Generation of antigen-specific t and b cells using engineered commensals

Abstract

Presenting antigens linked to commensal bacterial surface proteins results in a surprisingly high immune response triggered when the bacteria are applied to an epithelial surface of a mammal. In some embodiments, an antigenic polypeptide or immunomodulatory molecule can be expressed from bacteria as a fusion with a bacterial surface protein. In other aspects, a variety of ways are described for linking an antigenic molecule or immunomodulatory molecule to a bacterial surface protein, providing flexibility for linking a desired antigen to the bacterial surface protein.

Description

GENERATION OF ANTIGEN-SPECIFIC T AND B CELLS USING ENGINEERED COMMENSALSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 588,398 filed on October 6, 2023, the contents of which are hereby incorporated in their entirety by reference.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is hereby incorporated by reference in its entirety. The electronic copy, created on October 4, 2024, is named 59702_SeqListing.xml, and is 101,405 bytes in size.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0003] This invention was made with Government support under contract All 75642 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0004] The ubiquitous skin colonist Staphylococcus epidermidis (S. epidennidis) elicits an immune response including, for example, an induction of cytokines, a CD4 response, and a CD8+ T cell response pre-emptively, in the absence of an infection. However, the scope and purpose of this anti-commensal immune program are not well defined, limiting the ability to harness it therapeutically. If antigens or immunomodulatory molecules could be attached to cell surface proteins and this colonist could induce a potent, durable, and specific antibody response, an anti-commensal immune program could be therapeutically harnessed, potentially as a novel form of a topical vaccine.BRIEF SUMMARY OF THE INVENTION

[0005] Provided herein is a commensal bacterium comprising on the surface of the bacterium, an extracellular protein selected from the group consisting of Accumulation-associated protein (Aap), peptidoglycan, an M protein, Lysin Motif (LysM)-containing protein and a peptidoglycan-binding protein, or a fragment thereof linked to a heterologous antigenic molecule or immunomodulatory molecule.

[0006] In some embodiments, the extracellular protein is heterologous to the commensal bacterium. In some embodiments, the extracellular protein is endogenous to the commensal bacterium.

[0007] In some embodiments, the fragment comprises at least 5, 14, 25, 50, 75, 100, 150 or 200 amino acids of the extracellular protein.

[0008] In some embodiments, the commensal bacterium is a gram-positive bacterium.

[0009] In some embodiments, the heterologous antigenic molecule is an antigenic polypeptide. In some embodiments, one or more first affinity sequence is fused to the extracellular protein or fragment thereof and a second affinity sequence is fused to the heterologous antigenic polypeptide and the extracellular protein or fragment thereof is linked to the heterologous antigenic polypeptide via affinity or covalent linkage of the first and second affinity sequences.

[0010] In some embodiments, the first affinity tag and the second affinity tag are a pair selected from the following list of pairs: a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and a DogTag sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; a HaloTag sequence and a haloalkane moiety; a cysteine residue and a maleimide moiety, a free amine and an N-hydroxy succinimide ester.

[0011] In some embodiments, the extracellular protein or fragment thereof may be linked to the heterologous antigenic polypeptide via a transpeptidase recognition sequence. In some embodiments, the transpeptidase configured to recognize the transpeptidase recognition sequence may be sortase or Butelase 1.

[0012] In some embodiments, the extracellular protein or fragment thereof is linked to the heterologous antigenic polypeptide via at least one covalent non-peptide bond. In some embodiments, the at least one non-peptide bond may be formed via azide-alkyne cycloaddition.

[0013] In some embodiments, the extracellular protein or fragment thereof and the heterologous antigenic polypeptide may form a fusion protein.

[0014] In some embodiments, the antigenic molecule comprises a lipid or polysaccharide. In some embodiments, a first affinity sequence may be fused to the extracellular protein or fragment thereof, a second affinity sequence may be linked to the lipid or polysaccharide and the extracellular protein or fragment thereof may be linked to the heterologous antigenic molecule via affinity or covalent linkage of the first and second affinity sequences.

[0015] In some embodiments, the first affinity tag and the second affinity tag are a pair selected from the following list of pairs: a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and a DogTag sequence; a sequence comprising residues 565-587 of Clostridium perfiingens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; a HaloTag sequence and a haloalkane moiety; a cysteine residue and a maleimide moiety, a free amine and an N-hydroxy succinimide ester.

[0016] In some embodiments, the commensal bacterium comprises a nucleic acid encoding the fusion protein and the fusion protein is expressed in the commensal bacterium. In some embodiments, the commensal bacterium does not comprise a nucleic acid encoding the fusion protein.

[0017] In some embodiments, the heterologous antigenic polypeptide may be selected from a cancer antigen, a viral antigen, a bacterial antigen, a fungal antigen and an autoimmune antigen.

[0018] In some embodiments, the commensal bacterium is selected from the group consisting of Staphylococcus sp. (for example Staphylococcus epidermidis), Streptococcus sp., Lactobacillus spp., Micrococcus sp., Ruminococcus sp., Eubacterium spp., Dorea sp., Subdoligranulum sp., Roseburia sp., Blautia sp., Faecalibacterium sp., Corynebacterium sp., Cutibacterium sp. And Clostridium sp. In some embodiments, the commensal bacterium is selected from the groupconsisting of Neisseria sp., Veillonella sp., Enhydrobacter sp., Akkermansia sp., Bacteroides sp., Parabacteroides sp., and. Alcaligenes sp.

[0019] In some embodiments, the bacterium may not be genetically engineered and / or the bacterium does not comprise a polynucleotide encoding the antigenic polypeptide.

[0020] Also provided is a commensal bacterium comprising on the surface of the bacterium, an extracellular protein or a fragment thereof linked to a lipid nanoparticle containing an antigenic molecule or a polynucleotide encoding an antigenic polypeptide. In some embodiments, the polynucleotide may be an RNA. In some embodiments, the RNA may be a messenger RNA.

[0021] In some embodiments, one or more first affinity sequence is fused to the extracellular protein or fragment thereof, a second affinity sequence is linked to the lipid nanoparticle, and the extracellular protein or fragment thereof is linked to the lipid nanoparticle via affinity or covalent linkage of the first and second affinity sequences. In some embodiments, the first affinity tag and the second affinity tag are a pair selected from the following list of pairs: a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and aDogTag sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; a HaloTag sequence and a haloalkane moiety; a cysteine residue and a maleimide moiety, a free amine and an N- hydroxysuccinimide ester.

[0022] Also provided is a commensal bacterium non-covalently coated on the surface of the bacterium with an antigenic molecule or immunomodulatory molecule or a nanoparticle containing (i) an antigenic molecule or (ii) a polynucleotide encoding an antigenic polypeptide. In some embodiments, the antigenic molecule or nanoparticle is coated non-covalently on the bacterial cell surface by non-specific electrostatic interactions.

[0023] Also provided is a method of inducing an immune response to an antigen in a mammal. In some embodiments, the method comprises contacting the skin or a mucosal surface of the mammal with a bacterium as described herein, in an amount sufficient to induce an immune response to the antigenic polypeptide. In some embodiments, the immune response may comprise a B-cell response, a T-cell response or both a B-cell response and a T-cell response.

[0024] Also provided herein is a method of making a bacterium as described herein. In some embodiments, the method comprises providing a commensal bacterium expressing an extracellular protein or a fragment thereof on the surface of the bacterium and linking the antigenic molecule or immunomodulatory molecule to the extracellular protein fragment thereof.

[0025] In some embodiments, the antigenic molecule is an antigenic polypeptide and the extracellular protein on the surface of the bacterium comprises a transpeptidase recognition sequence. In some embodiments, the linking of the antigenic polypeptide and the extracellular protein comprises contacting the surface of the gram-positive bacterium with (i) the antigenic polypeptide, wherein the antigenic polypeptide comprises an amino terminal pentaglycine and (ii) transpeptidase, such that the transpeptidase cleaves the transpeptidase recognition sequence and links the antigenic polypeptide to the extracellular polypeptide or fragment thereof.

[0026] In some embodiments, the method comprises feeding the bacterium a source of azide- containing amino acids or alkyne-containing amino acids such that the azide-containing or alkyne-containing amino acids by the bacterium. In some embodiments, the antigenic molecule comprises an alkyne or azide moiety. In some embodiments, the linking of the extracellular protein to the antigenic molecule comprises inducing an azide-alkyne cycloaddition reaction to link the extracellular protein to the antigenic molecule.

[0027] In some embodiments, the providing a commensal bacterium expressing an extracellular protein or a fragment thereof on the surface of the bacterium comprises expressing in the commensal bacterium the extracellular protein or fragment thereof linked to a first affinity sequence. In some embodiments, the linking of the antigenic molecule or immunomodulatory molecule to the extracellular protein fragment thereof comprises contacting the extracellular protein or fragment thereof linked to a first affinity sequence, to a second affinity sequence that is fused to the heterologous antigenic polypeptide wherein affinity or covalent linkage of the first and second affinity sequences occurs. In some embodiments, the first affinity tag and the second affinity tag may be a pair selected from the following list of pairs: a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and a DogTag sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; a HaloTag sequence and ahaloalkane moiety; a cysteine residue and a mal eimide moiety, a free amine and an N- hydroxysuccinimide ester.

[0028] In some embodiments, the method may comprise producing in a first cell a recombinant fusion protein comprising (i) an extracellular protein or fragment thereof and (ii) the antigenic polypeptide; isolating the fusion protein from the first cell; contacting the isolated fusion protein to a second cell wherein the second cell is a gram-positive bacterium expressing the extracellular protein on the surface of the bacterium; and linking the fusion protein to the extracellular protein on the surface of the bacterium. In some embodiments, the first cell may be a bacterial cell (for example E. coll) or a mammalian cell.

[0029] Also provided herein is a fusion protein comprising (i) a bacterial extracellular protein selected from the group consisting of Accumulation-associated protein (Aap), peptidoglycan, an M protein, LysM and a peptidoglycan-binding protein or a fragment thereof and (ii) a heterologous antigenic polypeptide, immunomodulatory polypeptide, or an amino terminal pentaglycine or a transpeptidase recognition sequence or one or more affinity sequences. In some embodiments, the affinity sequence is a SpyCatcher sequence a SnoopCatcher sequence; a DogCatcher sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147; a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence; a biotin moiety; a HaloTag sequence; a haloalkane moiety; a cysteine residue; a maleimide moiety, a free amine; or an N-hydroxy succinimide ester.

[0030] In some embodiments, the fusion polypeptide comprises an amino terminal pentaglycine or a transpeptidase recognition sequence. In some embodiments, the fragment comprises at least 25, 50, 75, 100, 150 or 200 amino acids of the extracellular protein. In some embodiments, the heterologous antigenic polypeptide is selected from a cancer antigen, a viral antigen, a bacterial antigen a fungal antigen and a self-antigen.

[0031] Also provided is a nucleic acid encoding a fusion protein as described herein.

[0032] Also provided is an expression cassette comprising a promoter operably linked to the nucleic acid as described herein.

[0033] Also provided is a cell comprising the expression cassette as described herein.

[0034] Also provided herein is a commensal bacterium comprising on the surface of the bacterium an extracellular protein selected from the group consisting of Accumulation-associated protein (Aap), peptidoglycan, an M protein, Lysin Motif (LysM)-containing protein and a peptidoglycan-binding protein, or a fragment thereof, linked to a heterologous antigenic molecule or immunomodulatory molecule, wherein the heterologous antigenic molecule or immunomodulatory molecule is linked by any one of: (a) Heterologous expression, or (b) Chemical labeling.

[0035] In some embodiments, the chemical labeling linking the extracellular protein to the heterologous antigenic molecule comprises a first affinity tag coupled to the extracellular protein and a second affinity tag coupled to the heterologous antigenic molecule or immunomodulatory molecule.

[0036] In some embodiments, the chemical labeling linking the extracellular protein to the heterologous antigenic molecule is selected from the group consisting of chemical labeling with a genetic carrier, chemical labeling using a transpeptidase, and chemical labeling via covalent attachment.

[0037] In some embodiments, chemical labeling linking the extracellular protein to the heterologous antigenic molecule using the transpeptidase comprises the transpeptidase recognizing a c-terminal transpeptidase recognition sequence.

[0038] In some embodiments, the transpeptidase comprises sortase. In some embodiments, the c- terminal transpeptidase recognition sequence comprises an amino acid sequence motif of: (a) LPXTG (SEQ ID NO: 79); (b) LPXTA (SEQ ID NO: 98); or (c) LAXTG (SEQ ID NO: 90).

[0039] In some embodiments, the chemical labeling linking the extracellular protein to the heterologous antigenic molecule via covalent attachment comprises a click chemistry reaction or a free amine and N-hydroxysuccinimide ester reaction.

[0040] In some embodiments, the extracellular protein comprises a non-naturally-occurring amino acid comprising an alkyne moiety and the heterologous antigenic molecule or immunomodulatory molecule comprises an azide moiety, wherein the alkyne moiety and the azide moiety participate in the click chemistry reaction to link the extracellular protein to the heterologous antigenic molecule.

[0041] In some embodiments, the extracellular protein comprises a non-naturally-occurring amino acid comprising an azide moiety and the heterologous antigenic molecule or immunomodulatory molecule comprises an alkyne moiety, wherein the alkyne moiety and the azide moiety participate in the click chemistry reaction to link the extracellular protein to the heterologous antigenic molecule.

[0042] In some embodiments, the first affinity tag comprises a biotin moiety and the second affinity tag comprises a streptavidin sequence. In some embodiments, the first affinity tag comprises a SpyCatcher sequence, and the second affinity tag comprises a SpyTag sequence.DEFINITIONS

[0043] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0044] The term “a” and “an” as used herein mean “one or more” and include the plural unless the context is appropriate.

[0045] As used herein, the term “commensal” means a relationship between two or more organisms. In certain embodiments, commensal refers to a relationship between two or more organisms of different species in which one generally derives some benefit while another is generally unharmed. In certain embodiments, a commensal refers to a relationship between two or more organisms of different species in which one organism derives a benefit from another organism. In certain embodiments, a commensal refers to a relationship between two or more organisms of different species in which a first organism derives a benefit from a second organism and the second organism is unharmed. In certain embodiments, a commensal refers to a symbiotic relationship between two or more organisms. In certain embodiments, a commensal refers to a symbiotic relationship between two or more organisms wherein a first organism derives a benefit from a second organism and the second organism is unharmed. In certain embodiments, a commensal microbe may be one that is normally present as a non-pathogenic member of a host gut microbiome, a host skin microbiome, a host mucosal microbiome, or other host niche microbiome.

[0046] As used herein, the term “bacteria” includes both singular and plural forms, such as a bacterium (single bacterial cell) and bacteria (plural), and genetically modified (recombinant) bacterial cells, bacteria and bacterial strains thereof.

[0047] As used herein, the terms “commensal bacteria” and “commensal microbe” are used interchangeably herein and refer to a bacterium, bacteria (singular or plural), bacterial cell or bacterial strain that is commensal with an animal host or animal cell(s). In certain embodiments, commensal bacteria refers to a bacterium, bacteria (singular or plural), bacterial cell or bacterial strain that is commensal with a vertebrate host or vertebrate cells. In certain embodiments, commensal bacteria refers to a bacterium, bacteria (singular or plural), bacterial cell or bacterial strain that is commensal with a mammalian host or mammalian cells. In certain embodiments, commensal bacteria refers to a bacterium, bacteria (singular or plural), bacterial cell or bacterial strain that is commensal with a human host. In certain embodiments, commensal bacteria refers to a bacterium, bacteria (singular or plural), bacterial cell or bacterial strain that is commensal with human cells. In certain embodiments, the commensal bacterial act on the host’s immune system. In certain embodiments and as understood by one of ordinary skill in the art, most commensal bacteria are typically symbiotic, but a commensal strain can become pathogenic or cause pathology under certain conditions, such as host immunodeficiency, microbial dysbiosis or intestinal barrier impairment. In certain embodiments, for example, a commensal bacteria is present as a non-pathogenic member of a host gut microbiome, a host skin microbiome, a host mucosal microbiome, or other host niche microbiome.

[0048] As used herein, the terms “colonization,” “colonized,” or “colonize” refers to the occupation of a microbe, e.g., a live, recombinant, commensal bacteria, in a niche of a host. In certain embodiments, colonization can be persistent, e.g., lasting over 60 days, or transient, e.g., lasting between one to 60 days.

[0049] As used herein, the terms “heterologous” or “non-native” refer to a molecule (e.g., peptide or protein) that is not normally or naturally produced or expressed by a cell or organism.

[0050] The term “antigen” refers to a molecule (e.g., peptide or protein or non-protein such as a carbohydrate or lipid) or immunologically active fragment thereof that is capable of eliciting an immune response, for example, a B-cell or T-cell response, or both. Peptide antigens are typically presented by an APC to an immune cell, such as a T lymphocyte (also called a T cell).

[0051] The terms “heterologous antigen,” or, in reference to proteins or peptides, “non-native antigen”, refer to a peptide, protein, or antigen that is not normally expressed by a cell or organism. In certain embodiments, term includes antigens, or fragments thereof, that bind to a T cell receptor and induce an immune response. In certain embodiments, for example, protein or peptide antigens are digested by APCs into short peptides that are expressed on the cell surface of an APC in the context of a major histocompatibility complex (MHC) class I or MHC-II molecule. In certain embodiments, the term antigen includes the peptides presented by an APC and recognized by a T cell receptor. In certain embodiments, heterologous antigens or nonnative antigens may be host-derived antigens, or non-host derived antigens.

[0052] The term “fusion peptide” and “fusion protein” are used interchangeably herein and refer to a recombinant protein comprising two or more proteins or peptides expressed in the same amino acid chain in sequence. In certain embodiments, the two or more protein or peptide nucleic acid coding sequences can be expressed sequentially in a single open reading frame of a vector or expression plasmid. In certain embodiments, the resulting peptide or protein thus comprises a single amino acid chain with two or more proteins of interest connected via end-to- end fusion at the N- or C-termini.

[0053] In reference to microbial niches in a host, the term “native” refers to an environment in or on a host in which a commensal microorganism or host immune cell is naturally present under normal, non-pathogenic conditions.

[0054] In reference to proteins expressed by a microorganism, e.g., a bacterium, the term “native” refers to a protein, or portion thereof, that is normally expressed and present in a wildtype microorganism in nature.

[0055] The term “effective amount,” or “therapeutically effective amount,” refers to an amount of a composition sufficient to prevent, decrease or eliminate one or more symptoms of a medical condition or disease when administered to a subject in need of treatment.

[0056] As used herein, the term “operably linked” refers to a functional linkage between one or more nucleic acid sequences, such as between a regulatory or promoter sequence and a coding region sequence, where transcription of the coding region sequence is positively or negatively regulated by the linked regulatory sequence.

[0057] As used herein, the terms “engineered”, “recombinant” and “modified” are used interchangeably and refer to an organism, microorganism, cell, or bacteria that does not exist innature. In certain embodiments, the engineered bacteria is an engineered commensal bacteria (also referred to as “engineered commensal” or “engineered commensals” herein).

[0058] As used herein, “animal” refers to an animal or an animal cell. In certain embodiments, an animal is a mammal (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like). In certain embodiments, an animal is a human. In certain embodiments, an animal is an organism to be treated or treated with a recombinant commensal microbe. In certain embodiments, the commensal microbe is an engineered bacterium or a surface-labeled bacterium.

[0059] As used herein, “host” refers to a non-microbial organism in or on which a commensal microorganism colonizes. In certain embodiments, “host” refers to a non-microbial organism in or on which commensal bacteria colonizes. In certain embodiments, the host is an animal. In certain embodiments, the host is a mammal. In certain embodiments, the host is a human.

[0060] As used herein, the terms “subject” or “patient” are used interchangeably, and refer to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the subject is a human. In certain embodiments, a subject refers to an organism to which a modified bacteria as described herein is administered. In certain embodiments, a subject has an autoimmune or proliferative disease, disorder or condition.

[0061] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline (PBS) solution, water, emulsions (e.g., such as oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Martin, Remington’s Pharmaceutical Sciences, 15thEd. Mack Publ. Co., Easton, PA

[1975] ,

[0062] As used herein, “pharmaceutical formulation” and “pharmaceutical composition are used interchangeably and refer to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0063] The terms “identical,” “substantially identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of nucleotides or amino acid residues that are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using the BLAST and PSI-BLAST algorithms, which are described in Altschul et al. (J. Mol. Biol. 215:403-10, 1990), and Altschul et al. (Nucleic Acids Res., 25:3389-3402, 1997), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see the internet at ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) andN (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992).

[0064] All nucleic acid and amino acid sequences disclosed herein can include sequences that have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence or sequence identifier recited herein. All nucleic acid and amino acid sequences disclosed herein can include sequences that have about 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence or sequence identifier recited herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIGS. 1A-1E demonstrates that colonization with Staphylococcus epidermidis induces a potent B cell response. FIG. 1 A shows a graph of mouse antibody titers of specific pathogen-free (SPF) mice colonized for 6 weeks with S. epidermidis strain LM087 or LM088. The antibody titers were measured by ELISA and three independent experiments were pooled. FIGS. 1B-1D depict graphs of mouse antibody titers of SPF mice colonized for 2-9 weeks with LM088. Two independent experiments were pooled. FIG. IE depicts a graph measuring the frequency of germinal center B cells among live B cells (Live+CD19+CD4-) in the draining cervical lymph nodes of one representative mouse / group (left) and all mice analyzed (right).

[0066] FIGS. 2A-2E demonstrates that the predominant target of the antibody response is Accumulation-associated protein (Aap). FIG. 2A depicts a schematic of the S. epidermidis cell surface mutants used in FIG. 2B. FIG. 2B depicts a graph of a bacterial ELISA using the serum of mice colonized with LM087 for 6 weeks against a panel of cell surface mutants. Three independent experiments were pool. FIG. 2C depicts a graph of a bacterial ELISA using the serum of mice colonized with LM088 for 6 weeks. The ELISA was performed with LM088 (left), LMO88 tsaap (middle) or LM088 Osaap expressing Aap recombinantly (right). FIG. 2D depicts a schematic of Aap in LMO88. FIG. 2E depicts a graph of the percent of germinal center (GC) B cells among the live B cells (Live+CD19+CD4 ) in the draining cervical lymph nodes (middle) and the colony forming units (CFUs) recovered from all mice analyzed (right), where SPF mice were colonized for 6 weeks with LM088 or LM088Aaap.

[0067] FIGS. 3A-3G demonstrate that the B cell response can be redirected against a non-native immunogen. FIG. 3 A depicts a schematic of the approach used to redirect the B cell response towards a non-native antigen of interest. FIG. 3B depicts a graph of antibody titers against LM088, Aaap and Aaap expressing plasmid-borne Aap (Aaap+Aap) in mice colonized for 5 weeks with LM087 harboring an empty vector (LM087+empty vector) or expressing Aap on a plasmid (LM087+Aap). FIG. 3C depicts a strategy used to express a non-native immunogen in S. epidermidis: an engineered version of Aap is expressed in which the parallel -helix domain is replaced with the antigen. For FIG. 3D, SPF mice were colonized for 6 weeks with S.epidermidis LM088 Aaap engineered to express tetanus toxin fragment C (TTFC). TTFC was either targeted for secretion (sTTFC) or attached to the cell-wall by fusion to Aap (wTTFC). FIG. 3D depicts a schematic (left) of the TTFC constructs (e.g., LM088 tsaap engineered to express tetanus toxin fragment C (TTFC), a model immunogen, a graph of measured TTFC specific antibodies in serum (middle) or in nasal washes (right). FIG. 3E depicts a graph of IgG serum titers specific for diphtheria toxin (DT) in mice colonized with LM088 aap + Aap-TTFC (wTTFC) or + Aap fused to a catalytic mutant version of DT, Aap-DT (wDT) for 6 weeks. FIG. 3F depicts a graph of percent survival of mice colonized with either wTTFC or wDT that were challenged with a lethal dose of tetanus toxin (150ng / kg) 6 weeks later. FIG. 3G depicts a percent survival of mice injected with a lethal dose of tetanus toxin (1 lOng / kg) preincubated with the serum of mice colonized with either wTTFC or wDT.

[0068] FIGS. 4A-4E demonstrate that generation of immunity can accomplished using enzymatic conjugation. FIG. 4A depicts a schematic representing the difference in approached developed to elicit an antibody response against a non-native antigen using engineered S. epidermidis. FIG. 4B depicts a schematic representing the attachment of a non-native antigen to a strain expressing an Aap-SpyCatcher (Aap-sc) fusion protein. FIG. 4C depicts a graph of a histogram of S. epidermidis LM087 engineered to express either a fusion protein between Aap and SpyCatcher (Aap-sc, right) or a catalytically dead version of SpyCatcher (Aap-Sc*, left). Both strains were incubated with 0.1 mg / ml of sGFP-SpyTag for 2 min and visualized by flow cytometry. FIG. 4D depicts schematics of the experiments of FIG. 4E. Mice were colonized with either Aap-sc conjugated with tetanus toxin fragment C-SpyTag003 (Aap-sc-TTFC), the unconjugated strain (Aap-sc) or wTTFC). FIG. 4E depicts graphs (1, 2, 3, 4) depicts antibody titers specific for TTFC measured in the serum 6 weeks later. In parallel, another group of mice were immunized intramuscularly with SpyCatcher-based nanoparticles (mi3) conjugated with TTFC and antibody titers were measured at 6 weeks.

[0069] FIG. 5 provides a summary table describing different methods of attaching an antigen or immunomodulatory molecule to the cell surface of commensal bacteria that can be used to modulate an immune system.

[0070] FIG. 6 depicts a schematic of construct design for fusion proteins to lysM useful for heterologous expression of an antigen. TTFC=tetanus toxin fragment C, aa=amino acids.

[0071] FIGS. 6B-6C depicts a schematic of construct design for fusion proteins to M6 (FIG. 6B, top) and Aap (FIG. 6C, bottom), useful for heterologous expression of an antigen. SrtA= sortase A, TTFC=tetanus toxin fragment C, aa=amino acids, HA2= hemagglutinin fragment 2.

[0072] FIG. 7A depicts a schematic of an experiment to colonize mice with engineered S. epidermidis strains. FIG. 7B depicts a schematic of a bacterial surface of a S. epidermidis strain LM088 Aaap expressing the indicated antigen construct heterologously on a plasmid as seen in FIGS. 6A-6C.

[0073] FIGS. 7C-7D depicts graphs of antibody titers and absorbance of undiluted nasal washes against TTFC as measured by ELISA (enzyme-linked immunosorbent assay) of mice colonized with engineered S. epidermidis strains.

[0074] FIG. 8 depicts a schematic of step 1 of a method of using sortase A (SrtA) to attach antigen to a bacteria cell wall protein.

[0075] FIG. 9 depicts a schematic of the method of using sortase A to attach an antigen to a bacterial cell wall protein.

[0076] FIG. 10 depicts a histogram of sortase-based labeling in live S. epidermidis, to demonstrate that sortase-based labeling enables simple attachment of peptides (T cell epitopes) to the surface of live S. epidermidis.

[0077] FIG. 11 A depicts photographic copies of plates containing colonies of S. epidermidis labeled with peptides using sortase, demonstrating that peptide labeled S. epidermidis using sortase do not display any fitness defect. FIG. 1 IB depicts a graph of the median fluorescence intensity of fluorescently labeled S. epidermidis using sortase, demonstrating that labeling of bacteria is rapid and efficient.

[0078] FIG. 12A depicts histograms of in vitro labeling of S. epidermidis with a fluorescent peptide (e.g., either Cy5-LPETGG peptide or a scrambled version of the peptide Cy5-GEGTLP) with either 0 uM (top) or 1 uM (bottom) concentration of sortase A (SrtA).

[0079] FIG. 12B depicts histograms of in vitro labeling of S. epidermidis with a fluorescent peptide (e.g., either Cy5-LPETGG peptide or a scrambled version of the peptide Cy5-GEGTLP) with either 50 uM (top) or 100 uM (bottom) concentration of sortase A (SrtA).

[0080] FIGS. 13-15 depicts graphs of in vitro labeling of different gram-positive bacteria with a fluorescent peptide (e.g., either 100 uM Cy5-LPETGG peptide or a 100 uM scrambled version of the peptide Cy5-GEGTLP) and 50 uM concentration of sortase A (SrtA).

[0081] FIG. 16 depicts a graph of the median fluorescence of an intractable skin colonist (C. acnes) labeled with Cy5-LPETGG peptide or a scrambled version of the peptide Cy5-GEGTLP using Sortase A.

[0082] FIG. 17A-17D depict dot plots of a panel of S. epidermidis isolates labeled with an OT1 peptide using sortase to look at inducement of proliferation of antigen-specific CD8 T cells in vitro. This suggests that S. epidermidis labeled with an OT1 peptide using sortase can prime and induce proliferation of antigen-specific CD8 T cells in vitro.

[0083] FIG. 18A depicts a schematic of an in vivo colonization assay in mice with different bacteria labeled with an OT1 peptide using sortase.

[0084] FIG. 18B-18C depict a graph and dot plots of the results of the assay in FIG. 18A.

[0085] FIG. 19A-19D depict dot plots of a panel of S. epidermidis isolates labeled with an OT2 peptide using sortase to look at inducement of proliferation of antigen-specific CD4 T cells in vitro. This suggest that S. epidermidis labeled with an OT2 peptide using sortase can prime and induce proliferation of antigen-specific CD4 T cells in vitro.

[0086] FIG. 20A depicts a schematic of an in vivo colonization assay in mice with different bacteria labeled with an OT2 peptide using sortase.

[0087] FIG. 20B-20C depict a graph and dot plots of the results of the assay in FIG. 20A.

[0088] FIGS. 21A-21I depicts histograms of sortase-based labeling of T cell epitopes to a variety of species of anaerobic gut commensal bacteria.

[0089] FIGS. 22A-22B depicts histograms of sortase-based labeling of T cell epitopes to different species of anaerobic gut commensal bacteria.

[0090] FIG. 23A depicts a schematic of an in vivo activation of antigen-specific T cells in mice colonized with sortase-labelled S', epidermidis.

[0091] FIGS. 23B depicts graphs of in vivo activation of antigen-specific T cells using sortase- labelled S. epidermidis, of the assay in FIG. 23 A.

[0092] FIG. 23C depicts dot plots of the in vivo activation of antigen-specific T cells in FIG. 23B.

[0093] FIG. 23D depicts a graph of the percent of proliferating cells that contain the OT1 peptide, of the assay in FIG. 23 A.

[0094] FIG. 23E depicts a graph of the percent of proliferating cells that contain the OT2 peptide, of the assay of FIG. 23C.

[0095] FIGS. 24A-24D depicts the results of in vitro optimization of S. epidermidis Aap- SpyCatcher labeling. FIG. 24A depicts a schematic of the different strains tested. FIG.24Bdepicts a histogram of labeling efficiency as measured by flow cytometry of strains harboring an Aap-SpyCatcher construct (original=blue or 003=purple) or a catalytic mutant version (EQ mutant=light grey or 003 EQ mutant=dark grey) were labeled with O.lmg / ml of SpyTag-GFP for 15min at room temperature. FIG. 24C depicts off-set histograms showing the labeling efficiency of SpyCatcher EQ mutant (top) or SpyCatcher003 (bottom) with varying concentrations of SpyTag-GFP for different times (2, 5, 15, 60 min) as measured by flow cytometry. FIG. 24D depicts off-set histograms showing the labeling efficiency of the SpyCatcher original strain with varying concentrations of SpyTag-GFP for different times (2, 5, 15, 60 min) as measured by flow cytometry.

[0096] FIG. 25A depicts a schematic of an in vivo TTFC-specific immunity generation assay in mice using a Aap-SpyCatcher S. epidermdidis strain.

[0097] FIG. 25B depicts graphs of in vivo generation of TTFC-specific immunity in mice colonized with a S. epidermidis strain expressing an Aap-SpyCatcher S. epidermdidis labeled or not with TTFC-SpyTag using the indicated reaction conditions.

[0098] FIG. 26A depicts a schematic of the process for S. epidermidis to incorporate azide- containing non-canonical amino acids into cell surface proteins, useful in a click chemistry reaction.

[0099] FIGS. 26B-26C depict histograms of labeling efficiency of S. epidermidis strains (LM087 and LM088) using click chemistry.

[0100] FIG. 27depicts graphs of flow cytometry data demonstrating that S. epidermidis (S. epi) can incorporate azide-containing noncanonical amino acids L-homoalanine. (top) and 3-azido-D- alanine (bottom) into the bacterial cell surface via feeding, and that the azide group is accessible for a strain promoted azide-alkyne cycloaddition reaction to occur with the alkyne group of dibenzocyclooctyne (DBCO).

[0101] FIGS. 28A-28J demonstrates that metabolic labeling can be used to label S. epidermidis with OT1 and OT2 antigens that can be used to prime T cells in vivo. FIG. 28A depicts a schematic of the in vivo assay. FIGS. 28B-28E depict dot plots of primed T cells using S. epidermis labeled with an OT1 antigen. FIG. 28F depicts a graph of the % proliferation of FIGS. 28B-28E. FIGS. 28G-28I depict dot plots of primed T cells using S epidermis labeled with an OT2 antigen. FIG. 28 J depicts a graph of the % proliferation of FIGS. 28G-28I.

[0102] FIG. 29A depicts a schematic of the process to label S. epidermidis with antigens using an affinity tag of a streptavidin sequence and a biotin moiety. FIG. 29B depicts a histogram of labeling efficiency of a S. epidermidis strain (LM088) with biotin-PEG-NHS ester.

[0103] FIG. 30 depicts a histogram of labeling efficiency of a S. epidermidis strain (LM087) labeled with either proteins (SA) or small molecules (Cy5) by NHS esters. Labeling is compared to sortase labeling with peptides.

[0104] FIGS. 31A-31L depict histograms of labeling efficiency of different types of bacteria (Gram positive, gram negative, or anerobic) with NHS-biotin or streptavidin.

[0105] FIGS. 32A-32B depict histograms of labeling efficiency of S. epidermidis labeled with NHS-biotin followed by streptavidin (SA)-fusion protein, then anti-FLAG for OVA and TTFC. SA-fusion proteins are GFP, TTFC-FLAG, or OVA-FLAG.

[0106] FIG. 33 depicts a graph of antibody titers in mice colonized for 6 weeks with NHS ester labeled S. epidermidis, demonstrating that S. epidermidis labeled using NHS esters elicits an immune response in vivo.

[0107] FIGS. 34A-34B depict histograms of labeling efficiency of a S. epidermidis strain (LMO88) with amine reactive crosslinkers that can be used to attach clickable linkers to label bacterial surface with a small molecule (Cy5) (FIG. 34A) or intact protein (FIG. 34B).

[0108] FIG. 35 depicts a schematic of methods to increase valency through conjugating Aap to a nanoparticle.

[0109] FIG. 36 depicts histograms of labeling efficiencies of S. epidermidis strain (LM087 WT, bottom; or LM087-Aap-SpyTag003, top) with mi3-antigen particles.

[0110] FIGS. 37A-37C demonstrate that the incorporation of a nanoparticle within the labeling process can increase antibody titers in serum. FIG. 37A depicts a schematic of an in vivo assay to induce an immune response to a viral antigen. FIGS. 37B-37C depict graphs of neutralization antibody titers from the assay of FIG. 37 A.

[0111] FIGS. 38A-38I depicts schematics of exemplary construct designs to redirect the B cell response to S. epidermidis. Schematics include (FIG.38B) secreted tetanus toxin fragment C (sTTFC), (FIG. 38C) Protein A-TTFC, (FIG. 38D) M6-TTFC, (FIG. 38E) Aap-TTFC (wTTFC), (FIG. 38F) LysM-TTFC, (FIG. 38G) Aap-TTFC short amino acid repeats, (FIG. 38H) Aap-2x TTFC and (FIG. 381) Aap-TTFC 2x B domain.DETAILED DESCRIPTION OF THE INVENTION

[0112] Disclosed herein are compositions, methods of attaching antigens to cell surface proteins of engineered colonists and methods of use that demonstrate engineered colonists elicit potent neutralizing antibody responses by expressing recombinant immunogens that are conjugated with chimeric proteins. These engineered colonists may be directed against pathogens as a novel form of topical vaccination

[0113] As will be described in various embodiments herein, the present disclosure relates to linking antigens to commensal bacterial surface proteins, which results in a surprisingly high immune response triggered when the bacteria are applied to an epithelial surface of a mammal. In some embodiments, an antigenic polypeptide or immunomodulatory molecule may be expressed from bacteria as a fusion protein or chimeric protein with a bacterial surface protein.In other embodiments, the antigenic molecule or immunomodulatory molecule may be linked to a bacterial surface protein in other ways, providing flexibility for linking a desired antigen to the bacterial surface protein. In some embodiments, the bacteria may be genetically engineered while in other embodiments, the bacteria need not be or will not be genetically engineered. In some embodiments, exemplary bacterial surface proteins used herein may be for example extracellular proteins. In other embodiments, antigens may be linked to the commensal bacterial surface protein using a variety of different methods including enzymatic reactions (e.g., using transpeptidase, free amine and anN-hydroxysuccinimide ester, a streptavidin sequence and a biotin moiety, and affinity tags) and chemical reactions (e.g., using click chemistry), as will be described in more detail herein.Bacterial surface proteins

[0114] The methods and compositions described herein can allow for linkage of an antigen or immunomodulatory molecule on any bacterial surface protein or other bacterial wall molecule as desired. A bacterial surface protein is a protein that accumulates on the surface of bacteria, and for example is anchored in the gram-positive cell wall and / or bacterial cell membrane.Exemplary surface proteins are extracellular proteins. In some embodiments, the extracellular protein or fragment thereof comprises a transpeptidase (e.g., sortase) recognition sequence, for example in embodiments in which a transpeptidase is used to link the antigenic or immunomodulatory molecule. Fragments of extracellular proteins can be for example, at least 5, 14, 25, 50, 75, 100, 150 or 200 amino acids of the extracellular protein. In some cases, the antigenic polypeptide or immunomodulatory polypeptide is inserted within the extracellular protein such that a fragment of the extracellular protein is linked to each end of the antigenic polypeptide or immunomodulatory polypeptide. Optionally the extracellular protein or fragment thereof can be further fused directly or indirectly to a OTII epitope (T helper epitope to ensure antibody maturation), for example SEQ ID NO: 19 or a substantially identical sequence thereof.

[0115] In some embodiments, the bacterial surface protein is Accumulation-associated protein (Aap) or a fragment thereof. Exemplary Aaps can include, for example, AAP from Staphylococcus epidermidis. Aap can comprise an N-terminal A-repeat region with 11 degenerate 16 amino acid repeats, a putative globular domain (“a / p”, also referred to here and elsewhere as a “L-type lectin domain”), and a B -repeat region with a variable number (5 to 17)of nearly identical 128 amino acid repeats terminating in a conserved “half repeat” motif. For example, as described in Rohde, H. et al., Biomaterials 28:1711-20 (2007). The repeated sequence element within the B-repeat region has been described as a G5 domain, found in grampositive surface proteins, Zn2+ metalloproteases, and other bacterial virulence factors. The B- repeat region in Aap is followed by a collagen-like repeat and an amino acid sequence of LPXTG (SEQ ID NO: 79), a sortase recognition sequence and cell wall anchor sequence. A similar domain arrangement exists in the S. aureus homolog as an amino acid sequence of SasG as seen in Corrigan, R.M. et al., Microbiology 153:2435-46 (2007) or Shaeffer et al., Infection and Immunity Vol. 83, No. 1 (2014). The antigen polypeptide or immunomodulatory polypeptide can be linked to a fragment of the Aap polypeptide. For example, the Aap fragment can comprise one or more of a wall-attachment sequence, one or more (e.g., 2, 3, 4, 5, 6, or more) B-domain repeats, one or more L-type lectin domain (e.g., 2, 3, 4, or more) and one or more amino acid repeat as well as a signal sequence (e.g., SEQ ID NOS: 2, or 9 or an amino acid having at least an 80% sequence identity thereto ). In some embodiments, the fragment comprises a wall-attachment sequence, one or more (e.g., 2, 3, 4, 5, 6, or more) B-domain repeats (see, e.g., Bateman, et. Al., Bioinformatics, Volume 21, Issue 8, April 2005, Pages 1301— 1303, describing B-domains and their G5 portions), linked to the antigen polypeptide or immunomodulatory polypeptide, whose amino terminus can be further linked (e.g., as a polypeptide fusion) with one or more amino acid repeat sequences and a signal sequence. In these embodiments, the antigen polypeptide or immunomodulatory polypeptide is fused to a first fragment of Aap on one end and a different fragment of Aap on its other end. An exemplary Aap-antigen fusion is depicted as SEQ ID NO:1 and SEQ ID NO: 8, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the Aap (optionally derived from S. epi strain LM088) fragment is simply the Aap in which the N-terminal lectin domains have been removed and replaced with one or more antigenic or immunomodulatory polypeptide sequences. In some embodiments, the Aap fragment comprises its amino acid repeat (e.g., SEQ ID NO: 3, 10 or 27, or an amino acid sequence having at least 80% sequence identity thereto), one or more lectin domains (e.g., SEQ ID NO: 4, or an amino acid sequence having at least 80% sequence identity thereto), one or more B domains (e.g., any sequence of SEQ ID NO: 5, 13, 29, or an amino acid sequence having at least 80% sequence identity thereto, or one or more G5 or E spacer subsequences). In some embodiments, the Aap or a fragment thereof comprises atranspeptidase (e.g., sortase) recognition sequence, for example in embodiments in which a transpeptidase is used to link the antigenic or immunomodulatory molecule. As depicted in SEQ ID NOS: 1 and 8, the sortase recognition sequences may comprise an amino acid sequence of SEQ ID NO:6. In some embodiments, the Aap or a fragment thereof comprises an affinity sequence as described elsewhere herein (see, e.g., SEQ ID NO: 26 or an amino acid sequence having at least 80% sequence identity thereto. For example, in some embodiments, the Aap fragment comprises a signal sequence (e.g., SEQ ID NO: 2 or an amino acid sequence having at least 80% sequence identity thereto ), amino acid repeats (e.g., SEQ ID NO: 27 or an amino acid sequence having at least 80% sequence identity thereto and an affinity sequence as described elsewhere herein and a B-domain sequence (e.g., SEQ ID NO: 29 or an amino acid sequence having at least 80% sequence identity thereto). In some embodiments, a terminal region is included. Examples of such terminal regions include, e.g., SEQ ID NOS: 7 and 13 and 30 or an amino acid sequence having at least 80% sequence identity thereto. While some exemplary sequences provided herein comprise an HA sequence, such sequences may be omitted.

[0116] In some embodiments, the bacterial cell surface molecule may comprise a nonpolypeptide such as peptidoglycan. Peptidoglycan occurs in both gram-positive and gramnegative bacteria and has been described in many species as seen in Vollmer et al., FEMS Microbiology Reviews, Volume 32, Issue 2, March 2008, Pages 149-167. Peptidoglycan is made up of N-acetylglucosamine (GlcNAc or NAG) and N-acetylmuramic acid (MurNAc or NAM). The alternating sugars are connected by a 0-(l,4)-glycosidic bond. MurNAc can be attached to a short (e.g., 4- to 5 -residue) amino acid chain. In some embodiments, peptidoglycan or a fragment thereof comprises a transpeptidase (e.g., sortase) recognition sequence, for example in embodiments in which a transpeptidase is used to link the antigenic or immunomodulatory molecule. In some embodiments, the peptidoglycan or a fragment thereof comprises an affinity sequence as described herein.

[0117] In some embodiments, the bacterial cell surface protein is an M protein. M protein can be selected from many bacterial species, including for example, Streptococcus Protein M as described in Metzger et al., Virulence 2011 Sep-Oct;2(5):402-12. In some embodiments, the M protein or a fragment thereof comprises a transpeptidase (e.g., sortase) recognition sequence, for example in embodiments in which a transpeptidase is used to link the antigenic orimmunomodulatory molecule. In some embodiments, the M protein or a fragment thereof comprises an affinity sequence as described elsewhere herein.

[0118] In some embodiments, the bacterial cell surface protein may comprise a peptidoglycan- binding protein or a fragment thereof. In some embodiments, peptidoglycan-binding protein is peptidoglycan recognition protein 3. In some embodiments, the peptidoglycan-binding protein is a Lysin Motif (LysM)-containing protein. LysM typically ranges in length from 44 to 65 amino acid residues and binds to various types of peptidoglycan and chitin, for example in some cases the N-acetylglucosamine moiety. Most bacterial LysM-containing proteins are peptidoglycan hydrolases with various cleavage specificities as described for example in Buist et al., Mol Microbiol. 2008 May;68(4):838-47. In some embodiments, when peptidoglycan-binding protein comprise only fragments of a Lysin Motif (LysM)-containing protein, the fragments will generally comprise one, two, three or more LysM sequences. Exemplary LysM sequences can include, but are not limited to, SEQ ID NOS: 16, 17, 18 or an amino acid sequence having at least 80% sequence identity thereto. For example, SEQ ID NO: 14 comprises a signal sequence (e.g., SEQ ID NO: 15) three LysM sequences (SEQ ID NOS: 16-18) linked to an antigenic sequence. However, it can be appreciated that any antigenic or immunomodulatory amino acid sequence could be replaced with the specific antigenic sequence (SEQ ID NO: 12), shown in SEQ ID NO: 14. SEQ ID NO: 14 also comprises optional HA sequence (SEQ ID NO: 11) and an OTII (“OT2") epitope sequence (SEQ ID NO: 19). In some embodiments, the peptidoglycan- binding protein or a fragment thereof comprises an affinity sequence as described herein.

[0119] In some embodiments, the bacterial cell surface protein comprises a cell -wall attachment sequence, a coiled-coil amino acid sequence. Coiled-coil regions have been shown to enhance immune responses as shown for example in Bolken, et al., Infection and Immunity, May 2002, p. 2487-2491. In some embodiments, the bacterial cell surface protein is identical or substantially identical to SEQ ID NO:20 (optionally lacking the HA sequence therein and comprising any antigen or immunomodulatory sequence in place of the TTFC antigen (SEQ ID NO: 12). For example, the sequence can comprise a signal sequence (e.g., SEQ ID NO:21 or an amino acid sequence having 70% sequence identity thereto) and portions of an M6 sequence (e.g., SEQ ID NOS: 22, and 23, or an amino acid sequence having 70% sequence identity thereto), for example with an antigenic or immunomodulatory polypeptide sequence between the two listed M6fragments, and a proline glycine sequence (e.g., SEQ ID NO:24 or an amino acid sequence having 70% sequence identity thereto). In some embodiments, the bacterial cell surface protein as described herein, or a fragment thereof comprises an affinity sequence as also described elsewhere herein. While some exemplary sequences provided herein comprise an HA sequence, such sequences may be omitted.

[0120] In some embodiments, the bacterial cell surface protein comprises Protein A or a fragment thereof. Protein A is a protein found in the cell wall of the bacteria (for example, Staphylococcus aureus'). Exemplary Protein A sequences can include for example, SEQ ID NO:33 or 34 or an amino acid sequence having 70% sequence identity thereto. In some embodiments, both sequences are provided as a fragment of Protein A, optionally separated by a transpeptidase recognition sequence. As an example, SEQ ID NO:31 is provided, which presents a particular antigen (SEQ ID NO: 12), however it will be appreciated that other antigenic or immunomodulatory proteins could be inserted in its place. While SEQ ID NO:31 comprises an HA sequence (SEQ ID NO:11), such sequences may be omitted.

[0121] In some embodiments, a fragment of the bacterial surface protein is provided on the surface of the bacteria. The fragment can be expressed natively or heterologously from the bacteria, and the fragment can be generated by cleaving the full-length bacterial surface protein once it is anchored in the bacterial surface, or the fragment can be expressed first and then anchored in the bacterial surface. In can be appreciated that the length of the bacterial fragment can vary. In general, the fragment will include sufficient amino acid sequences to be anchored in the bacterial surface. In some embodiments, a longer fragment can be useful in that when linked to an antigen, the antigen may be better available to the immune system when the bacteria is contacted to an animal. In some embodiments the bacterial surface protein can comprise a fragment of at least 5, 14, 25, 50, 75, 100, 150, or 200 amino acids of a native or non-natural bacterial surface protein.

[0122] The bacterial surface protein, or fragment thereof, can be native or heterologous to the bacteria on which the bacterial surface protein occurs. For example, the bacterial surface protein and the bacteria on which the bacterial surface protein occurs can be from the same species or different bacterial species. In some embodiments, the heterologous surface protein can comprise addition amnio acid sequences heterologous to both the surface protein and the antigen to whichit is linked. For example, in some embodiments, the bacterial protein or fragment thereof is fused to one or two or more lectin domains, B lectin domains or other non-lectin amino acid sequences. These can be selected and introduced in the amino terminal direction from any membrane or cell-wall anchoring domain so that the resulting protein for example extends further from the bacterial surface, allowing for presentation of the antigen. The antigen can be presented at the amino terminus of the protein or as a sequence within the protein.

[0123] In some embodiments, the fusion protein further comprises a signal sequence peptide. In some embodiments, the signal sequence peptide directs tethering of the fusion protein to a cell wall of the bacterium following expression. In some embodiments, the signal sequence peptide that directs secretion comprises a tat signal sequence peptide. In some embodiments, the tat signal sequence peptide comprises an S. aureus derived signal sequence peptide. In some embodiments, the signal sequence peptide that directs secretion comprises a sec signal sequence peptide. In some embodiments, the sec signal sequence peptide comprises an S. epidermidis derived signal sequence peptide. In some embodiments, the S. epidermidis derived signal sequence peptide is derived from predicted sec-secreted S. epidermidis protein (gene locus HMPREF9993 06668).Methods of attachment of antigens to bacterial surface proteins

[0124] As noted above, the disclosure provides for bacterial surface proteins, or a fragment thereof, linked to one or more antigens or immunomodulatory molecules. The one or more antigens or immunomodulatory molecules may be linked, conjugated, attached covalently or non-covalently using any of the methods as illustrated in the table of FIG. 5. In some embodiments, the bacterial surface proteins, or a fragment thereof and the one or more antigens are produced as a recombinant fusion polypeptide, which optionally is encoding by the bacteria on which the fusion polypeptide occurs, as seen in FIG. 5, left column. In other embodiments, one of several methods can be used to link the one or more antigen to the bacterial surface proteins or a fragment, as seen in FIG. 5, right four columns.Using heterologous expression of polypeptide fusion proteins to express an antigen on bacterial cell surface

[0125] In certain embodiments, commensal bacteria can be engineered to express an extracellular protein or fragment thereof fused with an antigenic or immunomodulatory polypeptide using general molecular biology methods as described in Green, M.R. and Sambrook, J., eds., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), and Ausubel, F. M., et al. Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012), which are incorporated herein by reference.

[0126] In certain embodiments, to produce a live, recombinant, commensal bacterial strain that expresses an extracellular protein or fragment thereof fused with an antigenic or immunomodulatory polypeptide, the extracellular protein or fragment thereof fused with the antigenic or immunomodulatory polypeptide can be cloned into an expression vector.

[0127] In some embodiments, the live, recombinant commensal bacterium comprises a nonnative or heterologous nucleic acid that is used to express an extracellular protein or fragment thereof fused with heterologous polypeptide. In some embodiments, the heterologous nucleic acid may comprise an RNA that is translated to produce a heterologous protein, or antigenic fragment thereof. In some embodiments, the heterologous nucleic acid may comprise DNA that encodes a heterologous protein, or antigenic fragment thereof i.e., the DNA can be transcribed into mRNA that is translated to produce the heterologous protein or antigenic fragment thereof).

[0128] In certain embodiments, the heterologous nucleic acid typically includes regulatory sequences and coding region sequences. In some embodiments, the regulatory sequences are operably linked to the coding region sequences, such that the regulatory sequences control expression e.g., transcription or translation) of the coding region sequences. In certain embodiments, the regulatory sequences can include sequence elements such as promoters and enhancers that bind regulatory proteins such as transcription factors and influence the rate of transcription of operably linked sequences. In certain embodiments, the regulatory sequences can be located upstream (5’) or downstream (3’) of the coding region sequences, or both.

[0129] In some embodiments, the coding region sequences encode a heterologous protein that is useful for eliciting an immune response in a mammal. Various online servers can used to predict epitope-coding sequences of the heterologous protein that strongly bind to MHC-II and elicit a T cell response (for example, see Reynisson et al. NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res. 2020; 48(W1): W449-454.). In certain embodiments, the nucleic acid can also include sequences that, when transcribed and translated, provide signals for trafficking the heterologous protein to a specific cellular location or compartment (e.g., intracellular, secreted, or membrane bound).

[0130] In some embodiments, the heterologous nucleic acid is an expression vector comprising regulatory sequences that upregulate or downregulate transcription of the coding region sequence into RNA. In some embodiments, the engineered bacterium comprises the necessary components to translate the RNA into protein, such as amino acids and tRNA. In some embodiments, the modified microorganism is a live recombinant commensal bacterium that comprises the necessary components to translate the RNA into protein, such as amino acids and tRNA. In certain embodiments, the expression vector can contain regulatory elements that direct expression of the heterologous antigen anywhere in the live, recombinant commensal bacterium. In certain embodiments, the expression vector can contain regulatory elements that direct expression of the heterologous antigen in the cytoplasm (i.e., soluble, not in inclusion bodies), periplasm, fused to a cell surface protein, or secreted by the bacterium. Nucleic acid vectors for the expression of recombinant proteins in bacteria are well known by persons of skill in the art. In some embodiments, the expression vector may be any of: pNBU2-bla-ermGb, pNBU2-bla- tetQb, or pExchange-tdk as described for example in Wang J. et al. (2000). JBacteriol. 182. 3559-71; pMM668, Addgene; Mimee M. et al. (2015) Cell Syst. 1 (l):62-71 ; and Koropatkin N. et al. 2008. Structure. 16(7): 1105-1115.

[0131] In some embodiments, the expression vector is apWW3837 vector (Genbank# KY776532), which is used to integrate an antigenic epitope coding region into the bacterial genome, as described in Whitaker et al., “Tunable Expression Tools Enable Single-Cell Strain Distinction in the Gut Microbiome,” Cell 169, 538-546, April 20, 2017.

[0132] In some embodiments, the heterologous nucleic acid is stably integrated into the genome of the bacteria. In some embodiments, the heterologous nucleic acid is maintained as a plasmid in the bacteria. In some embodiments, the heterologous nucleic acid is an episomal plasmid.Using chemical labeling (e.g., affinity agent) and a genetic carrier to attach antigen to bacterial surface protein

[0133] In certain embodiments, commensal bacteria can be engineered to express an extracellular protein or fragment thereof fused with a first affinity agent. Subsequently, an antigenic or immunomodulatory molecule attached to a second affinity agent that has affinity for the first affinity agent can be contacted to the bacteria, thereby linking the first and second affinity agents, and thereby linking the extracellular protein or fragment thereof to the antigenic or immunomodulatory polypeptide, as seen depicted in FIGS. 5A-6. Advantageously, this linking method may allow a first bacterial strain to expresses the extracellular protein or fragment thereof fused with the first affinity agent, and a second bacterial strain can express antigenic or immunomodulatory molecules attached to a second affinity agent. In another embodiment, two or more different antigenic or immunomodulatory molecules, each linked to a copy of the second affinity agent can in combination be linked to the bacteria, generating a bacterium or mixture of bacteria that present two or more different antigenic or immunomodulatory molecules on their surface. It can be appreciated that in some embodiments, when using wild type bacteria, the wild type bacteria may be reacted with NHS-biotin to form a first affinity sequence, as may be described in more detail herein.

[0134] In some embodiments, the extracellular protein or fragment thereof will be fused to two or more copies of the first affinity agent such that more than one copies of the antigenic or immunomodulatory molecules can be attached to a single extracellular protein or fragment thereof.

[0135] In some embodiments, the first affinity agent(s) can be presented at the amino terminus of the protein or as a sequence within the protein.

[0136] Any affinity agents can be used so long as: (i) the first affinity agent can be linked to the extracellular protein or fragment thereof, (ii) the second affinity agent can be linked to the antigenic or immunomodulatory molecule, and (iii) the first and second affinity agents bind to each other. In this context “bind” and “link” can be covalent or non-covalent depending on the affinity agent used. Exemplary affinity agents can include, but are not limited to, a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and a DogTag sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; aHaloTag sequence and a haloalkane moiety; a cysteine residue and a mal eimide moiety, a free amine and an N-hydroxysuccinimide ester.

[0137] Various Spycatcher and Spytag systems are known and have been adequately described, for example, in Hatlem et al., IntJMol Sci. 2019 May; 20(9): 2129. Exemplary peptide tag / affinity agents include but are not limited to: SpyT / Spycatcher, ALFA / NbALFA and / or GFP11 / GFP1-10. In some embodiments, SpyT may comprise an amino acid sequence of AHIVMVDAYKPTK (SEQ ID NO: 72), as described in Zakeri et al., Proc Natl Acad Sci USA 2012 109:E690-697. Alternatively, SpyT002 may comprise an amino acid sequence of VPTIVMVDAYKRYK (SEQ ID NO: 73 / Spycatcher002 as described in Keeble et al., Angew. Chem. Int. Ed. 2017, 56, 16521 -16525. Alternatively, SpyTag003 may comprise an amino acid sequence of RGVPHIVMVDAYKRYK (SEQ ID NO: 74) / SpyCatcherOO3 as described in Keeble et al., Proc. Natl. Acad. Sci. USA 2019, 116(52), 26523-26533. SpyCatcher comprises an amino acid sequence ofAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWI SDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 75). SpyCatcher002 comprises an amino acid sequence of AMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTW ISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT (SEQ ID NO: 76). SpyCatcher003 comprises an amino acid sequence of AMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTW ISD GHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT (SEQ ID NO: 77). The ALFA NbALFA system comprising an amino acid sequence of (SRLEEELRRRLTE (SEQ ID NO: 78) is described in Gotzke et al., Nature Communications volume 10, Article number: 4403 (2019). NbALFA can be obtained commercially from, e.g., NanoTag Biotechnologies.

[0138] SnoopCatcher / SnoopTag pairs are described in Veggiani et al. Proc Natl Acad Sci USA 2016, 113(5):1202-7 and have also been described inWO-2022200804, which is incorporated by reference in its entirety herein. Dogcatcher and Dogtag sequences are described in WO2022214795, which is incorporated by reference in its entirety herein. Clostridium perfringens adhesin Cpe0147 fragments that bind to each other are described in WO2018093274,which is incorporated by reference in its entirety herein. Halotags are described in WO 04 / 072232, which is incorporated by reference in its entirety herein, and in Los et al., ACS Chem Biol. 2008 Jun 20;3(6):373-82.

[0139] Interactions of cysteine residue and maleimide moieties are described in, Gober et al., J Pept Sci. 2021 Jul; 27(7): e3323, which is incorporated by reference in its entirety herein. Peptides containing cysteine react readily with maleimides to form succinimidyl thioether conjugates. In some embodiments, a cysteine-malemimide reaction can be used to link a polypeptide affinity agent to a non-protein. As an example, maleimide-functionalized molecules can be linked to an affinity agent protein sequence ending in cysteine via cysteine-maleimide chemistry. In one example, a PEGylated lipid or other PEGylated molecule can include a maleimide moiety that is subsequently linked to an affinity agent protein sequence ending in cysteine via cysteine-maleimide chemistry. This can be used to link for example, a lipid nanoparticle comprising the PEGylated lipid to a protein affinity tag (e.g., a Spytag, Snooptag, Dogtag or other protein affinity agents). The resulting product will comprise a protein affinity agent that can then be linked to a second protein affinity agent that is linked to a bacterial surface protein, as seen for example, in FIG. 12 and the Examples.

[0140] An advantage of utilizing affinity agents to link an antigenic or immunomodulatory molecule to a surface protein of a bacterium is that essentially any molecule to which an affinity agent can be linked can be subsequently linked to the bacterial surface protein. Thus, for example, one can link an affinity agent to a non-protein. In addition to proteins, an affinity agent can be linked to a lipid, polysaccharide or even for example a lipid nanoparticle containing a desired payload. Exemplary payloads contained in a lipid nanoparticle can include but is not limited to, antigenic or immunomodulatory proteins, antigenic or immunomodulatory non- protein molecules, or polynucleotides (e.g., DNA or RNA) encoding antigenic or immunomodulatory proteins.Using a transpeptidase to attach an antigen to bacterial surface protein

[0141] In certain embodiments, a transpeptidase can be used to transfer an antigenic or immunomodulatory molecule to a polypeptide on the surface of the bacteria, as seen in the schematic of FIG.8. In some embodiments, the antigenic or immunomodulatory molecule may comprise a transpeptidase recognition sequence and the transpeptidase may link, transfer, orconjugate the antigenic or immunomodulatory molecule to the polypeptide on the surface of the bacteria using the transpeptidase recognition sequence. In some embodiments, the antigenic or immunomodulatory molecule may have an amino acid sequence or be linked to an amino acid sequence that can be transferred (conjugated) by the transpeptidase. This is depicted, for example, in column 3 of FIG. 1.

[0142] In some embodiments, the bacterial surface protein comprising the transpeptidase recognition sequence is an endogenous (meaning native, non-recombinant) polypeptide on the surface of the commensal bacteria. In these embodiments, the naturally occurring transpeptidase recognition sequences may be recognized by an exogenous transpeptidase that may be supplied by a user along with the antigenic or immunomodulatory molecule having an amino acid sequence or linked to an amino acid sequence that can be transferred (conjugated) by the transpeptidase. The resulting reaction, mediated by the transpeptidase, cleaves the recognition sequence on the surface protein and transfers the amino acid sequence (and linked antigenic or immunomodulatory molecule) to the cleaved surface protein, resulting in a fusion of the surface protein fragment with the antigenic or immunomodulatory molecule. As noted above, in some embodiments, this reaction occurs in a non-recombinant bacterium. Advantageously, this method can be used across a wide range of bacterial species, including but not limited to species that are currently difficult or impossible to genetically transform.

[0143] The recognition sequence for a transpeptidase used by exemplary native bacterial surface proteins may vary depending on the transpeptidase, and thus the recognition sequence will also vary. It need not be known what native bacterial surface protein is being modified. For example, a bacterium can be contacted with a transpeptidase, and antigenic or immunomodulatory molecule having an amino acid sequence or linked to an amino acid sequence that can be transferred (conjugated) by the transpeptidase. If there are recognition sequences present on the surface of the bacterium, under the appropriate conditions, the transpeptidase will link the antigenic or immunomodulatory molecule having an amino acid sequence or linked to an amino acid sequence to protein(s) on the surface of the bacterium.

[0144] In other embodiments, a bacterium can be genetically engineered to express one or more surface proteins with the transpeptidase recognition sequence, thereby generating a recombinant bacterium expressing a surface protein or fragment thereof fused to the transpeptidaserecognition sequence or a fusion protein comprising the surface protein or fragment thereof and the transpeptidase recognition sequence. The bacterium may be contacted with a transpeptidase, and an antigenic or immunomodulatory molecule having an amino acid sequence or linked to an amino acid sequence that can be transferred (conjugated) by the transpeptidase under appropriate conditions. The transpeptidase will link the antigenic or immunomodulatory molecule having an amino acid sequence or linked to an amino acid sequence to one or more surface proteins with the transpeptidase recognition sequence on the bacterium.

[0145] Exemplary transpeptidase enzymes that may be used in the embodiments or methods described herein may include but are not limited to sortases, butelase and trypsiligase. In some embodiments, a soluble, catalytic domain of the sortase enzyme sortase A SrtA of Staphylococcus aureus can be expressed recombinantly and used (Belscher, JG, et al. (2011) FASEB J. 25, 2650-2658). This transpeptidase enzyme recognizes the peptidic recognition amino acid sequence, -LPXTG, wherein the amino acid X may be any amino acid. For example, - LPXTG (SEQ ID NO: 79) may include both LPETG (SEQ ID NO: 80) or - LPATG (SEQ ID NO: 81). Other variants of the sortase are, for example, the sortase B srtB of Staphylococcus aureus or Bacillus anthracis, which may have a different peptide recognition amino acid sequence -NP(Q / K)TN (SEQ ID NO: 82 and SEQ ID NO: 83) than srtA (as seen in Maresso, A. W. et al., J. Bacteriol. 188, 8145-8152 and Mazmanian, S.K. et al. 0. Proc. Natl. Acad. Sci.U.S.A. 2002, 99, 2293-2298, each of which are incorporated by reference in their entirety herein).

[0146] Sortases may be divided into different classes (e.g., A, B, C, D, E, and the like) and each class may different peptide recognition sequences, as described in Spirig, T. et al., Mol. Microbiol., 2011, 82, 1044 and Bradshaw, W.J., et al., FEBS J. 2015, 282, 2097. Sortases of class C and D comprise a peptide recognition amino acid sequence of- (I / L)(P / A)XTG SEQ ID NOS: 84-87(class C) or-QVPTG (SEQ ID NO: 88) or -LPNTA (SEQ ID NO: 89) (class D), respectively. Class E sortases may comprise the peptide recognition amino acid sequence of - LAXTG (SEQ ID NO: 90). Sortase A can generally comprise a whole series of peptide recognition amino acid sequence motif of the general sequence X1X2X3X4G (SEQ ID NO: 91) wherein- a first amino acid (Xi) comprises M, L, or V, a second amino acid (Xi)of P, T, A, or S, a third amino acid (Xs)of X, where X can be any amino acid including in particular A, a fourthamino acid (X4) of A,L,T,S,V or I), and a fifth amino acid of G as described in Ning Li et al., Biochem Biophys Res Commun, 2017, 486 (2), 257-263. Other sortase-recognized peptide recognition sequences may comprise the amino acid sequences of: IPKTG (SEQ ID NO: 92), APKTG (SEQ ID NO: 93), DPKTG (SEQ ID NO: 94), SPKTG (SEQ ID NO: 95), APATG (SEQ ID NO: 96), LPECG (SEQ ID NO: 97), or LPXTA (SEQ ID NO: 98), as described in John M. Antos et al., Curr Opin. Struct. Biol., 2016, 38: 111-118, or US20230193291, the contexts of which are incorporated herein in their entirety.

[0147] Other non-sortase transpeptidases can include, for example, Butelase 1, an Asn / Asp (Asx) peptide ligase, as described inGK Nguyen, et al., Nat. Chem. Biol., 2014, 10, 732-738. This transpeptidase enzyme, Butelase 1, has the peptide recognition amino acid sequence of: - NHV or -DHV. Furthermore, alternatively or additionally, it is also possible to use trypsiligase as a transpeptidase. Trypsiligase may comprise the peptide recognition amino acid sequence and the nucleophilic sequence of Y-RH, where Y may be present, but need not be present. During the formation of the intermediate product between the transpeptidase and the peptide substrate, Y and RH if Y is present, resulting in an intermediate sequence -YRH- between two blocks linked by the trypsiligase, as described for example in US20230193291, or WO-2014140317, the contexts of which are incorporated herein in their entirety, and in Freund, et al., ChemBioChem Volume22, Issue 8 April 16, 2021.Using covalent attachment (e.g., click chemistry) to an attach antigen to bacterial surface protein

[0148] In certain embodiments, click chemistry can be used to transfer an antigenic or immunomodulatory molecule to a polypeptide on the surface of bacterium. In some embodiments, the antigenic or immunomodulatory molecule may comprise an azide or alkyne moiety or be linked to an azide or alkyne moiety. A click chemistry reaction may be used to link the antigenic or immunomodulatory molecule to the polypeptide on the surface of the bacterium, as seen in column 4 of FIG. 5. In some embodiments, the polypeptide on the surface of the bacterium may comprise an azide or alkyne moiety and a click chemistry reaction may be used to link the polypeptide on the surface of the bacterium to the antigenic or immunomodulatory molecule.

[0149] Click chemistry can involve an azide alkyne Huisgen cycloaddition reaction. An example of click chemistry includes copper-catalyzed azide-alkyne cycloaddition (CuAAC), as seen in Chemical Reviews: Click Chemistry, June 23, 2021, Volume 121, Issue 12, Pages 6697- 7248, or for example, in Fantoni et al., “A Hitchhiker’s Guide to Click-Chemistry with Nucleic Acids” Chem. Rev. 2021, 121, 7122-7154. Other click-chemistry compatible reactions may alternately be employed. For instance, in various embodiments, click-chemistry compatible reactions may include CuAAC, strain-promoted azide-alkyne cycloaddition (SPAAC), strain- promoted alkyne-nitrone cycloaddition (SPANC), strained alkene reactions such as alkene-azide cycloaddition.

[0150] An alkyne or azide moiety can be introduced into a polypeptide on the surface of the bacterium by providing a non-naturally-occurring amino acid comprising the alkyne or azide moiety to the bacterium, and allowing the bacterium to incorporate the non-naturally-occurring amino acid into proteins made by the bacterium, including surface proteins. Non-naturally- occurring amino acids comprising alkyne or azide moieties can include, for example, L- Azidohomoalanine (AHA), 4-Azido-L-phenylalanine, O-propargyl-puromycine (OPP), L- Homopropargylglycine (HPG) or 3 -Azido-d- Alanine, as described in Rodriguez et al, Mater Today Bio. 2023 Apr; 19: 100587.

[0151] Once bacteria express surface polypeptides comprising the non-naturally-occurring amino acids described above, click chemistry can be used to link an antigenic or immunomodulatory molecule comprising a complementary click chemistry moiety (e.g., an azide moiety if the surface protein has an alkyne moiety, or an alkyne moiety of the surface protein has an azide moiety), thereby linking the antigenic or immunomodulatory molecule to the surface polypeptides of the bacterium.

[0152] An advantage of utilizing click chemistry to link an antigenic or immunomodulatory molecule to a surface protein of a bacterium is that any molecule to which an appropriate click chemistry moiety can be linked can also be subsequently linked to the bacterial surface protein. For example, a non-protein that comprises an appropriate click chemistry moiety can be linked to the bacterial surface. In addition to proteins, click chemistry moieties (e.g., azide or alkyne) can be linked to a lipid, polysaccharide, or even for example a lipid nanoparticle containing a desired payload. Exemplary payloads contained in a lipid nanoparticle can include but is not limited to,antigenic or immunomodulatory proteins, antigenic or immunomodulatory non-protein molecules, or polynucleotides (e.g., DNA or RNA) encoding antigenic or immunomodulatory proteins.Using non-covalent attachment to attach an antigen to bacterial surface protein

[0153] In some embodiments, an antigenic or immunomodulatory molecule can be simply applied to the surface of a commensal bacterium, resulting in bacteria that are coated non- specifically on their surface with the antigenic or immunomodulatory molecule. In some embodiments, the antigenic or immunomodulatory molecule may be attached to the bacteria via non-covalent interactions, for example by non-specific electrostatic interactions.Exemplary antigens or immunomodulatory molecules

[0154] As described herein, an antigen or immunomodulatory molecule can be attached to a bacterial surface protein. In some embodiments, antigenic molecules can include, for example, antigenic proteins or fragments thereof, or non-protein molecules, such as lipids or polysaccharides. In some embodiments, the antigenic protein or fragment thereof may comprise a non-native protein. In some embodiments, the one antigenic peptide may comprise a size of but is not limited to, about 5 amino acid residues, about 6 amino acid residues, about 7 amino acid residues, about 8 amino acid residues, about 9 amino acid residues, about 10 amino acid residues, about 11 amino acid residues, about 12 amino acid residues, about 13 amino acid residues, about 14 amino acid residues, about 15 amino acid residues, about 16 amino acid residues, about 17 amino acid residues, about 18 amino acid residues, about 19 amino acid residues, about 20 amino acid residues, about 21 amino acid residues, about 22 amino acid residues, about 23 amino acid residues, about 24 amino acid residues, about 25 amino acid residues, about 26 amino acid residues, about 27 amino acid residues, about 28 amino acid residues, about 29 amino acid residues, about 30 amino acid residues, about 31 amino acid residues, about 32 amino acid residues, about 33 amino acid residues, about 34 amino acid residues, about 35 amino acid residues, about 36 amino acid residues, about 37 amino acid residues, about 38 amino acid residues, about 39 amino acid residues, about 40 amino acid residues, about 41 amino acid residues, about 42 amino acid residues, about 43 amino acid residues, about 44 amino acid residues, about 45 amino acid residues, about 46 amino acid residues, about 47 amino acid residues, about 48 amino acid residues, about 49 amino acidresidues, about 50 amino acid residues, about 60 amino acid residues, about 70 amino acid residues, about 80 amino acid residues, about 90 amino acid residues, about 100 amino acid residues, about 110 amino acid residues, about 120 or greater amino acid residues, and any range derivable therein. In specific embodiments the antigenic peptide molecule may be equal to or less than about 50 amino acids.

[0155] In some embodiments, a non-native protein or peptide comprises one or more T cell epitopes capable of presentation by MHC-I (e.g., a non-native protein or peptide engineered to elicit a CD8+ cytotoxic T cell response). In some embodiments, the T cell epitope may be typically 15 residues or less in length and may comprise a length of between about 8 amino acid residues and about 11 amino acid residues, including about 9 amino acid residues or about 10 amino acid residues. In some embodiments, the non-native protein or peptide comprises one or more epitopes capable of presentation by MHC-II (e.g., a non-native protein or peptide engineered to elicit a CD4+ T cell response). In some embodiments, the epitope may comprise a length of between about 6 amino acids and about 30 amino acid residues, inclusive. In some embodiments, the non-native protein or peptide is capable of undergoing antigen processing into one or more T cell epitopes capable of presentation by MHC-I and / or MHC-II. In some embodiments, the non-native protein or peptide comprises an epitope, or antigen capable of antigen processing, capable of being presented on one or more distinct HLA alleles, such as any one of: HLA-A, HLA-B, HLA-C, HLA-DQ, HLA-DR, and HLA-DP.

[0156] In some embodiments, the bacterium may be engineered to express a single non-native protein or peptide comprising one or more T cell epitopes capable of presentation by an MHC molecule and one or more B cell epitopes capable of eliciting an antibody response. In some embodiments, the bacterium may be engineered to display on its surface a single non-native protein or peptide comprising one or more T cell epitopes capable of presentation by an MHC molecule and one or more B cell epitopes capable of eliciting an antibody response. In some embodiments, the T cell epitopes and B cell epitopes may be derived from the same antigen protein, or the T cell epitopes and B cell epitopes may be derived from distinct antigenic proteins.

[0157] In some embodiments, the bacterium is engineered to express a single non-native protein or peptide comprising two or more T cell epitopes capable of presentation by an MHC molecule. In some embodiments, the bacterium is engineered to display on its surface a singlenon-native protein or peptide comprising two or more T cell epitopes capable of presentation by an MHC molecule. For example, a single non-native protein or peptide can comprise a T cell epitope capable of presentation by MHC-I and a T cell epitope capable of presentation by MHC- II. In some embodiments, a T cell epitope capable of presentation by MHC-I and a T cell epitope capable of presentation by MHC-II are each derived from the same antigenic protein, such as a single contiguous amino acid sequence derived from a naturally occurring antigen (e.g., a full- length protein or protein domain) or a non-natural peptide fusion (e.g., concatemer) of epitopeencoding amino acid sequences. In some embodiments, a T cell epitope capable of presentation by MHC-I and a T cell epitope capable of presentation by MHC-II are each derived from distinct antigenic proteins, such as a non-natural peptide fusion (e.g., concatemer) of epitope-encoding amino acid sequences from a first protein and epitope-encoding amino acid sequences from a second protein. In certain embodiments, the T cell epitope capable of presentation by MHC-I and a T cell epitope capable of presentation by MHC-II are encoded by a single non-native protein or peptide.

[0158] In some embodiments, the bacterium is engineered to express two or more non-native proteins or peptides. In some embodiments, the bacterium is engineered to display on its surface two or more non-native proteins or peptides. In some embodiments, an engineered microorganism is engineered to express two or more non-native proteins or peptides and each of the two or more non-native proteins independently comprise a T cell epitope capable of presentation by MHC-I, a T cell epitope capable of presentation by MHC-II, a B cell epitope, or combinations thereof.

[0159] In some embodiments, the bacterium is engineered to express two or more non-native proteins or peptides including at least a first non-native protein or peptide that comprises one or more T cell epitopes capable of presentation by an MHC molecule and at least a second non- native protein or peptide that comprises one or more B cell epitopes capable of eliciting an antibody response. In some embodiments, the bacterium is engineered to display on its surface two or more non-native proteins or peptides including at least a first non-native protein or peptide that comprises one or more T cell epitopes capable of presentation by an MHC molecule and at least a second non-native protein or peptide that comprises one or more B cell epitopes capable of eliciting an antibody response. T cell epitopes and B cell epitopes can be derived fromthe same antigenic protein. T cell epitopes and B cell epitopes can be derived from distinct antigenic proteins.

[0160] In some embodiments, the bacterium is engineered to express two or more non-native proteins or peptides including at least a first non-native protein or peptide that comprises one or more T cell epitopes capable of presentation by MHC-I and at least a second non-native protein or peptide that comprises one or more T cell epitopes capable of presentation by MHC-II. MHC- I T cell epitopes and MHC-II T cell epitopes can be derived from the same antigenic protein. In some embodiments, the bacterium is engineered to display on its cell surface two or more non- native proteins or peptides including at least a first non-native protein or peptide that comprises one or more T cell epitopes capable of presentation by MHC-I and at least a second non-native protein or peptide that comprises one or more T cell epitopes capable of presentation by MHC-II. MHC-I T cell epitopes and MHC-II T cell epitopes can be derived from the same antigenic protein. MHC-I T cell epitopes and MHC-II T cell epitopes can be derived from distinct antigenic proteins.

[0161] In some embodiments, two or more bacteria can be engineered to express one or more non-native proteins or peptides. In some embodiments, two or more bacteria can be engineered to display on its cell surface one or more non-native proteins or peptides

[0162] In some embodiments, two or more bacteria can be engineered to express one or more non-native proteins or peptides. The one or more non-native proteins or peptides may include at least a first bacterium engineered to express a first non-native protein or peptide that comprises one or more T cell epitopes capable of presentation by an MHC molecule and at least a second engineered bacterium engineered to express a second non-native protein or peptide that comprises one or more B cell epitopes capable of eliciting an antibody response.

[0163] In some embodiments, two or more bacteria can be engineered to display on its cell surface, one or more non-native proteins or peptides. The one or more non-native proteins or peptides to be displayed on the cell surface may include at least a first bacterium engineered to express a first non-native protein or peptide that comprises one or more T cell epitopes capable of presentation by an MHC molecule and at least a second engineered bacterium engineered to express a second non-native protein or peptide that comprises one or more B cell epitopes capable of eliciting an antibody response.

[0164] In certain embodiments, T cell epitopes and B cell epitopes expressed by distinct engineered bacteria or surface-labeled bacteria, can be derived from the same antigenic protein or can be derived from distinct antigenic proteins.

[0165] In some embodiments, two or more bacteria can be engineered to express one or more non-native proteins or peptides including at least a first engineered bacterium may be engineered to express a first non-native protein or peptide comprising one or more T cell epitopes capable of presentation by MHC-I and at least a second engineered bacterium may be engineered to express a second non-native protein or peptide comprising one or more T cell epitopes capable of presentation by MHC-II.

[0166] In some embodiments, two or more bacteria can be engineered to display on its cell surface one or more non-native proteins or peptides. For example, a first engineered bacterium may be engineered to display on its cell surface a first non-native protein or peptide comprising one or more T cell epitopes capable of presentation by MHC-I and at least a second engineered bacterium may be engineered to display on its cell surface a second non-native protein or peptide comprising one or more T cell epitopes capable of presentation by MHC-II.

[0167] In certain embodiments, MHC-I T cell epitopes and MHC-II T cell epitopes expressed by distinct engineered bacteria, or surface-labeled bacteria, can be derived from the same antigenic protein or may be derived from different antigenic proteins.

[0168] In some embodiments, the non-native protein or peptide may comprise an autoimmune (e.g., a self-) antigen. In some embodiments, the non-native protein or peptide may include any one or more of the following but is not limited to: myelin oligodendrocyte glycoprotein, insulin, chromogranin A, hybrid insulin peptides, proteolipid protein, myelin basic protein, villin, epithelial cellular adhesion molecule, collagen alpha- 1, aggrecan core protein, 60kDa chaperonin 2, vimentin, alpha-enolase, fibrinogen alpha chain, fibrinogen beta chain, chitinase-3 -like protein, 60kDa mitochondrial heat shock protein, matrix metalloproteinase- 16, thyroid peroxidase, thyrotropin receptor, thyroglobulin, gluten, TSHR protein, glutamate decarboxylase 2, receptor-type tyrosine-protein phosphatase-like N, glucose-6-phosphatase 2, insulin isoform 2, zinc transporter 8, glutamate decarboxylase 1, GAD65, UniProt:A2RGM0, integrin alpha-lib, integrin beta-3, EBV DNA polymerase catalytic subunit, 2’3’-cyclic-nucleotide 3’ phosphodiesterase, myelin associated oligodendrocyte basic protein, small nuclearribonucleoprotein, U1 small nuclear ribonucleoprotein, histone H2B, histone H2A, histone H3.2, beta-2-glycoprotein, histone H4, 60S ribosomal protein L7, TNF-alpha, myeloperoxidase, Cbirl, MS4A12, DNA topoisomerase, CYP2D6, O-phosphoseryl-tRNA selenium transferase, pyruvate dehydrogenase complex, spectrin alpha chain, steroid 21 -hydroxylase, acetylcholine receptor, MMP-16, keratin associated proteins, Chondroitin sulfate proteoglycan 4, myeloblastin, U1 small nuclear ribonucleoprotein 70 kDa, blood group Rh(D), blood group Rh(CE), myelin P2 protein, peripheral myelin protein 22, myelin protein P0, S-arrestin, collagen Alpha- 1, coagulation factor VIII, collagen alpha-3(IV), desmoglein-3, desmoglein-1, Insulin-2, major DNA-binding protein, tyrosinase, 5,6-dihydroxyindole-2-carboxylic acid oxidase, HLA-A2, aquaporin-4, myelin proteolipid protein, ABC transporter, HLA I B-27 alpha chain, HLA I B-7 alpha chain, retinol-binding protein 3, or antigenic fragments thereof.

[0169] In some embodiments, the non-native protein or peptide is an antigen that is associated with an autoimmune disease. In some embodiments, the non-native protein or peptide may be associated with any one or more of the following autoimmune diseases: multiple sclerosis, psoriasis, celiac disease, diabetes mellitus Type I, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, Graves’ disease, Hashimoto’s autoimmune thyroiditis, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, temporal arteritis, ulcerative colitis, Crohn’s disease, scleroderma, antiphospholipid syndrome, autoimmune hepatitis type 1, primary biliary cirrhosis, Sjogren’s syndrome, Addison’s disease, dermatitis herpetiformis, Kawasaki disease, sympathetic ophthalmia, HLA-B27 associated acute anterior uveitis, primary sclerosing cholangitis, discoid lupus erythematosus, polyarteritis nodosa, CREST Syndrome, myasthenia gravis, polymyositis / dermatomyositis, Still’s disease, autoimmune hepatitis type 2, Wegener’s granulomatosis, mixed connective tissue disease, microscopic polyangiitis, autoimmune polyglandular syndrome, Felty’s syndrome, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre Syndrome, Behcet’s disease, autoimmune neutropenia, bullous pemphigoid, essential mixed cryoglobulinemia, linear morphea, autoimmune polyglandular syndrome 1 (APECED), acquired hemophilia A, Batten disease / neuronal ceroid lipofuscinoses, autoimmune pancreatitis, Hashimoto’s encephalopathy, Goodpasture’s disease, pemphigus vulgaris, autoimmune disseminated encephalomyelitis, relapsing polychondritis, Takayasu arteritis, Churg-Strauss syndrome, epidermolysis bullosaacquisita, cicatricial pemphigoid, pemphigus foliaceus, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, autoimmune orchitis, autoimmune polyglandular syndrome, Cogan’s syndrome, encephalitis lethartica, erythema elevatum diutinum, Evans syndrome, immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX), Issac’s syndrome / acquired neuromyotonia, Miller Fisher syndrome, Morvan’s syndrome, PANDAS, POEMS syndrome, Rasmussen’s encephalitis, stiff-person syndrome, Vogt-Koyanagi-Harada syndrome, neuromyelitis optica, graft vs host disease, esophageal encephalitis, or autoimmune uveitis.

[0170] In some embodiments the non-native protein or peptide is myelin oligodendrocyte glycoprotein, or an antigenic fragment thereof, which is associated with multiple sclerosis (MS). In some embodiments, the non-native protein or peptide is a pancreatic antigen, or antigenic fragment thereof, that is associated with Type I Diabetes (e.g., insulin).

[0171] In some embodiments, the non-native protein or peptide may comprise a heterologous antigen or antigenic fragment thereof. In some embodiments, the heterologous antigen, or antigenic fragment thereof, may be linked to sialidase, endonuclease, secreted endoglycosidase, anti-sigma factor, thiol peroxidase, hypothetical protein BT 2621, hypothetical protein BT 3223, peptidase, Icc family phosphohydrolase, exo-poly-alpha-D-galacturonosidase, hypothetical protein BT 4428, or functional fragments thereof.

[0172] In some embodiments, the heterologous antigen is an antigen, or antigenic fragment thereof, associated with a proliferative disorder such as cancer. In some embodiments the heterologous antigen is associated with melanoma, basal cell carcinoma, squamous cell carcinoma, or testicular cancer. In some embodiments, the heterologous antigen is a melanocytespecific antigen such as PMEL, TRP2, or MART-1. In some embodiments, the heterologous antigen is a testis cancer antigen such as NY-ESO or MAGE-A. In some embodiments, the heterologous antigen is a neoantigen. In some embodiments, the heterologous antigen is not a neoantigen.

[0173] In some embodiments, the heterologous antigen is a protein or antigenic peptide fragment thereof that is not natively expressed by either commensal bacteria or a host. In some embodiments, the heterologous antigen is gluten, or an antigenic fragment thereof, which is associated with celiac disease in a host.

[0174] In some embodiments, the non-native protein or peptide is a neoantigen protein or peptide fragment thereof. Neoantigens are mutated peptide antigens that are specifically expressed by cancer cells and are not expressed by normal, healthy cells. A cancerous cell can express a single neoantigen or multiple neoantigens. Some neoantigens are common in various cancers and may be expressed by a significant number of patients while other neoantigens are rare and expressed by only a few patients. T cells can recognize neoantigens when they are displayed on MHCs of the cancer cell or by an APC. A description of neoantigen repertoire, identification, and their role in cancer immunotherapy is provided in “Neoantigens in cancer immunotherapy.” TN Schumacher et al., Science, 2015: Vol. 348, Issue 6230, pp. 69-74, DOI: 10.1126 / science.aaa4971, hereby incorporated by reference in its entirety.

[0175] In some embodiments, the neoantigen is associated with a proliferative disorder. In some embodiments, the proliferative disorder is cancer. In some embodiments, the neoantigen is associated with a cancer selected from the group consisting of: melanoma, kidney, hepatobiliary, head-neck squamous carcinoma (HNSC), pancreatic, colon, bladder, glioblastoma, prostate, lung, breast (mammary), ovarian, gastric, kidney, bladder, esophageal, renal, melanoma, leukemia, lymphoma, mesothelioma, basal cell carcinoma, squamous cell carcinoma, and testicular cancer.

[0176] In some embodiments, the neoantigen is selected from the group consisting of Intsl 1, Kifl8bp, T3 sarcoma neoantigens, and a neoantigen as expressed by the TRAMPC2 prostate cancer cell line. Intsl 1 and Kifl 8bp neoantigens are described in Castle et al., “Exploiting the Mutanome for Tumor Vaccination” Cancer Res. 2012; 72(5): 1081-1091 , T3 sarcoma neoantigens neoantigens are described in Alspach et al., “MHC-II neoantigens shape tumour immunity and response to immunotherapy” Nature. 2019; 574:696-701, and Tramp-C2 neoantigens are described in Fasso et al., “SPAS-1 (stimulator of prostatic adenocarcinomaspecific T cells) / SH3GLB2: A prostate tumor antigen identified by CTLA-4 blockade” PNAS. 2008; 105(9):3509-3514, each of which are incorporated by reference in their entirety herein.

[0177] Neoantigens and tumor-associated peptides that can serve as active pharmaceutical ingredients of vaccine compositions that stimulate an antitumor response are described in U.S. Patent No. 9,115,402, which is herein incorporated by reference in its entirety. In certain embodiments, a neoantigen can be selected by first identifying the available mutations that constitute a neoantigen or tumor-associated antigen in cancer cells from an individual cancersubject. In certain embodiments, once identified, the neoantigen, or immunogenic fragment thereof, can be expressed in a live, recombinant commensal bacterium described herein to elicit an adaptive T cell response in the cancer subject or in HLA-matched donor T cells that can be introduced into the cancer subject to recognize and kill the cancer cells.

[0178] In some embodiments, the at least one non-native protein or peptide is an antigen associated with an infectious disease-causing organism. In certain embodiments, an infectious disease-causing organism includes any infectious virus, bacteria, fungus, or parasite that infects and causes disease in a host. In some embodiments, the host is a mammal. In some embodiments, the host is a human. In some embodiments, the infectious disease-causing organism is a virus. In some embodiments, the infectious disease-causing organism is a bacterium. In some embodiments, the infectious disease-causing organism is a fungus. In some embodiments, the infectious disease-causing organism is a parasite.

[0179] In some embodiments, the infectious disease-causing organism is selected from the group consisting of: Influenza virus A, Influenza virus B, Influenza virus C, herpesviruses, herpes simplex virus (HSV-1, HSV-2), retroviruses, human immunodeficiency virus (HIV-1, HIV-2), human adenovirus (hAdV), parainfluenza viruses (PIV), respiratory syncytial virus (RSV), rhinoviruses, coronaviruses, SARS-coronavirus, COVID-19, measles virus , mumps virus, rubella virus, polio virus, varicella-zoster virus (VZV), dengue virus, flaviviruses, ebola virus, Epstein-Barr virus, norovirus, rotavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, West Nile virus, rabies virus, Staphylococcus aureus (MRSA), Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Clostridium tetani, Clostridium difficile, Mycobacterium tuberculosis, Borrelia burgdorferi, Yersinia pestis, Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, Clostridium botulinum, group A Streptococcus bacteria (strep throat causing bacteria), Listeria, Shigella, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Legionella pneumophila, Cryptococcus, Histoplasmosis, Pneumocystis jirovecii, Aspergillus, Trichophyton, Microsporum, Epidermophyton, Trichomonas vaginalis, Plasmodium, Toxoplasma gondii, Giardia lamblia, and Leishmania. In some embodiments, the at least one non-native protein or peptide is NP366-374, NP306-322, NA177- 193, M2 ectodomain, HA2 stem- HA 12-63, HA2 stem HA 76-130, gB glycoprotein, gd glycoprotein, and gB glycoprotein 498-505.

[0180] In some embodiments, the non-native protein or peptide comprises an amino acid sequence as listed in Table 1.TABLE 1: EXEMPLARY NON-NATIVE PEPTIDES AND AMINO ACID SEQUENCES.

[0181] In some embodiments, the SARS-CoV2 Spike protein epitope is substantially identical to SEQ ID NO:65 of PCT / US2021 / 065011 but has Lys986Pro and Val986Pro mutations to stabilize the protein in its pre-fusion conformation as used in the SARS-CoV-2 vaccines. For example, the epitope can comprise SEQ ID NO:39.Bacteria species that may be used in the attachment of antigens or immunomodulatory molecules thereto

[0182] The bacteria, including commensals and ubiquitous colonists, described herein can be from a variety of bacteria species. In some embodiments, the bacteria is derived from a commensal bacteria or bacterial strain. In some embodiments, the bacteria is derived from a commensal bacteria or bacterial strain in a mammal. In some embodiments, the bacteria or bacterial strain is derived from a commensal bacteria or bacterial strain in a human. In some embodiments, the bacteria or bacterial strain is derived from a commensal bacteria or bacterial strain native in a human niche, for example, a gastrointestinal tract, respiratory tract, urogenital tract, and / or skin.

[0183] In some embodiments, the bacteria is derived from a commensal bacteria that is native to the digestive tract of a mammal. The bacterium can be a gram-negative bacteria or a grampositive bacteria. In some embodiments, the bacterium is derived from a Bacteroi des spp., Clostridium spp., Faecalibacterium spp., Helicobacter spp., Parabacteroides spp., or Prevotella spp. In some embodiments, the live, recombinant bacterium is derived from Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides finegoldii, or Helicobacter hepaticus.

[0184] In some embodiments, the bacteria is derived from a commensal bacteria that is native to the skin of a mammal. For example, in some embodiments, the live, recombinant bacterium is derived from a Staphylococcus spp., or Corynebacterium spp. In some embodiments, the live, recombinant bacterium is derived from Staphylococcus epidermidis. For example, in some embodiments, the live, recombinant bacterium is derived from S. epidermidis NIHLM087.

[0185] In some embodiments, the bacteria is from a commensal bacteria or other bacteria that is Gram negative. For example, in some embodiments, the Gram negative bacteria is a Bacteroides spp., a Helicobacter spp., or a Parabacteroides spp. In some embodiments, the live, recombinant bacterium is B. thetaiotaomicron, B. vulgatus, B. finegoldii, or H. hepaticus.

[0186] In some embodiments, the bacteria is from a commensal bacteria or other bacteria that is Gram positive. For example, in some embodiments, the Gram positive bacteria is a Staphylococcus spp., a. Faecalibacterium spp., or a Clostridium spp. In some embodiments, the live, recombinant bacterium is S. epidermidis.

[0187] In some embodiments, the bacteria is from a commensal bacteria that is known to induce a Tregresponse in a mammalian host. In some embodiments, the live, recombinantbacteria is derived from a Bacteroides spp., Helicobacter spp., Parabacteroides spp., Clostridium spp., Staphylococcus spp., Lactobacillus spp., Fusobacterium spp., Enterococcus spp., Acenitobacter spp., Flavinofractor spp., Lachnospiraceae spp., Erysipelotrichaceae spp., Anaerostipes spp., Anaerotruncus spp., Coprococcus spp., Clostridiales spp., Odoribacter spp., Collinsella spp., Bifidobacterium spp., or Streptococcusor Prevotella spp.

[0188] In some embodiments, the bacterium is from Clostridium ramosum, Staphylococcus saprophyticus, Bacteroides thetaiotaomicron, Clostridium histolyticum, Lactobacillus rhamnosus, Parabacteroides johnsonii, Fusobacterium nucleatum, Enterococcus faecium, Lactobacillus casei, Acenitobacter Iwofii, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides uniformis, Bacteroides finegoldii, Clostridium spiroforme, Flavonifractor plautii, Clostridium hathewayi, Lachnospiraceae bacterium, Clostridium bolteae, Erysipelotrichaceae bacterium, Anaerostipes caccae, Anaerotruncus colihominis, Coprococcus comes , Clostridium asparagiforme, Clostridium symbiosum, Clostridium ramosum, Clostridium sp. D5, Clostridium scindens , Lachnospiraceae bacterium , Clostridiales bacterium , Bacteroides intestinalis, Bacteroides caccae, Bacteroides massiliensis, Parabacteroides distasonis, Odoribacter splanchnicus, Collinsella aerofaciens, Acinetobacter Iwoffii, Bifidobacterium breve, Bacteroides finegoldii, Bacteroides fragilis, Bacteroides massiliensis, Bacteroides ovatus, Bifidobacterium bifidum, Lactobacillus acidofilus, Lactobacillus casei, Lactobacillus reuteri, Streptococcus thermophilus, or Prevotella histicola.

[0189] In some embodiments, the bacterium is from Corynebacterium tuberculostearicum, Corynebacterium accolens, Corynebacterium accolens, Corynebacterium amycolatum, Corynebacterium aurimucosum, Corynebacterium aurimucosum, Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum, Corynebacterium granulosum, Cutibacterium acnes, Cutibacterium acnes, Cutibacterium avidum, Cutibacterium avidum, Dolosigranulum pigrum, Finegoldia magna, Moraxella catarrhalis, Moraxella nonliquefaciens, Haemophilus influenzae, Haemophilus aegyptius, Rothia mucilaginosa, Streptococcus pyogenes, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus agalactiae, Streptococcus gordonii, Neisseria lactamica, Neisseria inereal, Neisseria Mucosa:, Lactobacillus crispatus, Lactobacillus jensenii gasser, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus reuteri, Lactobacillus salivarius, Bifidobacterium breve, Bifidobacterium longum, Veillonellaparvula, Veillona parvula, Gardnerella vaginalis, Atopobium vaginae, Prevotella bivia, Mobiluncus mulieris, Mageeibacillus indolicus, Prevotella buccalis, Enterococcus faecium, Lactococcus lactis, Ruminococcus gnavus, or Eubacterium limosum. In some embodiments, the commensal bacterium is derived from a bacterium having ATCC accession number 35692, 49725, 49726, 49368, 700975, 700540, 51488, 10700, 25564, 51277, 11827, 25577, 49753,51524, 29328, 25238, 25240, 19976, 51907, 11116, 25296, 19615, 12344, BAA-611, 13813, 10558, 23970, 14685, 19696, 33820, 25258, 19992, 55195, 4356, 33200, 7469, 393, 7995D-5, 23272, 11741, 15700, 15697, 10790, 17745, 14018, BAA-55, 29303, 35243, BAA-2120, 35310, 19434, 19435, 29149, or 8486. Commensal bacteria useful for the methods and compositions described herein are shown in Table 2.

[0190] In some embodiments, the bacteria is derived from a commensal bacteria or other bacteria that is known to induce a Teffresponse in a mammalian host. In some embodiments, the bacteria is derived from a Staphylococcus spp., Parabacteroides spp., Alistipes spp., Bacteroides spp., Eubacterium spp., Runimococcaceae spp., Phascolarctobacterium spp., Fusobacterium spp., Klebsiella spp., Clostridium spp., Coprobacillus spp., Erysipelotrichaceae spp., Subdoligranulum spp., Ruminococcus spp., Firmicutes spp., or Bifidobacterium spp.

[0191] In some embodiments, the bacteria is derived from S. epidermidis, Parabacteroides distasonis, Parabacteroides gordonii, Alistipes senegalensis, Parabacteroides johnsonii, Paraprevotella xylaniphila, Bacteroides dorei, Bacteroides uniformis JCM 5828, Eubacterium limosum, Ruminococcaceae bacterium cv2, Phascolarctobacterium faecium, Fusobacterium ulcerans, Klebsiella pneumoniae , Clostridium bolteae 90B3, Clostridium cf. saccharolyticum K10, Clostridium symbiosum WAL-14673, Clostridium hathewayi 12489931, Ruminococcus obeum A2-162, Ruminococcus gnavus AGR2154, Butyrate-producing bacterium SSC / 2, Clostridium sp. ASF356, Coprobacillus sp. D6 contl.l , Eubacterium sp. 3 1 31 contl.l, Erysipelotrichaceae bacterium 21 _3 , Subdoligranulum sp. 4_3_54A2FAA, Ruminococcus bromii L2-63, Firmicutes bacterium ASF500, Bacteroides dorei 5 1 36 / D4 supercont2.3, Bifidobacterium animalis subsp. lactis ATCC 27673, or Bifidobacterium breve UCC2003.

[0192] Additional commensal and non-commensal bacterial strains that can be engineered to express or display non-native proteins or peptides are listed in Table 3.TABLE 3: ADDITIONAL COMMENSAL AND NON-COMMENSAL BACTERIAL STRAINS FOR ENGINEERING OR USE IN A SURFACE DISPLAY SYSTEM

[0193] In some embodiments, the commensal bacterium is selected from the group consisting of: Corynebacterium tuberculostearicum, Corynebacterium accolens, Corynebacterium amycolatum, Corynebacterium aurimucosum, Corynebacterium propinquum, Corynebacteriumpseudodiphtheriticum, Corynebacterium granulosum, Cutibacterium acnes, Cutibacterium avidum, Dolosigranulum pigrum, Finegoldia magna, Moraxella catarrhalis, Moraxella nonliquefaciens, Haemophilus influenzae, Haemophilus aegyptius, Rothia mucilaginosa, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus gordonii, Neisseria lactamica, Neisseria cinerea, Neisseria mucosa, Lactobacillus crispatus, Lactobacillus jensenii Gasser, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus reuteri, Lactobacillus salivarius, Bifidobacterium breve ATCC 15700, Bifidobacterium longum, Veillonella parvula, Gardnerella vaginalis, Atopobium vaginae, Prevotella bivia, Mobiluncus mulieris, Mageeibacillus indolicus, Prevotella buccalis, Enterococcus faecium, Lactococcus lactis, Ruminococcus gnavus JCM6515, and Eubacterium limosum ATCC 8486. In some embodiments, the commensal bacterium is selected from the group consisting of: a bacterium having ATCC accession number 35692, 49725, 49726, 49368, 700975, 700540, 51488, 10700, 25564, 51277, 11827, 25577, 49753, 51524, 29328, 25238, 25240, 19976, 51907, 11116, 25296, 19615, 12344, BAA-611, 13813, 10558, 23970, 14685, 19696, 33820, 25258, 19992, 55195, 4356, 33200, 7469, 393, 7995D-5, 23272, 11741, 15700, 15697, 10790, 17745, 14018, BAA-55, 29303, 35243, BAA-2120, 35310, 19434, 19435, 29149, and 8486. In some embodiments, the commensal bacterium is selected from the group consisting of: Lactobacillus casei, Lactococcus lactis, Streptococcus gordonii, Lactobacillus crispatus, Lactobacillus iners, Cutibacterium acnes, Streptococcus agalactiae, Ruminococcus gnavus JCM6515, Neisseria lactamica, Bifidobacterium breve ATCC 15700, and Bifidobacterium longum. In some embodiments, the commensal bacterium is selected from the group consisting of: a bacterium having ATCC accession number 393, 19435, 35105, 33820, 55195, 6919, 13813, 23970, 15700, and 15707, and a bacterium having an accession number JCM6515.Immunomodulatory molecules

[0194] As described herein, immunomodulatory molecules may be linked to polypeptides on the cell surface of bacteria using any of the methods described above. Immunomodulatory molecules are molecules that increase or decrease the activity of the immune system. Exemplary immunomodulatory agents can include for example cytokines or chemokines. Examples of immunomodulatory agents include, but are not limited to, proteinaneous agents such ascytokines, peptide mimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments or epitope binding fragments), small molecules, organic compounds, and inorganic compounds. In particular, immunomodulatory agents include, but are not limited to, methothrexate, leflunomide, cyclophosphamide, cyclosporin A, and macrolide antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti-CD5 antibodies (e.g. , an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies (Pharmingen), anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH IH (Ilex)), anti-CD2 antibodies, anti-CDl la antibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))), CTLA4- immunoglobulin LFA3TIP (Biogen), cytokines (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF- a, TNF-0, interferon (IFN)-a, IFN-0, IFN-y, and GM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL- 6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies), and anti -cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-a antibodies, anti-IL-10 antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)), anti-IL-9 antibodies, and anti-IL-12 antibodies).Nucleic acids and host cells

[0195] The present disclosure also provides nucleic acids that encode any of the polypeptide fusion protein described herein. Such nucleic acids are useful, for example, for transforming commensal bacteria to express the desired fusion protein. Exemplary polypeptides can include, for example, (i) a bacterial extracellular protein and (ii) a heterologous antigenic polypeptide, immunomodulatory polypeptide, or an amino terminal pentaglycine or a transpeptidase recognition sequence or one or more affinity sequences as described herein. Exemplary surface proteins can include, for example, Accumulation-associated protein (Aap), peptidoglycan, an Mprotein, LysM and a peptidoglycan-binding protein or a fragment thereof. Exemplary nucleic acids can be DNA, RNA, or can comprise one or more non-natural nucleotide.

[0196] In some embodiments, a nucleic acid encoding a fusion protein as described herein includes, for example from N-terminus to C-terminus, a signal sequence, an antigen, and a HA tag; a signal sequence, a cleavage site, a HA tag, an antigen, and a wall attachment protein such as protein A; a signal sequence, a cleavage site, Aa repeats sequence, a HA tag, an antigen, a B domain, and a wall attachment protein, a signal sequence, a cleavage site, LysM domains 1 and 2, a spacer sequence with LysM3, optionally a second spacer sequence, one or more antigen sequences, optionally a third spacer sequence, and a T helper epitope sequence, a signal sequence, a cleavage site, Aa repeats sequence, a HA tag, one or more antigen sequences, a B domain sequence, and a wall attachment protein sequence. FIGS. 38A-38I depict schematics of exemplary constructs including those described above

[0197] Also provided herein are expression cassettes comprising a promoter operably linked to the nucleic acids such that when the expression cassette is introduced into a bacterial cell, the polypeptide fusion protein encoded by the nucleic acids is produced in the cell. In some embodiments, the expression cassette may be included within a vector, for example an expression vector.

[0198] Also provided are host cells for expression of any of the polypeptide fusion proteins of the disclosure. In some embodiments, the host cells may comprise commensal bacteria as described herein or alternatively bacterial or non-bacterial (e.g., yeast, mammalian cells, etc.) useful for expression of the fusion polypeptides of the disclosure, which can in turn be purified from the host cells and applied, for example, to commensal bacteria.Pharmaceutical Compositions:

[0199] Also disclosed herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions may comprise any of the engineered bacterium described herein including any of the engineered bacteria comprises one or more antigenic or immunomodulatory molecules linked to a polypeptide on the surface of the bacteria. In some embodiments, the one or more antigenic or immunomodulatory molecules may be linked to a polypeptide on the surface of the bacteria using any of the methods described herein. In some embodiments, thepharmaceutical compositions may be configured for administration to a subject in need thereof and may comprise excipients configured for administration.

[0200] For the clinical use of the methods described herein, administration of the engineered bacteria can include formulation into pharmaceutical compositions, pharmaceutical formulations, or medicaments, for administration, e.g., topical, or other routs of administration. In some embodiments, the engineered bacteria can be administered along with any pharmaceutically acceptable carrier, excipient, or diluent, which results in an effective treatment and / or effective prophylaxis in the subject. Thus, in one aspect, the present disclosure provides pharmaceutical compositions comprising any of the engineered bacteria of the disclosure, in combination with one or more pharmaceutically acceptable carrier, excipient, or diluent.

[0201] The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, media, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of, an antibody or antigen binding fragment thereof of the present disclosure. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like, and may include other proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to mild chemical modifications or the like. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. The terms “excipient,” “carrier,” “pharmaceutically acceptable carrier,” and the like are used interchangeably herein. The compositions of the present disclosure may further comprise one or more pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like (herein collectively referred to as “pharmaceutically acceptable carriers or diluents”). A multitude of appropriateformulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA, 1998, J. Pharm. Sci. Technol. 52:238-311.

[0202] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

[0203] Optionally, the formulations comprising the compositions described herein contain a pharmaceutically acceptable salt, typically, e.g., sodium chloride, and preferably at about physiological concentrations. Optionally, the formulations of the disclosure can contain a pharmaceutically acceptable preservative. In some embodiments the preservative concentration ranges from 0.1 to 2.0%, typically v / v. Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben are examples of preservatives. Optionally, the formulations of the disclosure can include a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.

[0204] The compositions described herein can be specially formulated for administration of the engineered bacteria to a subject in solid, liquid or gel form, including those adapted for thefollowing: (a) parenteral administration, for example, by topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin. Compositions of the present disclosure can be in the form of, for example, granules, powders, syrup, suppositories, injections, emulsions, elixirs, suspensions, or solutions. As noted above, suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, com oil and olive oil. Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as, but not limited to, ethanol, iso-propyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol. Ethers, such as but not limited to, poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil and petrolatum; and water may also be used in suspension formulations.Methods of administration of pharmaceutical compositions comprising engineered bacteria:

[0205] Disclosed herein are methods of administration of the pharmaceutical compositions or engineered bacteria disclosed herein. In some embodiments, the bacterium, or pharmaceutical composition comprising the bacterium or bacteria, may be administered to a native host niche including a subject. For example, a commensal bacterium derived from a commensal bacterium native to a host gut niche, is administered to the same host gut niche for colonization. In another example, a bacterium derived from a commensal bacterium native to a host skin niche, is administered to the same host skin niche for colonization. In some embodiments, the commensal bacterium may be administered to the native host niche through any method of application appropriate including spreading, spraying, injecting, swallowing, or the like.

[0206] In some embodiments, the bacterium persistently colonizes a native host niche when administered to a subject. For example, in some embodiments, the bacterium persists in the native host niche for over 60 days, over 112 days, over 178 days, over 1 year, over 2 years, or over 5 years. As an illustrative non-limiting example, Staphylococcus epidermidis can colonize skin of mice for at least 180 days post-association.

[0207] In some embodiments, the bacterium transiently colonizes a native host niche when administered to a subject. For example, in some embodiments, the bacterium transiently colonizes the native host niche for between 1 and 60 days, 2 and 60 days, 10 and 60 days, 20 and60 days, 40 and 60 days, 1 and 40 days, 2 and 40 days, 10 and 40 days, 20 and 40 days, 1 and 20 days, 2 and 20 days, 10 and 20 days, 1 and 10 days, or 2 and 10 days. In some embodiments, the bacterium transiently colonizes the native host niche in the subject then migrates to a different niche within the host.Methods of inducing an antigen-specific immune response using engineered bacteria

[0208] The bacteria described herein are useful for inducing an antigen-specific immune response to a non-native protein or peptide (e.g., a non-native antigen), which results in a B-cell response or the generation or expansion of T cells that express a T cell receptor that specifically binds to the heterologous antigen or an immunologically active fragment thereof. Thus, the bacteria can be used to treat a disease or condition in a subject by administering a therapeutically effective amount of the bacteria, or a pharmaceutical composition comprising the bacteria, to a subject. Following administration, the subject’s immune system responds by producing antigenic molecule presented by the bacteria. In some embodiments, the immune system responds by producing antibodies and / or antigen-specific regulatory T cells (Treg), which reduce the host’s immune response against a self-antigen or other antigen corresponding to the expressed heterologous protein or peptide. In some embodiments, the immune system responds by producing antigen-specific T cells (Teff), which modulate an immune response against the expressed non-native protein or peptide, e.g, a tumor associated antigen, neoantigen, or an antigen associated with an infectious disease. In some embodiments, the immune system responds by producing antigen-specific THI cells, which modulate an immune response against the expressed heterologous antigen, such as through promoting cellular immunity (e.g., promoting an immune environment conducive to an antigen-specific CD8 cytotoxic T cell response). In some embodiments, the immune system responds by producing antigen-specific TH2 cells, which modulate an immune response against the expressed heterologous antigen, such as through promoting humoral immunity (e.g., promoting an immune environment conducive to an antigen-specific B cell response and production of antibodies). In some embodiments, the immune system responds by producing antigen-specific T helper 17 cells (THI 7), which modulate an immune response against the expressed heterologous antigen. In some embodiments, the immune system responds by producing antigen-specific T follicular helper cells (TFH), which modulate an immune response against the expressed heterologous antigen. In some embodiments, the immune system responds by producing antigen-specific B cells, whichmodulate an immune response (e.g., a humoral immune response) against the expressed heterologous antigen.

[0209] In some embodiments, antigen-specific immune responses induced by the bacteria described herein can be localized to a native host niche colonized by the bacteria (e.g., a specific organ, such as skin). In some embodiments, antigen-specific immune responses induced by the bacteria can be restricted to a native host niche colonized by the bacteria. In some embodiments, antigen-specific immune responses induced by the bacteria can be distal to a native host niche colonized by the bacteria (e.g., an antigen-specific immune response in an organ, or site in a subject, that is not colonized by the bacteria). For example, a distal antigen-specific immune response can include stimulation of immune cells at a native host niche colonized by the bacteria followed by migration of the immune cells to another site (e.g., another organ). As a non-limiting illustrative example, the bacteria that colonize the skin can induce an antigen-specific immune response that results in immune cells (e.g., antigen-specific T cells) carrying out their effector function in organs other than the skin. In certain embodiments, the organ other than the skin is the lungs, breasts, prostate, colon, bladder, uterus, kidney, liver, pancreas, thyroid, or ovaries. In some embodiments, antigen-specific immune responses can include both a localized and distal immune response relative to a native host niche. In certain embodiments, the antigen-specific immune response targets metastases, such as skin melanoma that has metastasized to other organs.

[0210] In some embodiments, distal antigen-specific immune responses are distal relative to the site of administration of the bacteria. In some embodiments, distal antigen-specific immune responses are distal relative to a host niche colonized by the bacteria. In some embodiments, distal antigen-specific immune responses are in the same organ as the site of administration of the bacteria and / or the native host niche colonized by the bacteria. In certain embodiments, the bacteria is applied to and / or colonizes one area of skin and produces an immune response in a separate part of the skin, such as a melanoma skin metastasis. In some embodiments, distal antigen-specific immune responses are in a different organ as the site of administration of the engineered commensals or other engineered bacteria and / or the native host niche colonized by the bacteria. In certain embodiments, the bacteria are applied to and / or colonizes the skin and produces an immune response in an organ other than skin, such as a melanoma that has metastasized to other organs. In some embodiments, distal antigen-specific immune responsesare in both the same organ and a different organ as the site of administration of the engineered commensals or other engineered bacteria and / or the native host niche colonized by the bacteria. In certain embodiments, the bacteria is applied to and / or colonizes the skin and produces an immune response both in the skin and in an organ other than skin, such as targeting skin melanoma and targeting melanoma that has metastasized to other organs.

[0211] In some embodiments, the bacteria described herein are useful for inducing an antigenspecific immune response to a non-native protein or peptide (e.g., a non-native antigen), which results in the generation or expansion of T cells that express a T cell receptor that specifically binds to the heterologous antigen or an immunologically active fragment thereof. Thus, the bacteria can be used to treat a disease or condition in a subject by administering a therapeutically effective amount of the bacteria, or a pharmaceutical composition comprising the bacteria, to a subject. Following administration, in some embodiments, the subject’s immune system responds by producing antigen-specific B-cells or T cells that bind the heterologous antigen expressed by the bacteria. In some embodiments, the immune system responds by producing antigen-specific regulatory T cells (Treg), which reduce the host’s immune response against a self-antigen or other antigen corresponding to the expressed heterologous protein or peptide. In some embodiments, the immune system responds by producing antigen-specific T cells (Teff), which modulate an immune response against the expressed non-native protein or peptide, e.g., a tumor associated antigen, neoantigen, or an antigen associated with an infectious disease. In some embodiments, the immune system responds by producing antigen-specific THI cells, which modulate an immune response against the expressed heterologous antigen, such as through promoting cellular immunity (e.g., promoting an immune environment conducive to an antigen-specific CD8 cytotoxic T cell response). In some embodiments, the immune system responds by producing antigen-specific TH2 cells, which modulate an immune response against the expressed heterologous antigen, such as through promoting humoral immunity (e.g., promoting an immune environment conducive to an antigen-specific B cell response and production of antibodies). In some embodiments, the immune system responds by producing antigen-specific T helper 17 cells (THI 7), which modulate an immune response against the expressed heterologous antigen. In some embodiments, the immune system responds by producing antigen-specific T follicular helper cells (TFH), which modulate an immune response against the expressed heterologous antigen. In some embodiments, the immune system responds by producing antigen-specific Bcells, which modulate an immune response (e.g., a humoral immune response) against the expressed heterologous antigen.

[0212] In some embodiments, antigen-specific immune responses induced by commensal bacteria can be localized to the site of administration of the bacteria. In some embodiments, antigen-specific immune responses induced by the bacteria can be restricted to the site of administration of the engineered commensals or other engineered bacteria. In some embodiments, antigen-specific immune responses induced by the bacteria can be distal to the site of administration of the bacteria. In some embodiments, antigen-specific immune responses can include both a localized and distal immune response relative to the site of administration of the bacteria.

[0213] In some embodiments, antigen-specific immune responses induced by the bacteria can be localized to a native host niche colonized by the bacteria (e.g., a specific organ, such as skin). In some embodiments, antigen-specific immune responses induced by the bacteria can be restricted to a native host niche colonized by the bacteria. In some embodiments, antigen-specific immune responses induced by the bacteria can be distal to a native host niche colonized by the engineered commensals or other engineered bacteria (e.g., an antigen-specific immune response in an organ, or site in a subject, that is not colonized by the bacteria). For example, a distal antigen-specific immune response can include stimulation of immune cells at a native host niche colonized by the bacteria followed by migration of the immune cells to another site (e.g., another organ). As a non-limiting illustrative example, the bacteria that colonize the skin can induce an antigen-specific immune response that results in immune cells (e.g., antigen-specific T cells) carrying out their effector function in organs other than the skin. In certain embodiments, the organ other than the skin is the lungs, breasts, prostate, colon, bladder, uterus, kidney, liver, pancreas, thyroid, or ovaries. In some embodiments, antigen-specific immune responses can include both a localized and distal immune response relative to a native host niche. In certain embodiments, the antigen-specific immune response targets metastases, such as skin melanoma that has metastasized to other organs.

[0214] In some embodiments, distal antigen-specific immune responses are distal relative to the site of administration of the bacteria. In some embodiments, distal antigen-specific immune responses are distal relative to a host niche colonized by the bacteria. In some embodiments, distal antigen-specific immune responses are in the same organ as the site of administration ofthe bacteria and / or the native host niche colonized by the bacteria. In certain embodiments, the bacteria is applied to and / or colonizes one area of skin and produces an immune response in a separate part of the skin, such as a melanoma skin metastasis. In some embodiments, distal antigen-specific immune responses are in a different organ as the site of administration of the bacteria and / or the native host niche colonized by the bacteria. In certain embodiments, the bacteria is applied to and / or colonizes the skin and produces an immune response in an organ other than skin, such as a melanoma that has metastasized to other organs. In some embodiments, distal antigen-specific immune responses are in both the same organ and a different organ as the site of administration of the bacteria and / or the native host niche colonized by the engineered commensals or other engineered bacteria. In certain embodiments, the bacteria is applied to and / or colonizes the skin and produces an immune response both in the skin and in an organ other than skin, such as targeting skin melanoma and targeting melanoma that has metastasized to other organs.

[0215] In another aspect, provided are methods for inducing an antigen-specific T cell or B cell response to a non-native protein, peptide, antigen, or antigenic fragment thereof, expressed or displayed by a bacterium as described herein. In certain embodiments, the methods can be performed in vitro or in vivo.T cell response

[0216] In some embodiments, the T cell response is a THI, TH2, TH17, Treg, CD8+, or T Follicular helper (TFH) response. In some embodiments, the live, recombinant commensal bacterium limits differentiation of THI T cells in the host. In some embodiments, the bacterium modulates the native host niche to limit differentiation of THI T cells in the host. In some embodiments, the bacterium promotes differentiation of TH2 T cells in the host. In some embodiments, the bacterium modulates the native host niche to promote differentiation of TH2 T cells in the host.

[0217] In certain embodiments, a T cell response after administration of a modified bacterium as described herein can include cytokine and / or chemokine expression, or cell killing. In some embodiments, the T cell response comprises a cytokine and / or chemokine response. In some embodiments, the T cell response comprises increased secretion of cytokines and / or chemokines. Increased secretion of cytokines and / or chemokines includes, but is not limited to, an increase in the number of T cells secreting cytokines and / or chemokines as compared to the administrationof a non-modified bacterium; an increase in the amount or volume of secreted cytokines and / or chemokines as compared to the administration of a non-modified bacterium; enhanced secretion of cytokines and / or chemokines by T cells as compared to the administration of a non-modified bacterium; or an induction of the secretion of cytokines and / or chemokines as compared to the administration of a non-modified bacterium. In some embodiments, the T cell response comprises a TH2 response.

[0218] In some embodiments, the T cell response comprises a cytotoxic T cell response. An increased cytotoxic T cell response includes, but is not limited to, an increase in the number of cytotoxic T cells as compared to the administration of a non-modified bacterium; an increase in the activation of cytotoxic T cells as compared to the administration of a non-modified bacterium; enhanced activation of cytotoxic T cells as compared to the administration of a nonmodified bacterium; or an induction of cytotoxic T cell activation as compared to the administration of a non-modified bacterium.

[0219] In some embodiments, the T cell response does not comprise a THI response. In certain embodiments, limiting, suppressing, or reducing a THI response, include, but is not limited to, a reduction or decrease in the number of THI T cells or activated THI T cells as compared to the administration of a non-modified bacterium.

[0220] Regulatory T cells (TregS) have pluripotent anti-inflammatory effects on multiple cell types. In particular, they control the activation of innate and adaptive immune cells. Tregs acting in an antigen-specific manner reduce effector T cell activation and function, for example, after effector T cells have successfully mounted an attack against an invading pathogen, or to suppress reactivity to self-antigen and thereby prevent autoimmune disease.

[0221] Tregcells play a major role in establishing and maintaining immune homeostasis in peripheral tissues, particularly at barrier sites where they stably reside. In the intestinal lamina propria, Tregcells not only maintain self-tolerance but also play a crucial role in mediating tolerance to commensal organisms. A large percentage of gut-resident Tregcells recognize commensal antigens, and thymically derived Tregcells support tolerance to intestinal microbes. In addition, certain bacterial species expand Tregcells in the lamina propria.

[0222] TregSare a subset of T helper (TH) cells, and are considered to be derived from the same lineage as naive CD4+ cells. Tregs are involved in maintaining tolerance to self-antigens, and preventing auto-immune disease. Tregsalso suppress induction and proliferation of effector Tcells (Teff). Tregsproduce inhibitory cytokines such as TGF-0, IL-35, and IL-10. Tregs express the transcription factor Foxp3. In humans, the majority of Tregcells are MHC-II restricted CD4+ cells, but there is a minority population that are FoxP3+, MHC-I restricted, CD8+ cells. Tregs can also be divided into subsets: “natural” CD4+ CD25+ FoxP3+ Tregcells (nTregs) that develop in the thymus, and “inducible” regulatory cells (iTregs) which arise in the periphery. Naturally occurring Tregs suppress self-reactive immune responses in the periphery. iTregs are also CD4+CD25+FoxP3+, and develop from mature CD4+ T cells in the periphery (z.e., outside of the thymus) from conventional CD4+ T cells to ensure tolerance to harmless antigens, including those derived from, for example, food and intestinal flora. Both subsets of Tregcells are characterized by expression of high levels of CD25 and the transcription factor Foxp3. Tregsare thought to inhibit the antigen-specific expansion and / or activation of self-reactive effector T cells and to secrete suppressive cytokines, including TGF-0 or IL- 10. iTregs can also express both RORyt and Foxp3. Research has shown that TGF-0 and retinoic acid produced by dendritic cells can stimulate naive T cells to differentiate into Tregs, and that naive T cells within the digestive tract differentiate into Tregsafter antigen stimulation. iTregscan also be induced in culture by adding TGF-0.

[0223] T effector (Teff) cells generally stimulate a pro-inflammatory response upon antigenspecific T Cell receptor (TCR) activation via the expression or release of an array of membranebound and secreted proteins that are specialized to deal with different classes of pathogen. Teffcells are usually divided into CD8+ cytotoxic T cells and T helper cells. T helper cells can be further classified as THI cells, TH2 cells, and TH17 cells.

[0224] CD 8+ cytotoxic T cells recognize and kill target cells that display peptide fragments of intracellular pathogens (e.g., viruses) presented in the context of MHC-I molecules at the cell surface. CD8+ cytotoxic T cells store preformed cytotoxins in lytic granules which fuse with the membranes of infected target cells. CD8+ cytotoxic T cells additionally express Fas ligand, which induces apoptosis in Fas-expressing target cells.

[0225] T helper (TH) cells are a class of CD4+ cells that function to regulate the proliferation of B cells and B cell responses. TH cells play an important role in humoral immunity and immunopathology. CD4 + T helper cells differentiate into either THI or TH2 cells. Both THI and TH2 cells express CD4 and recognize peptide fragments processed within intracellular vesicles and presented on the cell surface in the context of MHC-II molecules. THI cells can directly orindirectly activate a number of other immune cells, including macrophages and B cells, thereby promoting more efficient destruction and clearance of intracellular microorganisms. THI cells can also be involved in pathways that lead to activation of CD8+ cytotoxic T cells. TH2 cells stimulate the differentiation of B cells and promote the production of antibodies and other effector molecules of the humoral immune response. TH cells can differentiate into THI or TH2 T cells depending upon antigen stimulation and cytokine environment. T helper cells first activated by antigen in the presence of IL- 12 develop predominantly into THI cells, whereas those activated in the presence of IL-4 develop predominantly into TH2 cells. Progenitor T helper cells may require cellular divisions before becoming competent to synthesize the cytokines that are indicative of either the THI or TH2 pathway. THI and TH2 cell phenotypes are different from each other in early activation signal transduction pathways, especially in the different roles of TCR- related protein tyrosine kinases. TCR and its downstream protein tyrosine kinases such as Fyn, p56(Ick), and ZAP-70 are involved in the development and differentiation of THI and TH2 cells. TH17 cells are a subset of pro-inflammatory TH cells that express IL-17. TH17 cells are developmentally distinct from THI and TH2 cells. The signaling pathway that induces differentiation of TH cells into THI 7 cells inhibits Tregdifferentiation.

[0226] T follicular helper cells (TFH) are a subset of CD4+ cells. TFH cells are essential for helping cognate B cells form and maintain the germinal center (GC) reaction, and for development of humoral immune responses. These cells are defined by expression of the chemokine receptor CXCR5, which directs them to the B cell follicles via gradients of the chemokine CXCL131. TFH cells also express the transcription factor Bcl6 (which represses Blimp- 1 / Prdml) and high levels of the costimulatory receptor ICOS, which are both critical for their differentiation and maintenance. In addition, TFH cells secrete large amounts of IL-21, which aids in GC formation, isotype switching and plasma cell formation. In humans and mice, functionally similar TFH cells can be found in secondary lymphoid organs. CXCR5+ TFH cells are also present in peripheral blood and seen at elevated levels in individuals with autoantibodies.B Cell Response

[0227] In some embodiments, the antigen-specific response is a B cell response. A B cell response can include secretion of antibodies. In some embodiments, the B cell response is an IgA, IgG, IgM, or IgE producing plasma cell response.

[0228] In certain embodiments, a B cell response after administration of a modified bacterium, as described herein, is an increase in antibody production by B cells. In some embodiments, the B cell response comprises an IgA, IgG, IgM, or IgE producing plasma cell response. In some embodiments, the B cell response comprises an IgA, IgG, IgM, or IgE producing memory B cell response. In some embodiments, the B cell response comprises increased production of IgA, IgG, IgM, or IgE antibodies by plasma cells and / or memory B cells. In certain embodiments, increased secretion of IgA, IgG, IgM, or IgE antibodies includes, but is not limited to, an increase in the number of B cells secreting IgA, IgG, IgM, or IgE antibodies as compared to the administration of a non-modified bacterium; an increase in the amount or volume of secreted IgA, IgG, IgM, or IgE antibodies as compared to the administration of a non-modified bacterium; enhanced secretion of IgA, IgG, IgM, or IgE antibodies by plasma cells or memory B cells as compared to the administration of a non-modified bacterium; and / or an induction of the secretion of IgA, IgG, IgM, or IgE antibodies by plasma cells or memory B cells as compared to the administration of a non-modified bacterium.

[0229] B cells are a part of the humoral immunity component of the adaptive immune system and secrete antibodies. B cells can also act as APCs and secrete cytokines. Immature B cells travel from the bone marrow to secondary lymphoid organs such as the spleen and lymph nodes. B cells are activated in the secondary lymphoid organs when they bind an antigen via the B cell receptor (BCR). There are multiple types of B cells, including plasmablasts, plasma cells, lymphoplasmacytoid cells, memory B cells, follicular (FO) B cells, marginal zone (MZ) B cells, BI B cells, and regulatory B cells. FO B cells preferentially undergo T cell dependent activation. MZ B cells can undergo both T cell dependent and T cell independent activation. Once activated, B cells undergo a two-step differentiation process resulting in both short lived plasmablasts as well as long lived plasma cells and memory B cells. Plasma cells are long lived, nonproliferating cells that secrete antibodies that recognize a specific antigen. Memory B cells are a dormant B cell that function to provide a stronger, more rapid antibody response after a second encounter with an antigen or infection. TFH cells are involved in the activation and differentiation of memory B cells. B cell differentiation, memory B cells, and antibody secretion by B cells are generally described in “Dynamics of B cells in germinal centres,” Nilushi S. et al., doi:10.1038 / nri3804, Nature Reviews Immunology, 15, 137-148 (2015); “Memory B cells,” Tomohiro Kurosaki, Kohei Kometani & Wataru Ise, doi:10.1038 / nri3802, Nature ReviewsImmunology, 15, 149-159 (2015); and “The generation of antibody-secreting plasma cells,” Stephen L. Nutt, Philip D. Hodgkin, David M. Tarlinton & Lynn M. Corcoran, doi:10.1038 / nri3795, Nature Reviews Immunology, 15, 160-171 (2015).

[0230] Exemplary B-cell surface markers include the B cell receptor (BCR), CD10, CD19, CD20 (MS4A1), CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85, and CD86 leukocyte surface markers (for descriptions, see The Leukocyte Antigen Facts Book, 2ndEdition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other B-cell surface markers include RP105, FcRH2, B-cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRHl, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287.

[0231] In some embodiments, the B cell response is an IgA, IgG, IgM, or IgE producing plasma cell response.Antigen Presenting Cells (APC)

[0232] In some embodiments, a modified bacterium expressing or displaying a non-native protein or peptide of interest as described herein is contacted with an antigen presenting cell (APC), wherein the APC phagocytizes the modified bacterium and processes the heterologous antigen, or antigenic fragment thereof, for presentation on MHC-I or MHC-II molecules. In some embodiments, the modified bacterium comprises a live, recombinant commensal bacterium expressing or displaying a non-native protein or peptide of interest that is contacted with an APC, wherein the APC phagocytizes the recombinant bacterium and processes the heterologous antigen, or antigenic fragment thereof, for presentation on MHC-I or MHC-II molecules. In certain embodiments, examples of APCs include dendritic cells, macrophages, Langerhans cells, B cells, intestinal epithelial cells, and innate lymphoid cells, splenic dendritic cells, CD8+ dendritic cells, CD1 lb+ dendritic cells, plasmacytoid dendritic cells, follicular dendritic cells, monocytic cells, macrophages, bone marrow-derived macrophages, and Kupffer cells. In some embodiments, the APC is a dendritic cell, a splenic dendritic cell, a CD8+ dendritic cell, a CD1 lb+ dendritic cell, a plasmacytoid dendritic cell, a follicular dendritic cell, a monocytic cell, a macrophage, a bone marrow-derived macrophage, a Kupffer cell, a B-cell, a Langerhans cell, an innate lymphoid cell, a microglial cell, or an intestinal epithelial cell. In some embodiments,the APC is a dendritic cell, such as a CD 103+CD 1 lb+ dendritic cell. In some embodiments, the APC is an intestinal macrophage, such as a CX3CR1+ intestinal macrophage.

[0233] In some embodiments, the APC displaying the processed heterologous antigen in complex with an MHC molecule on its cell surface is then contacted with a T cell, such as a naive T cell. In some embodiments, binding of the processed heterologous antigen / MHC complex to the T Cell Receptor (TCR) on the naive T cell results in activation of the TCR and differentiation of the naive T cell into a Treg. In some embodiments, binding of the processed heterologous antigen / MHC complex to the T Cell Receptor (TCR) on the naive T cell results in differentiation of the naive T cell into an effector T cell (Teff).

[0234] In certain embodiments, the induction of an antigen-specific T cell response can be detected using a suitable assay, such as cell surface marker expression analysis (e.g., by flow cytometry analysis) for specific T cell sub-populations. In certain embodiments, suitable assays for detecting Tregand TH2 cells are described herein or known by one of skill in the art.

[0235] In certain embodiments, in an in vitro method of inducing an antigen-specific T cell response, modified bacterium expressing or displaying a heterologous antigen of interest are cultured with APCs in a suitable media under conditions that permit the APC to phagocytize the bacteria, process the heterologous antigen, and display the processed antigen on the cell surface. In certain embodiments, in an in vitro method of inducing an antigen-specific T cell response, live, recombinant commensal bacteria expressing or displaying a heterologous antigen of interest are cultured with APCs in a suitable media under conditions that permit the APC to phagocytize the bacteria, process the heterologous antigen, and display the processed antigen on the cell surface. In certain embodiments, naive T cells can be added to the in vitro culture of APCs and bacteria, or the APCs can be isolated from the bacteria and cultured with the naive T cells. In certain embodiments, the media can contain growth factors and cytokines that promote survival and differentiation of the T cells into a given T cell subset. In some embodiments, the media contains factors that promote the differentiation of Tregcells, such as TGF- . In some embodiments, the media contains factors that promote the differentiation of Teffcells, such as IL- 12, IL-2, and IFNy.

[0236] In some embodiments, the T cells are primary T cells. In some embodiments, the T cells are primary T cells isolated from the gut or spleen of a subject. In some embodiments, the isolated T cells include fully differentiated Tregs. In some embodiments, freshly isolated primaryT cells are cultured in basic medium (i.e., Dulbecco’s Modified Eagle’s Medium +5% Fetal Bovine Serum) without growth factors or cytokines.

[0237] In another embodiment of inducing an antigen-specific T cell response, the method is an in vivo method. In some embodiments, a subject is administered a pharmaceutical composition comprising a modified bacterium expressing or displaying a heterologous antigen of interest. In some embodiments, a subject is administered a pharmaceutical composition comprising a modified bacterium that is a live, recombinant commensal bacteria expressing or displaying a heterologous antigen of interest. The pharmaceutical composition can be administered by any suitable route, iurther described herein. In some embodiments the pharmaceutical composition is ingested by the subject for delivery of the recombinant bacteria to a native gastrointestinal niche in the subject. In some embodiments, the pharmaceutical composition is administered topically for delivery of the recombinant bacteria to an epidermal niche on the subject. In certain embodiments, upon administration of the pharmaceutical composition comprising a modified bacterium, the modified bacterium is phagocytized by an APC in the subject, processed, and presented to naive T cells in the subject, thereby inducing an antigen-specific T cell response. In certain embodiments, upon administration of the pharmaceutical composition comprising a live, recombinant commensal bacteria, the live, recombinant commensal bacteria is phagocytized by an APC in the subject, processed, and presented to naive T cells in the subject, thereby inducing an antigen-specific T cell response. In some embodiments, administration of the pharmaceutical composition elicits an antigen-specific Tregresponse. In some embodiments, administration of the pharmaceutical composition elicits a Teffresponse.

[0238] In some embodiments, differentiation into Tregs is influenced by the type of bacteria engulfed by an APC. In some embodiments, a heterologous antigen can induce the differentiation of different T cell populations depending on the bacterial strain the heterologous antigen is expressed in. In some embodiments, a live, recombinant commensal bacterium derived from a bacterial strain that is commensal to a mammalian gut niche can induce a Tregresponse specific for the heterologous antigen expressed by the recombinant bacterium, whereas the same heterologous antigen when expressed in a live, recombinant commensal bacterium derived from a bacterial strain that is commensal to a skin niche of a mammal induces the generation of an antigen-specific CD8+ Teffresponse.Methods of preventing or treating a disease, disorder or condition using engineered bacteria

[0239] Also provided are methods of preventing or treating a disease, disorder or condition in a subject with a pharmaceutical composition comprising a bacterium as described herein. In some embodiments, the disease, disorder or condition in a subject is an autoimmune disease, disorder or condition in a subject. In some embodiments, the disease, disorder or condition in a subject is an infectious disease. In some embodiments, the disease, disorder or condition in a subject is a cancer or proliferative disorder. In some embodiments, the administration of the bacterium or pharmaceutical composition comprising a recombinant bacterium or surface-labeled bacterium described herein induces a T cell or B cell response. In some embodiments, the administration of the bacterium or pharmaceutical composition comprising a recombinant bacterium or surface- labeled bacterium described herein induces a TeffT cell response. In some embodiments, the administration of the bacterium or pharmaceutical composition comprising a recombinant bacterium or surface-labeled bacterium described herein induces a TregT cell response. In some embodiments, the administration of the bacterium or pharmaceutical composition comprising a recombinant bacterium or surface-labeled bacterium described herein induces a TH2 T cell response. In some embodiments, the administration of the bacterium or pharmaceutical composition comprising a recombinant bacterium or surface-labeled bacterium described herein induces an immune response. In some embodiments, the immune response promotes differentiation of TH2 T cells in the host. In some embodiments, the immune response limits differentiation of THI T cells in the host.

[0240] In some embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a modified bacterium, e.g., a live recombinant commensal bacterial cell or strain, described herein to the subject. In certain embodiments, the pharmaceutical composition can be administered to the subject by any suitable route that does not trigger an adverse reaction in the subject. In certain embodiments, the pharmaceutical composition can be administered by oral, nasal, vaginal, rectal, topical, subcutaneous, intradermal or intramuscular routes. In some embodiments, the pharmaceutical composition is ingested orally by the subject, administered topically to the subject, inhaled by the subject, or injected into the subject. In some embodiments, the pharmaceutical composition is administered in a material, such as a delayed release enteric coating, that permits transit throughthe stomach to the small intestine before the pharmaceutical is released. In some embodiments, the pharmaceutical composition comprises an enteric-coated capsule containing a bacterium described herein.

[0241] In some embodiments, pharmaceutical compositions comprising a bacterium as described herein is used for the prevention or treatment of an autoimmune disease. In certain embodiments, examples of autoimmune diseases that can be treated by a modified microorganism disclosed herein include multiple sclerosis, psoriasis, celiac disease, diabetes mellitus Type I, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, Graves’ disease, Hashimoto's autoimmune thyroiditis, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, temporal arteritis, ulcerative colitis, Crohn's disease, scleroderma, antiphospholipid syndrome, autoimmune hepatitis type 1, primary biliary cirrhosis, Sjogren's syndrome, Addison's disease, dermatitis herpetiformis, Kawasaki disease, sympathetic ophthalmia, HLA-B27 associated acute anterior uveitis, primary sclerosing cholangitis, discoid lupus erythematosus, polyarteritis nodosa, CREST Syndrome, myasthenia gravis, polymyositis / dermatomyositis, Still's disease, autoimmune hepatitis type 2, Wegener's granulomatosis, mixed Connective tissue disease, microscopic polyangiitis, autoimmune polyglandular syndrome, Felty's syndrome, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre Syndrome, Behcet disease, autoimmune neutropenia, bullous pemphigoid, essential mixed cryoglobulinemia, linear morphea, autoimmune polyglandular syndrome 1 (APECED), acquired hemophilia A, Batten disease / neuronal ceroid lipofuscinoses, autoimmune pancreatitis, Hashimoto's encephalopathy, Goodpasture's disease, pemphigus vulgaris, autoimmune disseminated encephalomyelitis, relapsing polychondritis, Takayasu arteritis, Churg-Strauss syndrome, epidermolysis bullosa acquisita, cicatricial pemphigoid, pemphigus foliaceus, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, autoimmune orchitis, autoimmune polyglandular syndrome, Cogan's syndrome, encephalitis lethartica, erythema elevatum diutinum, Evans syndrome, immunodysregulation polyendocrinopathy enteropathy X- linked (IPEX), Issac's syndrome / acquired neuromyotonia, Miller Fisher syndrome, Morvan's syndrome, PANDAS, POEMS syndrome, Rasmussen's encephalitis, stiff-person syndrome,Vogt-Koyanagi-Harada syndrome, neuromyelitis optica, graft vs host disease, esophageal encephalitis, and autoimmune uveitis.

[0242] In some embodiments, pharmaceutical compositions comprising a bacterium as described herein is used for the prevention or treatment of a proliferative disorder. In some embodiments, the proliferative disorder is cancer. In some embodiments, the cancer is melanoma, kidney, hepatobiliary, head-neck squamous carcinoma (HNSC), pancreatic, colon, bladder, glioblastoma, prostate, lung, breast (mammary), ovarian, gastric, kidney, bladder, esophageal, renal, melanoma, leukemia, lymphoma, mesothelioma, basal cell carcinoma, squamous cell carcinoma, or testicular cancer.

[0243] In some embodiments, pharmaceutical compositions comprising a bacterium as described herein is used for the prevention or treatment of a proliferative disease. In certain embodiments, examples of proliferative diseases include melanoma, basal cell carcinoma, squamous cell carcinoma, and testicular cancer.

[0244] In some embodiments, pharmaceutical compositions comprise a bacterium as described herein engineered to express or surface-labeled to display a neoantigen or tumor-associated antigen identified in cancer cells from an individual cancer subject. In certain embodiments, a bacterium engineered to express or surface-labeled to display an identified neoantigen or tumor- associated antigen, can be administered in a pharmaceutical formulation to elicit an adaptive T cell response in the cancer subject or ex vivo cultured with HLA-matched donor T cells that can subsequently be introduced into the cancer subject to recognize and kill the cancer cells.

[0245] Any suitable animal model can be used to test the methods described herein. In some embodiments, the animal model is a mouse model, or a non-human primate model.

[0246] In some embodiments, pharmaceutical compositions comprising a bacterium as described herein is used for the prevention or treatment of a proliferative disease. In certain embodiments, examples of proliferative diseases include melanoma, basal cell carcinoma, squamous cell carcinoma, and testicular cancer.

[0247] In some embodiments, a commensal bacterium as described herein is co-administered with one or more additional agents. In certain embodiments, a therapeutically effective amount of one or more additional agents can be co-administered. In certain embodiments, coadministration generally refers to administering two or more agents (e.g., a recombinant commensal bacterium and a second agent), such that each agent is capable of exerting theirpharmacological effect during the same period of time; such co-administration can be achieved by either simultaneous, contemporaneous, or sequential administration of the two or more agents. In certain embodiments, agents that can be co-administered include immune checkpoint inhibitors, chemotherapeutic agents, and / or cell-based therapies. In certain embodiments, illustrative immune checkpoint inhibitors include, but are not limited to, Tremelimumab (CTLA- 4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (Anti-B7-Hl; MEDI4736), ipilimumab, MK-3475 (PD-1 blocker), Nivolumamb (anti-PDl antibody), CT-011 (anti-PDl antibody), BY55 monoclonal antibody, AMP224 (anti-PDLl antibody), BMS-936559 (anti- PDL1 antibody), MPLDL3280A (anti-PDLl antibody), MSB0010718C (anti-PDLl antibody) and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor). In certain embodiments, illustrative chemotherapeutic agents include, but are not limited to, alkylating agents such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine (DTIC), nitrosoureas, temozolomide (oral dacarbazine); anthracyclines, such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin; cytoskeletal disruptors, such as paclitaxel, nab-paclitaxel, docetaxel, abraxane, and taxotere; epothilones; histone deacetylase inhibitors such as vorinostat and romidepsin; inhibitors of topoisomerase I such as irinotecan and topotecan; inhibitors of topoisomerase II such as etoposide, teniposide and tafluposide; kinase inhibitors such as bortezomib, erlotinib, gefitinib, imatinib, vemurafenib and vismodegib; nucleotide analogs and precursor analogs such as azacitidine, azathioprine, capecitabine; peptide antibiotics such as bleomycin and actinomycin; platinum-based agents, such as carboplatin, cisplatin and oxaliplatin; retinoids such as tretinoin and alitretinoin; and vinca alkaloids and derivatives such as vinblastine, vincristine, vindesine and vinorelbine. In certain embodiments, cell-based therapies include, but are not limited to, immune cells engineered to express chimeric antigen receptors (e.g., CAR-T and / or CAR-NK therapies) or exogenous T cell receptors.EXAMPLESExample 1: S. epidermidis induces a potent B cell response

[0248] To determine whether immune response to S. epidermidis involves B cells in addition to T cells, S. epidermidis NIHLM087 (LM087), a primary isolate from a healthy donor that elicits antigen-specific CD8+ and CD4+ T cells under conditions of physiologic colonization, and S. epidermidis NIHLM088 (LM088), a strain isolated from the same donor and body site as LM087 but that does not induce CD8+ T cells were used to colonized the head of unprepped, conventionally housed C57BL / 6 mice gently, using a cotton swab. These conditions do not yield an infection, barrier breach, or inflammation; instead, they simulate physiologic microbial colonization of a host.

[0249] Mice were housed at Stanford University under specific pathogen-free conditions according to the guidelines established by the institutional animal care and use committee from Stanford University. C57BL / 6 (B6), C57BL / 6 CD45.1 (4007), Rag2- / - OT-I (2334), Rag2- / - OT-II (11490) mice were purchased from Taconic.

[0250] After colonizing mice for 6 weeks, serum was harvested, and S. epidermidis-specific antibodies were measured by ELISA against immobilized cells of the bacterial colonist. Colonized mice had higher levels of S. epidermidis-specific IgG in the serum than uncolonized controls; LM088 induced particularly high titers as seen in FIG. 1 A.

[0251] The magnitude of the antibody response over time was analyzed. Mice were colonized with LM088 by gentle topical application; at each timepoint a group of mice was euthanized, serum was collected, and antibody titers were measured. Increasing titers of LM088-specific IgG that reached ~2 x 104by six weeks were seen and remained steady for the duration of the nine- week experiment as seen in FIG. IB. Serum antibodies were dominated by IgGl and IgG2b, with lower levels of other isotypes (IgG2b>IgGl>IgG2c>IgA) as seen in FIG. 1C. Notably, increasing levels of S. epidermidis-specific IgA were observed in the nares as seen in FIG. ID.

[0252] Having observed a systemic B cell response involving class-switched antibodies, it was reasoned that a germinal center might be present in the draining lymph nodes. Two weeks after the initiation of colonization, B cells in the skin-draining lymph nodes were examined formarkers of germinal center induction. LM088 elicited germinal center B cells, whereas Corynebacterium accolens — a skin colonist that induces y8 T cells — did not as seen in FIG. IE.Example 2: The predominant target of the antibody response is Aap

[0253] To identify the molecular target of the antibody response against S. epidermidis, wildtype LM088 was incubated with serum from LM088-colonized mice and imaged by electron microscopy. Antibody labeling of the cell wall was observed, as seen in FIG. 2A, which was consistent with the view that the target(s) are surface molecules.

[0254] To determine what class of surface molecule was bound by the antibodies, a set of LM087 mutants was used with defined cell envelope alterations: Dlgt, which is deficient in lipoproteins; DdltA, which is missing the D-alanine modification on teichoic acid; DsrtA, which lacks cell-wall-attached proteins; and DtagO, which fails to elaborate wall teichoic acid.

[0255] Each of these mutants was immobilized on a plate and probed them with serum from LM087-colonized mice. Only DsrtA showed a ~10-fold reduction in antibody binding, indicating that a large fraction of the S. epidermidis-specific antibody pool binds to cell- wall-attached substrates of sortase as seen in FIG. 2C.

[0256] srtA encodes the enzyme sortase, a transpeptidase that recognizes a C-terminal sorting motif (LPXTG) in its substrates. Catalytic attack by an active site cysteine on the amide bond between T and G yields an acyl-S-enzyme intermediate that is captured by the pentaglycine bridge on lipid II, linking a protein to a peptidoglycan precursor that will be incorporated into the cell wall.

[0257] Two derivatives of LMO88 were constructed: one in which aap is cleanly deleted (Aaap) and the other in which Daap is complemented by a plasmid-borne copy of the gene (Aaap+Aap). These bacterial strains were immobilized on a plate and probed with serum from LMO88- colonized mice. Serum titers were reduced in Aaap and reappeared in Aaap+Aap.

[0258] Aap is a 1 ,431 amino acid protein composed of five domains: an N-terminal signal peptide; an amino-acid repeat domain (four complete and two partial 50-residue repeats); a large parallel b-helix domain of unknown function; a series of highly conserved G5-E repeat sequences (the B-domain) that form a fibril-like structure that extends upward from the cellwall27; and a C-terminal anchor domain that contains the sortase recognition sequence as seen in the schematic in FIG. 2D.

[0259] Noting that LM088 induces higher antibody titers than LM087, to determine whether deletion of aap impacts the strength of the B cell response against LM088 mice were colonized with wild-type LM088 or Aaap for 6 weeks as seen in FIG. 2E. Aaap colonizes with comparable efficiency, but mice colonized by Aaap had a reduced frequency of germinal center B cells, suggesting that the presence of Aap drives a more vigorous B cell response to S. epidermidis.

[0260] These results indicate that Aap is a predominant antigen on the surface of S. epidermidis that helps determine the magnitude of the anti-commensal B cell response.Example 3: Redirecting the B cell response against a non-native immunogen

[0261] By colonizing mice with a variant of LM087 in which a non-native epitope is expressed on the surface, the anti-commensal T cell response could be directed against the non-native epitope. This elicited a robust antitumor immune response, potentially constituting a novel form of vaccination.

[0262] An antibody against Aap might enable a parallel strategy to redirect the B cell response against an unrelated pathogen. By colonizing mice with an engineered variant of S. epidermidis in which Aap is fused to a non-native immunogen, it might be possible to elicit an antibody response against a target of interest, as indicated in the schematic of FIG. 3A.

[0263] LM087 was engineered to express Aap (LM087-Aap), which is not present in its genome. Mice were colonized with LM087-Aap and then their serum antibodies was tested for binding to wild-type LMO88, Aaap, and Aaap+Aap as seen in FIG. 3B. As a negative control, mice were colonized with LM087 harboring an empty vector. IgG from the mice colonized with LM087-Aap recognizes two Aap-expressing strains (LM088 and Aaap+Aap) but not an Aap- deficient mutant (Aaap), confirming a specific antibody response against Aap. In contrast, mice colonized with the negative control strain (LM087 + empty vector) did not have Aap-specific serum IgG. These data demonstrate that it is possible to elicit a B cell response against a non- native protein by expressing it on the surface of S. epidermidis.

[0264] A B cell epitope from a pathogen of interest was introduced as seen in FIG. 3C. As a model immunogen, tetanus toxin fragment C (TTFC) was chosen, a 52 kDa C-terminal domainthat is non-toxic but can elicit a protective antibody response against tetanus toxin. Chimeric versions of four carrier proteins bearing TTFC were created to determine the ideal scaffold for displaying the immunogen: the sortase substrates protein A (Staphylococcus aureus, 11 kDa), M6 (Streptococcus pyogenes, 27 kDa), and Aap; LysM (S. epidermidis, 18 kDa) which binds peptidoglycan non-covalently; and the signal sequence from FepB (S. aureus), targeting TTFC for secretion (sTTFC) into the culture medium.

[0265] To test whether these proteins elicit antibodies against TTFC in the setting of colonization, LMO88 Aaap and LM087 were engineered to express each TTFC fusion protein. Mice were then colonized for 6 weeks with these strains and the titer of TTFC-binding IgG was measured in serum and IgA in nasal washes as seen in FIG. 3D. Mice colonized with Aap-TTFC (wTTFC) had high titers of anti-TTFC IgG in serum and IgA in the nares, demonstrating that engineered S. epidermidis can elicit a robust systemic and mucosal antibody response against a non-native immunogen. Antibody titers elicited by the other chimeric proteins were lower or undetectable, indicating that the spatial and structural requirements for eliciting a B cell response are satisfied by Aap but not other carrier proteins. It is noted that Protein A and the secreted antigen scaffold can elicit a productive T cell response against non-native epitopes, suggesting that the antigen scaffolding requirements may be stricter for a B cell response than a T cell response.

[0266] Having seen that Aap is a productive scaffold, a series of variants of wTTFC were generated (i.e. smaller amino acid repeat domain, duplication of TTFC, or duplication of the B domain). While expressing the immunogen in place of the parallel b-helix domain gave rise to high antibody titers against TTFC as seen in FIG. 3E, other attempts to improve the carrier led to less robust B cell responses.

[0267] The parallel b-helix domain was replaced with a different immunogen as seen in FIG. 3E, to see if this strategy could be extended. A catalytically dead mutant of diphtheria toxin (DT) was cloned into the same site, engineered this transgene into LMO88 Aaap, and mice were colonized topically with the resulting strain (wDT). After six weeks, wDT-colonized mice had high titers of IgG against DT in circulation, showing that this strategy is not limited to a single immunogen, as seen in FIG. 3E.

[0268] It was observed that the antibody titers elicited by wTTFC were sufficiently high that they might be protective against a challenge with tetanus toxin. Mice were colonized for 6 weekswith wTTFC (or wDT as a negative control) and challenged with a lethal dose of tetanus toxin. wTTFC-colonized mice were completely protected from death as seen in percent survival curve in FIG. 3F. It was observed that symptoms of toxin exposure demonstrated that topical colonization with an engineered skin commensal can yield protection against a heterologous challenge. It was also observed that the mice colonized with wTTFC remained symptom-free for the duration of the experiment.

[0269] To determine if the protection was mediated by neutralizing antibodies, serum was isolated from colonized mice, incubated with tetanus toxin, and then mice were injected with the toxin as seen in FIG. 3G (mice injected at the timepoint indicated by *). Pre-treatment with wTTFC serum (but not wDT serum) was sufficient to prevent death from toxin exposure, suggesting that the protective effect of wTTFC is mediated, at least in part, by antibodies. These experiments indicate that the antibody response generated by S. epidermidis colonization is sufficiently potent to provide protection against a lethal dose of a bacterial neurotoxin.Example 4: Surface conjugation to expand the immunogen scope

[0270] Given these results, it was reasoned that the scope of immunogens that can be displayed on the surface of S. epidermidis could be expanded. The main limitation of the Aap chimera protein is that the immunogen needs to be functional when expressed in S. epidermidis — properly folded, secreted, and post-translationally modified if needed. Viral glycoproteins, which are large and heavily glycosylated, would be difficult to express in a functional form in bacteria.

[0271] An alternative approach was devised that would allow for expression and purification of a recombinant immunogen separately — e.g., in a mammalian cell — and then the immunogen could be attach it to the surface of S. epidermidis as seen in the schematic of FIG. 4A. The SpyCatcher / SpyTag system was used, which is derived from the extracellular Streptococcus pyogenes protein CnaB2, itself a sortase substrate. CnaB2 is stabilized by an autocatalytic isopeptide bond; by splitting this protein into two pieces, the globular SpyCatcher domain reacts quickly and irreversibly with the 16-aa SpyTag, forming a stable isopeptide bond, as seen in the schematic of FIG. 4B.

[0272] It was reasoned that a S. epidermidis strain expressing a chimeric version of Aap in which the parallel b-helix domain is replaced with SpyCatcher003 (a catalytically enhanced version of SpyCatcher39) could be conjugated with a recombinant protein bearing SpyTag003seen in the schematic of FIG. 4C, left, potentially allowing for a wide range of recombinant proteins to be conjugated to the surface of S. epidermidis.

[0273] Aap-SpyCatcher003 was expressed in S. epidermidis (LM087) hereafter, Aap-sc and , incubated with recombinant superfolder GFP (sGFP) bearing a C-terminal SpyTag003 and analyzed bacterial cells by flow cytometry. After 2 min, Aap-sc cells are conjugated robustly with sGFP-SpyTag003, whereas a variant of this strain expressing a catalytically dead version of SpyCatcher (Aap-sc*) is not efficiently conjugated despite having similar levels of surface expression as seen in the graph of FIG. 4C.

[0274] Looking in vivo, Aap-sc was conjugated with TTFC-SpyTag003 and colonized mice with the immunogen-conjugated cells for six weeks (seen in the schematic of FIG. 4e). As a point of comparison, a nanoparticle vaccine — mi3- SpyCatcher conjugated with TTFC- SpyTag003 (mi3-TTFC) was prepared, which contains up to 60 immunogens per particle — and used to immunize a second group of mice by intramuscular injection. Surprisingly, serum from Aap-sc-TTFC-colonized mice had very high titers of anti-TTFC IgG; at least as potent as wTTFC-colonized mice as seen in FIG. 4E (graphs 1-4), in spite of the fact that the antigen is transient and will dilute as the cells divide. Physical attachment of TTFC to the bacterial cell surface is required for this effect, while it was observed that mixed (but unconjugated) immunogen and bacterial cells yielded only a weak response

[0275] Remarkably, the serum antibody response elicited by Aap-sc-TTFC was of similar magnitude to the highly potent intramuscular mi3-TTFC vaccine. However, while mi3-TTFC induced an IgG response, both Aap-sc-TTFC and wTTFC also generated mucosal responses with high titers of TTFC-specific IgA in the nasal and pulmonary mucosa, as seen in FIG. 4F. Thus, display of antigens on the surface of S. epidermidis using the SpyCatcher system is an efficient strategy to elicit a potent antibody response in vivo, and immunogen dilution over rounds of cell division does not result in lower titers.Example 5: Using heterologous expression to label S. epidermdis

[0276] While Examples 3-4 demonstrate that an immunogen could be conjugated to a surface protein of bacteria and generate an immune response in vivo, there are other ways to attach or conjugate an antigen or immunomodulatory molecule to a surface protein of bacteria. One such way is through heterologous expression of fusion proteins with different substrates (e.g., sortasesubstrate, protein A, M proteins, or LysM). To that end, constructs were generated comprising a signal sequence, LysM domains 1 and 2 coupled to an antigen, as seen in the schematic of FIG.6.

[0277] All antigen vectors for expression in S. epidermidis were designed similarly to what was previously published. Briefly, all vectors were built on a replicative pLI50 plasmid backbone containing a constitutive promoter (Ppen) and a ribosome binding site ftom Staphylococcus aureus delta-hemolysin (hid) gene. Unless otherwise noted, all sequences were codon optimized for S. aureus using the IDT codon optimization tool. For repeated protein sequences, a simple algorithm was used to generate codon optimized DNA sequences that are distinct from each other to avoid recombination. Constructs where the antigen was fused to protein A fragment were based on previous publications. Briefly, the wall-spanning region of protein A XM domain was fused at the C-terminus to the antigens. The signal sequence from protein A was used as described. For secreted constructs, the N-terminal Tat secretion signal from S. aureus fepB (gene locus name SAOUHSC 00326) was fused to the antigen, with no carrier protein. To build fusions to the peptidoglycan binding protein LysM, the LysM protein from LMO88 (N-terminus) was fused to a fragment of LysM from S. epidermidis strain RP62A followed by the antigen domain. To build Aap-TTFC 2x B domain, the B domain from S. epidermidis LM088 on the original Aap-TTFC construct was replaced with the B domain of S. aureus COL strain, which is roughly twice as large. For plasmids for expression in E. coli, all constructs were cloned into a pET-30b vector, similarly to a previously described strategy. To map the epitope within the B domain of Aap, truncations fused to a superfolder GFP (sGFP) protein were cloned to visualize the fragments by immunoblotting. To generate pIMAY-Aap, ~1 kb upstream and ~1 kb downstream of Aap were cloned into pIMAY vector, similar to a previous method.

[0278] Genetic engineering of S. epidermidis with any of the constructed generated, was performed as previously described with minimal changes. Briefly, S. epidermidis was grown overnight in BHI (Difco Brain Heart Infusion media, BD 237200) containing 0.5 M sorbitol (BHIS) (Sigma, S6021) at 37°C shaking (220 rpm). The next day, the cultures were diluted 1 :200, grown to OD600 -0.5-0.8, centrifuged at 3,600 x g at 4°C, washed 5x with ice cold 10% glycerol using the same volume as the volume of cultures (i.e., for 40 ml of culture, wash 5x with 40 ml of 10% glycerol), and resuspended in 10% glycerol. During the washes, the DNA (previously isolated from E. coli DC10B strain) was desalted on 0.025 VSWP membrane filters(Sigma, VSWP02500). Briefly, 22.5 ml of MQ was added to a petri dish and one membrane was floated on the water. DNA was carefully added to the membrane for dialysis for 1 h at RT. Next, 100 ul of resuspended bacteria (see above) was added to Eppendorf tubes containing 0.2-1 ng of desalted DNA, gently mixed and left on ice for ~5 min. For the transformation, the bacteria and DNA were heat shocked for 2 min at 56°C and immediately transferred to a 0.1 cm cuvette (BioRad, 1652089) and electroporated with a pulse of 1.8 kV using a Bio-Rad Micropulser. The typical constant time was 2.3 -2.5 and anything below 2.2 generally did not yield any transformants. After the transformation, the bacteria were transferred into 3 ml of BHIS and moved to a 37°C shaker. After ~lh of recovery, the bacteria were plated on BHIS+chlorlO plates and grown overnight at 37°C. The next day, transformants were grown on BHI+chlorlO plates and confirmed by colony PCR. Single colonies of correct clones were picked and inoculated in BHI + chlorlO for overnight cultures with shaking at 37°C. The next day, the strains were banked, and the DNA sequence confirmed again by either miniprep (for LM087) or PCR (LMO88) to ensure no mutations were acquired during liquid growth.

[0279] As described above, constructs comprising the Tetanus Toxin Fragment C (TTFC) antigen were incorporated into the fusion protein constructs as depicted in FIG. 7B. These constructs were expressed in S. epidermidis engineered to lack Aap (Aaap). Mice were then colonized with the engineered S. epidermidis. Serum and nasal washes were collected and ELISAs were performed for antibody titers. wTTFC (Aap) demonstrated a significant difference compared to the other constructs as seen in FIG. 7C and FIG. 7D. This indicates that heterologous expression of fusion proteins may be used to attach antigens to bacterial surface proteins.Example 6: Using the Transpeptidase, Sortase, to label S. epidermidis with peptides.

[0280] Another effective way conjugate or attach an antigen to a bacterial surface protein is using a transpeptidase. FIG. 8 depicts a schematic for the mechanism of attaching an antigen comprising a C terminal sequence of LPETG (SEQ ID NO: 80) to abacterial surface protein. srtA encodes the enzyme sortase, a transpeptidase that recognizes a C-terminal sorting motif (LPXTG; SEQ ID NO: 79) in its substrates. Catalytic attack by an active site cysteine on the amide bond between T and G yields an acyl-S-enzyme intermediate that is captured by thepentaglycine bridge on lipid II, linking a protein to a peptidoglycan precursor that will be incorporated into the cell wall.

[0281] Peptides comprising T cell epitopes were generated. These peptides comprising T cell epitopes either expressed LPETG (SEQ ID NO: 80), a C-terminal sorting motif recognized by Sortase, coupled to Cy5 or GEGTLP (SEQ ID NO: 99), a scrambled version that would not be recognized by sortase coupled to Cy5 and were added to live S. epidermidis in the presence or absence of Sortase (SrtA) as seen in FIG. 10. When Sortase was present with the C-terminal sorting motif recognized by sortase, the live S. epidermidis demonstrated simple attachment of the peptide.

[0282] To determine if labeling of S. epidermidis with peptides impacted the fitness of the bacteria, labeled bacteria (Cy5-GEGTLP or Cy5-LPETGG) were plated at different densities on plates, as seen in FIG. HA. No appreciable fitness defects were identified compared to unlabeled for either the Cy5-GEGTLP or Cy5-LPETGG labeled bacteria. Looking at the MFI of the labeled bacteria over time, it was demonstrated in FIG. 1 IB, that the labeling of S. epidermidis by the peptide Cy5-LPETGG is rapid and efficient, lasting up to 8 hours.

[0283] To determine if Sortase labeling was concentration dependent, overnight cultures of S. epi strain LM087 were OD600 normalized (500pl of OD600=6 / sample). The bacteria were washed two times with 1ml of PBS and resuspended into lOOpl of staining solution (10, 100 or 500pM of peptide with 0, 1, 50 or 100 of sortase A) and stained for 15min at 4°C rotating. The samples were then washed 2x with ImL of PBS and resuspended into lOOpl of Syto9 staining buffer (1.5pl / lmL of PBS, ThermoFischer, L7012) and stained for 15min at room temperature. The cells were then washed 2 more times with PBS and resuspended into 200pl of PBS for flow cytometry analysis.

[0284] FIGS. 12A-12D depict the histograms of the labeling efficiency. The addition of 1 uM of Sortase with lOuM of the LPETG (SEQ ID NO: 80), a C-terminal sorting motif clearly demonstrates a shift in the histogram, indicating the bacterial surface was successfully labeled at the lowest concentration of either Sortase or the peptide. Increasing concentrations of either the peptide or sortase maintains the shift.

[0285] Labeling efficiency of a variety of different bacteria species was analyzed for incorporation of the peptides Cy5-GEGTLP or Cy5-LPETGG (lOOuM) in the presence of 50 uM Sortase, as seen in FIGS. 13-15. In a majority of the bacteria analyzed, Sortase was able tosuccessfully label the cell surface of the different bacteria species, indicated by a clear shift in the histogram between the scrambled peptide Cy5-GEGTLP and the C-terminal sorting motif Cy5-LPETGG. This same analysis was performed looking at intractable skin colonists such as C. acnes. Sortase was still able to successfully label the bacterial surface, indicated by the clear shift in the histogram between the scrambled peptide Cy5-GEGTLP and the C-terminal sorting motif Cy5-LPETGG as seen in FIG. 16.Example 7: In vivo Proliferation Assay with Sortase labeled bacteria

[0286] Although Example 6 clearly demonstrated the ability of sortase to label S. epidermidis, it was unclear if the peptide the labeled the cell surface could induce an antigen-specific immune response in vivo. To that end, mice were adoptively transferred with 300k Rag2- / - OT-I or 300k Rag2- / - OT-II prelabelled with CellTrace- Violet (ThermoFischer, C34557) (see Bousbaine et al., 2022 for full description). For the sortase labeling, the bacteria were either 1) OD600 normalized to OD600=0.3 and labelled overnight shacking at 37°C in BHI or 2) OD600 normalized to OD600=6 and labelled for 15min at 4°C in PBS. The bacteria were labelled with 0.5mg / ml of peptide (OT1, FIGS. 17-18 or OT2, FIGS. 19-20) and 16pM of Sortase A. After labeling, the bacteria were washed 3x with 50mL of PBS and resuspended into BHI for colonization. The mice were colonized on day 1 and day 3. On day 8, the mice were euthanized, and the cervical draining lymph nodes harvested and stained for flow cytometry analysis seen in FIGS. 17A-18D for OT1 and CD8 T cells and FIGS. 19A-20D for OT2 and CD4 T cells. For each, S. epidermidis labeled with OT1 or OT2 using sortase was able to price and induce proliferation of an antigenspecific CD8 or CD4 T cell response in vitro or in vivo.Example 8: Sortase-based labeling enables attachment of peptides (T cell epitopes) to a diverse community of over 100 live anaerobic gut commensals

[0287] To determine how successful the sortase based labeling could be for other types of bacteria, a diverse group of anaerobic gut commensals were studied for labeling efficiency using sortase and the peptides Cy5-GEGTLP or Cy5-LPETGG, as seen in FIGS. 21 A-22B. A majority of the anaerobic bacteria was successfully labeled using sortase, demonstrated by rightward shift in the peak with the combination of sortase and Cy5-LPETGG.Example 9: In vivo Activation of Antigen-Specific T cells in Mice

[0288] This experiment measures the proliferation of antigen-specific T cells in a mouse. Labeled T cells of known antigen specificity are transferred into a recipient mouse, and if the antigen-specific T cells encounter their cognate antigen (as presented to them by antigen- presenting cells), the labeled T cells will divide and proliferate in the draining lymph nodes, indicative of successful priming, which is the first step to inducing a potent systemic T cell response. FIG. 23 A depicts a schematic of an in vivo activation of antigen-specific T cells in mice colonized with sortase-labelled S. epidermidis. FIGS. 23B depicts graphs of in vivo activation of antigen-specific T cells using sortase-labelled S. epidermidis, of the assay in FIG. 23A. FIG. 23C depicts dot plots of the in vivo activation of antigen-specific T cells in FIG. 23B. FIG. 23D depicts a graph of the percent of proliferating cells that contain the OT1 peptide, of the assay in FIG. 23A. FIG. 23E depicts a graph of the percent of proliferating cells that contain the OT2 peptide, of the assay of FIG. 23C. The graphs demonstrate that success priming has occurred.Example 10: Using Chemical Labeling and a Genetic Carrier to label £ epidermidis

[0289] Another effective way conjugate or attach an antigen to a bacterial surface protein is using a chemical labeling approach together with a genetic carrier. The chemical labeling approach with the genetic carrier allows for pairs of affinity tags to be used, wherein the bacteria can be engineered to express one half of the pair of the affinity tag within a surface protein and the other half can be coupled to the antigen of interest. The interaction of the two affinity tags may couple the antigen of interest to the surface protein. One such example of this approach is the SpyCatcher / SpyTag system.

[0290] To determine if the SpyCatcher system could conjugate an antigen to the bacterial surface protein, four difference constructs were generated: Aap- SpyCatcher EQ mutant (catalytic mutant), Aap-SpyCatcher003 EQ mutant (catalytic mutant), Aap- SpyCatcher, and Aap- SpyCatcher003 as seen in the schematic of FIG. 24A.SpyCatcher conjugation for in vitro experiments

[0291] Overnight cultures of S. epidermidis LM087 + Aap-SpyCatcher003 (sc) or the catalytic mutant (sc*, E77Q mutant of SpyCatcher) were OD normalized (150 pl of OD600 = 8 / sample), washed 2x with 1 ml of RT PBS and incubated for the indicated times (2, 5, 15, or 60 min) with the indicated amount of sGFP-SpyTag003 (0.1, 0.3 and 1 mg / ml) in PBS at RT (totalvolume=150 pl / sample). The cells were washed with 1 ml of PBS, spun down and resuspended in 100 pl of Mycolight staining buffer (1:100 dilution in PBS, MycoLight™ Red JJ94, AAT Bioquest) and incubated for 15 min at RT. The cells were then washed two times with 1 ml of PBS and resuspended in 200 ml PBS for flow cytometry analysis.SpyCatcher labeling for in vitro experiment

[0292] Overnight cultures of S. epi strain LM087 + Aap-SpyCatcher003 were OD normalized (150pl of OD600=8 / sample), washed 2 times with 1ml of PBS and incubated for the indicated times (2, 5, 15, 60min) with the indicated amount of SpyTag003-GFP (0.1, 0.3 and Img / ml) in PBS at room temperature (total volume=150pl / sample). The cells were washed with 1ml of PBS, spun down and resuspended in lOOpl of Mycolight staining buffer (1 : 100 dilution, MycoLight™ Red JJ94, AAT Bioquest) and incubated for 15min at room temperature. The cells were then washed two times with 1ml of PBS and resuspend in PBS for flow cytometry analysis.Protein production and purification

[0293] SpyTag003-GFP, SpyTag003-TTFC were cloned into apET-30b expression vector. Sortase A heptamutant expression vector was acquired from Addgene (plasmid #51141). All expression vectors were transformed into BL21 (DE3) strain (NEB, C2527H) for protein purification. Single colonies of each construct were inoculated into LB+Kanamycin (50 pg / ml) for overnight growth at 37°C shacking at 250rpm. The next day, the cultures were back diluted (1:200 dilution) into IL of Terrific Broth (Fisher Scientific, AAJ75856A1) + Kanamycin (50 pg / ml). When the cultures reached an OD600 of 0.4-0.6, protein expression was induced by adding ImM IPTG and transferring the cultures to 28°C shacking at 250rpm. The next day, the cultures were spun down (3,600g for 8min at 4°C) and resuspended into prechilled 30mL of lysis buffer (50mM Tris-HCl + 150mM NaCl + lOmM imidazole) + DNAse and + Lysozyme + 1 tablet of protease inhibitor (Sigma, 11836170001). The resuspended pellet was sonicated 2-3 times for 5min (15sec sonication 40% output, stop 45 sec). The lysate was then cleared by spinning for 1.5h at 20,000g at 4°C. In the meantime, 6ml of Ni-NTA slurry was washed with 30mL of lysis buffer two times. The clarified lysate was then added to the prewashed Ni-NTA beads and incubated for Ih rotating at 4°C. The beads were then loaded on polypropylene columns (Qiagen, 34964, 2 columns / L of culture), washed two times with 25mL of lysis buffer and eluted in 4ml of elution buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 500mM Imidazole). The eluate was concentrated to ~2.5mL on a Amicon column (EMD Millipore, UFC901024).The eluate was then buffer exchanged using a PD10 column (according to manufacturer’s instructions) into lysis buffer containing 10% of glycerol, aliquoted, flash frozen and stored at - 80°C.

[0294] The bacterial strain S. epidermidis LM087 was engineered to express each of the 4 constructs and SpyTag-GFP was added to evaluate the labeling efficiency of the SpyCatcher approach, as seen in FIG. 24B., FIG. 24C, and FIG. 24.D.

[0295] Seen in FIG. 24B, within 2 minutes the Aap-SpyCatcher003 construct was able to attach the SpyTag-GFP to the cell surface, indicated by the rightward shift in the peak. The same experiment was repeated with different time points (2 min, 5 min, 15 min, 60 minutes) and with different concentrations of SpyTag-GFP (0.1, 0.3, and 1.0 mg / mL) for Aap-SpyCatcher EQ mutant (as seen in FIG. 24C top), Aap-SpyCatcher003 (as seen in FIG. 24C, bottom) or Aap- SpyCatcher (as seen in FIG. 24D). The catalytic mutant demonstrated minimal rightward shift only after an extended period of time and at higher concentrations of SpyTag-GFP. Both Aap- SpyCatcher003 and Aap-SpyCatcher demonstrated rightward shifts at all concentrations and timepoints, indicating that both SpyCatcher constructs could attach a mock antigen (SpyTag- GFP) to the bacterial cell surface protein (Aap).SpyCatcher labeling for in vivo colonizations

[0296] Overnight cultures of S. epi strain LM087 + Aap-SpyCatcher003 were OD normalized (5ml / cage of OD600=6), washed 2 times with 50ml of PBS and incubated for the indicated times (3 or 15min) with the indicated amount of SpyTag003-GFP (0.1 or Img / ml) in PBS at room temperature (total volume=5ml / sample). The cells were washed with 50ml of PBS 3 times and resuspended in 5ml of BHI + chloramphenicol (10 pg / ml). The mice were colonized as described above.TTFC ELISA

[0297] High-binding plates (Coming, 9018) were coated with 0.25pg of SpyTag-TTFC / well in PBS overnight at 4°C. The following day, the plates were washed five times with PBST (PBS + 0.05% Tween-20, Sigma, Pl 379), blocked with 200pl of 2% BSA (Sigma-Aldrich, A7030- 100G) in PBST at room temperature for 2h. The plates were then incubated with the indicated sera from mice in serial dilutions. Next, the plates were washed five times with PBST and incubated with secondary antibody at a dilution of 1 : 10,000 (SouthemBiotech, 1030-05) in 2% BSA / PBST. After five more PBST washes, the plates were revealed using TMB reagents (BD,555214) and the reaction stopped with stop solution (BioLegend, 423001). Antibody titers were calculated by logarithmic interpolation of the dilutions with absorbance at 450nm immediately above and below the absolute value 0.2, as previously described (Ersching et al., 2017).

[0298] To test the ability for the SpyCatcher labeled cells to induce an antigen specific immune response, TTFC- Spytag constructs were generated and purified. Aap-SpyCatcher003 constructs were expressed in S. epidermidis strain and TTFC-Spytag was added in two difference concentrations for either 15 minutes or 30 minutes. The bacteria was then used to colonize mice as depicted in FIG. 25 A. The serum of the mice was analyzed for IgG titers and mice colonized with Aap-SpyCatcher003+TTFC-Spytag Img / ml for 15 minutes or Aap-SpyCatcher003+TTFC- Spytag O.lmg / mL for 30 minutes demonstrated robust neutralizing antibody detection as seen in FIG. 25B. This suggests that chemical labeling and a genetic carrier (e.g., SpyCatcher / SpyTag) is an effective way to attach antigens or immunomodulatory molecules to bacterial surface proteins.Example 11: Using covalent attachment to attach antigens to bacterial surface protein

[0299] Covalent attachment can be used to attach antigens to bacterial surface proteins. One such example of covalent attachment is a click chemistry reaction. A click chemistry reaction can involve an azide alkyne Huisgen cycloaddition reaction. An example of click chemistry includes copper-catalyzed azide-alkyne cycloaddition (CuAAC).

[0300] An alkyne or azide moiety can be introduced into a polypeptide on the surface of the bacterium by providing a non-naturally-occurring amino acid comprising the alkyne or azide moiety to the bacterium, and allowing the bacterium to incorporate the non-naturally-occurring amino acid into its proteins made by the bacterium, which includes surface proteins. Antigens comprising a DBCO group can react with the azide moiety of the cell surface protein, linking the two in a click chemistry reaction, as seen in FIG. 26 A. To analyze whether a click chemistry reaction may be used to label S. epidermidis, S. epidermidis was inoculated with L- azidohomoalanine or 3-azido-D-alanine for incorporation into newly synthesized proteins, as described below.Metabolic incorporation of L-AHA or az-D-ala

[0301] For metabolic incorporation of L-azidohomoalanine (L-AHA), procedures from Shalizi et al., PLoS One, 2020 were utilized. Briefly, a single colony of S. epidermidis was inoculatedovernight in brain heart infusion (BHI) liquid media at 37 °C, with shaking. After 16 hours of growth, the culture was back-diluted 1 : 100 in luria broth (LB) media and grown to mid-log phase at 37 °C, with shaking. Once -OD600 0.5 was reached, 5 mM L-AHA was added to the culture. Cells were incubated at 37 °C with shaking for one hour for metabolic incorporation of L-AHA into newly synthesized proteins. Cultures were harvested by centrifugation and the cells were washed three times with phosphate buffered saline (PBS) before proceeding with click reaction

[0302] For metabolic incorporation of 3-azido-D-alanine (az-D-ala), procedures from Weidenbacher et al., ACS Chem. Bio., 2022 were followed. A single colony of S. epidermidis was grown to saturation in LB media at 37 °C with shaking. This culture was diluted 1 : 1000 into LB containing 1 mM az-D-ala, which was grown to saturation at 37 °C overnight. Cultures were harvested by centrifugation and the cells were washed three times with PBS before proceeding with click reaction.

[0303] Washed S. epidermidis cells containing the non-canonical amino acids (either L-AHA or az-D-ala) were used for a click chemistry attachment of dibenzocyclooctyne (DBCO) derivatized dye (Cy5) or DBCO-cpsl4. S. epidermidis cells (LM087 or LM088) containing the azide- containing amino acids were incubated with 100 mM Cy5-DBCO or 2 mg cpsl4-DBCO in PBS for one hour at room temperature. Cells were washed five times with PBS before proceeding with downstream analysis, seen in FIG. 26. For flow cytometry analysis, the cells were stained with the bacterial viability dye Styo9 (7.5 mM) for 15 minutes and then washed twice more. Cells were acquired on a Beckman Coulter CytoFLEX flow cytometer and data were analyzed using FlowJo software as seen in FIG. 27.

[0304] As seen in in FIGS. 26B and 26C, the bacteria that incorporated azido-d-alanine (az-D- ala) also demonstrated an increase in detection of Cy-5 due to the presence of DBCO indicating a successful click chemistry reaction. Interestingly, the results were the same whether the bacterial strain was LM087 or LM088, or the bacteria was grown in BHI or LB. This suggests that click chemistry may be a useful method to attach antigens to bacterial cell surface proteins.

[0305] The same experiment was repeated with either L-azidohomoalanine (L-AHA) or az-D- ala for metabolic incorporation into protein synthesis. Cells were also stained with the bacterial viability dye Styo9, as seen in FIG. 27. S. epidermidis cells that incorporated either L-AHA or az-D-ala and were treated with Cy5-DBCO demonstrated a successful click chemistry reactionand remained viable, indicated by a right upward shift as seen in FIG. 27 (Top right and bottom right) compared to controls.

[0306] To evaluate the in vivo metabolic labeling of a click chemistry reaction and the ability of the antigens to prime T cells, mice were injected with either OT1 peptide (CD8 T cell antigen) or OT2 peptide (CD4 T cell antigen). One day later, mice were colonized with a S. epidermidis strain (LM087) and az-D-ala alone, OT1-DBCO, or both az-D-ala and OT1-DBCO. After 8 days, serum was collected for analysis of primed T cells (CD8 as seen in FIGS. 28B-28E, and CD4, as seen in FIG. 28G and 281) and percent proliferation (as seen in FIG. 28F and 28J). Not only did successfiil click chemistry reactions take place with the incorporate of the OT1-DBCO peptide in the presence of az-D-ala, but this incorporation was able to prime CD8 T cells or CD4 T cells (FIG. 28E and FIG. 281). This incorporation was able to prime either CD8 T cells or CD4 T cells to at least the same level as administration of either the OT1 or OT2 peptides alone. This indicates that a click chemistry reaction can not only attach antigens in vitro but can attach antigens in vivo which go on to induce an immune response.Example 12: Using covalent attachment using surface exposed free amines and biotinstreptavidin

[0307] Another method to attach antigens to bacterial surface proteins is to use surface exposed free amines, a biotin-N-hydroxy succinimide (NHS) ester and strepadvidin. The surface exposed free amine of the bacterial surface can react with an NHS ester coupled to a biotin molecule. The streptavidin can then react with the biotin to join the streptavidin to the bacterial surface as seen in the schematic of FIG. 29 A. It can be appreciated that the biotin-NHS ester can comprise an antigen or immunomodulatory molecule or the streptavidin can comprise an antigen or immunomodulatory molecule or both.

[0308] To evaluate this labeling method in vitro, S. epidermidis was incubated with either streptavidin or different concentrations (5mM, ImM or 500 nM) of NHS-PEG24- biotin+streptavidin-BV421, wherein successful labeling would be detected by an increased presence (e.g., rightward shift) of a fluorescent tag (BV421). In the presence of any concentration of the NHS-PEG24-biotin and streptavidin, a rightward shift was detected as seen in FIG. 29B, indicating a successful labeling reaction.

[0309] To determine if there was a limit on the size or specificity that NHS esters could label bacteria surfaces, NHS esters were used to attempt to label S. epidermidis with proteins (streptavidin, “SA”) or small molecules (Cy5) using a similar experiment as described above. As seen in FIG. 30, not only were NHS esters able to label S. epidermidis with proteins and small molecules but this method had comparable efficiency to labeling S. epidermidis with sortase.

[0310] To determine if NHS-ester labeling working on different types of bacteria, a similar experiment was attempted as described above, except different types of bacteria were used including Gram-positive, Gram-negative, and anaerobic, as seen in FIGS. 28A-28L. NHS-ester labeling was effective at labeling each of Gram-positive bacteria, Gram-negative bacteria, and anaerobic bacteria.

[0311] NHS-ester labeling was compared with SpyCatcher labeling. S. epidermidis (LM087 or LM088) was labeled with NHS-biotin followed by one of a number of streptavidin (SA)-fusion proteins and was compared to S. epidermidis labeled with SpyCatcher, as seen in FIGS. 32A- 32B. SA-fusion proteins included SA-GFP, SA-TTFC-FLAG, and SA-OVA-FLAG. Successful labeling is demonstrated by an increase in detection (e.g., rightward shift) of GFP or FLAG-PE detected by flow cytometry. NHS-ester labeling was able to label S. epidermidis as successfully as Spycatcher was able to label S. epidermidis.

[0312] While NHS-ester labeling clearly demonstrated successful labeling in vitro, could bacteria labeled using NHS esters elicit an immune response? To that end, LMO88 was labeled with NHS-biotin and streptavidin, or Aap-Spycatcher and TBAg and mice were colonized for 6 weeks. After 6 weeks, serum was collected and analyzed for titers against streptavidin. As seen in FIG. 33, mice colonized with cateria labeled using NHS esters were able to successfully demonstrate an immune response, indicating that NHS-ester labeling could successfully label bacteria in vivo and could generate an immune response.

[0313] It can be appreciated that there are a number of molecules that could be attached to bacterial cell surface proteins using NHS-esters. To that end, it was investigated whether amine reactive crosslinkers could be used to attach linkers to bacterial surface proteins that could be used to label the bacterial surface with small molecules using a click chemistry reaction. For example, a free amine on the bacterial surface could react with NHS ester coupled to an azide. The azide could react with DBCO coupled to a small molecule in a click chemistry reaction to couple the small molecule to the cell surface.

[0314] NHS-esters coupled to azide or TFP-esters coupled to azide were incubated with S. epidermidis strain LM088. DBCO-Cy5 was added to the reaction and bacterial cells were analyzed by flow cytometry for presence of Cy5, as seen in FIG. 34A. Bacterial cells that had both the NHS ester or TFP-ester and the DBCO-Cy5 demonstrated successful labeling compared to bacterial cells lacking the NHS ester. As seen in FIG. 34. B, bacterial cells with TFP-ester were successfully labeled with DBCO-GFP (at both ImM and 500 uM concentrations) to almost the same level as LM087 labeled with SpyCatcher-SpyTag-GFP.Example 13: Increasing valency of covalent attachment by including nanoparticles

[0315] Each of the methods described herein have demonstrated that antigens could be directly coupled to bacterial surface proteins. Nanoparticles can self-assemble and express multiple copies of antigens on their cell surface. Coupling an assembled nanoparticle having multiple copies of antigens thereon to a bacterial cell surface protein could increase valency useful in a potential vaccine application, as seen in FIG. 35. To that end, the nanoparticle mi3 was used for further analysis to determine if mi-3 could self-assemble into a nanoparticle and be attached to a bacterial cell surface using the SpyCatcher attachment method.Production of mi3 nanoparticles

[0316] SpyCatcher003-mi3 particles were prepared as previously described9 by purification from E. coli BL21 (DE3) - RIPL competent cells (Agilent) transformed with a pET28a SpyCatcher-mi3 gene comprising an N-terminal hexa-histidine tag (Addgene, Plasmid #112255). Briefly, transformed cells were selected for with kanamycin and grown in LB until an OD600 of -0.6-0.8. Protein expression was induced by adding 420 pM IPTG and transferring the cultures to 20°C, shaking at 200 rpm for 16 h. Cells were harvested by centrifugation (4,000 x g), pellets were resuspended in lysis buffer (25 mM Tris-HCl pH 8.0, 300 mM NaCl) plus DNAse (Sigma, 10104159001), lysozyme (Sigma, L6876), and protease inhibitors (Sigma, 11836170001), and lysed with a cell disruptor. Clarified lysates were obtained by centrifugation at 21,000 x g for 30 min, and SpyCatcherOO3-mi3 particles were isolated by ammonium sulfate precipitation (25% saturation), followed by dialysis and size exclusion chromatography (SEC) purification using a HiLoad 16 / 600 Superose 6 column (Cytiva) equilibrated with 25 mM Tris-HCl pH 8.0 and 150 mM NaCl (TBS). SpyCatcherOO3-mi3 particles were stored at 4°C and used for conjugations after filtering with a 0.2-pm filter and centrifugation at 20,000 x g for 30 min.Preparation of TTFC-mi3 nanoparticles for immunization

[0317] Purified unconjugated SpyCatcherOO3-mi3 particles were incubated with 3 -fold molar excess of TTFC-SpyTag003 protein at 22°C for 1 h in TBS. Conjugated particles were separated from unconjugated proteins by SEC on a Superose 6 10 / 300 column (Cytiva) equilibrated with PBS. Fractions corresponding to conjugated SpyCatcherOO3-mi3 particles were collected and analyzed by SDS-PAGE (not shown). Concentrations of conjugated particles were determined using the absorbance at 280 nm as measured by a Nanodrop spectrophotometer (ThermoFisher). Conjugated particles were stored at 4°C for up to 1 month after conjugation in PBS buffer. On days 0 and 14, the mice were bled via cheek bleed. For immunization (day 0 and 28), conjugated particles were mixed with 50% v / v PBS / AddaVaxTM adjuvant (Invivogen) (5 mg / mouse in 50 ml) and injected intramuscularly in the thigh of preshaved mice. At the experimental endpoint (day 42), the blood was harvested via cardiac puncture and the serum isolated using coagulating tubes (BD, 365967), as seen in FIG. 32A.

[0318] S. epidermidis LM087 (WT or genetically engineered to express Aap-SpyTag003) was incubated in the presence of Mi3 alone, GFP-SpyTag003, or Mi3+ GFP-SpyTag003 and analyzed by flow cytometry. Only S. epidermidis Aap-SpyTag003 + Mi3+ GFP-SpyTag003 demonstrated a shift in GFP detection as seen in FIG. 36 top compared to controls.

[0319] To study this in vivo, bacteria was engineered to express SpyCatcher or SpyCatcher- RBD wherein the RBD was the receptor binding domain of SARS-CoV-2. Mice were colonized for 6 weeks with the bacteria. At day 0 and day 28, mice were injected with mi3 nanoparticles that also displayed the RBD from SARS-CoV-2, as seen in the schematic of FIG. 37A. After 14 days and 42 days serum was collected and analyzed for IgG titers (FIG. 37B) by ELISA and neutralizing antibodies for SARS-CoV-2 RBD (FIG. 37C). While mice colonized with bacteria displaying SpyCatcher+RBD, the serum IgG levels were inconsistent (FIG. 37B). However, mice injected with the mi3 nanoparticles demonstrated comparatively high serum IgG titers after 2 weeks and 6 weeks. The mice injected with the mi3 nanoparticles for 6 weeks demonstrated almost an order of magnitude higher in a neutralizing assay (FIG. 37C).

[0320] Materials for the nanoparticles production. A custom synthetic peptide containing a SpyTag sequence with a 6-carboxyfhiorescein moiety at the N-terminus and a cysteinecarboxamide residue at the C-terminus (“6FAM-SpyTag-Cys”, full sequence: [6-FAM]-GSGRGVPHIVMVDAYKRYK-GGGC-NH2) was synthesized by CPC Scientific (San Jose, CA) and delivered as the trifluoroacetic acid (TFA) salt at >95% purity. The 6- carboxyfluorescein moiety enables flow cytometric tracking of the attachment of the 6FAM- SpyTag-Cys labeled mRNA-LNPs to bacterial cells. Ovalbumin (OVA) mRNA containing 5- methoxyuridine (5moU) modification was obtained from TriLink Biotechnologies (San Diego, CA). The following antibodies were obtained from Abeam (Cambridge, UK): recombinant rabbit anti-PEG antibody RM105, catalog #abl90652; PE-Goat anti-rabbit IgG H&L, pre-adsorbed, catalog #ab72465. Lipids were obtained from the following sources: SM-102 from Cayman Chemical, l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC) and 1,2-dimyristoyl-rac-glycero- 3 -(methoxy )polyethylene glycol-2000 (DMG-PEG2000) from Avanti Polar Lipids, cholesterol from Sigma, and l,2-dimyristoyl-rac-glycero-3-(maleimide[polyethylene glycol-2000]) (DMG- PEG2000-Mal) from Nanocs Inc.

[0321] Synthesis of lipid nanoparticles containing OVA messenger RNA (mRNAfOVA]- LNPs). Stock solutions of lipids SM-102, cholesterol, DSPC, DMG-PEG2000, and DMG- PEG2000-Mal in ethanol were mixed and diluted in ethanol to form a solution containing 10 mg / mL total lipid and the following molar composition: 50 mol% SM-102; 38.5 mol% cholesterol; 10 mol% DSPC; 1.05 mol% DMG-PEG2000; 0.45% DMG-PEG2000-Mal. OVA mRNA was diluted in 1 mM aqueous sodium citrate buffer, pH 4.0, to a concentration of 150 pg / mL mRNA. The ethanolic lipid and aqueous mRNA solutions were mixed at a volumetric flow rate ratio of 1 :3 and a total flow rate of 3 mL / min using a NanoGenerator™ Flex-M microfluidic mixer (PreciGenome, San Jose, CA) with a Mix-4 disposable mixing chip. A total volume of 1.5 mL of mixed solution was dispensed into a tube containing 3.5 mL of phosphate- buffered saline (PBS), pH 7.4, creating an LNP solution with a final volume of 5 mL. This solution was dialyzed against 500 mL of PBS pH 7.4 at 4°C for 8 h. and then concentrated to approx. 1 mL at 5,000 x g and 4°C using an Amicon centrifugal ultrafiltration device (Millipore) with a molecular weight cutoff of 100 kDa.

[0322] Synthesis of 6FAM-SpyTag-mRNA[OVA]-LNPs by conjugation of 6FAM-SpyTag- Cys to mRNA[OVA]-LNPs. A 1.8-mM stock solution of 6FAM-SpyTag-Cys peptide in deionized water was combined with a solution of mRNA[OVA]-LNPs in PBS such that the total concentration of the DMG-PEG2000-Mal component in the combined solution was equal to its original concentration in the 1.5 mL of LNP solution dispensed from the microfluidic mixer(approx. 270 nmol / 1.5 mL = 18 pM) and the concentration of peptide was 180 pM (i.e. 10-fold excess relative to DMG-PEG2000-Mal). The reaction was incubated at 4°C in an inverting mixer at 24 rpm overnight. Following the incubation, the reaction was desalted using a 2-mL 40-kDa Zeba™ centrifugal desalting column (Pierce) equilibrated in ice-cold PBS according to the manufacturer’s instructions, followed by two rounds of dialysis at 4°C against 13.5 mL PBS in a 0.5-mL 20-kDa Slide-A-Lyzer™ Mini dialysis cassette (Pierce) to remove excess unreacted 6FAM-SpyTag-Cys peptide from the newly formed 6FAM-SpyTag-niRNA[OVA]-LNPs. The theoretical SpyTag concentration in the desalted, dialyzed 6FAM-SpyTag-mRNA[OVA]-LNP solution was calculated based on its final measured volume, assuming 1 equivalent of 6FAM- SpyTag label per DMG-PEG2000-Mal and 100% recovery of material during purification. The 6FAM-SpyTag-mRNA[OVA]-LNP solution was stored in the dark at 4°C.

[0323] Attachment of 6FAM-SpyTag-mRNA[OVA]-LNPs to Staphylococcus epidermidis LM087 cells expressing Aap-SpyCatcher fusion proteins. On the day prior to the attachment, two S. epidermidis LM087 strains (DBc528 and DBc553) containing plasmids encoding Aap fusion proteins with the lectin domain replaced by two different SpyCatcher sequences were picked as single colonies from agar plates and inoculated into overnight cultures of 4 mL Brain Heart Infusion (BHI) + 10 pg / mL chloramphenicol (Chlor). Strain DBc553 contains plasmid DBc504, which encodes an Aap protein with its lectin domain replaced by a SpyCatcher003 protein sequence. Strain DBc528 contains plasmid DBc503, which encodes an Aap protein with its lectin domain replaced by a SpyCatcherOOl E77Q mutant protein sequence having impaired SpyCatcher-SpyTag covalent bond formation activity. The cultures were incubated overnight at 37°C and 225 rpm. On the day of attachment, the dilution-corrected optical density at 600 nm (OD600) was measured for a 1 : 10 dilution of a sample each culture in BHI. Assuming a direct proportional relationship between OD600 and concentration of bacteria, the volume of each culture equivalent to 150 μL of bacteria from an OD600 = 8 culture was calculated and these volumes were harvested in a microcentrifuge at 14,000 * g for 3 min. Each cell pellet was washed by removal of the supernatant followed by resuspension in 1 mL PBS and recentrifugation as above. After removal of the PBS wash, each cell pellet was resuspended in 150 μL of PBS containing either a 1:10 dilution of the 6FAM-SpyTag-mRNA[OVA]-LNP solution or no LNPs. The reactions were allowed to proceed at room temperature for 15 min and quenched by addition of 1 mL of ice-cold PBS.

[0324] Flow cytometric analysis of 6FAM-SpyTag-mRNA[OVA]-LNP linkage to S. epidermidis cells. Immediately after the quenching procedure described above, reaction mixtures were centrifuged at 14,000 x g for 3 min. The cell pellets were resuspended in 100 μL of PBS containing 10 μM MycoLight™ Red JJ94 stain (AAT Bioquest, Pleasanton, CA) and stained at room temperature for 15 min. Cells were then washed with 200 μL of ice-cold staining buffer (PBS containing 10 mg / mL BSA) and stained for 12 min at room temperature with 100 μL of a solution of 2 pg / mL recombinant rabbit anti-PEG antibody RM 105 in staining buffer. After staining, cells were diluted with 1 mL of ice-cold staining buffer, centrifuged, washed with 200 μL of ice-cold staining buffer, and stained for 12 min at room temperature with 100 μL of a solution of 0.5 μg / mL of PE-Goat anti-rabbit IgG antibody in staining buffer. After this second staining, cells were washed with 2 x 100 μL ice-cold staining buffer and resuspended in 200 μL PBS. For flow cytometry, 100 μL of each cell suspension was added to 100 μL of PBS in different wells of a 96-well plate and the cells were analyzed using a CytoFLEX S flow cytometer (Beckman-Coulter) for front scatter, side scatter, MycoLight™ Red JJ94 fluorescence (APC channel), 6-carboxyfluorescein fluorescence (FITC channel), and phycoerythrin fluorescence (PE channel).

[0325] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0326] All publications, patents, and patent applications cited herein are hereby incorporated by reference with respect to the material for which they are expressly cited.

Claims

WHAT IS CLAIMED IS:

1. A commensal bacterium comprising on the surface of the bacterium an extracellular protein selected from the group consisting of Accumulation-associated protein (Aap), peptidoglycan, an M protein, Lysin Motif (LysM)-containing protein and a peptidoglycan-binding protein, or a fragment thereof linked to a heterologous antigenic molecule or immunomodulatory molecule.

2. The commensal bacterium of claim 1 , wherein the extracellular protein is heterologous to the commensal bacterium.

3. The commensal bacterium of claim 1, wherein the extracellular protein is endogenous to the commensal bacterium.

4. The commensal bacterium of claim 1, wherein the fragment comprises at least 5, 14, 25, 50, 75, 100, 150 or 200 amino acids of the extracellular protein.

5. The commensal bacterium of claim 1 , wherein the commensal bacterium is a gram-positive bacterium.

6. The commensal bacterium of claim 1, wherein the antigenic molecule is an antigenic polypeptide.

7. The commensal bacterium of claim 6, wherein one or more first affinity sequence is fused to the extracellular protein or fragment thereof and a second affinity sequence is fused to the heterologous antigenic polypeptide and the extracellular protein or fragment thereof is linked to the heterologous antigenic polypeptide via affinity or covalent linkage of the first and second affinity sequences.

8. The commensal bacterium of claim 3, wherein the first affinity tag and the second affinity tag are a pair selected from the following list of pairs: a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and aDogTag sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; a HaloTag sequenceand a haloalkane moiety; a cysteine residue and a maleimide moiety, a free amine and an N- hydroxysuccinimide ester.

9. The commensal bacterium of claim 6, the extracellular protein or fragment thereof is linked to the heterologous antigenic polypeptide via a transpeptidase recognition sequence.

10. The commensal bacterium of claim 9, wherein the transpeptidase is a sortase or Butelase 1.

11. The commensal bacterium of claim 6, wherein the extracellular protein or fragment thereof is linked to the heterologous antigenic polypeptide via at least one covalent non-peptide bond.

12. The commensal bacterium of claim 11, wherein the at least one non- peptide bond is formed via azide-alkyne cycloaddition.

13. The commensal bacterium of claim 6, wherein the extracellular protein or fragment thereof and the heterologous antigenic polypeptide form a fusion protein.

14. The commensal bacterium of claim 1, wherein the antigenic molecule comprises a lipid or polysaccharide.

15. The commensal bacterium of claim 14, wherein one or more first affinity sequence is fused to the extracellular protein or fragment thereof and a second affinity sequence is linked to the lipid or polysaccharide and the extracellular protein or fragment thereof is linked to the heterologous antigenic molecule via affinity or covalent linkage of the first and second affinity sequences.

16. The commensal bacterium of claim 15, wherein the first affinity tag and the second affinity tag are a pair selected from the following list of pairs: a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and aDogTag sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; a HaloTag sequenceand a haloalkane moiety; a cysteine residue and a maleimide moiety, a free amine and an N- hydroxysuccinimide ester.

17. The commensal bacterium of claim 13, wherein the commensal bacterium comprises a nucleic acid encoding the fusion protein and the fusion protein is expressed in the commensal bacterium.

18. The commensal bacterium of claim 13, wherein the commensal bacterium does not comprise a nucleic acid encoding the fusion.

19. The commensal bacterium of any one of claims 1-18, wherein the heterologous antigenic polypeptide is selected from a cancer antigen, a viral antigen, a bacterial antigen, a fungal antigen and a self-antigen.

20. The commensal bacterium of any one of claims 1-18, wherein the commensal bacterium is selected from the group consisting of Staphylococcus sp., Streptococcus sp., Lactobacillus spp., Micrococcus sp., Ruminococcus sp., Eubacterium spp., Dorea sp., Subdoligranulum sp., Roseburia sp., Blautia sp., Faecalibacterium sp., Corynebacterium sp., Cutibacterium sp. and Clostridium sp.

21. The commensal bacterium of any one of claims 1-18, wherein the commensal bacterium is selected from the group consisting of Neisseria sp., Veillonella sp., Enhydrobacter sp., Akkermansia sp., Bacteroides sp., Parabacteroides sp., and Alcaligenes sp.

22. The commensal bacterium of any one of claims 1-20, wherein the bacterium is not genetically engineered and / or the bacterium does not comprise a polynucleotide encoding the antigenic polypeptide.

23. A commensal bacterium comprising on the surface of the bacterium an extracellular protein or a fragment thereof linked to a lipid nanoparticle containing an antigenic molecule or a polynucleotide encoding an antigenic polypeptide.

24. The commensal bacterium of claim 23, wherein the polynucleotide is an RNA.

25. The commensal bacterium of claim 24, wherein the RNA is a messengerRNA.

26. The commensal bacterium of claim 23, wherein one or more first affinity sequence is fused to the extracellular protein or fragment thereof and a second affinity sequence is linked to the lipid nanoparticle and the extracellular protein or fragment thereof is linked to the lipid nanoparticle via affinity or covalent linkage of the first and second affinity sequences.

27. The commensal bacterium of claim 26, wherein the first affinity tag and the second affinity tag are a pair selected from the following list of pairs: a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and aDogTag sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; a HaloTag sequence and a haloalkane moiety; a cysteine residue and a maleimide moiety, a free amine and an N- hydroxysuccinimide ester.

28. A commensal bacterium non-covalently coated on the surface of the bacterium with an antigenic molecule or immunomodulatory molecule or a nanoparticle containing (i) an antigenic molecule or (ii) a polynucleotide encoding an antigenic polypeptide.

29. The commensal bacterium of claim 28, wherein the antigenic molecule or nanoparticle is coated non-covalently on the bacterial cell surface by non-specific electrostatic interactions.

30. A method of inducing an immune response to an antigen in a mammal, the method comprising, contacting the skin or a mucosal surface of the mammal with the bacterium of any one of claims 1-28 in an amount sufficient to induce an immune response to the antigenic polypeptide.

31. The method of claim 30, wherein the immune response is a B-cell response, a T-cell response or both a B-cell response and a T-cell response.

32. A method of making the bacterium of any one of claims 1 -27, the method comprising, providing a commensal bacterium expressing an extracellular protein or a fragment thereof on the surface of the bacterium, and linking the antigenic molecule or immunomodulatory molecule to the extracellular protein fragment thereof.

33. The method of claim 32, wherein the antigenic molecule is an antigenic polypeptide and the extracellular protein on the surface of the bacterium comprises a transpeptidase recognition sequence and the linking comprises contacting the surface of the gram-positive bacterium with (i) the antigenic polypeptide, wherein the antigenic polypeptide comprises an amino terminal pentaglycine and (ii) transpeptidase, such that the transpeptidase cleaves the transpeptidase recognition sequence and links the antigenic polypeptide to the extracellular polypeptide or fragment thereof.

34. The method of claim 32, comprising feeding the bacterium a source of azide-containing amino acids or alkyne-containing amino acids such that the azide-containing or alkyne-containing amino acids are incorporated in the extracellular protein expressed by the bacterium; and wherein the antigenic molecule comprises an alkyne or azide moiety, respectively; and wherein the linking comprises inducing an azide-alkyne cycloaddition reaction to link the extracellular protein to the antigenic molecule.

35. The method of claim 32, wherein the providing comprises expressing in the commensal bacterium the extracellular protein or fragment thereof linked to a first affinity sequence; and the linking comprises contacting the extracellular protein or fragment thereof linked to a first affinity sequence to a second affinity sequence that is fused to the heterologous antigenic polypeptide wherein affinity or covalent linkage of the first and second affinity sequences occurs.

36. The method of claim 35, wherein the first affinity tag and the second affinity tag are a pair selected from the following list of pairs: a SpyCatcher sequence and a SpyTag sequence; a SnoopCatcher sequence and a SnoopTag sequence; a DogCatcher sequence and a DogTag sequence; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147 and a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence and a biotin moiety; a HaloTag sequence and a haloalkane moiety; a cysteine residue and a maleimide moiety, a free amine and an N- hydroxysuccinimide ester.

37. The method of claim 32, comprising producing in a different cell a recombinant fusion protein comprising (i) an extracellular protein or fragment thereof and (ii) the antigenic polypeptide; isolating the fusion protein form the different cell; contacting the isolated fusion protein to the gram-positive bacterium expressing the extracellular protein on the surface of the bacterium; and linking the fusion protein to the extracellular protein on the surface of the bacterium.

38. The method of claim 37, wherein the different cell is a bacterial cell (for example E. coll) or a mammalian cell.

39. A fusion protein comprising (i) a bacterial extracellular protein selected from the group consisting of Accumulation-associated protein (Aap), peptidoglycan, an M protein, LysM and a peptidoglycan-binding protein or a fragment thereof and (ii) a heterologous antigenic polypeptide, immunomodulatory polypeptide, or an amino terminal pentaglycine or a transpeptidase recognition sequence or one or more affinity sequences.

40. The fusion protein of claim 39, wherein the affinity sequence is a SpyCatcher sequence a SnoopCatcher sequence; a DogCatcher sequence ; a sequence comprising residues 565-587 of Clostridium perfringens adhesin Cpe0147; a sequence comprising residues 439-563 of Clostridirum perfringens adhesin Cpe0147; a streptavidin sequence; a biotin moiety;a HaloTag sequence; a haloalkane moiety; a cysteine residue; a maleimide moiety, a free amine; and an N-hydroxy succinimide ester.

41. The fusion protein of claim 39, wherein the fusion polypeptide comprises an amino terminal pentaglycine or a transpeptidase recognition sequence.

42. The fusion protein of claim 39, wherein the fragment comprises at least 25, 50, 75, 100, 150 or 200 amino acids of the extracellular protein.

43. The fusion protein of any one of claims 39-42, wherein the heterologous antigenic polypeptide is selected from a cancer antigen, a viral antigen, a bacterial antigen a fungal antigen and a self-antigen.

44. A nucleic acid encoding the fusion protein of any one of claims 39-43.

45. An expression cassette comprising a promoter operably linked to the nucleic acid of claim 44.

46. A cell comprising the expression cassette of claim 45.

47. A commensal bacterium comprising on the surface of the bacterium an extracellular protein selected from the group consisting of Accumulation-associated protein (Aap), peptidoglycan, an M protein, Lysin Motif (LysM)-containing protein and a peptidoglycan-binding protein, or a fragment thereof, linked to a heterologous antigenic molecule or immunomodulatory molecule, wherein the heterologous antigenic molecule or immunomodulatory molecule is linked by any one of:(a) Heterologous expression, or(b) Chemical labeling.

48. The commensal bacterium of claim 47, wherein the chemical labeling linking the extracellular protein to the heterologous antigenic molecule comprises a first affinity tag coupled to the extracellular protein and a second affinity tag coupled to the heterologous antigenic molecule or immunomodulatory molecule.

49. The commensal bacterium of claim 48, wherein the chemical labeling linking the extracellular protein to the heterologous antigenic molecule is selected from the group consisting of chemical labeling with a genetic carrier, chemical labeling using a transpeptidase, and chemical labeling via covalent attachment.

50. The commensal bacterium of claim 49, wherein chemical labeling linking the extracellular protein to the heterologous antigenic molecule using the transpeptidase comprises the transpeptidase recognizing a c-terminal transpeptidase recognition sequence.

51. The commensal bacterium of claim 50, wherein the transpeptidase comprises sortase.

52. The commensal bacterium of claim 50, wherein the c-terminal transpeptidase recognition sequence comprises an amino acid sequence motif of:(a) LPXTG (SEQ ID NO: 79);(b) LPXTA (SEQ ID NO: 98); or(c) LAXTG (SEQ ID NO: 90).

53. The commensal bacterium of claim 49, wherein the chemical labeling linking the extracellular protein to the heterologous antigenic molecule via covalent attachment comprises a click chemistry reaction or a free amine and N-hydroxy succinimide ester reaction.

54. The commensal bacterium of claim 53, wherein the extracellular protein comprises a non-naturally-occurring amino acid comprising an alkyne moiety and the heterologous antigenic molecule or immunomodulatory molecule comprises an azide moiety, wherein the alkyne moiety and the azide moiety participate in the click chemistry reaction to link the extracellular protein to the heterologous antigenic molecule.

55. The commensal bacterium of claim 53, wherein the extracellular protein comprises a non-naturally-occurring amino acid comprising an azide moiety and theheterologous antigenic molecule or immunomodulatory molecule comprises an alkyne moiety, wherein the alkyne moiety and the azide moiety participate in the click chemistry reaction to link the extracellular protein to the heterologous antigenic molecule.

56. The commensal bacterium of claim 48, wherein the first affinity tag comprises a biotin moiety and the second affinity tag comprises a streptavidin sequence.

57. The commensal bacterium of claim 48, wherein the first affinity tag comprises a SpyCatcher sequence, and the second affinity tag comprises a SpyTag sequence.

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