM- FliD-His plasmids will be transformed into E.
  • Example 5 Materials and Methods Culture of bacterial strains Escherichia coli strains NEB® 10-beta Competent, NEB® Stable Competent, and CA434 were grown aerobically in Lysogeny broth (LB; Vegetable tryptone 10g/L, Yeast extract 5g/L, Sodium chloride 10g/L) supplemented with 15% (w/V) agar and/or antibiotics where appropriate at 30°C or 37°C depending on metabolic burden associated with plasmid propagation. Liquid cultures were agitated at 200 rpm during incubation.
  • Clostridium butyricum Strain DSM10702 (CHN-0) is deposited in the DSMZ depository (Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffen No 7B, 38124 Braunschweig, GERMANY). CHNR-0 is deemed an identical strain by NCIMB sequencing.
  • Clostridium butyricum strains were routinely grown in anoxic workstations (Don Whitley, 10% Hydrogen, 10% Carbon dioxide, 80% Nitrogen, 37°C) in Reinforced Clostridial growth medium (RCM; Yeast extract 13g/L, Vegetable peptone 10g/L, Soluble starch 1g/L, Sodium chloride 5g/L, Sodium acetate 3g/L, Cysteine hydrochloride 0.5g/L) supplemented with 10g/L Calcium carbonate, 2% (w/V) Glucose, 15% (w/V) agar and/or antibiotics where appropriate.
  • C for maintenance and selection of genetically engineered strains where genes have been integrated into the genome, C.
  • butyricum was grown in anoxic workstations in Clostridial Basal Medium (CBM, Iron sulphate heptahydrate 12.5mg/L, Magnesium sulphate heptahydrate 250mg/L, Manganese sulphate tetrahydrate 12.5mg/L, Casamino acids 2g/L, 4- aminobenzoic acid 1.25mg/L, Thiamine hydrochloride 1.25mg/L, Biotin 2.5 ⁇ g/L) supplemented with 10g/L Calcium carbonate, 2% (w/V) Glucose, 15% (w/V) agar, uracil and/or antibiotics where appropriate, respectively.
  • CBM Clostridial Basal Medium
  • CBM Iron sulphate heptahydrate 12.5mg/L
  • Magnesium sulphate heptahydrate 250mg/L Magnesium sulphate heptahydrate 250mg/L
  • FMC Fermentation Media for Clostridia
  • FMC Fermentation Media for Clostridia
  • yeast extract 2.5 g/L yeast extract, 2.5 g/L Tryptone, 0.025 g/L iron (II) sulphate heptahydrate (FeSO 4 .7H 2 O), 0.5 g/L Ammonium sulphate (NH 4 ) 2 SO 4 )) supplemented with 10g/L Calcium carbonate, 2% (w/V) Glucose, and antibiotics where appropriate.
  • butyricum spores were produced in 2L vessels of FerMac 320 Microbial culture batch bioreactor systems (ElectroLab Biotechnology Ltd) in RCM supplemented with 2% (w/V) Glucose. Vessels were sparged with nitrogen gas at a flow rate of 0.2 vvm, maintained at a pH of 6.5, temperature of 37°C and agitated at 100 rpm. Cell and spore mass were harvested, and spores were separated from cell matter by repeated washing in ice-cold sterile water. Spores were stored at 4°C until further use. Enumeration of spores was conducted by plating serial dilutions of spore stocks on pre- reduced RCM agar plates in triplicate. Plates were incubated for 24 hours in the anoxic workstation before colony forming units (CFU) were determined. Sequences of the disclosure

Abstract

A bacterium of the class Clostridia comprising a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein (i) the bacterium is capable of exporting the antigen such that It becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen; and wherein the at least one antigen comprises an infectious agent antigen or a tumour antigen; or (li) the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the SI subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orflab protein of SARS-CoV-2.

Description

Compositions and methods Field of Invention The invention relates to bacterial vaccines, particularly live bacterial vaccines suitable for oral administration and for stimulating humoral and/or cellular immunity. Background Vaccines play a leading role in disease prevention, particularly of infectious diseases, and show promise in therapy of existing infections and chronic diseases. Oral vaccines address some of the disadvantages of traditional injection-based formulations, providing improved safety and compliance and easier administration. Oral vaccines may stimulate humoral and cellular responses at both systemic and mucosal sites, but there are significant challenges in their development posed by the gastrointestinal (GI) tract, as reviewed in Vela Ramirez, J. E., Sharpe, L. A., & Peppas, N. A. (2017). Current state and challenges in developing oral vaccines. Advanced drug delivery reviews, 114, 116–131. https://doi.org/10.1016/j.addr.2017.04.008. For example, strategies are needed to avoid fragile antigens being degraded by proteolytic enzymes and the acidic environment of the stomach. Once an oral vaccine reaches the intestine, the presence of a mucus layer, the composition of the gastrointestinal fluid and the action of epithelial barriers limits the permeability of molecules to the lymphatic system. It is believed that antigens are sampled by specialised epithelial cells, “M cells”, in the Peyer’s patches of the gut-associated lymphoid tissue (GALT) of the small intestine and transcytosed and delivered to dendritic cells (DCs) that process and present antigenic fragments on their surface to activate naïve T-cells. Typical strategies in oral vaccines under development have relied on high antigen doses and potent adjuvants in order to trigger an immune response (Ramirez et al, supra). Some strategies make use of Gram-negative bacterial lipopolysaccharide, Salmonella lipid A derivatives or cholera toxin that may elicit adjuvant effects, but there is a trade- off in terms of toxicity. Bacterial vaccines offer promise, and live-attenuated vaccines for Vibrio cholera or Salmonella typhi vaccines have been licensed. Gram-positive bacteria such as Lactococcus, which avoid LPS and may be better tolerated, have been suggested as a potential vaccine platform (Bahey-El-Din, M and Gahan, CGM (2010) Lactococcus lactis based vaccines: ‘Current status and future perspectives’, Human Vaccines, 7:1, 106- 109, DOI:10.4161/hv.7.1.13631). An oral recombinant Lactobacillus vaccine is disclosed in WO 2001/021200 A1. Bacterial vaccines have to date been used to target the small intestine, where the mucosal immune system has been well studied. An attenuated Clostridium perfringens engineered to express high levels of antigen in inclusion bodies during sporulation has been proposed in Chen Y et al (2004) Use of a Clostridium perfringens vector to express high levels of SIV p27 protein for the development of an oral SIV vaccine, Virology 329: 226-233, ISSN 0042-6822, https://doi.org/10.1016/j.virol.2004.08.018. The mechanism seems to rely on the mother cell lysing after sporulation to deliver high levels of antigen directly to the Peyer’s patches located in the terminal ileum of the small intestine. US 5,800,821 (New England Medical Center Hospitals et al.) discloses immunisation strategies involving oral administration of engineered Bacillus subtilis spores for secretion or display of antigen on the bacterial surface. Bacillus subtilis is an aerobe, and therefore would not germinate in the lower GI tract due to lack of oxygen, as described in Tam NK, Uyen NQ, Hong HA, et al. The intestinal life cycle of Bacillus subtilis and close relatives. J Bacteriol. 2006;188(7):2692-2700. Typically, orally administered agents would pass through the upper GI tract in a few hours. US 5,800,821 discloses strategies to promote germination of Bacillus subtilis in the upper GI tract, namely pre-germinating spores before administration, and co-expression of invasin, which bind M cells and may promote colonization. A cell wall binding system cwbA specific for Bacillus is disclosed. An Example in US 5,800,821 discloses C. perfringens engineered to express is Shiga-like toxin B under control of the cpe promoter. As would be known to the skilled person, the cpe promoter is sporulation specific in Clostridium, and would not be active during vegetative growth (see Melville SB, Labbe R, Sonenshein AL. Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis. Infect Immun. 1994;62(12):5550-5558). Oral vaccines licensed to date are typically intended for prevention of infection rather than as therapeutic vaccines. Antibodies produced by B cells are the predominant correlate of protection for current vaccines, but cell-mediated immune functions are critical in protection against intracellular infections, and in almost all diseases, CD4+ cells are necessary to help B cell development (Stanley A. Plotkin (2008) Correlates of Vaccine-Induced Immunity, Clinical Infectious Diseases, Pages 47: 401–409, https://doi.org/10.1086/589862). For control of established infection, and tumour immunity, cellular immunity including CD8+ cytolytic T-cells, is generally perceived as more important. For many protein-based vaccines, the proteins are phagocytosed or endocytosed into endosomes and lysosomes by antigen presentation cells (APCs), whereby lysosomes degrade the protein into smaller peptides, some of which can (CD4 epitopes) bind to MHC class II molecules on lysosomal membranes and are presented to the cell surface to stimulate CD4+ T-cells, which in turn are required for B cells to produce antibodies (T cell help). Therefore, protein antigens have been mainly used to stimulate the body to produce antibodies. The main pathway for the presentation of antigenic peptides on MHC Class I molecules (required for stimulation of CD8+ cytotoxic T cells) relies on antigen that is expressed within the APC, such as following viral infection. However, studies have found that APC can also internalise antigens and present them on MHC Class I molecules to stimulate cytotoxic T lymphocytes (CTL) by a process called antigen cross presentation, which is typically an inefficient process. The delivery of exogenous peptides or proteins to the MHC class I pathway has been partially successful through use of chemical adjuvants such as Freund's adjuvant, and mixtures of squalene and detergents (Hilgers et al. (1999) VACCINE 17:219-228). EP3235831 (Oxford Vacmedix UK Ltd) demonstrates that an artificial multi-epitope fusion protein known as a recombinant overlapping peptide (ROP) is capable of simultaneously stimulating CD4+ and CD8+ T- cell responses. ROPs are made up of overlapping peptides linked by the cathepsin cleavage site target sequence and are more efficient in priming protective immunity than the whole protein from which the peptides are derived. Subcutaneous immunisation with ROPs has been shown to have protective effects in a viral model and a tumour model (Zhang H et al (2009) J. Biol. Chem. 284:9184–9191; and Cai L et al (2017) Oncotarget 8: 76516-76524); and has been shown to generate antibodies specific for SARS-CoV-2 antigens in a murine model in WO 2022/090679 (Oxford Vacmedix UK Ltd). There remains a need for effective bacterial vaccines that are suitable for oral administration, and for stimulating humoral and/or cellular immunity. WO 2018/055388 (CHAIN Biotechnology Limited) discloses Clostridium engineered to express (R)-3-hydroxybutyrate (R-3-HB) as an anti-inflammatory agent, including in a simulated colon environment. WO 2019/180441 (CHAIN Biotechnology Limited) discloses in vivo and pharmacokinetic profiling of R-3-HB engineered Clostridium butyricum. The engineered strain could be isolated from colon samples of mice that had been dosed orally with bacterial spores. The present inventors sought to exploit the ability of Clostridium to grow in anaerobic conditions to target the lower anaerobic regions of the GI tract, such as the large intestine, in order to develop a platform vaccine technology. Contrary to the anti- inflammatory effects of the R-3-HB engineered Clostridium, the present invention is based on the surprising discovery that Clostridium engineered for expression of antigen during anaerobic cell growth can stimulate antigen-specific immune responses. In PCT Patent Application No. PCT/GB2021/053264, the inventors describe the engineering of the wild-type C. butyricum strain used in the present Examples to express a heterologous antigen in the intracellular compartment during anaerobic cell growth; and the generation of an antigen-specific immune response in an oral immunisation mouse model corresponding to that used in the present Examples. Two heterologous FLAG-tagged antigens were tested, based on the HPV E7 and OVA ROP antigens described in Cai L et al (2017) Oncotarget 8: 76516-76524. The amino acid sequence and nucleic acid sequence of the HPV E7 ROP are provided in SEQ ID NOs: 1 and 2 respectively. The amino acid sequence and nucleic acid sequence of the OVA ROP are provided in SEQ ID NOs: 108 and 109 respectively. Nucleic acid cassettes including antigen genes were prepared using an appropriate nucleic acid sequence for either ORF under the control of the fdx promoter, and the antigen gene integrated into the bacterial chromosome using techniques as described in the present application. Intracellular expression of either antigen was detected in an amount of <80 ng/mg dry cell weight. Mice were dosed orally with 108 spores/mouse at days 0, 14 and 28 and sacrificed at day 42. Mice orally immunised with the strain expressing ROP-HPV developed both CD4+ and CD8+ T-cell response, while mice immunised with the strain expressing ROP-OVA developed CD4+ T-cell response specific to the respective antigen, according to IFN-^ ELISPOT evaluation of T-cells isolated from spleens and re- stimulated with antigen. The immunised mice did not develop a T-cell immune response against the C. butyricum strain itself. The technical rationale for the present disclosure is based on the discovery in PCT/GB2021/053264 that Clostridium engineered to express a heterologous antigen in the intracellular compartment during anaerobic cell growth is capable of inducing an antigen-specific immune response in a host following oral immunisation. The present disclosure provides Clostridium which expresses surface-tethered antigen or secretes antigen during anaerobic cell growth; or which expresses SARS-CoV-2 antigens in the intracellular compartment during anaerobic cell growth. Clostridium expressing surface-tethered antigen can be expected to be internalised by the same cells that internalise Clostridium expressing an antigen in the intracellular compartment. Likewise, secreted antigen may accumulate in the vicinity of such cells and be internalised by endocytosis; or may even be secreted into cells that have already internalised Clostridium. Furthermore, intracellular, secreted or surface-tethered forms of antigen may become suitably located for capture by B cells and generation of B cell antibody responses. The listing or discussion of a prior-published document in this specification should not be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Description of the invention A first aspect of the present invention is a bacterium of the class Clostridia comprising a heterologous nucleic acid molecule encoding at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region. The promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; and the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or is capable of secreting the antigen as a secreted antigen. The at least one antigen is an infectious agent antigen or a tumour antigen. A second aspect of the present invention is a bacterium of the class Clostridia comprising a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2. The infectious agent antigen or tumour antigen is heterologous to the bacterium. By “capable of causing expression of” an antigen, we mean that the promoter is capable of causing transcription and typically also translation in the bacterium, which results in the expression of the antigen. The expression of antigen by the bacterium occurs during anaerobic cell growth. Bacteria of the class Clostridia are obligately anaerobic bacteria, the majority of which have the ability to form spores (i.e., are spore-forming bacteria). Such bacteria may be in the form of a spore or in a vegetative form; in the latter form, the bacteria are metabolically active and typically growing. By targeting expression of the antigen to metabolically active forms of the Clostridia, it is possible to use the Clostridia as a vehicle to target antigen to the anaerobic portions of the gut. By administering the bacteria orally as spores, the bacteria remain dormant and viable during transit through the gastrointestinal tract, until they reach the anaerobic portions where they germinate and multiply. Antigens By “antigen”, we mean a molecule that binds specifically to an antibody or a T-cell receptor (TCR). Antigens that bind to antibodies are called B cell antigens. Suitable types of molecule include peptides, polypeptides, glycoproteins, polysaccharides, gangliosides, lipids, phospholipids, DNA, RNA, fragments thereof, portions thereof and combinations thereof. Peptide and polypeptide antigens, including glycoproteins, are preferred. TCRs bind only peptide fragments complexed with MHC molecules. The portions of an antigen that are recognised are termed “epitopes”. Where a B cell epitope is a peptide or polypeptide, it typically comprises 3 or more amino acids, generally at least 5 and more usually at least 8 to 10 amino acids. The amino acids may be adjacent amino acid residues in the primary structure of the polypeptide or may become spatially juxtaposed in the folded protein. T cell epitopes are normally short primary sequences from antigens. They may bind to MHC Class I or MHC Class II molecules. Typically, MHC Class I-binding T-cell epitopes are 8 to 11 amino acids long. Class II molecules bind peptides that may be 10 to 30 residues long or longer, the optimal length being 12 to 16 residues. Peptides that bind to a particular allelic form of an MHC molecule contain amino acid residues that allow complementary interactions between the peptide and the allelic MHC molecule. The ability of a putative T-cell epitope to bind to an MHC molecule can be predicted and confirmed experimentally (Peters et al. (2020) T Cell Epitope Predictions, Annual Reviews of Immunology, Vol. 38:123-145). According to the first aspect, the antigen expressed by the bacterium of the class Clostridia is an infectious agent antigen or a tumour antigen. By “infectious agent antigen”, we mean that the antigen derives from an infectious agent that is capable of infecting a susceptible host, such as a human, typically resulting in a pathology. By “derives from”, we include that the infectious agent antigen is encoded in the genome of an infectious agent, or is a variant of such an encoded antigen. By “tumour antigen”, we mean that the antigen derives from an antigen that is expressed predominantly, such as almost exclusively or exclusively by tumour cells, or acts as a marker that is used in the art to distinguish a tumour cell from a healthy cell. By “derives from”, we include that the tumour antigen is encoded in the genome of a cancer cell, or is a variant of such an encoded antigen. In some embodiments, the antigen may be an infectious agent antigen that is associated with a risk of cancer. An antigen may be a fragment or portion of a complete protein, which fragment includes an epitope. An “antigen segment” is a portion of an antigen, which antigen comprises an epitope. In the context of an antigen, a “fragment” typically comprises at least 5 amino acid residues, typically at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid residues of the antigen from which it is derived. Such fragment lengths may be sufficient to provide an epitope of the antigen. An antigen or fragment may comprise at least or up to 20 amino acid residues, at least or up to 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900 or 1,000 amino acid residues. Any range involving these values is envisaged, such as between 10 and 200 amino acid resides, between 20 and 100 etc. Antigens and fragments of up to about 100, 150, 200, 250, 300, 350, 400, 500 or 550 amino acid residues are typical. Typical fragments of an antigen will have an amino acid sequence comprising at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% of the corresponding native antigen or variant thereof. In the context of an antigen, a fragment may also be referred to as an “antigenic portion”, in other words, a portion of antigen which comprises an epitope. The term “antigenic portion” is also intended to include an entire antigen, since the entire antigen is rendered antigenic by virtue of the antigenic portion. A “variant” refers to a protein or peptide wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non- conservative. By “conservative substitutions” is intended combinations such as Val, Ile, Leu, Ala, Met; Asp, Glu; Asn, Gln; Ser, Thr, Gly, Ala; Lys, Arg, His; and Phe, Tyr, Trp. Preferred conservative substitutions include Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Typical variants of the antigen or portion thereof will have an amino acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 99.5% identical to the corresponding native antigen or portion thereof. The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson et al., (1994) Nucleic Acids Res., 22(22), 4673-80). The parameters used may be as follows: x Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. x Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. x Scoring matrix: BLOSUM. A “variant” may also refer to the nucleic acid molecule that encodes a variant antigen. In some embodiments, the at least one antigen comprises the complete amino acid sequence of a mature polypeptide which is an infectious agent antigen or a tumour antigen or variant thereof; or a fragment thereof. By “mature polypeptide” is intended to exclude a signal peptide present in the precursor of the mature polypeptide. In some embodiments, the at least one antigen comprises one or more T cell antigen segments and/or one or more B cell antigen segments. An antigen segment is a portion of an antigen, which antigen comprises an epitope. Typically, an antigen segment comprises an epitope. T cell antigen segments may be CD4+ T cell antigen segments or CD8+ T cell antigen segments. A CD4+ T cell antigen segment is an antigen or portion thereof comprising an epitope which is capable of being presented to a CD4+ T cell in the context of MHC II. A CD8+ T cell antigen segment is an antigen or portion thereof comprising an epitope which is capable of being presented to a CD8+ T cell in the context of MHC I. Different antigen segments can be provided in different antigens or the same antigen. Multiple antigens or portions/fragments thereof may be used. Suitably, an antigen segment is in the form of a fragment of an antigen, such as a fragment comprising or consisting of a B or T cell epitope. This is convenient where a natural antigen is particularly large. Where a polypeptide epitope is provided in the context of a larger molecule, it may be provided contiguous with cleavage sites to facilitate cleavage of the epitope from the larger molecule in an antigen presenting cell (APC). This is particularly useful in the context of CD8+ T cell epitopes, to facilitate exit of the epitope from endolysosomal compartments of the APC and entry into the cytosol for loading on MHC I. Alternatively, CD8+ T cell epitopes may be provided as antigen fragments of less than about 70 amino acids, such as less than 60, less than 50, less than 40. Suitably, the antigen is a multi-antigen fusion polypeptide comprising two or more antigen segments, such as ^3, ^4 ^5 or more or 10 or more antigen segments, optionally with an upper limit of ^30, ^20 or ^15. By “multi-antigen fusion polypeptide”, we mean a polypeptide comprising antigen segments such as epitopes which are linked together, either directly or separated by appropriate linking sequences, to form an artificial polypeptide; this may be referred to as a polyepitope, artificial polyepitope, or mosaic polyepitope. Intervening sequences that occur between antigen segments in an antigen may thereby be avoided in a multi-antigen fusion polypeptide. Each antigen segment may be from the same or different antigen. Suitable linking sequences may be included to facilitate cleavage of antigen segments or epitopes, particularly CD8+ T cell antigen segments or epitopes, from the multi- antigen fusion polypeptide, as described in the Examples, or in EP3235831. Suitably, the multi-antigen fusion polypeptide comprises at least one CD4+ T cell antigen segment and at least one CD8+ T cell antigen segment. The antigen segments in a multi-antigen fusion polypeptide may suitably be derived from polypeptide sequences that partially overlap in the antigen from which they are derived. Where two antigen segments partially overlap, the first will have an N- terminal sequence that is not shared by the second, and the second will have a C- terminal sequence that is not shared by the first, and the two antigen segments will share a common sequence. For example, one antigen may be split into overlapping peptides that altogether contain the entire sequence of said antigen. In cases where there are multiple antigens, each may be present as overlapping peptides. The term “overlapping peptides” encompasses recombinant overlapping peptides (ROPs), such as those described in EP3235831. By “overlapping peptides” and “ROP”, we mean that the antigen is a multi-antigen fusion polypeptide as defined above (also referred to herein as multi-antigen fusion protein) comprising two or more antigen segment sequences, i.e. peptide sequences, which partially overlap. Suitably, the antigen segments in a multi-antigen fusion protein are partially overlapping, and in combination encompass ^40%, ^50, ^60%, ^70%, ^80%, ^90%, more preferably 100% of the amino acid sequence of the antigen from which they are derived. In other words, a first polypeptide may partially overlap with a second polypeptide, and the second polypeptide may partially overlap with the third polypeptide, etc. In some embodiments, the multi-antigen fusion protein comprising overlapping peptides may comprise ^2, ^3, ^4, preferably ^5, more preferably ^10 antigen segments; optionally with an upper limit of ^30, 20 or 15 antigen segments. For example, a ROP may comprise 10 antigen segments, wherein all segments combined comprise 100% of the amino acid sequence for the whole antigen. It will be understood that not every antigen segment in a multi-antigen fusion protein necessarily contains an epitope. In some embodiments, each antigen segment comprises at least one (preferably at least 2) CD8+ epitope; at least one (preferably at least 2) CD4+ epitope; and/or at least one (such as at least 2) B cell epitope. In some embodiments, each antigen segment comprises at least one (preferably at least 2) amino acid sequence simultaneously serving as a CD8+ epitope and a CD4+ epitope. In some embodiments, each antigen segment comprises 8-50 amino acids, preferably 10-40 amino acids, more preferably 15-35 amino acids in length. In some embodiments, each antigen segment may comprise sequences of an exogenous protease cleavage site located between antigen segments. The exogenous protease is present in the host and may act extracellularly, or more typically intracellularly. For example, the sequence of a cleavage site may comprise a cleavage site of a cathepsin. In some embodiments, the cleavage site is selected from the group consisting of a cleavage site of cathepsin S (as described further in Lützner and Kalbacher, 2008, J. Biol. Chem., 283(52):36185-36194) (e.g., Leu-Arg-Met-Lys (SEQ ID NO: 32) or a similar cleavage site), a cleavage site of cathepsin B (e.g., Met-Lys-Arg-Leu (SEQ ID NO: 33) or a similar cleavage site), a cleavage site of cathepsin K (e.g., His-Pro-Gly- Gly (SEQ ID NO: 34) or a similar restriction site), or combinations thereof. In some embodiments, the cleavage site of cathepsin S is selected from a group consisting of X-Val/Met-X Ļ Val/Leu-X-Hydrophobic amino acid, Arg-Cys-Gly Ļ, -Leu, Thr-Val-Gly Ļ, -Leu, Thr-Val-Gln Ļ, -Leu, X-Asn-Leu-Arg Ļ (SEQ ID NO: 35), X-Pro-Leu-Arg Ļ (SEQ ID NO: 36), X-Ile-Val-Gln Ļ (SEQ ID NO: 37) and X-Arg-Met-Lys Ļ (SEQ ID NO: 38); wherein each X is independently any natural amino acid, and Ļ represents cleavage position. In some embodiments, the cleavage site of cathepsin S is Thr-Val-Lys-Leu- Arg-Gln (SEQ ID NO: 39). In some embodiments, each antigen segment is directly connected in the artificial multi-antigen fusion protein via said sequence of cleavage site. In some embodiments, the sequence of cleavage site used to connect each antigen segment is the same or different. In some embodiments, the sequence of cleavage site is not contained in each antigen segment; or the sequence of cleavage site is contained in the antigen segment, while at least one cleavage product (or some or all of the cleavage products) formed after the antigen segment is digested is still a CD8+ epitope or CD4+ epitope. Surface-tethered, secreted or intracellular antigens As provided by the first aspect of the invention, the bacterium of the class Clostridia is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen or is capable of secreting the antigen as a secreted antigen. By “exporting”, it is meant that a substrate – i.e. the antigen – is translocated across the bacterial plasma membrane out of the bacterial cytoplasm into the periplasmic space or extracellular milieu. Suitable mechanisms of export by bacteria are known to the person skilled in the art and include in Gram-positive bacteria export by the Sec- dependent secretion pathway; the Tat-dependent secretion pathway; and Wss/Esx pathway. Bacterial secretion systems and export pathways are reviewed in Green and Mecsas, 2006, Microbiol. Spectr., 4(1), doi: 10.1128/microbiolspec.VMBF-0012-2015. By “secretion” it is meant the translocation or export of the antigen from the cytoplasm of the bacterial cell across the plasma membrane into the extracellular milieu. Exemplary methods of verifying antigen secretion by Clostridia are disclosed in Kovács et al., 2013, Biotechnol. Biofuels, 6:117 (DOI: 10.1186/1754-6834-6-117). Secreted peptides are harvested by centrifugation to separate supernatant and subsequently concentrated through Trichloroacetic acid precipitation. Following this, detection of secreted proteins is performed by the use of SDS-PAGE and western blotting. By “tethered to the surface of the bacterium”, it is meant that the antigen is bound covalently or non-covalently to components of the surface of the bacterium, typically the bacterial cell wall or capsule; and hence is displayed on the surface of the bacterium. An antigen may suitably be tethered to peptidoglycan comprised within the bacterial cell wall via a peptidoglycan anchoring domain or via enzymatic linkage. Antigens that are tethered to the surface of the bacterium may be cleaved or degraded, thereby releasing the antigen or a fragment thereof into the extracellular milieu. The released antigen or fragment thereof is typically soluble. Typically, in order to become tethered to the surface of the bacterium, the antigen must first be exported before becoming tethered. Thus, where an antigen is cleaved or degraded after being exported, it may not become tethered or may be tethered only transiently, even though it originally included a domain or motif required for tethering. Exemplary methods of determining whether an antigen is cell-wall anchored in Clostridia are disclosed in Willson et al., 2016, Biotechnol. Biofuels, 9:109 (DOI: 10.1186/s13068-016-0526-x). Cell wall attachment is confirmed by cell fractionation isolating the supernatant through centrifugation, the cell wall and cytosolic fractions through lysozyme digestion of the cell wall and separation of protoplasts from cell wall components by centrifugation. Fractions, following optional trichloroacetic acid precipitation to concentrate, are subject to an SDS-PAGE and western blot analysis. By “secreted antigen” it is meant that the antigen has been secreted by the bacterium and is present in the extracellular milieu in a form that is not surface-tethered, and is typically soluble. The Examples demonstrate successful secretion and/or surface tethering of polypeptides having a size range of between 4 kDa and 60 kDa. Typically, surface- tethered or secreted antigens have a size range of up to 200 kDa, such as up to 150 kD, 100 kDa, 90 kDa, 80 kD, 70 kD or up to 60 kDa. A typical lower limit may be 1 kDa, 2 kDa, 3kDa or 4kDa. However, where a LysM tethering domain is included, this would add 23 kDa to the size of the antigen. Thus, in that scenario, a lower limit may be 24, 25, 26 or 27 kDa. An average 80-90 amino acid residue polypeptide may have a molecular weight of about 10kDa. Some Clostridia do not incorporate disulphide bonds into secreted proteins (Reardon-Robinson and Ton-That, 2016, J. Bacteriol., 198(5):746-754 (https://doi.org/10.1128/JB.00769-15)); nor do some obligately anaerobic bacteria lacking an electron transport chain (Landeta et al., 2018, Nat. Microbio., 3:270-280, DOI: 10.1038/s41564-017-0106-2). Accordingly, in some embodiments, the surface-tethered antigen or secreted antigen does not comprise disulphide bonds. In some embodiments, the surface-tethered antigen or secreted antigen does not comprise cysteine residues. It is known that, despite including motifs or domains that are known to be suitable for effecting secretion and/or surface- tethering of polypeptides, not all candidate polypeptides can be secreted by bacteria. For example, polypeptides encoded by nucleic acid molecules comprising repetitive regions (other than in the LysM coding portion for example) may not be efficiently expressed. Smaller polypeptides may be produced more efficiently than larger ones. Due to the redundancy of the genetic code, nucleic acid molecules encoding polypeptides may be re-designed such that the nucleic acid sequence is less repetitive; or may be codon optimised. Suitable alternative signal sequences and/or motifs or domains that are known to be suitable for effecting secretion and/or surface tethering may be incorporated into the antigen to address the absence of secretion of the antigen by the bacteria. Cysteine residues, if present, may be removed or substituted by re- designing the coding sequence. A suitable alternative promoter may be incorporated into the nucleic acid encoding the antigen, to optimise expression of the antigen by the bacteria. In embodiments where the antigen is a surface-tethered antigen, the antigen is typically expressed as a precursor comprising an N-terminal signal peptide; and the surface-tethered antigen typically comprises a domain or motif required for tethering to peptidoglycan, optionally wherein the domain or motif is (a) a peptidoglycan anchoring domain, suitably comprising at least one LysM motif, such as a LysM domain; or (b) a sequence required for enzymatic linkage to peptidoglycan. In embodiments where the antigen is a secreted antigen, the antigen is typically expressed as a precursor comprising an N-terminal signal peptide. By “precursor” is meant a polypeptide or portion thereof comprising an N-terminal signal peptide. The signal peptide is present during translation of the nascent polypeptide, and is typically cleaved off such that the mature polypeptide formed following completion of translation lacks the signal peptide. The signal peptide may typically comprise or be followed by a cleavage sequence at its C-terminal end to facilitate cleavage. In some embodiments, the signal peptide is capable of directing secretion of the antigen via the Sec-dependent secretion system. Export by the Sec pathway requires a hydrophobic signal sequence at the N-terminus of the secreted protein, which is typically 20 amino acids in length and contains 3 regions: a positively charged amino terminal, a hydrophobic core, and a polar carboxyl-terminal (Green and Mecsas, 2016, Microbiol. Specr., 4(1), doi:10.1128/microbiolspec.VMBF-0012-2015; Papanikou et al., 2007, Nat. Rev. Microbiol., 5(11):831-51; Borrero et al., 2011, Applied Genetics and Microbiology, 89:131-143).. Exemplary methods of predicting signal peptides that are capable of directing secretion of an antigen via the Sec secretion pathway are disclosed in Desvaux et al., 2005, Biochim. Biophys. Acta, 1745(2):223-253 (DOI: 10.1016/j.bbamcr.2005.04.006). Detection of signal peptides would utilise tools such as SignalP to identify proteins comprising N-terminal regions containing positively charged amino acid residues, followed by hydrophobic amino acid residues, and a carboxylic tail region containing polar amino acid residues; and comparing to a prediction of protein localisation utilising a predictive tool such as PSORT, or comparing to an appropriate mass spectrometry analysed secretome of the organism. Suitable signal peptides include the Usp45 signal peptide MKKKIISAILMSTILSAAAP (SEQ ID NO: 41), the Licheninase signal peptide MNKKKLKIMTFAMLVSTFLVGGLMQVPASA (SEQ ID NO: 144) or the LysM signal peptide MKKYYYLFLFTALISILLLSC (SEQ ID NO: 107). Accordingly, in some embodiments, the antigen comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 144, and/or SEQ ID NO: 107. A suitable cleavage sequence to be included directly after the Usp45 signal peptide is LSVGYA (SEQ ID NO: 63), and the cleavage site is predicted to be directly after the “A”. A suitable cleavage sequence to be included directly after the LysM signal peptide is “Q”, and the cleavage site is predicted to be between the final “C” of the LysM signal peptide and the “Q”. A suitable cleavage sequence to be included directly after the Licheninase signal peptide is “LTT”, and the cleavage site is predicted to be directly between the Licheninase signal peptide and the “LTT”. Suitable peptidoglycan anchoring domains include domains which anchor proteins to the cell surface through non-covalent attachment to the peptidoglycans found in the cell wall. Non-covalent peptidoglycan anchoring domains include the LysM domain and fragments thereof. In a preferred embodiment, the peptidoglycan anchoring domain comprises at least one LysM motif, suitably a LysM domain. LysM (Lysin Motif) is a widely distributed protein motif for binding to peptidoglycans and its function is reviewed in Buist et al., 2008, Mol. Microbiol., 68(4):838-847 (doi:10.1111/j.1365- 2958.2008.06211.x). The motif typically ranges in length from 44 to 65 amino acid residues. Naturally occurring LysM domains typically comprise multiple copies of the LysM motif separated by spacer sequences. Suitable LysM motifs may be identified from Gram positive bacteria, including Clostridia, suitably C. butyricum or C. perfringens. A suitable peptidoglycan anchoring domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 LysM motifs. In a preferred embodiment, the peptidoglycan anchoring domain comprises 2 LysM motifs, suitably from C. butyricum LysM. In one embodiment, the LysM domain comprises an amino acid sequence of SEQ ID NO: 42; or amino acids 22 to 200 of SEQ ID NO: 42; or a variant comprising at least 90%, at least 95%, at least 99% or at least 99.5% sequence identity to either sequence; or fragment comprising at least 90% of either sequence or variant. The first 21 amino acid residues of the corresponding native LysM domain of SEQ ID NO: 42 i.e. MKKYYYLFLFTALISILLLSC (SEQ ID NO: 107) constitute the predicted signal peptide. Thus, the LysM signal peptide and the LysM domain may conveniently be provided together, such that the precursor of the polypeptide comprises an N-terminal LysM signal peptide, and the mature polypeptide comprises the LysM domain. The LysM domain may be comprised at the N-terminus or C-terminus of the antigen; or may be comprised within the antigen sequence itself. Where the LysM domain is located other than at the N-terminus, the signal peptide found in the native LysM domain is typically omitted. In another embodiment, the surface-tethered antigen may comprise a sequence required for enzymatic linkage to peptidoglycan. The antigen may be anchored to the cell surface via covalent attachment to the peptidoglycans found in the cell wall. For example, the antigen may comprise a motif comprising a C-terminal Cell Wall Sortase signal (CWSS), and become anchored to the cell surface by the Sortase system, which covalently links peptides to peptidoglycan. The CWSS is typically followed (from N- to C-terminus) by a segment of hydrophobic amino acids and a tail composed primarily of positively charged amino acids. The Sortase system is reviewed in Dramsi et al., 2008, FEMS Microbio. Rev. 32(2):307-320 and Clancy et al., 2010, Biopolymers, 94(4):385-396. Six classes of Sortase systems are found in Gram positive bacteria: Class A, Class B, Class C, Class D, Class E, and Class F. Class A, C, D, E, and F Sortase system CWSSs have the consensus amino acid sequence LPXTG (SEQ ID NO: 43; where X is any amino acid), optionally LP[A/N/K]TG (SEQ ID NO: 112). The Class B Sortase system CWSS has the consensus amino acid sequence: NP(Q/K)(T/S)(N/G/S)(D/A) (SEQ ID NO: 111). Other exemplary CWSS include: SPXTG (SEQ ID NO: 44) and PPXTG (SEQ ID NO: 45). Accordingly, in one embodiment, the antigen further comprises a cell wall sorting signal (CWSS); and optionally comprises a cell wall sorting signal comprising an amino acid sequence that is encoded by SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 111 or SEQ ID NO: 112. In one embodiment, the antigen comprising a CWSS further comprises a C-terminal segment of hydrophobic amino acids, and a tail region composed primarily of positively charged residues. Exemplary tail region sequences are disclosed in Willson et al., 2016, Biotech. Biofuels, 9:109 (DOI: 10.1186/s13068- 016-0526-x), and include NKFKNKQKSVQ (SEQ ID NO: 145) and NKKKSSPTK (SEQ ID NO: 146). In one embodiment, the cell wall sorting signal comprises an amino acid sequence of SEQ ID NO: 145 or SEQ ID NO: 146. It will be appreciated that, in order for the antigen to be covalently attached to peptidoglycan by the Sortase system, the antigen must first be exported into the periplasmic space. By “periplasmic space” it is meant – in Gram-positive bacteria – the space between the cell membrane and cell wall. Accordingly, in some embodiments, the precursor antigen comprises an N-terminal signal sequence – for example a Sec secretion system signal sequence – and a C-terminal CWSS. In some embodiments, the precursor antigen comprising an N-terminal signal sequence and a C-terminal CWSS further comprises a C-terminal segment of hydrophobic amino acids, and a tail composed primarily of positively charged residues. As provided in the second aspect of the invention, the bacterium is capable of expressing the antigen as an intracellular antigen. Intracellular antigens may typically be designed without any special motifs or domains intended for interaction with the bacterial sorting systems. Thus, antigens intended for expression in the intracellular compartment of the bacterium are typically not provided with N-terminal signal peptides. An intracellular antigen is typically expressed as a soluble polypeptide or inclusion body in the bacterial cytoplasm, or as a combination of either form. The antigen may further comprise amino acid sequences that are suitable for use as purification tags and/or tags used for immunostaining. Accordingly, in some embodiments, the antigen comprises a FLAG tag, a His tag, a Strep tag, an HA tag, c- Myc. In some embodiments, the antigen comprises a tag comprising an amino acid sequence that is encoded by SEQ ID NO: 46 and/or SEQ ID NO: 47. In some embodiments, the antigen comprises linkers disposed between the elements comprised by said antigen – for example, an antigen may comprise a linker disposed between a LysM domain and the antigen. Suitable linkers include, for example, [Gly]4Ser linkers (SEQ ID NO: 48). Infectious agent antigens or cancer antigens Suitable infectious agent antigens may include a viral antigen, a bacterial antigen (including a chlamydial antigen or a mycoplasma antigen), a parasite antigen, a protozoan antigen, a helminth antigen, a nematode antigen, a fungal antigen, a prion, or any combination thereof. Combinations of an infectious agent antigen and a tumour antigen may also be used. In some cases, the antigen selected provides cross- immunity (also referred to as cross-protection) in that a single antigen or multiple antigens combined may confer immunity or protection against related infectious agents. Cross-immunity may occur where an antigen is conserved (i.e. shared or homologous) in multiple strains or species of infectious agents. Accordingly, it may be desirable to use antigens (either single antigens or multiple combined antigens) that provide such cross-immunity. In some embodiments, the infectious agent antigen is from an infectious agent which is capable of causing an infection at a mucosal site in a susceptible host (i.e. is a mucosal infectious agent). Mucosal infections may involve the following pathogens: Vibrio cholerae, coronaviruses e.g. SARS-CoV-2, influenza type A and B virus, poliovirus, rotavirus, Salmonella typhimurium, Salmonella sp. (including but not limited to S. enteria and subspecies including S. e. enterica, S. e. salamae, S. e. arizonae, S. e. diarizonae, S. e. houtenae, S. e. indica, S. enterica serovar Typhi, S. enterica serovar Typhimurium, S. enterica serovar Paratyphi; and S. bongori); adenovirus, respiratory syncytial virus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Helicobacter pylori, Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E coli (EPEC), Enterohaemorrhagic E. coli (EHEC), Enteroinvasive E coli (EIEC), Enteroaggregative E. coli (EAEC), Adherent-Invasive E. coli (AIEC), Shigella (including S. sonnei, S. flexneri, and S. boydii), Clostridium (difficile/perfringens), Treponema pallidum, rabies virus, Campylobacter jejuni, Neisseria gonorrhoeae, Herpes simplex virus 2, Human papillomavirus (HPV), Hepatitis B/C, HIV, bovine parainfluenza virus 3, bovine respiratory syncytial virus, Bordetella bronchiseptica, canine parainfluenza virus, African Swine Fever Virus, and Newcastle disease virus. The infectious disease associated with the infectious agent may be categorised based on the location. For example, the infectious agent may be SARS- CoV-2, seasonal influenza, respiratory syncytial virus (RSV) e.g. RSV-ALRI, Streptococcus pneumoniae or Mycobacterium tuberculosis, which are associated with the respiratory tract; rotavirus, Helicobacter pylori, enterotoxigenic Escherichia coli (ETEC), Salmonella, Shigella or Clostridium (difficile or perfringens), which are associated with the GI tract; or syphilis, gonorrhoea, herpes simplex virus 2, HPV, hepatitis B, hepatitis C or HIV, which are associated with the urogenital tract. In some embodiments, the infectious agent antigen is from an infectious agent which is capable of causing a respiratory tract infection in a susceptible host, optionally a virus such as a coronavirus, such as SARS-Cov-2; or optionally respiratory syncytial virus (RSV). In some embodiments, the infectious agent antigen is from an infectious agent which is capable of causing infection in the GI tract of susceptible host, such as gastroenteritis, optionally a virus such as a Human Rotavirus; a bacterium such as Vibrio cholerae, Campylobacter jejuni, Escherichia coli (including but not limited to ETEC, EHEC, EIEC, EPEC, EAEC, and AIEC), Shigella sp. (including S. flexneri, S. sonnei, S. boydii, and S. dysenteriae), or Clostridium perfringens; or a protozoan such as Cryptosporidium parvum, Giardia duodenalis, or Entamoeba histolytica. Examples of viral antigens include human papilloma virus (HPV) antigens; coronavirus antigens, such as SARS-CoV-2 coronavirus antigens, such as SARS-CoV-2 spike protein (for example, the coronavirus antigen may be an antigen or multiple combined antigens that confer cross-immunity to 229E, NL63, OC43 and HKU1 coronavirus strains, each of which are relevant for SARS-CoV2); human immunodeficiency virus (HIV) antigens such as products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis, e.g., hepatitis A, B, and C, hepatitis viral antigens such as the S, M, and L proteins of hepatitis, the pre-S antigen of hepatitis B virus; influenza viral antigens hemagglutinin and neuraminidase and other influenza viral antigens; measles viral antigens such as SAG-1 or p30; rubella viral antigens such as proteins El and E2 and other rubella virus components; rotaviral antigens such as VP7sc components and other rotaviral components (for example, VP4, found on the surface capsid of the virus, which is cleaved by intestinal proteases into VP8 and VP5); cytomegaloviral antigens such as envelope glycoprotein B and other cytomegaloviral proteins; respiratory syncytial viral antigens, such as the RSV fusion protein, the M2 protein; varicella zoster viral antigens such as gpl, gpll, and telomerase; antigens of flavivirus associated with Yellow fever; West Nile virus antigens; dengue virus antigens; Zika virus antigens; Japanese encephalitis virus antigens; African swine fever virus antigens; Porcine Reproductive and Respiratory Syndrome (PRRS) virus antigens; and foot-and-mouth disease virus (e.g. coxsackievirus A16) antigens. Antigens of viruses that cause chronic persistent infection may be preferred, such as human papillomavirus (HPV); hepatitis C; hepatitis B; human immunodeficiency virus (HIV); herpesviruses including herpes simplex virus 1, herpes simplex virus 2 and varicella zoster virus. In some embodiments the at least one antigen is an HPV antigen corresponding to an E1, E2, E4, E5, E6 and/or E7 protein, preferably an E1, E2, E4, E5, E6 and/or E7 protein that is conserved across one or more high-risk HPV genotypes, such as described in WO 2019/034887. In an embodiment, the HPV antigen comprises the amino acid sequence of amino acids 2 to 152 of SEQ ID NO: 1, or amino acids 2 to 140 of SEQ ID NO: 1, such as wherein the HPV antigen is encoded by nucleotides 19 to 477 of the nucleic acid sequence of SEQ ID NO: 2. Examples of bacterial antigens include clostridium bacterial antigens such as Clostridium difficile (renamed Clostridioides difficile) toxin A and B; pertussis bacterial antigens such as pertussis toxin; diptheria bacterial antigens such as diptheria toxin or toxoid erythematosis, and other diptheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other bacterial antigen components; streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; Gram-negative bacilli bacterial antigens, Mycobacterium tuberculosis bacterial antigens such as heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A and other mycobacterial antigen components; Vibrio cholerae bacterial antigens such as the Cholera toxin B-subunit (CtxB); Campylobacter bacterial antigen components such as FliD, FlaA, MOMP, PEB3, CadF, FlgK, FlgE, and Flagellin (FliC); Helicobacter pylori bacterial antigen components; pneumococcal bacterial antigens such as pneumolysin, pneumococcal bacterial antigen components; Haemophilus influenzae bacterial antigens including Haemophilus influenzae bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as rompA and other rickettsiae bacterial antigen component; or bovine tuberculosis antigens; or Brucella antigens. Also included with the bacterial antigens described herein are any other bacterial mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens. Antigens of bacteria which cause chronic persistent infection may be preferred, such as those of Mycobacterium tuberculosis, Borrelia species such as B. burgdorferi, Corynebacterium diphtheriae, Chlamydia, Vibrio cholerae, Salmonella enterica serovar Typhi; mycoplasma. Fungal antigens which can be used include but are not limited to Candida fungal antigen components; Histoplasma fungal antigens, Coccidiodes fungal antigens such as spherule antigens and other Coccidiodes antigens; cryptococcal fungal antigens and other fungal antigens. Examples of protozoal and other parasitic antigens include but are not limited to antigens from Plasmodium species which cause malaria, such as P. falciparum; Toxoplasma antigens; Cryptosporidium parvum and other Cryptosporidium antigens; Giardia duodenalis antigens; Entamoeba histolytica antigens; Schistosoma antigens; Leishmania major and other leishmaniae antigens; and Trypanosoma antigens. Cancer antigens or tumour antigens may be used, which may be categorised as tumour-associated antigens (e.g. overexpressed proteins, differentiation antigens or cancer/testis antigens), or as tumour-specific antigens (e.g. oncoviral antigens, shared neoantigens or private neoantigens). For example, cancer/testis antigens (also referred to as cancer/germline antigens) are normally expressed only in immune privileged germline cells (e.g. MAGE-A1, MAGE-A3, and NY-ESO-1); differentiation antigens refers to cell lineage differentiation antigens that are not normally expressed in adult tissue (e.g. tyrosinase, gp100, MART-1, prostate specific antigen (PSA)); and overexpressed antigens simply refer to antigens that are expressed in cancer cells above healthy or normal levels (e.g. hTERT, HER2, mesothelin, and MUC-1) (Hollingsworth & Jansen (2019), npj Vaccines, 4(7)). Accordingly, cancer antigens or tumour antigens may include, but are not limited to, K-Ras, survivin, dystroglycan, KS [1/4] pan-carcinoma antigen, ovarian carcinoma antigen (CA125), prostatic acid phosphate, PSA, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumour-associated antigens such as: CEA, TAG-72, CO17-1A; GICA 19-9, CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19, human B-lymphoma antigen-CD20, CD33, melanoma specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, tumour-specific transplantation type of cell-surface antigen (TSTA) such as virally-induced tumour antigens including T-antigen DNA tumour viruses and Envelope antigens of RNA tumour viruses, oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumour oncofetal antigen, differentiation antigens such as human lung carcinoma antigen L6, L20, antigens of fibrosarcoma, human leukemia T-cell antigen-Gp37, neoglycoprotein, sphingolipids, breast cancer antigens such as EGFR, EGFRvIII, FABP7, doublecortin, brevican, HER2 antigen, polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigen such as I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes, preimplantation embryos, I (Ma) found in gastric adenocarcinomas, M18, M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM- D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, E1 series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 found in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43, G49 found in EGF receptor of A431 cells, MH2 found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, T5A7 found in myeloid cells, R24 found in melanoma, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8 found in embryonal carcinoma cells, SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos, a T-cell receptor derived peptide from a Cutaneous T-cell Lymphoma, fibroblast activation protein alpha (FAP) found in carcinoma, and variants thereof. In some embodiments, the cancer antigen or tumour antigen is a multi-antigen fusion polypeptide or recombinant overlapping peptide (ROP) for K-Ras, PSA or survivin. Vibrio In some embodiments, the bacterial antigen is a Vibrio cholerae antigen. V. cholerae is a diarrhoeagenic intestinal pathogenic bacterium and is the etiological agent of Cholera. Suitable V. cholerae antigens include peptides or proteins associated with or secreted by the V. cholerae bacterium. During V. cholerae infection, the bacterium secretes the cholera toxin, a heteropolymeric holotoxin consisting of one copy of the A subunit, CtxA P01555 (UniProtKB); and five copies of the B subunit, CtxB P01556 (UniProtKB). The CtxA subunit catalyzes the ADP-ribosylation of Gs alpha, a GTP- binding regulatory protein, to activate the adenylate cyclase. This leads to an overproduction of cAMP and eventually to a hypersecretion of chloride and bicarbonate followed by water, resulting in the characteristic cholera stool. The CtxB subunit forms a pentameric ring that The B subunit pentameric ring directs the A subunit to its target by binding to the GM1 gangliosides present on the surface of the intestinal epithelial cells. It can bind five GM1 gangliosides. It has no toxic activity by itself. Accordingly, in an embodiment, the V. cholerae antigen is CtxB. During V. cholerae infection, the bacterium expresses the toxin-coregulated pilus (TCP) structure. TCP comprise long, thin, flexible homopolymers of the TcpA Q60153 (UniProtKB) subunit that self-associate to hold cells together in microcolonies and facilitate gut colonisation by the bacteria. The TCP also serves as the receptor for the cholera toxin phage CTXij, which comprises nucleic acid encoding the cholera toxin, thereby facilitating horizontal transmission of the cholera toxin gene. In some embodiments, the V. cholerae antigen is TcpA. Suitably the V. cholerae antigen is derived from a strain selected from the V. cholerae serotype O1 or O139, optionally strain ATCC 39315 / El Tor Inaba N16961. Suitable V. cholerae antigens include CtxB P01556 and TcpA Q60153. An exemplary CtxB antigen is VEVPGSQHIDSQKKAIERMKDTLRIA (SEQ ID NO: 149), which is derived from the P01556 native sequence. An exemplary CtxB antigen is VEVPGSQHIDSQKKAIERMKNTLRIA (SEQ ID NO: 110), which has a D21N substitution compared to the native P01556 sequence. A further exemplary CtxB antigen is ASLAGKREMAIITFKNGAIFQV (SEQ ID NO: 147), as disclosed in Guyon-Gruaz et al., 1986, Eur. J. Biochem., 159:525-528. Suitably, the antigen comprises a V. cholerae antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of CtxB, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 110, 147 or 149. The antigenic portion comprising SEQ ID NO: 147 or 149 may comprise a greater portion of the complete CtxB polypeptide as provided in SEQ ID NO: 29, such as the complete CtxB. The antigenic portion comprising SEQ ID NO: 110 may comprise a greater portion of the complete CtxB polypeptide as provided in SEQ ID NO: 150, such as the complete CtxB. The CtxB- protein sequence (SEQ ID NO: 29) was determined from the UniProtKB submission P01556 with removal of the signal sequence (MIKLKFGVFFTVLLSSAYAHG (SEQ ID NO: 148)). Thus, the V. cholerae antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of any antigenic portion of CtxB comprising SEQ ID NO: 110, 147 or 149, such as SEQ ID NO: 29 or 150. The antigen according to these embodiments may be secreted or surface-tethered. A secreted antigen comprising a V. cholerae antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 27 to 129 of SEQ ID NO: 205. The first 26 amino acid residues of SEQ ID NO: 205 is predicted to be the signal peptide (amino acids 1 – 20; SEQ ID NO: 41) and cleavage site (amino acids 21 – 26; SEQ ID NO: 63); and thus the precursor of the secreted antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 129 of SEQ ID NO: 205. A surface-tethered antigen comprising a V. cholerae antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 22 to 231 of SEQ ID NO: 30. The first 21 amino acid residues of SEQ ID NO: 30 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 231 of SEQ ID NO: 30. Human Rotavirus In some embodiments, the infectious agent antigen is a human rotavirus (HRV) antigen. Human Rotavirus is the most common cause of diarrhoeal disease among infants and young children, causing severe gastroenteritis and contributes to a significant number of infant deaths across the world, with higher prevalence in low- and middle-income countries. The HRV genome codes for six structural proteins (VP1- VP4, VP6 and VP7) and six non-structural proteins (NSP1-NSP6). The infectious virion particle is formed from 3 layers of protein, whereby the outer later (outer capsid) consists of the surface proteins, VP7 and VP4, which contribute to the classification of serotypes. The glycoprotein Viral Protein 7 (VP7) defines the G serotype and the protease sensitive VP4 defines the P serotype. The VP4 protein protrudes on the cell surface of the virion as a spike and is involved in binding the human cell receptors including sialoglycans (such as Gangliosides GM1 and GD1a) and histo-blood group antigens (HBGAs), driving entry into the cell. The VP4 is cleaved by trypsin (found in the human gut) into VP5 and VP8 to allow the virus particle to become infectious. The majority of the HRV virion capsid is made up of VP7 and VP4. Accordingly, these proteins are important in rotavirus immunity, containing epitopes for T-cell and B-cell activation as well as antigenic sites responsible for neutralisation of the virus. Suitably the HRV antigen is derived from a strain selected from HRV G1, HRV G2, HRV G3, HRV G4, HRV G8, HRV Wa (Taxonomy ID: 10962), HRV DS-1, and HRV 1076. Representative serotypes are described in https://doi.org/10.1016/j.vaccine.2011.09.111. The antigen may be derived from any one or more HRV structural proteins, i.e from VP1, VP2, VP3, VP4, VP5, VP6, VP7 and/or VP8, and/or any one or more HRV non- structural proteins NSP1-NSP6. VP7 and/or VP4 are preferred, particularly the VP8 fragment of VP4. Suitable antigens may be determined from the sequence of the full- length VP7 protein P03533 (UniProtKB; SEQ ID NO: 154) sequence or the full-length VP4 protein P11193 (UniProtKB; SEQ ID NO: 153) sequence. An exemplary VP8 antigen is SEQ ID NO: 78: LDGPYQPTTFTPPNDYWILINSNTNGVVYESTNNSDFWTAVVAIEPHVNPVDRQYTIFGE SKQFNVSNDSNKWKFLEMFRSSSQNEFYNRRTLTSDTRFVGILKYGGRVWTFHGETPRAT TDSSSTANLNNISITIHSEFYIIPRSQESKCNEYINNGLPPG An alternative VP8 antigen is SEQ ID NO: 155, identified in Mohanty et al., J. Biotechnol., 281:48-60 (DOI: 10.1016/j.jbiotec.2018.06.306) Suitably, the antigen comprises a VP8 antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of VP8, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 78 or SEQ ID NO: 155. The antigenic portion comprising SEQ ID NO: 78 or 155 may comprise a greater portion of the complete VP8 polypeptide as provided in SEQ ID NO: 25, such as the complete VP8, or of the complete VP4 (SEQ ID NO: 153). Thus, the VP8 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of any antigenic portion of VP8 comprising SEQ ID NO: 25. The antigen according to these embodiments may be secreted or surface-tethered. The precursor of a secreted antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 188 of SEQ ID NO: 26. The first 20 amino acids of this sequence are predicted to be the signal peptide, although an alternative signal peptide may be used. Thus, the secreted antigen would be based on the sequence of amino acid residues 21 to 188 of SEQ ID NO: 26. A secreted VP7 antigen precursor was designed from the UniProtKB submission P03533 with removal of the signal sequence (MYGIEYTTVLTFLISIILLNYILKSLTRIMDCIIYRLLFIIVILSPFLRA (SEQ ID NO:61)) and the first 27 amino acids of the VP7 sequence (QNYGINLPITGSMDTAYANSTQEETFL (SEQ ID NO:62)). The sequence was also modified by the addition of a 6 amino acid sequence (LSGVYA (SEQ ID NO:63)) at the N-terminus and a C-terminal FLAG tag (DYKDDDDK (SEQ ID NO: 46)). A sequence coding for the usp45 signal peptide (MKKKIISAILMSTILSAAAP (SEQ ID NO: 41) was added at the N-terminus contiguous with the sequence encoding the 6 amino acid sequence. A 5’ NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) was added and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA was added at the 3’ end. The nucleic acid sequence is provided in SEQ ID NO: 27 and the encoded recombinant protein in SEQ ID NO: 24. The nucleic acid sequence will be cloned into a pMTL plasmid for expression in CHN-0, under the control of a Clostridial promoter, and engineered strains tested for ability to express VP7 antigen. Promoters to be tested include the C. sporogenes pfdx promoter and the C. acetobutylicum pthl promoter disclosed in Heap et al., 2009, J. Microbiol. Meth., 78(1):79-85 (DOI: 10.1016/j.mimet.2009.05.004). Suitably the Clostridial promoter is not a C. acetobutylicum p0957 promoter. Thus, a VP7 antigen may or may not comprise SEQ ID NO: 79: TSTLCLYYPTEAATEINDNSWKDTLSQLFLTKGWPTGSVYFKEYTNIASFSVDPQLYCDYNVVLM KYDATLQLDMSELADLILNEWLCNPMDITLYYYQQTDEANKWISMGSSCTIKVCPLNTQTLGIGC LTTDATTFEEVATAEKLVITDVVDGVNHKLDVTTATCTIRNCKKLGPRENVAVIQVGGSDILDIT ADPTTAPQTERMMRINWKKWWQVFYTVVDYVDQIIQVMSKRSRSLNSAAFYYRVG The antigen may or may not comprise a VP7 antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of VP7, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 79. The antigenic portion comprising SEQ ID NO: 79 may or may not comprise a greater portion of the complete VP7 polypeptide as provided in SEQ ID NO: 23, such as the complete VP7. Thus, the VP7 antigen may or may not comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of any antigenic portion of VP7 comprising SEQ ID NO: 23. The antigen according to these embodiments may be secreted or surface-tethered. The precursor of a secreted antigen may or may not comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 276 of SEQ ID NO: 24. The first 20 amino acids of this sequence are predicted to be the signal peptide, although an alternative signal peptide may be used. Thus, the secreted antigen would be based on the sequence of amino acid residues 21 to 276 of SEQ ID NO: 24. The VP7 antigen may or may not be encoded by the nucleic acid sequence of SEQ ID NO: 27. Suitably, the HRV antigen is a multi-antigen fusion polypeptide, also referred to as a polyepitope. Suitable epitopes of VP7 are: TTTCTIRNCKKLGP (SEQ ID NO: 67, LDITADPTTNPQIE (SEQ ID NO: 68), KVCPLNTQALGIGC (SEQ ID NO: 69), KINLTTTTCTIRNC (SEQ ID NO: 70) and RNCKKLGPRENVAI (SEQ ID NO: 71). Further suitable VP7 epitopes are SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, or SEQ ID NO: 161 as described in Ghosh et al (doi: 10.1371/journal.pone.0040749). Suitably, the HRV antigen comprises one, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the epitopes of SEQ ID NOs: 67-71 and 156-161, such as one, 2, 3, 4 or 5 of the epitopes of SEQ ID NOs: 67-71; or variants or fragments comprising at least 90% sequence identity thereto. Suitably, the HRV antigen comprises a linker between each epitope, such as a flexible [Gly]4Ser linker (GGGGS; SEQ ID NO: 48). Alternatively, a protease cleavage site may be included between each epitope, such as a cathepsin S cleavage site or any other suitable protease cleavage site as provided herein. The epitopes may be provided in any arrangement in the antigen. Multiple copies of any one or more of the antigens may be provided, such as 2, 3 or 4 copies. The antigen according to these embodiments may be secreted or surface-tethered. A VP7 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 21 to 314 of SEQ ID NO: 77. The first 20 amino acid residues of SEQ ID NO: 77 are predicted to be the signal peptide. The precursor of a secreted antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 314 of SEQ ID NO: 77. The first 20 amino acids of this sequence are predicted to be the signal peptide, although an alternative signal peptide may be used. Thus, the secreted antigen would be based on the sequence of amino acid residues 21 to 314 of SEQ ID NO: 77. The precursor or surface-tethered antigen may comprise a LysM domain, such as a LysM domain comprising an amino acid sequence of SEQ ID NO: 42; or amino acids 22 to 200 of SEQ ID NO: 42 or variants or fragments thereof as provided above. Surface-tethered or secreted SARS-CoV-2 antigens In some embodiments, the infectious agent antigen is a coronavirus antigen. Coronavirus infection can result in the development of pathologies such as severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). In some embodiments of the first aspect of the invention, the viral antigen is a SARS-CoV-2 antigen. SARS-CoV-2 contains four structural proteins, including Spike (S), Envelope (E), membrane (M) and nucleocapsid (N) and at least 6 other non-structural open reading frames (ORFs) including Orf1ab. The S protein mediates viral attachment, fusion and entry into the host cell. The S protein contains 2 major subunits: the S1 subunit which contains the receptor binding domain (RBD), which binds to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell; and the S2 subunit, which allows fusion between the virus and the host cell membrane. Suitable antigens are disclosed in WO 2022/090679 (Oxford Vacmedix Ltd). In some embodiments, the selection of epitopes is based on a comparison with homologous SARS proteins and the top predicted B and T cell epitopes identified by Fast et al. (2020) Potential T-cell and B-cell Epitopes of 2019-nCoV, bioRxiv preprint, doi: https://doi.org/10.1101/2020.02.19.955484, on the basis of likely presentation across MHC alleles. Additional suitable epitopes are described in Li et al. (2020) Epitope-based peptide vaccines predicted against novel coronavirus disease caused by SARS-CoV-2, Virus Research, https://doi.org/10.1016/j.virusres.2020.198082. Suitably the SARS-CoV-2 antigen is derived from any one or more strains selected from the Wuhan strain (GenBank: NC_045512.2); SARS-CoV-2 variant strains including but not limited to the alpha variant (GenBank: MZ344997.1), the beta variant (GenBank: MW598419.1), the gamma variant (GenBank: MW642250.1), the delta variant (GenBank: MZ009823.1), the epsilon variant (GenBank: MW453103.1), the zeta variant (GenBank: MW523796.1), the eta variant (GenBank: MW560924.1), the theta variant, the iota variant (GenBank: MW643362.1), the kappa variant (GenBank: MW966601.1), the lambda variant (GenBank: MW850639.1), the mu variant, and the omicron variant (GenBank: OL672836.1); and variants thereof. By “SARS-CoV-2 variant” strain it is meant a SARS-CoV-2 strain comprising a genome wherein at one or more positions there have been nucleotide insertions, deletions, or substitutions; and includes transitions and transversions. The insertion, deletion, or substitution may be in a nucleotide sequence that encodes a polypeptide or may be in a non-coding sequence. Where the insertion, deletion, or substitution is in a nucleotide sequence that encodes a polypeptide, the mutation may be conservative or non-conservative. Of particular interest are Variants Of Concern (VOC), which may be identified by WHO. Suitable SARS-CoV-2 antigens are derived from any one or more of S, E, M, or N protein or Orf1ab polyprotein. The complete amino acid sequence of the S polypeptide of the Wuhan strain is SEQ ID NO: 3. The amino acid sequence of the S1 subunit of said strain is SEQ ID NO: 4 and the amino acid sequence of the S2 subunit is SEQ ID NO: 5. The amino acid sequences of the E polypeptide of said strain is SEQ ID NO: 15; of the M polypeptide of said strain is SEQ ID NO: 14; of the N polypeptide of said strain is SEQ ID NO: 12; and of the Orf1ab polypeptide of said strain is SEQ ID NO: 16. A suitable antigen may be derived from the receptor binding domain (RBD) of the S1 subunit, such as SEQ ID NO: 6. It may for example include the epitopes TEIYQAGSTPCNGVEGFN (SEQ ID NO: 162) and/ or NLDSKVGGNYNYLYRLFRKSN (SEQ ID NO:178) disclosed in Tai et al., 2020, Cell Mol. Immunol., 16:613-620, DOI: 10.1038/s41423-020-0400-4, or the antigenic fragment SEQ ID NO: 163 disclosed therein. A suitable antigen may be derived from the S1 subunit, such as SEQ ID NO: 8, or derived from the S2 subunit, such as SEQ ID NO: 10. Suitably, the antigen comprises a SARS-CoV-2 antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of an antigenic portion of S, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 6, 8, 10, 162, 178 or 163. The antigenic portion comprising SEQ ID NO: 6, 8, 162, 178 or 163 may comprise a greater portion of the S polypeptide as provided in SEQ ID NO: 4 or 3, such as the complete sequence of SEQ ID NO: 4 or 3. The antigenic portion comprising SEQ ID NO: 10 may comprise a greater portion of the S polypeptide as provided in SEQ ID NO: 5 or 3, such as the complete sequence of SEQ ID NO: 5 or 3. Thus, the SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of any antigenic portion of S comprising SEQ ID NO: 6, 8, 10 or 163. The antigen according to these embodiments may be secreted or surface-tethered. A surface-tethered antigen comprising a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 22 to 401 of SEQ ID NO: 7, or amino acid residues 22 to 339 of SEQ ID NO: 9, or amino acid residues 22 to 273 of SEQ ID NO: 11. The first 21 amino acid residues of SEQ ID NOs: 7 and 9 are predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 401 of SEQ ID NO: 7, or 1 to 339 of SEQ ID NO: 9. Suitably, the SARS-CoV-2 antigen is a multi-antigen fusion polypeptide, also referred to as a polyepitope. Suitable epitopes derived from the nucleoprotein are: NTASWFTALTQHGKED (SEQ ID NO: 81), DDQIGYYRRATRRIRGGDG (SEQ ID NO: 82), KMKDLSPRWYFYYLGTGPEA (SEQ ID NO: 83), NKDGIIWVATEGALNTPK (SEQ ID NO: 84), LPKGFYAEGSRGGSQASSRSSSRSRNS (SEQ ID NO: 85), AALALLLLDRLNQLESKMSGKGQQQQG (SEQ ID NO: 86), ATKAYNVTQAFGRRGPE (SEQ ID NO: 87), KHWPQIAQFAPSASAFFGMSRI (SEQ ID NO: 88), LTYTGAIKLDDKDPNF (SEQ ID NO: 89), KDQVILLNKHIDAYKTFPPTEPKKD (SEQ ID NO: 90), and FSKQLQQSMSSADSTQ (SEQ ID NO: 91). Further suitable SARS-CoV-2 epitopes include SQSIIAYTMSLGAEN (SEQ ID NO: 93), NPTTFHLDGEVITFD (SEQ ID NO: 94), IINLVQMAPISAMVR (SEQ ID NO: 95), SYGFQPTNGVGYQPY (SEQ ID NO: 96), APHGVVFLHVTYVPA (SEQ ID NO: 97), DGEVITFDNLKTLLS (SEQ ID NO: 98), VKPSFYVYSRVKNLN (SEQ ID NO: 99), IPTNFTISVTTEILP (SEQ ID NO: 100), VAAIFYLITPVHVMS (SEQ ID NO: 101), ATKAYNVTQAFGRRG (SEQ ID NO: 102), EVRTIKVFTTVDNIN (SEQ ID NO: 103), IASFRLFARTRSMWS (SEQ ID NO: 104), and AAAYYVGYLQPRTFL (SEQ ID NO: 105). Suitably, the SARS-CoV-2 antigen comprises one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the epitopes of SEQ ID NOs: 81-91, 93 to 105; or variants or fragments comprising at least 90% sequence identity thereto. Suitably, the SARS-CoV-2 antigen comprises a protease cleavage site between each epitope, such as a cathepsin S cleavage site such as TVKLRQ (SEQ ID NO: 39) or any other suitable protease cleavage site as provided herein. The epitopes may be provided in any arrangement in the antigen. Multiple copies of any one or more of the antigens may be provided, such as 2, 3 or 4 copies. The antigen according to these embodiments may be secreted or surface-tethered. A SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 80. A surface-tethered antigen comprising a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 22 to 496 of SEQ ID NO: 13. The first 21 amino acid residues of SEQ ID NO: 13 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 496 of SEQ ID NO: 13. A SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 92. A surface-tethered antigen comprising a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 474 of SEQ ID NO: 17. The first 21 amino acid residues of SEQ ID NO: 17 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 474 of SEQ ID NO: 17. A SARS-CoV-2 antigen may comprise a recombinant overlapping peptide, as disclosed in WO 2022/090679. A suitable antigen disclosed therein as SEQ ID NO: 21 and provided herein as SEQ ID NO: 106 comprises 9 overlapping peptides from the S1 receptor binding domain, and 3 peptides from the C-terminal end of S2 HR2 region and proximal region of S2. Each peptide may be regarded as an antigenic portion and is linked to the next by a LRMK sequence. Immunisation studies in mice with an N- terminally His tagged version of the ROP showed high antibody responses. Accordingly, a SARS-CoV-2 antigen may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the antigenic portions of SARS-CoV-2 present in SEQ ID NO: 106, or fragments or variants comprising at least 90% sequence identify thereto, and a protease cleavage site between each antigenic portion, such as a cathepsin S cleavage site, such as LRMK. Suitably, the SARS-CoV-2 antigen is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 106. Secreted or surface-tethered forms may be prepared with appropriate signal peptide and surface-tethering domains or motifs, as provided herein. Intracellular SARS-CoV-2 antigens Suitable intracellular SARS-CoV-2 antigens are derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or E, M, N, or Orf1ab protein. In this aspect of the invention, the S1 and S2 subunits are regarded separately, such that an antigen spanning S1 and S2 subunits (i.e. comprising a S1 sequence which is contiguous with a S2 sequence in the S protein) is excluded. Where a SARS-CoV-2 antigen comprises only one epitope, it is typically an epitope which has not been disclosed in Fast et al. (2020) Potential T-cell and B-cell Epitopes of 2019-nCoV, bioRxiv preprint, doi: https://doi.org/10.1101/2020.02.19.955484; or Li et al. (2020) Epitope-based peptide vaccines predicted against novel coronavirus disease caused by SARS-CoV-2, Virus Research, https://doi.org/10.1016/j.virusres.2020.198082. SARS-CoV-2 antigens comprising more than one antigen portion or epitope may or may not exclude any epitope disclosed in either document. For example, SARS-CoV-2 antigens comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more antigen portions or epitopes may or may not comprise one or more epitopes disclosed in Li et al. or Fast et al., for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more epitopes. Thus, where the intracellular SARS- CoV-2 antigen comprises or consists of a sequence X or fragment or variant thereof having at least 90% sequence identity thereto, and either document discloses a sequence Y falling within that scope, it may be excluded that the antigen is Y. This may be the case for each such epitope in a polyepitope antigen. Where a SARS-CoV- 2 antigen comprises an antigen portion or epitope not disclosed in either document, it may comprise one or more further epitopes disclosed in either document. Where a SARS-CoV-2 antigen comprises an antigen portion or epitope disclosed in one document, it may or may not comprise one or more further epitopes disclosed in the other document. The following epitopes are disclosed in Fast et al supra: KAYNVTQAF (SEQ ID NO: 164), SIIAYTMSL (SEQ ID NO: 165), HLDGEVITF (SEQ ID NO: 166), VQMAPISAM (SEQ ID NO: 167), YGFQPTNGV (SEQ ID NO: 168), VVFLHVTY (SEQ ID NO: 169), ITFDNLKTL (SEQ ID NO: 170), YVYSRVKNL (SEQ ID NO: 171), FTISVTTEI (SEQ ID NO: 172), YLITPVHVM (SEQ ID NO: 173), RTIKVFTTV (SEQ ID NO: 175), RLFARTRSM (SEQ ID NO: 176), VGYLQPRTF (SEQ ID NO: 177), ATKAYNVTQAFGRRG (SEQ ID NO: 102), SKQLQQSMSSADSTQ (SEQ ID NO: 186), TASWFTALTQHGKED (SEQ ID NO: 187), KAYNVTQAFGRRGPE (SEQ ID NO: 188), SEQ ID NOs: 93-101 and NOs: 103-105. The following epitopes are disclosed in Li et al supra: TESNKKFLPFQQ (SEQ_ID_NO:179), HADQLTPTWRVY (SEQ_ID_NO:180), FGRDIADTTDAV (SEQ ID NO: 193), RDPQTLEILDIT (SEQ ID NO: 194), PCSFGGVSVITP (SEQ ID NO: 195), GTNTSNQVAVLY (SEQ ID NO: 196), QDVNCTEVPVAI (SEQ ID NO: 197), STGSNVFQTRAG (SEQ ID NO: 198), CLIGAEHVNNSY (SEQ ID NO: 199), ECDIPIGAGICA (SEQ ID NO: 200), SYQTQTNSPRRA (SEQ ID NO: 201), NRALTGIAVEQD (SEQ ID NO: 202), KNTQEVFAQVKQ (SEQ ID NO: 203), IYKTPPIKDFGG (SEQ ID NO: 189), FNFSQILPDPSK (SEQ ID NO: 190), PSKRSFIEDLLF (SEQ ID NO: 191), and IEDLLFNKVTLA (SEQ ID NO: 192). Suitable intracellular SARS-CoV-2 antigen amino acid sequences are provided in SEQ ID NOs: 129 (RBD), 131 (nuc), 133 (polyepitope), 135 (SB1) and 137 (SB2). Corresponding coding sequences are provided in SEQ ID NOs: 128, 130, 132, 134 and 136. Suitably, the intracellular antigen comprises a SARS-CoV-2 antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of an antigenic portion of S1, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 6, 8, 162, 178 or 163. The antigenic portion comprising SEQ ID NO: 6, 8, 162, 178 or 163 may comprise a greater portion of the S1 polypeptide as provided in SEQ ID NO: 4, such as the complete sequence of SEQ ID NO: 4. Suitably, the intracellular antigen comprises a SARS-CoV- 2 antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of an antigenic portion of S2, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 10. The antigenic portion comprising SEQ ID NO: 10 may comprise a greater portion of the S2 polypeptide as provided in SEQ ID NO: 5, such as the complete sequence of SEQ ID NO: 5. An intracellular antigen comprising a SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of SEQ ID NO: 129, or amino acid residues 4 to 197 of SEQ ID NO: 129, or SEQ ID NO: 134, or amino acid residues 4 to 135 of SEQ ID NO: 134, or SEQ ID NO: 137 or amino acid residues 4 to 69 of SEQ ID NO: 137. Suitably, the intracellular SARS-CoV-2 antigen is a multi-antigen fusion polypeptide, also referred to as a polyepitope. Suitable epitopes derived from the nucleoprotein are: NTASWFTALTQHGKED (SEQ ID NO: 81), DDQIGYYRRATRRIRGGDG (SEQ ID NO: 82), KMKDLSPRWYFYYLGTGPEA (SEQ ID NO: 83), NKDGIIWVATEGALNTPK (SEQ ID NO: 84), LPKGFYAEGSRGGSQASSRSSSRSRNS (SEQ ID NO: 85), AALALLLLDRLNQLESKMSGKGQQQQG (SEQ ID NO: 86), ATKAYNVTQAFGRRGPE (SEQ ID NO: 87), KHWPQIAQFAPSASAFFGMSRI (SEQ ID NO: 88), LTYTGAIKLDDKDPNF (SEQ ID NO: 89), KDQVILLNKHIDAYKTFPPTEPKKD (SEQ ID NO: 90), and FSKQLQQSMSSADSTQ (SEQ ID NO: 91). Further suitable SARS-CoV-2 epitopes include SQSIIAYTMSLGAEN (SEQ ID NO: 93), NPTTFHLDGEVITFD (SEQ ID NO: 94), IINLVQMAPISAMVR (SEQ ID NO: 95), SYGFQPTNGVGYQPY (SEQ ID NO: 96), APHGVVFLHVTYVPA (SEQ ID NO: 97), DGEVITFDNLKTLLS (SEQ ID NO: 98), VKPSFYVYSRVKNLN (SEQ ID NO: 99), IPTNFTISVTTEILP (SEQ ID NO: 100), VAAIFYLITPVHVMS (SEQ ID NO: 101), ATKAYNVTQAFGRRG (SEQ ID NO: 102), EVRTIKVFTTVDNIN (SEQ ID NO: 103), IASFRLFARTRSMWS (SEQ ID NO: 104), and AAAYYVGYLQPRTFL (SEQ ID NO: 105). Suitably, the intracellular SARS-CoV-2 antigen comprises one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the epitopes of SEQ ID NOs: 81-91, 93 to 105; or variants or fragments comprising at least 90% sequence identity thereto. Suitably, the SARS-CoV-2 antigen comprises a protease cleavage site between each epitope, such as a cathepsin S cleavage site such as TVKLRQ (SEQ ID NO: 39) or any other suitable protease cleavage site as provided herein. The epitopes may be provided in any arrangement in the antigen. Multiple copies of any one or more of the antigens may be provided, such as 2, 3 or 4 copies. An intracellular SARS-CoV-2 antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 80, SEQ ID NO: 92, SEQ ID NO: 131, amino acid residues 4 to 292 of SEQ ID NO: 131, SEQ ID NO: 133 or amino acid residues 4 to 270 of SEQ ID NO: 133. An intracellular SARS-CoV-2 antigen may comprise a recombinant overlapping peptide, as disclosed in WO 2022/090679, suitably SEQ ID NO: 106. An intracellular SARS- CoV-2 antigen may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the antigenic portions of SARS-CoV-2 present in SEQ ID NO: 106, or fragments or variants comprising at least 90% sequence identify thereto, and a protease cleavage site between each antigenic portion, such as a cathepsin S cleavage site, such as LRMK. Suitably, the SARS-CoV-2 antigen is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 106. Campylobacter In some embodiments, the infectious agent antigen is a Campylobacter jejuni antigen. In some embodiments, the infectious agent antigen is a Campylobacter coli antigen. C. jejuni and C. coli are diarrhoeagenic intestinal pathogenic bacteria. Suitable C. jejuni antigens include peptides or proteins associated with or secreted by the C. jejuni or C. coli bacterium. During C. jejuni and C. coli infection, the bacteria express flagella, which are complexes comprising a basal membrane-bound secretion body and motor; and an extracellular flagellum comprising the major flagellin, FlaA, the flagella cap, FliD, the hook protein, FlgE, and the hook-filament protein, FlgK. In C. jejuni and C. coli, the major outer membrane protein (MOMP) is a water-filled porin through which nutrients and antibiotics transit, portions of which are displayed on the bacterial cell surface. CadF is a fibronectin-binding protein, portions of which are displayed on the bacterial cell surface, that promotes binding of C. jejuni and C. coli bacteria to the surface of cells in the gastrointestinal tract. FliD contains several immunodominant epitopes, which sera from broilers react to, suggesting application in vaccines (Hung-Yueh Yeh et al Comparative Immunology, Microbiology and Infectious Diseases, Volume 49, 2016, Pages 76-81, ISSN 0147- 9571, https://doi.org/10.1016/j.cimid.2016.10.003). FlaA, MOMP, PEB3, CadF peptides have been determined via in silico predictions to generate potent B cell and T cell epitopes (Yasmin et al., 2016 In Silico Pharmacol., 4:5, DOI: 10.1186/s40203- 016-0020-y). FlgK, FlgE flagellar antigens with extracellular locations which produce antibody reactions in chicken sera and reduced bacterial load in chickens immunised by the recombinant proteins (Yeh et al., 2015, Arch. Microbiol., 197:353-358, DOI: 10.1007/s00203-014-1062-3). Flagellin used as a vaccine candidate can produce antigen specific antibody responses (IgY, IgM and IgA) as well as reduce bacterial loads in challenge models (Lee et al., 1999, Infect. Immun., 67(11):5799-5805, DOI: 10.1128/iai.67.11.5799-5805.1999; Chintoan-Uta et al., 2016, Vaccine, 34(15):1734- 1743, DOI: 10.1016/j.vaccine.2016.02.052). Exemplary antigens have been designed taking into account these disclosures. Accordingly, in some embodiments, the C. jejuni antigen is selected from: FlicC, FlaA, FliD, FlgE, FlgK, MOMP, and CadF. In preferred embodiments, the C. jejuni antigen is FlaA or FliD. Suitably the C. jejuni antigen is derived from a strain selected from: C. jejuni NCTC11168 (GenBank: NZ_CP046317), C. jejuni 81-176 (GenBank: AY681239 to AY681296), C. jejuni ST50, C. jejuni ST-257, and C. jejuni ST51. Suitably, the C. coli antigen is derived from a strain selected from C. coli (GenBank: NZ_CP046317). Suitable C. jejuni antigens include FlaA (WP_011812789.1; SEQ ID NO: 151) and FliD (WP_002864504; SEQ ID NO: 152). An exemplary FlaA antigen is SEQ ID NO: 113: GFRINTNVAALNAKANSDLNAKSLDASLSRLSSGLRINSAADDASGMAIADSLRSQANTLGQAISNGNDALG ILQTADKAMDEQLKILDTIKTKATQAAQDGQSLKTRTMLQADINKLMEELDNIANTTSFNGKQLLSGNFTNQ EFQIGASSNQTVKATIGATQSSKIGVTRFETGAQSFTSGVVGLTIKNYNGIEDFKFDNVVISTSVGTGLGAL AEEINKSADKTGVRATYDVKTTGVYAIKEGTTSQEFAINGVTIGKIEYKDGDGNGSLISAINAVKDTTGVQA SKDENGKLVLTSADGRGIKITGDIGVGSGILANQKENYGRLSLVKNDGRDINISGTNLSAIGMGTTDMISQS SVSLRESKGQISATNADAMGFNSYKGGGKFVFTQNVSSISAFMSAQGSGFSRGSGFSVGSGKNLSVGLSQGI QIISSAASMSNTYVVSAGSGFSSGSGNSQFAALKTTAANTTDETAGVTTLKGAMAVMDIAETAITNLDQIRA DIGSIQNQVTSTINNITVTQVNVKAAESQIRDVDFASESANYSKANILAQSGSYAMAQANSSQQNVLRLLQ Suitably, the antigen comprises a C. jejuni antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of FlaA, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 113. The antigen according to these embodiments may be secreted or surface-tethered. A surface-tethered antigen comprising a C. jejuni antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 780 of SEQ ID NO: 114. The first 21 amino acid residues of SEQ ID NO: 114 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 780 of SEQ ID NO: 114. An exemplary FliD antigen is SEQ ID NO: 115: AFGSLSSLGFGSGVLTQDTIDKLKEAEQKARIDPYTKKIEENTTKQKDLTEIKTKLLSFQTAVSSLADATVF AKRKVVGSISDNPPASLTVNSGVALQSMNINVTQLAQKDVYQSKGLANDGGFVNAQLNGTADLTFFSNGKEY TVTVDKNTTYRDLADKINEASGGEIVAKIVNTGEKGTPYRLTLTSKETGEDSAISFYAGKKDSNGKYQKDIN AEKIFDDLGWGLDVSASIDPDKDKKGYGIKDASLHIQTAQNAEFTLDGIKMFRSSNTVTDLGVGMTLTLNKT 5 GEINFDVQQDFEGVTKAMQDLVDAYNDLVTNLNAATDYNSETGTKGTLQGISEVNSIRSSILADLFDSQVVD GTTEDANGNKVNTKVMLSMQDFGLSLNDAGTLSFDSSKFEQKVKEDPDSTESFFSNITKYEDINHTGEVIKT GSLSKYLNSNGGNTNGLEFKPGDFTIVFNNQTYDLSKNSDGTNFKLTGKTEEELLQNLANHINSKGIEGLKV KVESYNQNNVTGFRLNFSGDGSSDFSIKGDANILKELGLSDVNITSKPIEGKGIFSKLKATLQEMTGKDGSI TKYDESLTNDIKSLNTSKDSTQAMIDTRYDTMANQWLQYESILNKLNQQLNTVTNMINAANN Suitably, the antigen comprises a C. jejuni antigen comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of FliD, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 115. The antigen according to these embodiments may be secreted or surface-tethered. A surface-tethered antigen comprising a C. jejuni antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 846 of SEQ ID NO: 116. The first 21 amino acid residues of SEQ ID NO: 116 is predicted to be the signal peptide; and thus the precursor of the surface-tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 846 of SEQ ID NO: 116. Suitably, the C. jejuni antigen is a multi-antigen fusion polypeptide, also referred to as a polyepitope. Suitable epitopes of FlaA are: LQTADKAMDEQLKILDTIKTKATQAAQDGQSLKTRTMLQADIN (SEQ ID NO: 117), VVISTSVGTGLGALA (SEQ ID NO: 118), ENYGRLSLVKNDGRDIN (SEQ ID NO: 119), and AGVTTLKGAMAVMDIAETAITNLDQIRADIGS (SEQ ID NO: 120). Suitable epitopes of FliD are: NSGVALQSMNINVTQ (SEQ ID NO: 121), DLGVGMTLTLNKTGE (SEQ ID NO: 122), DGTTEDANGNKVNTK (SEQ ID NO: 123), EDANGNKVNTKVMLS (SEQ ID NO: 124), and ESILNKLNQQLNTVT (SEQ ID NO: 125). Suitably, the C. jejuni antigen comprises one, 2, 3, or all 4 of the epitopes of SEQ ID NOs: 117-120; or variants or fragments comprising at least 90% sequence identity thereto. Suitably, the C. jejuni antigen comprises one, 2, 3, 4, or all 5 of the epitopes of SEQ ID NOs: 121-125; or variants or fragments comprising at least 90% sequence identity thereto. Suitably, the C. jejuni antigen comprises one, 2, 3, or all 4 of the epitopes of SEQ ID NOs: 117-120; or variants or fragments comprising at least 90% sequence identity thereto, and one, 2, 3, 4, or all 5 of the epitopes of SEQ ID NOs: 121-125; or variants or fragments comprising at least 90% sequence identity thereto. Suitably, the C. jejuni antigen comprises a linker between each epitope, such as a flexible [Gly]4Ser linker (GGGGS; SEQ ID NO: 48). Alternatively, a protease cleavage site may be included between each epitope, such as a cathepsin S cleavage site or any other suitable protease cleavage site as provided herein. The epitopes may be provided in any arrangement in the antigen. Multiple copies of any one or more of the antigens may be provided, such as 2, 3 or 4 copies. The antigen according to these embodiments may be secreted or surface-tethered. A FlaA antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 142. A surface tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 454 of SEQ ID NO: 126. The first 21 amino acids of this sequence are predicted to be the signal peptide; and thus the precursor of the surface- tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 454 of SEQ ID NO: 126. A FliD antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 143. A surface tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 400 of SEQ ID NO: 127. The first 21 amino acids of this sequence are predicted to be the signal peptide; and thus the precursor of the surface- tethered antigen may comprise an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 400 of SEQ ID NO: 127. Bacteria and methods of preparation The bacterium of the first aspect of the invention and the second aspect of the invention is of the class Clostridia. Clostridia includes the orders Clostridiales, Halanaerobiales and Thermoanaerobacteriales. The order Clostridiales includes the family Clostridiaceae, which includes the genus Clostridium. Clostridium is one of the largest bacterial genera. The genus is defined by rod-shaped, Gram-positive bacteria that are obligate anaerobes and capable of producing spores. Preferably the Clostridial bacterium or Clostridium species is capable of forming spores. Certain Clostridium species are known to be responsible for human diseases due to the formation of toxins, https://doi.org/10.1533/9781845696337.2.820. These include C. difficile, C. botulinum, C. novyi and C. perfringens. C. difficile is included in Clostridium cluster XIa. Preferably, the species is not a pathogenic Clostridium species. It may or may not be an attenuated strain from such a pathogenic species, such a natural or engineered strain which does not produce toxins. Typically, attenuated strains from pathogenic species may undesirably colonise the GI tract, and may therefore preferably be avoided. Several Clostridium species are found in the human lower gastrointestinal tract. The predominant Clostridia detected in lower GI tract include Clostridium cluster XIVa (also known as the Clostridium Coccoides group), and Clostridium cluster IV (also known as the Clostridium leptum group), Lopetuso et al. Gut Pathogens 2013, 5:23. The Clostridium cluster XIVa includes species belonging to the Clostridium, Eubacterium, Ruminococcus, Coprococcus, Dorea, Lachnospira, Roseburia and Butyrivibrio genera. Clostridium cluster IV is composed by the Clostridium, Eubacterium, Ruminococcus and Anaerofilum genera. Preferably, the Clostridial bacterium is a Cluster I Clostridium. The Clostridium cluster I includes species present in the gut microbiota (Alou et al., 2018, New Microbes New Infect., 21:128-139; https://doi.org/10.1016/j.nmni.2017.11.003) while others are predominantly found in soil and other such environmental niches and represent useful industrial chassis for the production of solvents and acids (Minton et al., 2016, Anaerobe, 41:104-112; DOI: 10.1016/j.anaerobe.2016.05.011). Non-pathogenic Cluster I species include: C. aceticum, C. acetobutylicum, C. aurantibutyricum, C. autoethanogenum, C. baratii, C. beijerinckii, C. butyricum, C. cadaveris, C. cellulovorans, C. colicanis, C. drakei, C. estertheticum, C. fallax, C. formicaceticum, C. kluyveri, C. ljungdahlii, C. paraputrificum, C. pasteurianum, C. ragsdalei, C. roseum, C. saccharoperbutylacetonicum, C. scatologenes, C. sporogenes, C. tyrobutyricum, C. saccharobutylicum, C. carboxidovorans, and C. autoethanogenum. Pathogenic Cluster I species include C. botulinum, C. noyyi, C. perfringens, C. septicum, C. tertium, C. chauvoei and C. tetani. A minority of Clostridium cluster I species found in the human gut are associated with disease whilst the majority are generally considered to contribute to health and wellbeing. Preferably the bacteria selected from Cluster I are species associated with health benefits. These species include C. sporogenes and C. butyricum. Preferably the bacterium is from cluster I, IV and/or XIVa of Clostridia, typically a non- pathogenic species. Preferably the bacterium is detectable in the lower gastrointestinal tract, for example of a human, but not considered to permanently colonise or form part of the resident microbiota in the lower GI tract, for example of a human. Preferably, the bacterium does not colonise the GI tract. It has been observed that Clostridium that are capable of colonising the GI tract – for example, C. perfringens – are also capable of binding and/or degrading mucins (Deplancke et al., 2002, Am. J. Clin. Nutr., 76(5):1117-1125; Li and McClane, 2018, Infect. Immun. 86(2):e00547- 17; Low et al., 2021, Glycobiology, 31(6):681-690). Accordingly, in some embodiments, the bacterium is not capable of binding and/or degrading mucins. Exemplary methods of determining mucin binding and/or degradation are disclosed in Deplancke et al. (2002), Li and McClane (2018), and Low et al, 2021. A suitable non- colonising species is C. butyricum. However, C. butyricum is capable of binding mucin despite not colonising the GI tract. Preferably the bacterium is capable of growing in an anaerobic section of the lower gastrointestinal tract and/or the bacterium is saccharolytic and can utilise di- and tri- saccharides in the colon. The ability to grow in an anaerobic section of the lower GI tract, such as colon or terminal ileum, may be related to saccharolytic metabolism. A suitable saccharolytic species is C. butyricum, which can utilise di- and tri-saccharides found in the colon. Further saccharolytic species include C. acetobutylicum, C. rouseum, C. sacchrolyticum, C. saccharoperbutylacetonicum, C. pasteurianum, C. aurantybutylicum, and C. beijerinckii. Due to the solventogenic nature of these further saccharolytic species, C. acetobutylicum, C. rouseum, C. sacchrolyticum, C. saccharoperbutylacetonicum, C. pasteurianum, C. aurantybutylicum, and C. beijerinckii are not usually found in the human gut. In contrast C. perfringens is proteolytic and may rely more on amino acids which perhaps are more readily available in the terminal small intestine. Saccharolytic Clostridia are capable of fermenting organic sugars to acids and/or solvents, and can further be distinguished from proteolytic strains by lack of ability to generate ATP via Stickland reactions, as described in in Mitchell, 1992, Res. Microbiol., 143(3):245-250 (DOI: 10.1016/0923- 2508(92)90016-H). In embodiments in which the bacterium is capable of secreting the antigen as a secreted antigen, the bacterium comprises a secretion system or accessory factors allowing for secretion of the antigen. Suitable secretion systems are disclosed in Green and Mecsas, 2016, Microbiol. Specr., 4(1), doi:10.1128/microbiolspec.VMBF-0012- 2015. Accordingly, in some embodiments the bacterium expresses a Sec-dependent secretion pathway, comprising SecYEG, SecA, and SecB. This system is found naturally in all Clostridia. In some embodiments, the bacterium expresses Tat-dependent secretion pathway, comprising TatA, TatB, and TatC. In embodiments in which the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, the bacterium may comprise accessory factors allowing for such tethering. These may include a secretion system or accessory factors allowing for secretion of the antigen as provided above. In embodiments involving sortase-dependent tethering, the bacterium expresses a Sortase system, comprising SrtA, SrtB, SrtC, and/or SrtD. The Sortase system is reviewed in Dramsi et al., 2008, FEMS Microbio. Rev.32(2):307- 320. Clostridia sp. carry Class A (StrA), Class B (StrB), Class C (StrC), and Class D (StrD) Sortase systems; however, Sortase systems may be species-specific. For example, Clostidium difficile has a single sortase, StrB (van Leeuwen et al., 2014, FEBS Lett., 588(23):4325-33, DOI: 10.1016/j.febslet.2014.09.041; Donahue et al., 2014, BMC Microbiol., 14:219, doi: 10.1186/s12866-014-0219-1); whereas Clostridium perfringens carries both Class A and Class B Sortase systems (Tami et al., 2021, Biochem. Biophys. Res Commun., 554:138-144, DOI: 10.1016/j.bbrc.2021.03.106; Suryadinata et al., 2015, Acta Crystallogr. D. Biol. Crystallogr., 71(7):1505-13, DOI: 10.1107/S1399004715009219). Butyrate production is widely distributed among anaerobic bacteria belonging to the Clostridial sub-phylum and in particular, to the Clostridial clusters XIVa and IV. Butyrate-producing species are found within two predominant families of commensal human colonic Clostridia, Ruminococcaceae and Lachnospiraceae. https://doi.org/10.1111/1462-2920.13589. Within the Lachnospiraceae are included: Eubacterium rectale, Roseburia inulinivorans, Roseburia intestinalis, Dorea longicatena, Eubacterium hallii, Anaerostipes hadrus, Ruminococcus torques, Coprococcus eutactus, Blautia obeum, Dorea formicigenerans, Coprococcus catus, Within the Ruminococcaceae are included: Faecalibacterium prausnitzii, Subdoligranulum variabile, Ruminococcus bromii, Eubacterium siraeum. Preferably, the bacterial species produces butyric acid. Butyrate-producing species, not considered to permanently colonise in the human lower GI tract, include Clostridium butyricum. Preferably, the species is amenable to genetic engineering techniques such as transformation by electroporation or conjugation, and is typically a non-pathogenic strain. Known transformable strains include industrial solvent strains including C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and C. saccharolyticum and pathogenic species including C. difficile. Preferably the species is C. butyricum. Suitable strains include the ‘Rowett’ strain, also referred to as (DSM10702/ATCC19398/NCTC 7423). In some embodiments, the Clostridial bacterium is capable of producing short-chain fatty acids (SCFAs), such as butyrate. SCFAs include lactate, acetate and butyrate and are believed to enhance T cell responses. Butyrate can stimulate CD8+ T cells and increase their effector functionality (Luu et al, 2018, Scientific Reports, 8:14430, and Trompette et al, 2018, Immunity, 48(5): 992-1005), and high concentration of faecal butyrate has been associated with longer progression-free survival following treatment with Nivolumab or Pembrolizumab in patents with solid cancer tumours (Nomura et al, 2020, Oncology, 3(4):e202895). Furthermore, butyrate enhanced memory potential of activated CD8+ T cells, and short-chain fatty acids (SCFAs) were required for optimal recall responses upon antigen re-encounter (Bachem et al, 2019, Immunity, 51(2):285-297). Therefore, there is a role for the microbiota, including Clostridium, in promoting CD8+ T cell long-term survival as memory cells. Production of SCFAs can be determined by metabolism of carbohydrate substrates, such as glucose, to SCFAs such as butyrate, i.e. through anaerobic metabolism, typically during vegetative cell growth. The bacterium according to the first and second aspects comprise a heterologous nucleic acid molecule encoding an antigen as described herein. In other words, it is an engineered bacterium. By “heterologous nucleic acid molecule”, we mean that the nucleic acid molecule comprises one or more non-native sequences such as in the open reading frame (ORF) encoding an antigen, although it is alternatively envisaged that a native antigen coding-sequence could be provided under the control of non-native control sequences, such as to facilitate an increased level of gene expression of a native antigen during anaerobic cell growth. The heterologous nucleic acid molecule comprises a gene, i.e. an ORF operatively linked to a promoter, which drives transcription of the gene. Other control sequences may also be present, as known in the art (Minton et al. (2016) A roadmap for gene system development in Clostridium, Anaerobe, 41:104-112). The heterologous nucleic acid molecule may comprise a non- native gene. The term “non-native gene” refers to a gene that is not in its natural environment and includes a gene from one species of a microorganism that is introduced into another species of the same genus. As used herein, the term “cassette” includes any heterologous nucleic acid molecule as described herein, optionally where the heterologous nucleic acid molecule comprises one or more non-native sequences including but not limited to an ORF encoding an antigen; an ORF operatively linked to a promoter; other control sequences; a non-native gene; or any combination thereof. The heterologous nucleic acid molecule may be codon optimised for Clostridia. The promoter is selected to enable expression of the antigen during anaerobic cell growth, such as following spore germination in anoxic conditions and/or during anaerobic vegetative cell metabolism. By “anaerobic cell growth”, we mean that the Clostridial bacterium is in the form of a cell, rather than a spore, and is capable of undergoing vegetative growth i.e. cell division. Clostridial bacteria are only capable of growing under anaerobic conditions. The growth may be recognised by increase in colony forming units. Anaerobic vegetative cell metabolism may be assessed by production of SCFAs, such as butyrate, acetate, lactate or combinations thereof from an available carbohydrate source. For example, a fermentable substrate, such as a carbohydrate substrate like glucose, can be supplied to the bacteria, and the production of SCFAs, such as butyrate, acetate, lactate or combinations thereof, can be measured, indicative of metabolism. The expressions “anaerobic cell growth” and “anaerobic vegetative cell metabolism” may be used interchangeably. Thus, the promoter is selected to be active in metabolically active or growing cells. Suitable promoters are active during cell growth and may be constitutive promoters. Promoters of genes that are essential to primary metabolism may be suitable constitutive promoters. The expression level of the antigen can be optimised by controlling gene expression using a promoter having a selected strength, such as a strong promoter. Promoter activity can be assessed using a gene expression reporter system, such as described in Tummala et al (1999) App. Environ. Microbiol. 65: 3793- 3799. Suitably, a native Clostridia promoter is used. Suitable promoters include the p0957 promoter of C. acetobutylicum described in the Examples which is based on SEQ ID NO: 181, and which further includes the ribosome binding site (RBS) from fdx as provided in SEQ ID NO: 182. The canonical ribosome binding site (RBS) sequence is AGGAGG, and is typically separated from the start codon of the nucleic acid sequence encoding the antigen by a spacer, for example a spacer comprising up to 5, up to 10, or up to 15 nucleotides. The RBS sequence may be a non-canonical RBS sequence, comprising a variant sequence as described herein. Thus, a promoter is typically provided in combination with a RBS and the term “promoter” may encompass the downstream sequence including RBS and spacer up to the start codon of the open reading frame. Thus the promoter may be SEQ ID NO: 181 or 182. Other suitable promoters include the fdx gene promoter of C. perfringens (Takamizawa et al (2004) Protein Expression Purification 36: 70-75); the ptb, and the thl promoters of C. acetobutylicum (Tummala et al (1999) App. Environ. Microbiol. 65: 3793-3799) and the thiolase promoter from C. acetobutylicum (Winzer et al (2000) J. Mol. Microbiol. Biotechnol. 2: 531-541). Other suitable promoters may be C. acetobutylicum promoters hbd, crt, etfA, etfB amd bcd (Alsaker and Papoutsakis (2005) J Bacteriol 187:7103-7118); and the fdx promoter from C. sporogeneses (NCIMB 10696), which can be obtained from the pMTL80000 modular shuttle plasmid (Heap et al. (2009) A modular system for Clostridium shuttle plasmids, Journal of Microbiological Methods, 78:79-85). The heterologous nucleic acid molecule can be introduced into Clostridia using methods known in the art. Typically, the heterologous nucleic acid molecule is integrated into the genome as a single copy or is present on a low copy plasmid. Alternatively, it may be present on a high copy plasmid. A high copy plasmid may be present at a copy number of about 8 to 14, or greater, for example as described in SY, Mermelstein LD, Papoutsakis ET. Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824. FEMS Microbiol Lett. 1993 Apr 15;108(3):319-23. Suitable plasmids include those that are stably maintained by the Clostridia. Suitably plasmids contain a suitable origin of replication and any necessary replication genes to allow for replication in the Clostridia. Plasmid transformation is typically achieved in Clostridia by conjugation or transformation. Methods of transformation and conjugation in Clostridia are provided in Davis, I, Carter, G, Young, M and Minton, NP (2005) “Gene Cloning in Clostridia”, In: Handbook on Clostridia (Durre P, ed) pages 37-52, CRC Press, Boca Raton, USA. The heterologous nucleic acid molecule may be integrated into the genome, typically the chromosome of Clostridia, using gene integration technology, such as by Allele Coupled Exchange (ACE) as described in WO 2010/084349 and Minton et al (2016) Anaerobe 41: 104-112; or CRISPR gene editing (Atmadjaja et al. (2019) CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT), FEMS Microbiol. Lett. 366(6)). It is believed that Allele Coupled Exchange can be used to engineer any clostridial species, and is reliant on the initial creation of a pyrE deletion mutant that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent insertion of a DNA fragment by allelic exchange using a heterologous pyrE allele as a counter-/negative- selection marker in the presence of FOA. Following modification of the insertion site, the strain created may be rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild- type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. The initial creation of the pyrE deletion may be performed by a special form of ACE, as described in Minton et al, supra, or by means of retargeting mobile group II introns as described in WO 2007/148091. CRISPR gene editing also has wide application in Clostridia for integration of large DNA fragments and has been successfully applied in a number of Clostridial strains, including C. acetobutylicum (Li et al. (2016) CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Biotechnol J. 11:961–72), C. beijerinckii (Li et al. (2016) and Wang et al. (2015) Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J Biotechnol. 200:1–5), C. pasteurianum (Pyne et al. (2016) Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium. Sci Rep. 6:25666) and C. saccharoperbutylacetonicum (Wang et al. (2017) Genome editing in Clostridium saccharoperbutylacetonicum N1-4 using CRISPR-Cas9 system. Appl Environ Microbiol. 83:e00233–17). Where the heterologous nucleic acid molecule is integrated into the genome as a single copy or is present on a low copy plasmid, the amount of antigen expressed will typically be lower than if the heterologous nucleic acid molecule is present on a high copy number plasmid. The inventors have found in PCT Patent Application No. PCT/GB2021/053264 that an antigen-specific immune response can be effectively stimulated even when the heterologous nucleic acid molecule is integrated into the genome as a single copy. The amount of antigen expressed per cell weight of clostridial cells undergoing anaerobic cell growth may typically be in the range of up to 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 ng/mg, 1μg/ mg, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10 or 20 μg/ mg dry cell weight (but greater than 0 ng/ mg dry cell weight, typically greater than 10 ng/mg, 20 ng/mg or 40 ng/mg). Any range between any two of these values is envisaged. For example, the amount of antigen expressed per cell weight of clostridial cells undergoing anaerobic cell growth may be from 10 to 400 ng/ mg dry cell weight; 20 to 200 ng/ mg dry cell weight; 40 to 100 ng/ mg dry cell weight; 100 ng to 5 μg/ mg dry cell weight; 200 ng to 2.5 μg/ mg dry cell weight; 400-1500 ng/ mg dry cell weight; or about 800 ng/ mg dry cell weight; or it may be between 1 and 5 μg/ mg dry cell weight, or 2 and 4 μg/ mg dry cell weight, such as about 3 μg/ mg dry cell weight; or any other combination. The amount can be determined by culturing clostridial cells, obtaining antigen typically comprising a detection tag such as FLAG, and quantifying the antigen by detection means such as ELISA or western blotting. Antigen may be obtained by extraction from bacterial cells, or recovery from the culture medium as appropriate. Protein standards, such as FLAG- tag standards available from Sigma, may be used in such assays to construct a standard curve. Typically, the amount of antigen is determined for a specific volume of cells cultured to a specific density e.g. OD1.0. The amount of antigen per dry weight of bacteria in that amount of culture is then estimated assuming the cell density in OD1.0 is 0.3 g/L. The amount of antigen produced may be varied depending on the strength of the promoter, the number of copies of the heterologous nucleic acid molecule per cell etc. In any of the embodiments, the bacterial cell may comprise a further heterologous nucleic acid molecule encoding an immunostimulatory agent or adjuvant, which is capable of being co-expressed with the antigen. Typical immunostimulatory agents may be polypeptides, such as cytokines, such as IL-12, IL-18 or GM-CSF, IFN-ڛ, IL-2, IL-15. For example, HPV16 and HPV18 E6/E7 antigens have been combined with IL- 12 in clinical trials (Hasan et al. (2020) A Phase 1 Trial Assessing the Safety and Tolerability of a Therapeutic DNA Vaccination Against HPV16 and HPV18 E6/E7 Oncogenes After Chemoradiation for Cervical Cancer, Int J Radiat Oncol Biol Phys. 107(3):487-498). A corresponding aspect of the invention provides a method for preparing a bacterium according to the first aspect and the second aspect comprising introducing at least one heterologous nucleic acid molecule into the bacterium. It will be appreciated that where accessory factors such as sortase systems etc are not already present, these may also be introduced by means of at least one heterologous nucleic acid molecule. A related aspect of the invention provides a nucleic acid molecule suitable for propagation in a bacterium of the class Clostridia comprising an antigen gene comprising a region encoding at least one surface-tethered antigen precursor and a promoter operably linked to said region, which promoter is capable of causing expression of the surface-tethered antigen precursor by a bacterium of the class Clostridia; wherein the antigen precursor comprises an N-terminal signal peptide and a domain or motif required for tethering of the antigen to peptidoglycan in a bacterium of the class Clostridia. Suitably, according to this aspect, the domain or motif is a peptidoglycan anchoring domain or a sequence required for enzymatic linkage to peptidoglycan, such as wherein the peptidoglycan anchoring domain comprises at least one LysM motif, such as a LysM domain. Suitably, the promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth, such as wherein the promoter is the p0957 promoter of C. acetobutylicum, for example selected from SEQ ID NO: 181 and SEQ ID NO: 182, fdx promoter of C. perfringens, the ptb, thl hbd, crt, etfA, etfB or bcd promoter of C. acetobutylicum and/or wherein the nucleic acid molecule is suitable for propagation in a bacterium of the class Clostridia by virtue of comprising a traJ conjugal transfer function such as described in Zatyka, et al., 1998, FEMS Microbiol. Rev., 21(4):291- 319, DOI: 10.1016/j.mimet.2009.05.004); and/or a pBP1 Gram+ replicon such as described in Heap et al., 2009, J. Microbiol. Meth., 78(1):79-85, DOI: 10.1016/j.mimet.2009.05.004. The features of the antigen and domain or motif required for tethering of the antigen to peptidoglycan are suitably as described in relation to the first and second aspects of the invention. Pharmaceutical compositions and methods of preparation A third aspect of the invention is a pharmaceutical composition comprising a bacterium according to the first aspect or comprising the bacterium according to the second aspect. A corresponding aspect of the invention provides a method for preparing a pharmaceutical composition according to the third aspect comprising formulating the bacteria with one or more pharmaceutically acceptable diluents or excipients. While it is possible for the bacterium to be administered alone, it is preferable for it to be present in a pharmaceutical composition. The present invention includes pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier, excipient or further component such as therapeutic and/or prophylactic ingredient (such as adjuvant). A “pharmaceutically acceptable carrier” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. The carrier may include one or more excipients or diluents. The Clostridia can be prepared by fermentation carried out under suitable conditions for growth of the bacteria. After fermentation, the bacteria can be purified using centrifugation and prepared to preserve activity. The bacteria in the composition are provided as viable organisms. The composition can comprise bacterial spores and/or vegetative cells. The bacteria can be dried to preserve the activity of the bacteria. Suitable drying methods include freeze drying, spray-drying, heat drying, and combinations thereof. The obtained powder can then be mixed with one or more pharmaceutically acceptable excipients as described herein. The spores and/or vegetative bacteria may be formulated with the usual excipients and components for oral administration, as described herein. In particular, fatty and/or aqueous components, humectants, thickeners, preservatives, texturing agents, flavour enhancers and/or coating agents, antioxidants, preservatives and/or dyes that are customary in the pharmaceutical and food supplement industry. Suitable pharmaceutically acceptable carriers include microcrystalline cellulose, cellobiose, mannitol, glucose, sucrose, lactose, polyvinylpyrrolidone, magnesium silicate, magnesium stearate and starch, or a combination thereof. The bacteria can then be formed into a suitable orally ingestible forms, as described herein. Suitable orally ingestible forms of probiotic bacteria can be prepared by methods well known in the pharmaceutical industry. Suitable pharmaceutical carriers, excipients and formulations are described in Remington: The Science and Practice of Pharmacy 22nd Edition, The Pharmaceutical Press, London, Philadelphia, 2013. Pharmaceutical compositions of the invention can be placed into dosage forms, such as in the form of unit dosages. Pharmaceutical compositions include those suitable for oral or rectal administration. The compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. Preferably, administration is oral using a convenient dosage regimen. Suitable oral dosage forms include tablet, capsule, powder (e.g. a powder in sachet) and liquid. Where the bacterium is for administering orally, it is suitably provided in the form of a spore; or in the form of a vegetative cell in a delayed release pharmaceutical composition. Pharmaceutical compositions of the invention can also be formulated for rectal administration including suppositories and enema formulations. In the case of suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized moulds, allowed to cool, and to solidify. Enema formulations can be semi-solid including gels or ointments or in liquid form including suspensions, aqueous solutions or foams, which are known to those skilled in the art. The pharmaceutical compositions of the invention are administered such that an effective amount of bacterium is delivered to an anaerobic section of the gut. By “effective amount of bacterium” we include the meaning that the bacterium results in the delivery of an amount of antigen effective to induce a suitable immune response to said antigen; or to prevent, ameliorate or treat a disease. For example, for a viral infection where a CTL response may be suitable, the antigen will be in an amount effective to induce a CD8+ CTL response against that antigen. Suitably the bacteria may be present in the pharmaceutical composition in an amount equivalent to between 1x105 to 1x1011 colony forming units/g (CFU/g) of dry composition. Suitably, the bacteria may be present in an amount of 1x106 to 1x1010 CFU per unit dosage form, preferably from about 1x107 to 1x109 CFU per unit dosage form, such as about 1x108 CFU per unit dosage form. Pharmaceutical compositions may include adjuvants or immunostimulatory molecules, particularly pharmaceutical compositions that are formulated for delayed release. However, it is envisaged that an adjuvant may not be necessary, or may be necessary only in a quantity that is lower than would be required if the antigen were provided in a conventional polypeptide antigen vaccine formulation, or that a less toxic adjuvant only may be required. Thus, pharmaceutical compositions which lack an adjuvant are envisaged, as are those which contain only an adjuvant which is appropriate for human use, such as alum. Adjuvants are any substance whose admixture into the pharmaceutical composition increases or otherwise modifies the immune response to an antigen. Adjuvants can include but are not limited to AlK(SO4)2, AlNa(SO4)2, AlNH(SO4)4, silica, alum, AI(OH)3, Ca3(PO4)2, kaolin, carbon, aluminium hydroxide, muramyl dipeptides, N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D- isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-s-n-glycero-3-hydroxphosphoryloxy)- ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80(R) emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see U.S. Pat. Nos. 58,767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21. Additional adjuvants or compounds that may be used to modify or stimulate the immune response include ligands for Toll-like receptors (TLRs). In mammals, TLRs are a family of receptors expressed on DCs that recognize and respond to molecular patterns associated with microbial pathogens. Several TLR ligands have been intensively investigated as vaccine adjuvants. Bacterial lipopolysaccharide (LPS) is the TLR4 ligand and its detoxified variant mono-phosphoryl lipid A (MPL) is an approved adjuvant for use in humans. TLR5 is expressed on monocytes and DCs and responds to flagellin whereas TLR9 recognizes bacterial DNA containing CpG motifs. Oligonucleotides (OLGs) containing CpG motifs are potent ligands for, and agonists of, TLR9 and have been intensively investigated for their adjuvant properties. Other agents that stimulate the immune response (immunostimulatory agents) can included, such as cytokines that are useful as a result of their lymphocyte regulatory properties. Suitable cytokines may include interleukin-12 (IL-12), GM-CSF or IL-18. Pharmaceutical compositions of the invention can be formulated as capsules comprising viable cells, such as vegetative cells or spores, wherein the capsules comprise a delayed-release layer or coating that allows for the release of the viable cells, typically vegetative cells in an anaerobic section of the lower GI tract following oral administration. By “delayed-release” or “delayed release”, we mean that release of the bacterium is delayed for a certain period of time after administration or application of the dosage (the delay is also known as the lag time). This modification is achieved by special formulation design and/or manufacturing methods. The subsequent release can be similar to that of an immediate release dosage form. Excipients and formulations for delayed release are well known in the art and described, for example in WO 2018/055388 or WO 2019/180441. Suitably, pharmaceutical compositions of the invention are formulated to deliver the bacterium according to the first aspect or the second aspect to the GI tract, preferably by oral administration. The human GI tract consists of digestive structures stretching from the mouth to the anus, including the oesophagus, stomach, and intestines. The GI tract does not include the accessory glandular organs such as the liver, biliary tract or pancreas. The intestines include the small intestine and large intestine. The small intestine includes the duodenum, jejunum and ileum. The large intestine includes the cecum, colon, rectum and anus. The upper GI tract includes the buccal cavity, pharynx, oesophagus, stomach, and duodenum. The lower GI tract includes the small intestine (below the duodenum) and the large intestine. Preferably, the pharmaceutical compositions of the invention deliver the bacterium according to the first aspect or the second aspect to the lumen or mucosal surface of the GI tract, more preferably the lumen or mucosal surface of the large intestine, and more preferably the lumen or mucosal surface of the colon. Preferably, the pharmaceutical compositions of the invention deliver bacterium according to the first aspect or the second aspect to anaerobic sections of the lower GI tract, preferably the colon and/or terminal small intestine (ileum, also referred to as the “terminal ileum”). A steep oxygen gradient exists within the human intestinal tract, as reviewed in Zheng, Kelly and Colgan, American Journal of Physiology-Cell Physiology 2015 309:6, C350- C360. Breathable air at sea level has a Po2 of ^145 mmHg (^21% O2). Measurements of the healthy lung alveolus have revealed a Po2 of 100–110 mmHg. By stark contrast, the most luminal aspect of the healthy colon exists at a Po2 below 10 mmHg (1.4% O2). Such differences reflect a combination of oxygen sources, local metabolism, and the anatomy of blood flow. The Po2 drops precipitously along the radial axis from the intestinal submucosa to the lumen, which is home to trillions of anaerobic microbes. Where the bacterium is delivered orally as a spore, it will transit through the GI tract until it reaches the anaerobic portions, where it will germinate and grow. Anaerobic sections of the lower GI tract include the terminal ileum and colon. The colon may have a lower PO2 than the terminal ileum, in view of Zheng, supra, and bacterial growth may therefore be more efficient in the colon. PO2 required to trigger spore germination and anaerobic metabolism or growth may be in the range of 0 to 2%. The human colon volume (sum of ascending/descending and transverse) is around 600ml (Pritchard, S. E. et al. (2-14) Neurogastroenterol. Motil. 26, 124-130) whereas the entire intestine of a mouse is around 1 ml in volume (McConnell, E. L., Basit, A. W. & Murdan, S. (2008) J. Pharm. Pharmacol. 60, 63-70). The approximate total GI transit time is around 5-6 hours in a mouse (Padmanabhan, P., et al. (2013) EJNMMI Res. 3, 60 and Kashyap, P. C. et al. (2013) Gastroenterology 144, 967-977) and the colon transit times have been estimated to be between 23 and 40 hours in humans (Degen, L. P. & Phillips, S. F. (1996) Gut 39, 299-305 and Wagener, S., et al (2004) J. Pediatr. Surg. 39, 166-169-169). Since transit time in the human gut is five times longer than in mouse, fewer spores are needed (e.g. by a factor of five) to achieve the same concentration of antigen if the colon volumes were the same. Further, because the bacteria are resident in the human colon approximately five time longer than the mouse colon, there will be a longer duration for cell division (by a factor of five), therefore resulting in more cell numbers and in an increase in production of antigen. The lab fermentation based doubling time of the bacterial strain CHN0 is similar to that for E. coli and E. coli have a gut doubling time of about 3 hours (Myhrvold, C., et al (2015) Nat. Commun.6, 10039). CHN0 may undergo 10 doublings of cells during gut transit, equating to a three order of magnitude increase in cell numbers. In the mouse there is only sufficient time for around two doublings of cells equating to less than a 10-fold increase in cell numbers. Approximately 100 times more cells will grow from each spore delivered to the human gut relative to the mouse gut. When accounting for gut volume differences, colon transit times and cell division within the gut, approximately the same dose delivered to a mouse and a human will result in approximately the same content of antigen within the gut lumen. Although the above specifies the difference between the human gut and murine gut, this can be readily adapted to other hosts based on what is known in the art (e.g. to adapt the delivery of the bacterium to the intended host, for example to other mammals or birds. A pharmaceutical composition taken on an empty stomach is likely to arrive in the ascending colon about 5 hours after dosing, with the actual arrival dependent largely on the rate of gastric emptying. Drug delivery within the colon is greatly influenced by the rate of transit through this region. In healthy men, capsules pass through the colon in 20-30 hours on average. Solutions and particles usually spread extensively within the proximal colon and often disperse throughout the entire large intestine.Suitably, the pharmaceutical compositions are for administration between meals or with food. Growth of the bacterium according to the first aspect of the invention or the second aspect of the invention upon arrival in the anaerobic portion of the gut can be verified by culture, including stool culture. In experimental models, bacteria may be cultured from portions of the GI tract obtained from the experimental animal. Growth of the bacterium according to the first aspect of the invention or the second aspect of the invention upon arrival in the anaerobic portion of the gut can also be verified by immunohistological approaches known to the skilled person, for example by using antibodies that recognise the bacteria. The genetically engineered anaerobic bacteria that produce antigen can also be incorporated as part of a food product, i.e. in yoghurt, milk or soy milk, or as a food supplement; or as part of a beverage. Such food products, food supplements and beverages can be prepared by methods well known in the food, supplement and beverage industry. The compositions can be incorporated into animal feed products as a feed additive. The growth and degree of colonisation in the gut of the genetically engineered bacteria can be controlled by the species and strain choice and/or by providing specific substrates that the bacteria thrive on as a prebiotic, either within the same dose that contains the probiotic or as a separately ingested composition. Accordingly, the composition may also further comprise or be for administering with a prebiotic to enhance the growth of the administered probiotic. The prebiotic may be administered sequentially, simultaneously or separately with a bacterium as described herein. The prebiotic and bacterium can be formulated together into the same composition for simultaneous administration. Alternatively, the bacteria and prebiotic can be formulated separately for simultaneous or sequential administration. Prebiotics are substances that promote the growth of probiotics in the intestines. They are food substances that are fermented in the intestine by the bacteria. The addition of a prebiotic provides a medium that can promote the growth of the probiotic strains in the intestines. One or more monosaccharides, oligosaccharides, polysaccharides, or other prebiotics that enhances the growth of the bacteria may be used. Preferably, the prebiotic may be selected from the group comprising of oligosaccharides, optionally containing fructose, galactose, mannose; dietary fibres, in particular soluble fibres, soy fibres; inulin; or combinations thereof. Preferred prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides, xylo-oligosaccharides, oligosaccharides of soy, glycosylsucrose (GS), lactosucrose (LS), lactulose (LA), palatinose-oligosaccharides (PAO), malto-oligosaccharides, pectins, hydrolysates thereof or combinations thereof. Medical uses A fourth aspect of the invention provides the bacterium of the first aspect or the second aspect; or the pharmaceutical composition of the third aspect for use in medicine. A fifth aspect of the invention provides a bacterium of the class Clostridia for use in generating an antigen-specific response in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen. A corresponding aspect provides a method of generating an antigen-specific immune response in a subject, comprising administering a bacterium of the class Clostridia, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen. A sixth aspect of the invention provides a bacterium of the class Clostridia for use in generating an antigen-specific immune response in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2. A corresponding aspect provides a method of generating an antigen-specific immune response in a subject, comprising administering a bacterium of the class Clostridia, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2. A seventh aspect of the invention provides a bacterium of the class Clostridia for use in the preventive or therapeutic treatment of a disease in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen; wherein the antigen is an infectious agent antigen and the disease is the infectious disease, or the antigen is a tumour antigen and the disease is cancer. A corresponding aspect provides a method of preventing, ameliorating or treating a disease in a subject, comprising administering a bacterium of the class Clostridia, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen; wherein the antigen is an infectious agent antigen and the disease is the infectious disease, or the antigen is a tumour antigen and the disease is cancer. An eighth aspect of the invention provides a bacterium of the class Clostridia for use in the preventive or therapeutic treatment of COVID-19 in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2. A corresponding aspect provides a method of preventing, ameliorating or treating COVID-19 in a subject, comprising administering a bacterium of the class Clostridia, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2. Typically, in any of these aspects of the invention, the subject is a mammal or bird, typically a mammal, preferably a human. Suitable mammals for veterinary vaccination include agricultural animals, such as ungulates, including cows, sheep or goats; or horses; or pigs; or domestic animals such as cats or dogs. Suitable birds include chickens or turkeys. Typically, where the antigen is an infectious agent antigen, the subject is of a species which is susceptible to a disease caused by the infectious agent. Typically, where the antigen is a tumour antigen, the subject is of a species for which the tumour antigen is characteristic of a tumour. These uses involve vaccination. Appropriate doses for vaccination, and schedules of administration (e.g. primary and one or more booster doses) are described in Vaccines: From concept to clinic, Paoletti and McInnes, eds, CRC Press, 1999. For example, vaccination may be effective after a single dose, or one to three inoculations may be provided about 2 weeks to six months apart. In some embodiments, the vaccination may be provided in a vaccination regimen with a different vaccine, such as a prime – boost regimen in which the vaccine of the invention is either the prime or booster vaccine, and the other of those is a different vaccine. There may be more than one booster. Typically, such regimens will be directed at the same infectious agent or the same cancer. Medical uses in generating an antigen-specific immune response In the fifth aspect, the antigen may be any antigen as defined herein, not limited to tumour antigen or infectious agent antigen. For example, the antigen may include an artificial sequence (i.e., artificially designed sequence, which is not present in nature). In the sixth aspect, the antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2. Suitable antigens are as defined herein. By “antigen-specific immune response” we include any cellular or humoral immune response that is antigen-specific, i.e. T cell responses such as CD4+, CD8+ T-cell responses, or B cell (antibody) responses. In a typical immune response, antigen is delivered to antigen presenting cells (APCs), especially dendritic cells (DC), which then stimulate and elicit antigen specific cytotoxic CD8+ (CTL) and/or helper CD4+ T lymphocytes. Also known as professional APCs, DCs sample antigens in the microenvironment and process them intracellularly (for example, following the antigen being phagocytosed). Upon DC activation (e.g. due to an inflammatory signal), they migrate to the lymph nodes whereby they can activate the adaptive immune response. Without wishing to be bound by theory, bacteria of the class Clostridia, or antigens secreted by them, may be internalised by APCs, particularly DCs in the intestine, such as mucosal DCs. For example, a DC that has taken up (e.g. phagocytosed) an antigen by virtue of having internalised a bacterium of the class Clostridia and/or secreted antigen may become activated, and may migrate to the lymph node and activate T-cells that have specificity to said antigen, and thence B cells. The APC may be exposed to a further activating signal in addition to the bacterium of the class Clostridia, such as provided by an adjuvant, lipopolysaccharide (LPS), or inflammatory cytokine. T cells express a T-cell receptor that recognises antigenic peptides that are presented by major histocompatibility complex (MHC), referred to as human leukocyte antigen (HLA) in humans. Helper CD4+ T-cells can effectively stimulate and amplify cytotoxic CD8+ T-cells and help B cells to produce antibodies. A CD4+ response can be categorised by the type of CD4+ T-cell that is induced/activated. For example, a CD4+ response may be that of a T helper (Th) 1, Th2, and/or Th17. Th1, Th2 and Th17 cells can be categorised by markers (e.g. cell surface markers), cytokine secretion and/or functional assays that are known to the skilled person. The type of CD4+ response (or combination thereof) achieved may depend on the antigen being used and/or adjuvants or other immunomodulatory molecules, which may be selected depending on the desired outcome. For example, Th2 responses are more suitable than Th1 responses for protecting against helminth infection. Th1 responses, which are often associated with IFN-Υ production, are more suitable than Th2 responses for protecting against intracellular parasites. Th1 cells stimulate CD8+ killer T cells, Th2 cells stimulate B cells; and Th17 cells facilitate inflammation. CD8+ T-cells can specifically recognise and induce apoptosis of target cells containing target antigens. Activation of specific CD8+ T-cells depends on the antigen being efficiently presented to MHC class I molecule (HLA-I antigen in humans). CD8+ cytotoxic T lymphocytes (CTLs) are the main cell type targeted by prophylactic and therapeutic cellular immune vaccines because they can directly recognise and destroy tumour cells or cells infected by intracellular infectious agents, such as viruses. Therefore, for the purposes of targeting tumour antigens and antigens of intracellular infectious agents such as viruses, it can be advantageous to mount a CD8+ response as these cells are capable of directly recognising these antigens presented on MHC class I molecules on the cell surface. CTLs are also associated with anti-tumour responses. A combination of CD4+ and CD8+ responses may be beneficial, as subsets of CD4+ cells may support and/or enhance the activity of CD8+ cells by releasing cytokines into the local microenvironment. Accordingly, in some embodiments, a combination of T-cell responses is induced by the antigen. The efficiency of single peptide antigens to stimulate an immune response may differ between subjects and populations based on their expression profiles for MHC (or in the case of humans, HLA). MHC/HLA haplotypes differ between subjects, with each haplotype of MHC/HLA being capable of binding and thereby presenting particular types of peptide fragments. For example, for the same antigen, the peptide fragments presented by the MHC/HLA of a first subject may differ in sequence to those presented by the MHC/HLA of a second subject. These MHC/HLA subtypes may differ in their ability to induce an immune response, resulting in differences within populations for responsiveness to a particular antigen. This is a major drawback of single peptide- based vaccines, as not all subjects will be capable of processing and presenting the peptide adequately to induce the required immune response. This limitation of single- peptide vaccines can be overcome by using multi-antigen fusion proteins, such as polyepitopes and/or polypeptides comprising overlapping peptides as described above, including the ROPs described herein and in EP 3235831 A1. ROPs have been shown to be capable of simultaneously inducing CD4+ and CD8+ T-cell responses and comprise multiple peptide segments that vastly increases the likelihood of there being a segment that suits a particular subject. This overcomes the MHC/HLA restriction of a population. A B cell response is characterised by antibodies (i.e. “immunoglobulins” or “Ig”) that target specific antigens. B cells are able to internalise components, such as polypeptides, and present fragments of polypeptide molecules on the cell surface in complex with MHC class I or II molecules. B cells may also express on their cell surface antigen specific B cell receptors (BCR). Unlike T-cells and the TCR, which rely upon antigen being presented by MHC, the BCR can recognise antigenic epitopes without them being presented by MHC (i.e. BCR can also recognise soluble antigen). Antigen activates B cells bearing appropriate surface immunoglobulin directly to produce IgM. In some instances, B cells rely upon T-cells for activation by presenting antigen loaded to MHC class II. CD4+ T cells, having responded to processed Ag, may induce immunoglobulin class-switching from IgM to IgG. However, some antigens are able to activate B cells in a T-cell independent manner. Therefore, in some embodiments, the induction of a B cell response may be in conjunction with the induction of a T-cell response (CD4+ and/or CD8+). Suitable antibody responses may include different isotypes, such as IgA and/or IgG isotypes. The type of antibody response achieved may depend on the antigen being used and/or adjuvants or other immunomodulatory molecules, which may be selected depending on the desired outcome. IgA, also referred to as sIgA in its secretory form is an antibody that plays a crucial role in the immune function of mucous membranes. The amount of IgA produced in association with mucosal membranes is greater than all other types of antibody combined. In absolute terms, between three and five grams are secreted into the intestinal lumen each day. This represents up to 15% of total immunoglobulins produced throughout the body. IgA has two subclasses (IgA1 and IgA2) and can be produced as a monomeric as well as a dimeric form. The IgA dimeric form is the most prevalent and is also called secretory IgA (sIgA). sIgA is the main immunoglobulin found in mucous secretions, including tears, saliva, sweat, colostrum and secretions from the genitourinary tract, gastrointestinal tract, prostate and respiratory epithelium. It is also found in small amounts in blood. The secretory component of sIgA protects the immunoglobulin from being degraded by proteolytic enzymes; thus, sIgA can survive in the harsh gastrointestinal tract environment and provide protection against microbes that multiply in body secretions. sIgA can also inhibit inflammatory effects of other immunoglobulins. IgA is a poor activator of the complement system and opsonizes only weakly. There are several subtypes of IgG. In humans, IgG1 and IgG3 are associated with T helper 1-type responses, complement fixation, phagocytosis by high affinity FcRs and are indicative of protective immunity, whereas IgG2 and IgG4 responses tend to be less effective. In some embodiments, the antigen-specific immune response induced by the antigen is a B-cell response. In some embodiments, the antigen-specific immune response includes the generation of antigen-specific antibodies, i.e., the antigen induces the production of antigen-specific antibodies that are specific for (i.e., bind to) said antigen. In some embodiments, the antigen-specific antibody belongs to an antibody serotype selected from the group comprising or consisting of: IgA, IgM, IgG, or any combination thereof. In some embodiments, the antigen-specific antibody is a secreted antibody, for example secretory IgA (sIgA), secretory IgM, or secretory IgG. Accordingly, in some embodiments, the antigen induces the production of antigen-specific IgA, antigen-specific IgM, antigen-specific IgG, or any combination thereof. A bacterium comprising antigen, as described herein, can be tested for capability for inducing an antigen-specific immune response, such as following oral immunisation in a mouse model. The bacterial spores (e.g. C. butyricum) encoding an antigen of interest can be administered to a group of mice by oral gavage. A negative control of spores from the same bacterium but without antigen may be administered to a separate group of mice. A comparison of the bacterium with antigen and such a negative control will attribute any differences as being antigen specific. A suitable positive control for this experiment includes the parenteral administration by subcutaneous injection of the antigen, which will be taken up by DCs resulting in activation of an immune response to said antigen. Therefore, a comparison with this positive control will give an indication as to whether the immune response induced by the bacterium comprising antigen is equivalent to the administration of the antigen itself. The administration of the bacterium comprising antigen, the negative control and the positive control may be done as an immunisation regimen. For example, mice may be immunised 3 times at fortnightly intervals. Following the immunisation regimen, for example after 42 days of the regimen, the mice are sacrificed. As will be appreciated, other immunisation regimens may suitably be employed. For example, mice may be immunised over consecutive days with intervals between immunisations. Various numbers of consecutive days of immunisation, the duration of intervals between immunisation doses, and number of times the mice are immunised may be varied. For example, the mice may be immunised for 2 consecutive days, 3 consecutive days, 4 consecutive days, or 5 consecutive days. The interval between immunisation doses may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. The interval between immunisation doses may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more weeks. The mice may be immunised 1 time, 2 times, 3 times, 4 times, 5 times or 6 times. An immune response can be detected in a number of ways known to the skilled person. For example, splenocytes from homogenised spleens and peripheral blood mononuclear cells (PBMCs) from blood samples can collected and mononuclear cell isolates obtained using standard methods. These mononuclear cell isolates can then be subjected to various sorting protocols to isolate cell populations of interest, for example by using magnetic associated cell sorting (MACS) or Ficoll-Hypaque gradient (density) separation to obtain lymphocytes. For example, T cells (CD4+ and CD8+) may be enriched from the mononuclear cell isolates based on a T cell specific marker (e.g. CD8a for CD8+ T cells). Alternatively, or additionally, other cell populations may be isolated, such as B cells. Upon obtaining an enriched population of a cell type of interest, the cell type can be tested for the secretion of cytokines associated with activation of an immune response (e.g. IFN-Υ and/or TNFĮ) or for the expression of markers (e.g. cell surface markers and/or intracellular markers) indicative of an activated cellular phenotype. Cytokines, such as IFN-Υ and/or TNFĮ, can be tested by ELISPOT by culturing T cells in plates in the presence of anti-IFN-Υ or anti-TNFĮ antibodies, respectively, and re-stimulating the cells with wildtype antigen protein, with vegetative bacterial cells that comprise the antigen, or with antigen presenting cells that have previously been exposed to the antigen. If T cells are present that have specificity for the antigen of interest, then re- stimulation with that antigen will induce the T cells to secrete IFN-Υ and/or TNFĮ. The use of vegetative bacterial cells that do not comprise antigen is a negative control, which can be compared with the test condition to identify to what extent the IFN-Υ and/or TNFĮ secretion is antigen specific. IFN-Υ and/or TNFĮ standards (i.e. aliquots of these cytokines at varying concentrations) can be used as a positive control and to establish a dose response curve. For ELISPOT, spot forming units (SPU) can be assessed, wherein an SPU for a test condition (e.g. a vaccinated group) that is at least two standard deviations higher than the average of a control group would indicate a positive result for the test condition. Alternatively, or additionally, an immune response can be tested by intracellular cytokine staining, such as that described in Zhang et al., 2009. In brief, splenocytes obtained from the mice subjected to the above-described immunisation regimen can be cultured with the antigen and same negative and positive controls as the ELISPOT. The cells can then be labelled with antibodies (e.g. phycoerythrin-conjugated monoclonal rat anti-mouse CD8 or CD4 antibody) or an immunoglobulin isotype control. Splenocytes can then be fixed and permeabilised using a fix/perm protocol (e.g. the Cytofix/Cytoperm kit by BD Pharmingen) and incubated with a detection antibody for intracellular antigen (e.g. fluorescein isothiocyanate-conjugated anti-IFN- DŽ antibody). Samples can then be assessed by flow cytometry, with fluorescence above that of the isotype control indicative of the antigen specific activation of the cells. The co-staining with CD8 or CD4 and the IFN-Υ will attribute the antigen specific expression of IFN-Υ to either CD8+ or CD4+ T cells. Alternatively, or additionally, an immune response can be tested by detecting the expression of T cell-surface receptors or receptor ligands, typically after re-stimulation of T cells with APCs. For example, cell surface CD40 ligand expression can be assessed on CD4+ T cells, as described in Hegazy et al (2017) Gastroenterology 153: 1320- 1337. The % of CD4+ T-cells expressing CD40L (CD154) following defined antigen stimulation may be determined, and non-parametric analyses performed between experimental and control groups to identify any difference in the population average antigen-specific T-cell percentage. A positive result for the test condition would be indicated by a higher percentage antigen-specific (i.e., CD40L upregulated) CD4+ T- cells versus negative control group, for example at least 1% higher, at least 2% higher, at least 5% higher and/or up to 10% higher or more. Cytotoxicity of CTL responses may be assessed using a chromium-51 (51Cr) release assay (see B. Paige Lawrence, 2004, Current Protocols in Toxicology, 22(1):18.6.1- 18.6.27). For example, target cells expressing an antigen of interest for CTLs (e.g. cancer cells expressing a tumour antigen) may be labelled with 51Cr, which is released from the target cells upon cytolysis. Accordingly, the cytotoxicity of CTLs derived from vaccinated subjects (which would be expected to be able to mount an antigen specific response) may be compared with CTLs derived from a control, naïve subject (which would not be expected to have antigen specific CTLs). An increase in 51Cr detection for CTLs derived from a vaccinated group would indicate a positive result for inducing an antigen specific response. Typically, a positive result for a test group is indicated where the mean is at least two standard deviations higher than the mean for a control group. Another suitable assay for assessing cytotoxicity is the CyQUANT LDH Cytotoxicity Assay. Lactase dehydrogenase (LDH) is a cytosolic enzyme that is released upon damage to the plasma membrane. Accordingly, LDH levels can be tested in a coculture of CTLs and target cells, using the same conditions as described for the 51Cr release assay, to identify whether the vaccinated group has higher LDH indicative of increased cytotoxicity compared with the control group. Typically, a positive result for a test group is indicated where the mean is at least two standard deviations higher than the mean for a control group. Alternatively, or additionally, T cell proliferation can be tested using 3H thymidine. 3H is incorporated into new strands of chromosomal DNA during mitotic cell division, and so accumulates intracellularly as cells divide. T cells (or subsets of T cells) isolated from vaccinated subjects may be compared with T cells (or subsets of T cells) isolated from control, naïve subjects. Isolated T cells can be cocultured with PBMCs or activated DCs loaded with antigen in a mixed lymphocyte reaction (MLR), and their proliferation assessed over time. If the vaccination regime results in antigen specific T cells, these would proliferate at a higher rate when cocultured with antigen presenting cells expressing said antigen. Accordingly, an increase in T cell proliferation based on a higher amount of 3H thymidine incorporation is indicative of a positive finding for vaccinated subjects. Typically, a positive result for a test group is indicated where the mean is at least two standard deviations higher than the mean for a control group. Another suitable assay for assessing cellular proliferation is the carboxyfluorescein succinimidyl ester (CFSE) assay. CFSE is a fluorescent cell staining dye that reacts with intracellular free amines to generate covalent dye-protein conjugates. This results in live cells that can be detected based on the CFSE fluorescence by flow cytometry or fluorescent microscopy. As cells with CFSE divide, the level of CFSE fluorescence divides between the cells, allowing the visualisation of peaks corresponding to generations of cellular division. Accordingly, the same conditions as for 3H thymidine described above can be assessed in a CFSE assay. In this assay, an increase in T cell proliferation is based on the detection of additional emission peaks for fluorescein, which would indicate cell division as a positive finding for vaccinated subjects. Typically, a positive result for a test group is indicated where the mean is at least two standard deviations higher than the mean for a control group. B cell responses may be assessed by quantifying the levels of antibodies in sera or other appropriate samples collected during the immunisation regimen or following termination. An antibody titre is a measurement of how much antibody an organism has produced that recognizes a particular epitope, expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result. Antibody titre may be tested using ELISA. Therefore, sera obtained from mice subjected to the above-described immunisation regimen can be assessed for antibody titre and compared with the same controls. A higher antibody titre, such as at least two standard deviations higher compared with the negative control would be indicative of B cell activation in an antigen specific manner. In some embodiments, vaccination may result in incremental increases, such as 2-fold to 200-fold (e.g., 20- or 200-fold), in antigen-specific antibodies, relative to an unimmunised control. In immunisation strategies against viruses, such as HRV or SARS-CoV-2, neutralizing antibody responses are preferred. Neutralizing antibodies are those antibodies which can inhibit or block a key component of the viral replication cycle. Viral replication may thereby be lessened and/or prevented. A neutralization titer may typically be expressed as the highest serum dilution required to achieve a 50% reduction in the number of virus plaque forming unit, pfu. In some embodiments, vaccination may result in incremental increases, such as 2-fold to 200-fold (e.g., 20- or 200-fold), in neutralizing antibodies against the virus, relative to an unimmunised control. For example, the increase in neutralizing antibody titre compared to an unimmunised control may be between 10-fold and 200-fold, such as about 50-fold, about 100-fold or about 200-fold. Exemplary assays for detecting and/or quantifying neutralising HRV or SARS-CoV-2 antibodies are disclosed in Morita et al., 1988, J Gen. Virol., 69(2):451- 458 (DOI: 10.1099/0022-1317-69-2-451), Padilla-Noriega et al., 1995, Virology, 206(1):148-154 (DOI: 10.1016/s0042-6822(95)80029-8), Xue et al., 2016, Hum. Vaccin. Immunother., 12(11):2959-2968 (DOI: 10.1080/21645515.2016.1204501), Dogan et al., 2021, Commun. Biol., 4:129 (DOI: 10.1038/s42003-021-01649-6). The IgA antibody titre is indicative of mucosal immunity, and so the levels of antigen- specific IgA may specifically be tested to assess the induction of mucosal immunity. Suitable samples for testing for IgA include sera, faeces, contents of the colon or gut, or ileal wall extract. Additionally, or alternatively, the antigen-specific IgG titre, which is indicative of systemic immunity and/or antigen-specific IgM may be tested. Typically, samples of faeces or sera will be tested. The ratio of antigen-specific to total IgA may be measured, and may be indicative of a B cell response. For example, total IgA and antigen-specific IgA may be determined by ELISA. Non-parametric analyses may be performed between experimental and control groups to look for a difference in the population average antigen-specific IgA/IgA ratio. A positive result would be the identification of a statistically significant difference in the average antigen-specific IgA/IgA ratio between experimental and control groups. Generally accepted animal models (such as those described in Ireson et al. (2019) British J Cancer 121: 101-108) can be used for testing of immunisation against cancer using a tumour or cancer antigen. For example, cancer cells (human or murine) can be introduced into a mouse to create a tumour, and a bacterium comprising a tumour antigen as described herein may be delivered to a subject harbouring a tumour associated with said antigen. Cancer cells can be introduced by subcutaneous injection to form a xenograft or syngeneic tumour associated with an antigen of interest. The effect on the cancer cells (e.g., reduction of tumour size or reduction in tumour progression (i.e., the rate at which a tumour continues to grow), which can be measured using callipers) can be assessed as a measure of the effectiveness of the immunisation. More complex models include the use of patient-derived xenograft (PDX) models, in which an antigen associated with the cancer of said patient is implanted into mice (e.g. humanised mice) that have undergone an immunisation regimen as described herein. Suitable mouse models are disclosed, for example, in Zottnick et al., "Inducing Immunity Where It Matters: Orthotopic HPV Tumor Models and Therapeutic Vaccinations", Front. Immunol., 11:1750. Alternatively, or additionally, the levels and activity of anti-tumour CTLs may be tested, for example taking a tumour biopsy and testing the levels of CTLs (including tumour antigen specific CTLs) in the tumour microenvironment. Antigen specific CTLs may be identified using MHC tetramers specific to the MHC-loaded tumour antigen, and CTLs in the tumour microenvironment can then be quantified, for example by flow cytometry. A biopsy may also be tested for cytokines, by measuring those associated with an inflammatory response and T cell activation (e.g. IL-2, IFN-Υ, GM-CSF). The tests also can be performed in humans, where the end point is to test for the presence of enhanced levels of circulating cytotoxic T lymphocytes against cells bearing the antigen, to test for levels of circulating antibodies against the antigen, to test for the presence of cells expressing the antigen and so forth. A suitable test is described in Cai et al., 2017, which demonstrated that immunisation with ROP-survivin or ROP-HPV-E7 generated specific cellular immune responses and protected mice from inoculation with melanoma B16 cells expressing survivin or HPV- E7 proteins. In these experiments, C57BL/10 mice were primed subcutaneously with ROP-antigen (ROP-survivin or ROP-HPV-E7), which was compared with the wildtype antigens as a positive control, both conditions having the antigen emulsified in monophosphoryl lipid A (MPL). Immunisation was boosted subcutaneously twice at 3- week intervals with the same vaccine emulsified with MPL. Three weeks following the final boost, mice were challenged with B16-E7 or B16-survivin and subsequently assessed in ELISPOT assays. ELISPOT assays were performed on PBMCs and splenocytes, as described above, with re-stimulation performed with ROP-HPV or ROP- survivin in anti-IFN-Υ-Ab precoated plates. The data in Cai et al., 2017 demonstrate a mouse system where ROP-antigen and wildtype antigen can be used to immunise mice for anti-tumour immune responses. Therefore, the immunisation strategy of Cai et al., 2017 can be deployed as a positive control. In a test of IFN-Υ secretion, an SFU (Spot Forming Unit) count for a test condition (e.g. a vaccinated group) that is at least two standard deviations higher than the average of a control group would indicate a positive result for the test condition. Accordingly, the skilled person can readily assess whether a bacterium of the class Clostridia encoding an antigen, such as an infectious agent antigen and/or a tumour antigen induces an immune response to said antigen. These systems are not limited to the type of antigen nor the bacterium. Therapeutic or preventive treatment of an infectious disease or cancer in a subject In this sixth aspect, the antigen is an infectious agent antigen and the disease is the disease caused by the infectious agent, or the antigen is a tumour antigen and the disease is cancer. Suitable antigens are as described in relation to the first aspect of the invention. In the eight aspect, the antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2; and the disease is COVID-19 disease. Suitable antigens are as described in relation to the second aspect of the invention. By “ameliorating” or “treating” a disease, particularly cancer, we mean slowing, arresting or reducing the development of the disease or at least one of the clinical symptoms thereof; alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient; modulating the disease, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both; or preventing or delaying the onset or development or progression of the disease or disorder or a clinical symptom thereof. In the case of an infectious disease “ameliorating” or “treating” may be interpreted accordingly and may also include reducing the burden of viable infectious agent in the subject, or preventing or reducing the recurrence of dormant infectious agents into actively growing forms. “Therapeutic treatment” is to be interpreted accordingly. By “preventing”, we include that the agents described herein are prophylactic. A preventative or prophylactic use or treatment includes a use or treatment that reduces or removes the risk of a subject contracting a disease or being infected by an infectious agent, or which reduces the risk of the subject contracting a severe form of the disease, or which reduces the severity of the disease in the event that the subject does contract the disease, for example by vaccination. “Preventive treatment” is to be interpreted accordingly. A subject is in need of a treatment if the subject would benefit biologically, medically or in quality of life from such treatment. Treatment will typically be carried out by a physician or a veterinary surgeon who will administer a therapeutically effective amount of the bacterium or composition. A therapeutically effective amount of bacterium according to the first aspect or second aspect; or composition according to the third aspect refers to an amount that will be effective for the treatments described herein, for example slowing, arresting, reducing or preventing the disease or symptom thereof. The therapeutically effective amount may depend on the antigen (e.g. the capability of the antigen to provoke a particular type or strength of immune response thereto), the efficiency of production of the antigen by the clostridial cell, the subject being treated, the severity and type of the affliction etc. Typically, a subject in need of therapeutic treatment is presenting symptoms of the disease. Alternatively, a subject may be susceptible to the disease or has been tested positive for the disease but has not yet shown symptoms or is asymptomatic. Typically, a subject in need of preventive treatment does not have the disease but may be at risk of developing it. Preventive treatment is particularly appropriate for infectious disease. The infectious disease to be treated is suitably one which may respond to an antigen specific immune response directed at the infectious agent. The infectious disease, disorder or condition can be selected from those associated with the infectious agent antigens listed herein. The cancer to be treated can be any cancer associated with a tumour antigen, such as those tumour antigens listed herein, particularly a cancer that has been shown to respond to immunotherapy utilising the tumour antigen. Preventive treatment is particularly advantageous in relation to infectious agents which cause acute disease, although it may also be used in relation to infectious agents which cause chronic diseases. Acute infections may include gastroenteritis, such as caused by a virus such as a Human Rotavirus; a bacterium such as Vibrio cholerae, Campylobacter jejuni, Escherichia coli (including but not limited to ETEC, EHEC, EIEC, EPEC, EAEC, and AIEC), Salmonella sp. (including but not limited to S. enteria and subspecies including S. e. enterica, S. e. salamae, S. e. arizonae, S. e. diarizonae, S. e. houtenae, S. e. indica, S. enterica serovar Typhi, S. enterica serovar Typhimurium, S. enterica serovar Paratyphi; and S. bongori); Shigella sp. (including S. flexneri, S. sonnei, S. boydii, and S. dysenteriae), Clostridium difficile, or Clostridium perfringens; or a protozoan such as Cryptosporidium parvum, Giardia duodenalis, or Entamoeba histolytica. Acute infections may include respiratory tract infections, such as caused by a virus such as a coronavirus, such as SARS-CoV-2; or respiratory syncytial virus (RSV), influenza A or B, or human parainfluenza virus (HPIV). SARS-CoV-2 causes COVID-19 disease, which may be classified as asymptomatic or presymptomatic infection (individuals who test positive for SARS-CoV-2 using a virologic test but who have no symptoms that are consistent with COVID-19); mild illness (individuals who have any of the various signs and symptoms of COVID-19 e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell, but who do not have shortness of breath, dyspnea, or abnormal chest imaging); moderate illness (individuals who show evidence of lower respiratory disease during clinical assessment or imaging and who have an oxygen saturation (SpO2) ^94% on room air at sea level); severe illness (individuals who have SpO2 <94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) <300 mm Hg, a respiratory rate >30 breaths/min, or lung infiltrates >50%); or critical illness (individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction), as defined in https://www.covid19treatmentguidelines.nih.gov/overview/clinical-spectrum/. Preventive treatment of COVID-19 disease is intended to cover reduction in the severity of COVID-19 illness, such as a lower proportion of infected individuals experiencing severe illness or death; or reduction in the proportion of individuals who go on to develop symptomatic or asymptomatic infection. Thus, preventive treatment of COVID-19 disease is also intended to mean preventive treatment of SARS-CoV-2 infection. Therapeutic treatment is particularly advantageous in relation to cancer, or chronic infectious diseases. Chronic infectious diseases include those that are perpetuated for months or years by the infectious agent, or which exhibit periods of active growth of the infectious agent and/or symptoms, and periods of dormancy. Chronic persistent infection may be caused by viruses including human papillomavirus (HPV); hepatitis C; hepatitis B; human immunodeficiency virus (HIV); herpesviruses including herpes simplex virus 1, herpes simplex virus 2 and varicella zoster virus; flavivirus associated with Yellow fever; West Nile virus; dengue virus; Zika virus; Japanese encephalitis virus; African swine fever virus; Porcine Reproductive and Respiratory Syndrome (PRRS) virus and foot-and-mouth disease virus (e.g. coxsackievirus A16). SARS-CoV- 2 may cause persistent infection, such as in immunocompromised subjects (Nakajima Y et al. Prolonged viral shedding of SARS-CoV-2 in an immunocompromised patient. J Infect Chemother. 2021 Feb;27(2):387-389. doi: 10.1016/j.jiac.2020.12.001). The duration of viral shedding of SARS-CoV-2 in acute COVID-19 disease is usually less than 10 days. Therapeutic treatment of COVID-19 disease is intended to encompass treatment of patients who have already contracted SARS-CoV-2 infection, and continue to shed SARS-CoV-2 for longer than is typical in acute SARS-CoV-2 infection, e.g. longer than 10 days, or are at risk of such prolonged infection, e.g. by virtue of being immunocompromised. Chronic persistent infection may be also caused by bacteria, including Mycobacterium tuberculosis, Mycobacterium bovis, Brucella, Borrelia species such as B. burgdorferi, Corynebacterium diphtheriae, Chlamydia, Vibrio cholerae, Salmonella enterica serovar Typhi; mycoplasma; fungi including Candida albicans; and various parasites including helminths and protozoa. Suitable cancers to be treated include melanoma and renal cell carcinoma, which are considered to be two of the most immunogenic solid tumours and have been studied extensively in vaccine development or cancers of the colon, lung, cervix, pancreas, stomach, liver, intestine, bladder, ovary, prostate, bone, brain, or head and neck. Preventive treatment typically requires the establishment of immunological memory, such that the immunised subject is protected or partially protected from subsequent challenge, typically with the infectious agent antigen. Immunological memory is an important consequence of adaptive immunity, as it enables a more rapid immune response to be mounted to pathogens that have been previously encountered to prevent them from contracting a disease. Immunological memory may also be important in therapeutic treatments. Immunological memory in T cells can be tested using MHC tetramers that identify whether memory T cells exist for a particular antigen. MHC tetramers have specificity to MHC-loaded antigen, and so an MHC tetramer can be used that is specific to an antigen of interest. These can be used on samples isolated from a subject (e.g. a blood sample or splenocytes) to measure the frequency of antigen specific T cells. MHC tetramers are available for MHC class I and II, meaning that both CD4+ and CD8+ cells can be measured using MHC tetramers. Furthermore, the MHC tetramers can be used in conjunction with fluorescent antibodies for other T cell markers to assess the proportion of antigen specific T cell subsets (e.g. antigen specific Th1, Th2 and/or Th17 cells). The proportion of antigen specific T cells can be assessed by flow cytometry, comparing immunised and non-immunised subjects. For example, samples obtained from mice that have undergone the immunisation regimen described herein may have blood samples and/or splenocytes assessed for MHC tetramer binding and a panel of fluorescent markers for T cell subsets. Compared with non-immunised mice, the immunised mice should have a higher proportion of cells that can be bound with an MHC tetramer, and thus identified as antigen-specific T cells, which can be further assessed by T cell subset to identify the type of T cell response induced. For example, a positive result for a test condition may be indicated by a higher percentage antigen- specific (i.e., CD40L upregulated) CD4+ T-cells versus negative control group, or the higher presence of the MHC tetramer reagent on the cell surface compared to negative control groups. Suitable MHC tetramers may be generated using the methods disclosed in Ramachandiran et al., 2007, J. Immunol. Methods, 319:1-2):13-20 (DOI: 10.1016/j.jim.2006.08.014). Exemplary MHC tetramers for detecting SARS-CoV-2- specific T cells are disclosed in Poluektov et al., 2021, Vaccine, 39(15):2110-2116 (DOI: 10.1016/j.vaccine.2021.03.008). Immunological memory in B cells can be tested in vitro by isolating B cells from immunised and non-immunised mice (e.g. as per the immunisation regimen described herein) and re-stimulating the B cells in the presence of helper T cells specific for the same antigen. B cells from immunised mice respond both quantitatively and qualitatively better, the former of which can be assessed by comparing the frequency of B cells (i.e. count the number of cells in a cell suspension) following re-stimulation. Due to affinity maturation of B cells, the memory B cell antibodies produced would also have a higher affinity compared with naïve B cells from non-immunised mice, which can be tested by purifying the produced antibodies (from immunised and non- immunised mice) and comparing their affinity for the antigen (or epitope thereof). If the antibodies produced from the immunised mice have a higher affinity, then a B cell memory response has been established that may indicate protective immunity. Corresponding in vivo studies would use such mice and challenge them with the pathogen from which the antigen is derived (e.g. infection with HPV if the antigen is an HPV antigen, for example, as described in Longet et al, 2011, Journal of Virology, 85:13253-13259; infection with HRV if the antigen is an HRV antigen such as VP8, for example as described in Lappalainen et al., 2015, Arch. Virol., 160(8):2075-2078 (DOI: 10.1007/s00705-015-2461-8); and infection with SARS-CoV-2 if the antigen is a SARS-CoV-2 antigen, for example as described in Amanat et al., 2021, PLoS Biology, 19(1):e3001384 (DOI: 10.1371/journal.pbio.3001384)) to assess infection burden compared with mice challenged for the first time with the pathogen. Suitable challenge models also include, for example, the Syrian Hamster challenge model disclosed in Johnson et al., 2022, J. Infect. Dis., 225(1):34-41 (DOI: 10.1093/infdis/jiab561). The cellular systems described above may be supplemented with in vivo mouse systems, wherein the mice are challenged with the pathogen associated with the antigen. Due to the existence of T and/or B cell immunity, immunised mice should have reduced infection burden, such as increased rates of partial or complete protection from infection compared with naïve mice. Accordingly, a suitable in vivo system would include a challenge regimen following the immunisation regimen to assess infection burden. Suitable animal models are described in Bakaletz (2004) Developing animal models for polymicrobial diseases, Nature Reviews Microbiology, 2:552-568). Immunological memory may also be tested in in vivo tumour models, including tumour challenge models, such as described in Cai et al., 2017, supra and Ireson et al., 2019, supra as described in relation to the fifth aspect of the invention. In this specification and the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. All publications mentioned herein are incorporated herein by reference to the fullest extent possible for the purpose of describing and disclosing those components that are described in the publications which might be used in connection with the presently described invention. The present invention will be further illustrated in the following non-limiting Examples and Figures. Figure Legends Figure 1: Alignment to demonstrate presence of 50-75 peptide in full CtxB protein (P01556) (a) and in truncated CtxB protein, i.e. 50-75 alignment (b), single amino acid exchange noted by # Figure 2: LysM-CtxB50-75 nucleic acid sequence information showing 5’ upstream LysM domain including signal peptide, 3’ downstream FLAG tag and stop codon (SEQ ID NO: 31). LysM-CtxB50-75- amino acid sequence is shown as SEQ ID NO: 30 and the LysM amino acid sequence as SEQ ID NO: 42. NdeI (5’) and NheI (3’) restriction sites flank the coding region. Figure 3: Western blot detection of FLAG-tag linked to Usp45-CtxB (12.7kDa). The blot shows plasmid based Usp45-CtxB-FLAG expression (denoted “F” or “CtxB-Full”) at OD1.0 and OD 4.0 time points. CHN-0 wild-type (WT) was used as the negative control. Bands demonstrating expressed Usp45-CtxB-FLAG can be seen in both whole cell lysate (WCL) and supernatant (S/N) samples, for both time points (arrow). A maximum of 20μL protein was loaded and assessed via SDS-PAGE and Western blot, and anti- FLAG (A9469) was used to probe for the FLAG-tagged protein and stain was developed using SIGMAFAST BCIP/NBT (3 min). Figure 4: Western blot detection of FLAG-tag linked to LysM-CtxB50-75- (27 kDa) expressed by C. butyricum engineered to express antigens associated with the cell wall. CHN-0 wild-type (WT) was used as the negative control. (A) Whole cell lysates of CADD + pMTL82151-pfdx-LysM-CtxB50-75-FLAG, CADD + pMTL82151-pfdx-LysM-FLAG and WT CHN-0. Samples were removed from the growth culture at the OD600 time points indicated on the blot. (B) Whole cell lysates from samples of triplicate CADD + pMTL82151-pfdx-LysM-CtxB50-75--FLAG grown to OD6001.0 and assessed against FLAG- BAP™ standards (1 μg – 50 ng). (C) Whole cell lysates of CADD + LysM-CtxB50-75- FLAG (integrated), CADD + LysM-FLAG (integrated) and WT CHN-0 as a control. Samples were removed from the growth culture at the OD600 time points indicated on the blot. For all gels, 30 μl protein loaded, blots blocked 5% milk, anti-FLAG (A9469) 1:5000 in TBS-T 2 h, developed using SIGMAFAST BCIP/NBT 20 min. Figure 5: Western blot detection of FLAG-tag linked to LysM-CtxB50-75- (27 kDa) and LysM only (24 kDa) located in the cell wall fraction. CHN-0 wild-type was used as the control. Bands demonstrating expressed LysM (light arrow), and LysM-CtxB50-75- (dark arrow) can be seen in both the cell wall fractions and the protoplast fractions once separated, demonstrating extracellular localisation of the antigenic peptide and intracellular protein production respectively. A doublet of banding appears in the protoplast due to pre- and processed protein expression, i.e. cleavage of the signal peptide. This is not observed in the cell wall fraction which has been processed for extracellular localisation and association with the peptidoglycan layer. 30 μl protein was loaded, blocked 5% milk, anti-FLAG (A9469) 1:5000 in TBS-T 2 h, developed using SIGMAFAST BCIP/NBT 20 min. Figure 6: High resolution fluorescence microscopy to demonstrate extracellular location of the CtxB50-75-FLAG antigen. Brightfield (bottom) images were taken to identify bacteria, before fluorescently labelled FLAG images (top) were taken to show the LysM-CtxB50-75--FLAG antigen on the cell surface of the CHN-0 cells. The –ve control shows bacteria that have not been exposed to the anti-FLAG antibody to detect any background staining or auto-fluorescence. CHN-0 wild-type was also used as a control to demonstrate any non-specific binding of the anti-FLAG antibody. An increased level of puncta is observed in the LysM-CtxB50-75--FLAG image which demonstrates localisation of the antigen to the cell surface. Figure 7: Nucleic and amino acid sequences of Usp45-VP8. The Usp45 amino acid sequence is shown (SEQ ID NO: 41). Usp45-VP8 nucleic acid sequence (SEQ ID NO: 26) consists of the Usp45 secretion signal followed by VP8 sequence, followed by a FLAG tag and stop codon. NdeI (5’) and NheI (3’) restriction sites flank the coding region. Figure 8: Nucleic and amino acid sequences of Usp45-VP7 polyepitope. The nucleic acid sequence is shown as SEQ ID NO: 76 and consists of Usp45 secretion signal followed by VP7 polyepitope, followed by FLAG tag and a stop codon. NdeI (5’) and NheI (3’) restriction sites flank the coding region. Usp45-VP7 polyepitopes are separated by flexible [Gly]4Ser linkers, shown in SEQ ID NO: 77. Figure 9: Western blot detection of FLAG-tag linked to the HRV-VP8 (20 kDa) antigen secreted by engineered C. butyricum. CHN-0 wild-type was used as the control. FLAG- BAP™ standards used at a known concentration, with test samples of HRV-VP8 antigen TCA precipitated from the equivalent of 10 mL culture supernatant, at OD6001.0, 2.0 and 4.0 in the growth phase. A 10 mL sample of wild-type CHN-0 grown to OD6002.0 was used as a control. 40 μl protein loaded, blots blocked 5% milk, anti-FLAG (A9469) 1:5000 in TBS-T 2 h, developed using SIGMAFAST BCIP/NBT 4.5 - 5 min. Figure 10: Western blot detection of FLAG tag linked to HPV-VP8 in C. butyricum engineered to secrete HRV proteins either from an integrated single gene copy (sample lanes 1-3) or from a multicopy plasmid (sample lanes 4-6) indicated by the arrow. CHN-0 wild-type was used as the control (sample lanes 7-9). 40 μl protein loaded, blots blocked 5% milk, anti-FLAG (A9469) 1:5000 in TBS-T 2 h, developed using SIGMAFAST BCIP/NBT 2.5 min. Figure 11: Nucleic and amino acid sequences of LysM-Cov-2-RBD, LysM-CoV-2_Nuc, LysM-CoV- 2-Comb, LysM-CoV-2-SB1 and LysM-CoV-2-SB2. All nucleic acid sequences in this figure (SEQ ID NOs: 18, 19, 20, 21, 22) consist of the LysM peptidoglycan association domain, followed by a flexible [Gly]4Ser linker, followed by the LysM-CoV-2-XX sequence, followed by a 6X His tag and stop codon. All sequences in this figure are flanked by 5’ NdeI and 3’ NheI restriction sites. Amino acid sequences (SEQ ID NOs: 7, 9, 11, 13, 17) are shown with LysM peptidoglycan association domain highlighted in bold. LysM-CoV-2-Nuc amino acid sequence (SEQ ID NO: 13) and LysM-CoV-2-Comb amino acid sequence (SEQ ID NO: 17) contain multiple epitopes with predicted antigenicity, separated by cathepsin cleavage sites (underlined). Figure 12: Nucleic and amino acid sequences of intracellular CoV-2-XX antigens. All compositions are identical to the LysM-CoV-2-XX antigens shown in Figure 11, but do not contain a LysM peptidoglycan anchoring domain or adjacent [Gly]4Ser linker. As for LysM-CoV- 2-Nuc and LysM-CoV-2-Comb, amino acid sequences of CoV-2-Nuc (SEQ ID NO: 131) and CoV-2-Comb (SEQ ID NO: 133) contain multiple epitopes with predicted antigenicity separated by cathepsin cleavage sites (underlined). Figure 13: Western blot detection of 6XHis tag linked to LysM-SARS-CoV-2 antigens. (A) RBD, 47kDa (B) Nucleocapsid, 56 kDa (C) Combined antigen, 54 kDa (D) spike B cell epitopes 1 (SB1), 38 kDa (E) spike B cell epitopes 2 (SB2), 31 kDa. All antigens in C. butyricum engineered to express antigens located extracellularly via association with the peptidoglycan cell wall. CHNR-0 wild-type was used as the control. Gels had 40 μl protein loaded, blots blocked 5% milk, anti-6XHis (1:1000) in TBS-T 1 h, anti- mouse IgG (1:5000) in TBS-T 1h, developed using SIGMAFAST BCIP/NBT for 2-6 min. Dark arrows indicate expected MW size, whereas light arrows indicate the possible cleavage between the LysM and the antigen, resulting in detection of the antigen alone (as estimated from the MW band size). Figure 14: Western blot detection of 6XHis tag linked to LysM-SARS-CoV-2- Nucleocapsid-His antigen in the supernatant of a culture of C. butyricum engineered to express the antigens located extracellularly via association with the peptidoglycan cell wall. CHNR-0 wild-type was used as the control. Either whole cell lysates (2 mL of culture at OD6001.0 pelleted) or proteins extracted from 10 mL culture at OD6001.0 using TCA precipitation were assessed. Gels had 40 μl protein loaded, blots blocked 5% milk, anti-6XHis (1:1000) in TBS-T 1 h, anti-mouse IgG (1:5000) in TBS-T 1h, developed using SIGMAFAST BCIP/NBT for 6 min. Uncleaved LysM-Nucleocapsid antigen expected at 56 kDa, however, significant cleavage and release into the extracellular environment (supernatant) was observed. Figure 15: Nucleic and amino acid sequences of C. jejuni antigens, FlaA and FliD, for extracellular expression via the LysM domain (SEQ ID: 42). Amino acid sequences of LysM-FlaA recombinant (SEQ ID NO: 114) and LysM-FliD recombinant (SEQ ID NO: 116) contain sequences for expression of the recombinant protein downstream of the LysM protein separated by a flexible [Gly]4Ser linker, whereas LysM-FlaA polyepitope (SEQ ID NO: 126) and LysM-FliD polyepitope (SEQ ID NO: 127) contain multiple epitopes with predicted antigenicity, also downstream of the LysM protein separated by a flexible [Gly]4Ser linker. All nucleic acid sequences in this figure (SEQ ID NOs: 138, 139, 140, and 141) consist of the LysM peptidoglycan association domain (in bold), followed by a flexible [Gly]4Ser linker, followed by the LysM-C. jejuni antigenic sequence, followed by a 6X His tag and stop codon (underlined). All sequences in this figure are flanked by 5’ NdeI and 3’ NheI restriction sites (in bold underlined italics). Examples Example 1: Immunisation of mice using an extracellular CtxB peptide antigen and a secreted CtxB recombinant antigen expressed by Clostridium The Cholera enterotoxin subunit B (CtxB) is a 13 kDa subunit protein that makes up the pentameric ring of the Cholera enterotoxin of Vibrio cholerae. Together with the A subunit, it forms the holotoxin (choleragen). The holotoxin consists of a pentameric ring of B subunits whose central pore is occupied by the A subunit. The A subunit contains two chains, A1 and A2, linked by a disulfide bridge. The B subunit pentameric ring directs the A subunit to its target by binding to the GM1 gangliosides present on the surface of the intestinal epithelial cells. It can bind five GM1 gangliosides. It has no toxic activity by itself. Secreted expression of the CtxB protein involves expression of a truncated CtxB protein coupled to the Usp45 secretion signal. The CtxB protein has been shown to provide adjuvant activities in many vaccines - Stratmann, T. Cholera Toxin Subunit B as Adjuvant–An Accelerator in Protective Immunity and a Break in Autoimmunity. Vaccines 2015, 3,579-596. https://doi.org/10.3390/vaccines3030579. Extracellular expression of the CtxB protein involves the expression of a shorter 25 amino acid peptide (4 kDa) found within the CtxB protein, coupled to cell wall- associated moiety known as LysM. The immunogenicity of the shorter 25 amino acid peptide (4 kDa) from CtxB has previously been demonstrated by Guyon-Gruaz et al., 1986, Eur. J. Biochem., 159:525-528. The shorter 25 amino acid peptide (4 kDa) also has an almost 100% homology (one amino acid substitution of N>D) to an Enterotoxigenic Escherichia coli toxin, the heat-labile (LT) toxin, and hence may provide cross protection against V. cholerae infection and ETEC infection. The LysM protein domain is a ubiquitous protein domain found in a wide variety of extracellular proteins and receptors, and anchors proteins to the cell surface through non-covalent attachment to the peptidoglycans found in the cell wall. Gene constructs and plasmids For the secreted CADD-Usp45-CtxB oral vaccine development the truncated amino acid sequence was determined from UniProtKB submission P01556 with the isolation of amino acids 22-124 (SEQ ID 29). An addition of a C-terminal FLAG tag (DYKDDDDK (SEQ ID NO: 46)) was made, alongside a sequence encoding the Usp45 secretion signal (SEQ ID NO: 41) and cleavage site (SEQ ID NO: 63), which was incorporated 5’ upstream of the CtxB22-124 genetic element. Further modifications included for genetic engineering include an NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA (i.e. in the order 5’- FLAG-TAA-NheI-3’). The Usp45-CtxB-FLAG construct was codon optimised for genetic engineering into C. butyricum and synthesised by GeneWiz (Azenta), who cloned into an intermediate pUC-GW-Amp vector CHAIN Biotechnology completed vector assembly by restriction digest of the construct between NheI and NdeI sites and ligation into the pMTL82121_p0957 empty vector. The plasmid was transformed into Thermo Fisher DH5Į Competent E. coli for propagation and storage, with colonies positive for plasmid uptake determined using LB agar + 25 μg/mL chloramphenicol for selection. Plasmids were isolated as before and sequenced using GeneWiz sequencing services using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers (Table 1) to confirm the correct insertion of cassettes. The nucleic acid sequence of the p0957-Usp45-CtxB-FLAG construct from the p0957 promoter to the stop codon is SEQ ID NO: 206. The polypeptide sequence is SEQ ID NO: 205. Following sequence confirmation in E. coli DH5Į, cells were grown overnight in LB supplemented with 12.5 μg/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Sequence confirmed plasmid pMTL82121_p0957-Usp45-CtxB-FLAG was then transformed into E. coli CA434 conjugation donor strain. Following sequence confirmation as above, E. coli CA434 were grown overnight in LB supplemented with 50 μg/mL kanamycin and 12.5 μg/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. For the cell wall-associated CADD-LysM-CtxB50-75- oral vaccine development (Figure 2), the 25 amino acid CtxB-encoding peptide sequence was determined from the UniProtKB submission P01556 with the isolation of the amino acids 71-96 (or 50-75 once the signal sequence has been discounted) with the exception of one amino acid exchange (Asp, D to Asn, N) at position 96 (or position 70 in the sequence without the signal sequence) as shown in Figure 1, and as described in Guyon-Gruaz et al supra. An addition of a C-terminal FLAG tag (DYKDDDDK (SEQ ID NO: 46)) was made, alongside a sequence encoding the peptidoglycan-binding moiety, LysM (SEQ ID NO: 42), which was incorporated 5’ upstream of the CtxB50-75 genetic element and with a flexible [Gly]4Ser linker separating the LysM-encoding gene from the CtxB50-75 sequence. Further modifications included for genetic engineering include an NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA (i.e. in the order 5’-FLAG-TAA-NheI-3’). The LysM- CtxB50-75-FLAG construct (SEQ ID NO: 31) was codon optimised for genetic engineering into C. butyricum and synthesised as a GeneArt String from ThermoFisher Scientific. Once received, the GeneArt String was cloned into a pCR™Blunt II-TOPO™ vector using the Zero Blunt™ TOPO™ PCR Cloning Kit (ThermoFisher Scientific) as per the manufacturer’s instructions. For cloning into the correct plasmid for plasmid-based expression within the cell wall of C. butyricum, the pCR™Blunt II-TOPO™ vector containing the LysM-CtxB50-75--FLAG construct was first transformed into NEB® 10-beta Competent E. coli cells (New England Biolabs) according to the manufacturer’s instructions and colonies were selected using LB Agar plates containing 50 μg/mL kanamycin. Colonies positive for the plasmid as determined by kanamycin resistance were selected and the plasmid was isolated using Wizard Plus SV Miniprep DNA Purification kit (Promega) following the manufacturer’s instructions and confirmed for correct insertion using primers CH478 (SEQ ID NO: 49) and CH479 (SEQ ID NO: 50) denoted in Table 1. The LysM-CtxB50-75-- FLAG construct was excised from the pCR™Blunt II-TOPO™ vector using restriction endonucleases NdeI and NheI in CutSmart® buffer (all New England Biolabs Inc) according to the manufacturer’s instructions. The isolated cassette was introduced into pMTL82151 (pBP1 Gram+ replicon, catP antibiotic marker, ColE1 Gram- replicon, traJ conjugal transfer function, and multiple cloning site (MCS)) already containing a pfdx (C. sporogenes ferrodoxin) promoter (SEQ ID NO: 183). The plasmid was transformed into NEB® 10-beta Competent E. coli for propagation and storage, with colonies positive for plasmid uptake determined using LB agar + 25 μg/mL chloramphenicol for selection. Plasmids were isolated as before and sequenced using GeneWiz sequencing services using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers (Table 1) to confirm the correct insertion of cassettes. Following sequence confirmation in E. coli NEB® 10-beta, cells were grown overnight in LB supplemented with 12.5μg/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Sequence confirmed plasmid pMTL82151_pfdx-LysM-CtxB50-75--FLAG was then transformed into E. coli CA434 conjugation donor strain. Following sequence confirmation as above, E. coli CA434 were grown overnight in LB supplemented with 50 μg/mL kanamycin and 12.5μg/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. For cloning into the correct plasmid for cell wall-associated expression from a single copy of the LysM-CtxB50-75--FLAG integrated into the genome, the LysM-CtxB50-75--FLAG construct was excised from the pMTL82151 vector using restriction endonucleases NotI and NheI in CutSmart® buffer (all New England Biolabs Inc) according to the manufacturer’s instructions. This excises the pfdx promoter alongside the LysM-CtxB50- 75-FLAG construct. The isolated cassette + promoter was introduced into pMTL83151 (pCB102 Gram+ replicon, catP antibiotic marker, ColE1 + tra Gram- replicon, traJ conjugal transfer function, and multiple cloning site (MCS)) already containing homologous arm sequences for the repair of the pyrE gene which is the site that is targeted for genetic insertion on the genome of the CHN-0.1 strain (ƩpyrE derivative of WT CHN-0). Plasmids were transformed into E. coli NEB® 10-beta for propagation. Plasmids were isolated as before and sequenced using GeneWiz sequencing services using CH22 (SEQ ID NO: 51) and CH648 (SEQ ID NO: 53) primers (Table 1) to confirm the correct insertion of cassettes. Sequence confirmed plasmids pMTL83151_pyrE repair_pfdx_LysM-CtxB50-75--FLAG were transformed into E. coli CA434 conjugation donors. Following sequence confirmation as above, E. coli CA434 were grown over night in LB supplemented with 50 μg/mL kanamycin and 12.5μg/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Genetic engineering of C. butyricum For introduction of the plasmid for plasmid based expression, fresh colonies of revived E. coli CA434 harbouring the pMTL82151-LysM-CtxB50-75-FLAG plasmid or pMTL82121- Usp45-CtxB-FLAG plasmid were used to inoculate LB broth supplemented with 50 μg/mL kanamycin and 12.5μg/mL chloramphenicol. After overnight incubation, cultures were used to inoculate fresh supplemented medium 1:10 and incubated until an OD600 of 0.5-0.7 was reached. A volume of 1 mL of culture was removed and centrifuged at 5,000×g for 3 minutes. The supernatant was discarded, and the pellet re-suspended in 500 μL phosphate buffered saline (PBS) solution. The culture was centrifuged as above, and the supernatant discarded. Fresh colonies of revived C. butyricum CHN-0 were used to inoculate a serial dilution series in fresh pre-reduced RCM broth supplemented with 2% glucose and 1% CaCO3. After overnight incubation in anoxic conditions, the most dilute culture showing growth was used to inoculate fresh supplemented medium 1:10 and incubated until an OD600 of 0.5-0.7 was reached. A volume of 1 mL of culture was removed and heat treated for 10min at 50°C. Both E. coli CA434 and C. butyricum CHN-0 such treated were transferred into the anoxic workstation and mixed at a ratio of 5:1 (OD600:OD600), usually 1 mL E. coli to 0.2 mL C. butyricum. The conjugation mixture was spotted onto pre-reduced non- selective RCM agar plates and incubated upright overnight. Following incubation, the mixture was harvested into 500 μL fresh pre-reduced RCM broth and spread in 100 μL volume onto fresh pre-reduced RCM agar plates supplemented with 250 μg/mL D- cycloserine and 15 μg/mL thiamphenicol. To select for C. butyricum CHN-0 carrying either plasmid, colonies that were thiamphenicol resistant were patch plated reiteratively onto RCM + 15 μg/mL thiamphenicol agar plates. Genomic DNA of thiamphenicol resistant colonies was isolated using the GenElute™ Bacterial Genomic DNA kit (SIGMA-Aldrich) as per the manufacturer’s instructions and used for sequencing to confirm presence of the pMTL82151_pfdx-LysM-CtxB50-75--FLAG plasmid or the pMTL82121-p0957-Usp45-CtxB-FLAG plasmid using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers spanning the MCS (Table 1). Table 1: Primers used for sequence confirmation for LysM-CtxB50-75-FLAG incorporation into pCR™Blunt II-TOPO™ vectors, and pMTL82151_pfdx-LysM-CtxB50-75--FLAG, pMTL82121-p0957-Usp45-CtxB-FLAG, and pMTL83151_pyrE repair_pfdx_LysM- CtxB50-75-FLAG plasmid generation. CH478 (SEQ ID F GTAAAACGACGGCCAG M13F NO: 49)
Figure imgf000079_0001
Figure imgf000080_0001
The introduction of the pMTL82151_pfdx-LysM-CtxB50-75-FLAG plasmid into C. butyricum CHN-0 leads to a high expression of the CtxB50-75-FLAG peptide by C. butyricum from a multicopy plasmid. Similarly, the introduction of the pMTL82121- p0957-Usp45-CtxB-FLAG plasmid into C. butyricum CHN-0 leads to a high expression and secretion of the CtxB-FLAG protein by C. butyricum from a multicopy plasmid into the culture supernatant. For integration of a single copy of the LysM-CtxB50-75-FLAG cassette into the genome, conjugation was carried out as above for pMTL82151_pfdx-LysM-CtxB50-75-FLAG plasmid introduction, except the base strain used was CHN-0.1 (ƩpyrE derivative of WT CHN-0), the plasmid was pMTL83151_pyrE repair_pfdx_LysM-CtxB50-75--FLAG and additional steps were included after selection on RCM agar plates supplemented with 250μg/mL D-cycloserine and 15μg/mL thiamphenicol. The additional steps include: colonies were then patch plated reiteratively onto Clostridium Basal Medium (CBM) agar plates and cross-checked for plasmid loss on thiamphenicol-containing selective RCM agar plates to select for mutants with restored uracil prototrophy. Genomic DNA of prototroph colonies that had lost the plasmid was isolated using the GenElute™ Bacterial Genomic DNA kit (SIGMA-Aldrich) as per the manufacturer’s instructions and used for sequencing to confirm presence of the LysM-CtxB50-75--FLAG cassette in the chromosome of C. butyricum using primers spanning the integration region, the promoter and respective gene construct sequence (Table 2). Table 2: Primers used for sequence confirmation of CADD-LysM-CtxB50-75- cassette integration.
Figure imgf000080_0002
The integration of the LysM-CtxB50-75--FLAG cassette into the chromosome introduced a single copy under the control of a constitutive promoter. This leads to a lower expression and production of protein which can be adjusted by use of stronger promoters and/or insertion of multiple copies of the gene. Confirmation of expression of Usp45-CtxB-FLAG in C. butyricum Fresh colonies of revived C. butyricum CHN-0 + pMTL82121_p0957Usp45-CtxB-FLAG were used to inoculate fresh pre-reduced supplemented RCM broth + 15 μg/mL thiamphenicol in serial dilution and grown overnight. The most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth + 15 μg/mL thiamphenicol at a starting OD600 of 0.05. When cultures were grown to an OD600 of 1.0 and 4.0 post initial inoculation (as indicated in Figure 3), the equivalent of OD600 of 10 was centrifuged at 9500×g for 6 min. Supernatants were separated from the pellets and filtered using a 0.22 μm syringe filter (Sartorius). Proteins precipitated by Trichloroacetic acid (TCA) precipitation whereby 100% TCA was added to a final volume of 10% to the supernatants. The 10% TCA-supernatant samples were incubated at - 20°C overnight (~20 h), before being thawed and centrifuged once again at 9,400 r.p.m for 30 minutes at 4°C. Supernatants were discarded and all residual TCA removed, before protein pellets were washed thrice in ice-cold acetone and dried. Dried supernatant protein pellets were then resuspended in 20 μL 5× SDS loading dye (20% (V/V) 0.5 Tris hydrochloride pH 6.8, 23% (V/V) Glycerol, 40% (V/V) of a 10% (w/V) Sodium dodecyl sulphate (SDS) solution, 10% (V/V) 2-Mercaptoethanol, 10 mL dH2O, Bromophenol blue) + 1-5 μL 1 M Tris/HCl, pH 7.4 buffer until the protein pellets turned blue, and heat treated at 98°C for 15 minutes. A maximum of 20 μL/well of the re-suspended pellets was loaded onto a NovexTM Wedgewell™ 16% Tricine 1.0 mm mini gel (ThermoFisher Scientific) and run in 1×Novex™ Tricine SDS Running Buffer (ThermoFisher Scientific) using 150 V at room temperature. Spectra™ Prestained Protein Ladder (ThermoFisher Scientific) was loaded at 5 μL/well as a marker. Separated proteins were blotted onto PVDF membranes using the Tran-Blot® TurboTM blotting system (BioRad) with the Trans-Blot® TurboTM packs as per the manufacturer’s instructions. To detect FLAG-tagged proteins, PVDF membranes were first incubated in TBS-T blocking buffer (50 mM Tris hydrochloride, 150 mM Sodium chloride, 0.1% Tween20, pH 7.4, 5% (w/V) milk powder) for 1 h at room temperature on a shaking platform. The membrane was then washed once for 5 min in TBS-T buffer (50 mM Tris hydrochloride, 150 mM Sodium chloride, 0.1% Tween20, pH 7.4), before TBS-T containing Anti-FLAG tag® antibody Alkaline phosphatase conjugate (1:5,000; Sigma) was added for incubation at room temperature for 2 h on a shaking platform. The membrane was washed twice for 5 min at room temperature in TBS-T buffer and once for 5 min at room temperature in TBS buffer (50mM Tris hydrochloride, 150 mM Sodium chloride, pH 7.4). Alkaline phosphatase detection was performed using SIGMAFAST BCIP®/NBT substrate (SIGMA Aldrich) as per the manufacturer’s instructions. Expression can be seen in Figure 3. For plasmid-based expression, the secreted CtxB-FLAG protein was detectable to high levels on a Western blot, with a reduction of protein observed at the later time point. Confirmation of expression of LysM-CtxB50-75-FLAG in C. butyricum Fresh colonies of revived C. butyricum CHN-0 + pMTL82151_pfdx-LysM-CtxB50-75--FLAG were used to inoculate fresh pre-reduced supplemented RCM broth + 15 μg/mL thiamphenicol in serial dilution and grown overnight. The most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth + 15 μg/mL thiamphenicol at a starting OD600 of 0.05. When cultures were grown to an OD600 of 0.5, 1.0, 2.0 or at 24 h post initial inoculation (as indicated in Figure 4 demonstrating protein expression), the equivalent of OD600 of 2/mL was centrifuged at 13,000×g for 2 min. Supernatants were discarded and the pellets prepared for SDS-PAGE analysis as below. The same method was repeated for integrated LysM-CtxB50-75--FLAG, except fresh colonies of revived C. butyricum CHN-0 + pfdx-LysM-CtxB50-75--FLAG were used to inoculate fresh pre-reduced supplemented CBM broth with no antibiotics. The pellets were re-suspended in 40 μL 5× SDS loading dye (20% (V/V) 0.5 Tris hydrochloride pH 6.8, 23% (V/V) Glycerol, 40% (V/V) of a 10% (w/V) Sodium dodecylsulphate (SDS) solution, 10% (V/V) 2-Mercaptoethanol, 10mL dH2O, Bromophenol blue) and heat treated at 98°C for 15 minutes. A maximum of 30 μL/well of the re-suspended pellets was loaded onto a NovexTM Wedgewell™ 14% Tris Glycine 1.0 mm mini gel (ThermoFisher Scientific) and run in 1×Novex™ Tris Glycine SDS Running Buffer (ThermoFisher Scientific) using 180V at room temperature. PageRuler™ Plus Prestained Protein Ladder (ThermoFisher Scientific) was loaded at 10 μL/well as a marker. Either the E. coli Positive Control Whole cell lysate ab5395 (abcam) was used as FLAG tag positive control in a 1:5 dilution or Carboxy-terminal FLAG-BAP™ Fusion Protein (FLAG-BAP™, Sigma Aldrich) standards were used, diluted to a range of concentrations from 1000 ng – 50 ng. Separated protein were blotted onto PVDF membranes using the Tran-Blot® TurboTM blotting system (BioRad) with the Trans-Blot® TurboTM packs as per the manufacturer’s instructions. To detect FLAG-tagged proteins, PVDF membranes were first incubated in TBS-T blocking buffer (50 mM Tris hydrochloride, 150 mM Sodium chloride, 0.1% Tween20, pH7.4, 5% (w/V) milk powder) for 1 h at room temperature on a shaking platform. The blocking buffer was then replaced by TBS-T buffer (50mM Tris hydrochloride, 150mM Sodium chloride, 0.1% Tween20, pH7.4) containing Anti-FLAG tag® antibody Alkaline phosphatase conjugate (1:5,000; Sigma) for incubation at room temperature for 2 h on a shaking platform. The membrane was washed twice for 5 min at room temperature in TBS-T buffer and once for 5 min at room temperature in TBS buffer (50mM Tris hydrochloride, 150 mM Sodium chloride, pH7.4). Alkaline phosphatase detection was performed using SIGMAFAST BCIP®/NBT substrate (SIGMA Aldrich) as per the manufacturer’s instructions. Expression can be seen in Figure 4A-C. For plasmid-based expression, the LysM-CtxB50-75--FLAG protein was detectable to high levels on a Western blot, corresponding to ~270 ng in a specific volume of cells cultured to OD1.0. Assuming the cell density in OD1.0 is 0.3 g/L, it is estimated that the protein is therefore 0.9 μg / mg dry cell weight. When the LysM- CtxB50-75-FLAG cassette was integrated, expression was observed to be lower than from a multi-copy gene, but still expressed to a relatively high level (Figure 4C). Confirmation of cell wall-association of LysM-CtxB50-75-FLAG protein Cell fractionation assays (as detailed in https://doi.org/10.1186/s13068-016-0526-x) were carried out on liquid cultures CHN-0 + pMTL82151-pfdx-LysM-CtxB50-75-FLAG to determine correct insertion of the LysM-CtxB50-75-FLAG into the peptidoglycan layer of the vegetative CHN-0 cell. In brief, fresh colonies of revived C. butyricum wild-type, CHN-0 + pMTL82151_pfdx-LysM-CtxB50-75--FLAG and CHN-0 + pMTL82151_pfdx-LysM- FLAG were used to inoculate fresh pre-reduced supplemented Clostridial Growth Medium (CGM) broth (5 g/L yeast extract, 1 g/L sodium chloride (NaCl), 0.75 g/L potassium phosphate dibasic (K2HPO4), 0.75 g/L potassium phosphate monobasic, 0.4 g/L magnesium sulphate heptahydrate (Mg2SO4.7H2O), 0.01 g/L iron (II) sulphate heptahydrate (FeSO4.7H2O), 0.01 g/L manganese(II) sulphate tetrahydrate (MnSO4.4H2O), 2 g/L ammonium sulphate ((NH4)2SO4) and 2 g/L asparagine) supplemented with 2% glucose, 1% CaCO3 + 15 μg/mL thiamphenicol in serial dilution and grown overnight. The most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented CGM broth + 2% glucose, 1% CaCO3 and 0.4% glycine at a starting OD600 of 0.05. When cultures were grown to an OD600 of 0.7-0.9, the equivalent of OD600 of 5/mL was centrifuged at 5,000×g for 10 min. The supernatant as discarded and the pellet was washed once with 5 mL pre-reduced filter- sterilised lysis buffer (50 mM TBS with 25 mM CaCl2, 25 mM MgCl2, and 0.3 M sucrose). The pellet was then resuspended in 5 mL of the pre-reduced filter sterilised lysis buffer supplemented with 3 mg/mL lysozyme (Lysozyme from chicken egg white, Sigma Aldrich) and incubated at 37°C for 3 h in an anoxic environment. The digested cell suspensions were then centrifuged for 3 minutes at 5,000xg (4°C) to pellet out any undigested cells, and then the supernatant centrifuged at 9,000xg for 20 minutes to separate the cell wall fraction (supernatant) from the protoplast (pellet). Proteins found in the cell wall fraction were then precipitated by Trichloroacetic acid (TCA) precipitation whereby 100% TCA was added to a final volume of 10% to the supernatants previously filtered using a 0.22 μm syringe filter (Sartorius). The 10% TCA-supernatant samples were incubated at -20°C overnight (~20 h), before being thawed and centrifuged once again at 9,400 r.p.m for 30 minutes at 4°C. Supernatants were discarded and all residual TCA removed, before protein pellets were washed thrice in ice-cold acetone and dried. Dried cell wall protein pellets were then resuspended in 100 μL 5× SDS loading dye + 10 μL 1 M Tris/HCl, pH7.4 buffer until the protein pellets turned blue. The samples were then heat treated at 98°C for 15 minutes. A maximum of 30 μL/well of the re-suspended pellets was loaded onto a NovexTM Wedgewell™ 14% Tris Glycine 1.0 mm mini gel (ThermoFisher Scientific) and run in 1×Novex™ Tris Glycine SDS Running Buffer (ThermoFisher Scientific) using 180V at room temperature. PageRuler™ Plus Prestained Protein Ladder (ThermoFisher Scientific) was loaded at 10 μL/well as a marker. The E. coli Positive Control Whole cell lysate ab5395 (abcam) was used as FLAG-tag positive control in a 1:5 dilution and then subject to Western blotting as described for LysM-CtxB50-75-FLAG whole cell lysate detection as above. Cell wall fractions containing the LysM-CtxB50-75--FLAG can be seen in Figure 5, demonstrating the extracellular location of the CtxB50-75 peptide. For further confirmation of cell wall localisation, high-resolution structured illumination microscopy (SIM) was carried out to demonstrate the surface display of fluorescently labelled LysM-CtxB50-75-FLAG antigens. Overnight cultures of CHN-0 wild-type and CHN-0 + pMTL82151-pfdx-LysM-CtxB50-75-FLAG were prepared in a serial dilution in pre-reduced RCM broth supplemented with + 15 μg/mL thiamphenicol as previously described. Fresh day cultures of pre-reduced RCM broth were inoculated from the lowest growing overnight to a starting OD600 of 0.05. When cultures reached an OD600 of 1.0, a volume equivalent to 5 mL was harvested by centrifugation at 4,500xg for 10 minutes. The resulting pellet was washed twice in TBS and then fixed in 1 mL 4% paraformaldehyde in PBS (Alfa Aesar) overnight at 4°C. After fixing, the cells were then washed twice in TBS and then blocked in PBS containing 1% bovine serum albumin (BSA) for 1 h. The pellets were then incubated for 1 h at room temperature with monoclonal anti-FLAG® M2 antibody (Sigma Aldrich) at a 1:100 dilution in PBS + 1% BSA. Pellets were washed three times before incubating with a fluorescence- conjugated secondary antibody, goat anti-mouse-Alexa-Fluor® 647 (Cell Signalling) at 1:1000 dilution (in PBS + 1% BSA) overnight without light at 4°C. Pellets were then washed five times in PBS before 10 μL of the fluorescently-labelled cells were mounted onto coverslips using Vectashield H-1700 supplemented with 10 mM MEA (Cysteamine Hydrochloride, Sigma) and left to dry before imaging. Samples were imaged using structured illumination microscopy techniques and as the antibodies cannot permeate through the peptidoglycan cell wall of the CHN-0 cells, detection of the fluorescently-labelled FLAG tag on the LysM-CtxB50-75--FLAG protein antigen demonstrates decoration of the antigen on the vegetative cell surface, as seen in Figure 6. Immunogenicity testing In vivo immunisation experiments will be performed to assess delivery of antigen and induction of immune responses, with a focus on cellular and humoral responses, using oral delivery of spores of engineered C. butyricum expressing the LysM-CtxB50-75--FLAG peptidoglycan-anchored antigen or the secreted Usp45-CtxB antigen. C. butyricum spores will be generated as set out previously. For assessment of antigen-specific immune responses to Usp45-CtxB-FLAG and LysM- CtxB50-75-FLAG cell wall-associated antigen, C57BL/6 mice will be administered an immunisation regimen as described herein. For example, the mice may be administered 1 x 108 CFU/dose orally in 3 doses, 2 weeks apart. Alternatively, mice may be administered the dose orally in two or more immunisations comprising two or more consecutive days, with intervals of one or more days in between immunisations. Groups of mice will be administered individual strains selected from a wild-type CHN- 0 strain (negative control) or the CHN-0 vaccine strain expressing the antigens from the pMTL82121-P0957-Usp45-CtxB-FLAG, pMTL82151-pfdx-LysM-CtxB50-75--FLAG plasmid and/or the integrated LysM-CtxB50-75--FLAG constructs. Two final groups will be administered a commercially obtained cholera holotoxin (C-TX) as a positive control in either oral gavage or intraperitoneal (i.p.) injection. Clinical observations will be taken throughout to determine tolerability of the test articles (weight changes and physical appearances such as hunching or coat piloerection). Table 3 – Dosing groups for CHN-0-based oral vaccines against Vibrio Cholerae
Figure imgf000086_0001
At sacrifice, spleens will be harvested and processed to a single cell suspension and CD4+ and CD8+ cells purified individually to determine CD4+/CD8+-specific T cell response via IFN-Υ release in ELISPOT assays (described below). CD4+ T cell response will also be analysed in gut-specific tissues (small intestine and colon), where the tissue will be extracted, treated with mucolytic enzymes + EDTA and digested to a single cell suspension, as described in Di Luccia et al (2020) Cell Host & Microbe 27: 899-908. Isolated CD4+ T cells from this suspension will be re-stimulated with antigen presenting cells (APCs, previously exposed to a commercial C-TX antigen) and the change in CD40 ligand expression on the cell surface will be assessed via Flow Cytometry. Faecal pellets will be assessed for the antigen-specific humoral response via ELISA assays to determine CtxB-specific secretory IgA (sIgA) production as a percentage of the total IgA, as described in Di Luccia et al (2020) Cell Host & Microbe 27: 899-908. Materials and Methods Isolation of mononuclear cells Harvested spleens at termination will be homogenised and splenocytes isolated, whilst PBMCs will be isolated from the terminal whole blood samples. Isolation of cells will be performed using Ficoll-Paque 1.084 density gradient (GE healthcare) according to manufacturer’s instructions. Cell suspension or whole blood will be layered on Ficoll- Paque media and centrifuged at 400 × g for 20-30min at RT. The mononuclear cells isolates will then be washed in balanced salt solution to remove residual contaminants. For T-cell purification, mononuclear cell isolates will be purified using CD8a (Ly-a) MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions. A volume of 90μL of MACS buffer (PBS, 0.5% bovine serum albumin, 2mM EDTA, pH 7.2) will be used to resuspend 1 × 107 cells before addition of MicroBeads and incubation at 4°C for 10 min. Cell suspensions will then be applied to MACS LS columns in a magnetic field for retention of CD8+ T-cells. The flow through will be collected twice and used for CD4+ T-cell specific experiments. CD8+ T-cells will then be eluted subsequently by application of buffer without magnetic field. Both CD4+ and CD8+ T-cells will be resuspended in RPMI medium before use in ELISPOT experiments. IFN-Υ T-cell ELISPOT The Mouse IFN-Υ T-cell ELISPOT kit (U-CyTech Bioscience) will be used for detection of IFN-Υ release according to manufacturer’s instructions. A total of 2.5 × 105 T-cells in 100μL RPMI/well will be added to plates precoated with Anti-IFN-Υ antibodies and re-stimulated with C-TX holotoxin (each at 5μg/well; SIGMA) or CHN-0 vegetative cells at 0.5 × 105 CFU/well. Concanavalin A (Sigma Aldrich) will be added as positive control at a concentration of 5mg/mL. Plates will then be incubated overnight at 37°C and 5% CO2 before addition of biotinylated detection antibody followed by incubation with GABA conjugate and incubation with Activator I/II solution to allow for spot formation. Spots will then be scanned using a Celigo Image Cytometer and quantified using ImageJ software. Expected results When mice are immunised with LysM-CtxB50-75-FLAG and Usp45-CtxB-FLAG CHN-0 oral vaccine strains, we expect the ELISPOT assays of CD4+/CD8+ T-cells to show mice CtxB-antigen-specific T-cell response, with a stronger emphasis on the CD4+ response. We do not expect to see mice immunised with the CHN-0 wild type strain developing a T-cell response. A detectable CD4+ T-cell response would likely indicate a stronger, more long-lasting, and more specific vaccine response in account of the T-cell help to antibody production and recall responses. In the gut-specific tissue assessment, enumeration of antigen-specific T cells will be undertaken using flow cytometry with two separate approaches i) detecting recall response based on CD40L upregulation following stimulation with soluble antigen (presented on pulsed antigen presenting cells), ii) Class 2 MHC tetramer (reagent in development) response. A positive result for the test condition would be indicated by a higher percentage antigen-specific (i.e., CD40L upregulated) CD4+ T-cells versus negative control group, or the higher presence of the MHC tetramer reagent on the cell surface compared to negative control groups. The sIgA antibody response is also known to be important in protective immunity against infections by V. cholerae, and therefore we also seek to determine the humoral response for mucosal immunity via assessment of the production of CtxB-specific secretory IgA (sIgA). Through ELISAs, we expect to see an increase in antigen-specific sIgA in response to administration of the CHN-0 CtxB oral vaccines (LysM and Usp45), compared to the wild-type CHN-0 platform alone. Example 2: Immunisation of mice using secreted Human Rotavirus antigens, VP7 and VP8, expressed by Clostridium Human Rotavirus is the most common cause of diarrhoeal disease among infants and young children, causing severe gastroenteritis and contributes to a significant number of infant deaths across the world, with higher prevalence in low- and middle-income countries. Humans are primarily infected by the species rotavirus A within which several different serotypes exist. The HRV genome codes for six structural proteins (VP1-VP4, VP6 and VP7) and six non-structural proteins (NSP1-NSP6). The infectious virion particle is formed from 3 layers of protein, whereby the outer later (outer capsid) consists of the surface proteins, VP7 and VP4, which contribute to the classification of serotypes. The glycoprotein Viral Protein 7 (VP7) defines the G serotype and the protease sensitive VP4 defines the P serotype. The VP4 protein protrudes on the cell surface of the virion as a spike and is involved in binding the human cell receptors including sialoglycans (such as Gangliosides GM1 and GD1a) and histo-blood group antigens (HBGAs), driving entry into the cell. The VP4 is cleaved by trypsin (found in the human gut) into VP5 and VP8 to allow the virus particle to become infectious. As the majority of the virion capsid is made up of VP7 and VP4, these proteins are important in rotavirus immunity, containing epitopes for T-cell and B-cell activation as well as antigenic sites responsible for neutralisation of the virus (specifically in the VP8 fragment of VP4) and as such, were chosen as target immunogens for the oral CHN-0 vaccine against Human Rotavirus. The HRV-VP7 and -VP8 protein expression is coupled to a Sec-dependent secretion system signal peptide known as Usp45, which is a native secretion signal peptide found within C. butyricum. Gene constructs and plasmids For the secreted HRV protein oral vaccine development, two different approaches were taken for antigen design depending on the antigenic target. Antigenic targets were selected for their external location on the virion outer capsid and therefore increased likelihood of generating an antigenic immune response. The VP7 antigen was derived from in silico prediction for surface accessible conserved region in the VP7 protein (Ghosh et al, 2012, PLoS One, 7(7):e40749, DOI: 10.1371/journal.pone.0040749) and the regions determined were then applied to the epitope prediction servers Immune Epitope Database and Analysis Resource (IEDB; Vita et al., 2010, Nucleic Acids Res., 38:D854-862, DOI: 10.1093/nar/gkp1004) and ABCpred (Saha & Raghava, 2006, Proteins, 65:40-48, DOI: 10.1002/prot.21078) for their epitopic properties towards possible T-cell and B-cell activation. The VP8 protein (as part of the VP4 protein) is another surface exposed antigen, which has previously been shown to be suitable for inducing immune responses (Xue et al., 2016, Hum. Vaccin. Immunother., 12(11):22959-2968, DOI: 10.1080/21645515.2016.1204501; Wen et al., 2012, Vaccine, 30(43):6121-6126, DOI: 10.1016/j.vaccine.2012.07.078). For the secreted HRV-VP8 (Figure 7) protein, the sequence was determined from the UniProtKB submission P11193 for the outer capsid protein VP4, whereby amino acids 1-225 (SEQ ID NO: 25) relate to the VP8 domain. The first 63 amino acids including the signal sequence (MASLIYRQLLTNSYSVDLHDEIEQIGSEKTQNV TINPSPFAQTRYAPVNWGHGEINDSTTVEPI (SEQ ID NO: 57)) were removed, a 6 amino acid sequence (LSGVYA (SEQ ID NO: 63)) was added at the N-terminus and a C- terminal FLAG tag (DYKDDDDK (SEQ ID NO: 46)) was also added. Further modifications included for genetic engineering include an NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA. The HRV-VP8-FLAG construct was codon optimised for genetic engineering into C. butyricum and synthesised by GeneWiz and cloned into pMTL82121_p0957 (pBP1 Gram+ replicon, catP antibiotic marker, p15 Gram- replicon, traJ conjugal transfer function, p0957 promoter and adjacent multiple cloning site (MCS)) vector submitted to GeneWiz for subcloning. Once plasmids were received and sequences confirmed via PCR using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers detailed in Table 1, cloning was undertaken to introduce a signal peptide (MKKKIISAILMSTILSAAAP, known as Usp45 (SEQ ID NO: 41)) between the p0957 promoter (SEQ ID NO: 182 - from C. acetobutylicum ATCC824, p0957 is the promoter for the locus tag Ca_c0957) and the HRV-VP8 open reading frame. The usp45 nucleic acid sequence was amplified using primers CH754 and CH758 (see Table 4), which were designed to overlap with the RBS region of the pMTL82121_p0957 vector digested with NdeI at the 5’ and a VP8 fragment at the 3’ utilising a design that preserved the NdeI site. The FLAG-tagged VP8 fragment was then amplified using primers CH758 (SEQ ID NO: 58) and CH757 (SEQ ID NO: 59) (Table 4), from the Genewiz-generated pMTL82121-p0957-HRV-VP8-FLAG plasmid, to create a fragment which contained an overlap at the 5’ to the usp45 sequence and at the 3’ to a pMTL82121-p0957 vector digested with NheI utilising a design that preserves the NheI site. Hi-Fi assembly was used to assemble the entire pMTL82121-p0957-Usp45- HRV-VP8-FLAG construct from the templates Usp45 fragment and the HRV-VP8-FLAG fragment generated via PCR described above, plus an NdeI and NheI digested pMTL82121-p0957 vector. The resulting constructs transformed into NEB® Stable Competent E. coli (New England Biolabs) for propagation and storage, with colonies positive for plasmid uptake determined using LB agar + 25 μg/mL chloramphenicol for selection. Plasmids were isolated as before and sequenced using GeneWiz sequencing services to confirm the correct insertion of cassettes. Following sequence confirmation in NEB® Stable Competent E. coli, cells were grown overnight in LB supplemented with 12.5μg/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. The Usp45-HRV-VP8-FLAG cassette was also introduced into the pMTL83151_pyrE repair plasmid previously mentioned in Example 1 for insertion of the p0957-Usp45- HRV-VP8-FLAG construct into the genome as a single copy. The p0957-Usp45-HRV- VP8 construct was excised from the pMTL82151_p0957-Usp45-HRV-VP8-FLAG plasmid using restriction endonucleases NotI and NheI in CutSmart® buffer (all New England Biolabs Inc) according to the manufacturer’s instructions. The isolated cassette + promoter was introduced into the pMTL83151_pyrE repair vector. Plasmids were transformed into NEB® Stable Competent E. coli for propagation. Plasmids were isolated as before and sequenced using GeneWiz sequencing services to confirm the correct insertion of cassettes using primers CH22 (SEQ ID NO: 51) and CH648 (SEQ ID NO: 60). For the secreted HRV-VP7 protein, a polyepitope antigenic protein has been designed for expression and secretion (Figure 8). This polyepitope consists of 5 identified immunogenic epitopes, repeated 3 times and separated by flexible linkers and has been constructed as below for future incorporation into the CHN-0 strain. Table 4: Primers used in Usp45 cloning introduction to HRV-VP8 construct. CH754 (SEQ attcaaggaggtgtgttacatATGAAAAAAAAAATCA Usp45 for HRV-VP8 ID NO: 64) F TTTCAGCTAT Hifi
Figure imgf000091_0001
The Usp45-HRV-VP7-FLAG polyepitope construct was designed by identifying 5 immunogenic epitopes within the VP7 protein (TTTCTIRNCKKLGP (SEQ ID NO: 67, LDITADPTTNPQIE (SEQ ID NO: 68), KVCPLNTQALGIGC (SEQ ID NO: 69), KINLTTTTCTIRNC (SEQ ID NO: 70) and RNCKKLGPRENVAI (SEQ ID NO: 71)) and linking them with a flexible [Gly]4Ser linker (GGGGS; SEQ ID NO: 48). The epitopes were repeated 3 times within the construct to maximise antigen presentation, with the Usp45 secretion signal (MKKKIISAILMSTILSAAAP (SEQ ID NO: 41)) and a 6 amino acid sequence (LSGVYA (SEQ ID NO: 63)) added 5’ to the polyepitope construct, and also a C-terminal FLAG tag (DYKDDDDK (SEQ ID NO: 46)) was added. Further modifications included for genetic engineering include an NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA. The HRV-VP7-FLAG polyepitope construct was codon optimised for genetic engineering into C. butyricum, synthesised by GeneWiz and cloned into a pMTL82121_p0957 vector submitted to GeneWiz for subcloning. Once plasmids are received, the plasmids will be cloned into NEB® Stable Competent E. coli for propagation and storage, and then sequences will be confirmed via PCR using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 52) primers detailed previously in Table 1. If the Usp45-VP7-polyepitope construct expresses to a detectable level (via Western blot as previously described), the CHN-0 strain carrying this antigen will be subject to the same in vivo analysis as detailed below for HRV-VP8-FLAG carrying CHN-0 strains. Genetic engineering of C. butyricum Correct plasmids for pMTL82121-p0957-Usp45-HRV-VP8-FLAG were transformed into E. coli CA434 conjugation donor strain and conjugated into C. butyricum CHN-0 for plasmid-based expression as described previously for pMTL82151-pfdx-LysM-CtxB50- 75–FLAG constructs in Example 1. Primers used to determine correct uptake of the pMTL82121-p0957-Usp45-HRV-VP8-FLAG plasmid are listed in Table 1. The plasmid pMTL83151-pyrE repair-p0957-Usp45-VP8-FLAG for integration of p0957- HRV-VP8-FLAG into the genome was also transformed into E. coli CA434 conjugation donor strain and conjugated into C. butyricum CHN-0.1 (ƩpyrE derivative of WT CHN- 0) for single copy integration expression as described previously for pMTL83151-pyrE repair-pfdx-LysM-CtxB50-75-–FLAG constructs in Example 1. Primers used to determine correct integration of the p0957-Usp45-HRV-VP8-FLAG construct into the genome are listed in Table 5 below. Table 5: Primers used for confirmation of integration of Usp45-HRV-VP8 into the CHN- 0 genome.
Figure imgf000092_0001
The introduction of the pMTL82151_p0957-Usp45-HRV-VP8 plasmid into C. butyricum CHN-0 leads to a high expression of the secreted VP8 protein by C. butyricum from a multicopy plasmid, with a lower expression of HRV-VP8-FLAG when integrated into the genome as it is single copy. Confirmation of expression and secretion of the HRV-VP8 candidate in C. butyricum Fresh colonies of revived C. butyricum CHN-0 + pMTL82121_p0957-Usp45-HRV-VP8- FLAG were used to inoculate fresh pre-reduced supplemented Fermentation Media for Clostridia (FMC) broth + 15 μg/mL thiamphenicol in serial dilution and grown overnight. The most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented FC broth + 15 μg/mL thiamphenicol at a starting OD600 of 0.05. When cultures were grown to an OD600 of 1, 2, and 4, 10mL of culture per growth phase was harvested before centrifugation at 13,000×g for 10 min at 4°C. Supernatants were removed and filtered using a 0.22 μm syringe filter (Sartorius) before TCA precipitation of the protein was carried out as described previously. Dry pellets were re-suspended in 40 μL 5× SDS loading dye and 4-5 μL 1M Tris/HCl, pH7.4 buffer until the protein pellets turned blue. The samples were then heat treated at 98°C for 15 minutes. A maximum of 40 μL/well of the re-suspended pellets was loaded onto a NovexTM Wedgewell™ 14% Tris Glycine 1.0 mm mini gel (ThermoFisher Scientific) and run in 1×Novex™ Tris Glycine SDS Running Buffer (ThermoFisher Scientific) using 180V at room temperature. PageRuler™ Plus Prestained Protein Ladder (ThermoFisher Scientific) was loaded at 10 μL/well as marker. Carboxy-terminal FLAG-BAP™ Fusion Protein (FLAG-BAP™, Sigma Aldrich) standards were used, diluted to a range of concentrations from 2 μg – 50 ng. The separated proteins were subject to Western blotting as described for LysM-CtxB50- 75-FLAG in Example 1 and shown in Figure 9. The secreted HRV-VP8 protein was detectable to high levels on a Western blot, corresponding to 2500 ng in a specific volume of supernatant culture (10 mL) when cells reach OD1.0 in their growth phase. Assuming the cell density in OD1.0 is 0.3 g/L, it is estimated that the secreted protein is therefore ~0. 825 μg/mg cell weight. Expression of integrated Usp45-HRV-VP8-FLAG was also assessed similar to plasmid- based expression above. Fresh colonies of revived C. butyricum CHN-0 + Usp45-HRV- VP8-FLAG were used to inoculate fresh pre-reduced supplemented FMC broth (no antibiotics) at a starting OD600 of 0.05. When cultures were grown to an OD600 of 1, 2, and 4, 10mL of culture was harvested before centrifugation at 13,000×g for 10 min at 4°C and the supernatants kept for protein assessment. Proteins were extracted from supernatants using TCA precipitation and pellets prepared as described for plasmid- based expression of HRV-VP7 and HRV-VP8, before SDS-PAGE analysis and Western blotting as described above. Expression from integrated Usp45-HRV-VP8-FLAG was compared to plasmid-based expression from pMTL82121-p0957-Usp45-HRV-VP8 and wild-type CHN-0 as seen in Figure 10. Based on densitometric comparison with the amount of protein secreted from the plasmid based strain, it is estimated that the strain having a single copy in the genome produces secreted protein at 40-80 ng/mg cell weight. Immunogenicity testing In vivo immunisation experiments will be performed to assess delivery of antigen and induction of immune responses, with a focus on cellular and humoral responses using spores of engineered C. butyricum expressing the HRV-VP7 / HRV-VP8 secreted antigens. C. butyricum spores will be generated as set out above in Example 1. For assessment of antigen-specific immune responses to secreted HRV-VP7 and HRV-VP8 antigens, C57BL/6 mice will be administered an immunisation regimen as described herein. For example, the mice may be administered 1 x 108 CFU/dose orally in 3 doses, 2 weeks apart. Alternatively, mice may be administered the dose orally in two or more immunisations comprising two or more consecutive days, with intervals of one or more days in between immunisations. Groups of mice will be administered individual strains selected from a wild-type CHN-0 strain (negative control), the CHN-0 oral vaccine strain expressing the antigens from the pMTL82151-p0957-Usp45-HRV-VP7- polyepitope-FLAG plasmid (once constructed and analysed for expression), the pMTL82151-p0957-Usp45-HRV-VP8-FLAG and/or the integrated CADD+Usp45-HRV- VP8-FLAG strain. A final group will be administered the recombinant HRV-VP7 or HRV- VP8 via subcutaneous or i.p. injection (+adjuvant) as a positive control. Clinical observations will be taken throughout to determine tolerability of the test articles (weight changes and physical appearances such as hunching or coat piloerection). Table 6 – example dosing groups for CHN-0-based oral vaccines against Human Rotavirus
Figure imgf000094_0001
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At sacrifice, spleens will be harvested and processed to a single cell suspension and CD4+ and CD8+ cells purified individually to determine CD4+/CD8+-specific T cell response via IFN-Υ release in ELISPOT assays following antigen restimulation using either the control HRV-VP7/8 protein or CHN-0 vegetative cells and detected using the Mouse IFN-Υ T-cell ELISPOT kit (U-CyTech Bioscience) . CD4+ T cell response will also be analysed in gut-specific tissues (small intestine and colon), where the tissue will be extracted, treated with mucolytic enzymes + EDTA and digested to a single cell suspension, as described in Di Luccia et al (2020) Cell Host & Microbe 27: 899-908. Isolated CD4+ T cells from this suspension will be re-stimulated with antigen presenting cells (APCs, previously exposed to a recombinant HRV-VP7/8 antigen) and the change in CD40 ligand expression on the cell surface will be assessed via Flow Cytometry. Faecal pellets will be assessed for the antigen-specific humoral response will be assessed via ELISA assays to determine HRV antigen-specific secretory IgA (sIgA) production as a percentage of the total IgA, as described in Di Luccia et al (2020) Cell Host & Microbe 27: 899-908. Expected results As with the LysM-CtxB50-75-FLAG CHN oral vaccine strains, we expect the ELISPOT assays of CD4+/CD8+ T-cells to show mice immunised with the CADD strain expressing the secreted HRV-VP7 and HRV-VP8 antigens to develop an antigen-specific T-cell response, with a stronger emphasis on the CD4+ response. Again, we do not expect to see mice immunised with the CHN-0 wild type strain developing a T-cell response. A detectable CD4+ T-cell response would likely indicate a stronger, more long-lasting, and more specific vaccine response in account of the T-cell help to antibody production and recall responses. In the gut-specific tissue assessment, enumeration of antigen-specific T cells will be undertaken using flow cytometry with two separate approaches i) detecting recall response based on CD40L upregulation following stimulation with soluble antigen (presented on pulsed antigen presenting cells), ii) Class 2 MHC tetramer (reagent in development) response. A positive result for the test condition would be indicated by a higher percentage antigen-specific (i.e., CD40L upregulated) CD4+ T-cells versus negative control group, or the higher presence of the MHC tetramer reagent on the cell surface compared to negative control groups. The sIgA antibody response is also known to be important in protective immunity against mucosal infections, and therefore we also seek to determine the humoral response for mucosal immunity via assessment of the production of HRV antigen- specific secretory IgA (sIgA). Through ELISAs, we expect to see an increase in antigen- specific sIgA in response to administration of the CHN-0-HRV oral vaccines, compared to the wild-type CHN-0 platform alone. Example 3: Immunisation of mice using extracellular and intracellular SARS- CoV-2 peptide antigens expressed by Clostridium COVID-19 is caused by the severe acute respiratory syndrome coronavirus (SARS- CoV-2). SARS-CoV-2 contains four structural proteins, including Spike (S), Envelope (E), membrane (M) and nucleocapsid (N) and at least 6 other open reading frames (ORFs). Amongst the structural proteins, the S protein plays the most important role in viral attachment, fusion and entry into the host cell and thereby serves as a strong target for vaccine development. The S protein contains 2 major subunits, the S1 subunit which contains the receptor binding domain (RBD) which binds to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell, and the S2 subunit which allows fusion between the virus and the host cell membrane. SARS-CoV-2 epitopes were selected based on the methods described in Fast et al. (2020), bioRxiv preprint, doi: https://doi.org/10.1101/2020.02.19.955484; Li et al. (2020), Virus Research, https://doi.org/10.1016/j.virusres.2020.198082; and/or Tai et al., 2020, Cell Mol. Immunol., 16:613-620, DOI: 10.1038/s41423-020-0400-4, which disclose the successful selection of antigenic T- and B-cell epitopes in SARS- CoV-2 proteins. Li et al used peptide microarrays where 200+ peptides in the S protein were synthesised and screened for IgG and IgM responses in convalescent COVID-19 patient sera, followed by neutralisation assays. We used a SB1 antigen comprising eleven of the epitopes described in Li, and an SB2 antigen comprising of six epitopes described in Li. Fast et al used antigen presentation prediction artificial neural network algorithms (NetMHCpan4 and MARIA) to determine T cell epitopes & homology modelling to determine B cell epitopes (e.g. Discopte2 tool). We designed 15mer epitopes that were larger than the 9mer minimal epitopes described in Fast (although the full 15mer sequences are present in Fast et al). Tai et al used alignment with known epitopes from SARS-CoV to find conserved regions and then generated recombinant proteins of the identified epitopes. They used fluorescence staining for interaction with the ACE2 receptor and neutralisation assays to demonstrate efficacy. We used a RBD antigen comprising of the RBD antigenic sequence (amino acids 331- 524 of Genbank ID QHR63250.1; SEQ ID NO: 6) described in Tai et al. with an additional 6XHis tag. Selected epitopes were identified from the Spike protein (including in the RBD) which had good predicted antigen presentation scores for MHC I and MHC II, and also linear epitopes that show stimulation of neutralising antibodies. Other epitopes were identified within the other structural proteins (M, N, E) and in a non-structural protein, Orf1ab, which have the potential to elicit a strong T cell response. Chosen epitopes were linked with a cathepsin S cleavage site for higher probability of epitope presentation via the MHCs, and as with the CtxB50-75-FLAG antigen in Example 1, these polyepitopes were linked to the LysM peptidoglycan-anchoring domain for extracellular expression. Gene constructs and plasmids In total, five LysM-SARS-CoV-2-His constructs (Figure 11) were designed for extracellular expression of COVID-19 antigenic polyepitopes, denoted LysM-CoV-2- RBD (immunogenic epitopes determined in the RBD protein sequence of the S1 subunit of the spike protein, SEQ ID NO: 7), LysM-CoV-2-Nuc (immunogenic epitopes determined in the nucleocapsid protein sequence, SEQ ID NO: 13), LysM-CoV-2-Comb (immunogenic epitopes determined from the nucleocapsid, membrane, envelope, and spike protein sequences, as well as immunogenic epitopes determined in the Orf1ab non-structural protein, SEQ ID NO: 17), LysM-CoV-2-SB1 (neutralising (B cell) linear epitopes determined in the spike protein, SEQ ID NO: 9), and LysM-CoV-2-SB2 (neutralising (B cell) linear epitopes determined in the spike protein, SEQ ID NO: 11). Chosen epitopes for the in silico predicted or in vitro and in vivo confirmed antigenic properties were modified by connection of the individual epitopes in some cases via a Cathepsin S cleavage sequence (TVKLRQ (SEQ ID NO: 39) (in LysM-CoV-2-Nuc, -CoV- 2-Comb) and all polyepitopes had a C-terminal 6xHis tag (HHHHHH (SEQ ID NO: 47)) added for expression identification and purification. The LysM peptidoglycan-anchoring domain sequence (SEQ ID NO: 42) was also added upstream of all SARS-CoV-2 polyepitopes for extracellular localisation and the LysM domain was separated from the SARS-CoV-2 polyepitope by a flexible [Gly]4Ser linker. Further modifications included for genetic engineering include two NdeI cleavage sites (CATATG) either side of the LysM encoding domain incorporating the nucleotide signal for aa methionine (M, ATG) and allowing removal of the LysM domain, and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the His-tag by the stop codon TAA. The LysM- SARS-CoV-2-His antigenic constructs were codon optimised for genetic engineering into C. butyricum and synthesised behind the p0957 promoter by GeneWiz and cloned into pMTL82121 vector submitted to GeneWiz for subcloning. Once plasmids were received, they were transformed into NEB® Stable Competent E. coli (New England Biolabs) for propagation and storage, with colonies positive for plasmid uptake determined using LB agar + 25 μg/mL chloramphenicol for selection. Plasmids were isolated as before and sequenced using GeneWiz sequencing services and using CH22 (SEQ ID NO: 51) and CH54 (SEQ ID NO: 42) primers spanning the MCS (Table 1) to confirm the correct sequence of constructs. Following sequence confirmation in NEB® Stable Competent E. coli, cells were grown overnight in LB supplemented with 12.5μg/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Cloning of intracellular expressed SARS-COV-2-X-His antigens was performed through NdeI digestion of sequence confirmed pMTL82121_p0957-LysM-SARS-COV-2-X-His constructs to excise the LysM domain (Figure 12). Following this linear plasmid was isolated by gel electrophoresis, extracted, and circularised with the use of T4 DNA ligase. These constructs were transformed into NEB® Stable Competent E. coli as isolated as described before. Sequence confirmed pMTL82121_p0957-LysM-SARS-CoV-2-X-His and intracellular pMTL82121_p0957-SARS-CoV-2-X-His plasmids (where X denotes any one of the 5 CoV candidates, -RBD, -Nuc, -Comb, -SB1, -SB2) were transformed into E. coli CA434 conjugation donors and positive colonies were isolated for their resistance to 50 μg/mL kanamycin and 25 μg/mL chloramphenicol on LB agar plates. Following plasmid isolation and sequence confirmation as above, E. coli CA434 were grown over night in LB supplemented with 50 μg/mL kanamycin and 12.5μg/mL chloramphenicol and stored at -80°C as 15% glycerol stocks. Genetic engineering of C. butyricum Correct plasmids for all pMTL82121-p0957-LysM-SARS-CoV-2-X-His antigens and pMTL82121_p0957-SARS-CoV-2-X-His in E. coli CA434 conjugation donor strain were then conjugated into C. butyricum CHNR-0 (an identical strain to CHN-0) for plasmid- based expression as described previously for pMTL82151-pfdx-LysM-CtxB50-75-–FLAG constructs in Example 1. Primers used to determine correct uptake of the pMTL82121- p0957-LysM-CoV-2-RBD-His, pMTL82121-p0957-LysM-CoV-2-Nuc-His, pMTL82121- p0957-LysM-CoV-2-Comb-His, pMTL82121-p0957-LysM-CoV-2-SB1-His and pMTL82121-p0957-LysM-CoV-2-SB2-His plasmids are listed in Table 7. Intracellular plasmids used the same primer set however primers CH733 and CH734 were not required as the sequence had been truncated. Table 7: Primers for LysM-SARS-CoV-2-X-His construct sequencing in pMTL82121 vectors.
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Confirmation of expression of SARS-CoV-2 candidates in C. butyricum Fresh colonies of revived C. butyricum CHNR-0 + pMTL82121_p0957-LysM-SARS-CoV- 2-X-His or intracellular pMTL82121_p0957-SARS-CoV-2-X-His were used to inoculate fresh pre-reduced supplemented RCM broth + 15 μg/mL thiamphenicol in serial dilution and grown overnight. The most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth + 15 μg/mL thiamphenicol at a starting OD600 of 0.05. When cultures were grown to an OD600 of 1.0, 2.0 and 4.0, the equivalent of OD600 of 2/mL was centrifuged at 13,000×g for 2 min. Supernatants were discarded and the pellets prepared for SDS-PAGE analysis as below. This process was identical for intracellular expression plasmids pMTL82121_p0957-SARS-CoV-2-X-His. The pellets were re-suspended in 40 μL 5× SDS Loading dye and heat treated at 98°C for 15 minutes. A maximum of 40 μL/well of the re-suspended pellets was loaded onto a NovexTM Wedgewell™ 14% Tris Glycine 1.0 mm mini gel (ThermoFisher Scientific) and run in 1×Novex™ Tris Glycine SDS Running Buffer (ThermoFisher Scientific) using 180V at room temperature. PageRuler™ Plus Prestained Protein Ladder (ThermoFisher Scientific) was loaded at 10 μL/well as a marker. 10 μl E. coli Positive Control Whole Cell Lysate - expressing 6X His tag protein (ab2431; abcam) was used as His-tag positive control. The separated proteins were subject to Western blotting as described for LysM-CtxB50- 75-FLAG in Example 1, except different antibodies for detection were used. An anti- 6XHis primary antibody (1:1000 incubated for 1 h in TBS-T, Abcam) and an HRP- conjugated anti-mouse IgG secondary antibody (1:5000 incubated for 1 h in TST-T, Sigma Aldrich) were used instead of the anti-FLAG-HRP conjugated antibody. Resultant blots are shown in Figure 13. The introduction of the pMTL82121_p0957-LysM-SARS-CoV-2-X-His plasmids into C. butyricum CHNR-0 leads to varied levels of expression of the peptidoglycan-associated SARS-CoV-2-X-His antigenic proteins by C. butyricum from multicopy plasmids. A level of cleavage presumed to be between the LysM and the SARS-CoV-2 antigen is observed in most antigens, whereby a higher band at the correct expected MW is observed and a lower band at the expected size of the antigenic peptide without a LysM domain is also observed. In the SARS-CoV-2-LysM-Nuc-His antigenic peptide expression (Figure 13B), a ladder of bands appeared on the Western blot and therefore assessment of the supernatant of this strain was carried out. Briefly, overnight and fresh day cultures were set up as previously described, and a 10 mL sample was removed from the culture when growth reached OD6001.0. The sample was centrifuged at 6,000 r.p.m. for 10 minutes and the culture supernatant filter sterilised using a 0.22 μm filter (Sartorius). The proteins in the supernatant were then precipitated using TCA precipitation methods previously described, before pellets were resuspended in 40 μL 5× SDS Loading dye and heat treated at 98°C for 15 minutes. A maximum of 40 μL/well of the re-suspended pellets was loaded onto a NovexTM Wedgewell™ 14% Tris Glycine 1.0 mm mini gel (ThermoFisher Scientific) and run in 1×Novex™ Tris Glycine SDS Running Buffer (ThermoFisher Scientific) using 180V at room temperature. PageRuler™ Plus Prestained Protein Ladder (ThermoFisher Scientific) was loaded at 10 μL/well as a marker. 10 μl E. coli Positive Control Whole cell lysate ab2431 (abcam) was used as His tag positive control. The separated proteins were subject to Western blotting as described preciously and the anti-6XHis primary antibody (1:1000 incubated for 1 h in TBS-T, Abcam) and an HRP-conjugated anti-mouse IgG secondary antibody (1:5000 incubated for 1 h in TST-T, Sigma Aldrich) were used. The secreted protein profile can be seen compared to CHNR-0 wild type in Figure 14. The further cleavage of the peptidoglycan-associated nucleocapsid polyepitope into several peptides that are then released into the extracellular environment may be due to the pH basic nature of the protein, and the exposure of multiple epitopes from the CoV-2-Nuc-His antigen may increase the immunogenicity of the candidate leading to a better immune response. Immunogenicity testing In vivo immunisation experiments will be performed to assess delivery of antigen and induction of immune responses, with a focus on cellular responses, using oral delivery of spores of engineered C. butyricum to express cell wall-associated SARS-CoV-2-X- His antigens (Table 8). For assessment of antigen-specific cellular immune responses, C57BL/6 mice will be administered an immunisation regimen as described herein. For example, the mice may be administered 1 x 108 CFU/dose orally in 3 doses, 2 weeks apart. Alternatively, mice may be administered the dose orally in two or more immunisations comprising two or more consecutive days, with intervals of one or more days in between immunisations. Groups of mice will be administered individual strains selected from a wild-type CHNR-0 strain (negative control) or the CHNR-0 vaccine strain expressing the antigens from the pMTL82121-p0957-LysM-SARS-CoV-2-X-His antigen series. A positive control group will be administered recombinant spike protein, nucleocapsid protein or a polyepitope protein of membrane, spike, envelope, nucleocapsid and Orf1ab protein antigens via subcutaneous or i.p. injection (+ adjuvant) as a positive control. Clinical observations will be taken throughout to determine tolerability of the test articles (weight changes and physical appearances such as hunching or coat piloerection). Table 8: Dosing groups for CHN-based oral vaccines against SARS-CoV-2
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At sacrifice, spleens will be harvested and processed to a single cell suspension and CD4+ and CD8+ cells purified individually to determine CD4+/CD8+-specific T cell response via IFN-Υ release in ELISPOT assays following antigen restimulation using the appropriate control protein or CHNR-0 vegetative cells and detected using the Mouse IFN-Υ T-cell ELISPOT kit (U-CyTech Bioscience). Expected results We expect the ELISPOT assays of CD4+/CD8+ T-cells to show mice immunised with the CADD strain expressing the extracellular SARS-CoV-2 antigens to develop an antigen- specific T-cell response, with a stronger emphasis on the CD4+ response. Importantly, we do not expect to see mice immunised with the CHNR-0 wild type strain developing a T cell response. A detectable CD4+ T-cell response would likely indicate a stronger, more long-lasting, and more specific vaccine response. Example 4 – Constructing strains for expression of Campylobacter peptide antigens by Clostridium Two Campylobacter antigens – FlaA and FliD – were selected for expression by Clostridia. FlaA is the major component of flagellin, and FliD is the flagellar cap protein. Both FlaA and FliD are readily accessible on the surface of Campylobacter bacteria; and have previously been shown to comprise T-cell and/or B-cell epitopes (Yasmin et al, 2016, In silico pharmacol., 4:5 (DOI: 10.1186/s40203-016-0020-y); Lee et al., 1999, Infect. Immun., 67(11):5799-5805 (DOI: 10.1128/iai.67.11.5799-5805.1999); Chintoan-Uta et al., 2019, Vaccine, 34(15):1739-1743 (DOI: 10.1128/iai.67.11.5799- 5805.1999). Two LysM-FlaA-His constructs and two LysM-FliD-His constructs (Figure 14) were designed for extracellular expression of C. jejuni antigenic epitopes, denoted LysM-FlaA-recombinaint (recombinant FlaA, SEQ ID NO: 114), LysM-FlaA-polyepitope (immunogenic epitopes determined in the FlaA protein sequence, SEQ ID NO: 126), LysM-FliD-recombinant (recombinant FliD, SEQ ID NO: 115), and LysM-FliD- polyepitope (Immunogenic epitopes determined in the FliD sequence, SEQ ID NO: 127). Chosen epitopes were modified by connection of the individual epitopes in some cases via a Cathepsin S cleavage sequence (TVKLRQ (SEQ ID NO: 39) (in LysM-FlaA- polyepitope, LysM-FliD-polyepitope) and all antigens had a C-terminal 6xHis tag (HHHHHH (SEQ ID NO: 47)) added for expression identification and purification. The LysM peptidoglycan-anchoring domain sequence (SEQ ID NO: 42) was also added upstream of all Campylobacter antigens for extracellular localisation and the LysM domain was separated from the Campylobacter antigens by a flexible [Gly]4Ser linker. Further modifications included for genetic engineering include two NdeI cleavage sites (CATATG) either side of the LysM encoding domain incorporating the nucleotide signal for aa methionine (M, ATG) and allowing removal of the LysM domain, and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the His-tag by the stop codon TAA. The LysM-FlaA-His and LysM-FliD-His antigenic constructs will be codon optimised for genetic engineering into C. butyricum and synthesised behind the p0957 promoter by GeneWiz and cloned into pMTL82121 vector submitted to GeneWiz for subcloning. Plasmids will be transformed into NEB® Stable Competent E. coli (New England Biolabs) for propagation and storage as set out in Examples 1-3. Sequence confirmed pMTL82121_p0957-LysM-FlaA-His and pMTL82121_p0957-LysM- FliD-His plasmids will be transformed into E. coli CA434 conjugation donors as described above and conjugated into C. butyricum CHNR-0 (an identical strain to CHN- 0) for plasmid-based expression as described above. Immunisation experiments will be performed as set out in Examples 1-3, with comparable results expected. Example 5 – Materials and Methods Culture of bacterial strains Escherichia coli strains NEB® 10-beta Competent, NEB® Stable Competent, and CA434 were grown aerobically in Lysogeny broth (LB; Vegetable tryptone 10g/L, Yeast extract 5g/L, Sodium chloride 10g/L) supplemented with 15% (w/V) agar and/or antibiotics where appropriate at 30°C or 37°C depending on metabolic burden associated with plasmid propagation. Liquid cultures were agitated at 200 rpm during incubation. Clostridium butyricum Strain DSM10702 (CHN-0) is deposited in the DSMZ depository (Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, GERMANY). CHNR-0 is deemed an identical strain by NCIMB sequencing. Clostridium butyricum strains were routinely grown in anoxic workstations (Don Whitley, 10% Hydrogen, 10% Carbon dioxide, 80% Nitrogen, 37°C) in Reinforced Clostridial growth medium (RCM; Yeast extract 13g/L, Vegetable peptone 10g/L, Soluble starch 1g/L, Sodium chloride 5g/L, Sodium acetate 3g/L, Cysteine hydrochloride 0.5g/L) supplemented with 10g/L Calcium carbonate, 2% (w/V) Glucose, 15% (w/V) agar and/or antibiotics where appropriate. For maintenance and selection of genetically engineered strains where genes have been integrated into the genome, C. butyricum was grown in anoxic workstations in Clostridial Basal Medium (CBM, Iron sulphate heptahydrate 12.5mg/L, Magnesium sulphate heptahydrate 250mg/L, Manganese sulphate tetrahydrate 12.5mg/L, Casamino acids 2g/L, 4- aminobenzoic acid 1.25mg/L, Thiamine hydrochloride 1.25mg/L, Biotin 2.5μg/L) supplemented with 10g/L Calcium carbonate, 2% (w/V) Glucose, 15% (w/V) agar, uracil and/or antibiotics where appropriate, respectively. For growth in low-protein broths for assessment of protein secretion, strains were grown in Fermentation Media for Clostridia (FMC) broth (2.5 g/L yeast extract, 2.5 g/L Tryptone, 0.025 g/L iron (II) sulphate heptahydrate (FeSO4.7H2O), 0.5 g/L Ammonium sulphate (NH4)2SO4)) supplemented with 10g/L Calcium carbonate, 2% (w/V) Glucose, and antibiotics where appropriate. C. butyricum spores were produced in 2L vessels of FerMac 320 Microbial culture batch bioreactor systems (ElectroLab Biotechnology Ltd) in RCM supplemented with 2% (w/V) Glucose. Vessels were sparged with nitrogen gas at a flow rate of 0.2 vvm, maintained at a pH of 6.5, temperature of 37°C and agitated at 100 rpm. Cell and spore mass were harvested, and spores were separated from cell matter by repeated washing in ice-cold sterile water. Spores were stored at 4°C until further use. Enumeration of spores was conducted by plating serial dilutions of spore stocks on pre- reduced RCM agar plates in triplicate. Plates were incubated for 24 hours in the anoxic workstation before colony forming units (CFU) were determined. Sequences of the disclosure
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Claims

Claims 1. A bacterium of the class Clostridia comprising a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen; and wherein the at least one antigen comprises an infectious agent antigen or a tumour antigen; and wherein the bacterium is of a non-pathogenic species.
2. The bacterium of Claim 1, wherein the antigen is a surface-tethered antigen which is tethered to the bacterial cell wall.
3. The bacterium of Claim 2, wherein the antigen is expressed as a precursor comprising an N-terminal signal peptide; and wherein the surface-tethered antigen comprises a domain or motif required for tethering to peptidoglycan, optionally wherein the domain or motif is a peptidoglycan anchoring domain or a sequence required for enzymatic linkage to peptidoglycan.
4. The bacterium of Claim 3, wherein the peptidoglycan anchoring domain comprises at least one LysM motif, such as a LysM domain, optionally wherein the LysM domain is a Clostridium LysM domain, such as a Clostridium butyricum LysM domain.
5. The bacterium of Claim 1, wherein the antigen is a secreted antigen.
6. The bacterium of Claim 5, wherein the secreted antigen is expressed as a precursor comprising an N-terminal signal peptide, suitably a Sec secretion system signal peptide, suitably Usp45.
7. The bacterium of any preceding claim, wherein the at least one antigen comprises the complete amino acid sequence of a mature polypeptide which is an infectious agent antigen or a tumour antigen; or a fragment or variant thereof.
8. The bacterium of any preceding claim, wherein the at least one antigen comprises one or more B cell antigen segments and/or one or more T-cell antigen segments.
9. The bacterium of Claim 8, wherein the one or more T-cell antigen segments are CD4+ T-cell antigen segments and/or CD8+ T-cell antigen segments.
10. The bacterium of any one of Claims 1 to 6, wherein the at least one antigen comprises one or more B cell antigen segments and/or one or more T-cell antigen segments and is a multi-antigen fusion polypeptide comprising two or more antigen segments, such as three or more, five or more or 10 or more antigen segments; optionally wherein the multi-antigen fusion polypeptide comprises at least one CD4+ T-cell antigen segment and at least one CD8+ T-cell antigen segment; optionally wherein the antigen segments are partially overlapping, and in combination encompass ^40%, ^50, ^60%, ^70%, ^80%, ^90%, more preferably 100% of the amino acid sequence of the antigen from which they are derived.
11. The bacterium of any preceding claim, wherein the infectious agent antigen is a viral antigen, a bacterial antigen, a parasite antigen, a prion antigen, a helminth antigen, a nematode antigen, a protozoan antigen, fungal antigen, or any combination thereof.
12. The bacterium of any preceding claim, wherein the infectious agent antigen is from an infectious agent which is capable of causing gastroenteritis in a susceptible host, optionally a virus such as a Human Rotavirus; a bacterium such as Vibrio cholerae, Campylobacter jejuni, Escherichia coli (including but not limited to ETEC, EHEC, EIEC, EPEC, EAEC, and AIEC), Salmonella sp. (including but not limited to S. enterica and subspecies including S. e. enterica, S. e. salamae, S. e. arizonae, S. e. diarizonae, S. e. houtenae, S. e. indica, S. enterica serovar Typhi, S. enterica serovar Typhimurium, S. enterica serovar Paratyphi; and S. bongori); Shigella sp. (including S. flexneri, S. sonnei, S. boydii, and S. dysenteriae), Clostridium difficile, or Clostridium perfringens; or a protozoan such as Cryptosporidium parvum, Giardia duodenalis, or Entamoeba histolytica.
13. The bacterium of Claim 12, wherein the antigen comprises a Vibrio cholerae antigen, optionally derived from a cholera toxin, optionally CtxB, optionally wherein (a) the V. cholerae antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of CtxB, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 110, SEQ ID NO: 150, or a portion of SEQ ID NO: 150 comprising SEQ ID NO: 110; or wherein the antigenic portion comprises or consists of SEQ ID NO: 147, 149 or 29, or a portion of SEQ ID NO: 29 comprising SEQ ID NO: 147 or 149; and/or (b) the antigen comprising a V. cholerae antigen is a surface-tethered antigen and comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 231 of SEQ ID NO: 30; and/or wherein the precursor of the surface-tethered antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 231 of SEQ ID NO: 30.
14. The bacterium of Claim 12, wherein the antigen comprises a Human Rotavirus (HRV) antigen, optionally derived from any one or more HRV structural proteins, optionally VP8 and/or VP7, optionally wherein: the HRV antigen comprises a VP8 antigen and: (a) the HRV antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of VP8, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 78, SEQ ID NO: 155, SEQ ID NO: 25, or a portion of SEQ ID NO: 25 comprising SEQ ID NO: 78 or SEQ ID NO: 155; and/or (b) the antigen comprising a HRV antigen is a secreted antigen and the precursor comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to amino acid residues 1 to 188 of SEQ ID NO: 26; or the HRV antigen comprises a VP7 antigen and: (c) the HRV antigen comprises one, 2, 3 or 4 copies of one, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the VP7 epitopes of SEQ ID NOs: 67-71 or 156-161 or variants or fragments comprising at least 90% sequence identity thereto; and a linker or protease cleavage site between each epitope, such as wherein the VP7 antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 21 to 314 of SEQ ID NO: 77; and/or (d) the antigen comprising a HRV antigen is a secreted antigen and the precursor comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 314 of SEQ ID NO: 77.
15. The bacterium of any one of Claims 1 to 11, wherein the infectious agent antigen is from an infectious agent which is capable of causing a respiratory tract infection in a susceptible host, optionally a virus such as a coronavirus, such as SARS-Cov-2; or respiratory syncytial virus (RSV). 16. The bacterium of Claim 15, wherein the wherein the antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of S, E, M, N or Orf1ab protein of SARS-CoV-2. 17. The bacterium of Claim 16, wherein: (a) the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of S, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 6, 8, 10, 162, 178 or 163, or a portion of SEQ ID NO: 4 or 3 comprising SEQ ID NO: 6, 8, 162, 178 or 163; or a portion of SEQ ID NO: 5 or 3 comprising SEQ ID NO: 10 and/or (b) the antigen comprising a SARS-CoV-2 antigen is a surface-tethered antigen and comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 401 of SEQ ID NO: 7, or amino acid residues 22 to 339 of SEQ ID NO: 9, or amino acid residues 22 to 273 of SEQ ID NO: 11; and/or wherein the precursor of the surface- tethered antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 401 of SEQ ID NO: 7, or 1 to 339 of SEQ ID NO: 9. 18. The bacterium of Claim 16, wherein: (a) the SARS-CoV-2 antigen comprises at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the epitopes of SEQ ID NO: 81-91, 93 to 105, or variants or fragments comprising at least 90% sequence identity thereto; and a protease cleavage site between each epitope, optionally wherein the protease cleavage site is a cathepsin S cleavage site, such as wherein the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 80 or 92; or (b) the SARS-CoV-2 antigen is a surface-tethered antigen and comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 22 to 496 of SEQ ID NO: 13; and/or wherein the precursor of the surface-tethered antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 496 of SEQ ID NO: 13; or (c) the SARS-CoV-2 antigen is a surface-tethered antigen and comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 474 of SEQ ID NO: 17; and/or wherein the precursor of the surface-tethered antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 474 of SEQ ID NO: 17. 19. The bacterium of Claim 16, wherein the SARS-CoV-2 antigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the antigenic portions of SARS-CoV-2 present in SEQ ID NO: 106, or fragments or variants comprising at least 90% sequence identify thereto, and a protease cleavage site between each antigenic portion, such as a cathepsin S cleavage site, such as LRMK; such as wherein the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 106. 20. The bacterium of Claim 12, wherein the antigen is a Campylobacter antigen, optionally derived from any one or more C. jejuni proteins, optionally derived from FlaA or FliD, optionally wherein: (a) the C. jejuni antigen comprises a FlaA antigen and: (i) the C. jejuni antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of FlaA, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 113; and/or (ii) the antigen comprising a C. jejuni antigen is a surface-tethered antigen and comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 780 of SEQ ID NO: 114; and/or wherein the precursor of the surface-tethered antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 780 of SEQ ID NO: 114; or (b) the C. jejuni antigen comprises a FlaA antigen and: (iii) the C. jejuni antigen comprises one, 2, or 3 copies of one, 2, 3, or 4 of the FlaA epitopes of SEQ ID NOs: 117-120 or variants or fragments comprising at least 90% sequence identity thereto; and a linker or protease cleavage site between each epitope, such as wherein the FlaA antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 142; and/or (iv) the antigen comprising a C. jejuni antigen is a surface-tethered antigen and the precursor comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 454 of SEQ ID NO: 126; or (c) the C. jejuni antigen comprises a FliD antigen and: (v) the C. jejuni antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of an antigenic portion of FlaA, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 115; and/or (vi) the antigen comprising a C. jejuni antigen is a surface-tethered antigen and comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or at least 99.5% identical to an amino acid sequence of amino acid residues 22 to 846 of SEQ ID NO: 116; and/or wherein the precursor of the surface-tethered antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 1 to 846 of SEQ ID NO: 116; or (d) the C. jejuni antigen comprises a FliD antigen and: (vii) the C. jejuni antigen comprises one, 2, 3, or 4 copies of one, 2, 3, 4, or 5 of the FliD epitopes of SEQ ID NOs: 121-125 or variants or fragments comprising at least 90% sequence identity thereto; and a linker or protease cleavage site between each epitope, such as wherein the FliD antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 143; and/or (viii) the antigen comprising a C. jejuni antigen is a surface-tethered antigen and the precursor comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of amino acid residues 22 to 400 of SEQ ID NO: 127. 21. A bacterium of the class Clostridia comprising a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2; and wherein the bacterium is of a non-pathogenic species. 22. The bacterium of Claim 21, wherein: (a) the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of S1, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 6, 8, 162, 178 or 163, or a portion of SEQ ID NO: 4 comprising SEQ ID NO: 6, 8, 162, 178 or 163; or (b) the antigen comprising a SARS-CoV-2 antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to an amino acid sequence of an antigenic portion of S2, optionally wherein the antigenic portion comprises or consists of SEQ ID NO: 10, or a portion of SEQ ID NO: 5 comprising SEQ ID NO: 10. 23. The bacterium of Claim 21, wherein the SARS-CoV-2 antigen comprises at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16,
17,
18,
19,
20,
21,
22,
23 or 24 of the epitopes of SEQ ID NO: 81-91, 93 to 105, SEQ ID NO: 162-180, or variants or fragments comprising at least 90% sequence identity thereto; and a protease cleavage site between each epitope, optionally wherein the protease cleavage site is a cathepsin S cleavage site, such as wherein the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 80 or 92.
24. The bacterium of Claim 21, wherein the SARS-CoV-2 antigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the antigenic portions of SARS-CoV-2 present in SEQ ID NO: 106, or fragments or variants comprising at least 90% sequence identify thereto, and a protease cleavage site between each antigenic portion, such as a cathepsin S cleavage site, such as LRMK; such as wherein the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 106.
25. The bacterium of any preceding claim, wherein the amount of antigen expressed per cell weight of clostridial cells undergoing anaerobic cell growth is greater than 10 ng/mg, 20 ng/mg or 40 ng/mg and up to 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 ng/mg, 1μg/ mg, 1.5, 2.0, 2.5, 5.0, 10 or 20 μg/ mg dry cell weight, such as from 10 to 400 ng/ mg dry cell weight; 20 to 200 ng/ mg dry cell weight; 40 to 100 ng/ mg dry cell weight; 100 ng to 5 μg/ mg dry cell weight; 200 ng to 2.5 μg/ mg dry cell weight; 400-1500 ng/ mg dry cell weight; or about 800 ng/ mg dry cell weight.
26. The bacterium of any preceding claim, wherein the heterologous nucleic acid molecule is integrated into the genome as a single copy or on a low copy plasmid or on a high copy plasmid.
27. The bacterium of any preceding claim, wherein the bacterium comprises a further heterologous nucleic acid molecule encoding an immunostimulatory agent or adjuvant, which is capable of being co-expressed with the antigen; and/or wherein the bacterium is capable of producing short-chain fatty acids (SCFAs) such as butyrate.
28. The bacterium of any preceding claim, wherein the bacterium is from cluster I, IV and/or XlVa of Clostridia, such as wherein the bacterium is from the genus Clostridium, such as wherein the bacterium is Clostridium butyricum.
29. The bacterium of any preceding claim, wherein (i) the bacterium is not capable of colonising the gastrointestinal tract; and/or (ii) the bacterium in not capable of binding to or degrading mucin; (iii) the bacterium is capable of growing in an anaerobic section of the lower gastrointestinal tract; and/or (iv) the bacterium is saccharolytic and can utilise di- and tri-saccharides in the colon.
30. The bacterium of any preceding claim in the form of a spore or a vegetative cell.
31. A pharmaceutical composition comprising bacteria as defined in any preceding claim, optionally wherein the pharmaceutical composition is added to a food or to a beverage.
32. A bacterium as defined in any of Claims 1 to 30 or pharmaceutical composition as defined in Claim 31 for use in medicine.
33. A bacterium of the class Clostridia for use in generating an antigen-specific immune response in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen; and wherein the bacterium is of a non-pathogenic species.
34. A bacterium of the class Clostridia for use in generating an antigen-specific immune response in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2; and wherein the bacterium is of a non-pathogenic species.
35. The bacterium for use of Claim 33, wherein the bacterium is as defined in any one of Claims 1 to 20; or Claims 25 to 29 insofar as they depend from Claims 1 to 20.
36. The bacterium for use of Claim 34, wherein the bacterium is as defined in any one of Claims 21 to 24; or Claims 25 to 30 insofar as they depend from Claims 21 to 24.
37. The bacterium for use of any of Claims 33 to 36, wherein the antigen-specific immune response is a B cell response; and/or is a cell-mediated immune response, such as a CD4+ or CD8+ T-cell response.
38. A bacterium of the class Clostridia for use in the preventive or therapeutic treatment of a disease in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of exporting the antigen such that it becomes tethered to the surface of the bacterium as a surface-tethered antigen, or wherein the bacterium is capable of secreting the antigen as a secreted antigen; wherein the antigen is an infectious agent antigen and the disease is the infectious disease, or the antigen is a tumour antigen and the disease is cancer; wherein the bacterium is of a non-pathogenic species.
39. A bacterium of the class Clostridia for use in the preventive or therapeutic treatment of COVID-19 in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule; wherein the heterologous nucleic acid molecule comprises at least one antigen gene comprising a region encoding at least one antigen and a promoter operably linked to said region, which promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth; wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium; and wherein the at least one antigen is a SARS-CoV-2 antigen comprising an amino acid sequence derived from any one or more of the S1 subunit of the S protein, the S2 subunit of the S protein, or the E, M, N, or Orf1ab protein of SARS-CoV-2; and wherein the bacterium is of a non-pathogenic species.
40. The bacterium for use of any one of Claims 33 to 39, wherein the bacterium is for administering orally.
41. The bacterium for use of Claim 40, wherein the bacterium is in the form of a spore or in the form of the pharmaceutical composition of Claim 31.
42. The bacterium for use of any one of Claims 33 to 41, wherein the subject is human.
43. A method for preparing a bacterium as defined in any one of Claims 1 to 30 comprising introducing the heterologous nucleic acid molecule into the bacterium.
44. A method for preparing a pharmaceutical composition as defined in Claim 31, comprising formulating the bacteria with one or more pharmaceutically acceptable diluents or excipients.
45. A nucleic acid molecule suitable for propagation in a bacterium of the class Clostridia comprising an antigen gene comprising a region encoding at least one surface-tethered antigen precursor and a promoter operably linked to said region, which promoter is capable of causing expression of the surface-tethered antigen precursor by a bacterium of the class Clostridia; wherein the antigen precursor comprises an N-terminal signal peptide and a domain or motif required for tethering of the antigen to peptidoglycan in a bacterium of the class Clostridia.
46. The nucleic acid molecule of Claim 45 wherein the domain or motif is a peptidoglycan anchoring domain or a sequence required for enzymatic linkage to peptidoglycan, such as wherein the peptidoglycan anchoring domain comprises at least one LysM motif, such as a LysM domain, optionally wherein the LysM domain is a Clostridium LysM domain, such as a Clostridium butyricum LysM domain.
47. The nucleic acid molecule of Claim 45 or 46 wherein the promoter is capable of causing expression of the antigen by the bacterium during anaerobic cell growth, such as wherein the promoter is a p0957 promoter of C. acetobutylicum, fdx promoter of C. perfringens, the ptb, thl hbd, crt, etfA, etfB or bcd promoter of C. acetobutylicum and/or wherein the nucleic acid molecule is suitable for propagation in a bacterium of the class Clostridia by virtue of comprising a traJ conjugal transfer function and/or a pBP1 Gram+ replicon.
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