Oral vaccine

Abstract

The present invention relates to a non-viable bacterial cell in which an immunogenic polypeptide fused to an autotransporter comprising a transmembrane linker and a transporter domain is displayed on the surface of the cell, as well as to formulations comprising such non-viable cells. The formulations are useful as oral vaccines.

Description

Technical Field

[0001] The present invention relates to a non-viable bacterial cell in which an immunogenic polypeptide fused to an autotransporter comprising a transmembrane linker and a transporter domain is displayed on the cell surface, and to a preparation comprising such a non-viable cell, wherein the preparation is useful as an oral vaccine. Background Art

[0002] Jong et al. (Microbial Cell Factories 13 (2014), 162) described an autotransporter display platform for the development of recombinant bacterial vector vaccines. They demonstrated the simultaneous secretion and display of Mycobacterium tuberculosis antigens in Escherichia coli. Furthermore, they showed the stable multivalent display of these antigens in attenuated Salmonella typhimurium strains upon chromosomal integration and demonstrated the expression of multiple antigenic fragments from Chlamydia trachomatis and influenza A virus on the surface of Salmonella cells. They proposed the use of this autotransporter display platform to generate multivalent recombinant bacterial live vaccines and also for derived non-live vaccines based on outer membrane vesicles or bacterial ghosts. Hjelm et al. described an autotransporter-based antigen display in bacterial ghosts obtained by expression of the lytic gene E (Appl. Env. Microbiol. 81 (2015), 726-735).

[0003] However, the vaccines proposed by Jong et al. or Hjelm et al. have significant drawbacks. On the one hand, the administration of live bacterial vaccines is associated with potential health risks. On the other hand, the administration of outer membrane vesicles or bacterial ghosts may not be as effective as the administration of intact bacterial cells. Furthermore, there is a lack of evidence demonstrating long-term immunogenicity and stability.

[0004] Therefore, the object of the present invention is to provide a highly effective, stable and safe vaccine. Summary of the Invention

[0005] According to a first aspect, the present invention relates to a non-viable bacterium cell, wherein an immunogenic polypeptide fused to an autotransporter comprising a transmembrane linker and a transporter domain is displayed on the cell surface.

[0006] In certain embodiments, the immunogenic polypeptide is attached to the surface of the outer membrane of the Gram-negative bacterial cell via a β-barrel autotransporter, which acts as an anchor within the outer membrane. In certain embodiments, the immunogenic polypeptide is located at the N-terminus of the β-barrel autotransporter. In other embodiments, the immunogenic polypeptide is located at the C-terminus of the β-barrel autotransporter.

[0007] In specific embodiments, the immunogenic polypeptide is from a coronavirus (e.g., MERS, SARS-CoV-1, or SARS-CoV-2), or from a Plasmodium organism (e.g., Plasmodium falciparum), from a human papillomavirus (HPV), or from bovine viral diarrhea virus (BVDV).

[0008] In some embodiments, the bacterial cell does not comprise a gene encoding an antibiotic resistance polypeptide.

[0009] In specific embodiments, the bacterial cell comprises a metabolic selectable marker gene.

[0010] In specific embodiments, the non-viable bacterial cells are lyophilized.

[0011] Another aspect of the present invention is a preparation comprising non-viable bacterial cells as described above, wherein the preparation does not contain living bacterial cells.

[0012] In specific embodiments, the formulation is a dry formulation, such as a lyophilized formulation.

[0013] In other specific embodiments, the formulation is an effervescent powder.

[0014] In some embodiments, the preparation comprises bacterial cells that do not contain a gene encoding an antibiotic resistance polypeptide.

[0015] In specific embodiments, the preparation comprises bacterial cells comprising a metabolic selectable marker gene.

[0016] In certain embodiments, the non-viable bacterial cells or preparations are suitable for medical use.

[0017] Another aspect of the present invention is an oral vaccine comprising the non-viable bacterial cells or preparation as described above.

[0018] In certain embodiments, the bacterial cell is a Gram-negative bacterial cell. In specific embodiments, the bacterial cell is a Gram-negative bacterial cell, such as a Salmonella cell, an Escherichia coli cell, or a Vibrio cholerae cell, which is suitable for oral administration to a subject, such as a human subject.

[0019] In a specific embodiment, the bacterial cell is a Salmonella typhi Ty 21a cell (Germanier & Fürer, J. Infect. Dis. 131 (1975), 553-558; Bumann et al., Vaccine 20 (2002), 845-852; Kopecko et al., Int. J. Med. Microbiol. 209 (2009), 233-246), the contents of which are incorporated herein by reference. This embodiment also includes genetic variants, particularly genetic variants that lack genes encoding polypeptides necessary for the production and / or degradation of metabolites in the cell, such as ΔthyA (thymidylate synthase) variants.

[0020] In other specific embodiments, the bacterial cell is an Escherichia coli Nissle 1917 cell (Schultz, Inflammatory Bowel Dis. 14 (2008), 1012-1018; Yu et al., Microb. Biotechnol. 13 (2020), 629-636), the contents of which are incorporated herein by reference. This embodiment also includes genetic variants, particularly genetic variants that lack a gene encoding a polypeptide necessary for the production and / or degradation of a metabolite in the cell, such as a ΔthyA variant.

[0021] In other specific embodiments, the bacterial cell is a Vibrio cholerae Vaxchora cell (Cabrera et al., Ann. Pharmacother. 51 (2017), 584-589), the contents of which are incorporated herein by reference. This embodiment also includes genetic variants, particularly genetic variants that lack genes encoding polypeptides necessary for the production and / or degradation of metabolites in the cell, such as ΔthyA variants.

[0022] The immunogenic polypeptide displayed on the bacterial cell surface is part of a recombinant autotransporter fusion protein located on the outer membrane of the host cell. The recombinant autotransporter typically comprises (i) an N-terminal signal peptide (which may be cleaved); (ii) a passenger domain, which is an immunogenic polypeptide; and (iii) an autotransporter comprising a linker domain and a β-barrel domain. The passenger domain can be located at the N-terminus or C-terminus of the autotransporter domain.

[0023] In certain embodiments, the recombinant autotransporter comprises, from N- to C-terminus, (i): a signal peptide (which may be cleaved); (ii) a passenger domain, which is an immunogenic polypeptide; and (iii) an autotransporter comprising a linker domain and a transporter domain (particularly a β-barrel domain). In these embodiments, the autotransporter is typically a classical autotransporter, such as EhaA. In other embodiments, the recombinant autotransporter comprises, from N- to C-terminus, (i): a signal peptide (which may be cleaved); (ii) an autotransporter comprising a transporter domain (particularly a β-barrel domain) and a linker; and (iii) a passenger domain, which is an immunogenic polypeptide. In these embodiments, the autotransporter is typically a reverse autotransporter, such as YeeJ.

[0024] The autotransporter fusion protein is encoded by a recombinant nucleic acid molecule that is present in the extrachromosomal or intrachromosomal portion of the bacterial cell. The recombinant nucleic acid molecule is operably linked to an expression control sequence that comprises a promoter and optionally other sequences required for gene expression in the corresponding host cell. The expression control sequence can be homologous or heterologous to the host cell. Preferably, the nucleic acid molecule is located on a recombinant vector (e.g., a plasmid vector). Expression control sequences are understood by those skilled in the art, particularly for expression in host cell species as described herein.

[0025] In certain embodiments, the recombinant nucleic acid molecule is operably linked to an inducible promoter, such as the arabinose inducible araBAD promoter (Greenfield et al., Proc. Natl. Acad. Sci. USA 75 (1978), 4724-4728), the contents of which are incorporated herein by reference. In other embodiments, the inducible promoter is a rhamnose promoter, a Lac promoter, a Tac promoter, a pRox promoter (Meyers et al., Microb Biotechnol, 12 (2019), 1003-1013). Alternatively, a constitutive promoter, such as a pGB35 ​​promoter (Zobel et al., ACS Synthetic Biology, 4 (2015), 1341-1351) can be used.

[0026] The sequence of the nucleic acid molecule can have a codon usage preference that is compatible with the host cell. Specifically, the codon usage preference of the transmembrane linker sequence, the transporter domain, or / and the passenger can be compatible with the host cell. More specifically, the codon usage preference of the transmembrane linker sequence or / and the transporter domain can be compatible with the host cell. This can improve expression if the transporter domain is heterologous to the host cell. Optimization of codon usage does not usually affect the amino acid sequence.

[0027] In certain embodiments, the recombinant nucleic acid molecule comprises: (i) a portion encoding a signal peptide, (ii) a portion encoding the immunogenic polypeptide to be displayed, and (iii) a portion encoding an autotransporter containing the transmembrane linker and transporter domains.

[0028] In other embodiments, the recombinant nucleic acid molecule comprises: (i) a portion encoding a signal peptide, (ii) a portion encoding an autotransporter comprising a transporter domain and a transmembrane linker; and (iii) a portion encoding the immunogenic polypeptide to be displayed.

[0029] The recombinant nucleic acid molecule comprises a portion encoding an N-terminal signal peptide, preferably a portion encoding a Gram-negative signal peptide that allows transport across the inner cell membrane into the periplasm. The signal peptide may be a signal peptide homologous to the host cell. The signal peptide may also be a signal peptide heterologous to the host cell. An example of a suitable signal peptide is the cholera toxin B (CtxB) signal peptide. In other embodiments, PeIB, OmpA, YeeJ, or OprF signal peptides may be used. The signal peptide may be cleaved during polypeptide maturation.

[0030] The recombinant nucleic acid molecule also includes a portion encoding an immunogenic polypeptide. The term "immunogenic polypeptide" refers to any polypeptide capable of eliciting an immune response (e.g., an antibody response and / or a T cell response) in a host organism to which the bacterial cells are administered. The host organism is typically a vertebrate organism, such as a mammal, including a human.

[0031] In certain embodiments, the immunogenic polypeptide is a polypeptide from a pathogen (e.g., a virus, bacteria, fungus, or parasite). In specific embodiments, the immunogenic polypeptide is from a pathogen selected from the group consisting of a coronavirus, such as MERS, SARS-CoV-1, or SARS-CoV-2; a malarial organism, such as Plasmodium falciparum; Mycobacterium tuberculosis; Mycobacterium leprae; Trypanosoma brucei; Trypanosoma cruzi; Neisseria meningitidis; Haemophilus influenzae; Streptococcus pneumoniae; dengue virus; Ebola virus, or human papillomavirus (HPV), preferably an oncogenic HPV type, such as HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, or bovine viral diarrhea virus (BVDV).

[0032] In other embodiments, the pathogen is a multidrug resistant bacterium, such as Staphylococcus aureus (MRSA), Escherichia coli EHEC, Enterobacter or Streptococcus pneumoniae. In other embodiments, the pathogen is selected from Borrelia burgdorferi or Campylobacter jejuni.

[0033] In even more specific embodiments, the immunogenic polypeptide is a SARS-CoV-2 spike glycoprotein or an immunogenic fragment thereof, a SARS-CoV-2 receptor binding domain or an immunogenic fragment thereof (optionally including a trimerization domain), or a SARS-CoV-2 nucleocapsid protein or an immunogenic fragment thereof.

[0034] In other even more specific embodiments, the immunogenic polypeptide is the surface-associated 6-cysteine ​​domain of the Plasmodium falciparum sexual stage protein Pfsi230 or an immunogenic fragment thereof, particularly Pfs230D1M; the surface-associated circumsporozoite protein of Plasmodium falciparum or an immunogenic fragment thereof, particularly RV21.

[0035] In other even more specific embodiments, the immunogenic polypeptide is the L1 protein of human papillomavirus or an immunogenic fragment thereof, in particular HPV L1 from HPV type 18 or other oncogenic HPV types such as types 16, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59.

[0036] In other even more specific embodiments, the immunogenic polypeptide is the bovine viral diarrhea virus E2 protein or an immunogenic fragment thereof, particularly BVDV344.

[0037] In specific embodiments, the immunogenic polypeptide is a SARS-CoV-2 nucleocapsid protein comprising amino acids 40-457 of SEQ ID NO: 2, or a polypeptide having at least 70%, at least 90%, at least 95%, or at least 98% identity thereto throughout the entire sequence. The immunogenic polypeptide can be encoded by a nucleotide sequence of nucleotides 118-1371 of SEQ ID NO: 1, or a nucleotide sequence within the degeneracy of the genetic code.

[0038] In other specific embodiments, the immunogenic polypeptide is a SARS-CoV-2 spike protein comprising amino acids 40-1237 of SEQ ID NO: 4, or a polypeptide having at least 70%, at least 90%, at least 95%, or at least 98% identity thereto throughout the entire sequence. The immunogenic polypeptide can be encoded by a nucleotide sequence of nucleotides 118-3711 of SEQ ID NO: 3, or a nucleotide sequence within the degeneracy of the genetic code.

[0039] In other specific embodiments, the immunogenic polypeptide is a SARS-CoV-2 receptor binding domain (RBD) comprising amino acids 40-262 of SEQ ID NO: 5 or SEQ ID NO: 7, or a polypeptide having at least 80%, at least 90%, or at least 95% identity thereto over the entire sequence. The immunogenic polypeptide may be encoded by a nucleotide sequence of nucleotides 118-786 of SEQ ID NO: 6 or SEQ ID NO: 8, or a nucleotide sequence within the degeneracy of the genetic code.

[0040] In certain embodiments, an immunogenic polypeptide, such as a SARS-CoV-2 RBD, may comprise a multimerization domain at its N-terminus or C-terminus, such as a dimerization or trimerization domain. In specific embodiments, the multimerization domain is a trimerization domain comprising amino acids 263-289 of SEQ ID NO: 5, or a polypeptide having at least 70%, at least 90%, at least 95%, or at least 98% identity thereto over the entire sequence. The trimerization domain may be encoded by a nucleotide sequence of nucleotides 787-867 of SEQ ID NO: 6, or a nucleotide sequence within the degeneracy of the genetic code.

[0041] In other specific embodiments, the immunogenic polypeptide is a Plasmodium falciparum Pfs 230 D1M polypeptide comprising amino acids 1-195 of SEQ ID NO: 9, or a polypeptide at least 70%, at least 90%, at least 95%, or at least 98% identical thereto over the entire sequence.

[0042] In other specific embodiments, the immunogenic polypeptide is a Plasmodium falciparum RV21 polypeptide comprising amino acids 1-268 of SEQ ID NO: 10, or a polypeptide that is at least 70%, at least 90%, at least 95%, or at least 98% identical thereto over the entire sequence.

[0043] In other specific embodiments, the immunogenic polypeptide is an HPV L1 polypeptide comprising amino acids 1-507 of SEQ ID NO: 11, or a polypeptide having at least 70%, at least 90%, at least 95%, or at least 98% identity thereto over the entire sequence.

[0044] In other specific embodiments, the immunogenic polypeptide is an N-terminal fragment of the BVDV E2 polypeptide comprising amino acids 1-344 of SEQ ID NO: 12, or a polypeptide having at least 70%, at least 90%, at least 95%, or at least 98% identity thereto over the entire sequence.

[0045] Suitable methods known to those skilled in the art are used to determine the degree of identity of nucleic acid sequences and amino acid sequences. Known algorithms, such as NBLAST (for nucleic acids) or BLASTp (for amino acid sequences) can be used. Nucleic acids or polypeptides are comprised in sequences or fragments of given nucleic acids or polypeptides that have at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identity to a given sequence over the entire sequence.

[0046] The recombinant nucleic acid molecule further comprises a portion encoding an autotransporter, which is located C-terminally or N-terminally to the portion encoding the immunogenic polypeptide.

[0047] This portion contains the transmembrane linker, which is necessary for displaying the passenger polypeptide on the external surface of the host cell outer membrane. The transmembrane linker is usually located between the autotransporter domain and the immunogenic polypeptide.

[0048] In one embodiment, the transmembrane linker domain can be used to form a transmembrane linker homologous to the autotransporter domain, that is, the transmembrane linker domain is encoded by the nucleic acid portion located at the 3' end or 5' end of the autotransporter domain. In other embodiments, the transmembrane linker domain can be used to form a transmembrane linker heterologous to the autotransporter domain. The length of the transmembrane linker is preferably 30-250 amino acid. The transmembrane linker is preferably a transmembrane linker obtained from EhaA protein as described herein. In a specific embodiment, the transmembrane linker comprises the 484-687 amino acid of SEQ ID NO: 1, or has a polypeptide of at least 70%, at least 90%, at least 95% or at least 98% identity with it over the entire sequence. The transmembrane linker can be encoded by the nucleotide sequence of the 1450-2061 nucleotide of SEQ ID NO: 2 or a nucleotide sequence within the scope of the genetic code degeneracy.

[0049] The recombinant nucleic acid molecule further comprises a portion of a transporter domain encoding an autotransporter. Examples of suitable autotransporters are described in Celik et al. (PLoS ONE 7 (2012), e43245), Leo et al. (Int J Med Microbiol. 305 (2015), 276-82), and Meuskens et al. (Front Microbiol. 31 (2019), 1163), the contents of which are incorporated herein by reference.

[0050] In certain embodiments, the autotransporter is AIDA-1 (Jose et al., Microbiol. Mol. Biol. 71 (2007), 600-619) or hemoglobin protease (Hbp) (Jong et al., supra), the contents of which are incorporated herein by reference.

[0051] In certain embodiments, the autotransporter is an antiporter, such as YeeJ, intimate, or invasin (Tian et al., Microb. Biotechnol. 15 (2022), 2235-2249; Martinez-Gil et al., Sci Rep. 7 (2017), 11326), the contents of which are incorporated herein by reference.

[0052] In a specific embodiment, the autotransporter is EhaA, an autotransporter derived from Escherichia coli strain O157:H7 (Wells et al., Environ. Microbiol. 10 (2008), 589-604), the contents of which are incorporated herein by reference.

[0053] In a specific embodiment, the autotransporter domain is an EhaA autotransporter β-barrel domain comprising amino acids 688-972 of SEQ ID NO: 2, or a polypeptide having at least 70%, at least 90%, at least 95%, or at least 98% identity thereto over the entire sequence. The autotransporter domain can be encoded by a nucleotide sequence of nucleotides 2062-2916 of SEQ ID NO: 1, or a nucleotide sequence within the degeneracy of the genetic code.

[0054] The EhaA transporter domain also includes variants that can be obtained by altering the amino acid sequence in the β-barrel loops that are not involved in the transmembrane domain. Alternatively, the nucleic acid portion encoding the surface loops can be completely deleted. In addition, conservative amino acid exchanges can occur within the amphipathic β-sheet, i.e., exchanging a hydrophilic amino acid for another hydrophilic amino acid or / and exchanging a hydrophobic amino acid for another hydrophobic amino acid.

[0055] The recombinant nucleic acid molecule can also comprise at least one nucleic acid sequence encoding an affinity tag. The nucleic acid sequence encoding the affinity tag can flank the part of the immunogenic polypeptide to be displayed. For example, the affinity tag can be a His epitope tag, a Myc tag and / or a PEYVK tag. In the present embodiment, the nucleic acid sequence encoding the affinity tag can be separated from the part encoding the immunogenic polypeptide by encoding at least one protease recognition sequence (for example, a recognition sequence of a bacterial protease and / or a recognition sequence of a eukaryotic organism (particularly a mammalian protease). For example, at least one protease recognition sequence is independently selected from the group consisting of a factor Xa cleavage site, an OmpT cleavage site and a TEV protease cleavage site.

[0056] In addition, the recombinant nucleic acid molecule may further comprise at least one restriction enzyme recognition site to facilitate cloning of the portion encoding the immunogenic polypeptide to be displayed.

[0057] In certain embodiments, the non-viable bacterial cells comprise a first immunogenic polypeptide and a second immunogenic polypeptide that is different from the first immunogenic polypeptide. In specific embodiments, the first immunogenic polypeptide and the second immunogenic polypeptide can be derived from a pathogen as described above, e.g., from the same pathogen or from different pathogens. In these embodiments, the first and second immunogenic polypeptides can be fused to first and second autotransporters, respectively, and optionally the first autotransporter is different from the second autotransporter.

[0058] In other specific embodiments, the first immunogenic polypeptide is from a pathogen and the second immunogenic polypeptide is an adjuvant polypeptide, wherein the first immunogenic polypeptide is different from the second immunogenic polypeptide. In these embodiments, the first and second immunogenic polypeptides can be fused to first and second autotransporters, respectively, and optionally the first autotransporter is different from the second autotransporter, and optionally the first autotransporter is different from the second autotransporter. Alternatively, the extracellular portion of the autotransporter itself can contain the second adjuvant immunogenic polypeptide.

[0059] Examples of adjuvant polypeptides for use herein include: cholera toxin or a subunit thereof, such as cholera toxin B subunit; enterotoxin or a subunit thereof, such as type IIb heat-labile enterotoxin subunit (LT-IIb-B5); Brucella cell surface protein 31 (rBCSP31); endopeptidase O (PepO) and / or DnaJ, as described in Holmgren et al. (Immunol Lett. 97(2) (2005), 181-188), Li et al. (Cell. Mol. Immunol. 11 (2014), 477-494), Liang et al. (Vaccine 27(2009), 4302-4308), Zhang et al. (Front. Cell. Infect. Microbiol. 6 (2016), 23) and Su et al. (Front. Immunol. 8 (2017), 686), the contents of which are incorporated herein by reference.

[0060] In specific embodiments, the present invention relates to a mixture of one or more different non-viable bacterial cells. In certain embodiments, the mixture comprises a first non-viable bacterial cell and a second non-viable bacterial cell, the first non-viable bacterial cell comprising a first immunogenic polypeptide and the second non-viable bacterial cell comprising a second immunogenic polypeptide, wherein the first immunogenic polypeptide is different from the second immunogenic polypeptide. In specific embodiments, the first immunogenic polypeptide and the second immunogenic polypeptide can be from a pathogen as described above, for example, from the same pathogen or from different pathogens. In these embodiments, the first and second immunogenic polypeptides are fused to a first and a second autotransporter, respectively, and optionally the first autotransporter is different from the second autotransporter.

[0061] In other embodiments, the first immunogenic polypeptide is derived from a pathogen and the second immunogenic polypeptide is an adjuvant polypeptide as described above.

[0062] The recombinant nucleic acid molecule encoding the fusion polypeptide can be located on an extrachromosomal vector (eg, a plasmid).

[0063] The recombinant bacterial cell may comprise a selectable marker gene for proliferation. In certain embodiments, the selectable marker gene is located on an extrachromosomal vector (eg, a vector that also has the recombinant nucleic acid molecule).

[0064] In certain embodiments, the vector comprises a metabolic selection marker gene, such as an antibiotic resistance gene, such as an ampicillin resistance gene or a kanamycin resistance gene.

[0065] In other embodiments, the bacterial cell does not comprise a gene encoding an antibiotic resistance polypeptide. In these embodiments, the bacterial cell is auxotrophic for essential metabolites (particularly thymidylate, aspartate, etc.). Thus, in a preferred embodiment, the vector comprising the recombinant nucleic acid molecule described herein does not contain an antibiotic resistance gene, but rather contains an essential gene, such as a gene encoding an enzyme required for providing the bacterial cell with essential metabolites, in particular thymidylate synthase, aspartate semialdehyde dehydrogenase, KDPG-aldolase, orotidine-5'-phosphate decarboxylase / pyrroline-5-carboxylic acid reductase, as described in Bumann et al. (Vaccine 20(5-6) (2001), 845-852), Curtis et al. (Res Microbiol. 141(7-8) (1990), 797-805), Xin et al. (Infect Immun. 80(10) (2012), 3621-33), Voss and Steinbüchel (Metab Eng. 8(1) (2006), 66-78) and Schneider et al. (Biotechnol Prog. 21(2)). (2005), 343-8), the contents of which are incorporated herein by reference.

[0066] Specifically, the essential gene is deleted from the chromosomal DNA and placed on an extrachromosomal vector, which results in the cell requiring the vector for survival. Growth of bacterial cells containing the vector indicates that the plasmid has integrated into the bacterial cells.

[0067] Non-viable bacterial cells can be prepared by a method comprising the following steps: (a) culturing living bacterial cells in a culture medium, wherein the bacterial cells are transformed or transfected with a recombinant nucleic acid molecule comprising: (i) a portion encoding a signal peptide, (ii) a portion encoding the immunogenic polypeptide to be displayed, (iii) a portion encoding an autotransporter comprising a transmembrane linker and a transporter domain of the autotransporter, or (i) a portion encoding a signal peptide, (ii) a portion encoding an autotransporter comprising the transporter domain and the transmembrane linker of the autotransporter; and (iii) a portion encoding the immunogenic polypeptide to be displayed, Under conditions where the recombinant nucleic acid molecule is expressed; (b) obtaining a bacterial cell in which an immunogenic polypeptide of an autotransporter fused to an autotransporter domain comprising a transmembrane linker and encoding the recombinant nucleic acid molecule of (a) is displayed on the cell surface; (c) subjecting the bacterial cells of (b) to conditions whereby non-viable bacterial cells are obtained.

[0068] Steps (a) and (b) can be performed using standard methods known in the art. Those skilled in the art will appreciate suitable culture conditions for efficiently expressing the passenger protein on the surface of host cells (particularly Escherichia coli or other Gram-negative bacterial cells), achieving up to 100,000 or more molecules per cell, by using a liquid culture medium composed of: 5 g / l to 20 g / l (preferably approximately 10 g / l) tryptone, 2 g / l to 10 g / l (preferably approximately 5 g / l) yeast extract, 5 g / l to 20 g / l (particularly approximately 10 g / l) NaCl, and the remainder water. The culture medium should contain as few divalent cations as possible. Furthermore, the liquid culture medium may preferably contain EDTA at a concentration of 2 µM to 20 µM (particularly 10 µM). Furthermore, it preferably contains a reducing agent, such as 2-mercaptoethanol, dithiothreitol, or dithioerythritol, preferably at a concentration of 2 mM to 20 mM. Reducing agents help maintain the unfolded structure of the polypeptide during transport. The liquid culture medium may also contain an additional carbon source, preferably glucose, for example in an amount of up to 10 g / l, to facilitate secretion, ie the transport of passengers to the surrounding culture medium.

[0069] In a specific embodiment, the expression system described herein that utilizes the EhaA autotransporter domain is referred to as the MATE (Maximized Autotransporter Expression) system of the pMATE system. Plasmids used in the MATE system, particularly expression plasmids comprising the nucleic acid fusions described herein, are also designated with the prefix "pMATE." WO2014 / 139862 describes the MATE / pMATE system, the contents of which are incorporated herein by reference.

[0070] Step (c) includes subjecting the bacterial cells to conditions that render them no longer viable. In certain embodiments, the bacterial cells are rendered non-viable by lyophilization. In a specific embodiment, step (c) includes lyophilizing the bacterial cells of step (b) in the presence of an aqueous medium containing phosphate ions (e.g., an aqueous medium containing phosphate buffered saline (PBS)). In certain embodiments, lyophilization includes freezing the cell suspension to a temperature of approximately -80°C, for example, for at least 6 hours or overnight. In certain embodiments, the cells are stored at a temperature of -40°C or -80°C for at least 1 day, for example, at least 2 days, after freezing.

[0071] In other embodiments, non-viable bacterial cells can be obtained by ultraviolet irradiation, heating, or treating intact bacterial cells with chemical reagents (eg, NaNO2 or β-propiolactone).

[0072] In embodiments where a mixture of different non-viable bacterial cells comprising a first immunogenic polypeptide and non-viable bacterial cells comprising a second immunogenic polypeptide is prepared, the individual components of the mixture can be obtained separately in step (b) and mixed in step (c) before or after lyophilization.

[0073] The bacterial cells of the present invention are non-viable cells. In certain embodiments, the non-viable bacterial cells are intact cells, such as cells comprising a cell membrane and cytoplasmic components, wherein the cell membrane may be permeable. The morphology of the non-viable cells after lyophilization is similar or identical to that of viable cells as measured by flow cytometry.

[0074] In certain embodiments, the bacterial cells of the present invention are freeze-dried non-viable bacterial cells. In specific embodiments, the cells have been freeze-dried in a medium containing phosphate and / or sodium, potassium, and chloride (e.g., a PBS (phosphate-buffered saline) buffer as described herein, i.e., 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2.0 mM KH2PO4, pH 7.4). Surprisingly, the freeze-dried bacterial cells under these conditions are non-viable, i.e., cannot be recultured by the steps described herein. After incubation at 37°C for 16 or 24 hours, no cell growth was observed.

[0075] Another aspect of the present invention is a formulation comprising non-viable bacterial cells as described above, particularly lyophilized and optionally reconstituted non-viable bacterial cells, wherein the formulation does not contain viable bacterial cells. This is an advantageous feature for medical applications, particularly those involving oral delivery. In certain embodiments, the formulation comprises about 10 8 to 10 12 cells.

[0076] In certain embodiments, the formulation is a solid formulation, such as a tablet or powder. In specific embodiments, the formulation is an effervescent powder. In other embodiments, the formulation is a liquid formulation, such as an aqueous or oily suspension.

[0077] In certain embodiments, the formulation comprises bacterial cells and other components from the lyophilization step, such as phosphate ions and / or sodium ions and chloride ions. In certain embodiments, the formulation further comprises phosphate ions, sodium ions and chloride ions.

[0078] In certain embodiments, the formulation further comprises at least one pharmaceutically acceptable excipient, such as a filler, a diluent, a lubricant, etc. These excipients are typically added after the freeze-drying step. Examples of suitable excipients include excipients from tablet or powder formulations known to those skilled in the art, such as cellulose or cellulose derivatives, lactose, starch, sorbitol, and / or mannose. In a specific embodiment, the formulation comprises an acidic compound and a carbonate or bicarbonate-containing compound to provide an effervescent formulation.

[0079] The formulation of the invention is stable, ie, it maintains immunogenicity when stored for extended periods of time. In certain embodiments, it retains at least 50%, at least 70%, or at least 90% of its immunogenicity when measured in vitro in a suitable antigen assay.

[0080] In a specific embodiment, the formulation is stable as a lyophilisate in a sealed vial for at least 5 months or at least 12 months at a temperature of 25° C., in particular at a temperature of 25° C. In other specific embodiments, the formulation is stable as a lyophilisate in a sealed vial for at least 1 month or at least 12 months at a temperature of 37° C., in particular at a temperature of 37° C.

[0081] The non-viable bacterial cells or preparations are suitable for medical use. In certain embodiments, the non-viable bacterial cells or preparations are used as vaccines, particularly oral vaccines. In specific embodiments, the preparations are used as vaccines for immunizing a subject against infection by a pathogen, particularly a pathogen from which the immunogenic polypeptide is derived. Presentation of immunogenic polypeptides on the surface of bacterial cells is expected to provide a strong immune response in vaccinated subjects, such as vaccinated human subjects, because components of bacterial cells, particularly autotransporter components presented on the cell surface, are effective as intrinsic adjuvants.

[0082] Therefore, another aspect of the present invention is a vaccine comprising the non-viable bacterial cells or preparations described herein. The inventors have found that the immunogenicity of the immunogenic peptides is maintained over a long period of time even at room temperature or higher. Furthermore, the inventors have found that administering the preparation comprising non-viable bacterial cells in a dried form, particularly a lyophilized form, provides an additional immunostimulatory effect.

[0083] The vaccine may comprise a single type of non-viable bacterial cell or a plurality of different types of non-viable bacterial cells, ie bacterial cells that present different immunogenic polypeptides on their surface. The different immunogenic polypeptides may be from a single pathogen or from different pathogens as described above.

[0084] In a preferred embodiment, the vaccine is a multivalent HPV vaccine, such as a nine-valent HPV vaccine that protects against HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, or a four-valent HPV vaccine that protects against HPV types 6, 11, 16, and 18, or a two-valent HPV vaccine that protects against HPV types 16 and 18.

[0085] The present invention is further illustrated by the following examples. DETAILED DESCRIPTION

[0086] The pMATE expression plasmid was constructed according to the method described in WO2014 / 139862. Figure 1 Plasmid pMATEAra NP SARS-CoV2 is shown, Figure 2 Plasmid pMATE Ara SP SARS-CoV2 is shown, Figure 3 Plasmid pMATE Ara3RBD SARS-CoV2 (containing the trimerization domain) is shown, Figure 4 Plasmid pMATE Ara RBD SARS-CoV2 (without trimerization domain) is shown.

[0087] Figure 5a shows the nucleotide sequence encoding the nucleocapsid autotransporter fusion protein from pMATE Ara NP SARS-CoV2 (SEQ ID NO: 1).

[0088] Annotation of SEQ ID NO: 1

[0089] FIG5 b shows the amino acid sequence of the nucleocapsid autotransporter fusion protein (SEQ ID NO: 2).

[0090] Annotation of SEQ ID NO: 2

[0091] Figure 6a shows the nucleotide sequence encoding the spike autotransporter fusion protein from pMATE Ara SP SARS-CoV2 (SEQ ID NO: 3).

[0092] Annotation of SEQ ID NO: 3

[0093] Figure 6b shows the amino acid sequence of the spike autotransporter fusion protein (SEQ ID NO: 4).

[0094] Annotation of SEQ ID NO: 4

[0095] Figure 7a shows the nucleotide sequence encoding the receptor binding domain (RBD) autotransporter fusion protein including the trimerization domain from pMATE Ara 3RBD SARS-CoV2 (SEQ ID NO: 5).

[0096] Annotation of SEQ ID NO: 5

[0097] Figure 7b shows the amino acid sequence of a receptor binding domain autotransporter fusion protein comprising a trimerization domain (SEQ ID NO: 6).

[0098] Annotation of SEQ ID NO: 6

[0099] Figure 8a shows the nucleotide sequence encoding the receptor binding domain autotransporter fusion protein without the trimerization domain from pMATE Ara RBD SARS-CoV2 (SEQ ID NO: 7).

[0100] Annotation of SEQ ID NO: 7

[0101] FIG8 b shows the amino acid sequence of the receptor binding domain autotransporter fusion protein without the trimerization domain (SEQ ID NO: 8).

[0102] Annotation of SEQ ID NO: 8

[0103] Figure 9Shown are the expression and surface display of the SARS-CoV-2 nucleocapsid protein in Salmonella Typhimurium Ty21a by Coomassie Brilliant Blue gel electrophoresis (left) and Western blot (right). The nucleocapsid protein is shown in the box in lane 1. Addition of arabinose induced expression of the fusion protein. Proteinase K was added to digest the surface-exposed protein.

[0104] Figure 10 Shown are the expression and surface display of the SARS-CoV-2 spike protein in Salmonella Typhimurium Ty21a by Coomassie brilliant blue gel electrophoresis (left) and Western blot (right). The spike protein is shown in the box in lane 1. Addition of arabinose induced expression of the fusion protein. Proteinase K was added to digest the surface-exposed protein.

[0105] Figure 11 Expression and surface display of the SARS-CoV-2 3RBD, containing the trimerization domain, in Salmonella Typhimurium Ty 21a are shown in Coomassie Brilliant Blue gel electrophoresis (left) and Western blot (right). The trimeric RBD (without dithiothreitol) is shown in the box in lane 1. The monomeric RBD (with dithiothreitol) is shown in the box in lane 2. Arabinose was added to induce expression of the fusion protein. Proteinase K was added to digest the surface-exposed protein.

[0106] Figure 12 Expression and surface display of the SARS-CoV-2 RBD containing the trimerization domain in Salmonella Typhimurium Ty 21a are shown in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right). The trimeric RBD (without dithiothreitol) is shown in the box in lane 1. The monomeric RBD (with dithiothreitol) is shown in the box in lane 2. Arabinose was added to induce expression of the fusion protein. Proteinase K was added to digest the surface-exposed protein.

[0107] Salmonella typhi cells expressing the recombinant fusion protein were harvested and lyophilized.

[0108] The lyophilization step was performed as follows: - Bacterial cells were grown in 20 ml of LB medium (containing 50 ng / ml kanamycin) in a shake flask at 200 rpm and 37°C to an OD of 578 is 0.5; - Add 0.2% (w / v) arabinose and continue incubation at 200 rpm and 37°C for 2 hours; - Centrifuge the cells at 5000 rpm and 4°C for 5 minutes; - Resuspend the cells in phosphate buffered saline and dilute to OD 578 is 10; - Freeze cells overnight at -80°C; and - The cells are lyophilized at -40°C, for example under a vacuum of 0.220 mbar in a freezer (Alpha 1-2LDplus, Martin Christ, Osterode am Harz, Germany) for 4 days.

[0109] Figure 13a shows a vial of suspended Salmonella enterica Ty21a bacterial cells after lyophilization. As shown in Figure 13b, when the lyophilized cells were plated on LB agar plates, the lyophilized cells were found to be non-viable. Top left: cells containing pMATE Ara SP SARS-CoV2 (spike protein); top right: cells containing pMATE Ara 3RBD (RBD containing the trimerization domain); bottom left: wild-type cells; bottom right: cells containing pMATE Ara NP SARS-CoV2 (nucleoprotein). No cell growth was observed after overnight.

[0110] The cell plating procedure was performed as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Spread 200µl of the suspended lyophilized cells onto LB agar plates without antibiotics.

[0111] Lyophilized non-viable cells containing pMATE Ara NP SARS-CoV2 (nucleoprotein) and lyophilized Salmonella Typhi Ty21a wild-type cells were tested for SARS-CoV-2 antigen. Cells containing nucleoprotein tested positive.

[0112] The detection steps are as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Test 4 drops of the suspension.

[0113] Immunogenicity is maintained even after long-term storage at 25°C (currently up to 6 months or 12 months) and 37°C (currently up to 2 months or 12 months).

[0114] Figure 14 Shown in a Coomassie Brilliant Blue gel are the expression and surface display of the SARS CoV-2 nucleocapsid protein and SARS CoV-2 RBD in the vaccine strain Escherichia coli Nissle 1917. Arabinose was added to induce expression of the fusion protein. Proteinase K was added to digest the surface-exposed proteins.

[0115] E. coli Nissle 1917 cells displaying the nucleoprotein tested positive for SARS CoV-2 antigen (not shown).

[0116] The pMATE expression plasmid was constructed according to the method described in WO2014 / 139862. Figure 15A The plasmid of pMATEAra spike protein SARS CoV-2 (spike (S) glycoprotein) and the corresponding protein are shown. Figure 15B The plasmid of pMATE Ara3RBD SARS CoV-2 (receptor binding domain containing N-terminal trimerization domain) and the corresponding protein are shown. Figure 15C The plasmid of pMATE Ara nucleocapsid protein SARS CoV-2 (nucleocapsid protein) and the corresponding protein are shown.

[0117] Figure 16 Shown are the expression and surface display of the SARS CoV-2 spike protein (a), the trimerization domain-containing 3RBD (b), and the nucleocapsid (c) in Salmonella Typhimurium Ty21a in the presence or absence of dithiothreitol on Coomassie brilliant blue gel electrophoresis. Arabinose was added to induce expression of the fusion protein. Proteinase K was added to digest the surface-exposed proteins.

[0118] The molecular weight of the protein is as follows: • Spike protein-EhaA: 191.4 kDa • 3RDD-EhaA: 82.8 kDa • Nucleocapsid protein - EhaA: 103.2 kDa Figure 17A -C shows the expression and surface display of the SARS-CoV-2 nucleocapsid protein in Salmonella Typhimurium Ty21a in the presence of dithiothreitol in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right). The nucleocapsid protein is shown in the box in lane 1. Arabinose was added to induce expression of the fusion protein. Proteinase K was added to digest the surface-exposed protein. Figure 17D -E shows the amounts of induced (ind.) and non-induced (ni) nucleocapsid protein and proteinase K in S. typhimurium Ty21a measured by FACS.

[0119] The following results are obtained: Salmonella Typhimurium Ty21a pMATE nucleocapsid protein ni: average DyLight 633: 261 Salmonella typhimurium Ty21a pMATE nucleocapsid protein ind.: Average DyLight 633: 3741 Salmonella Typhimurium Ty21a pMATE nucleocapsid protein + proteinase K: average DyLight 633: 389 Figure 18A -C shows the expression and surface display of the SARS-CoV-2 spike protein in Salmonella Typhimurium Ty21a in the presence of dithiothreitol in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right). The spike protein is shown in the box in lane 1. Arabinose was added to induce expression of the fusion protein. Proteinase K was added to digest the surface-exposed protein. Figure 18D -F shows the amount of induced (ind.) and non-induced (ni) spike protein and spike protein + proteinase K in Salmonella typhimurium Ty21a measured by FACS.

[0120] The following results are obtained: Salmonella Typhimurium Ty21a pMATE spike protein ni: average DyLight 633: 462 Salmonella Typhimurium Ty21a pMATE spike protein ind.: average DyLight 633:759 Salmonella Typhimurium Ty21a pMATE Spike Protein + Proteinase K: Average DyLight 633:229.

[0121] Figure 19A Expression and surface display of the SARS-CoV-2 3RBD containing the trimerization domain in Salmonella Typhimurium Ty21a are shown in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right). The trimeric RBD (without dithiothreitol) is shown in the box in lane 1. The monomeric RBD (with dithiothreitol) is shown in the box in lane 2. Arabinose was added to induce expression of the fusion protein. Proteinase K was added to digest the surface-exposed protein. Figure 19B Expression and surface display of the SARS-CoV-2 RBD containing the trimerization domain in Salmonella Typhimurium Ty 21a are shown in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right). The trimeric RBD (without dithiothreitol) is shown in the box in lane 1. The monomeric RBD (with dithiothreitol) is shown in the box in lane 2. Arabinose was added to induce expression of the fusion protein. Proteinase K was added to digest the surface-exposed protein. Figure 19C -E shows the amount of induced (ind.) and non-induced (ni) RBD protein and RBD+Proteinase K in S. Typhimurium Ty21a measured by FACS.

[0122] The following results are obtained: Salmonella Typhimurium Ty21a pMATE RBD ni: Average DyLight 633: 248 Salmonella Typhimurium Ty21a pMATE RBD ind.: Average DyLight 633: 1577 Salmonella Typhimurium Ty21a pMATE RBD + Proteinase K: Average DyLight 633: 268 Salmonella typhi cells expressing the recombinant fusion protein were harvested and lyophilized.

[0123] The lyophilization step was as described above.

[0124] Figure 20A and Figure 20B Shown is a vial / pan of suspended bacterial cells of Salmonella typhimurium Ty21a after lyophilization. Figure 20C As shown, the freeze-dried cells were found to be non-viable when plated on LB agar plates. Figure 20C Top left: cells containing pMATE Ara SP SARS-CoV2 (spike protein); top right: cells containing pMATE Ara 3RBD (RBD containing a trimerization domain); bottom left: wild-type cells; bottom right: cells containing pMATE Ara NP SARS-CoV2 (nucleoprotein). No cell growth was observed after overnight incubation.

[0125] The cell plating procedure was performed as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Spread 200µl of the suspended lyophilized cells onto LB agar plates without antibiotics.

[0126] Lyophilized non-viable cells containing pMATE Ara NP SARS-CoV2 (nucleoprotein) and lyophilized Salmonella Typhi Ty21a wild-type cells were tested for SARS-CoV-2 antigen. Cells containing nucleoprotein tested positive.

[0127] The detection steps are as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for testing.

[0128] Immunogenicity is maintained even after long-term storage at 25°C (currently up to 6 months) and 37°C (currently up to 2 months).

[0129] Figure 21Covid-19 antigen detection is shown for the following Ty21a vaccine strains: Salmonella Typhimurium pMATE Ara -Spike protein (a), Salmonella typhimurium pMATE Ara -3RBD(b), Salmonella typhimurium pMATE Ara -nucleocapsid (c) and Salmonella typhi (d). The detection steps are as follows: - Adjust cells to OD 578 1 - Use 4 drops of the suspension for antigen detection.

[0130] Figure 22 Covid-19 antigen detection is shown for the following lyophilized Ty21a vaccine strains: Lyophilized Salmonella Typhi pMATE Ara -Spike protein (a), lyophilized Salmonella typhimurium pMATE Ara -3RBD (b), freeze-dried Salmonella typhi pMATE Ara -nucleocapsid (c) and freeze-dried Salmonella typhi (d). The test steps are as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for antigen detection.

[0131] Figure 23A Shown is the Covid-19 antigen detection of the lyophilized Ty21a vaccine strain 1 day after harvest; Figure 23B Shown are the Covid-19 antigen detection of the Ty21a vaccine strain after 2 days of freeze-drying: nucleocapsid (a), spike protein (b) and RBD (c). The detection steps are as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for antigen detection.

[0132] Figure 24 Shown are the following Covid-19 antigen detections of the lyophilized Ty21a vaccine strain after 1 week: nucleocapsid (a), spike protein (b), and RBD (c). The assay was performed at room temperature (RT), 4°C, -20°C, and -80°C. The assay steps were as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for antigen detection.

[0133] Figure 25Shown are the following Covid-19 antigen detections of the lyophilized Ty21a vaccine strain after 4 weeks: nucleocapsid (a), spike protein (b), and RBD (c). The assay was performed at room temperature (RT), 4°C, -20°C, and -80°C. The assay steps were as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for antigen detection.

[0134] Figure 26 Shown are Covid-19 antigen detection after 2 months using the following lyophilized Ty21a vaccine strains: nucleocapsid (a), spike protein (b), and RBD (c). The assay was performed at room temperature (RT), 4°C, -20°C, and -80°C. The assay steps were as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for antigen detection.

[0135] Figure 27 Shown are Covid-19 antigen detection after 3 months using the following lyophilized Ty21a vaccine strains: nucleocapsid (a), spike protein (b), and RBD (c). The assay was performed at room temperature (RT), 4°C, -20°C, and -80°C. The assay steps were as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml dd H2O; - Use 4 drops of the suspension for antigen detection.

[0136] Figure 28 Figure 1 shows the results of Covid-19 antigen detection after 12 months using the following lyophilized Ty21a vaccine strains: nucleocapsid (a), spike protein (b), and RBD (c). The assay was performed at room temperature (RT), 4°C, -20°C, and -80°C. The assay steps were as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for antigen detection.

[0137] Figure 29 Shown is the Covid-19 antigen detection after 12 months at 37°C using the following lyophilized Ty21a vaccine strains: nucleocapsid (a), spike protein (b) and RBD (c). The detection steps are as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for antigen detection.

[0138] Figure 30 Shown are Covid-19 antigen detection after 8 months using the following lyophilized E. coli Nissle vaccine strain: nucleocapsid (a), spike protein (b), and RBD (c). The assay was performed at 37°C, room temperature (RT), 4°C, -20°C, and -80°C. The assay steps were as follows: - Freeze-dried cells (1 ml cell culture, OD 578 10) was suspended in 1 ml of ddH2O; - Use 4 drops of the suspension for antigen detection.

[0139] In conclusion, the storage (and transportation) of the lyophilized formulation does not require a cold chain (at least 12 months). In two strains, Salmonella Ty21a and E. coli nissle, no loss of antigenicity was observed with long-term storage of the lyophilized formulation.

[0140] Figure 31A A timeline of the immunoassay steps for BALB / c hACE2tg mice is shown. On November 21, 2023, December 5, 2023, and December 19, 2023, mice were vaccinated intranasally. Blood was collected before (November 20, 2023) and after (December 4, 2023, December 18, 2023; January 2, 2024, and January 23, 2024) intranasal vaccination and analyzed for absorbance at 450 nm to 620 nm. "Control" refers to cells without antigen; "negative" refers to the negative control provided by the ELISA manufacturer. Figure 31B and Figure 31C Shown are nucleocapsid-specific IgG responses in sera from mice treated with S. typhi nucleocapsid. Figure 31D and Figure 31E Shown are RBD-specific IgG responses in sera from mice treated with S. typhi RBD. Figure 31F and Figure 31G Anti-Spike IgG responses in sera from mice treated with Salmonella Typhimurium Spike protein are shown. ELISA Spike protein is associated with a blank column. Figure 31H and Figure 31I Anti-nucleocapsid IgG responses in sera from mice treated with Salmonella typhi nucleocapsids are shown. ELISA spike proteins are associated with negative bars in the graph. Figure 31J The infection LD is shown 50Survival rate (%) of mice infected with SARS-CoV-2 after treatment with Salmonella typhi vaccine. 50 :2.5×10 3 SARS-CoV-2 virus particles.

[0141] Immunization of BALB / c hACE2tg mice with spike protein, RBD, and nucleocapsid protein resulted in differential IgG responses. The vaccine is available as a sterile effervescent powder (no water required). The effervescent powder formulation does not eliminate antigenicity.

[0142] Figure 32 The preparation of effervescent powder (ePowder) is shown. The left vial shows the effervescent powder without cells and water. In the right vial, water is added to the effervescent powder (without cells).

[0143] Figure 33 ePowder (left: 0.05 g NaHCO3, 0.05 g citric acid and 490 µL ddH2O) or a formulation according to German Pharmacopoeia (NRF) 20.2 (right: 0.05 g NaHCO3, 0.05 g citric acid, 0.091 g sorbitol, 0.83*10 -3 gSyloid 244 FP and 490µL ddH2O) for Covid-19 antigen detection.

[0144] Figure 34A Shown is Covid-19 antigen detection of Salmonella Typhimurium Ty21a pMATE nucleocapsid with ddH2O, ePowder, or the formulation. Figure 34B Shown is the Covid-19 antigen detection of Salmonella Typhi Ty21a with ddH2O, ePowder, or the formulation. Figure 34C Comparison of Covid-19 antigen detection in the following ePowder conditions: (1) ddH2O; (2) Salmonella Typhi Ty21a; (3) Salmonella Typhi Ty21a nucleocapsid. ePowder: 0.05g NaHCO3, 0.05g citric acid, 490µL ddH2O, and lyophilized cells (from OD1 in 500µL PBS). Figure 34D Comparison of Covid-19 antigen detection using the following formulations is shown: (1): ddH2O; (2): Salmonella Typhi Ty21a; (3): Salmonella Typhi Ty21a with nucleocapsid. Formulation: 0.05g NaHCO3, 0.05g citric acid, 0.091g sorbitol, 0.83*10 -3 g syloide 244 FP, 490 µL ddH2O, and lyophilized cells (from OD1 in 500 µL PBS).

[0145] exist Figure 34C and Figure 34D In the case of : the same treatment as the previous experiment, but diluted 1:5 in ddH2O before detection. No problems such as dye adhesion were observed in the antigen detection.

[0146] Figure 35A Shown is the amino acid sequence of Pfs230D1M (corresponding to SEQ ID NO: 9), which is part of the surface-associated 6-cysteine ​​domain of the Plasmodium falciparum sexual stage protein Pfs230 (malaria). Figure 35B Shown is the amino acid sequence of RV21 (corresponding to SEQ ID NO: 10), which is part of the surface-associated circumsporozoite protein, the sporozoite motor field, of Plasmodium falciparum (malaria). Figure 35C The amino acid sequence of the human papillomavirus L1 protein is shown (corresponding to SEQ ID NO: 11). Figure 35D The amino acid sequence of the N-terminal amino acids (22 kDa) of the BVDV E2 protein (BVDV344) is shown (corresponding to SEQ ID NO: 12).

[0147] Figure 36 The expression of four additional antigens (two malaria antigens, one HPV antigen and one BVD antigen) in E. coli is shown.

[0148] The size is calculated as follows: - MATE-BVDV344: 99.96 kDa - MATE-HPVL1-18: 117.15 kDa - MATE-Pfs230D1M: 82.47 kDa - MATE-RV21: 85.62 kDa Figure 36 The spontaneous display of two malarial antigens, one HPV antigen, and one BVD antigen in E. coli was demonstrated.

[0149] The pMATE expression plasmid was constructed according to the method described in WO2014 / 139862. Figure 37A The plasmid of pMATEBVDV344 is shown. Figure 37B The pMATE HPVL1 plasmid for HPV type 18 is shown. Figure 37C The plasmid of pMATEPfs230D1M is shown. Figure 37D The plasmid of pMATE RV21 is shown.

[0150] Figure 38The expression and surface display of BVDV, HPV and malaria antigens in Salmonella Typhi Ty21a are shown in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right). The numbers are as follows (Salmonella Typhi Ty21a pMATE): 1: BVDV344 ind. 2: HPVL1 ind. of PHV type 18. 3:RV21 ind. 4: Pfs230D1M ind. 5:BVDV344 ni 6: HPVL1 ni of HPV type 18 7:RV21 ni 8: Pfs230D1M ni (ni = not induced; ind. = induced) S. typhi cells expressing the recombinant fusion protein can be harvested and lyophilized as described above.

[0151] Figure 39 Demonstration of surface display of Salmonella Typhimurium Ty21a pMATE RV21. Left: Salmonella Typhimurium Ty21a pMATE RV21 ni; Right: Salmonella Typhimurium Ty21a pMATE RV21 ind. In this experiment, 50,000 cells were labeled, treated with primary (mouse anti-6xHis) and secondary (goat anti-mouse DyLight 633) antibodies, and measured using a BD FACSAria™ III flow cytometer. For Salmonella Typhimurium Ty21a pMATE RV21 ni, the average DyLight 633 count was 297; for Salmonella Typhimurium Ty21a pMATE RV21 ind., the average DyLight 633 count was 1254 (ni = uninduced; ind. = induced).

[0152] Figure 40 The expression and surface display of E. coli Nissle pMATE BVDV344 in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right) are shown. The numbers are as follows (E. coli Nissle): 1 - pMATE without passengers 2 - pMATE BVDV344 3 - pMATE BVDV344 + Proteinase K 1 (pMATE without passenger) contains the CtxB signal peptide, 6xHis, linker, and β-barrel and represents the “empty pMATE vector” as a control.

[0153] Figure 41 The expression and surface display of HPV type 18 in E. coli Nissle pMATE HPVL are shown in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right). The numbers are as follows (E. coli Nissle): 1 - pMATE without passengers 2 - pMATE HPVL1-18 3 - pMATE HPVL1-18 + Proteinase K 1 (pMATE without passenger) contains the CtxB signal peptide, 6xHis, linker, and β-barrel and represents the “empty pMATE vector” as a control.

[0154] Figure 42 The expression and surface display of E. coli Nissle pMATE Pfs230D1M in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right) are shown. The numbers are as follows (E. coli Nissle): 1 - pMATE without passengers 2 - pMATE Pfs230D1M 3 - pMATE Pfs230D1M + Proteinase K 1 (pMATE without passenger) contains the CtxB signal peptide, 6xHis, linker, and β-barrel and represents the “empty pMATE vector” as a control.

[0155] Figure 43 The expression and surface display of E. coli Nissle pMATE RV21 in Coomassie brilliant blue gel electrophoresis (left) and Western blot (right) are shown. The numbers are as follows (E. coli Nissle): 1 - pMATE without passengers 2 - pMATE RV21 3 - pMATE RV21 + Proteinase K 1 (pMATE without passenger) contains the CtxB signal peptide, 6xHis, linker, and β-barrel and represents the “empty pMATE vector” as a control.

Claims

1. A preparation comprising dried non-viable bacterial cells wherein an immunogenic polypeptide fused to an autotransporter comprising a transmembrane linker and a transporter domain is displayed on the cell surface.

2. The preparation according to claim 1, wherein the non-viable bacterial cells are transformed or transfected with a recombinant nucleic acid molecule comprising: (i) a portion encoding a signal peptide, (ii) a portion encoding the immunogenic polypeptide to be displayed, (iii) a portion encoding an autotransporter comprising a transmembrane linker and a transporter domain, or (i) a portion encoding a signal peptide, (ii) a portion encoding an autotransporter comprising a transporter domain and a transmembrane linker; and (iii) a portion encoding the immunogenic polypeptide to be displayed. 3 . The preparation according to claim 1 , wherein the autotransporter comprises the transporter domain of the EhaA protein or the autotransporter domain of the YeeJ protein.

4. The preparation according to any one of claims 1 to 3, wherein the bacterial cells are Gram-negative bacterial cells, such as Salmonella typhi Ty21a, Escherichia coli Nissle 1917, or Vibrio cholerae Vaxchora.

5. The formulation of any one of claims 1 to 5, wherein the immunogenic polypeptide is derived from a pathogen, such as a virus, bacteria, fungus or parasite.

6. The formulation of any one of claims 1 to 5, wherein the immunogenic polypeptide is from a coronavirus, such as MERS, SARS-CoV-1 or SARS-CoV-2, or from a Plasmodium organism, such as Plasmodium falciparum, or from a human papillomavirus, or from bovine viral diarrhea virus.

7. The formulation of any one of claims 1 to 6, wherein the immunogenic polypeptide is a SARS-CoV-2 spike glycoprotein or an immunogenic fragment thereof, a SARS-CoV-2 receptor binding domain or an immunogenic fragment thereof, optionally comprising a trimerization domain, or a SARS-CoV-2 nucleocapsid protein or an immunogenic fragment thereof.

8. The formulation according to any one of claims 1 to 7, wherein the immunogenic polypeptide is the surface-associated 6-cysteine ​​domain of the sexual stage protein Pfs230 of Plasmodium falciparum or an immunogenic fragment thereof, in particular Pfs230D1M; the surface-associated circumsporozoite protein of Plasmodium falciparum or an immunogenic fragment thereof, in particular RV21; the L1 protein of human papillomavirus or an immunogenic fragment thereof, in particular HPV L1 of HPV type 18; a mixture of oncogenic HPV types, in particular a mixture of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58; or the E2 protein of bovine viral diarrhea virus or an immunogenic fragment thereof, in particular BVDV344.

9. The preparation according to any one of claims 1 to 8, wherein the preparation does not contain viable bacterial cells.

10. The formulation of any one of claims 1 to 9, wherein the bacterial cells are lyophilized.

11. The formulation according to claims 1 to 10, further comprising phosphate ions, and optionally further comprising sodium, potassium and / or chloride ions.

12. The preparation of any one of claims 1 to 11, wherein the non-viable bacterial cells comprise a first immunogenic polypeptide and a second immunogenic polypeptide, wherein the first immunogenic polypeptide is different from the second immunogenic polypeptide.

13. The formulation of claim 12, wherein the first immunogenic polypeptide and the second immunogenic polypeptide are from the same pathogen or from different pathogens.

14. The formulation of claim 12 or 13, wherein the first immunogenic polypeptide and the second immunogenic polypeptide are fused to first and second autotransporters, respectively, and optionally the first autotransporter is different from the second autotransporter.

15. The formulation of claim 12, wherein the first immunogenic polypeptide is from a pathogen and the second immunogenic polypeptide is an adjuvant polypeptide.

16. The formulation of claim 15, wherein the adjuvant polypeptide is fused to a second autotransporter, or wherein the adjuvant polypeptide is the extracellular portion of an autotransporter or a fragment thereof.

17. The preparation according to any one of claims 1 to 16, comprising a mixture of non-viable bacterial cells containing a first immunogenic polypeptide and non-viable bacterial cells containing a second immunogenic polypeptide, wherein the first immunogenic polypeptide is different from the second immunogenic polypeptide.

18. The formulation of claim 16, wherein the first immunogenic polypeptide and the second immunogenic polypeptide are from the same pathogen or from different pathogens.

19. The formulation of claim 16, wherein the first immunogenic polypeptide is from a pathogen and the second immunogenic polypeptide is an adjuvant polypeptide.

20. The formulation according to claims 1-19, wherein the formulation is an effervescent powder.

21. A method for preparing non-viable bacterial cells of the preparation according to any one of claims 1 to 20, comprising: (a) culturing living bacterial cells in a culture medium, wherein the bacterial cells are transformed or transfected with a recombinant nucleic acid molecule comprising: (i) a portion encoding a signal peptide, (ii) a portion encoding the immunogenic polypeptide to be displayed, (iii) the portion encoding the transmembrane linker, and (iv) a portion encoding the transporter domain of an autotransporter; Under conditions where the recombinant nucleic acid molecule is expressed; (b) obtaining a bacterial cell in which an immunogenic polypeptide fused to an autotransporter comprising a transmembrane linker and an autotransporter domain encoded by the recombinant nucleic acid molecule of (a) is displayed on the cell surface; and (c) subjecting the bacterial cells of step (b) to conditions whereby non-viable bacterial cells are obtained, wherein in step (c), the bacterial cells are rendered non-viable by lyophilization.

22. The method of claim 21, wherein step (c) comprises lyophilizing the bacterial cells of step (b) in the presence of an aqueous medium containing phosphate ions, such as an aqueous medium containing phosphate buffered saline (PBS).

23. The method according to claims 21-22, wherein non-viable bacterial cells containing a first immunogenic polypeptide are mixed with non-viable bacterial cells containing a second immunogenic polypeptide, and wherein in step (b) non-viable bacterial cells containing the first immunogenic polypeptide and non-viable bacterial cells containing the second immunogenic polypeptide are obtained separately.

24. The method according to claim 23, wherein in step (c), the non-viable bacterial cells are mixed before lyophilization or after lyophilization.

25. Use of the formulation according to any one of claims 1 to 20 in medicine.

26. An oral vaccine comprising the formulation according to any one of claims 1-20.

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