Compositions and methods

Abstract

A bacterium of the class Clostridium comprising a heterologous nucleic acid molecule encoding at least one antigen, the bacterium being capable of expressing the antigen in an intracellular compartment of the bacterium during anaerobic cell growth, the at least one antigen being an infectious pathogen antigen or a tumor antigen.

Description

[Technical field]

[0001] The present invention relates to bacterial vaccines, in particular live bacterial vaccines suitable for oral administration and for stimulating cell-mediated immunity. [Background technology]

[0002] Vaccines have a leading role in disease prevention, especially in the prevention of infectious diseases, as well as the promise of treating existing infections and chronic diseases. Oral vaccines address some of the shortcomings of traditional injection-based formulations, improving safety and compliance and facilitating administration. Oral vaccines can stimulate humoral and cellular responses at both systemic and mucosal sites, but their development faces major challenges posed by the gastrointestinal (GI) tract, as reviewed in Vela Ramirez, JE, Sharpe, LA, & Peppas, NA (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 prevent fragile antigens from being degraded by proteolytic enzymes and the acidic environment of the stomach. Once oral vaccines reach the intestine, the presence of a mucus layer, the composition of gastrointestinal juices, and the action of the epithelial barrier limit the permeability of molecules to the lymphatic system. It is believed that antigens are taken up by specialized epithelial cells called M cells in the Peyer's patches of the gut-associated lymphoid tissue (GALT) in the small intestine, transcytosed and delivered to dendritic cells (DCs), where they are processed and antigen fragments are presented on their surface, activating naive T cells.

[0003] Typical strategies for oral vaccines under development have relied on high doses of antigen and strong adjuvants to induce an immune response (Ramirez et al., supra). Some strategies utilize Gram-negative bacterial lipopolysaccharides, Salmonella lipid A derivatives, or cholera toxin, which can induce adjuvant effects, but with trade-offs in terms of toxicity.

[0004] Bacterial vaccines are promising, with live attenuated vaccines of Vibrio cholerae or Salmonella typhi licensed. Gram-positive bacteria such as Lactococcus may avoid LPS and be more tolerant, suggesting their potential as a 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 has been disclosed in WO2001 / 021200A1. Bacterial vaccines have been used so far to target the small intestine, where the mucosal immune system has been well studied. An attenuated Clostridium perfringens genetically modified 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 is thought to be the delivery of high concentrations of antigen directly to the Peyer's patches at the terminal ileum of the small intestine by lysis of the mother cells after sporulation.

[0005] Oral vaccines licensed to date have generally been intended to prevent infection rather than serve as a therapeutic vaccine. Although current vaccines primarily correlate with protection with antibodies produced by B cells, cell-mediated immune function is important in defense against intracellular infections, and for almost all diseases, CD4 + CD8 cells are necessary to support the differentiation and maturation of B cells (Stanley A. Plotkin (2008) Correlates of Vaccine-Induced Immunity, Clinical Infectious Diseases, Pages 47:401-409, https: / / doi.org / 10.1086 / 589862). CD8 cells are necessary to control established infections and tumor immunity. + It is generally accepted that cell-mediated immunity, including cytotoxic T cells, is more important.

[0006] In many protein-based vaccines, proteins are phagocytosed or endocytosed by antigen-presenting cells (APCs) and taken up into endosomes and lysosomes, where the lysosomes degrade the proteins into smaller peptides, some of which (CD4 epitopes) can bind to MHC class II molecules on the lysosomal membrane and are presented on the cell surface, thereby promoting CD4 + Stimulates T cells and CD4 + T cells are necessary for the subsequent production of antibodies by B cells (with the help of T cells), therefore protein antigens have been primarily used to stimulate the body to produce antibodies.

[0007] Presentation of antigen peptides on MHC class I molecules (CD8 +The main pathway for the stimulation of cytotoxic T lymphocytes (required for the stimulation of cytotoxic T lymphocytes) relies on antigens expressed within APCs, such as after viral infection. However, studies have shown that APCs can also internalize antigens and present them on MHC class I molecules to stimulate cytotoxic T lymphocytes (CTLs) by a process called antigen cross-presentation, but this has generally proven to be an inefficient process. Delivery of exogenous peptides or proteins to the MHC class I pathway has been partially successful through the 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) has reported that artificial multi-epitope fusion proteins known as recombinant overlapping peptides (ROPs) can be used to stimulate CD4 + and CD8 + ROPs consist of overlapping peptides linked by target sequences for cathepsin cleavage sites and are more efficient at priming protective immunity than the entire protein from which they are derived. Subcutaneous immunization with ROPs has been shown to have protective effects in viral and tumor models (Zhang H et al (2009) J. Biol. Chem. 284:9184-9191; and Cai L et al (2017) Oncotarget 8:76516-76524).

[0008] There remains a need for effective bacterial vaccines suitable for oral administration and stimulation of cell-mediated immunity.

[0009] WO2018 / 055388 (CHAIN ​​Biotechnology Limited) discloses genetically modified Clostridium to express (R)-3-hydroxybutyrate (R-3-HB) as an anti-inflammatory factor, for example in a simulated colonic environment. WO2019 / 180441 (CHAIN ​​Biotechnology Limited) discloses in vivo and pharmacokinetic profiling of R-3-HB genetically modified Clostridium butyricum. The genetically modified strain could be isolated from colon samples of mice orally administered bacterial spores.

[0010] The present inventors have attempted to exploit the ability of Clostridium to grow under anaerobic conditions to target the lower anaerobic regions of the gastrointestinal tract, such as the large intestine, to develop a platform vaccine technology. In contrast to the anti-inflammatory effects of R-3-HB genetically modified Clostridium, the present invention is based on the surprising discovery that Clostridium genetically modified to express antigens intracellularly during anaerobic cell growth can stimulate antigen-specific immune responses.

[0011] The listing or discussion of a prior-published document in this specification should not be construed as an acknowledgement that the document is part of the state of the art or is common general knowledge. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] A first aspect of the present invention is a bacterium of the class Clostridium comprising a heterologous nucleic acid molecule encoding at least one antigen, which bacterium is capable of expressing the at least one antigen in an intracellular compartment of the bacterium during anaerobic cell growth, wherein the at least one antigen is an infectious pathogen antigen or a tumor antigen.

[0013] The infectious agent antigen or tumor antigen is heterologous to the bacterium. By "capable of expressing" the antigen, it is meant that the heterologous nucleic acid molecule is transcribed, and typically translated, within the bacterium, resulting in expression of the antigen.

[0014] Expression of antigens by bacteria occurs in intracellular compartments of bacteria during anaerobic cell growth. Clostridia bacteria are obligate anaerobes, the majority of which have the ability to form spores (i.e., they are spore-forming bacteria). Such bacteria may be in the form of spores or in the form of vegetative forms; in the latter form, the bacteria are metabolically active and typically multiply. By targeting expression of antigens to the metabolically active form of Clostridia, Clostridia can be used as a vehicle to target antigens to the anaerobic portion of the gut. Oral administration of bacteria as spores allows the bacteria to survive in a dormant state during their passage through the gastrointestinal tract and germinate and multiply once they reach the anaerobic portion. antigen

[0015] By "antigen" is meant a molecule that specifically binds to an antibody or a T cell receptor (TCR). Antigens that bind to antibodies are called B cell antigens. Suitable types of molecules include peptides, polypeptides, glycoproteins, polysaccharides, gangliosides, lipids, phospholipids, DNA, RNA, fragments thereof, parts thereof, and combinations thereof. Peptide and polypeptide antigens, including glycoproteins, are preferred. TCRs bind only to peptide fragments complexed with MHC molecules. The site of the antigen that is recognized is called an "epitope." When a B cell epitope is a peptide or polypeptide, it typically contains three or more amino acids, generally at least five, and more generally at least 8-10 amino acids. The amino acids may be adjacent amino acid residues in the primary structure of the polypeptide or may be spatially juxtaposed in the folded protein. T cell epitopes are usually short primary sequences derived from an antigen. They may bind to MHC class I or MHC class II molecules. Typically, MHC class I-binding T cell epitopes are 8-11 amino acids in length. Class II molecules may bind peptides that may be 10-30 residues or more in length, with the optimal length of the peptide being 12-16 residues. Peptides that bind to a particular allelic MHC molecule contain amino acid residues that allow for 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).

[0016] According to a first aspect, the antigen expressed by the bacterium of the class Clostridium is an infectious agent antigen or a tumor antigen. By "infectious agent antigen" is meant an antigen derived from an infectious agent capable of infecting a susceptible host, such as a human, and typically causing symptoms. "Derived from" includes that the infectious agent antigen is encoded in the genome of the infectious agent, or is a variant of such an encoded antigen. By "tumor antigen" is meant that the antigen is derived from an antigen that is expressed predominantly, almost exclusively or exclusively, for example, by tumor cells, or is derived from an antigen that serves as a marker used in the art to distinguish tumor cells from normal cells. "Derived from" includes that the tumor 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 associated with risk of cancer. The antigen may be a fragment or a portion of a complete protein, the fragment comprising the epitope. An "antigen segment" is a portion of an antigen, the antigen comprising the epitope.

[0017] "Variants" refer to proteins or peptides in which conservative or non-conservative amino acid insertions, deletions, or substitutions are present at one or more positions. "Conservative substitutions" refers to 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. Exemplary variants of antigens or portions thereof will have an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.5% identical to the corresponding native antigen or portions thereof.

[0018] The percent sequence identity between two polypeptides can be determined using a suitable computer program, such as the GAP program of the University of Wisconsin Genetic Computing Group, and it will be understood that the percent identity is calculated relative to the polypeptides whose sequences are optimally aligned.

[0019] Alternatively, the alignment can be performed using the Clustal W program (Thompson et al., (1994) Nucleic Acids Res., 22(22), 4673-80). The parameters used can be as follows: Fast pairwise alignment parameters: K-tuple (word) size; 1, window size; 5, gap penalty; 3, number of upper diagonals; 5. Scoring method: x percent. Multiple alignment parameters: Gap opening penalty; 10, Gap extension penalty; 0.05. · Scoring matrix: BLOSUM.

[0020] "Mutant" may also refer to a nucleic acid molecule that encodes a mutant antigen.

[0021] Suitable infectious agent antigens may include viral antigens, bacterial antigens (including chlamydia or mycoplasma antigens), parasitic antigens, protozoan antigens, helminth antigens, nematode antigens, fungal antigens, prions, or any combination thereof. Combinations of infectious agent antigens and tumor antigens may also be used. In some cases, selected antigens provide cross-immunity (also called cross-protection), whereby a single antigen or multiple antigens in combination may confer immunity or protection against related infectious agents. Cross-immunity may occur when an antigen is conserved (i.e., common or homologous) in multiple strains or species of an infectious agent. Therefore, it may be desirable to use an antigen (either a single antigen or multiple antigens in combination) that provides such cross-immunity.

[0022] Examples of viral antigens include human papillomavirus (HPV) antigens; coronavirus antigens, such as SARS-CoV-2 coronavirus antigens, e.g., SARS-CoV-2 spike protein (e.g., the coronavirus antigen can be one antigen or a combination of antigens that confer cross-immunity to 229E, NL63, OC43, and HKU1 coronavirus strains, each of which is related to SARS-CoV2); human immunodeficiency virus (HIV) antigens, such as the products of the gag, pol, and env genes, Nef protein, reverse transcriptase, and other HIV components; hepatitis, e.g., hepatitis A, B, and C, hepatitis virus antigens, such as the hepatitis S, M, and L proteins, hepatitis B virus pre-S antigen; influenza virus antigens hemagglutinin and neuraminidase, and other influenza virus antigens; SAG-1 or Examples of suitable antigens include measles virus antigens such as p30; rubella virus antigens such as E1 and E2 proteins and other rubella virus components; rotavirus antigens such as the VP7sc component and other rotavirus components (e.g., VP4 found on the surface capsid of the virus, which is cleaved into VP8 and VP5 by a protease in the intestinal tract); cytomegalovirus antigens such as envelope glycoprotein B and other cytomegalovirus proteins; respiratory syncytial virus antigens such as the RSV fusion protein, M2 protein; varicella-zoster virus antigens such as gpI, gpII and telomerase; antigens of flaviviruses 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 infections may be preferred. For example, human papillomavirus (HPV); hepatitis C; hepatitis B; human immunodeficiency virus (HIV); herpes viruses include herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus.

[0023] In some embodiments, the viral antigen is an HPV antigen. Persistent HPV infection can lead to the development of warts or precancerous lesions, the latter of which increases the risk of cervical, vulvar, vaginal, penile, anal, oral, or throat cancer, depending on the site of infection. HPV genotypes 16, 18, 31, 52, 53, and 58 are high-risk HPV genotypes, meaning that they are strains associated with risk of cancer. Thus, in some embodiments, the at least one antigen is an HPV antigen from a high-risk genotype, such as E1, E2, E4, E5, E6, and / or E7 protein, preferably at least one antigen corresponding to E1, E2, E4, E5, E6, and / or E7 protein that is conserved across one or more high-risk HPV genotypes. Suitable antigens are described in WO2019 / 034887, which describes a nucleic acid encoding a polypeptide composed of multiple peptide sequences that are conserved across one or more HPV genotypes (i.e., strains). Other suitable antigens include antigens derived from the L1 and / or L2 capsid proteins, as described in Finnen et al. (2003) Interactions between Papillomavirus L1 and L2 Capsid Proteins, Journal of Virology, pages 4818 to 4826. In one embodiment, the HPV antigen comprises the amino acid sequence of SEQ ID NO:4, or amino acids 1 to 140 of SEQ ID NO:4, e.g., where the HPV antigen is encoded by nucleotides 19 to 477 of the nucleic acid sequence of SEQ ID NO:3.

[0024] In some embodiments, the viral antigen is a coronavirus antigen. Coronavirus infection can cause the appearance of symptoms such as severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). In some embodiments, the selection of epitopes is based on comparison of homologous SARS proteins with 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 based on likely representation across MHC alleles. Further 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.

[0025] Examples of bacterial antigens include clostridial antigens, such as Clostridium difficile (renamed Clostridioides difficile) toxins A and B; Bordetella pertussis antigens, such as pertussis toxin; Corynebacterium diphtheriae antigens, such as diphtheria toxin or toxoid erythematosis, and other Corynebacterium diphtheriae antigenic components; Clostridium tetanus antigens, such as tetanus toxin or toxoid and other bacterial antigenic components; Streptococcus antigens, such as M protein and other streptococcal antigenic components; Gram-negative bacillus antigens, Mycobacterium tuberculosis antigens, such as fever syncytial antigens, and other gram-negative bacillus antigens. Antigens include heat shock protein 65 (HSP65), 30 kDa major secretory protein, antigen 85A and other mycobacterial antigen components; Vibrio cholerae antigens, such as cholera toxin B subunit (CtxB); Helicobacter pylori antigen components; Pneumococcal antigens, such as pneumolysin, pneumococcal antigen components; Haemophilus influenzae antigens, such as Haemophilus influenzae antigen components; Bacillus anthracis antigens, such as Bacillus anthracis protective antigen and other Bacillus anthracis antigen components; Rickettsia antigens, such as rompA and other Rickettsia antigen components; or Mycobacterium bovis antigens; or Brucella antigens. Bacterial antigens as described herein also include any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydia antigens. Antigens of bacteria that cause chronic persistent infections, e.g., Mycobacterium tuberculosis, Borrelia species such as Borrelia burgdorferi, Corynebacterium diphtheriae, Chlamydia, Vibrio cholerae, Salmonella typhi; mycoplasma antigens may be preferred.

[0026] In some embodiments, the bacterial antigen is a Vibrio cholerae antigen. Vibrio cholerae is a diarrheagenic enteropathogen and the causative agent of cholera. Suitable Vibrio cholerae antigens include peptides or proteins associated with or secreted by Vibrio cholerae. During Vibrio cholerae infection, the bacterium secretes cholera toxin, a heteropolymeric holotoxin consisting of one copy of the A subunit (CtxA) and five copies of the B subunit (CtxB). The CtxA subunit catalyzes the ADP-ribosylation of Gsα, a GTP-binding regulatory protein, and activates adenylate cyclase. This leads to the overproduction of cAMP, which ultimately leads to the excessive secretion of chloride and bicarbonate, followed by water, resulting in the characteristic cholera stool. The CtxB subunit forms a pentameric ring, and the pentameric ring of the B subunit guides the A subunit to its target by binding to GM1 ganglioside present on the surface of intestinal epithelial cells. It can bind five GM1 gangliosides. It has no toxic activity by itself. Thus, in one embodiment, the Vibrio cholerae antigen is CtxB. In some embodiments, the Vibrio cholerae antigen comprises an amino acid sequence selected from the amino acid sequence encoded by SEQ ID NO:21 or amino acids 1-104 of SEQ ID NO:21, SEQ ID NO:24, and / or SEQ ID NO:25; or the amino acid sequence encoded by nucleotides 270-581 of SEQ ID NO:20.

[0027] In some embodiments, the infectious agent infects the host through a mucosal site (i.e., is a mucosally transmitted pathogen). Mucosal infections can involve the following pathogens: Vibrio cholerae, SARS-CoV-2, influenza A and B viruses, poliovirus, rotavirus, Salmonella typhimurium, adenovirus, respiratory syncytial virus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Helicobacter pylori, enterotoxigenic Escherichia coli (ETEC), Shigella, Clostridium difficile / perfringens, syphilis, rabies virus, Campylobacter jejuni, gonorrhea, herpes simplex virus type 2, human papillomavirus (HPV), hepatitis B / C, HIV, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, Bordetella bronchiseptica, canine parainfluenza virus, and Newcastle disease virus. Infectious diseases associated with infectious agents can be classified based on the site. For example, the infectious agent may be SARS-CoV-2, seasonal influenza, RSV-ALRI, Streptococcus pneumoniae, or Mycobacterium tuberculosis associated with the respiratory tract; rotavirus, Helicobacter pylori, enterotoxigenic Escherichia coli (ETEC), Salmonella, Rhodophyton, or Clostridium (difficile or perfringens) associated with the gastrointestinal tract; or syphilis, gonorrhea, herpes simplex virus type 2, HPV, hepatitis B, hepatitis C, or HIV associated with the genitourinary tract.

[0028] Fungal antigens that can be used include, but are not limited to, Candida fungal antigen components; Histoplasma fungal antigens, Coccidioides fungal antigens such as spherule antigens and other Coccidioides antigens; Cryptococcus fungal antigens, and other fungal antigens.

[0029] Examples of protozoan and other parasitic antigens include, but are not limited to, antigens from Plasmodium species that cause malaria, such as Plasmodium falciparum; Toxoplasma antigens; Schistosoma antigens; Leishmania moribund and other Leishmania antigens; and Trypanosoma antigens.

[0030] Cancer or tumor antigens may be used, which may be classified as tumor-associated antigens (e.g., overexpressed proteins, differentiation antigens, or cancer / testis antigens) or as tumor-specific antigens (e.g., oncoviral antigens, common neoantigens, or private neoantigens). For example, cancer / testis antigens (also called cancer / germline antigens) are typically expressed only in immune-privileged germline cells (e.g., MAGE-A1, MAGE-A3, and NY-ESO-1); differentiation antigens refer to lineage differentiation antigens that are not normally expressed in adult tissues (e.g., tyrosinase, gp100, MART-1, prostate-specific antigen (PSA)); and overexpressed antigens simply refer to antigens expressed in cancer cells above healthy or normal levels (e.g., hTERT, HER2, mesothelin, and MUC-1) (Hollingsworth & Jansen (2019), npj Vaccines, 4(7)).

[0031] Thus, cancer antigens or tumor antigens include, but are not limited to, K-Ras, survivin, dystroglycan, KS[1 / 4] pan-carcinoma antigen, ovarian cancer antigen (CA125), prostatic acid phosphatase, 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 tumor associated antigens, such as: CEA, TAG-72, CO17-1A; GICA19-9, CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19, human B-cell lymphoma antigen-CD20, CD33, melanoma-specific antigens, such as ganglioside GD2, ganglioside GD3, ganglioside GM2, and ganglioside GM3, tumor-specific transplantation type of cell-surface antigens (TSTA), such as T-antigen DNA tumor virus (T-antigen virus) and virally-induced tumor antigens including envelope antigens of RNA-type tumor viruses, carcinoembryonic antigen-alpha-fetoprotein, e.g., CEA in colon, bladder tumor carcinoembryonic antigen, differentiation antigens, e.g., human lung cancer antigen L6, L20, antigens in fibrosarcoma, human leukemia T cell antigen-Gp37, neoglycoproteins, sphingolipids, breast cancer antigens such as EGFR, EGFRvIII, FABP7, doublecortin, brevican, HER2 antigen, polymorphous epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigens such as I antigen found in fetal erythrocytes, early endoderm, I antigen found in adult erythrocytes, I(Ma), found in preimplantation embryos, gastric adenocarcinoma, M18, M39 found in breast epithelium, SSEA-1, VEP8, VEP9 found in myeloid cells , Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood type H), C14 found in colon adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinoma cells, TL5 (blood type A), EGF receptor found in A431 cells, E1 series found in pancreatic cancer (blood type B), FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 found in adenocarcinoma, NS-10 found in adenocarcinoma, CO-43, G49 found in EGF receptor of A431 cells, MH2 found in colon adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucin, T5A7 found in myeloid cells, R24 found in melanoma, 4.2 found in embryonal carcinoma cells, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8, SSEA-3 and SSEA-4 found in 4- to 8-cell stage embryos, T-cell receptor-derived peptides from cutaneous T-cell lymphoma, fibroblast activation protein alpha (FAP) found in cancer, and their variants.

[0032] In some embodiments, the cancer or tumor antigen is a multi-antigen fusion polypeptide or recombinant overlapping peptide (ROP) of K-Ras, PSA, or survivin.

[0033] In some embodiments, 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, and an antigen comprises an epitope. Typically, an antigen segment comprises an epitope. A T cell antigen segment is a segment that binds to a CD4 + T cell antigen segments or CD8 + It may be a T cell antigen segment. CD4 + T cell antigen segments are expressed in the context of MHCII and are expressed by CD4 + An antigen or a portion thereof that contains an epitope that can be presented to T cells. + T cell antigen segments are associated with MHCI and are CD8 + The antigen or part thereof comprises an epitope that can be presented to T cells. Different antigen segments can be provided on different antigens or on the same antigen. Multiple antigens or parts / fragments thereof may be used. Suitably, the antigen segment is in the form of a fragment of the antigen, such as a fragment comprising or consisting of a B cell epitope or a T cell epitope. This is convenient when the native antigen is particularly large. When the polypeptide epitope is provided in the context of a larger molecule, it may be provided adjacent to a cleavage site to facilitate cleavage of the epitope from the larger molecule in antigen presenting cells (APCs). This is useful for the detection of CD8 +In the context of T cell epitopes, it is particularly useful to promote the egress of epitopes from the endolysosomal compartment of APCs and their entry into the cytosol for loading into MHCI. + The T cell epitope may be provided as an antigenic fragment of less than about 70 amino acids, for example less than 60, less than 50, less than 40 amino acids.

[0034] Suitably, the antigen is a multi-antigen fusion polypeptide comprising two or more antigen segments, for example three or more, five or more, or ten or more antigen segments, optionally with a limit of ≦200, preferably ≦100, more preferably ≦50 antigen segments. By "multi-antigen fusion polypeptide" is meant a polypeptide comprising antigen segments, such as epitopes, which are either directly linked to form an artificial polypeptide or separated by suitable linking sequences and linked to form an artificial polypeptide; this may be referred to as a polyepitope, artificial polyepitope, or mosaic polyepitope. This allows for the avoidance of intervening sequences occurring between antigen segments within an antigen in a multi-antigen fusion polypeptide. Each antigen segment may be from the same or different antigens. As described in EP3235831, antigen segments or epitopes, in particular CD8 + To facilitate cleavage of the T cell antigen segment or epitope from the multi-antigen fusion polypeptide, the multi-antigen fusion polypeptide may include an appropriate linking sequence. + T cell antigen segment and at least one CD8 + Contains a T cell antigen segment.

[0035] The antigen segments in the multi-antigen fusion polypeptide can be suitably derived from the polypeptide sequences that overlap in the antigens from which they are derived. When two antigen segments overlap, the first antigen segment has an N-terminal sequence that is not shared with the second antigen segment, and the second antigen segment has a C-terminal sequence that is not shared with the first antigen segment, and the two antigen segments will share a common sequence. For example, an antigen can be divided into overlapping peptides that completely contain the entire sequence of the antigen. When multiple antigens are present, each may be present as an overlapping peptide.

[0036] The term "overlapping peptide" encompasses recombinant overlapping peptides (ROPs) as described in EP3235831. By "overlapping peptide" and "ROP" is meant that the antigen is a multi-antigen fusion polypeptide (also referred to herein as a multi-antigen fusion protein) as defined above, comprising two or more partially overlapping antigen segment sequences, i.e. peptide sequences. Suitably, the antigen segments in the multi-antigen fusion protein overlap in part and in combination encompass ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, ≧90%, more preferably 100% of the amino acid sequence of the antigens from which they are derived. That is, a first polypeptide may overlap with a second polypeptide, which may overlap with a third polypeptide, etc.

[0037] In some embodiments, a multi-antigen fusion protein comprising overlapping peptides may comprise >3, preferably >5, more preferably >10 antigen segments; optionally with a limit of <200, preferably <100, more preferably <50 antigen segments. For example, a ROP may comprise 10 antigen segments, all segments together constituting 100% of the amino acid sequence of the entire antigen. It will be understood that all antigen segments in a multi-antigen fusion protein necessarily comprise an epitope.

[0038] In some embodiments, each antigen segment binds at least one (preferably at least two) CD8 + epitopes; at least one (preferably at least two) CD4 + epitope; and / or at least one (such as at least two) B cell epitopes. In some embodiments, each antigen segment comprises a CD8 + Epitopes and CD4 + It contains at least one (preferably at least two) amino acid sequences which simultaneously function as an epitope.

[0039] In some embodiments, each antigen segment is 8-50 amino acids long, preferably 10-40 amino acids long, more preferably 15-35 amino acids long. In some embodiments, each antigen segment may include a cleavage site sequence located between the antigen segments. For example, the cleavage site sequence may include a cathepsin cleavage site. In some embodiments, the cleavage site is selected from the group consisting of a cathepsin S cleavage site (further described in Luetzner and Kalbacher, 2008, J. Biol. Chem., 283(52):36185-36194) (e.g., Leu-Arg-Met-Lys (SEQ ID NO: 26) or similar cleavage site), a cathepsin B cleavage site (e.g., Met-Lys-Arg-Leu (SEQ ID NO: 27) or similar cleavage site), a cathepsin K cleavage site (e.g., His-Pro-Gly-Gly (SEQ ID NO: 28) or similar restriction site), or a combination thereof. In some embodiments, the cleavage site of cathepsin S is selected from the 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:29), X-Pro-Leu-Arg↓ (SEQ ID NO:30), X-Ile-Val-Gln↓ (SEQ ID NO:31) and X-Arg-Met-Lys↓ (SEQ ID NO:32); where each X is independently any naturally occurring amino acid and ↓ denotes the cleavage position. In some embodiments, each antigen segment is directly connected in the artificial multi-antigen fusion protein via the above cleavage site sequence. In some embodiments, the cleavage site sequences used to connect each antigen segment are identical or different. In some embodiments, a cleavage site sequence is not included in each antigen segment; alternatively, a cleavage site sequence is included in the antigen segment, but at least one cleavage product (or some or all of the cleavage product) formed after the antigen segment is digested still binds to CD8. + Epitope or CD4 + It is an epitope.

[0040] In some embodiments, the multi-antigen fusion protein (optionally including overlapping peptides) further comprises: (a) a tag sequence (e.g., FLAG for detection); (b) a membrane-permeable sequence (e.g., a cell-penetrating peptide (CPP)) (c) a cathepsin cleavage site (e.g., LRMK (SEQ ID NO: 33)); and / or (d) Cell necrosis-inducing factor sequence The array includes one or more arbitrary elements selected from the group consisting of:

[0041] In some embodiments, the artificial multi-antigen fusion protein is 100-2000 amino acids in length, preferably 150-1500 amino acids in length, more preferably 200-1000 amino acids in length or 300-800 amino acids in length.

[0042] In some embodiments, the fusion protein has Formula I: Y-(AC)nZ (I) It is shown in the structure: During the ceremony, A is an antigenic segment; C is the sequence of the cathepsin cleavage site; n is a positive integer ≧3; Y is absent or a sequence represented by "Y0-B" (wherein Y0 is an adjuvant element sequence, a cell necrosis-inducing element sequence, or a combination thereof, and B is absent or a cleavage site sequence); Z is absent or is an adjuvant element sequence, a cell necrosis-inducing element sequence, or a combination thereof; However, if Z does not exist, the C in the last "AC" may not exist.

[0043] In some embodiments, the cleavage site sequence is different from C (i.e., B≠C). In some embodiments, the cleavage site sequence is identical to C (i.e., B=C). In some embodiments, n is any integer from 5 to 100, preferably 6 to 50, and more preferably 7 to 30. Bacteria and their preparation methods

[0044] The bacterium of the first embodiment of the present invention is a bacterium of the class Clostridia. The Clostridia include the orders Clostridiales, Halanaerobiales, and Thermoanaerobacteriales. The Clostridiales include the family Clostridiaceae, which includes the genus Clostridium. The genus Clostridium is one of the largest genera of bacteria. The genus is defined by rod-shaped, gram-positive bacteria that are obligate anaerobes and can produce spores.

[0045] Preferably the species of the order Clostridiale or genus Clostridium is capable of forming spores.

[0046] Certain Clostridium species are known to cause human disease due to the production of toxins (https: / / doi.org / 10.1533 / 9781845696337.2.820). These include Clostridioides difficile, Clostridium botulinum, Clostridium novyi, and Clostridium perfringens.

[0047] Preferably, the species is not a pathogenic Clostridium species, which may or may not be an attenuated strain from such a pathogenic species.

[0048] Several Clostridium species are present in the human lower gastrointestinal tract. The main Clostridium species detected in the lower gastrointestinal 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). Clostridium cluster XIVa includes species belonging to the genera Clostridium, Eubacterium, Ruminococcus, Coprococcus, Dorea, Lachnospira, Roseburia, and Butyrivibrio. Clostridium cluster IV is composed of the genera Clostridium, Eubacterium, Ruminococcus, and Anaerofilum.

[0049] Cluster I of Clostridia includes species present in the gut microbiota (https: / / doi.org / 10.1016 / j.nmni.2017.11.003), while other species are found primarily in soil and other such environmental niches and are representative of industrial chassis useful for solvent and acid production (DOI:10.1016 / j.anaerobe.2016.05.011). Cluster I includes: Clostridium aceticum, Clostridium acetobutylicum, Clostridium aerotolerans, Clostridium autoethanogenum, Clostridium baratii, Clostridium beijerinckii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium cellulolyticum, Clostridium cellulovorans, Clostridium chavoei, Clostridium clostridioforme, Clostridium colicanis, Clostridium difficile (now Clostridioides difficile), and Clostridium difficile (now Clostridioides difficile). (renamed to C. fisile), Clostridium drakei, Clostridium estertheticum, Clostridium fallax, Clostridium feseri, Clostridium formicaceticum, Clostridium glycolicum, Clostridium histolyticum, Clostridium innocuum, Clostridium kluyveri, Clostridium ljungdahlii, Clostridium lavalense, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium nobyi, Clostridium spp.novyi, Clostridium oedematiens, Clostridium paraputrificum, Clostridium pasteurianum, Clostridium perfringens, Clostridium phytofermentans, Clostridium piliforme, Clostridium ragsdalei, Clostridium ramosum, Clostridium roseum, Clostridium saccharoperbutylacetonicum, Clostridium scatologenes, Clostridium These include Clostridium septicum, Clostridium sordellii, Clostridium sporogenes, Clostridium sticklandii, Clostridium tertium, Clostridium tetani, Clostridium thermocellum, Clostridium thermosaccharolyticum, Clostridium tyrobutyricum, Clostridium paprosolvens, Clostridium saccharobutylicum, Clostridium carboxydovorans, Clostridium scindens, and Clostridium autoethanogenum. Although only a small number of the Clostridium cluster I species found in the human gut are associated with disease, the majority are generally considered to contribute to health and well-being. Preferably, the bacteria selected from Cluster I are species associated with health benefits. These species include Clostridium sporogenes, Clostridium scindens, and Clostridium butyricum.

[0050] Preferably, the bacterium is from cluster I, IV and / or XIVa of the genus Clostridium.

[0051] Preferably, the bacterium is detectable in the lower gastrointestinal tract, e.g., the lower gastrointestinal tract of humans, but is not thought to permanently colonize or form part of the resident microbiota in the lower gastrointestinal tract, e.g., the lower gastrointestinal tract of humans, or is an attenuated strain from such a resident species.

[0052] Butyrate production is widespread among anaerobic bacteria belonging to the subphylum Clostridia, and in particular to Clostridium clusters XIVa and IV. Butyrate-producing species are found within the two predominant families of the genus Clostridia, Ruminococcaceae and Lachnospiraceae, which are normal inhabitants of the human colon. https: / / doi.org / 10.1111 / 1462-2920.13589. The Lachnospiraceae family includes Eubacterium rectale, Roseburia inulinivorans, Roseburia intestinalis, Dorea longicatena, Eubacterium hallii, Anaerostipes hadrus, Ruminococcus torques, Coprococcus eutactus, Blautia obeum, Dorea formicigenerans, and Coprococcus catus. The family Ruminococcaceae includes Faecalibacterium prausnitzii, Subdoligranulum variabile, Ruminococcus bromii, and Eubacterium siraeum.

[0053] Preferably, the bacterial species produces butyrate. Butyrate-producing species that are not thought to colonize the human lower gastrointestinal tract persistently include Clostridium butyricum.

[0054] 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 Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum and Clostridium saccharolyticum, as well as pathogenic species including Clostridium difficile.

[0055] Preferably, the species is Clostridium butyricum. Suitable strains include the "Rowett" strain, also referred to as (DSM10702 / ATCC19398 / NCTC7423).

[0056] In some embodiments, the Clostridium bacteria can produce short chain fatty acids (SCFAs), such as butyrate. SCFAs include lactate, acetate, and butyrate, which are believed to enhance T cell responses. Butyrate is a potent stimulator of CD8 + It can stimulate T cells and enhance their effector function (Luu et al., 2018, Scientific Reports, 8:14430, and Trompette et al., 2018, Immunity, 48(5):992-1005), and high concentrations of fecal butyrate are associated with longer progression-free survival after treatment with nivolumab or pembrolizumab in patients with solid tumors (Nomura et al., 2020, Oncology, 3(4):e202895). Moreover, butyrate inhibits activated CD8 + Short-chain fatty acids (SCFAs) were required to enhance the memory capacity of T cells and for optimal recall responses upon antigen re-encounter (Bachem et al., 2019, Immunity, 51(2):285-297). Therefore, the microbiota, including Clostridium, is required for CD8+ They play a role in promoting the long-term survival of T cells as memory cells. The production of SCFAs can typically be determined by the metabolism of carbohydrate substrates such as glucose to SCFAs such as butyrate during vegetative cell proliferation, i.e., by anaerobic metabolism.

[0057] The bacterium according to the first aspect comprises a heterologous nucleic acid molecule encoding an antigen, as described herein. That is, it is a genetically modified bacterium. By "heterologous nucleic acid molecule" is meant that the nucleic acid molecule comprises one or more non-native sequences, such as an open reading frame (ORF) encoding the antigen, although it is contemplated that alternatively, a native antigen coding sequence can be provided under the control of a non-native control sequence to promote increased gene expression levels of the native antigen during anaerobic cell growth. The heterologous nucleic acid molecule comprises a gene, i.e., an ORF operably linked to a promoter that initiates 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, including a gene from one species of a microorganism that has been introduced into another species of the same genus. As used herein, the term "cassette" includes any heterologous nucleic acid molecule described herein, where appropriate, the heterologous nucleic acid molecule includes one or more non-native sequences, including, but not limited to, an ORF encoding an antigen, an ORF operably linked to a promoter, other control sequences, a non-native gene, or any combination thereof. The heterologous nucleic acid molecule may be codon-optimized for Clostridium.

[0058] The promoter is selected to allow expression of the antigen during anaerobic cell growth, such as after spore germination under anoxic conditions, and / or during anaerobic vegetative cell metabolism. "Anaerobic cell growth" means that the Clostridium bacteria are in the form of cells, not spores, and are capable of vegetative growth, i.e., cell division. Clostridium bacteria can only grow under anaerobic conditions. Growth can be recognized by an increase in colony forming units. Anaerobic vegetative cell metabolism can be evaluated by the production of SCFAs, such as butyrate, acetate, lactate, or combinations thereof, from available carbohydrate sources. For example, the bacteria can be fed with a fermentable substrate, such as a carbohydrate substrate, such as glucose, and the production of SCFAs, such as butyrate, acetate, lactate, or combinations thereof, can be measured to indicate metabolism. The terms "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.

[0059] The suitable promoter is active during cell growth and may be a constitutive promoter. The promoter of the gene essential for primary metabolism may be a suitable constitutive promoter. The expression level of the antigen may be optimized by controlling gene expression using a promoter with selected strength, such as a strong promoter. Suitably, a native Clostridium promoter is used. Suitable promoters include the fdx gene promoter of Clostridium perfringens (Takamizawa et al (2004) Protein Expression Purification 36:70-75); the ptb and thl promoters of Clostridium acetobutylicum (Tummala et al (1999) App. Environ. Microbiol. 65:3793-3799) and the cpe promoter of Clostridium perfringens (Melville, Labbe and Sonenshein (1994) Infection and Immunity 62: 5550-5558) and the thiolase promoter from Clostridium acetobutylicum (Winzer et al (2000) J. Mol. Microbiol. Biotechnol. 2:531-541). Other suitable promoters may be the hbd, crt, etfA, etfB and bcd promoters of Clostridium acetobutylicum (Alsaker and Papoutsakis (2005) J Bacteriol 187:7103-7118), and the p0957 promoter; and the fdx promoter from Clostridium sporogenes (NCIMB10696), which can be obtained from the pMTL80000 modular shuffle plasmid (Heap et al. (2009) A modular system for Clostridium shuttle plasmids, Journal of Microbiological Methods, 78:79-85).Preferably, the promoter of the selectable marker gene is the promoter of the thl gene of Clostridium acetobutylicum, the promoter of the fdx gene of Clostridium perfringens, or the promoter of the fdx gene of Clostridium sporogenes.

[0060] Heterologous nucleic acid molecules can be introduced into Clostridium 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, the heterologous nucleic acid molecule may be present on a high copy plasmid. High copy plasmids can be present at copy numbers of about 8 to 14 or more, 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.

[0061] Suitable plasmids include those that are stably maintained by Clostridium. Suitable plasmids include suitable origins of replication and any necessary replication genes to allow replication in Clostridium. Plasmid transformation is typically carried out in Clostridium by conjugation or transformation. Methods of transformation and conjugation in Clostridium are described 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.

[0062] Heterologous nucleic acid molecules can be integrated into the genome, typically the chromosome of the Clostridium, using gene integration techniques such as Allele Coupled Exchange (ACE) as described in WO 2010 / 084349 and Minton et al (2016) Anaerobe 41:104-112; or by 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)). Allele coupled exchange can be used to genetically modify any Clostridium species and is believed to rely on the initial creation of a pyrE deletion mutant that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This allows the insertion of a DNA fragment by allelic exchange using a heterologous pyrE allele as a counter / negative selection marker in the presence of FOA. After alteration of the insertion site, the resulting strain may be rapidly reverted to uracil prototrophy using ACE, allowing characterization of the mutant phenotype in a pyrE-proficient background. Importantly, a wild-type copy of the inactivated gene may be introduced into the genome using ACE simultaneously with correction of the pyrE allele. The initial creation of the pyrE deletion is performed by a special form of ACE, as described in Minton et al., supra, or by means of retargeting a mobile group II intron, as described in WO 2007 / 148091.CRISPR gene editing has also been widely applied in Clostridium for the integration of large DNA fragments, including Clostridium acetobutylicum (Li et al.(2016)CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii.Biotechnol J.11:961-72), Clostridium 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), and Clostridium pasteurianum (Pyne et al.(2016)Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium.Sci Rep. 6:25666), and Clostridium saccharoperbutylacetonicum (Wang et al. (2017) Genome editing in Clostridium saccharoperbutylacetonicum N1-4 using CRISPR-Cas9 system. Appl Environ Microbiol. 83:e00233-17).

[0063] When a heterologous nucleic acid molecule is integrated into the genome as a single copy or present on a low copy plasmid, the amount of antigen expressed will typically be less than when the heterologous nucleic acid molecule is present on a high copy number plasmid. The inventors have found that even when a heterologous nucleic acid molecule is integrated into the genome as a single copy, antigen-specific immune responses can be effectively stimulated. The amount of antigen expressed per cell weight of Clostridium cells during anaerobic cell growth can typically range up to 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900ng / mg, 1μg / mg, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10 or 20μg / mg dry cell weight (but more than 0ng / mg dry cell weight, typically more than 10ng / mg, 20ng / mg or 40ng / mg). Any range between any two of these values ​​is contemplated. For example, the amount of antigen expressed per cell weight of Clostridium cells during anaerobic cell growth may be 10-400 ng / mg dry cell weight; 20-200 ng / mg dry cell weight; 40-100 ng / mg dry cell weight; 100 ng-5 μg / mg dry cell weight; 200 ng-2.5 μg / mg dry cell weight; 400-1500 ng / mg dry cell weight; or about 800 ng / mg dry cell weight; or 1-5 μg / mg dry cell weight, 2-4 μg / mg dry cell weight, e.g., about 3 μg / mg dry cell weight; or any other combination. The amount may be determined by culturing Clostridium cells, extracting the antigen, which typically includes a detection tag such as FLAG, and quantitating by detection means such as ELISA or Western blotting. Protein standards, such as FLAG-tag standards available from Sigma, may be used in such assays to generate a calibration curve. In Example 1, FLAG-ROP was barely detectable (corresponding to <25 ng of specific volume of cells cultured to OD 1.0), which corresponds to <80 ng / mg dry cell weight, assuming a cell density of 0.3 g / L at OD 1.0.The amount of antigen produced can vary depending on the strength of the promoter, the copy number of the heterologous nucleic acid molecule per cell, and the like.

[0064] In any of the embodiments, the bacterial cell may contain an additional heterologous nucleic acid molecule encoding an immunostimulant or adjuvant that can be co-expressed with the antigen. Exemplary immunostimulants may be polypeptides, such as cytokines such as IL-12, IL-18 or GM-CSF, IFN-γ, IL-2, IL-15, etc. 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). Thus, a suitable immunostimulant for combination with any HPV antigen is IL-12.

[0065] A corresponding aspect of the invention provides a method of preparing a bacterium according to the first aspect, comprising introducing at least one heterologous nucleic acid molecule into the bacterium. Pharmaceutical compositions and methods of preparation

[0066] A second aspect of the invention is a pharmaceutical composition comprising the bacterium according to the first aspect.

[0067] A corresponding aspect of the invention provides a method of preparing a pharmaceutical composition according to the second aspect, comprising formulating the bacteria together with one or more pharma- ceutically acceptable diluents or excipients.

[0068] Although the bacteria can be administered alone, it is preferred that they are present in a pharmaceutical composition. The present invention includes pharmaceutical compositions containing at least one pharma- ceutically acceptable carrier, excipient, or additional component, such as a therapeutic and / or prophylactic component (such as an adjuvant). As used herein, "pharmaceutically acceptable carrier" refers to any known compound or combination of known compounds known to those skilled in the art to be useful in formulating a pharmaceutical composition. A carrier can include one or more excipients or diluents.

[0069] The Clostridia can be prepared by fermentation under conditions suitable for bacterial growth. After fermentation, the bacteria can be purified using centrifugation and prepared to retain activity. The bacteria in the composition are provided as viable organisms. The composition can contain bacterial spores and / or vegetative cells. The bacteria can be dried to retain bacterial activity. Suitable drying methods include freeze drying, spray drying, heat drying, and combinations thereof. The resulting powder can then be mixed with one or more pharma- ceutically acceptable excipients as described herein.

[0070] The spores and / or vegetative forms of the bacteria may be formulated with useful excipients and ingredients for oral administration as described herein. In particular, fatty and / or aqueous ingredients, humectants, thickeners, preservatives, texturizing agents, flavor enhancers and / or coating agents, antioxidants, preservatives, and / or dyes that are commonly used in the pharmaceutical and dietary supplement industries. Suitable pharmaceutically acceptable carriers include crystalline cellulose, cellobiose, mannitol, glucose, sucrose, lactose, polyvinylpyrrolidone, magnesium silicate, magnesium stearate and starch, or combinations thereof. The bacteria may then be formulated into a suitable orally ingestible form as described herein. The live bacteria in a suitable orally ingestible form may 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.

[0071] The pharmaceutical composition of the present invention can be in a dosage form, such as a unit dose form. Pharmaceutical compositions include those suitable for oral or rectal administration. The composition of the present invention can be administered once or continuously as part of a treatment plan. Preferably, administration is oral administration using a convenient dosing plan.

[0072] Suitable oral dosage forms include tablets, capsules, powders (e.g., powders in sachets) and liquids. When the bacterium is for oral administration, it is suitably provided in the form of a spore; or in the form of a vegetative cell in a delayed release pharmaceutical composition.

[0073] The pharmaceutical composition of the present invention can also be formulated for rectal administration, including suppository and enema preparation.For suppository, low melting wax such as mixture of fatty acid glyceride or cocoa butter is first melted, and active ingredient is dispersed homogeneously, for example by stirring.The homogeneous mixture is then poured into a mold of convenient size and cooled to solidify.Enema preparation can be semi-solid, including gel or ointment, or liquid form, including suspension, aqueous solution or foam, which are known to those skilled in the art.

[0074] The pharmaceutical composition of the present invention is administered such that an effective amount of the bacteria is delivered to the anaerobic portion of the intestine. By "effective amount of the bacteria" we mean that the bacteria induces an appropriate immune response to the antigen; or results in the delivery of an amount of antigen effective to prevent, ameliorate, or treat a disease. For example, in the case of a viral infection where a CTL response may be appropriate, the antigen may induce CD8 + This will be an amount effective to induce a CTL response.

[0075] Suitably, the bacteria are present in the pharmaceutical composition at a concentration of 1×10 of the dry composition. 5 ~1×10 11 The bacteria may be present in an amount equivalent to colony forming units / g (CFU / g). Suitably, the bacteria may be present in an amount equivalent to 1×10 per unit dosage form. 6 ~1×10 10 CFU, preferably about 1×10 per unit dosage form 7 ~1×10 9 CFU, e.g., about 1 x 10 per unit dosage form 8 It may be present in amounts of CFU.

[0076] The pharmaceutical composition may include an adjuvant or an immunostimulatory molecule, especially a pharmaceutical composition formulated for delayed release.However, it is envisaged that an adjuvant may not be required, or may only be required in a smaller amount than would be required if the antigen were provided in a conventional polypeptide antigen vaccine formulation, or only a less toxic adjuvant may be required.Therefore, pharmaceutical compositions that do not include an adjuvant are envisaged, as well as pharmaceutical compositions that include only an adjuvant suitable for human use, such as alum.

[0077] An adjuvant is any substance that, when incorporated into a pharmaceutical composition, increases or otherwise alters the immune response to an antigen. Adjuvants 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, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP11687, also known as nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1-methylpropyl)-2-propanol ( ... '2'-Dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP19835A, also called MTP-PE), RIBI in 2% squalene / Tween-80® emulsion (MPL+TDM+CWS), lipopolysaccharide and its various derivatives (including lipid A), Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck's adjuvant 65, polynucleotides (e.g., poly IC and poly AU acid), wax derived from Mycobacterium tuberculosis, D, substances found in Corynebacterium parvum, Bordetella pertussis, and Brucella bacteria, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see U.S. Patents 58,767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrices or GMDP, interleukin 1, interleukin 2, Montanide ISA-51, and QS-21.

[0078] Additional adjuvants or compounds that can be used to modify or stimulate immune responses include ligands of 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 studied as vaccine adjuvants. Bacterial lipopolysaccharide (LPS) is a TLR4 ligand and its detoxified variant monophosphoryl lipid A (MPL) is an adjuvant approved for human use. TLR5 is expressed on monocytes and DCs and responds to flagellin, while TLR9 recognizes bacterial DNA containing CpG motifs. Oligonucleotides (OLGs) containing CpG motifs are potent ligands and agonists of TLR9 and have been intensively studied for their adjuvant properties.

[0079] Other agents that stimulate the immune response (immunostimulants) may be 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.

[0080] The pharmaceutical composition of the present invention can be formulated as a capsule containing live cells, such as vegetative cells or spores, where the capsule includes a delayed release layer or coating that allows the release of live cells, typically vegetative cells, in the anaerobic portion of the lower gastrointestinal tract after oral administration. "Delayed release" means that the release of the bacteria is delayed for a period of time after administration or ingestion (the delay is also known as 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.

[0081] Excipients and formulations for delayed release are well known in the art and certain technologies are commercially available.

[0082] Various strategies have been proposed to target orally administered drugs to the colon, including coating with pH-sensitive polymers, formulation of timed-release systems, use of carriers that are specifically degraded by colonic bacteria, bioadhesive systems, and osmotically controlled drug delivery systems. Biodegradable polymers, especially azo-crosslinked polymers, have been investigated for use as coatings for colon-targeted drugs.

[0083] Certain plant polysaccharides, such as amylose, inulin, pectin, and guar gum, are unaffected in the presence of gastrointestinal enzymes and therefore have been investigated as coatings for drugs for the formulation of colon-targeted drug delivery systems. Furthermore, the combination of plant polysaccharides with shellfish extracts (including chitosan or its derivatives) has proven to be interesting in the development of colonic delivery systems.

[0084] Examples of excipients for delayed release formulations include hydrogels, which can swell rapidly in water and retain large amounts of water within their swollen structure. Different types of hydrogels have different drug release patterns, so the use of hydrogels to enhance colonic delivery has been investigated. For example, hydrogels have been prepared using Eudispert hv, a high viscosity acrylic resin gel, which has excellent long-term retention in the lower rectum. Eudragit® polymers (Evonik Industries) offer various forms of coatings, including gastroresistant, pH-controlled drug release, colonic delivery, protection of active substances, and protection from active substances.

[0085] The pharmaceutical composition may be prepared by coating the bacteria and one or more pharma- ceutically acceptable carriers, excipients and / or diluents with a delayed release layer or coating using techniques known in the art. For example, the coating may be formed by compression using any of the known press coaters. Alternatively, the pharmaceutical composition may be prepared by granulation and agglomeration techniques, or may be constructed and subsequently dried using spray drying techniques.

[0086] The thickness of the coating can be precisely controlled by using any of the techniques described above. One skilled in the art can select the coating thickness as a means to obtain a desired lag time and / or a desired rate at which the bacteria are released after the lag time.

[0087] pH-dependent systems take advantage of the generally accepted view that the pH of the human gastrointestinal tract increases gradually from the stomach (where the pH may be about 1-2, rising to pH 4 during digestion) through the small intestine at the site of digestion (where the pH may be about 6-7), and further increases in the distal ileum. Coating the tablet, capsule or pellet with a pH-sensitive polymer provides delayed release and protects the active agent from gastric juices.

[0088] The pharmaceutical composition of the present invention may be formulated to deliver the bacteria according to the first aspect to the GI tract at a specific pH. Commercially available excipients include Eudragit® polymers that can be used to deliver bacteria to specific sites in the gastrointestinal tract. For example, the pH in the duodenum may be greater than about 5.5. Eudragit® L 100-55 (powder), Eudragit® L 30 D-55 (aqueous dispersion), and / or Acryl-EZE® (Powder) can be used, for example, as a ready-to-use enteric coating based on Eudragit® L 100-55. The pH in the jejunum may be about 6 to about 7, and Eudragit® L 100 (powder) and / or Eudragit® L 12,5 (organic solution) can be used. Colonic delivery can be achieved at a pH above about 7.0, so Eudragit® S 100 (powder), Eudragit® S 12,5 (organic solution), and / or Eudragit® FS 30 D (aqueous dispersion) can be used. PlasACRYL™ T20 glidant and plasticizer premix, specially designed for the Eudragit® FS 30 D formulation, can also be used.

[0089] Suitably, the pharmaceutical composition of the invention is formulated to deliver the bacteria according to the first aspect to the gastrointestinal tract, preferably by oral administration.

[0090] The human gastrointestinal tract is composed of the structures involved in digestion, such as the esophagus, stomach, and intestines, which extend from the mouth to the anus. The gastrointestinal tract does not include accessory glandular organs, such as the liver, bile duct, and pancreas. The intestines include the small intestine and the large intestine. The small intestine includes the duodenum, jejunum, and ileum. The large intestine includes the cecum, colon, rectum, and anus. The upper gastrointestinal tract includes the buccal cavity, pharynx, esophagus, stomach, and duodenum. The lower gastrointestinal tract includes the small intestine (below the duodenum) and the large intestine. Preferably, the pharmaceutical composition of the invention delivers the bacteria according to the first aspect to the lumen or mucosal surface of the gastrointestinal tract, more preferably to the lumen or mucosal surface of the large intestine, more preferably to the lumen or mucosal surface of the colon. Preferably, the pharmaceutical composition of the invention delivers the bacteria according to the first aspect to the anaerobic portion of the lower gastrointestinal tract, preferably to the colon and / or the terminal small intestine (terminal ileum, also called "terminal ileum").

[0091] As reviewed in Zheng, Kelly and Colgan, American Journal of Physiology-Cell Physiology 2015 309:6, C350-C360, a steep oxygen gradient exists within the human intestinal tract. At sea level, breathable air has a Po2 of around 145 mmHg (around 21% O2). Measurements of healthy alveoli reveal a Po2 of 100-110 mmHg. In stark contrast, the most luminal portion of a healthy colon has a Po2 of 10 mmHg (1.4% O2) or less. These differences reflect a combination of oxygen sources, local metabolism, and anatomic morphology of blood flow. The Po2 drops sharply along the radial axis from the intestinal submucosa to the lumen, where trillions of anaerobic microorganisms live.

[0092] When bacteria are delivered orally as spores, they pass through the gastrointestinal tract to the anaerobic portion where they germinate and grow. The anaerobic portion of the lower gastrointestinal tract includes the terminal ileum and colon. According to Zheng's observation above, the colon may have a lower Po2 than the terminal ileum, so bacterial growth may be more efficient in the colon. The Po2 required to cause spore germination and anaerobic metabolism or growth may range from 0 to 2%.

[0093] The volume of the human colon (the sum of the ascending, descending and transverse colon) is approximately 600 ml (Pritchard, SE et al. (2-14) Neurogastroenterol. Motil. 26, 124-130), whereas the total intestinal volume of a mouse is approximately 1 ml (McConnell, EL, Basit, AW & Murdan, S. (2008) J. Pharm. Pharmacol. 60, 63-70). The approximate total gastrointestinal transit time is about 5-6 hours in mice (Padmanabhan, P., et al. (2013) EJNMMI Res. 3, 60 and Kashyap, PC et al. (2013) Gastroenterology 144, 967-977), while colonic transit time is estimated to be 23-40 hours in humans (Degen, LP & Phillips, SF (1996) Gut 39, 299-305 and Wagener, S., et al (2004) J. Pediatr. Surg. 39, 166-169-169). Since the transit time in the human intestine is 5 times longer than in mice, fewer spores (e.g., 5 times fewer) are needed to obtain the same concentration of antigen for the same colon volume.

[0094] Furthermore, the bacteria reside in the human colon for approximately 5 times longer than in the mouse colon, resulting in more time for cell division (5 times), resulting in more cells and more antigen production. The doubling time based on laboratory fermentation of strain CHN1 is similar to that of E. coli, which has an intestinal doubling time of approximately 3 hours (Myhrvold, C., et al (2015) Nat. Commun. 6, 10039). CHN1 can undergo 10 cell doublings during its passage through the intestine, which corresponds to a three-order increase in cell number. In mice, there is only enough time for approximately two cell doublings, which corresponds to a less than 10-fold increase in cell number. Approximately 100 times more cells are grown from each spore delivered to the human intestine compared to the mouse intestine. Taking into account the difference in intestinal volume, colonic transit time, and cell division in the intestine, the amount of antigen in the intestinal lumen will be approximately the same if approximately the same dose is delivered to mice and humans.

[0095] The differences between the human and murine intestines are detailed above, but this can be readily adapted to other hosts based on what is known in the art (e.g., delivery of bacteria can be adapted to the intended host, e.g., other mammals or birds).

[0096] Pharmaceutical compositions taken on an empty stomach are expected to reach the ascending colon approximately 5 hours after ingestion, but actual arrival is highly dependent on the rate of gastric emptying. Drug delivery in the colon is greatly influenced by the rate of passage through this region. In healthy men, a capsule will pass through the colon in an average of 20-30 hours. Solutions and particles are usually distributed extensively in the proximal colon and often disperse throughout the large intestine.

[0097] The pharmaceutical composition of the invention may be formulated for time-controlled delivery to the gastrointestinal tract, i.e. to deliver the bacteria according to the first aspect, thereby providing the antigen after a certain time (lag time) after administration.

[0098] Commercially available excipients for time-controlled delivery include Eudragit® RL 30 D (aqueous dispersion) and Eudragit® RL 12,5 (organic solution). These excipients are insoluble, highly permeable, pH-independent swelling excipients that can be combined with Eudragit® RS in different ratios to provide customized release profiles. Eudragit® RS 30 D (aqueous dispersion) and Eudragit® RS 12,5 (organic solution) are insoluble, poorly permeable, pH-dependent swelling excipients that can be combined with Eudragit® RL in different ratios to provide customized release profiles. Eudragit® NE 30 D (aqueous dispersion), Eudragit® NE 40 D (aqueous dispersion), and Eudragit® NM 30 D (aqueous dispersion) are insoluble, low-permeability, pH-dependent swelling excipients and can be matrix formers.

[0099] Preferably, the pharmaceutical composition may be formulated to deliver the bacteria according to the first aspect to the gastrointestinal tract about 4 hours after administration (i.e. after oral administration). Preferably, the pharmaceutical composition may be formulated to deliver the bacteria according to the first aspect about 4 to 48 hours after administration, preferably about 5 to 40 hours after administration, such as about 5, 10, 15, 20 or 24 hours after administration; preferably about 5 to 10 hours, 5 to 15 hours, 5 to 20 hours, or about 10 to 24 hours, 15 to 24 hours, or 20 to 24 hours after administration.

[0100] Suitably the pharmaceutical composition is for administration between meals or with a meal.

[0101] The proliferation of the bacteria according to the first aspect of the invention upon reaching the anaerobic part of the intestine can be confirmed by culture, such as fecal culture. In experimental models, the bacteria can be cultured from parts of the gastrointestinal tract obtained from experimental animals. The proliferation of the bacteria according to the first aspect of the invention upon reaching the anaerobic part of the intestine can also be confirmed by immunohistological approaches known to those skilled in the art, for example, using antibodies that recognize the bacteria.

[0102] The genetically modified anaerobic bacteria producing the antigens can also be incorporated as part of a food product, i.e., in yogurt, milk or soy milk, or as a dietary supplement. Such foods and dietary supplements can be prepared by methods well known in the food and dietary supplement industry.

[0103] The composition can be incorporated into animal feed products as a feed additive.

[0104] The extent of growth and colonization of the genetically modified bacteria in the intestine can be controlled by the choice of species and strain and / or by providing specific substrates on which the bacteria grow as prebiotics, either in the same dose with probiotics or as separate compositions taken.

[0105] Therefore, the composition may also contain or be administered with prebiotics to enhance the growth of administered probiotics. Prebiotics may be administered sequentially, simultaneously, or separately with the bacteria described herein. Prebiotics and bacteria may be formulated together in the same composition for simultaneous administration. Alternatively, bacteria and prebiotics may be formulated separately for simultaneous or sequential administration.

[0106] Prebiotics are substances that promote the growth of probiotics in the intestine. They are food substances that are fermented by bacteria in the intestine. The addition of prebiotics provides a medium that can promote the growth of probiotic strains in the intestine. One or more monosaccharides, oligosaccharides, polysaccharides, or other prebiotics that promote bacterial growth may be used.

[0107] Preferably, the prebiotic may be selected from the group consisting of oligosaccharides, optionally containing fructose, galactose, mannose; dietary fiber, especially soluble fiber, soy fiber; inulin; or combinations thereof. Preferred prebiotics are fructooligosaccharides (FOS), galactooligosaccharides (GOS), isomaltooligosaccharides, xylooligosaccharides, soybean oligosaccharides, glycosylsucrose (GS), lactosucrose (LS), lactulose (LA), palatinose oligosaccharides (PAO), maltooligosaccharides, pectin, their hydrolysates, or combinations thereof. medical use

[0108] A third aspect of the invention provides the bacterium of the first aspect or the pharmaceutical composition of the second aspect for use in medicine.

[0109] A fourth aspect of the invention provides a bacterium of the class Clostridium for use in generating an antigen-specific response in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule encoding an antigen and is capable of expressing the antigen in an intracellular compartment of the bacterium during anaerobic cell growth.

[0110] A corresponding embodiment provides a method of generating an antigen-specific immune response in a subject comprising administering a bacterium of the class Clostridium, where the bacterium comprises a heterologous nucleic acid molecule encoding the antigen and is capable of expressing the antigen in an intracellular compartment of the bacterium during anaerobic cell growth.

[0111] A fifth aspect of the invention provides a bacterium of the class Clostridium for use in the therapeutic or prophylactic treatment of a disease in a subject, wherein the bacterium comprises a heterologous nucleic acid molecule encoding an antigen, and wherein the bacterium is capable of expressing said antigen in an intracellular compartment of the bacterium during anaerobic cell growth, and wherein the antigen is an infectious pathogen antigen and the disease is a disease caused by an infectious pathogen, or wherein the antigen is a tumour antigen and the disease is cancer.

[0112] A corresponding aspect provides a method of preventing, ameliorating, or treating a disease in a subject comprising administering a bacterium of the class Clostridium, wherein the bacterium comprises a heterologous nucleic acid molecule encoding an antigen, and wherein the bacterium is capable of expressing said antigen in an intracellular compartment of the bacterium during anaerobic cell growth, and wherein the antigen is an infectious pathogen antigen and the disease is a disease caused by an infectious pathogen, or wherein the antigen is a tumor antigen and the disease is cancer.

[0113] Typically, in any of these aspects of the invention, the subject is a mammal or bird, typically a mammal, preferably a human.The mammals suitable for animal vaccination include agricultural animals, such as ungulates, including cows, sheep, or goats; or horses; or domestic animals, such as cats or dogs.Suitable birds include chickens or turkeys.Typically, when the antigen is an infectious pathogen antigen, the subject is a species susceptible to the disease caused by the infectious pathogen.Typically, when the antigen is a tumor antigen, the subject is a species of which the tumor antigen is a tumor characteristic.

[0114] These uses include vaccination. Suitable doses and administration schedules for vaccination (e.g., a primary dose 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 may involve one to three doses spaced about three weeks to six months apart. In some embodiments, vaccination may be provided in a vaccination regimen with different vaccines, such as a prime-boost regimen, in which the vaccine of the invention is either a prime vaccine or a booster vaccine, the other of which is a different vaccine. There may be two or more booster doses. Typically, such regimes are directed to the same infectious agent or the same cancer. Medical applications in generating antigen-specific immune responses

[0115] In a fourth embodiment, the antigen may be any antigen as defined herein, including but not limited to a tumor antigen or an infectious agent antigen. For example, the antigen may include an artificial sequence (i.e., an artificially designed sequence that does not exist in nature).

[0116] An "antigen-specific immune response" includes any cellular or humoral immune response that is antigen-specific, i.e., CD4 + T cell response, CD8 + This includes T cell responses, such as T cell responses, or B cell (antibody) responses.

[0117] In a typical immune response, antigens are delivered to antigen-presenting cells (APCs), particularly dendritic cells (DCs), which then express antigen-specific cytotoxic CD8 + (CTL) and / or helper CD4 +Stimulate and induce T lymphocytes. DCs, also known as professional APCs, sample antigens in the microenvironment and process them intracellularly (e.g., after the antigens are phagocytosed). When DCs are activated (e.g., due to inflammatory signals), they can migrate to lymph nodes, thereby activating adaptive immune responses. Without being bound by theory, Clostridium bacteria can be internalized by APCs, particularly intestinal DCs (such as mucosal DCs). For example, DCs that have internalized Clostridium bacteria and thus taken up (e.g., phagocytosed) antigens can be activated, migrate to lymph nodes, and activate T cells with specificity for the antigen, and subsequently activate B cells. In addition to Clostridium bacteria, APCs can be exposed to further activation signals, such as those provided by adjuvants, lipopolysaccharide (LPS), and inflammatory cytokines.

[0118] T cells express T cell receptors that recognize antigenic peptides presented by major histocompatibility complexes (MHC), which in humans are called human leukocyte antigens (HLA). Helper CD4 + T cells are cytotoxic CD8 + It effectively stimulates and amplifies T cells and helps B cells produce antibodies. + The response is induced / activated CD4 + They can be classified according to the type of T cell. For example, CD4 + The response can be a T helper (Th)1, Th2, and / or Th17 response. Th1, Th2, and Th17 cells can be classified by markers (e.g., cell surface markers), cytokine secretion, and / or functional assays known to those of skill in the art. + The type of response (or combination) may depend on the antigen and / or adjuvant or other immunomodulatory molecules used, which may be selected depending on the desired outcome. For example, a Th2 response is more suitable than a Th1 response for protection against helminth infections. Th1 responses, which are often associated with IFN-γ production, are more suitable than Th2 responses for protection against intracellular parasites. Th1 cells are CD8+ Th1 cells stimulate killer T cells, Th2 cells stimulate B cells; Thl7 cells promote inflammation.

[0119] CD8 + T cells can specifically recognize target cells containing the target antigen and induce apoptosis. + T cell activation depends on efficient presentation of antigens on MHC class I molecules (HLA-I antigens in humans). CD8 + Cytotoxic T lymphocytes (CTLs) are the main cell type targeted by prophylactic and therapeutic cell-mediated immune vaccines because they can directly recognize and destroy tumor cells and cells infected with intracellular infectious agents such as viruses. Therefore, for the purpose of targeting tumor cell antigens and antigens of intracellular infectious agents such as viruses, these cells directly recognize these antigens presented on the cell surface of MHC class I molecules and thus act against infectious agents such as viruses by expressing CD8 + It may be advantageous to initiate a response. CTLs have also been associated with anti-tumor responses.

[0120] CD4 + A subset of cells releases cytokines into the local microenvironment, resulting in CD8 + It may support and / or enhance the activity of CD4 + Responses and CD8 + Combining responses can be beneficial. Thus, in some embodiments, a combination of T cell responses is induced by the antigen.

[0121] The efficiency with which a single peptide antigen stimulates an immune response may vary between subjects and populations based on its expression profile for MHC (or HLA in humans). MHC / HLA haplotypes vary between subjects, with each haplotype of MHC / HLA able to bind and thereby present a particular type of peptide fragment. For example, for the same antigen, the peptide fragment presented by the MHC / HLA of a first subject may differ in sequence from that 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 a population in responsiveness to a particular antigen. This is a major drawback of single peptide-based vaccines, since not all subjects can properly process and present peptides 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 containing overlapping peptides as described above, including the ROPs described herein and in EP3235831A1. ROPs are a type of fusion protein that binds to CD4 + and CD8 + They have been shown to be capable of inducing T cell responses simultaneously and are composed of multiple peptide segments, greatly increasing the likelihood that a suitable segment exists for a particular subject, thus overcoming population MHC / HLA restriction.

[0122] B cell responses are characterized by antibodies (i.e., "immunoglobulins" or "Ig") that target specific antigens. B cells can also internalize components such as polypeptides and present fragments of polypeptide molecules on the cell surface in complexes with MHC class I or II molecules. B cells may also express an antigen-specific B cell receptor (BCR) on their cell surface. Unlike T cells and TCRs, which rely on antigens presented by MHC, BCRs can recognize antigen epitopes without being presented by MHC (i.e., BCRs can also recognize soluble antigens). Antigens directly activate B cells bearing the appropriate surface immunoglobulin to produce IgM. In some cases, B cells rely on T cells for activation by presenting antigens loaded on MHC class II. CD4 in response to processed antigens + T cells can be induced to class switch from IgM to IgG immunoglobulins. However, some antigens can activate B cells independently of T cells. Thus, in some embodiments, induction of a B cell response is achieved by inducing a T cell response (CD4 + and / or CD8 + ) may be linked to the induction of

[0123] Suitable antibody responses may include various isotypes, such as IgA and / or IgG isotypes. The type of antibody response achieved may depend on the antigen and / or adjuvant or other immunomodulatory molecule used and may be selected depending on the desired result.

[0124] IgA, also called slgA in its secretory form, is an antibody that plays a key role in mucosal immune function. The amount of IgA produced in association with mucosa is greater than all other types of antibodies combined. In absolute terms, 3-5 grams are secreted into the intestinal lumen daily. This represents up to 15% of the total immunoglobulin produced throughout the body. There are two subclasses of IgA (IgA1 and IgA2) that can be produced as both monomers and dimers. The dimeric form of IgA is the most common and is also called secretory IgA (slgA). slgA is the major immunoglobulin found in mucosal secretions, including tears, saliva, sweat, colostrum, and secretions from the urogenital tract, gastrointestinal tract, prostate, and respiratory epithelium. It is also found in small amounts in blood. The secretory component of slgA protects the immunoglobulin from degradation by proteolytic enzymes; thereby, slgA can survive the harsh gastrointestinal environment and protect against microorganisms that grow in the body's secretions. slgA can also inhibit the inflammatory effects of other immunoglobulins. IgA activates the complement system poorly and has poor opsonization.

[0125] There are several subtypes of IgG: in humans, IgG1 and IgG3 are associated with T helper type 1 responses, complement fixation, and phagocytosis via the high affinity FcR, and confer protective immunity, whereas IgG2 and IgG4 responses tend to be less effective.

[0126] 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 including or consisting of IgA, IgM, IgG, or any combination thereof. In some embodiments, the antigen-specific antibody is a secretory antibody, such as secretory IgA (slgA), secretory IgM, or secretory IgG. Thus, in some embodiments, the antigen induces the production of antigen-specific IgA, antigen-specific IgM, antigen-specific IgG, or any combination thereof. In some embodiments, the Vibrio cholerae antigen described herein induces a Vibrio cholerae antigen-specific immune response, e.g., a Vibrio cholerae antigen-specific B cell immune response, or the production of Vibrio cholerae antigen-specific antibodies. The Vibrio cholerae antigen-specific antibody may be IgA, IgM, or IgG. In some embodiments, the Vibrio cholerae antigen-specific antibody is IgA, optionally secretory IgA (slgA). In some embodiments, the Vibrio cholerae antigen is CtxB. In some embodiments, CtxB induces a CtxB-specific response, e.g., a CtxB-specific B cell response or the production of a CtxB-specific antibody. The CtxB-specific antibody may be IgA, IgM, or IgG. In some embodiments, the CtxB-specific antibody is IgA, optionally secretory IgA (slgA).

[0127] Bacteria containing the antigens described herein may be tested for their ability to induce antigen-specific immune responses, such as after oral immunization in a mouse model. Bacterial spores (e.g., Clostridium butyricum) containing the antigen of interest (e.g., a Vibrio cholerae antigen such as HPV, OVA, or CtxB) or a ROP corresponding to the antigen (e.g., ROP-HPV or ROP-OVA) can be administered by oral gavage to a group of mice. A negative control of spores derived from the same bacteria but not containing the antigen (or ROP antigen) may be administered to another group of mice. The antigen-containing bacteria are compared to such a negative control, and any differences are considered to be antigen-specific. Suitable positive controls for this experiment include parenteral administration by subcutaneous injection of the antigen (e.g., ROP-HPV or ROP-OVA, or a Vibrio cholerae antigen such as CtxB), which is taken up by DCs and leads to the activation of an immune response against said antigen. Comparison with this positive control would therefore provide an indication of whether the immune response induced by the antigen-containing bacteria is equivalent to the administration of the antigen itself.

[0128] Thus, this type of system can be used to identify antigen-specific responses and is not limited to a particular type of antigen. Indeed, the negative control is left as is (bacteria without antigen), the positive control is changed to parenteral administration by subcutaneous injection of the antigen of interest (e.g., any of the infectious pathogen antigens or tumor antigens described herein), and the test conditions only require preparation of bacteria containing the antigen of interest (or ROP of said antigen).

[0129] Furthermore, the bacteria is not limited to the exemplified Clostridium butyricum as it acts as a delivery vehicle for the antigen of interest. The exemplified Clostridium butyricum acts to deliver the antigen to the anaerobic portion of the gastrointestinal tract where the antigen can mediate an immune response. Therefore, other bacteria of the class Clostridium that are also known to be able to reach the anaerobic portion of the gastrointestinal tract are suitable delivery vehicles as well. In this case, the negative control is changed to the Clostridium bacterium of interest (such as those described herein), the positive control remains parenteral administration by subcutaneous injection of the antigen of interest, and the test conditions simply require that the selected bacterium is genetically modified to express the antigen of interest, in a manner similar to that described herein for Clostridium butyricum.

[0130] The administration of bacteria containing antigen, negative control and positive control can be carried out in an immunization regimen.For example, mice can be immunized three times at intervals of two weeks.After the immunization regimen, the mice are sacrificed, for example, after 42 days of the regimen.This regimen is carried out to evaluate whether the genetically modified bacteria spores containing antigen can deliver the antigen in a way that induces immune response.

[0131] Immune responses can be detected in a number of ways known to those skilled in the art. For example, splenocytes from homogenized spleens and peripheral blood mononuclear cells (PBMCs) from blood samples can be collected using standard methods to obtain mononuclear cell isolates. These mononuclear cell isolates can then be processed with various sorting protocols to isolate cell populations of interest, for example, using magnetic associated cell sorting (MACS) or Ficoll-Hypaque gradient (density) separation to obtain lymphocytes. For example, T cells (CD4 + and CD8 + ) are T cell specific markers (e.g., CD8 + T cells may be enriched from the mononuclear cell isolate based on CD8a. Alternatively, or in addition, other cell populations may be isolated, such as B cells.

[0132] Once an enriched population of a cell type of interest is obtained, the cell type can be tested for secretion of cytokines associated with activation of an immune response (e.g., IFN-γ and / or TNFα) or for expression of markers indicative of an activated cell phenotype (e.g., cell surface and / or intracellular markers). Cytokines such as IFN-γ and / or TNFα can be tested in ELISPOT by culturing T cells in plates in the presence of anti-IFN-γ or anti-TNFα antibodies, respectively, and restimulating the cells with wild-type antigen protein (e.g., HPV protein or Vibrio cholerae antigen such as CtxB), ROP of the antigen (e.g., ROP-HPV or ROP-OVA), or vegetative bacterial cells containing the antigen. If T cells with specificity for the antigen of interest (e.g., HPV-specific T cells) are present, then when restimulated with the antigen, the T cells will be induced to secrete IFN-γ and / or TNFα. The use of vegetative bacterial cells that do not contain antigen serves as a negative control, which can be compared with the test condition to determine how antigen-specific the secretion of IFN-γ and / or TNFα is. IFN-γ and / or TNFα standards (i.e., aliquots of these cytokines at various concentrations) can be used as positive controls and to establish dose-response curves. For ELISPOT, spot-forming units (SPUs) can be evaluated, where an SPU of the test condition (e.g., vaccinated group) that is at least two standard deviations higher than the mean of the control group indicates a positive result for the test condition.

[0133] Alternatively, or in addition, immune responses can be tested by intracellular cytokine staining as described in Zhang et al., 2009. Briefly, splenocytes obtained from mice that received the above immunization regimen can be cultured with antigen (e.g., ROP antigen), as well as the same negative and positive controls as in ELISPOT. The cells can then be labeled with antibodies (e.g., phycoerythrin-conjugated monoclonal rat anti-mouse CD8 or CD4 antibodies) or immunoglobulin isotype controls. The splenocytes can then be fixed and permeabilized using a fix / perm protocol (e.g., Cytofix / Cytoperm kit by BD Pharmingen) and incubated with a detection antibody for the intracellular antigen (e.g., fluorescein isothiocyanate-conjugated anti-IFN-γ antibody). Samples can then be evaluated by flow cytometry, where antigen-specific activation of cells is indicated by fluorescence above that of the isotype control. Co-staining of CD8 or CD4 with IFN-γ can be used to detect antigen-specific expression of IFN-γ by CD8. + or CD4 + The T cells will be assigned to one of the following groups:

[0134] Alternatively, or in addition, the immune response can be tested by detecting expression of T cell surface receptors or receptor ligands, typically after restimulation of T cells with APCs. For example, cell surface CD40 ligand expression can be detected by detecting CD4 + It can be assessed on T cells that express CD40L (CD154) after defined antigenic stimulation. + The percentages of T cells can be determined and non-parametric analysis can be performed between experimental and control groups to identify differences in the population mean antigen-specific T cell percentages. A positive result for a test condition is a higher percentage of antigen-specific CD4 T cells compared to the negative control group. +As indicated by a higher percentage of T cells (ie, upregulation of CD40L), for example, at least 1% higher, at least 2% higher, at least 5% higher, and / or up to 10% or more higher.

[0135] The cytotoxicity of the CTL response was evaluated using chromium 51 ( 51 The efficacy of CTLs can be assessed using a Cr) 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 tumor antigens) can be subjected to a cytolysis assay using a Cr) release assay. 51 The cytotoxicity of CTLs from vaccinated subjects (expected to be able to mount an antigen-specific response) can therefore be compared to CTLs from control naive subjects (not expected to have antigen-specific CTLs). 51 An increase in Cr detection indicates a positive result for induction of an antigen-specific response. Typically, a positive result for the test group is indicated when the mean of the test group is at least two standard deviations higher than the mean of the control group.

[0136] Another suitable assay for assessing cytotoxicity is the CyQUANT LDH cytotoxicity assay. Lactate dehydrogenase (LDH) is a cytosolic enzyme that is released upon cell membrane damage. Thus, 51 Using the same conditions as described for the Cr release assay, LDH levels can be tested in co-cultures of CTLs and target cells to see if the vaccinated group has higher LDH, indicative of increased cytotoxicity, compared to the control group. Typically, a positive result for the test group is indicated when the mean value of the test group is at least two standard deviations higher than the mean value of the control group.

[0137] Alternatively, or in addition, 3 H thymidine can be used to test for T cell proliferation.3 H is incorporated into new strands of chromosomal DNA during mitotic cell division and therefore accumulates in cells as cells divide. T cells (or subsets of T cells) isolated from vaccinated subjects can be compared to T cells (or subsets of T cells) isolated from control naive subjects. Isolated T cells can be co-cultured with PBMCs or antigen-loaded activated DCs in a mixed lymphocyte reaction (MLR) and their proliferation assessed over time. If the vaccination regimen results in antigen-specific T cells, these will proliferate at a higher rate when co-cultured with antigen-presenting cells expressing the antigen. Thus, a greater number of T cells can be obtained. 3 An increase in T cell proliferation based on H thymidine incorporation indicates a positive finding for the vaccinated subject. Typically, a positive result for the test group is indicated when the mean of the test group is at least two standard deviations higher than the mean of the control group.

[0138] Another suitable assay for assessing cell proliferation is the carboxyfluorescein succinimidyl ester (CFSE) assay. CFSE is a fluorescent cell staining dye that reacts with free amines within cells to generate covalent dye-protein complexes. This results in live cells that can be detected on the basis of CFSE fluorescence by flow cytometry or fluorescence microscopy. As cells bearing CFSE divide, the level of CFSE fluorescence divides between cells, allowing the visualization of peaks corresponding to generations of cell division. Thus, the above 3 The same conditions as for H-thymidine can be assessed with a CFSE assay, in which an increase in T cell proliferation is based on the detection of an additional emission peak of fluorescein, which indicates cell division as a positive finding for vaccinated subjects. Typically, a positive result for the test group is indicated when the mean value of the test group is at least two standard deviations higher than the mean value of the control group.

[0139] B cell responses may be assessed by quantifying the levels of antibodies in serum or other suitable samples collected during or after the immunization regimen. Antibody titers are a measure of how much an organism has produced antibodies that recognize a particular epitope and are expressed as the reciprocal of the highest dilution (in a serial dilution) that gives a positive result. Antibody titers may be tested using ELISA. Thus, serum obtained from mice that have undergone the above immunization regimen may be assessed for antibody titers and compared to the same control. Higher antibody titers, such as at least two standard deviations higher compared to a negative control, would indicate that B cells have been activated in an antigen-specific manner. Since IgA antibody titers are indicative of mucosal immunity, levels of antigen-specific IgA may be specifically tested to assess induction of mucosal immunity. Suitable samples for testing IgA include serum, feces, colon or intestinal contents, or ileal wall extracts. Additionally or alternatively, antigen-specific IgG titers may be tested, indicative of systemic immunity and / or antigen-specific IgM. Typically, serum samples will be tested.

[0140] The ratio of antigen-specific IgA to total IgA may be measured to indicate the B cell response. For example, total IgA and antigen-specific IgA may be determined by ELISA. Non-parametric analysis may be performed between the experimental and control groups to explore differences in the population mean antigen-specific IgA / IgA ratios. A positive result would be the identification of a statistically significant difference in the population mean antigen-specific IgA / IgA ratio between the experimental and control groups.

[0141] Generally accepted animal models (such as those described in Ireson et al. (2019) British J Cancer 121:101-108) can be used to test immunization against cancer using tumor or cancer antigens. For example, cancer cells (human or murine) can be introduced into mice to produce tumors, and bacteria containing tumor antigens as described herein can be delivered to subjects with tumors associated with said antigens. Cancer cells can be introduced by subcutaneous injection to form xenografts or syngeneic tumors associated with the antigen of interest. The effect on the cancer cells (i.e., reduction in tumor size, which can be measured using calipers, or reduction in tumor progression (i.e., the rate at which the tumor continues to grow)) can be evaluated as a measure of the efficacy of immunization. More complex models include the use of patient-derived xenograft (PDX) models, in which antigens associated with the patient's cancer are administered to mice (e.g., humanized mice) that have undergone the immunization regimen described herein. Alternatively, or in addition, the level and activity of anti-tumor CTLs may be tested, for example, by taking tumor biopsies and testing the level of CTLs (including tumor antigen-specific CTLs) in the tumor microenvironment. Antigen-specific CTLs may be identified using MHC-loaded tumor antigen-specific MHC tetramers, and then CTLs in the tumor microenvironment may be quantified, for example, by flow cytometry. Biopsies may be tested for cytokines by measuring cytokines associated with inflammatory responses and T cell activation (e.g., IL-2, IFN-γ, GM-CSF). This test may also be performed in humans, with endpoints including testing for elevated levels of circulating cytotoxic T lymphocytes against antigen-bearing cells, testing for levels of circulating antibodies against the antigen, and testing for the presence of cells expressing the antigen.

[0142] A suitable study was described in Cai et al., 2017, which demonstrated that immunization with ROP-survivin or ROP-HPV-E7 generated a specific cellular immune response and protected mice against inoculation with melanoma B16 cells expressing survivin or HPV-E7 proteins. In this experiment, C57BL / 10 mice were subcutaneously primed with ROP antigens (ROP-survivin or ROP-HPV-E7) and compared with wild-type antigen as a positive control. In both conditions, the antigens were emulsified in monophosphoryl lipid A (MPL). The same vaccine emulsified in MPL was boosted subcutaneously twice, 3 weeks apart. Three weeks after the last boost, mice were challenged with B16-E7 or B16-survivin and subsequently assessed by ELISPOT analysis. ELISPOT analysis was performed on PBMCs and splenocytes as described above, with restimulation with ROP-HPV or ROP-survivin on anti-IFN-γ-Ab precoated plates.

[0143] The data in Cai et al., 2017 show a mouse system in which mice can be immunized with ROP and wild-type antigens for anti-tumor immune responses. Hence, the immunization strategy of Cai et al., 2017 can be expanded with the wild-type antigen corresponding to the ROP or tumor antigen as a positive control. Clostridium bacteria can then be genetically modified to express the tumor antigen and used in a parallel immunization regimen. The negative control is immunization with the same bacteria but without the antigen. Hence, this system can be used to determine whether bacteria containing tumor-specific antigens can induce anti-tumor responses by restimulating PBMCs or splenocytes with tumor antigens and comparing IFN-γ secretion to the positive and negative controls as described in Cai et al., 2017. A positive result for a test condition (e.g., vaccinated group) with SFU (spot-forming unit) counts at least two standard deviations higher than the mean of the control group indicates a positive result for that test condition.

[0144] Thus, one skilled in the art can readily assess whether a Clostridium bacterium containing an antigen, such as an infectious pathogen antigen and / or a tumor antigen, induces an immune response against said antigen. These systems are not limited to the type of antigen or bacterium. Therapeutic or prophylactic treatment of infectious disease or cancer in a subject

[0145] In this fifth embodiment, the antigen is an infectious pathogen antigen and the disease is a disease caused by an infectious pathogen, or the antigen is a tumor antigen and the disease is cancer.

[0146] "Ameliorating" or "treating" a disease, particularly cancer, means slowing, arresting, or reducing the progression of at least one of the disease or its clinical symptoms; alleviating or restoring at least one physical parameter, including those that may not be discernible to the patient; modulating the disease physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both; or preventing or delaying the onset or development or progression of a disease or disorder or its clinical symptoms. In the case of infectious diseases, "ameliorating" or "treating" may be interpreted accordingly and may also include reducing the burden of live infectious agents in a subject, or preventing or reducing the recurrence of dormant infectious agents becoming actively multiplying forms. "Therapeutic treatment" should be interpreted accordingly. "Preventing" includes that the agent described herein is prophylactic. Protective or prophylactic uses or treatments include uses or treatments that reduce or eliminate the risk of a subject contracting a disease, for example by vaccination. "Prophylactic treatment" should be interpreted accordingly.

[0147] A subject is in need of treatment if he or she would benefit biologically, medically, or in quality of life from such treatment. Treatment is typically performed by a physician or veterinarian, and a therapeutically effective amount of the bacteria or composition may be administered. A therapeutically effective amount of the bacteria according to the first aspect or the composition according to the second aspect refers to an amount that would be effective for treating as described herein, such as delaying, arresting, alleviating, or preventing a disease or its symptoms. A therapeutically effective amount may depend on the antigen (e.g., the ability of the antigen to elicit a particular type or strength of immune response thereagainst), the efficiency of production of the antigen by the Clostridium cells, the subject being treated, the severity and type of affliction, and the like. Typically, a subject in need of therapeutic treatment is exhibiting symptoms of the disease. Alternatively, the subject may be susceptible to the disease or have tested positive for the disease but not yet exhibiting symptoms. Typically, a subject in need of prophylactic treatment is not afflicted by the disease, but may be at risk of developing the disease. Prophylactic treatment is particularly suitable for infectious diseases.

[0148] The infectious disease to be treated is suitably one that can respond to an antigen-specific immune response directed to 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 tumor antigens, such as the tumor antigens listed herein, particularly those that have been shown to respond to immunotherapy that utilizes tumor antigens.

[0149] Therapeutic treatment is particularly beneficial for cancer or chronic infectious diseases. Chronic infectious diseases include those that persist for months or years due to infectious agents, or those that exhibit active growth and / or symptomatic and dormant phases of infectious agents. Chronic persistent infections can be caused by viruses including human papillomavirus (HPV); hepatitis C; hepatitis B; human immunodeficiency virus (HIV); herpesviruses such as herpes simplex virus type 1, herpes simplex virus type 2, and varicella zoster virus; yellow fever-related flaviviruses; 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). Chronic persistent infections can also be caused by bacteria including Mycobacterium tuberculosis, Borrelia species such as Mycobacterium bovis, Brucella, Borrelia burgdorferi, Corynebacterium diphtheriae, Chlamydia, Vibrio cholerae, Salmonella typhi; mycoplasma; fungi including Candida albicans; and various parasites including helminths and protozoa. Suitable cancers to treat include melanoma and renal cell carcinoma, which are considered two of the most immunogenic solid tumors and have been extensively studied in vaccine development, or cancers of the colon, lung, cervix, pancreas, stomach, liver, intestine, bladder, ovary, prostate, bone, brain, or head and neck.

[0150] Preventive treatment typically requires the establishment of immune memory, which protects or partially protects the immunized subject from subsequent attacks, typically by infectious pathogen antigens.Immunological memory is an important result of adaptive immunity, and can mount a more rapid immune response against previously encountered pathogens, preventing disease.Immunological memory may also be important in therapeutic treatment.

[0151] The immune memory of T cells can be tested using MHC tetramers, which identify whether memory T cells exist for a particular antigen. MHC tetramers have specificity for the antigens loaded with MHC, so MHC tetramers specific for the antigen of interest can be used (e.g., HPV-specific MHC tetramers). These can be used in samples isolated from subjects (e.g., blood samples or splenocytes) to measure the frequency of antigen-specific T cells. MHC tetramers can be used for MHC class I and II, which means that MHC tetramers can be used to measure the frequency of CD4 + and CD8 + This means that both MHC tetramers and T cell subsets can be measured. Additionally, MHC tetramers can be used in conjunction with fluorescent antibodies for other T cell markers to assess the percentage of antigen-specific T cell subsets (e.g., antigen-specific Th1, Th2 and / or Th17 cells). The percentage of antigen-specific T cells can be assessed by flow cytometry in immunized versus non-immunized subjects. For example, samples obtained from mice subjected to the immunization regimen described herein can include blood samples and / or spleen cells assessed for MHC tetramer binding, and a fluorescent marker panel of T cell subsets. Compared to non-immunized mice, immunized mice should have a higher percentage of binding to MHC tetramers, which can be further assessed by T cell subsets to identify the type of T cell response induced. In the case of HPV infection, a higher percentage of antigen-specific CD8 + The T cells are believed to represent the establishment of protective T cell immunity by immunization with Clostridium bacteria containing HPV antigens.

[0152] B cell immune memory can be tested in vitro by isolating B cells from immunized and non-immunized mice (e.g., following the immunization regimen described) and restimulating the B cells in the presence of helper T cells specific for the same antigen. B cells from immunized mice show better responses both quantitatively and qualitatively, the former can be assessed by comparing the frequency of B cells after restimulation (i.e., counting the number of cells in the cell suspension). Due to affinity maturation of B cells, the memory B cell antibodies produced will also have a higher affinity compared to naive B cells of non-immunized mice. This can be tested by purifying the antibodies produced (from immunized and non-immunized mice) and comparing their affinity for the antigen (or its epitope). If the antibodies produced from immunized mice have a higher affinity, a B cell memory response that may indicate protective immunity has been established. Corresponding in vivo studies use such mice to be challenged with the pathogen from which the antigen is derived (e.g., infection with HPV if the antigen is an HPV antigen; e.g., as described in Longet et al., 2011, Journal of Virology, 85:13253-13259) and assess the infectious burden in comparison to mice challenged for the first time with that pathogen.

[0153] The above cell system may be complemented with an in vivo mouse system in which mice are challenged with a pathogen related to the antigen. The presence of T cell immunity and / or B cell immunity should result in a reduced infection burden in immunized mice compared to naive mice, such as an increased rate of partial or complete protection from infection. Thus, a suitable in vivo system would involve an immunization regime followed by a challenge regime to assess infection burden. Suitable animal models are described in Bakaletz (2004) Developing animal models for polymicrobial diseases, Nature Reviews Microbiology, 2:552-568. Immune memory may also be tested in in vivo tumor models, including tumor challenge infection models, as described in Cai et al., 2017 (see above) and Ireson et al., 2019 (see above) in connection with the fourth aspect of the invention.

[0154] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0155] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening value in the stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included within the range and are also encompassed within the invention, subject to any specific 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.

[0156] Preferences and options for any given aspect, feature or parameter of the invention should be considered as disclosed in combination with all preferences and options for all other aspects, features and parameters of the invention, unless the context dictates otherwise.

[0157] All publications mentioned in this specification are incorporated by reference to the maximum extent possible for the purpose of describing and disclosing the components described in the publications that may be used in connection with the invention(s) described in this application.

[0158] The invention is further illustrated in the following non-limiting examples and figures. [Brief description of the drawings]

[0159] [Figure 1] Figure 1: Design of ROP proteins and presentation of cleaved ROPs on antigen-presenting cells (APCs). Known T cell epitopes are split into overlapping peptide fragments linked into a single-chain protein by the minimal cleavage signal of cathepsin S (LRMK (SEQ ID NO: 33)). ROPs are cleaved in endosomes within APCs, and individual peptide epitopes can be presented via MHC molecules and recognized by receptors on T cells. [Diagram 2] Figure 2: Western blot detection of FLAG tag bound to ROP-HPV and ROP-OVA in Clostridium butyricum genetically engineered to express ROP protein intracellularly. CHN-0 wild type was used as a control. In (A), the arrow indicates the ROP-HPV band in CADD-HPV-ROP but not in CHN-0 wild type cells. (A) Contrast enhancement throughout the image - red arrow indicates the ROP-HPV band in CADD-HPV-ROP but not in CHN-0 wild type. (B) 35 μl of protein was loaded (~175 mg), blocked with 5% milk, reacted with anti-FLAG (A9469) at 1:5000 dilution in TBS-T for 2 h, and developed using SIGMAFAST BCIP / NBT for 7 min. [Diagram 3]Figure 3: CADD-ROP-OVA1 construct for genetic modification of Clostridium butyricum. [Figure 4] Figure 4: CADD-ROP-HPV constructs for genetic modification of Clostridium butyricum. [Diagram 5] Figure 5: pHRodo fluorescence observed within DC2.4 cell cultures exposed to CHN-0 vegetative cells at a ratio of 1000:1 (CHN-0:DC2.4 cells). Scale bar is 500 μm. [Figure 6] Figure 6: IFN-γ ELISPOT evaluation of CD8+ and CD4+ T cells isolated from the spleens of mice immunized by oral gavage with spores of CHN-0 wild type (orange bars), genetically modified CADD-HPV (gray bars), or by subcutaneous injection with purified ROP-HPV proteins (yellow bars) together with adjuvant. As a negative control, PBS was administered both orally and subcutaneously (light and dark blue bars). Isolated T cells were plated at a density of 2.5 × 105 / well and restimulated with purified ROP-HPV or wild type HPV proteins (both at 5 μg / well) or CHN-0 wild type vegetative cells (0.5 × 105 / well). [Figure 7] Figure 7: IFN-γ ELISPOT evaluation of CD8+ and CD4+ T cells isolated from the spleens of mice immunized by oral gavage with spores of CHN-0 wild type (orange bars), genetically modified CADD-OVA (gray bars), or by subcutaneous injection with purified ROP-OVA protein (yellow bars) together with adjuvant. As negative controls, both oral and subcutaneous administration of PBS was performed (light and dark blue bars). Isolated T cells were seeded at a density of 2.5 × 105 / well and restimulated with either purified ROP-OVA (5 μg / well) or CHN-0 wild type vegetative cells (0.5 × 105 / well). [Figure 8] FIG. 8: Diagram of the immunization strategy of Example 3. [Figure 9]Figure 9: (A) Nucleic acid sequence and translated protein sequence of FLAG-tagged antigen of cholera toxin CtxB. The underlined sequences in the nucleic acid sequence are, in order, NotI, NdeI, and NheI sites. The promoter region is between NotI and NdeI and is not translated, while the translated antigen region is between the NdeI and NheI sites. (B) Sequence alignment between the native CtxB protein sequence (P01556) and the FLAG-tagged antigen sequence of CtxB (CHAIN_CtxB). [Figure 10] Figure 10: Western blot detection of FLAG tag bound to CtxB in C. butyricum genetically engineered to express CtxB intracellularly from the pMTL82151 plasmid (CtxB-full plasmid). CHN-0 wild type was used as a control. The red arrow indicates a significant band at the expected MW (~13 kDa) in the CHN-0 strain expressing CtxB but not in the CHN-0 wild type. (B) 20 μl of protein was loaded, blocked with 5% milk, reacted with anti-FLAG (A9469) at a 1:5000 dilution in TBS-T for 2 h, and developed using SIGMAFAST BCIP / NBT for <3 min.

[0160] Example 1: Construction and preparation of genetically modified Clostridium butyricum The DSM10702 strain of Clostridium butyricum, a spore-forming anaerobic bacterium that can be present in soil and animal (including human) feces, was genetically modified to express antigens in the bacterial cytoplasm. Selected antigens were genetically modified based on recombinant overlapping peptide (ROP) technology as described in WO2007 / 125371A2.

[0161] The ROP protein sequence is composed of overlapping peptides linked by a cathepsin cleavage site targeting sequence (LRMK (SEQ ID NO: 33)) (see Figure 1). Cathepsins reside in the endosomes of dendritic cells (DCs) and, after endocytosis, cleave the ROP protein into its component peptides. This approach allows the delivery of a broad range of T cell epitopes, facilitating the induction of cellular immunity across a range of HLA alleles and maximizing population coverage. Naive CD8 + and CD4 + Effective antigen presentation by DCs is required for T cell priming. CD8 + Cytotoxic T cells are involved in immune defense against intracellular pathogens and tumor surveillance. + Helper T cells (e.g., Th1, Th2, and Th17) shape and control a wide range of immune functions, and play a key role in controlling adaptive immunity in particular. The use of ROP has previously been shown to be superior to the use of wild-type antigens in generating protective cellular immunity. They are expressed by CD4 T cells through the classical MHC class II presentation pathway. + In addition to stimulating T cells, ROP stimulates the expression of foreign antigens presented on APCs by MHC class I receptors, resulting in CD8 + Through cross-presentation, a process that activates T cells, + It can induce T cell responses (Cai et al, Oncotarget 2017, 8(44) pp76516-76524).

[0162] Previously, we have constructed a pyrE gene-disrupted strain of Clostridium butyricum for use in genetic modification by the ACE technique. In this study, we successfully integrated the ROP protein coding sequence under the control of a constitutive promoter into the pyrE locus in the chromosome of this strain.

[0163] We developed two different ROP protein coding sequences based on the human papillomavirus (HPV) type 16 E7 envelope protein and ovalbumin (OVA). Using these sequences, we designed cassettes for introduction into the pMTL80000 vector series for genetic modification by introducing the necessary enzyme cleavage sites and an additional cathepsin cleavage signal at the N-terminal site that links the FLAG tag to the ROP protein. The genetically modified pyrE-deficient strain of Clostridium butyricum expresses HPV- or ovalbumin-derived ROPs intracellularly (see Figure 2).

[0164] After confirming expression and production of ROP proteins, we used a previously developed spore fermentation protocol to generate spores and vegetative cell pellets of the genetically modified strains, and then evaluated these materials for in vitro baseline testing in DCs and for use in in vivo immune experiments in mice. Materials and Methods Cultivation of bacterial strains

[0165] E. coli strains BL21, DH5α 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 at 30° C. or 37° C. depending on the metabolic burden associated with propagation of the plasmid, as required. Liquid cultures were agitated at 200 rpm during incubation.

[0166] Clostridium butyricum strain DSM10702 has been deposited at the DSMZ depository (Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures, InhoffenstraSSe 7B, 38124 Braunschweig, Germany). Clostridium butyricum strains were routinely grown in enriched Clostridium 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 as required in an anaerobic workstation (Don Whitley, 10% hydrogen, 10% carbon dioxide, 80% nitrogen, 37 °C). For maintenance and selection of genetically modified strains, C. butyricum was grown in an anaerobic workstation in Clostridium basal medium (CBM; ferrous sulfate heptahydrate 12.5 mg / L, magnesium sulfate heptahydrate 250 mg / L, manganese sulfate 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 μg / L) supplemented with 10 g / L calcium carbonate, 2% (w / v) glucose, 15% (w / v) agar, uracil, and / or antibiotics as required. For detection of colony-forming units in mouse feces, homogenized fecal samples were plated on modified Clostridium 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 dihydrogen phosphate 7 g / L, dipotassium phosphate 7 g / L, ferrous sulfate 0.01% (w / v), biotin 0.00005% (w / v), cysteine ​​hydrochloride 0.5 g / L, glucose 2% (w / v), agar 15% (w / v), and D-cycloserine 250 mg / L).

[0167] Clostridium butyricum spores were prepared in RCM supplemented with 2% (w / V) glucose in 2 L vessels in FerMac 320 Microbial culture batch bioreactor systems (ElectroLab Biotechnology Ltd). The vessels were sparged with nitrogen gas at a flow rate of 0.2 vvm, maintained at pH 6.5, temperature 37 °C and stirred at 100 rpm. Cell and spore masses were harvested and spores were separated from cellular material by repeated washing with ice-cold sterile water. Spores were stored at 4 °C until further use. Spore counts were performed by plating serial dilutions of spore stocks in triplicate onto pre-reduced RCM agar plates. Plates were incubated in an anaerobic workstation for 24 h before colony forming units (CFU) were determined. Gene constructs and plasmids

[0168] For the ovalbumin construct, the amino acid (aa) sequence of wild-type ovalbumin ranging from aa241 to aa340 (SMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGR; SEQ ID NO: 5) was divided into four overlapping sequences and linked by a minimal cathepsin cleavage site (LRMK (SEQ ID NO: 33)) to generate a 142 aa recombinant overlapping peptide designated ROP-OVA (SMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMELRMKTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMALRMKKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISLRMKISSAESLKISQAVHAAHAEINEAGR; SEQ ID NO: 6).

[0169] ROP-OVA was further modified for genetic modification of Clostridium butyricum to include a nucleotide sequence for an NdeI cleavage site (CATATG) incorporating a nucleotide signal for the aa methionine (M, ATG) at position 2 of ROP-OVA, an additional cathepsin cleavage site at the N-terminal site followed by a signal for a FLAG-tag (DYKDDDDK (SEQ ID NO: 18)), and an NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA (Figure 3).

[0170] For the human papillomavirus type 16 construct, the aa sequence of the wild-type E7 protein ranging from aa1 to aa98 (MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP; sequence number 7) was divided into four overlapping sequences and linked by the minimal cathepsin cleavage site (LRMK (sequence number 33)) to create a 140aa recombinant overlapping peptide represented by ROP-HPV (MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEELRMKEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKLRMKHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGLRMKIRTLEDLLMGTLGIVCPICSQKP; sequence number 8).

[0171] ROP-HPV was further modified for genetic modification of Clostridium butyricum to include a nucleotide sequence for an NdeI cleavage site (CATATG) incorporating a nucleotide signal for the aa methionine (M, ATG) at position 1 of ROP-HPV, an additional cathepsin cleavage site at the N-terminal site followed by a signal for a FLAG-tag (DYKDDDDK (SEQ ID NO: 18)), and an NheI cleavage site (GCTAGC) separated from the FLAG-tag by the stop codon TAA (Figure 4).

[0172] ROP-OVA and ROP-HPV constructs were obtained from GeneArt Thermo Fisher Scientific as synthetic genes in pMK vectors.

[0173] The CADD-ROP-OVA1 and CADD-ROP-HPV constructs were obtained as synthetic genes without further codon usage optimization in the plasmid pMK-RQ from Life Technologies Ltd. The pMK-RQ plasmids containing the synthetic gene constructs were transformed into E. coli DH5α, grown overnight in LB supplemented with 50 μg / mL kanamycin, and stored at −80°C as stocks in 15% (V / V) glycerol. Expression of ROP protein standards in E. coli

[0174] Synthetic ROP-OVA and ROP-HPV constructs were excised from archived plasmids and cloned into BsaI restriction enzyme-linearized plasmid pNIC28-Bsa4 (Structural Genomic Consortium, Oxford) using ligation-independent cloning. Vector amplicons were transformed into E. coli BL21 (Thermo Fischer Scientific) according to the manufacturer's instructions.

[0175] For ROP-HPV expression, BL21 harboring pNIC28-Bsa4-ROP-HPV was cultured in LB broth supplemented with 50 μg / mL kanamycin. Protein production was induced with 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 sonicated at 600 W for 5 s with 20 cycles of 7 s intervals. Inclusion bodies containing the recombinant protein were collected by centrifugation at 20,000 × g for 45 min. The inclusion body pellet was resuspended in denaturing buffer (8 M urea) and incubated for 2 h with vigorous shaking. The solution was centrifuged to separate the protein from the debris.

[0176] The supernatant containing the protein fraction was loaded onto a nickel affinity column (GE Healthcare) and eluted with elution buffer (50 mM PB, 200 mM NaCl, 8 M urea, 300 mM imidazole, pH 7.4). Refolding of the purified protein was achieved by progressive dialysis with PBS (pH 7.4).

[0177] For ROP-OVA expression, BL21 harboring pNIC28-Bsa4-ROP-OVA was grown in LB medium supplemented with 50 μg / mL kanamycin. Protein production was induced with 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 sonicated for 10 min at 35% amplitude for 5 s with 15 s intervals. Inclusion bodies containing the recombinant protein were collected by centrifugation at 20,000 × g for 45 min. The inclusion body pellet was solubilized in 50 mM HEPES buffer containing 6 M guanidine hydrochloride, incubated on ice for 1 hour, and then filtered through a 0.2 μm filter.

[0178] The filtrate containing the protein fraction was loaded onto a Ni-NTA affinity column and eluted with elution buffer (50 mM HEPES, 6 M guanidine hydrochloride, 500 mM imidazole). After dilution with cold dilution buffer (50 mM HEPES, 500 mM NaCl, 10% glycerol, 0.5% sarkosyl), the proteins were concentrated using a Vivaspin column with a molecular weight cutoff of 10 kDa and the guanidine hydrochloride was removed by desalting through a PD-10 column with desalting buffer (50 mM HEPES, 500 mM NaCl, 10% glycerol).

[0179] Endotoxin was removed using the Pierce Endotoxin Removal Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Samples were filtered through a 0.2 μM filter and stored at 4° C. until further use. Genetic modification of Clostridium butyricum

[0180] To prepare plasmids for gene modification of Clostridium butyricum, CADD-ROP-OVA1 and CADD-ROP-HPV constructs were first propagated in E. coli DH5α with pMK-RQ. Plasmids were extracted using the Wizard Plus SV Miniprep DNA Purification kit (Promega) according to the manufacturer's instructions, and the constructs were excised from the plasmids using the restriction enzymes NdeI and NheI (all, New England Biolabs Inc) in CutSmart® buffer according to the manufacturer's instructions. The isolated cassettes were then cloned into a plasmid containing the pyrE repair cassette and the constitutive promoter P fdx The cassette was introduced into pMTL83151 (pCB102 Gram-positive replicon, catP antibiotic marker, ColE1 Gram-negative replicon, traJ conjugal transfer function, and multiple cloning site (MCS)) which additionally contains the pCB102 Gram-positive replicon, catP antibiotic marker, ColE1 Gram-negative replicon, traJ conjugal transfer function, and multiple cloning site (MCS) before the MCS. Plasmids were transformed into E. coli DH5α for propagation. Plasmids were isolated as before and sequenced using GeneWiz sequencing service to confirm the cassette was correctly inserted.

[0181] Sequence confirmed plasmid pMTL83151_pyrERepair_P fdx _CADD-ROP-OVA1 and pMTL83151_pyrE repair_P fdx_CADD-ROP-HPV was transformed into E. coli CA434 conjugation donor. After sequence verification as described above, E. coli CA434 was 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.

[0182] Fresh colonies of resurrected E. coli CA434 carrying the respective plasmids were used to inoculate LB broth supplemented with 50 μg / mL kanamycin and 12.5 μg / mL chloramphenicol. After overnight incubation, the cultures were used to inoculate 1:10 into fresh supplemented medium and incubated at OD 600 The cultures were incubated until the OD reached 0.5-0.7. A volume of 1 mL of culture was removed and centrifuged at 5,000 × g for 3 min. The supernatant was discarded and the pellet was resuspended in 500 μL of phosphate-buffered saline (PBS) solution. The cultures were centrifuged as above and the supernatant was discarded.

[0183] A fresh colony of resurrected Clostridium butyricum CHN-0.1 (ΔpyrE derivative of wild-type CHN-0) was used to inoculate a serial dilution series into fresh pre-reduced RCM broth supplemented with 2% glucose and 1% CaCO3. After overnight incubation in anaerobic conditions, the highest diluted culture showing growth was used to inoculate fresh supplemented medium at 1:10 and OD was measured. 600 The culture was incubated until the pH reached 0.5-0.7. A volume of 1 mL of the culture was removed and heat-treated at 50 °C for 10 min.

[0184] Both the so-treated E. coli CA434 and Clostridium butyricum were transferred to an anaerobic workstation and incubated at 5:1 (OD 600 :OD 600) ratio. The mating mixture was spotted onto pre-reduced non-selective RCM agar plates and grown upright overnight. After incubation, the mixture was taken up in 500 μL of fresh pre-reduced RCM broth and plated in a volume of 100 μL onto fresh pre-reduced RCM agar plates supplemented with 250 μg / mL D-cycloserine and 15 μg / mL thiamphenicol. To select mutants with restored uracil prototrophy, thiamphenicol-resistant colonies were repeatedly patch plated onto CBM agar plates and cross-checked for plasmid loss on selective RCM agar plates containing thiamphenicol. Genomic DNA from protist colonies that had lost the plasmid was isolated using the GenElute™ Bacterial Genomic DNA Kit (SIGMA-Aldrich) according to the manufacturer's instructions and used for sequencing to confirm the presence of the CADD-ROP cassette in the chromosome of Clostridium butyricum using primers spanning the integration region, the promoter, and the respective ROP sequences (Table 3). Table 3: Primers used to confirm the sequence of the CADD-ROP cassette TIFF2024502939000002.tif126170

[0185] Integration of the ROP cassette into the chromosome introduces a single copy under the control of a constitutive promoter, which reduces the expression and production of the protein in the cell, but this can be tuned by using stronger promoters and / or by inserting multiple copies of the gene. Confirmation of expression of ROP in Clostridium butyricum

[0186] Fresh colonies of resurrected Clostridium butyricum CHN-2 (CADD-ROP-HPV) and CHN-3 (CADD-ROP-OVA1) were used to inoculate serial dilutions into fresh pre-reduced supplemented RCM broth and grown overnight. The highest dilution culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth to a starting OD of 0.05. 600 The seeds were seeded at OD600 When grown to OD of 1, 2, and 4, and after 24 h incubation, the OD of 1 / mL 600 An equivalent volume of 1000 ml was centrifuged at 13,000 × g for 2 min. The pellet was resuspended in 45 μL of 5× SDS loading dye (20% (V / V) 0.5 Tris-HCl pH 6.8, 23% (V / V) glycerol, 40% (V / V) of 10% (w / V) sodium dodecyl sulfate (SDS) solution, 10% (V / V) 2-mercaptoethanol, 10 mL dH2O, bromophenol blue) and heat-treated at 98 °C for 15 min.

[0187] Up to 40 μL / well of the resuspended pellet was loaded onto Novex™ WedgeWell™ 12% Tris-Glycine Mini Gels (Thermo Fischer Scientific) and run in 1×SDS buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS) at room temperature using 200 V. PageRuler™ pre-stained protein ladder (Thermo Fischer Scientific) was loaded at 5 μL / well as a marker and E. coli Positive Control Whole cell lysate ab5395 (abcam) was used as a FLAG tag positive control at a 1:5 dilution.

[0188] The separated proteins were blotted onto PVDF membranes by using the Trans-Blot® Turbo™ Blotting System (BioRad) with the Trans-Blot® Turbo™ pack according to the manufacturer's instructions. To detect FLAG-tagged proteins, the PVDF membranes were first incubated in TBS-T blocking buffer (50 mM Tris-hydrochloride, 150 mM sodium chloride, 0.1% Tween 20, pH 7.4, 5% (w / V) milk powder) for 1 h at room temperature on a shaking platform. The blocking buffer was then replaced with TBS-T buffer (50 mM Tris-hydrochloride, 150 mM sodium chloride, 0.1% Tween 20, pH 7.4) containing Anti-FLAG tag® antibody alkaline phosphatase complex (1:5,000; Sigma) and incubated on a shaking platform for 2 h at room temperature. 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, pH 7.4). Detection of alkaline phosphatase was performed using SIGMAFAST BCIP® / NBT substrate (SIGMA Aldrich) according to the manufacturer's instructions. Example 2: Phagocytosis of Clostridium butyricum by a dendritic cell line and induction of cytokine responses

[0189] Baseline studies in murine DC2.4 cell cultures demonstrated that these cells can phagocytose vegetative cells and spores of Clostridium butyricum, a prerequisite for successful delivery of ROP proteins expressed in these bacterial cells.

[0190] Cell cultures of DC2.4 cells were exposed to vegetative cells and spores of the wild-type strain CHN-0, which was taken up by DC2.4 cells by phagocytosis in either the vegetative or spore form (see FIG. 5).

[0191] The cytokine profile of these exposed DC2.4 cell cultures was then assessed using R&D systems' Proteome Profiler® Mouse Cytokine Array Panel (Tables 1 and 2). Differences were observed in the response to CHN-0 and media control. Table 1: Spot density in the Proteome Profiler® mouse cytokine panel TIFF2024502939000003.tif220170Table 2: Selected cytokines and their functions TIFF2024502939000004.tif190170

[0192] From these preliminary experiments, it was concluded that the CHN-0 wild-type strain was able to induce the release of cytokines from cultured DC2.4 cells when they were exposed to either vegetative cells or spores, which may be related to the recruitment of leukocytes (NK cells) and the activation of innate and adaptive immunity. Materials and Methods cell line

[0193] DC2.4 cells (ATCC® Number: CRL-11904™) were maintained in RPMI1640 medium supplemented with 10% (V / V) fetal bovine serum, 1× MEM non-essential amino acids, and 1× 1M HEPES buffer (all Sigma Aldrich) at 37° C. under 5% CO2. Phagocytosis assay

[0194] DC2.4, 2 x 10 in a 96-well cell culture plate 4 CHN-0 vegetative cells were stained with pHrodo Red (Life Technologies) according to the manufacturer's instructions and plated at a density of 2 × 10 cells / well. 7 DC2.4 cells were incubated with CHN-0 cells for 3 hours and then imaged using a Celigo Image Cytometer. DC2.4 cell baseline study

[0195] Cytokine profiles were evaluated using the Proteome Profiler Mouse Cytokine Array Kit (R&D systems) according to the manufacturer's instructions. DC2.4 cells were cultured at 5 × 10 5 DC2.4 cells were seeded at a density of 1 × 10 7 The membranes were incubated with CHN-0 cells / well overnight. The cell culture supernatant was used for subsequent analysis. The cells were detached from the cell culture plate and centrifuged. A volume of 700 μL of the supernatant was incubated with the Detection Antibody Cocktail provided in the Proteome Profiler kit for 1 h at room temperature. This mixture was added to the pretreated membrane and gently shaken overnight at 4°C on a shaking platform. The membrane was then rinsed with Wash buffer, followed by development with Streptavidin-HRP conjugation and Chemi Reagent mixture. The membrane was exposed to X-ray film for 10 min, and the spot intensity was quantified with ImageJ software. Example 3: Oral immunization of mice with genetically modified Clostridium butyricum

[0196] We focused on exploring T cell responses by performing in vivo immunization experiments to evaluate whether ROP antigens could be delivered and induced immune responses using genetically modified Clostridium butyricum spores expressing ROP protein mutants. Mice were gavaged with spores or injected subcutaneously with purified ROP protein every other week for 28 days, and sacrificed after 42 days.

[0197] IFN-γ ELISPOT assays using splenocytes isolated after sacrifice demonstrated that mice immunized by oral gavage with antigen-expressing C. butyricum strains generated antigen-specific T cell responses. Specifically, mice immunized with ROP-HPV-expressing strains generated CD4 + and CD8 + Although mice immunized with the ROP-OVA expressing strain generated both CD4 T cell responses specific for each antigen (see Figure 6), mice immunized with the ROP-OVA expressing strain generated CD4 T cell responses specific for each antigen (see Figure 6). + Generate a T cell response (see Figure 7). Importantly, mice do not generate a T cell immune response directed against the Clostridium butyricum strain itself.

[0198] In-house evaluation of fecal samples from mice immunized with wild-type and genetically modified Clostridium butyricum spores demonstrated that strains could be detected in the feces as early as 7 hours after the initial immunization event. Materials and Methods In vivo experiments

[0199] Animals were housed in individually ventilated cages with nesting material. Mice had free access to food (provided as pellets) and water. All procedures were carried out in accordance with protocols under Home Office Licence 30 / 3197, in accordance with the Animal Scientific Procedures Act 1986 and Oxford University Committee guidelines.

[0200] For the immunization experiments, 6-week-old female mice were randomly divided into groups of 5. Immunization through the digestive tract was performed with 1 × 10 spores of CADD-ROP-HPV or CADD-ROP-OVA, i.e., the genetically modified CHN strain (referred to as CHN-ROP-HPV or CHN-ROP-OVA), in 100 μL of PBS. 8CFU were administered by oral gavage. CHN-0 wild-type spores and PBS were given under the same conditions as controls. Parenteral immunization was performed by subcutaneous injection of 100 μg ROP-HPV or ROP-OVA protein in 100 μL Freund's adjuvant (prime immunization, day 0) or incomplete Freund's adjuvant (boost immunization, days 14 and 28). Mice in each group were immunized three times on days 0, 14, and 28 and sacrificed 42 days later. Fecal samples were collected 3 and 7 hours after each dosing event. Whole blood and serum samples were collected at each dosing event and at sacrifice. Spleens were isolated at sacrifice. Table 3 TIFF2024502939000005.tif153170 Isolation of mononuclear cells

[0201] Splenocytes and PBMCs were isolated from homogenized spleen and peripheral whole blood samples, respectively, using a Ficoll-Paque 1.084 density gradient (GE healthcare) according to the manufacturer's instructions. Cell suspensions or whole blood were layered onto Ficoll-Paque medium and centrifuged at 400 × g for 20–30 min at room temperature. Mononuclear cell isolates were washed with balanced salt solution to remove residual contaminants.

[0202] For purification of T cells, mononuclear cells isolated from one immune group were pooled and purified using CD8a (Ly-a) MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. 1 × 10 7 The cells were resuspended, then MicroBeads were added and incubated for 10 min at 4°C. + To retain the T cells, the cell suspension was applied to a MACS LS column in a magnetic field. The flow-through was collected twice and CD4 + The CD8 T cells were then isolated by applying a buffer solution without the use of a magnetic field. + T cells were eluted. CD4 +and CD8 + Both T cells were resuspended in RPMI medium before use in ELISPOT experiments. IFN-γ T cell ELISPOT

[0203] For detection of IFN-γ release, a mouse IFN-γ T cell ELISPOT kit (U-CyTech Bioscience) was used according to the manufacturer's instructions. A total of 2.5 × 10 5 T cells were added to plates precoated with anti-IFN-γ antibody and treated with wild-type HPV protein, ROP-HPV protein, ROP-OVA protein (5 μg / well each), or 0.5 × 10 5 CFU / well of CHN-0 vegetative cells were either restimulated with 0.01 mg / mL of GABA or 0.01 mg / mL of GABA. Concanavalin A (Sigma Aldrich) was added at a concentration of 5 mg / mL as a positive control. Plates were incubated overnight at 37°C and 5% CO2 before the addition of biotinylated detection antibody, followed by incubation with GABA conjugate and activator I / II solution to form spots. Spots were scanned using a Celigo Image Cytometer and quantified using ImageJ software. Example 4: Immunization of mice with intracellular CtxB antigen in Clostridium

[0204] Cholera enterotoxin subunit B (CtxB) is a 13 kDa subunit protein that constitutes the pentameric ring of cholera enterotoxin from Vibrio cholerae. Cholera enterotoxin subunit B forms the holotoxin (choleragen) together with the A subunit. The holotoxin is composed of a pentameric ring of B subunits, the central pore of which is occupied by the A subunit. The A subunit contains two chains, A1 and A2, linked by disulfide bridges. The pentameric ring of the B subunit guides the A subunit to its target by binding to GM1 ganglioside present on the surface of intestinal epithelial cells. It can bind to five GM1 gangliosides. It has no toxic activity by itself. Gene constructs and plasmids

[0205] For CADD-CtxB oral vaccine development, the protein sequence encoding CtxB (SEQ ID NO: 24) was determined from UniProtKB submission P01556 by removing the signal sequence (MIKLKFGVFFTVLLSSAYAHG (SEQ ID NO: 19)) and adding a C-terminal FLAG tag (DYKDDDDK (SEQ ID NO: 18)). Additional modifications included for genetic modification include an NdeI cleavage site (CATATG) incorporating a nucleotide signal for aa methionine (M, ATG) and the nucleotide sequence of an NheI cleavage site (GCTAGC) separated from the FLAG tag by a stop codon TAA (Figure 9). The CtxB_FLAG construct was codon-optimized by GeneWiz for genetic modification into Clostridium butyricum and synthesized behind the p0957 promoter and cloned into the pMTL83151-pyrE repair vector for submission to GeneWiz for subcloning. Genetic modification of Clostridium butyricum

[0206] The pMTL83151-pyrE repaired_p0957_CtxB-FLAG plasmid was transformed into E. coli DH5α, grown overnight in LB supplemented with 12.5 μg / mL chloramphenicol, and stored at −80°C as 15% (V / V) glycerol stocks.

[0207] To clone into the correct plasmid for plasmid-based intracellular expression in Clostridium butyricum, the pMTL83151-pyrE repair_p0957_CtxB-FLAG plasmid was extracted from DH5α using the Wizard Plus SV Miniprep DNA Purification kit (Promega) according to the manufacturer's instructions, and the p0957-CtxB-FLAG construct was excised from the plasmid using the restriction enzymes NotI and NheI in CutSmart® buffer (all New England Biolabs Inc) according to the manufacturer's instructions. The isolated cassette (containing the p0957 promoter) was introduced into pMTL82151 (pBP1 Gram-positive replicon, catP antibiotic marker, ColE1 Gram-negative replicon, traJ conjugal transfer function, and multiple cloning site (MCS)). The plasmid was transformed into E. coli DH5α for propagation. Plasmids were isolated as before and sequenced using GeneWiz sequencing services to confirm correct insertion of the cassette.

[0208] The sequence-verified plasmid pMTL82151_p0957-CtxB-FLAG was transformed into E. coli CA434 conjugative transfer donor. After sequence verification as described above, E. coli CA434 was 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.

[0209] A fresh colony of resurrected E. coli CA434 harboring the CtxB-FLAG plasmid was used to inoculate LB broth supplemented with 50 μg / mL kanamycin and 12.5 μg / mL chloramphenicol. After overnight incubation, the culture was used to inoculate 1:10 into fresh supplemented medium and grown at OD 600The cultures were incubated until the OD reached 0.5-0.7. A volume of 1 mL of culture was removed and centrifuged at 5,000 × g for 3 min. The supernatant was discarded and the pellet was resuspended in 500 μL of phosphate-buffered saline (PBS) solution. The cultures were centrifuged as above and the supernatant was discarded.

[0210] A fresh colony of revived Clostridium butyricum CHN-0 was used to inoculate a serial dilution series into fresh pre-reduced RCM broth supplemented with 2% glucose and 1% CaCO3. After overnight incubation in anaerobic conditions, the highest diluted culture showing growth was used to inoculate fresh supplemented medium at 1:10 and OD was measured. 600 The culture was incubated until the pH reached 0.5-0.7. A volume of 1 mL of the culture was removed and heat-treated at 50 °C for 10 min.

[0211] Both the so-treated E. coli CA434 and Clostridium butyricum CHN-0 were transferred to an anaerobic workstation and incubated at 5:1 (OD 600 :OD 600 ), usually at a ratio of 0.2 mL of C. butyricum to 1 mL of E. coli. The mating mixture was spotted onto pre-reduced non-selective RCM agar plates and grown upright overnight. After incubation, the mixture was taken up in 500 μL of fresh pre-reduced RCM broth and plated in a volume of 100 μL onto fresh pre-reduced RCM agar plates supplemented with 250 μg / mL D-cycloserine and 15 μg / mL thiamphenicol. Thiamphenicol-resistant colonies were repeatedly patch-plated onto RCM + 15 μg / mL thiamphenicol agar plates to select for C. butyricum CHN-0 carrying the plasmid. Genomic DNA of thiamphenicol-resistant colonies was isolated using the GenElute™ Bacterial Genomic DNA Kit (SIGMA-Aldrich) according to the manufacturer's instructions and used for sequencing to confirm the presence of the pMTL82151_p0957-CtxB-FLAG plasmid by using primers spanning the MCS (Table 4). Table 4:Primers used to confirm the sequence of Clostridium butyricum CHN-0 colonies containing the pMTL82151_p0957-CtxB-FLAG plasmid TIFF2024502939000006.tif34170

[0212] Introduction of the pMTL82151-p0957-CtxB-FLAG plasmid into Clostridium butyricum CHN-0 resulted in high expression of the complete CtxB protein from the multicopy plasmid in the Clostridium butyricum cytoplasm. Confirmation of expression of CtxB in Clostridium butyricum

[0213] Fresh colonies of resurrected Clostridium butyricum CHN-0 and pMTL82151-p0957-CtxB-FLAG were used to inoculate serial dilutions into fresh pre-reduced supplemented RCM broth supplemented with 15 μg / mL thiamphenicol and grown overnight. The highest dilution culture showing growth was used to inoculate fresh pre-reduced supplemented RCM broth supplemented with 15 μg / mL thiamphenicol to a starting OD of 0.05. 600 The seeds were seeded at OD 600 When grown to OD of 1, 2, and 4, the OD of 2 / mL 600 An equivalent volume of 1000 ml was centrifuged at 13,000 × g for 2 min. The pellet was resuspended in 40 μL of 5× SDS loading dye (20% (V / V) 0.5 Tris-HCl pH 6.8, 23% (V / V) glycerol, 40% (V / V) of 10% (w / V) sodium dodecyl sulfate (SDS) solution, 10% (V / V) 2-mercaptoethanol, 10 mL dH2O, bromophenol blue) and heat-treated at 98 °C for 15 min.

[0214] Up to 20 μL / well of the resuspended pellet was loaded onto a Novex™ 16% Tricine Minigel (ThermoFischer Scientific) and run in 1× Novex™ Tricine SDS Running Buffer (ThermoFisher Scientific) at room temperature using 140 V. Spectra™ Multicolor Low Range Protein Ladder (Thermo Fischer Scientific) was loaded at 10 μL / well as a marker and E. coli Positive Control Whole cell lysate ab5395 (abcam) was used as a FLAG tag positive control at a 1:5 dilution.

[0215] The separated proteins were blotted onto PVDF membranes by using the Trans-Blot® Turbo™ Blotting System (BioRad) with the Trans-Blot® Turbo™ pack according to the manufacturer's instructions. To detect FLAG-tagged proteins, the PVDF membranes were first incubated in TBS-T blocking buffer (50 mM Tris-hydrochloride, 150 mM sodium chloride, 0.1% Tween 20, pH 7.4, 5% (w / V) milk powder) for 1 h at room temperature on a shaking platform. The blocking buffer was then replaced with TBS-T buffer (50 mM Tris-hydrochloride, 150 mM sodium chloride, 0.1% Tween 20, pH 7.4) containing Anti-FLAG tag® antibody alkaline phosphatase complex (1:5,000; Sigma) and incubated on a shaking platform for 2 h at room temperature. The membrane was washed twice in TBS-T buffer for 5 min at room temperature and once in TBS buffer (50 mM Tris-hydrochloride, 150 mM sodium chloride, pH 7.4) for 5 min at room temperature. Detection of alkaline phosphatase was performed using SIGMAFAST BCIP® / NBT substrate (SIGMA Aldrich) according to the manufacturer's instructions. Expression is shown in Figure 10. CtxB-FLAG protein was detected at high levels by Western blot, corresponding to 900 ng of specific volume of cells grown to OD 1.0. Thus, assuming a cell density of 0.3 g / L at OD 1.0, the protein is estimated to be 3 μg / mg dry cell weight. Immunogenicity testing

[0216] In vivo immunization experiments were performed to evaluate whether genetically modified Clostridium butyricum spores expressing the CtxB antigen could deliver antigens and induce immune responses, focusing on cellular and humoral responses. Clostridium butyricum spores were generated as described above. C57BL / 6 mice were infected with 1 × 10 spores of either the wild-type CADD strain (negative control) or the CADD vaccine strain expressing CtxB from the pMTL82151-p0957-CtxB-FLAG plasmid.8 CFU / dose will be administered orally three times at weekly intervals. A third group will receive the currently available oral cholera vaccine as a positive control. Clinical observations will be made throughout to determine tolerability of the test article (changes in body weight and physical appearance such as hunched back or piloerection of the coat). Table 5 TIFF2024502939000007.tif69170

[0217] At the time of sacrifice, spleens were harvested, processed into single cell suspensions, and CD4 + Cells and CD8 + Cells were individually purified and assayed for CD4 mediated IFN-γ release in an ELISPOT assay. + / CD8 + Determine specific T cell responses (described in Materials and Methods above). CD4 + T cell responses were also analyzed in gut-specific tissues (small intestine and colon), where tissues were extracted, treated with mucolytic enzymes and EDTA, and digested into a single cell suspension as described by Di Luccia et al. (2020) Cell Host & Microbe 27:899-908. CD4 T cells isolated from this suspension were analyzed using ELISA kits. + T cells are restimulated with antigen-presenting cells (APCs, previously exposed to commercially available CtxB antigen) and changes in cell surface CD40 ligand expression are assessed by flow cytometry as described in Hegazy et al (2017) Gastroenterology 153:1320-1337.

[0218] At termination, intestinal contents will be extracted, antigen-specific humoral responses will be assessed by ELISA assay, and CtxB-specific secretory IgA (sIgA) production will be determined as a percentage of total IgA as described in Di Luccia et al (2020) Cell Host & Microbe 27:899-908. Expected Results

[0219] As shown by the intracellular ROP expressed by the CADD platform, CD4 + / CD8 + In T cell ELISPOT assays, mice immunized with CADD strain expressing the intracellular CtxB antigen showed CD4 + Responses to CHN-0 wild-type strains are expected to generate more pronounced antigen-specific T cell responses. Importantly, mice immunized with CHN-0 wild-type strains are not expected to generate T cell responses.

[0220] Intestine-specific tissue evaluation showed that CD4 + CD40 ligand is used because it is rapidly upregulated by T cells. + Upon restimulation of the cells, expression of CD40 ligand was increased in the CADD-treated group expressing CtxB compared with the wild-type CADD group, and CtxB-specific CD4 + It is expected that a T cell response will be demonstrated.

[0221] Classically, stimulation of B cells and production of antibodies requires the CD4 + T cell stimulation is required, so a strong CD4 + T cell responses are generally accepted as a good correlate of protection. As sIgA antibody responses are also known to be important in protective immunity against V. cholerae, we also aim to determine the humoral response of mucosal immunity by assessing the production of CtxB-specific secretory IgA (sIgA). We expect that ELISA will confirm an increase in antigen-specific sIgA in response to administration of the CADD-CtxB oral vaccine compared to the wild-type CADD platform alone.

Claims

1. 1. A bacterium of the class Clostridium comprising a heterologous nucleic acid molecule encoding at least one antigen, said bacterium being capable of expressing said antigen in an intracellular compartment of said bacterium during anaerobic cell growth, said at least one antigen being an infectious pathogen antigen or a tumor antigen, and said bacterium being a species that is not a pathogenic Clostridium species.

2. The bacterium according to 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 one or more T cell antigen segments are ＋ T cell antigen segments and / or CD8 ＋ The bacterium according to claim 2 , which is a T cell antigen segment.

4. wherein said at least one antigen is a multi-antigen fusion polypeptide comprising two or more antigen segments, e.g., three or more, five or more, or ten or more antigen segments; optionally (a) the multi-antigen fusion polypeptide comprises at least one CD4 ＋ T cell antigen segment and at least one CD8 ＋ and / or (b) each antigenic segment is 8 to 50 amino acids in length and / or optionally, a cleavage site, such as a cathepsin site, optionally a cathepsin S site, is located between each antigenic segment.

5. 5. The bacterium of claim 4, wherein the antigenic segments are optionally overlapping and in combination encompass ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, ≧90%, more preferably 100% of the amino acid sequences of the antigens from which the antigenic segments are derived.

6. The amount of antigen expressed per cell weight of Clostridium cells during anaerobic cell growth is greater than 10 ng / mg, 20 ng / mg or 40 ng / mg and is up to 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 ng / mg, 1 μg / mg, 1.5, 2.0, 2.5, 5.0, 10 or 20 μg / mg dry cell.

6. The bacterium according to any one of claims 1 to 5, wherein the cell weight of said bacterium is, for example, 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 to 1500 ng / mg dry cell weight; or about 800 ng / mg dry cell weight. (a) the heterologous nucleic acid molecule is integrated into the genome as a single copy or is integrated into a low copy or high copy plasmid; and / or (b) the heterologous nucleic acid molecule encoding at least one antigen is operably linked to a promoter for expression during anaerobic growth, optionally for expression after spore germination in anoxic conditions and / or during anaerobic vegetative cell metabolism, optionally the promoter being selected from the fdx promoter and the p0957 promoter from C. sporogenes.

8. The bacterium according to any one of claims 1 to 7, wherein the bacterium is provided with a further heterologous nucleic acid molecule encoding an immunostimulant or adjuvant which can be co-expressed with the antigen; and / or the bacterium is capable of producing short chain fatty acids (SCFAs), such as butyric acid.

9. The bacterium according to any one of claims 1 to 8, wherein the infectious pathogen antigen is a viral antigen, a bacterial antigen such as a chlamydia antigen or a mycoplasma antigen, a parasitic antigen, a prion antigen, a helminth antigen, a nematode antigen, a protozoan antigen, a fungal antigen, or any combination thereof.

10. The infectious agent antigen is a. an HPV antigen, optionally comprising the amino acid sequence of SEQ ID NO: 4, or amino acids 1-140 of SEQ ID NO: 4, e.g., the HPV antigen is encoded by nucleotides 19-477 of the nucleic acid sequence of SEQ ID NO: 3; or b. A Vibrio cholerae antigen, optionally CtxB, optionally comprising the amino acid sequence of SEQ ID NO:21, or amino acids 1-104 of SEQ ID NO:21, or encoded by nucleotides 270-581 of SEQ ID NO:20; The bacterium according to any one of claims 1 to 9.

11. The bacterium (a) from cluster I, IV and / or XIVa of Clostridium, e.g., the bacterium is from the genus Clostridium, e.g., the bacterium is Clostridium butyricum; and / or (b) a bacterium according to any one of claims 1 to 10 which is detectable in the lower gastrointestinal tract but does not permanently colonise or form part of the resident microflora in the lower gastrointestinal tract.

12. The bacterium according to any one of claims 1 to 11, wherein the bacterium is capable of expressing the antigen as a soluble polypeptide or inclusion bodies in the cytoplasm of the bacterium.

13. The bacterium according to any one of claims 1 to 12, which is in the form of a spore or a vegetative cell.

14. A pharmaceutical composition comprising the bacteria according to any one of claims 1 to 13, said pharmaceutical composition being optionally added to a food product.

15. 15. The pharmaceutical composition of claim 14, comprising a capsule comprising spores or vegetative cells of the bacteria, the capsule comprising a delayed release layer or coating that enables release of the spores or vegetative cells in the anaerobic portion of the lower gastrointestinal tract following oral administration.

16. A bacterium according to any one of claims 1 to 13, or a pharmaceutical composition according to claim 14 or claim 15, for use in medicine.

17. A pharmaceutical comprising a bacterium of the class Clostridium for generating an antigen-specific immune response in a subject, said bacterium comprising a heterologous nucleic acid molecule encoding an antigen, said bacterium being capable of expressing said antigen in an intracellular compartment of said bacterium during anaerobic cell growth, and said bacterium not being a pathogenic Clostridium species.

18. The antigen-specific immune response is ＋ T cell responses and CD8 ＋ The medicament according to claim 17, which is a cellular immune response, such as a T cell response; and / or a B cell response.

19. 1. A medicament comprising a bacterium of the class Clostridium for the therapeutic or prophylactic treatment of a disease in a subject, said bacterium comprising a heterologous nucleic acid molecule encoding an antigen, said bacterium being capable of expressing said antigen in an intracellular compartment of said bacterium during anaerobic cell growth, said antigen being an infectious pathogen antigen and said disease being an infectious disease, or said antigen being a tumor antigen and said disease being cancer, and said bacterium is not a pathogenic Clostridium species.

20. The pharmaceutical composition according to any one of claims 17 to 19, wherein the bacterium is a bacterium according to any one of claims 1 to 13.

21. The pharmaceutical composition according to any one of claims 17 to 20, wherein the bacterium is for oral administration.

22. The method of claim 21, wherein the bacterium is in the form of a spore or in the form of a pharmaceutical composition according to claim 15.

23. The pharmaceutical composition according to any one of claims 17 to 22, wherein the subject is a human.

24. A method for preparing a bacterium according to any one of claims 1 to 13, comprising the step of introducing said heterologous nucleic acid molecule into said bacterium.

25. 16. A method of preparing a pharmaceutical composition according to claim 14 or claim 15, comprising formulating the bacteria with one or more pharma- ceutically acceptable diluents or excipients.

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