COMPOSITIONS AND METHODS

Abstract

A bacterium of the class Clostridia comprising a heterologous nucleic acid molecule encoding at least one antigen, wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium during anaerobic cell growth, and wherein at least one antigen is an infectious agent antigen or a tumour antigen.

Claims

1. A bacterium of the class Clostridia comprising a heterologous nucleic acid molecule encoding at least one antigen, wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium during anaerobic cell growth, a wherein the at least one antigen is an infectious agent antigen or a tumour antigen, and wherein the bacterium is of a species which is not a pathogenic Clostridium species.

2. The bacterium of claim 1, wherein the at least one antigen comprises one or more T-cell antigen segments and/or one or more B cell antigen segments.

3. The bacterium of claim 2, wherein the one or more T-cell antigen segments are CD4.sup.+ T-cell antigen segments and/or CD8.sup.+ T-cell antigen segments.

4. The bacterium of claim 1, wherein the at least one antigen 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.sup.+ T-cell antigen segment and at least one CD8.sup.+ T-cell antigen segment.

5. The bacterium of claim 4, 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.

6. The bacterium of claim 1, 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, 1 g/mg, 1.5, 2.0, 2.5, 5.0, 10 or 20 g/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 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.

7. The bacterium of claim 1, 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.

8. The bacterium of claim 1, 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.

9. The bacterium of claim 1, wherein the infectious agent antigen is a viral antigen, a bacterial antigen such as a chlamydial antigen or a mycoplasma antigen, a parasite antigen, a prion antigen, a helminth antigen, a nematode antigen, a protozoan antigen, fungal antigen, or any combination thereof.

10. The bacterium of claim 1, wherein the infectious agent antigen is a) an HPV antigen, optionally wherein the HPV antigen comprises the amino acid sequence of SEQ ID NO: 4, or amino acids 1 to 140 of SEQ ID NO: 4, such as wherein the HPV antigen is encoded by nucleotides 19 to 477 of the nucleic acid sequence of SEQ ID NO: 3; or b) a Vibrio cholerae antigen, optionally CtxB, optionally wherein the V. cholerae antigen comprises the amino acid sequence of SEQ ID NO: 21, or amino acids 1 to 104 of SEQ ID NO: 21, or is encoded by nucleotides 270 to 581 of the nucleic acid sequence of SEQ ID NO: 20.

11. The bacterium of claim 1, wherein the bacterium is from cluster I, IV and/or XIVa of Clostridia, such as wherein the bacterium is from the genus Clostridium, such as wherein the bacterium is Clostridium butyricum.

12. The bacterium of claim 1, wherein the bacterium is capable of expressing the antigen as a soluble polypeptide or inclusion body in the bacterial cytoplasm.

13. The bacterium of claim 1 in the form of a spore or a vegetative cell.

14. A pharmaceutical composition comprising the bacterium of claim 1 and a pharmaceutically acceptable carrier, excipient, diluent, or adjuvant.

15. The pharmaceutical composition of claim 14, further comprising capsules comprising spores or vegetative cells of the bacteria, wherein the capsules comprise a delayed-release layer or coating which allows for the release of the spores or vegetative cells in an anaerobic section of the lower gastrointestinal tract following oral administration.

16. (canceled)

17. A method for generating an antigen-specific immune response in a subject comprising administering to the subject an effective amount of a bacterium comprising a heterologous nucleic acid molecule encoding an antigen, wherein the bacterium is capable of expressing the antigen in an intracellular compartment of the bacterium during anaerobic cell growth, and wherein the bacterium is of a species which is not a pathogenic Clostridium species.

18. The method of claim 17, wherein the antigen-specific immune response is a cell-mediated immune response, such as a CD4.sup.+, CD8.sup.+ T-cell response; and/or is B cell response.

19. A method for treating or preventing an infectious disease or cancer in a subject comprising administering an effective amount of the bacterium of claim 1 to the subject.

20. (canceled)

21. The method of claim 17, wherein the bacterium is administered orally.

22. The method of claim 21, wherein the bacterium is in the form of a spore or in the form of a pharmaceutical composition.

23. (canceled)

24. A method for preparing the bacterium of claim 1 comprising introducing the heterologous nucleic acid molecule into the bacterium.

25. (canceled)

Description

FIGURE LEGENDS

[0177] FIG. 1: Design of ROP proteins and presentation of cleaved ROP on antigen presenting cells (APC). A known T-cell epitope is divided into overlapping peptide fragments linked into a single chain protein by the minimal cleavage signal (LRMK (SEQ ID NO: 33)) for cathepsin S. The ROP is cleaved in endosomes inside the APC and individual peptide epitopes are presented via MHC molecules that can be recognised by receptors present on T-cells.

[0178] FIG. 2: Western blot detection of FLAG tag linked to ROP-HPV and ROP-OVA in C. butyricum engineered to express ROP proteins intracellularly. CHN-0 wildtype was used as the control. In A, arrows indicate ROP-HPV bands in CADD-HPV-ROP but not in CHN-0 wild-type cells. (A) Contrast enhanced over entire imagered arrows indicate ROP-HPV bands in CADD-HPV-ROP but not in CHN-0 wild-type. (B) 35 l protein loaded (175 mg), blocked 5% milk, anti-FLAG (A9469) 1:5000 in TBS-T 2 h, developed using SIGMAFAST BCIP/NBT 7 min.

[0179] FIG. 3: CADD-ROP-OVA1 construct for genetic engineering of C. butyricum.

[0180] FIG. 4: CADD-ROP-HPV construct for genetic engineering of C. butyricum.

[0181] FIG. 5: pHRodo fluorescence observed inside DC2.4 cell culture exposed to CHN-0 vegetative cells in a ratio of 1000:1 CHN-0 to DC2.4 cells. Scale bar is 500 m

[0182] FIG. 6: IFN- ELISPOT evaluation of CD8.sup.+ and CD4.sup.+ T-cells isolated from spleens of mice immunised by oral gavage of spores of CHN-0 wildtype (orange bar), engineered CADD-HPV (grey bar), or subcutaneous injection of purified ROP-HPV protein with adjuvant (yellow bar). PBS was given as negative control both orally and subcutaneously (light and dark blue bars). Isolated T-cells were seeded at a density of 2.510.sup.5/well and re-stimulated with either purified ROP-HPV or wildtype HPV protein (both 5 g/well) or vegetative cells of CHN-0 wildtype (0.510.sup.5/well).

[0183] FIG. 7: IFN- ELISPOT evaluation of CD8.sup.+ and CD4.sup.+ T-cells isolated from spleens of mice immunised by oral gavage of spores of CHN-0 wildtype (orange bar), engineered CADD-OVA (grey bar), or subcutaneous injection of purified ROP-OVA protein with adjuvant (yellow bar). PBS was given as negative control both orally and subcutaneously (light and dark blue bars). Isolated T-cells were seeded at a density of 2.510.sup.5/well and re-stimulated with either purified ROP-OVA (5 g/well) or vegetative cells of CHN-0 wildtype (0.510.sup.5/well).

[0184] FIG. 8: A diagram to represent the immunisation strategy of Example 3.

[0185] FIG. 9: (A) Cholera toxin CtxB FLAG-tagged antigen nucleic acid sequence and translated protein sequence. The underlined sequences in the nucleic acid sequence are, in order, the NotI site, the NdeI site and NheI site. The promoter region is between NotI and NdeI and is not translated, whereas the antigen region that is translated is between the NdeI and the NheI sites. (B) Sequence alignment between the native CtxB protein sequence (P01556) and the CtxB FLAG-tagged antigen sequence (CHAIN_CtxB).

[0186] FIG. 10: Western blot detection of FLAG tag linked to CtxB in C. butyricum engineered to express CtxB intracellularly from the pMTL82151 plasmid (CtxB-full plasmid). CHN-0 wildtype was used as the control. The red arrow indicates significant bands in CtxB-expressing CHN-0 strains at expected MW (13 kDa) but not in CHN-0 wild-type. (B) 20 l protein loaded, blocked 5% milk, anti-FLAG (A9469) 1:5000 in TBS-T 2 h, developed using SIGMAFAST BCIP/NBT <3 min.

EXAMPLE 1: CONSTRUCTION AND PRODUCTION OF ENGINEERED CLOSTRIDIUM BUTYRICUM

[0187] Strain DSM10702 of Clostridium butyricum, a spore forming anaerobic bacterium that can be found in soil and animal (including human) faeces, was engineered to express antigen in the bacterial cytoplasm. Selected antigens were engineered based on recombinant overlapping peptide (ROP) technology, as described in WO 2007/125371A2.

[0188] The ROP protein sequence is made up of overlapping peptides linked by the cathepsin cleavage site target sequence (LRMK (SEQ ID NO: 33)) (see FIG. 1). Cathepsin is found in endosomes of dendritic cells (DCs) and following endocytosis the ROP protein is cleaved into its constituent peptides. This approach allows for the delivery of a wide range of T-cell epitopes which can facilitate induction of cellular immunity across a range of HLA alleles maximising population coverage. Effective antigen presentation by DCs is required to prime nave CD8.sup.+ and CD4.sup.+ T-cells. CD8.sup.+ cytotoxic T-cells are involved in immune defence against intracellular pathogens and tumour surveillance. CD4.sup.+ helper T-cells (e.g. Th1, Th2 and Th17) shape and control a wide range of immune functions and play a particularly important role in regulating adaptive immunity. The use of ROPs has been shown previously to be superior to using wildtype antigens in generating protective cellular immunity. In addition to stimulating CD4+ T-cells through the classical MHC class II presentation pathway, ROPs can lead to robust CD8.sup.+ T-cell responses through cross-presentation, a process by which exogenous antigen is presented by MHC class I on APCs to activate CD8.sup.+ T-cells (Cai et al., Oncotarget 2017, 8(44) pp 76516-76524).

[0189] Previously, a strain of C. butyricum was created with a disrupted pyrE gene for use in genetic engineering by ACE technology. We have now stably integrated ROP protein coding sequences under control of a constitutive promoter into the pyrE gene locus in the chromosome of this strain.

[0190] Two different ROP protein coding sequences have been developed, based on Human Papilloma Virus (HPV) type 16 E7 envelope protein and ovalbumin (OVA). These sequences were used to design cassettes for introduction into the pMTL80000 vector series for genetic engineering by introducing the required enzymatic cleavage sites and an additional cathepsin cleavage signal at the N-terminal site linking the FLAG tag to the ROP protein. The engineered pyrE deficient strain of C. butyricum expresses ROP derived from HPV or ovalbumin, intracellularly (see FIG. 2).

[0191] Following confirmation of expression and production of ROP proteins, spores of the engineered strains were produced using a previously developed spore fermentation protocol as well as vegetative cell pellets. The materials were then assessed for in vitro baseline studies in DCs and use for in vivo immunisation experiments in mice.

[0192] Materials and Methods

[0193] Culture of Bacterial Strains

[0194] Escherichia coli strains BL21, DH5 and CA434 were grown aerobically in Lysogeny broth (LB; Vegetable tryptone 10 g/L, Yeast extract 5 g/L, Sodium chloride 10 g/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.

[0195] Clostridium butyricum Strain DSM10702 is deposited in the DSMZ depository (Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstrale 7B, 38124 Braunschweig, GERMANY). 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 13 g/L, Vegetable peptone 10 g/L, Soluble starch 1 g/L, Sodium chloride 5 g/L, Sodium acetate 3 g/L, Cysteine hydrochloride 0.5 g/L) supplemented with 10 g/L Calcium carbonate, 2% (w/V) Glucose, 15% (w/V) agar and/or antibiotics where appropriate. For maintenance and selection of genetically engineered strains, C. butyricum was grown in anoxic workstations in Clostridial Basal Medium (CBM, Iron sulphate heptahydrate 12.5 mg/L, Magnesium sulphate heptahydrate 250 mg/L, Manganese sulphate tetrahydrate 12.5 mg/L, Casamino acids 2 g/L, 4-aminobenzoic acid 1.25 mg/L, Thiamine hydrochloride 1.25 mg/L, Biotin 2.5 g/L) supplemented with 10 g/L Calcium carbonate, 2% (w/V) Glucose, 15% (w/V) agar, uracil and/or antibiotics where appropriate, respectively. For detection of colony forming units in mice faeces, homogenised faecal samples were plated onto modified C. butyricum basal isolation medium (Sodium chloride 0.9 g/L, Calcium chloride 0.02 g/L, Magnesium chloride hexahydrate 0.02 g/L, Manganese chloride tetrahydrate 0.01 g/L, Cobalt chloride hexahydrate 0.001 g/L, Potassium phosphate monobasic 7 g/L, Potassium phosphate dibasic 7 g/L, Iron sulphate 0.01% (w/V), Biotin 0.00005% (w/V), Cysteine hydrochloride 0.5 g/L, Glucose 2% (w/V), Agar 15% (w/V), D-cycloserine 250 mg/L).

[0196] C. butyricum spores were produced in 2 L 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.

[0197] Gene Constructs and Plasmids

[0198] For the ovalbumin construct, the wildtype ovalbumin amino acid (aa) sequence ranging from aa241-aa340 (SMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGIT DVFSSSANLSGISSAESLKISQAVHAAHAEINEAGR; SEQ ID NO: 5) was split into four overlapping sequences and linked by the minimal cathepsin cleavage site (LRMK (SEQ ID NO: 33)) to form a 142aa recombinant overlapping peptide denoted ROP-OVA (SMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMELRMKTEWTSSNVMEERKIKVYLPRMKMEE KYNLTSVLMALRMKKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISLRMKISSAESLKISQA VHAAHAEINEAGR; SEQ ID NO: 6).

[0199] ROP-OVA was further modified for genetic engineering into C. butyricum to include a NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) found in position 2 of the ROP-OVA, a further cathepsin cleavage site at the N-terminal site followed by the signal for the FLAG-tag (DYKDDDDK (SEQ ID NO: 18)) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA (FIG. 3).

[0200] For the Human Papillomavirus type 16 construct, the wildtype E7 protein aa sequence ranging from aa1-aa98 (MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDS TLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP; SEQ ID NO: 7) was split into four overlapping sequences and linked by the minimal cathepsin cleavage site (LRMK (SEQ ID NO: 33)) to form a 140aa recombinant overlapping peptide denoted ROP-HPV (MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEELRMKEQLNDSSEEEDEIDGPAGQAEPDR AHYNIVTFCCKLRMKHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGLRMKIRTLEDLLMGT LGIVCPICSQKP; SEQ ID NO: 8).

[0201] ROP-HPV was further modified for genetic engineering into C. butyricum to include a NdeI cleavage site (CATATG) incorporating the nucleotide signal for aa methionine (M, ATG) found in position 1 of the ROP-HPV, a further cathepsin cleavage side at the N-terminal site followed by the signal for the FLAG-tag (DYKDDDDK (SEQ ID NO: 18)) and the nucleotide sequence for a NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA (FIG. 4).

[0202] ROP-OVA and ROP-HPV constructs were ordered as synthetic genes from GeneArt Thermo Fisher Scientific in pMK vectors.

[0203] CADD-ROP-OVA1 and CADD-ROP-HPV constructs were ordered as synthetic genes without further codon usage optimisation from Life Technologies Ltd in plasmid pMK-RQ. pMK-RQ plasmids containing the synthetic gene constructs were transformed into E. coli DH5, grown over night in LB supplemented with 50 g/mL kanamycin and stored at 80 C. as 15% (V/V) glycerol stocks.

[0204] Expression of ROP Protein Standards in E. coli

[0205] The synthetic ROP-OVA and ROP-HPV constructs were excised from storage plasmids and cloned into BsaI restriction endonuclease linearized plasmid pNIC28-Bsa4 (Structural Genomic Consortium, Oxford) using ligation independent cloning. The vector amplicon was transformed into E. coli BL21 (Thermo Fischer Scientific) following the manufacturer's instructions.

[0206] For ROP-HPV expression, BL21 harbouring pNIC28-Bsa4-ROP-HPV was cultured in LB broth supplemented with 50 g/mL Kanamycin. Protein production was induced using 0.2 mM IPTG. Cell pellets were harvested by centrifugation and resuspended in lysis buffer (PB, 0.5% Triton X-100, 1 mM DTT, pH 8.0). Resuspended cells were subjected to 20 cycles of sonication at 600 W for 5 sec in 7 sec intervals. Inclusion bodies containing the recombinant protein were harvested by centrifugation at 20,000g for 45 min. The inclusion body pellet was resuspended in denaturing buffer (8M urea) and incubated for 2 hr with vigorous shaking. The solution was centrifuged to separate the proteins from debris.

[0207] Supernatant containing the protein fraction was loaded onto a Nickel affinity column (GE Healthcare) and eluted using elution buffer (50 mM PB, 200 mM NaCl, 8M urea, 300 mM imidazole, pH 7.4). Refolding of the purified protein was achieved by gradual dialysis with PBS, pH 7.4.

[0208] For ROP-OVA expression, BL21 harbouring pNIC28-Bsa4-ROP-OVA was cultured in LB broth supplemented with 50 g/mL Kanamycin. Protein production was induced using 0.1 mM IPTG at 18 C. Cell pellets were harvested by centrifugation and resuspended in lysis buffer (50 mM HEPES, 500 mM NaCl, 10% glycerol, 1:30,000 Benzonase, 0.5 mg/mL lysozyme, 0.1% DDM, 0.1% protease inhibitor cocktail, pH 8.0). Resuspended cells were subjected to sonication for 10 min at 35% amplitude for 5 sec in 15 sec intervals. Inclusion bodies containing the recombinant protein were harvested by centrifugation at 20,000g for 45 min. The inclusion body pellet was solubilised in 50 mM HEPES buffer containing 6M guanidine hydrochloride and incubated on ice for 1 hr before filtration through 0.2 m filter.

[0209] The filtrate containing the protein fraction was loaded onto a Ni-NTA affinity column and eluted using elution buffer (50 mM HEPES, 6M guanidine hydrochloride, 500 mM imidazole). Guanidine hydrochloride was removed by dilution in cold dilution buffer (50 mM HEPES, 500 mM NaCl, 10% glycerol, 0.5% sarkosyl) followed by concentration of protein using a 10 kDa molecular weight cut off Vivaspin column (Sigma Aldrich) and desalting through a PD-10 column using desalting buffer (50 mM HEPES, 500 mM NaCl, 10% glycerol).

[0210] Endotoxin was removed using the Pierce Endotoxin removal kit (Thermo Fisher Scientific) according to manufacturer's instructions. Samples were filtered using a 0.2 M filter and stored at 4 C. until further use.

[0211] Genetic Engineering of C. butyricum

[0212] To prepare plasmids for engineering of C. butyricum, CADD-ROP-OVA1 and CADD-ROP-HPV constructs were first propagated in pMK-RQ in E. coli DH5. The plasmid was extracted using the Wizard Plus SV Miniprep DNA Purification kit (Promega) following the manufacturer's instructions and constructs were cut from the plasmids using restriction endonucleases NdeI and NheI in CutSmart buffer (all New England Biolabs Inc) according to the manufacturer's instructions. The isolated cassettes were introduced into pMTL83151 (pCB102 Gram+ replicon, catP antibiotic marker, ColE1 Gram-replicon, traJ conjugal transfer function, and multiple cloning site (MCS)) additionally containing a pyrE repair cassette and the constitutive promoter P.sub.fdx in front of the MCS. Plasmids were transformed into E. coli DH5 for propagation. Plasmids were isolated as before and sequenced using GeneWiz sequencing services to confirm the correct insertion of cassettes.

[0213] Sequence confirmed plasmids pMTL83151_pyrErepair_P.sub.fdx_CADD-ROP-OVA1 and pMTL83151_pyrErepair_P.sub.fdx_CADD-ROP-HPV 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.

[0214] Fresh colonies of revived E. coli CA434 harbouring the respective plasmids were used to inoculate LB broth supplemented with 50 g/mL Kanamycin and 12.5 g/mL Chloramphenicol. After overnight incubation, cultures were used to inoculate fresh supplemented medium 1:10 and incubated until an OD.sub.600 of 0.5-0.7 was reached. A volume of 1 mL of culture was removed and centrifuged at 5,000g for 3 minutes. The supernatant was discarded, and the pellet re-suspended in 500 L phosphate buffered saline (PBS) solution. The culture was centrifuged as above, and the supernatant discarded.

[0215] Fresh colonies of revived C. butyricum CHN-0.1 (pyrE derivative of wt CHN-0) were used to inoculate a serial dilution series in fresh pre-reduced RCM broth supplemented with 2% glucose and 1% CaCO.sub.3. 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 OD.sub.600 of 0.5-0.7 was reached. A volume of 1 mL of culture was removed and heat treated for 10 min at 50 C.

[0216] Both E. coli CA434 and C. butyricum such treated were transferred into the anoxic workstation and mixed at a ratio of 5:1 (OD.sub.600:OD.sub.600). 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 mutants with restored uracil prototrophy, thiamphenicol resistant colonies were patch plated reiteratively onto CBM agar plates and cross-checked for plasmid loss on thiamphenicol containing selective RCM agar plates. 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 CADD-ROP cassettes in the chromosome of C. butyricum using primers spanning the integration region, the promoter and respective ROP sequence (Table 3).

TABLE-US-00001 TABLE3 PrimersusedforsequenceconfirmationofCADD-ROPcassettes. CH142 F GTGTAGTAGCCTGTGAAATAAG(SEQIDNO:9) ForwardofPfdx CH324 R CCCATGTTGGATCTCCTGAG(SEQIDNO:10) GenomicDNA downstreamofpyrE CH332 F GCAAGTGCGGTGCAGATTGG(SEQIDNO:11) GenomicDNA upstreamofpyrE CH620 R TTACTTATCGTCGTCATCCTTGTAATCTTTCATTCTT FLAGtag AA(SEQIDNO:12) CH647 R AAATATTAACAAGTAATGATTATCCAAAAC(SEQ pyrErepairlong IDNO:13) homologyarm CH654 R GCAATGTAGGTGTATCTCCATGCATATGTAACACA Pfdx-overhang CCTCCTTAAAAA(SEQIDNO:14) intoHPV-ROP CH655 F TTTAAGGAGGTGTGTTACATATGCATGGAGATACA HPV-ROP- CCTAC(SEQIDNO:15) overhangofPfdx CH656 R CATCAGGCAACAGCACCAACATATGTAACACACCT Pfdx-overhangof CCTTAAAAA(SEQIDNO:16) OVA1 CH657 F TTTAAGGAGGTGTGTTCATATGTTGGTGCTGTTGC OVA1-overhangof CTGATG(SEQIDNO:17) Pfdx

[0217] The integration of the ROP cassette into the chromosome introduced a single copy under the control of a constitutive promoter. This leads to a low expression and production of protein inside the cell, which can be adjusted by use of stronger promoters and/or insertion of multiple copies of the gene.

[0218] Confirmation of Expression of ROPs in C. butyricum

[0219] Fresh colonies of revived C. butyricum CHN-2 (CADD-ROP-HPV) and CHN-3 (CADD-ROP-OVA1) were used to inoculate fresh pre-reduced supplemented RCM broth in serial dilution and grown overnight. The most diluted culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth at a starting OD.sub.600 of 0.05. When cultures were grown to an OD.sub.600 of 1, 2, and 4, and after 24 hr incubation, the equivalent of OD.sub.600 of 1/mL was centrifuged at 13,000g for 2 min. The pellet was re-suspended in 45 L 5SDS 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, 10 mL dH.sub.2O, Bromophenol blue) and heat treated at 98 C. for 15 minutes.

[0220] A maximum of 40 L/well of the re-suspended pellets was loaded onto a Novex WedgeWell 12% Tris Glycine mini gel (Thermo Fischer Scientific) and run in 1SDS buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS) using 200V at room temperature. PageRuler pre-stained protein ladder (Thermo Fischer Scientific) was loaded at 5 L/well as marker and the E. coli Positive Control Whole cell lysate ab5395 (abcam) was used as FLAG tag positive control in a 1:5 dilution.

[0221] Separated protein were blotted onto PVDF membranes using the Tran-Blot Turbo blotting system (BioRad) with the Trans-Blot Turbo 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 (50 mM Tris hydrochloride, 150 mM 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 (50 mM 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.

EXAMPLE 2: PHAGOCYTOSIS OF C. BUTYRICUM BY A DENDRITIC CELL LINE AND INDUCTION OF CYTOKINE RESPONSES

[0222] Baseline studies in murine DC2.4 cell culture showed that these cells can phagocytose vegetative cells and spores of C. butyricum, a prerequisite for successful delivery of the ROP proteins expressed within these bacterial cells.

[0223] Cell cultures of DC2.4 cells were exposed to vegetative cells and spores of the wildtype strain CHN-0. CHN-0, in either vegetative or spore form, was taken up by phagocytosis into the DC2.4 cells (see FIG. 5).

[0224] The cytokine profile of these exposed DC2.4 cell cultures was subsequently assessed using R&D systems Proteome Profiler Mouse cytokine Array Panel (Table 1 and 2). There was a differential response to CHN-0 and the medium control.

TABLE-US-00002 TABLE 1 Spot density in Proteome Profiler Mouse cytokine panel Cytokine CHN0 Media BLC 279 1 C5/C5a 37 19 G-CSF 1342 1 GM-CSF 181 1 I-309 778 17 Eotaxin 86 51 sICAM-1 13831 4050 IFN- 355 41 IL-1 150 3 IL-1 99 311 IL-1ra 941 425 IL-2 109 1 IL-3 1 1 IL-4 153 8 IL-5 165 132 IL-6 1 192 IL-7 94 1 IL-10 1 1 IL-13 448 318 IL-12 164 467 p70 IL-16 304 370 IL-17 21 178 IL-23 53 346 IL-27 1 1 IP-10 13025 1126 I-TAC 40 1 KC 1 66 M-CSF 1360 33 JE 17948 11165 MCP-5 6375 328 MIG 3907 1 MIP-1 24962 14433 MIP-1 26644 13591 MIP-2 13606 700 RANTES 7295 143 SDF-1 4546 317 TARC 125 1 TIMP-1 203 147 TNF 9000 9 TREM-1 52 1

TABLE-US-00003 TABLE 2 Selected cytokines and their function. Cytokine Function G-CSF Differentiation and activation of granulocytes C5/C5a Part of complement system - involved in Chemotaxis and formation of complement membrane attack complex Eotaxin Chemoattractant for eosinophils, basophils and Th2 lymphocytes; Th2 allergic response - Chemotaxis of eosinophils IFN- Th1 - Activator of macrophages and induced of MHC-II expression IL-1B Key mediator of inflammation, leucocyte activation factor IL-3 Differentiation of HSCs into myeloid progenitor and lymphoid progenitor cells IL-4 Regulates maturation of nave T helper cells into Th2; Differentiation of nave T-cells to Th2 cells IL-6 Important mediator of acute phase response, antagonistic to Treg IL-10 Anti-inflammatory cytokine, down-regulated Th1 and MHC Class II expression IL-12 Naturally produced by DC in response to antigen, differentiation of nave T cells to Th1 IL-17 Mediates pro-inflammatory responses IL-23 Proinflammatory cytokine, involved in Th17 maintenance and expansion IL-27 Member of IL-12 family - differentiation of Th1 and inhibition of Th2 cells. Can also promote anti- inflammatory IL-10 production I-TAC Chemotactic for T lymphocytes; Interferon-inducible T-cell alpha chemoattractant KC Chemotactic for T neutrophils MIG Migration, differentiation and activation of CTLs (CD8+), NK cells and macrophages TARC Inducer of chemotaxis in T cells

[0225] From these preliminary experiments, it was concluded that the CHN-0 wildtype strain can trigger the release of cytokines from cultured DC2.4 cells when these are exposed to either vegetative cells or spores. These cytokines seem to be associated with leukocyte recruitment (NK cells), activation of innate and adaptive immunity.

[0226] Materials and Methods

[0227] Cell Line

[0228] DC2.4 cells (ATCC Number: CRL-11904) were maintained in RPMI1640 medium supplemented with 100/(V/V) foetal calf serum, 1MEM non-essential amino acid and 11M HEPES buffer solution (all Sigma Aldrich) at 37 C. under 50/C02.

[0229] Phagocytosis Assay

[0230] DC2.4 were seeded at a density of 210.sup.4 cells/well into 96 well cell culture plate. CHN-0 vegetative cells were stained with pHrodo Red (Life Technologies) according to manufacturer's instructions and added at a concentration of 210.sup.7 cells/well. DC2.4 cells were incubated with CHN-0 cells for 3 hr before being imaged using a Celigo Image Cytometer.

[0231] DC2.4 Cell Baseline Studies

[0232] Cytokine profiles were evaluated using the Proteome Profiler Mouse Cytokine Array Kit (R&D systems) according to manufacturer's instructions. DC2.4 cells were seeded in 12 well cell culture plates at a density of 510.sup.5 cells/well. DC2.4 cells were incubated with 110.sup.7 CHN-0 cells/well overnight. The cell culture supernatant was used for subsequent analysis. Cells were detached from the cell culture plate and centrifuged. A volume of 700 L of the supernatant was then incubated with the Detection Antibody Cocktail provided with the Proteome Profiler kit for 1 hr at RT. This mixture was added to the pre-treated membranes and incubated on a shaking platform at gentle rocking overnight at 4 C. Membranes were then rinsed with Wash buffer, followed by Streptavidin-HRP conjugation and colour development by Chemi Reagent mixture. The membranes were exposed to X-ray film for 10 min and spot intensities were quantified by ImageJ software.

EXAMPLE 3: ORAL IMMUNISATION OF MICE WITH ENGINEERED C. BUTYRICUM

[0233] In vivo immunisation experiments were performed to assess whether spores of engineered C. butyricum expressing the ROP protein variants can be used to deliver the ROP antigen and induce an immune response, with a focus on exploring T-cell responses. Mice were dosed by oral gavage with spores or injected subcutaneously with purified ROP protein fortnightly over a 28-day period and sacrificed after 42 days.

[0234] IFN- ELISPOT assays using splenocytes isolated after sacrifice demonstrated that mice immunised with the antigen-expressing C. butyricum strains by oral gavage develop antigen-specific T-cell responses. Specifically, mice immunised with the strain expressing ROP-HPV develop both CD4.sup.+ and CD8.sup.+ T-cell response (see FIG. 6), while mice immunised with the strain expressing ROP-OVA develop CD4.sup.+ T-cell response specific to the respective antigen (see FIG. 7). Importantly, mice do not develop a T-cell immune response aimed at the C. butyricum strain itself.

[0235] In-house assessment of faecal samples derived from mice immunised with spores of wildtype and genetically engineered C. butyricum has demonstrated that strains can be detected in faeces from 7 hours after the first immunisation event.

[0236] Materials and Methods

[0237] In Vivo Experimentation

[0238] Animals were housed in individually ventilated cages with nesting material. Food (provided as pellets) and water were available to mice ad libitum. All procedures were carried out according to protocols under Home Office license 30/3197 in accordance with the Animal Scientific Procedures Act 1986 and the University of Oxford Committee guidelines.

[0239] For immunisation experiments, six-week old female mice were randomly divided into groups of five animals. Immunisation through the alimentary canal was performed by oral gavage of 110.sup.8 CFU of spores of CADD-ROP-HPV or CADD-ROP-OVA in 100 L PBS, i.e. the engineered CHN strains, which may also be referred to as CHN-ROP-HPV or CHN-ROP-OVA. CHN-0 wildtype spores and PBS were given as controls at the same conditions. Parenteral immunisation was performed by subcutaneous injection of 100 g ROP-HPV or ROP-OVA protein in 100 L Freund's adjuvant (prime immunisation, day 0) or Incomplete Freund's adjuvant (boost immunisation, days 14 and 28). Mice of each group were immunised 3 times at days 0, 14 and 28 and sacrificed after 42 days. Faecal samples were collected 3 h and 7 h after each dosing event. Whole blood and serum samples were collected at each dosing event and at sacrifice. Spleens were isolated at sacrifice.

TABLE-US-00004 TABLE 3 Immunized Group Antigen Tissue Collect G1. PBS Oral gavage 1. Bleeding (14 days after G2. CHN0 spores prime and boost) G3. CADD-ROP-HPV 2. Spleens (terminal) spores 3. Sera (terminal) G4. CADD-OVA spores G5. ROP-HPV + Subcutaneous 4. Faeces (3 h and 7 h after adjuvant injection oral gavage) G6. ROP-OVA + adjuvant G7. PBS Notes: 1. CADD-based vaccines: Clostridium spores, 10.sup.8 cells/mouse; 2. ROP peptides: recombinant overlapping peptides, 100 g/mouse; 3. Adjuvant: prime (complete Freund's Adjuvant); boost (incomplete Freund's Adjuvant) 4. Bleeding: 100 l/mouse, detection of antibody titer. 5. Sera: 0.8 ml/mouse, detection of cytokines; 6. Faeces: quantification of clostridium spores retained; 7. Spleens: IFN- ELISPOT assays.

[0240] Isolation of Mononuclear Cells

[0241] Splenocytes and PBMCs were isolated from homogenised spleens and terminal whole blood samples, respectively, using Ficoll-Paque 1.084 density gradient (GE healthcare) according to manufacturer's instructions. Cell suspension or whole blood were layered on Ficoll-Paque media and centrifuged at 400g for 20-30 min at RT. The mononuclear cells isolates were washed in balanced salt solution to remove residual contaminants.

[0242] For T-cell purification, mononuclear cell isolates from one immunisation group were pooled and 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, 2 mM EDTA, pH 7.2) was used to resuspend 110.sup.7 cells before addition of MicroBeads and incubation at 4 C. for 10 min. Cell suspensions were applied to MACS LS columns in a magnetic field for retention of CD8.sup.+ T-cells. The flow through was collected twice and used for CD4.sup.+ T-cell specific experiments. CD8.sup.+ T-cells were eluted subsequently by application of buffer without magnetic field. Both CD4.sup.+ and CD8.sup.+ T-cells were resuspended in RPMI medium before use in ELISPOT experiments.

[0243] IFN- T-Cell ELISPOT

[0244] The Mouse IFN- T-cell ELISPOT kit (U-CyTech Bioscience) was used for detection of IFN- release according to manufacturer's instructions. A total of 2.510.sup.5 T-cells in 100 L RPMI/well were added to plates precoated with Anti-IFN- antibodies and re-stimulated with either wildtype HPV protein, ROP-HPV protein, ROP-OVA protein (each at 5 g/well) or CHN-0 vegetative cells at 0.510.sup.5 CFU/well. Concanavalin A (Sigma Aldrich) was added as positive control at a concentration of 5 mg/mL. Plates were 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 were scanned using a Celigo Image Cytometer and quantified using ImageJ software.

EXAMPLE 4: IMMUNISATION OF MICE USING AN INTRACELLULAR CTXB ANTIGEN IN CLOSTRIDIUM

[0245] 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.

[0246] Gene Constructs and Plasmids

[0247] For the CADD-CtxB oral vaccine development, the CtxB-encoding protein sequence (SEQ ID NO: 24) was determined from the UniProtKB submission P01556 with removal of the signal sequence (MIKLKFGVFFTVLLSSAYAHG (SEQ ID NO: 19)) and the addition of a C-terminal FLAG tag (DYKDDDDK (SEQ ID NO: 18)). Further modifications included for genetic engineering include a 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 (FIG. 9). The CtxB_FLAG construct was codon optimised for genetic engineering into C. butyricum and synthesised behind the p0957 promoter by GeneWiz and cloned into a pMTL83151-pyrErepair vector submitted to GeneWiz for subcloning.

[0248] Genetic Engineering of C. butyricum

[0249] The pMTL83151-pyrErepair_p0957_CtxB-FLAG plasmid was transformed into E. coli DH5a, grown overnight in LB supplemented with 12.5 g/mL chloramphenicol and stored at 80 C. as 15% (V/V) glycerol stocks.

[0250] For cloning into the correct plasmid for plasmid-based intracellular expression in C. butyricum, the pMTL83151-pyrErepair_p0957_CtxB-FLAG plasmid was extracted from the DH5 using the Wizard Plus SV Miniprep DNA Purification kit (Promega) following the manufacturer's instructions and the p0957-CtxB-FLAG construct was cut from the plasmids using restriction endonucleases NotI and NheI in CutSmart buffer (all New England Biolabs Inc) according to the manufacturer's instructions. The isolated cassette (including p0957 promoter) was introduced into pMTL82151 (pBP1 Gram+ replicon, catP antibiotic marker, ColE1 Gram-replicon, traJ conjugal transfer function, and multiple cloning site (MCS). The plasmid was transformed into E. coli DH5 for propagation. Plasmids were isolated as before and sequenced using GeneWiz sequencing services to confirm the correct insertion of cassettes.

[0251] Sequence confirmed plasmid pMTL82151_p0957-CtxB-FLAG was then transformed into E. coli CA434 conjugation donors. Following sequence confirmation as above, E. coli CA434 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.

[0252] Fresh colonies of revived E. coli CA434 harbouring the CtxB-FLAG plasmid were used to inoculate LB broth supplemented with 50 g/mL Kanamycin and 12.5 g/mL Chloramphenicol. After overnight incubation, cultures were used to inoculate fresh supplemented medium 1:10 and incubated until an OD.sub.600 of 0.5-0.7 was reached. A volume of 1 mL of culture was removed and centrifuged at 5,000g for 3 minutes. The supernatant was discarded, and the pellet re-suspended in 500 L phosphate buffered saline (PBS) solution. The culture was centrifuged as above, and the supernatant discarded.

[0253] 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 OD.sub.600 of 0.5-0.7 was reached. A volume of 1 mL of culture was removed and heat treated for 10 min at 50 C.

[0254] 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 (OD.sub.600:OD.sub.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. butyricum CHN-0 carrying the plasmid, colonies that were thiamphenicol resistant were patch plated reiteratively onto RCM+15 g/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_p0957-CtxB-FLAG plasmid using primers spanning the MCS (Table 4).

TABLE-US-00005 TABLE4 PrimersusedforsequenceconfirmationofpMTL82151_p0957- CtxB-FLAGplasmid-containingC.butyricumCHN-0colonies. CH22(SEQID F GTACATCACCGACGAGCAAG Forwardplasmidbackbone NO:22) primer,5ofp0957 CH54(SEQID R GACTTATCCAGGGTGCTATCTTCG Reverseplasmidbackbone NO:23) primer,3ofFLAG-TAA

[0255] The introduction of the pMTL82151-p0957-CtxB-FLAG plasmid into C. butyricum CHN-0 leads to a high expression of the CtxB full protein in the C. butyricum cytoplasm from a multicopy plasmid.

[0256] Confirmation of Expression of CtxB in C. butyricum

[0257] Fresh colonies of revived C. butyricum CHN-0+pMTL82151-p0957-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.sub.600 of 0.05. When cultures were grown to an OD.sub.600 of 1, 2, and 4, the equivalent of OD.sub.600 of 2/mL was centrifuged at 13,000g for 2 min. The pellet was re-suspended in 40 L 5SDS 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, 10 mL dH.sub.2O, Bromophenol blue) and heat treated at 98 C. for 15 minutes.

[0258] A maximum of 20 L/well of the re-suspended pellets was loaded onto a Novex 16% Tricine mini gel (ThermoFisher Scientific) and run in 1Novex Tricine SDS Running Buffer (ThermoFisher Scientific) using 140V at room temperature. Spectra Multicolor Low Range Protein Ladder (ThermoFisher Scientific) was loaded at 10 L/well as marker and the E. coli Positive Control Whole cell lysate ab5395 (abcam) was used as FLAG tag positive control in a 1:5 dilution.

[0259] Separated protein were blotted onto PVDF membranes using the Tran-Blot Turbo blotting system (BioRad) with the Trans-Blot Turbo 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 (50 mM Tris hydrochloride, 150 mM 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 (50 mM 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 FIG. 10. The CtxB-FLAG protein was detectable to high levels on a Western blot, corresponding to 900 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 3 g/mg dry cell weight.

[0260] Immunogenicity Testing

[0261] In vivo immunisation experiments will be performed to assess whether spores of engineered C. butyricum expressing the CtxB antigen can deliver the antigen and induce an immune response, with a focus on cellular and humoral responses. C. butyricum spores will be generated as set out above. C57BL/6 mice will be administered 110.sup.8 CFU/dose orally in 3 doses, 1 week apart from either a wild-type CADD strain (negative control) or the CADD vaccine strain expressing CtxB from the pMTL82151-p0957-CtxB-FLAG plasmid. A third group will be administered a current marketed oral cholera vaccine 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-US-00006 TABLE 5 Immunized Group Antigen Tissue Collect G1. CHN-0 spores Oral gavage 1. Spleens (terminal) G2. CADD + CtxB spores 2. GIT tissue (terminal) 3. Gut wash (terminal) G3. OCV Notes: 1. CADD-based vaccines: Clostridium spores, 10.sup.8 cells/mouse/dose; 2. Spleens: IFN- ELISPOT assays. 3. GIT tissue: CD40 ligand upregulation in Flow Cytometry 4. Gut wash: sIgA/IgA ELISA

[0262] At sacrifice, spleens will be harvested and processed to a single cell suspension and CD4.sup.+ and CD8.sup.+ cells purified individually to determine CD4.sup.+/CD8.sup.+-specific T cell response via IFN- release in ELISPOT assays (described in materials and methods, pages above). CD4.sup.+ 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.sup.+ T cells from this suspension will be re-stimulated with antigen presenting cells (APCs, previously exposed to a commercially obtained CtxB antigen) and the change in CD40 ligand expression on the cell surface will be assessed via Flow Cytometry as described in Hegazy et al (2017) Gastroenterology 153: 1320-1337.

[0263] Gut contents will be extracted at termination and the antigen-specific humoral response will be assessed 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.

EXPECTED RESULTS

[0264] As shown with intracellular ROPs expressed by the CADD platform, we expect the ELISPOT assays of CD4.sup.+/CD8.sup.+ T-cells to show mice immunised with the CADD strain expressing the intracellular CtxB antigen to develop an antigen-specific T-cell response, with a stronger emphasis on the CD4.sup.+ response. Importantly, we do not expect to see mice immunised with the CHN-0 wild type strain developing a T cell response.

[0265] In the gut-specific tissue assessment, the CD40 ligand is used as it is rapidly upregulated by CD4.sup.+ T cells after stimulation, so it is expected that upon re-stimulation of the CD4.sup.+ cells via APCs there will be an increase in the CD40 ligand expression in the groups administered with CADD expressing CtxB compared to the wild-type CADD group, indicating a CtxB-specific CD4.sup.+ T-cell response.

[0266] A strong CD4.sup.+ T-cell response is generally accepted as a good correlate of protection in a cholera vaccine, as classically, CD4.sup.+ T-cell stimulation is necessary for B-cell stimulation and production of antibodies. The sIgA antibody response is also known to be important in protective immunity against V. cholera, 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 CADD-CtxB oral vaccine, compared to the wild-type CADD platform alone.