Oral vaccine compositions and related methods

Abstract

Provided herein are recombinant phage, probiotic vaccines and therapeutics comprising recombinant phage; and related methods of using same. In some embodiments, it includes recombinant phage (e.g., filamentous) and / or probiotic vaccines comprising a recombinant filamentous phage genome; and therapeutic methods of using the invention recombinant phage and / or probiotic vaccines. The invention recombinant filamentous phage: probiotic vaccines comprising said recombinant phage are useful wherein, for example, in therapeutic methods for preventing, treating, and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, among others).

Description

[0001] ORAL VACCINE COMPOSITIONS AND RELATED METHODS

[0002] BACKGROUND

[0003]

[0001] Field of the invention

[0004]

[0002] The present invention relates recombinant phage, probiotic vaccines and methods of using same.

[0005]

[0003] Related Art

[0006]

[0004] Cancer is more common today as a result of genetic abnormalities as well as epigenetic alterations in cell development and apoptotic pathways. Tumor development and growth are caused by the overexpression of genes associated with cell growth and proliferation in cancer and by the underexpression of genes that regulate cell death activities. Changes in cellular pathways are frequently produced by random mutations and unhealthy lifestyle habits. Immunocompetent individuals are vulnerable to natural immune alterations, which involve the activation of diverse immune responses to destroy competent tumor cells. Tumor-causing cells are going through apoptosis and necrosis as well as surrounding tissues are going through stress, and the microbiota in the gut all produce anti-cancer immune responses and signals.

[0007]

[0005] Immunotherapy is a form of cancer treatment that activates the immune system to attack and eradicate cancer cells. Cytotoxic T lymphocytes (“CTL”) are critical to a successful antitumor immune response. T cells that attack cancer cells require the presentation of tumor antigens to naive T cells that undergo activation, clonal expansion, and ultimately exert their cytolytic effector function. Effective antigen presentation is essential to successful CTL effector function. Thus, the development of a successful strategy to initiate presentation of tumor antigens to T ceils can be important to an immunotherapeutic strategy for cancer treatment.

[0008]

[0006] Likewise, B cells can inhibit tumor development through the production of tumor-reactive antibodies, promoting tumor killing by NK cells, phagocytosis by macrophages, and the priming of CD4+ and CD8+ T ceils. B cells can promote tumor development through the production of autoantibodies and tumor growth factors.

[0009]

[0007] Living microorganisms that enter gastrointestinal tract and exert a beneficial effect on the host are referred to as probiotics. With the clinical outcome of many types of cancers being from poor to lethal, there exists a significant need for the development of novel prophylactic and / or therapeutic probiotic treatments. SUMMARY

[0010]

[0008] Provided herein are recombinant phage (e.g., filamentous) and / or probiotic vaccines comprising a recombinant filamentous phage genome; and therapeutic methods of using the invention recombinant phage and / or probiotic vaccines. The invention recombinant filamentous phage; probiotic vaccines comprising said recombinant phage are useful herein, for example, in therapeutic methods for preventing, treating, and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, among others).

[0011]

[0009] It is known that the immune response within the circulatory system and the mucous membrane differs by response as SlgA antibodies exist in the domain of the mucous membranes and IgA and IgG antibodies exist in the circulatory domain. Monomeric IgA is present in serum, whereas in mucosal secretions is found secretory IgA (SlgA). it is different from the structure of IgA present in the serum because SlgA generally occurs in a polymeric form stabilized by joining chain (J chain), in particular in dimeric or tetrameric setup. Additionally, SlgA contains a secretory component (SC) derived from polymeric Ig receptor (plgR) utilized for transcytosis through epithelial cells during secretion. In humans, there are two subclasses of IgA: lgA1 and lgA2. In serum subclass lgA1 dominates, whereas in mucosal secretions the proportion between lgA1 and lgA2 depends on the site of production, e.g., up to: 60% lgA1 in saliva, 90% lgA1 in nasal and 60% lgA2 in intestinal secretions. In the human colostrum approximately 48% of immunoglobulins correspond to lgA2 and 40% to the lgA1 subclass that confers an adaptation to protect against potentially harmful pathogens, and which is also a way to regulate the colonization of the microbiota in newborns.

[0012]

[0010] Mucosal membranes lining gastrointestinal, respiratory and genitourinary tracts are exposed to permanent contact with a vast variety of microorganisms. The gastrointestinal tract (GIT) is colonized by numerous and diverse microbial communities, up to --1014 microbial cells per gram of colonic content represented by -500-1000 bacterial species, archaea and fungi. To prevent the invasion of pathogenic microbes and to regulate interactions between host and bacteria, the mucosal immune system is stimulated to produce SlgA. Abundant production of SlgA in the intestines is one of the mechanisms that gut-associated lymphoid tissue (GALT) runs in response to constant contact of gut mucosal membranes with a large number of diverse microorganisms, mainly bacteria and other food-derived antigens.

[0013]

[0011] In particular embodiments, the invention probiotic vaccines comprising invention recombinant bacteriophage, have demonstrated the ability to penetrate the mucous layer and generate a significant secretory IgA (SlgA) immune response allowing the body to respond to antigens and malignancies in the mucous membranes (see Figure 3; and Example 10). in other embodiments, the invention recombinant bacteriophage (e.g, filamentous phage), when delivered via injection and / or intravenously (IV), have demonstrated the ability to generate a significant IgA and IgG immune response (see Figure 2A and 2B; and Example 9).

[0014]

[0012] Accordingly, provided herein are therapeutic methods comprising administering an invention probiotic vaccine (e.g., via oral delivery, and the like), for preventing, treating, and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, among others). Also provided herein are therapeutic methods comprising administering an invention recombinant bacteriophage (e.g., via injection, and the like), for preventing, treating, and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, among others). In other aspects, also provided herein are therapeutic methods comprising administering a combination of an invention probiotic vaccine (e.g., via oral delivery, and the like) with an invention recombinant bacteriophage (e.g., via injection, and the like) for preventing, treating, and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, among others).

[0015]

[0013] For example, in particular embodiments, the invention recombinant filamentous phage; probiotic vaccines and therapeutic methods have shown significant impact on tumor growth in preclinical animal studies and the invention therapeutic approach is contemplated herein to address cancers native to the mucous membrane as well as slowing the metastasis of tumors originating elsewhere.

[0016]

[0014] Accordingly, provided herein are recombinant phage, comprising a recombinant phage genome comprising a nucleic acid encoding a polypeptide comprising an exogenous peptide dual-epitope, or fragments or variants thereof, wherein the exogenous peptide dual-epitope is selected from the group consisting of: SEQ ID NO:52-70; or wherein the exogenous peptide dual-epitope is represented by the formula: a first peptide epitope selected from SEQ ID NO: 1-32 or 71-74; -linker x- ; a second peptide epitope selected from SEQ ID NO:1-32 or 71-74, wherein the first peptide epitope is different from the second peptide epitope (e.g., the first and second peptide epitopes are different), in some embodiments, the linker x is 1 to 50 amino acids. In another embodiment, the linker x is a single glycine. In particular embodiments, the exogenous peptide dual-epitope is selected from SEQ ID NO:33- 51. in some embodiments, the exogenous peptide epitope is functionally expressed on a coat protein of said phage selected from the group consisting of: pill, pVI , pVII, pVIII and pIX. In other embodiments, the coat protein is pill or pVIII.

[0017]

[0015] In certains embodiments, the phage is selected from the group of filamentous phage consisting of: M13, fd, I Ke, CTX-tp, Pfl, Pf2, Pf3, f1 , MKE; M13KE; type 8; type f88; f88-4; Myoviridae (Pl-like viruses; P2-like viruses; Mu-like viruses; SPOI-like viruses; phiH- like viruses); Siphoviridae (A-like viruses, y-like viruses, Tl-like viruses; T5- like viruses; c2-like viruses; L5-like viruses; psiMI-like viruses; phiC31 -like viruses; N15-like viruses); Podoviridae (phi29-like viruses; P22-like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Aiphalipothrixvirus, Betalipothrixvirus, Gammaiipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fuseilovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, fd-like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus, Chlamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus). In other embodiments, the phage is a filamentous phage selected from the group consisting of: M13, fd, I Ke, CTX- q>, Pfl, Pf2, Pf3, f1 , MKE, M13KE, type 8, type f88, and f88-4. In particular embodiments, the phage is M13KE or f88-4.

[0018]

[0016] Also provided herein is a recombinant filamentous phage, said phage comprising a recombinant phage genome comprising a nucleic acid encoding a polypeptide comprising an exogenous peptide epitope, or fragments or variants thereof, selected from the group consisting of SEQ ID NOs: 1-74.

[0019]

[0017] In particular embodiments of the recombinant phage, the exogenous peptide epitope is functionally expressed on a coat protein selected from the group consisting of: pill, pVI, pVH, pVIII and pIX. in a particular embodiment, of the recombinant phage, the coat protein is pill. In another embodiment, the coat protein is pVIII. In further embodiments, the phage is selected from the group consisting of: filamentous phage, including, M13, fd, IKe, CTX-q>, Pfl, Pf2, Pf3, f 1 , MKE; M13KE; type 8, type f88, f88-4, Myoviridae (Pl-iike viruses; P2-like viruses; Mu-like viruses; SPOI-like viruses; phiH- like viruses); Siphoviridae (A-like viruses, y-iike viruses, Tl-like viruses; T5-like viruses; c2-like viruses; L5-like viruses; psiMI-like viruses; phiC31-like viruses; N15-like viruses); Pooviridae (phi29-like viruses; P22-like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, fd-like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus, Chiamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus). In yet further embodiments, the phage is a filamentous phage selected from the group consisting of: M13, fd, IKe, CTX-cp, Pfl, Pf2, Pf3, f 1 , MKE, and M13KE. In a particular embodiment, the phage is M13KE. In another embodiment, the phage is f88 or f88-4. In yet other embodiments, the recombinant phage generates IgG and / or IgA antibodies (e.g., SigA) that bind to the exogenous peptide epitope.

[0020]

[0018] Also provided herein is a probiotic vaccine, said vaccine comprising, a recombinant filamentous phage, wherein said phage comprises a nucleic acid encoding a polypeptide comprising an exogenous peptide epitope, or exogenous peptide dual-epitope, or fragments or variants thereof, selected from the group consisting of:

[0021] WGQPHGGG (SEQ ID NO:1), chronic wasting disease, PVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRN (SEQ ID NO:2), Norovirus,

[0022] NNYDPTEEIPAPLGTPDF (SEQ ID NO:3), Norovirus,

[0023] WIRNNF (SEQ ID NO:4), Norovirus,

[0024] YLQRDISEM (SEQ ID NO:5), MUC1 ; pancreatic cancer, ELQRDISEM (SEQ ID NO:6), MUC1 ; pancreatic cancer, LEEPNRVQL (SEQ ID NO:7), MUC1 ; pancreatic cancer, ATADLELAY (SEQ ID NO:8), Bacillus Calmette-Guerin, Liver Cancer, AQRMTTQLLLL (SEQ ID NO:9), folate receptor, Cancer, SLLMQITQC (SEQ ID NO: 10), Chondro Sarcoma, DPQGVTCGAATLSAERV (SEQ ID NO:11), IL-12, IBD, VELMYPPPYYLGIGN (SEQ ID NO:12), CTLA-4, EGGVAMPGAEDDVV (SEQ ID NO:13), podoplanin (PDPN), mucosal melanoma, NSELLSLINDMPITNDQKKLMSNN (SEQ ID NO:14), RSV, f protein, NSELLSLINDMPITNDQKKLMSNNV (SEQ ID NO:15), RSV, f protein, NSELLSLINDMPITNDQKKLMSNNVQ (SEQ ID NO:16), RSV, f protein, NSELLSLINDMPITNDQKKLMSNNVQ! (SEQ ID NO:17), RSV, f protein, GFEIGISQEPFDP (SEQ ID NO:18), Porcine Epidemic Diarrhea, SLPQDVTRC (SEQ ID NO: 19), Porcine Epidemic Diarrhea, DGVCNGAAV (SEQ ID NO:20), Porcine Epidemic Diarrhea, FDLDDGFYPISS (SEQ ID NO:21), Porcine Epidemic Diarrhea, ITGTPKPLEG (SEQ ID NO:22), Porcine Epidemic Diarrhea, SVYDPASGRVVQKR (SEQ ID NO:23), Porcine Epidemic Diarrhea, DQLPDVIPDYID (SEQ ID NO:24), Porcine Epidemic Diarrhea, PNRTGPSL (SEQ ID NO:25), Porcine Epidemic Diarrhea, YSNIGVCK (SEQ ID NO:26), Porcine Epidemic Diarrhea, NLLSHEQP (SEQ ID NO:27), Porcine Epidemic Diarrhea, ATAVIKTGTCPFSFDKLNNY (SEQ ID NO:28), Transmissible Gastroenteritis, PFSFDKLNNYLTFNKFCLSL (SEQ ID NO:29), Transmissible Gastroenteritis, VVRSLYVIYEEGCNIVGVPS (SEQ ID NO:30), Transmissible Gastroenteritis, LHLDSCTDYNIYGRTGVGII (SEQ ID NO:31), Transmissible Gastroenteritis, LYYTSLSGDLLGFKNVSDGV (SEQ ID NO:32), Transmissible Gastroenteritis, ALLEIASCLgAGNNWAKGHYTEGAELVD (SEQ ID NO:33), Mucosal Melanoma, ALLEIASCLgTFSVVPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:34), Mucosal Melanoma,

[0025] ALLEIASCLgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:35), Mucosal Melanoma,

[0026] ALLEIASCLgYLSDNHILI (SEQ ID NO:36), Mucosal Melanoma / CD3, AGAFAPSAAVAgAGNNWAKGHYTEGAELVD (SEQ ID NO:37), Prostate Cancer, AGAFAPSAAVAgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:38), Prostate Cancer, AGAFAPSAAVAgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:39), Prostate Cancer, AGAFAPSAAVAgYLSDNHILI (SEQ ID NO:40), Prostate Cancer / CD3, FLAEDALNTVgAGNNWAKGHYTEGAELVD (SEQ ID NO:41), Breast Cancer, FLAEDALNTVgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:42), Breast Cancer,

[0027] FLAEDALNTVgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:43), Breast Cancer,

[0028] FLAEDALNTVgYLSDNHILI (SEQ ID NO:44), Breast Cancer / CD3,

[0029] AQRMTTQLLLLgAGNNWAKGHYTEGAELVD (SEQ ID NO:45), Folate Receptor, AQRMTTQLLLLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:46), Folate Receptor,

[0030] AQRMTTQLLLLgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:47), Folate Receptor,

[0031] AQRMTTQLLLLgYLSDNHILI (SEQ ID NO:48), Folate Receptor / CD3,

[0032] SPRMSGLLSQTgAGNNWAKGHYTEGAELVD (SEQ ID NO:49), DLLS Receptor, SPRMSGLLSQTgTFSWPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:50), DLLS Receptor,

[0033] SPRMSGLLSQTgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:51), DLLS Receptor,

[0034] ALLEIASCLxAGNNWAKGHYTEGAELVD (SEQ ID NO:52), Mucosal Melanoma, ALLEIASCLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:53), Mucosal Melanoma,

[0035] ALLEIASCLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:54), Mucosal Melanoma,

[0036] ALLEIASCLxYLSDNHILI (SEQ ID NO:55), Mucosal Melanoma / CD3,

[0037] AGAFAPSAAVAxAGNNWAKGHYTEGAELVD (SEQ ID NO:56), Prostate Cancer, AGAFAPSAAVAxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:57), Prostate Cancer,

[0038] AGAFAPSAAVAxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:58), Prostate Cancer,

[0039] AGAFAPSAAVAxYLSDNHILI (SEQ ID NO:59), Prostate Cancer / CD3, FLAEDALNTVGAGNNWAKGHYTEGAELVD (SEQ ID NO:60), Breast Cancer, FLAEDALNTVxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:61), Breast Cancer, FLAEDALNTVxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:62), Breast Cancer,

[0040] FLAEDALNTVxYLSDNHiU (SEQ ID NO:63), Breast Cancer / CD3,

[0041] AQRMTTQLLLLxAGNNWAKGHYTEGAELVD (SEQ ID NO:64), Folate Receptor, AQRMTTQLLLLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:65), Folate Receptor,

[0042] AQRMTTQLLLLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:66), Folate Receptor,

[0043] AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67), Folate Receptor / CD3, SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68), DLL3 Receptor, SPRMSGLLSQTxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:69), DLL3 Receptor,

[0044] SPRMSGLLSQTxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:70),

[0045] DLL3 Receptor,

[0046] KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), MEK1 , RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), MEK1 , QUYNLTLCELNGTDWL (SEQ ID NO:73), PRRS, and / or KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR

[0047] I (SEQ ID NO:74) CD3; and a bacterium infected with said recombinant phage.

[0048]

[0019] In particular embodiments of the invention vaccine, the exogenous peptide epitope is functionally expressed on a coat protein selected from the group consisting of: pill, pVI , pVII, pVIII and pIX. In a particular embodiment, the coat protein is pill. In another embodiment, the coat protein is pVIII. In a particular embodiment the peptide sequence -TSGSGSGSGSGSGSG- is used as a linker between the coat protein and the exogenous peptide epitope.

[0049]

[0020] In further embodiments, the phage is selected from the group consisting of: filamentous phage, including, M13, fd, IKe, CTX-<p, Pfl, Pf2, Pf3, f 1 , MKE; M13KE; type 8, type f88, f88-4 (GenBank Accession # AF218363), Myoviridae (Pl-like viruses; P2-like viruses; Mu-like viruses; SPOI-like viruses; phiH-like viruses); Siphoviridae (A- like viruses, y-like viruses, Tl-like viruses; T5-like viruses; c2-like viruses; L5-like viruses; psiMI-like viruses; phiC31-like viruses; N15-like viruses); Podoviridae (phi29- like viruses; P22-iike viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, fd-iike viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus, Chlamydiamicrovirus); Leviviridae (Levivirus, Aiioievivirus) and Cystoviridae (Cystovirus). In a particular embodiment, the phage is M13KE. In another embodiment, the phage is f88 or f88-4.

[0050]

[0021] In yet other embodiments, the bacteria (e.g., probiotic bacteria) which is infected by the invention recombinant phage provided herein, can be selected from: E. coli Nissle 1917, E. coll ER2738, Bacillus amyloliquefaciens; Bacillus polyfermenticus, strain Bispan; Bifidobacterium animalis subsp. Lactis, strain BB-12; Bifidobacterium animalis subsp. Lactis, strain GPS1209; Bifidobacterium animalis subsp. Lactis, strain HN019 (DR1064); Bifidobacterium bifidum, strain BB-12; Bifidobacterium bifidum, strain Rosell-71 ; Bifidobacterium breve, strain M-16V; Bifidobacterium iongum; Bifidobacterium thermophilum; Lactobacillus acidophilus, strain La-1 ; Lactobacillus brevis, strain HA-112; Lactobacillus fermentum, strain HA- 179; Lactobacillus helveticus, strain Lafti L10; Lactobacillus helveticus, strain Rosell- 52; Lactobacillus paracasei, strain Lafti L26; Lactobacillus paracasei subsp. paracasei, strain 431 ; Lactobacillus rhamnosus, strain HN001 (DR20); Streptococcus salivarius, strain DSM 13084; Streptococcus thermophilus; Bacillus coagulans GBI- 30, 6086, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis, Escherichia coli Nissle 1917, E. coli ER2738, Lactobacillus acidophilus NCFM, Lactobacillus paracasei Stl 1 (or NCC2461), Lactobacillus johnsonii Lai (also referred to as Lactobacillus LCI, Lactobacillus johnsonii NCC533), Lactobacillus plantarum 299v, Lactobacillus reuteri ATCC 55730 (Lactobacillus reuteri SD2112), Lactobacillus reuteri Protectis (DSM 17938, daughter strain of ATCC 55730), Lactobacillus reuteri Prodentis (DSM 17938 / ATCC 55730 and ATCC PTA 5289 in combination), Lactobacillus rhamnosus GG, Saccharomyces boulardii, mixture of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, a mixture of Lactobacillus acidophilus NCFM and Bifidobacterium bifidum BB-12, a mixture of Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R, a mixture of Lactobacillus plantarum HEAL 9 and Lactobacillus paracasei 8700:2, Lactobacillus bulgaricus, Lactococcus thermophiles and Lactobacillus bifidus.

[0051]

[0022] In a particular embodiment, the probioitic vaccine bacteria, which is infected by the invention recombinant phage provided herein, is F-factor positive. In another embodiment, the bacteria is F-factor positive seiected from E. coii Nissie 1917 or E. coli ER2738. in a particular embodiment the bacteria is E. coii Nissie 1917. In another embodiment the bacteria is E. coii ER2738. Escherichia coll Nissie 1917 (EcN) is a nonpathogenic strain of the Enterobacteriaceae family (Creative Biolabs).

[0052]

[0023] In particular embodiments, the probiotic vaccine and / or recombinant phage generates both IgG and secretory IgA (SlgA) antibodies that bind to the exogenous peptide epitope.

[0053]

[0024] Also provided herein, is a composition comprising coconut water and a probiotic vaccine, wherein the probiotic vaccine comprises a recombinant bacteriophage, wherein the recombinant phage comprises at least one exogenous peptide epitope corresponding to SEQ ID NO:1-74, or fragments or variants thereof. In certain embodiments, the coconut water further comprises one or more excipients selected from the group consisting of buffer, tonicity adjusting agent, preservative, demulcent, viscosity modifier, osmotic agent, surfactant, antioxidant, alkaiinizing agent, acidifying agent antifoaming agent, and colorant. In a particular embodiment, the coconut water further comprises a buffer. In another embodiment, the coconut water further compises a salt. In certain embodiments, the composition has a pH of 7.0-8.0. In a particular embodiment, the composition has a pH of 7.5.

[0054]

[0025] Also provided herein are methods for preventing, treating, and / or reducing the risk of cancer, autoimmune disease, respiratory disease, neurological disease, and / or infectious disease, said method comprising administering to a patient in need thereof a combination of: a. an injectable composition comprising a recombinant phage or probiotic vaccine comprising at least one exogenous peptide epitope corresponding to SEQ ID NO:1- 74, or fragments or variants thereof; and b. an oral composition comprising a recombinant phage or probiotic vaccine comprising at least one exogenous peptide epitope corresponding to SEQ ID NO:1- 74, or fragments or variants thereof.

[0055] Also provided, is a method of preventing, treating, and / or reducing the risk of cancer, autoimmune disease, respiratory disease, neurological disease, and / or infectious disease, said method comprising administering, to a patient in need thereof, an invention probiotic vaccine provided herein, in certain embodiments, the probiotic vaccine is administered orally. In other embodiments, the method further comprises administering an invention recombinant bacteriophage provided herein. In certain embodiments, the recombinant phage is administered intravenously or intramuscularly. In yet other embodiments, secretory IgA (SlgA) is generated. In yet other embodiments, wherein sectretory IgA (SigA) is generated in amount compared to a control, that is 2- to 100-fold, 2- to 90-fold, 2- to 80-fold, 2- to 70-fold, 2- to 60-fold, 2- to 50-fold, 2- to 40-fold, 2- to 30-fold, 2- to 20-fold, 2- to 10-fold, 2- to 9-fold, 2- to 8- fold, 2- to 7-fold, 2- to 6-fold, 2- to 5-fold, 2- to 4-fold, greater than a control.

[0056]

[0026] Also provided herein are methods of preventing, treating, and / or reducing the risk of cancer, comprising administering to a patient in need thereof, one or more of the probiotic vaccines provided herein; and / or one or more of the recombinant phages provided herein, wherein the exogenous peptide epitope is selected from the group consisting of: SEQ ID NOs:5-10, 12, 13, and 33-72, and 74. In particular embodiments, the cancer is selected from the group consisting of: multiple myeloma, epithelial cancer, epithelial ovarian cancer, mucosal melanoma, non-small cell lung cancer, melanoma, head and neck cancer, renal cell cancer, Hodgkin’s lymphoma, Cutaneous Squamous Cell Carcinoma, glioblastoma, esophageal cancer, gastric cancer, duodenal cancer, small intestinal cancer, appendiceal cancer, large bowel cancer, colon cancer, rectum cancer, colorectal cancer, anal cancer, pancreatic cancer, liver cancer, gallbladder cancer, spleen cancer, renal cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, breast cancer, pulmonary cancer, thyroid cancer, thymus cancer, brain cancer, nervous system cancer, gliomas, oral cavity cancer, skin cancer, blood cancer, lymphomas, eye cancer, bone cancer, bone marrow cancer, muscle cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), urothelial carcinoma, non-muscie invasive bladder cancer [NMIBC]), colon or rectal cancer, esophageal or certain gastroesophageal junction (GEJ) carcinomas, cervical cancer, renal cell carcinoma (ROC), advanced endometrial carcinoma, cutaneous squamous cell carcinoma (cSCC), and / or triple-negative breast cancer (TNBC), and the like. In a particular embodiment, the cancer is pancreatic cancer, liver cancer, and / or chondro sarcorma.

[0057]

[0027] Also provided herein is a method of preventing, treating, and / or reducing the risk of breast, bladder, pancreatic, liver, chondro sarcoma, ovarian, and / or stomach cancer, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: YLQRDISEM (SEQ ID N0:5), ELQRDISEM (SEQ ID N0:6), LEEPNRVQL (SEQ ID N0:7), ATADLELAY (SEQ ID N0:8), AQRMTTQLLLL (SEQ ID N0:9), SLLMQITQC (SEQ ID NO:10), VELMYPPPYYLGIGN (SEQ ID N0:12), EGGVAMPGAEDDVV (SEQ ID N0:13), ALLEIASCLgAGNNWAKGHYTEGAELVD (SEQ ID NO:33), ALLEIASCLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:34):ALLEIASCLgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:35), ALLEIASCLgYLSDNHILI (SEQ ID NO:36),

[0058] AGAFAPSAAVAgAGNNWAKGHYTEGAELVD (SEQ ID NO:37), AGAFAPSAAVAgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:38), AGAFAPSAAVAgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:39, AGAFAPSAAVAgYLSDNHILI (SEQ ID NO:40), FLAEDALNTVgAGNNWAKGHYTEGAELVD (SEQ ID N0:41), FLAEDALNTVgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:42), FLAEDALNTVgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:43), FLAEDALNTVgYLSDNHILI (SEQ ID NO:44), AQRMTTQLLLLgAGNNWAKGHYTEGAELVD (SEQ ID NO:45),

[0059] AQRMTTQLLLLgTFSWPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:46), AQRMTTQLLLLgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:47), AQRMTTQLLLLgYLSDNHILI (SEQ ID NO:48), SPRMSGLLSQTgAGNNWAKGHYTEGAELVD (SEQ ID NO:49), SPRMSGLLSQTgTFSWPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:50), SPRMSGLLSQTgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID N0:51), ALLEIASCLxAGNNWAKGHYTEGAELVD (SEQ ID NO:52), ALLEIASCLxTFSWPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:53), ALLEIASCLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:54), ALLEIASCLxYLSDNHILI (SEQ ID NO:55),

[0060] AGAFAPSAAVAxAGNNWAKGHYTEGAELVD (SEQ ID NO:56), AGAFAPSAAVAxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:57), AGAFAPSAAVAxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:58), AGAFAPSAAVAxYLSDNHILI (SEQ ID NO:59),

[0061] FLAEDALNTVxAGNNWAKGHYTEGAELVD (SEQ ID NQ:60), FLAEDALNTVxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID N0:61), FLAEDALNTVxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:62), FLAEDALNTVxYLSDNHILI (SEQ ID NO:63), AQRMTTQLLLLxAGNNWAKGHYTEGAELVD (SEQ ID NO:64), AQRMTTQLLLLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:65) AQRMTTQLLLLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:66), AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67), SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68), SPRMSGLLSQTxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:69), SPRMSGLLSQTxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:70), KKKPTPIQLNPAPAGSAVNG (SEQ ID N0:71), RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), QLIYNLTLCELNGTDWL (SEQ ID NO:73), and / or

[0062] KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR

[0063] I (SEQ ID NO:74) CDS.

[0064]

[0028] In a particular embodiment, the recombinant phage and / or probiotic vaccine comprises YLQRDISEM (SEQ ID NO:5), ELQRDISEM (SEQ ID NO:6), and / or LEEPNRVQL (SEQ ID NO:7), and is used to prevent, treat, or reduce the risk of pancreatic cancer, in another embodiment, the recombinant phage and / or probiotic vaccine comprises ATADLELAY (SEQ ID NO:8), and is used to prevent, treat, or reduce the risk of liver cancer. In a further embodiment, the recombinant phage and / or probiotic vaccine comprising SLLMQITQC (SEQ ID NO:10), is used to prevent, treat, or reduce the risk of chondro sarcoma. In yet another aspect, the recombinant phage and / or probiotic vaccine comprising VELMYPPPYYLGIGN (SEQ ID NO: 12) and / or EGGVAMPGAEDDW (SEQ ID NO: 13), is used to prevent, treat, or reduce the risk of mucosal melanoma, prostate cancer, pulmonary fibrosis, lung cancer, non-small cell lung cancer, and the like.

[0065]

[0029] In particular embodiments, provided herein are methods of preventing, treating, and / or reducing the risk of mucosal melanoma, prostate cancer, pulmonary fibrosis, lung cancer, non-small cell lung cancer, and the like, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: VELMYPPPYYLGIGN (SEQ ID N0:12) and / or EGGVAMPGAEDDW (SEQ ID NO:13).

[0066]

[0030] In another embodiment, as set forth above, provided herein is a method of preventing, treating, and / or reducing the risk of virus infection (e.g., TT virus) and / or chronic wasting disease manifestation, comprising administering, to a subject or patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: WGQPHGGG (SEQ ID NO:1).

[0067]

[0031] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Norovirus infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:

[0068] PVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRN (SEQ ID NO:2), NNYDPTEEIPAPLGTPDF (SEQ ID NO:3), and / or

[0069] WIRNNF (SEQ ID NO:4).

[0070]

[0032] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Respiratory Syncytial Virus (RSV) infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: NSELLSLINDMPITNDQKKLMSNN (SEQ ID NO:14), NSELLSLINDMPITNDQKKLMSNNV (SEQ ID NO:15), NSELLSLINDMPITNDQKKLMSNNVQ (SEQ ID NO:16) and / or NSELLSLINDMPITNDQKKLMSNNVQI (SEQ ID NO:17).

[0071]

[0033] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of inflammatory bowel disease (IBD), comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from DPQGVTCGAATLSAERV (SEQ ID NO: 11), KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), and / or RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72). in yet another embodiment, provided herein are methods of preventing, treating, and / or reducing the risk of ankylosing spondylitis, primary sclerosing cholangitis and Takayasu’s arteritis, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at ieast one exogenous peptide epitope, or fragment or variant thereof, selected from DPQGVTCGAATLSAERV (SEQ ID NO:11), KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), and / or RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72). In yet another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of inflammatory disease or type 1 diabetes, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR I (SEQ ID NO:74).

[0072]

[0034] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Porcine Epidemic Diarrhea viral infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:

[0073] GFEIGISQEPFDP (SEQ ID NO:18), SLPQDVTRC (SEQ ID NO: 19), DGVCNGAAV (SEQ ID NQ:20), FDLDDGFYPISS (SEQ ID NO:21), ITGTPKPLEG (SEQ ID NO:22), SVYDPASGRWQKR (SEQ ID NO:23), DQLPDVIPDYID (SEQ ID NO:24), PNRTGPSL (SEQ ID NO:25), YSNIGVCK (SEQ ID NO:26), and / or NLLSHEQP (SEQ ID NO:27).

[0074]

[0035] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Transmissible Gastroenteritis viral infection and / or disease manifestation, comprising administering, to a subject or patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:

[0075] ATAVIKTGTCPFSFDKLNNY (SEQ ID NO:28), PFSFDKLNNYLTFNKFCLSL (SEQ ID NO:29), VVRSLYVIYEEGCNIVGVPS (SEQ ID NO:30), LHLDSCTDYNIYGRTGVGII (SEQ ID N0:31), and / or LYYTSLSGDLLGFKNVSDGV (SEQ ID NO:32).

[0076]

[0036] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Porcine Reproductive Respiratory Syndrome (PRRS) and / or disease manifestation, comprising administering, to a subject or patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: QLIYNLTLCELNGTDWL (SEQ ID NO:73).

[0077]

[0037] As set forth herein, in particular embodiments of the invention methods, secretive IgA (SigA) is generated. In certain embodiments, secretive IgA (SigA) is generated in amount compared to a control, that is 2- to 100-fold, 2- to 90-fold, 2- to 80-fold, 2- to 70-fold, 2- to 60-fold, 2- to 50-fold, 2- to 40-fold, 2- to 30-fold, 2- to 20- fold, 2- to 10-fold, 2- to 9-fold, 2- to 8-fold, 2- to 7-fold, 2- to 6-fold, 2- to 5-fold, 2- to 4- fold, greater than a control.

[0078]

[0038] Other features and advantages of the present invention will become more readily apparent to those of ordinary skill in the art after reviewing the following detailed description.

[0079] BRIEF DESCRIPTION OF THE DRAWINGS

[0080]

[0039] Figure 1A indicates the physiological effect of oral administration of the invention probiotic vaccine to produce slgA.

[0081]

[0040] Figure 1 B indicates the physiological effect of injection administration of epitope targets via the invention recombinant bacteriophage to produce an IgA and IgG immune response.

[0082]

[0041] Figure 2A shows the resulting serum IgA levels of the formulations administered as set forth in Example 9.

[0083]

[0042] Figure 2B shows the resulting serum IgG levels of the formulations administered as set forth in Example 9.

[0084]

[0043] Figure 3 shows the resulting serum secretory IgA (SigA) levels of the formulations administered as set forth in Example 10.

[0085]

[0044] Figure 4 shows the resulting average tumor volumes of the formulations administered in the Prostate Cancer Xenograft Athymic Mouse Study set forth in Example 11.

[0045] Figure 5 shows the heterologous peptide epitope insertion site in the M13KE- SALV-0004 recombinant phage from Example 1.

[0086]

[0046] Figure 6 shows the heterologous peptide epitope insertion site in the M13KE- SALV-0013 recombinant phage from Example 3.

[0087] DETAILED DESCRIPTION

[0088]

[0047] Provided herein is a probiotic vaccine, said vaccine comprising, a recombinant phage (e.g., filamentous phage), wherein said phage comprises a nucleic acid (e.g., genome) encoding a polypeptide comprising an exogenous peptide epitope, or fragments or variants thereof, selected from the group consisting of: WGQPHGGG (SEQ ID NO:1), chronic wasting disease, PVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRN (SEQ ID NO:2), Norovirus,

[0089] NNYDPTEEIPAPLGTPDF (SEQ ID NO:3), Norovirus, WIRNNF (SEQ ID NO:4), Norovirus,

[0090] YLQRDISEM (SEQ ID NO:5), MUC1 ; pancreatic cancer, ELQRDISEM (SEQ ID NO:6), MUC1; pancreatic cancer, LEEPNRVQL (SEQ ID NO:7), MUC1; pancreatic cancer, ATADLELAY (SEQ ID NO:8), Bacillus Calmette-Guerin, Liver Cancer, AQRMTTQLLLL (SEQ ID NO:9), folate receptor, Cancer, SLLMQITQC (SEQ ID NO: 10), Chondro Sarcoma, DPQGVTCGAATLSAERV (SEQ ID NO:11), IL-12, IBD, VELMYPPPYYLGIGN (SEQ ID NO:12), CTLA-4,

[0091] EGGVAMPGAEDDW (SEQ ID NO:13), podoplanin (PDPN), mucosal melanoma, NSELLSLINDMPITNDQKKLMSNN (SEQ ID NO:14), RSV, f protein, NSELLSLINDMPITNDQKKLMSNNV (SEQ ID NO:15), RSV, f protein, NSELLSLINDMPITNDQKKLMSNNVQ (SEQ ID NO:16), RSV, f protein, NSELLSLINDMPITNDQKKLMSNNVQI (SEQ ID NO:17), RSV, f protein, GFEIGISQEPFDP (SEQ ID NO:18), Porcine Epidemic Diarrhea, SLPQDVTRC (SEQ ID NO: 19), Porcine Epidemic Diarrhea, DGVCNGAAV (SEQ ID NO:20), Porcine Epidemic Diarrhea, FDLDDGFYPISS (SEQ ID NO:21), Porcine Epidemic Diarrhea, ITGTPKPLEG (SEQ ID NO:22), Porcine Epidemic Diarrhea, SVYDPASGRVVQKR (SEQ ID NO:23), Porcine Epidemic Diarrhea, DQLPDVIPDYID (SEQ ID NO:24), Porcine Epidemic Diarrhea, PNRTGPSL (SEQ ID NO:25), Porcine Epidemic Diarrhea, YSNIGVCK (SEQ ID NO:26), Porcine Epidemic Diarrhea, NLLSHEQP (SEQ ID NO:27), Porcine Epidemic Diarrhea, ATAVIKTGTCPFSFDKLNNY (SEQ ID NO:28), Transmissible Gastroenteritis, PFSFDKLNNYLTFNKFCLSL (SEQ ID NO:29), Transmissible Gastroenteritis,

[0092] VVRSLYVIYEEGCNIVGVPS (SEQ ID NQ:30), Transmissible Gastroenteritis, LHLDSCTDYNIYGRTGVGII (SEQ ID NO:31), Transmissible Gastroenteritis, and / or LYYTSLSGDLLGFKNVSDGV (SEQ ID NO:32), Transmissible Gastroenteritis, ALLEIASCLgAGNNWAKGHYTEGAELVD (SEQ ID NO:33), Mucosal Melanoma, ALLEIASCLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:34), Mucosal Melanoma,

[0093] ALLEIASCLgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:35), Mucosal Melanoma,

[0094] ALLEIASCLgYLSDNHILI (SEQ ID NO:36), Mucosal Melanoma / CD3, AGAFAPSAAVAgAGNNWAKGHYTEGAELVD (SEQ ID NO:37), Prostate Cancer, AGAFAPSAAVAgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:38), Prostate Cancer,

[0095] AGAFAPSAAVAgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:39), Prostate Cancer,

[0096] AGAFAPSAAVAgYLSDNHILI (SEQ ID NQ:40), Prostate Cancer / CD3, FLAEDALNTVgAGNNWAKGHYTEGAELVD (SEQ ID NO:41), Breast Cancer, FLAEDALNTVgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:42), Breast Cancer,

[0097] FLAEDALNTVgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:43), Breast Cancer,

[0098] FLAEDALNTVgYLSDNHILI (SEQ ID NO:44), Breast Cancer / CD3, AQRMTTQLLLLgAGNNWAKGHYTEGAELVD (SEQ ID NO:45), Folate Receptor, AQRMTTQLLLLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:46), Folate Receptor,

[0099] AQRMTTQLLLLgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:47), Folate Receptor,

[0100] AQRMTTQLLLLgYLSDNHILI (SEQ ID NO:48), Folate Receptor / CD3, SPRMSGLLSQTgAGNNWAKGHYTEGAELVD (SEQ ID NO:49), DLL3 Receptor, SPRMSGLLSQTgTFSWPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:50), DLL3 Receptor,

[0101] SPRMSGLLSQTgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:51), DLL3 Receptor,

[0102] ALLEIASCLxAGNNWAKGHYTEGAELVD (SEQ ID NO:52), Mucosal Melanoma, ALLEIASCLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:53), Mucosal Melanoma,

[0103] ALLEIASCLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:54), Mucosal Melanoma,

[0104] ALLEIASCLxYLSDNHILI (SEQ ID NO:55), Mucosal Melanoma / CD3,

[0105] AGAFAPSAAVAxAGNNWAKGHYTEGAELVD (SEQ ID NO:56), Prostate Cancer, AGAFAPSAAVAxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:57), Prostate Cancer,

[0106] AGAFAPSAAVAxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:58), Prostate Cancer,

[0107] AGAFAPSAAVAxYLSDNHILI (SEQ ID NO:59), Prostate Cancer / CD3, FLAEDALNTVxAGNNWAKGHYTEGAELVD (SEQ ID NO:60), Breast Cancer, FLAEDALNTVxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:61), Breast Cancer,

[0108] FLAEDALNTVxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:62), Breast Cancer,

[0109] FLAEDALNTVxYLSDNHILI (SEQ ID NO:63), Breast Cancer / CD3,

[0110] AQRMTTQLLLLxAGNNWAKGHYTEGAELVD (SEQ ID NO:64), Folate Receptor, AQRMTTQLLLLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:65), Folate Receptor,

[0111] AQRMTTQLLLLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:66), Folate Receptor,

[0112] AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67), Folate Receptor / CD3, SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68), DLL3 Receptor, SPRMSGLLSQTxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:69), DLLS Receptor,

[0113] SPRMSGLLSQTxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:70), DLL3 Receptor,

[0114] KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), MEK1 , RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), MEK1 , QUYNLTLCELNGTDWL (SEQ ID NO:73), PRRS, and / or KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR I (SEQ ID NO:74) CD3; and a bacterium infected with said recombinant phage.

[0115]

[0048] As set forth herein, the invention probiotic vaccines are particularly useful in methods for preventing, treating, and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neuroligical diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesteroal, among others). Unlike traditional vaccines, probiotic-based vaccines do not require critical storage conditions and transportation. Traditional live vaccines usually demand lower storage temperatures before administration. In addition, traditional vaccinations are developed using attenuated strains of pathogenic bacteria, such as Salmonella, Mycobacterium, and Bacillus, which have a number of drawbacks, including the possibility of attenuated strains reverting to virulent forms inside the body. The attenuated forms of vaccines also result in eliciting additional immune response in individual, hence weakening the effect of vaccine. The survival rates under the harshly acidic conditions of the human gut are another issue that contributes to the failure of traditional vaccines as compared with probiotic oral vaccines. The traditional forms of vaccines are generally not able to survive the harsh acidic conditions, and, as a result, they fail to reach the inner mucosal layer. On the other hand, probiotics are well versed with the acidic environmental conditions. Because probiotics are acid- and bile- tolerant, they offer a good choice for developing vaccines.

[0116]

[0049] In addition, immune response within the circulatory system and the mucous membrane differs by response as SlgA antibodies exist in the domain of the mucous membranes and IgA and IgG antibodies exist in the circulatory domain. Monomeric IgA is present in serum, whereas in mucosal secretions is found secretory IgA (SlgA). It is different from the structure of IgA present in the serum because SlgA generally occurs in a polymeric form stabilized by joining chain (J chain), in particular in dimeric or tetrameric setup. Additionally, SlgA contains a secretory component (SC) derived from polymeric Ig receptor (plgR) utilized for transcytosis through epithelial cells during secretion. In humans, there are two subclasses of IgA: lgA1 and lgA2. In serum subclass lgA1 dominates, whereas in mucosal secretions the proportion between lgA1 and lgA2 depends on the site of production, e.g., up to: 60% lgA1 in saliva, 90% lgA1 in nasal and 60% lgA2 in intestinal secretions. In the human colostrum approximately 48% of immunoglobulins correspond to lgA2 and 40% to the lgA1 subclass that confers an adaptation to protect against potentially harmful pathogens, and which is also a way to regulate the colonization of the microbiota in newborns.

[0117]

[0050] Mucosal membranes lining gastrointestinal, respiratory and genitourinary tracts are exposed to permanent contact with a vast variety of microorganisms. The gastrointestinal tract (GIT) is colonized by numerous and diverse microbial communities, up to -1014 microbial cells per gram of colonic content represented by -500-1000 bacterial species, archaea and fungi. To prevent the invasion of pathogenic microbes and to regulate interactions between host and bacteria, the mucosal immune system is stimulated to produce SlgA. Abundant production of SlgA in the intestines is one of the mechanisms that gut-associated lymphoid tissue (GALT) runs in response to constant contact of gut mucosal membranes with a large number of diverse microorganisms, mainly bacteria and other food-derived antigens.

[0118]

[0051] An intravenous administration with invention recombinant phage (e.g., MK13E- SALV-0013, and the like), results in IgA and IgG response, whereas oral administration of the invention N13 probiotic bacterium vaccine therapy (MK13E- SALV-0013 infected EcN, and the like) results in an SlgA response (see Example 10 herein). Current therapeutic monoclonal antibodies do not have the ability to act as SlgA and as such have limited impact in the mucous membranes. Mucous membranes are key to many diseases and serve as a pathway for metastasis.

[0119]

[0052] As used herein, the phrase “probiotic vaccine” refers to a bacterium comprising a recombinant phage, such as those described herein.

[0120]

[0053] As used herein, the phrase “recombinant bacteriophage” refers to a phage having an exogenous peptide region encoded therein.

[0121]

[0054] As used herein the term “filamentous” in the context of phage, refers to a non- lytic, male-specific bacteriophages which infect Escherichia coli cells carrying an F- episome (for review see 19). Filamentous phage particles contain a circular ssDNA genome (the + strand). The phage binds to the F-pilus of the bacterium followed by entry of the ssDNA genome into the host. The dsDNA replicating form is generated by initiating DNA synthesis at the phage ori(-) structure. Single-strand (+) DNAs are formed by additional rounds of synthesis, starting at the ori(+) structure, and are packaged into phage particles without causing lysis or apparent damage to the host.

[0055] As used herein the term "vaccine”, refers to a composition capable of stimulating the immune system of a living organism so that protection against a harmful antigen is provided, either through prophylaxis or through therapy. Preferably, a vaccine or a vaccine composition further comprises one or more immuno-adjuvant substances.

[0122]

[0056] As used herein, the term "preventing", "prevention", "prophylaxis" or "prevent" generally means to avoid or minimize the onset or development of a disease or condition before its onset. The term "preventing" encompasses "reducing the likelihood of occurrence of” or "reducing the likelihood of reoccurrence".

[0123]

[0057] As used herein, the term "treat" or "treating", or grammatical variations thereof, refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorders) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder's) being treated; (d) limiting or preventing recurrence of the disorders) in patients that ha ve previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorders). Accordingly, the term "treating, "treatment" or "treat", or grammatical variations thereof, encompasses reducing, ameliorating or curing a disease (e.g., cancer, and the like) or condition (or symptoms of a disease or condition) after its onset.

[0124]

[0058] As used herein, the phrase “reduce the risk of” refers to prophylactic use to reduce the occurrence of one getting or acquiring a disorder (e.g., cancer or disease), or limiting the development of a disorder, such as prophylactic administration using an invention probiotic vaccine and / or recombinant phage to avoid becoming infected with a virus, or developing cancer, and the like, as described herein.

[0125]

[0059] An "effective amount" or "effective dose" as used herein is an amount which provides the desired effect. For therapeutic purposes, an effective amount is an amount sufficient to provide a beneficial or desired clinical result. The preferred effective amount for a given application can be easily determined by the skilled person taking into consideration, for example, the size, age, weight of the subject, the type of disease / disorder to be prevented, treated, and / or reduction of risk of and the amount of time since the disease / disorder began. In the context of the present invention, in terms of prevention, treatment, and / or reducing the risk, an effective amount of the composition is an amount that is sufficient to induce a humoral and / or cell-mediated immune response directed against the disease / disorder.

[0126]

[0060] According to the different aspects and embodiments of the invention described herein, a "subject" or "host" preferably refers to a mammal, and most preferably to a human being. Said subject may have, been suspected of having, or be at risk of developing cancer and / or an infectious disease (e.g. via the Korean Fever Virus infection, and the like).

[0127] Exogenous Peptide Epitope

[0128]

[0061] As used herein, the phrase “exogenous peptide epitope” refers to any peptide or sequence that is not native or natural to the host phage strain being used to produce the invention recombinant phage. In other words, the exogenous peptide epitope, and the nucleic acid encoding it, is heterologous (i.e., foreign) to the peptide sequences of the particular phage strain being utilized. In several particular embodiments, the length of the exogenous peptide epitopes can be in a range selected from the group consisting of: 4-20 amino acids, 4-25 amino acids, 5-20 amino acids, 5-25 amino acids, 5-30 amino acids, 5-35 amino acids, 5-40 amino acids, 5-45 amino acids and 5-50 amino acids. In a particular embodiment, the length of the exogenous peptide epitopes is in a range of 5-20 amino acids.

[0129]

[0062] in accordance with the present invention, an exogenous peptide epitope (e g., an antigenic peptide) for use in the recombinant phage and / or probiotic vaccines provided herein comprise or consist of an amino acid sequence as set forth in any one of SEQ ID NOs:1-74, or fragments or variants thereof.

[0130]

[0063] The epitopes used for culturing the recombinant phage with the probiotic bacterial cells will depend on the type of disease (e.g., cancer, and the like) to be treated or prevented; or infectious disease to be prevented.

[0131]

[0064] For example, in particular embodiments, the following exogenous peptide epitopes can be used for preventing, treating, and / or reducing the risk of cancer, wherein the cancer is selected from the group consisting of: multiple myeloma, epithelial cancer, epithelial ovarian cancer, mucosal melanoma, non-small cell lung cancer, melanoma, head and neck cancer, renal cell cancer, Hodgkin’s lymphoma, Cutaneous Squamous Cell Carcinoma, glioblastoma, esophageal cancer, gastric cancer, duodenal cancer, small intestinal cancer, appendiceal cancer, large bowel cancer, colon cancer, rectum cancer, colorectal cancer, anal cancer, pancreatic cancer, liver cancer, gallbladder cancer, spleen cancer, renal cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, breast cancer, pulmonary cancer, thyroid cancer, thymus cancer, brain cancer, nervous system cancer, gliomas, oral cavity cancer, skin cancer, blood cancer, lymphomas, eye cancer, bone cancer, bone marrow cancer, muscle cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), urothelial carcinoma, non-muscle invasive bladder cancer [NMIBC]), colon or rectal cancer, esophageal or certain gastroesophageal junction (GEJ) carcinomas, cervical cancer, renal cell carcinoma (RCC), advanced endometrial carcinoma, cutaneous squamous cell carcinoma (cSCC), and / or triple-negative breast cancer (TNBC); and wherein the exogenous peptide epitope is selected from: SEQ ID NOs: 5-10, 12, 13, and 33-72, and 74.

[0132]

[0065] In another embodiment, the exogenous peptide epitope comprises five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution(s) within SEQ ID NOs: 5-10, 12, 13, and 33-72, and 74.

[0133] Dual-epitope embodiments

[0134]

[0066] Also provided herein are recombinant phage and / or probiotic vaccines comprising all dual-epitope permutations of the individual epitope target sequences set forth herein in combination with other single-epitope target sequences for recombination into the respective phage coat protein (e.g., pill or pVIII). Accordingly, provided herein a recombinant phage, comprising a recombinant phage genome comprising a nucleic acid encoding a polypeptide comprising an exogenous peptide dual-epitope, or fragments or variants thereof, wherein the exogenous peptide dualepitope is selected from the group consisting of: SEQ ID NO:52-70; or wherein the exogenous peptide dual-epitope is represented by the formula: a first peptide epitope selected from SEQ ID NO: 1-32 or 71-74; -linker x- ; a second peptide epitope selected from SEQ ID NO:1-32 or 71-74, wherein the first peptide epitope is different from the second peptide epitope (e.g., the first and second peptide epitopes are different). In some embodiments, the linker x is 1 to 50 amino acids. In another embodiment, the linker x is a single glycine. In particular embodiments, the exogenous peptide dual-epitope is selected from SEQ ID NO:33- 51. In some embodiments, the exogenous peptide epitope is functionally expressed on a coat protein of said phage seiected from the group consisting of: pill, pVI, pVli, pVIII and p!X. In other embodiments, the coat protein is pill or pVIII.

[0135]

[0067] In certain embodiments, the phage is selected from the group of filamentous phage consisting of: M13, fd, I Ke, CTX-cp, Pfl, Pf2, Pf3, f1 , MKE; M13KE; type 8; type f88; f88-4; Myoviridae (Pl-like viruses; P2-like viruses; Mu-like viruses; SPOI-like viruses; phiH- like viruses); Siphoviridae (A-like viruses, y-like viruses, Tl-like viruses; T5- like viruses; c2-like viruses; L5-like viruses; psiMI-like viruses; phiC31 -like viruses; N15-like viruses); Podoviridae (phi29-like viruses; P22-like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, fd-like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus, Chlamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus). In other embodiments, the phage is a filamentous phage selected from the group consisting of: M13, fd, I Ke, CTX- (p, Pfl, Pf2, Pf3, f1 , MKE, M13KE, type 8, type f88, and f88-4. In particular embodmients, the phage is M13KE or f88-4.

[0136]

[0068] In particular embodiments, exemplary invention dual-epitope recombinant phage comprise 2 separate single-epitopes corresponding to a first and second respective SEQ ID NO selected from SEQ ID NO; 1-32 and 71-74, separated by a “linker x”. In other embodiments, the order of the single-epitopes can be reversed, such that the previous second SEQ ID NO is now the first and the previous first SEQ ID NO is now second. In other embodiments, the dual-epitope sequences set forth herein as SEQ ID NOs:33-70, and the like, are used within the invention recombinant phage and / or probiotic vaccines provided herein. For example, in SEQ ID NOs:33- 51 , two separate and independent epitopes are set forth that are separated by a single glycine residue, which is depicted as a small case “g” residue.

[0137]

[0069] In another embodiment, e.g., SEQ ID NOs:52-70, two separate and independent epitopes are set forth that are separated by a linker (or spacer) comprising one or more amino acid linker (or spacer) residues, which “linker x” or spacer is depicted as a small case “x” residue. In one embodiment, x can be any length of linker from 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, and 1 to 10 amino acids, so long as the dual epitope is expressed fused to a coat protein of a filamentous bacteriophage such that the dual-epitope peptides are expressed on the surface of the virion and are available to present as immunogens; or to interact with target molecules or cell surface receptors. In a particular embodiment, the length of “linker x” is from 1 to 50 amino acids. In another embodiment, the length of “linker x” is from 1 to 20 amino acids. In yet another embodiment, the length of “linker x” is from 1 to 10 amino acids.

[0138]

[0070] The “linker” or “spacer” group (“x”) is optional. When it is present, it is not critical what its chemical structure is, since it serves primarily as a spacer. The linker should be chosen so as not to interfere with the biological activity of the dual-epitopes. The linker is made up of amino acids linked together by peptide bonds. Thus, in one embodiment, the linker comprises Yn, wherein Y is a naturally occurring amino acid or a stereoisomer thereof and “n” is any one of 1 through 20. The linker is therefore made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally-occurring amino acids. In another embodiment, the 1 to 20 amino acids are selected from Gly, Ala, Pro, Asn, Gin, Cys, Lys. In other embodiments, the linker (“x” in SEQ ID NOs:52-70) is made up of a majority of amino acids that are sterically un-hindered, such as Gly, Gly-Gly [(G!y)2], Gly-Gly-Gly [(Gly)s] . . . (Glyjso, Ala, Gly-Ala, Ala-Gly, Ala-Ala, etc. To explain the above nomenclature, for example, (Gly)3Lys(Gly)4 means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Linker / spacer ombinations of Gly and Ala are also contemplated for use herein.

[0139]

[0071] In another embodiment, linker or spacer “x” comprises (Gly)n, wherein n is 1 through 50, 1 through 40, 1 through 30, 1 through 20, or 1 through 10, and when n is greater than 1 , up to half of the Gly residues may be substituted by another amino acid selected from the remaining 19 natural amino acids or a stereoisomer thereof. Although a linker or spacer “x” may be depicted, in particular embodiments, when x = 0, there is no linker or spacer between the two epitope amino acid sequences, such that the first and second epitope sequences are contiguous.

[0140]

[0072] In other embodiments, the single-epitope target sequences set forth as SEQ ID NOs:1-133 in WO2023 / 158883 and WO2024 / 178160 (each of which is incorporated herein by reference in its entirety for all purposes) can be used to generate the dualepitopes for recombination into the respective phage coat protein (e.g., pill or pVIII) to produce invention recombinant phage and probiotic vaccines.

[0141]

[0073] As used herein, the phrase “recombinant phage genome” refers to a native phage genome that has been modified to contain nucleic acid encoding an exogenous peptide region.

[0074] As used herein, the phrase “functionally expressed” refers to a single or dual epitope being expressed as a recombinant fusion to a coat protein of a filamentous bacteriophage such that the single or dual-epitope peptides are expressed on the surface of the virion and are available to present as immunogens; or to interact with target molecules or cell surface receptors.

[0142]

[0075] As used herein, the term “fragments”, or grammatical variations thereof, refers to a smaller peptide (e g., subset of amino acids) relative to the complete reference peptide sequence from which it is derived. For example, the fragment can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids smaller than the complete reference peptide.

[0143]

[0076] As used herein, the phrase "variants thereof’, “variant”, or grammatical variations thereof, refers to a biologically active polypeptide having at least about 80% amino acid sequence identity with the reference sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N — or C-terminus of the polypeptide. In some embodiments, a variant will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid sequence identity. In some embodiments, a variant will have at least about 85% amino acid sequence identity. In some embodiments, a variant will have at least about 90% amino acid sequence identity. In some embodiments, a variant will have at least about 95% amino acid sequence identity with the native sequence polypeptide.

[0144]

[0077] As used herein, “Percent (%) amino acid sequence identity” and “homology” with respect to a peptide or polypeptide sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0145]

[0078] In particular embodiments, amino acid substitutions may Include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary conservative substitutions are shown in Table 1. In other embodiments, conservative amino acid substitutions may be introduced into a peptide of interest and the products screened for a desired activity, for example, increased immunogenicity, or the like.

[0146]

[0079] In some embodiments, the modifying can include introducing at least one conservative substitution into the peptide or protein, in which at least one property such as the size, shape or charge of the amino acid is conserved. A "conservative amino acid substitution" refers to substitution of a structurally and / or functionally similar amino acid that may be made without not substantially altering the function of a protein. An example of conservative substitution is the exchange of an amino acid in one of the following groups for another amino acid of the same group (U.S. Patent No. 5,767,063 ; Kyte and Doolittle, J. Mol. Biol. 157: 105-132 (1982)):

[0147] ■ (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie;

[0148] • (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0149] » (3) acidic: Asp, Glu;

[0150] » (4) basic: His, Lys, Arg;

[0151] » (5) residues that influence chain orientation: Gly, Pro;

[0152] » (6) aromatic: Trp, Tyr, Phe.

[0153]

[0154]

[0080] For example, substitutions can be made by changing, e.g., Vai to Leu; Ser to Thr; or Asp to Glu. Other substitutions can aiso be considered conservative, depending on the environment of the particular amino acid and its role in the three- dimensional structure of the protein. For example, when it is desired to alter the pKa of an amino acid side chain while retaining the size and structure of the side chain, Glu may be substituted by Gin, and Asp may be substituted by Asn.

[0081] In some embodiments, the amino acid residues selected for modification (e.g., replacement with another amino acid residue) are selected from the group consisting of: His, Glu, Asp, Cys, Lys and Tyr. In some embodiments, the amino acid residues selected for modification include His and Glu residues. These amino acid residues typically have pKa values (even in the whole-protein context) of between about pH 6 to about pH 8, and therefore possess high buffering capacities In some embodiments, the at least one conservative amino acid substitution is selected from the group consisting of His to Arg, Glu to Gin, Asp to Asn, Lys to Arg, and Tyr to Phe.

[0155]

[0082] In some embodiments, at least one of the one or more amino acid modifications includes a substitution of an amino acid with an alanine residue. Substitution or replacement of amino acid residues having high buffering capacities with alanine residues can be advantageous in applications in which it is desirable to reduce the buffering capacity of the peptide or protein. It should be noted that Nor Leucine is not coded but modified after substitution.

[0156] Bacterium

[0157]

[0083] As set forth above, provided herein are invention probiotic vaccines comprising a recombinant phage and a bacterium infected with said recombinant phage. In particular embodiments, any bacteria with F-factor (F+cell), i.e., sex factor, that produces pili and a sex pilus for conjugation with other bacteria is suitable for use herein. When an F+ cell conjugates / mates with an F- cell, the result is two F+ cells, both capable of transmitting the plasmid to other F- cells by conjugation. A pilus on the F+ cell interacts with the recipient cell allowing formation of a mating junction, the DNA is nicked on one strand, unwound and transferred to the recipient. Exemplary F- factor-containing bacteria, include Escherichia coli Nissle 1917, E. coll ER2738, and the like.

[0158]

[0084] Accordingly in particular embodiments, examples of probiotic bacteria for use in accordance with the present invention include, without limitation, Escherichia coli Nissle 1917, E. coli ER2738, Bacillus coagulans GBI-30, 6086, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis, Lactobacillus acidophilus NCFM, Lactobacillus paracasei Stl 1 (or NCC2461), Lactobacillus johnsonii Lai (also referred to as Lactobacillus LCI, Lactobacillus johnsonii NCC533), Lactobacillus plantarum 299v, Lactobacillus reuteri ATCC 55730 (Lactobacillus reuteri SD2112), Lactobacillus reuteri Protectis (DSM 17938, daughter strain of ATCC 55730), Lactobacillus reuteri Prodentis (DSM 17938 / ATCC 55730 and ATCC PTA 5289 in combination), Lactobacillus rhamnosus GG, Saccharomyces boulardii, mixture of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, a mixture of Lactobacillus acidophilus NCFM and Bifidobacterium bifidum BB-12, a mixture of Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R, a mixture of Lactobacillus plantarum HEAL 9 and Lactobacillus paracasei 8700:2, Lactobacillus bulgaricus, Lactococcus thermophiles and / or Lactobacillus bifidus. In some embodiments, probiotic bacteria for use in accordance with the present disclosure may be a mixture of any two or more of the foregoing strains set forth herein. In particular embodiments, the probiotic bacteria is Escherichia coli Nissle 1917 or E. coli ER2738. in another embodiment, the probiotic bacteria is Escherichia coli Nissle 1917. In yet another embodiment, the probiotic bacteria is E. coli ER2738.

[0159]

[0085] In other embodiments, when the peptide is displayed as part of pVIII, suitable phage systems include type 8, type 88 (also referred to herein as f88), and type 8+8 described (see, e.g., Zhong et al., J Biol. Chem. 269:24183-24188, 1994; Smith and Scott, Methods in Enzymology, 217:228-257, 1993; Smith, Gene, 128:1-2, 1993, each of which are incorporated herein by reference in their entirety). When pili is utilized, suitable phage systems include type 3, type 33 and type 3+3. When the peptide is inserted into pVI, suitable phage systems included type 6, type 66 and type 6+6. In addition, phage T7 and phage 8 vector systems can be used. In one particular embodiment, the peptides of the library are expressed fused to a coat protein of a filamentous bacteriophage so that the peptides are expressed on the surface of the virion and are available to present as immunogens; or to interact with target molecules or cell surface receptors.

[0160]

[0086] In one particular embodiment, the f88-4 library is used in which the selected peptide is fused to the pVIII coat protein, using the filamentous phage display vector f88-4 (GenBank Accession # AF218363). The PVIII protein is the major coat protein of the phage present in about 2700 copies per phage. PVIII is synthesized as a precoat protein containing a 23 amino acid leader peptide, which is cleaved to yield a mature 50 residue transmembrane protein. Two systems have been used that enable the generation of mosaic phages; the ‘type 8+8’ and ‘type 88’ systems (see Smith G.P. (1993) Surface display and peptide libraries. Gene, 128, 1-2; which is incorporated herein by reference in its entirety). The ‘type 88’ (e.g., f88-4) system benefits by containing the two pVIII genes in one and the same infectious phage genome. This obviates the need for a heiper phage and superinfection, where oniy one type of mosaic phage is produced.

[0161]

[0087] in other embodiments, bacteria suitable for use herein are small (typical linear dimensions of around 1 micron), non- compartmentalized organisms, with at least one circular DNA chromosomes and ribosomes of 70S. As used herein, the term "bacteria" encompasses all variants of bacteria (e.g., endogenous bacteria, which naturally reside in a closed system, environmental bacteria or bacteria released for bioremediation or other efforts).

[0162]

[0088] In the invention probiotic vaccines provided herein, the invention recombinant phage are loaded (e.g., infected) into donor bacteria (e.g., probiotic bacteria and / or commensal bacteria) for delivery to subjects in need thereof (e.g., human patients). Probiotic bacteria, for example, are live bacteria that can confer a health benefit on the host and / or, at the very least, are not harmful (e.g., not pathogenic) to the host, such as human patients. Thus, aspects of the invention contemplate the use of donor bacteria (e.g., probiotic bacteria and / or commensal bacteria) that is loaded or infected with non-lytic or inducible bacteriophage that can express and generate bacteriophage-based delivery particles in situ. Probiotics have also been found to penetrate the inner mucosal layer of the gut and aid in invasion of harmful bacteria, as well as protect against numerous enteric infections.

[0163]

[0089] In particular embodiments, examples of probiotic bacteria for use herein for infection by the invention recombinant phage, include those set forth in the “PROBIO” database (“ / / bidd.group / probio / homepage.htm”; Shamekhi et al., Clin Transl Oncol (2020) 22(8): 1227-39. doi:10.1007 / s12094-019-02270-0; which is incorporated herein by reference in its entirety for all purposes), which database includes 329 probiotics that are currently commercially available, 115 probiotic bacteria that are undergoing clinical trials. In particular embodiments, the invention recombinant phage are infected or loaded into particular probiotic bacteria with immunomodulatory capabilities selected from the group consisting of: Bacillus amyloliquefaciens; Bacillus polyfermenticus, strain Bispan; Bifidobacterium animalis subsp. Lactis, strain BB-12; Bifidobacterium animalis subsp. Lactis, strain GPS1209; Bifidobacterium animalis subsp. Lactis, strain HN019 (DR1064); Bifidobacterium bifidum, strain BB-12; Bifidobacterium bifidum, strain Rosell-71; Bifidobacterium breve, strain M-16V; Bifidobacterium longum; Bifidobacterium thermophilum; Lactobacillus acidophilus, strain La-1 ; Lactobacillus brevis, strain HA-112; Lactobacillus fermentum, strain HA- 179; Lactobacillus helveticus, strain Lafti L10; Lactobacillus helveticus, strain Resell- 52; Lactobacillus paracasei, strain Lafti L26; Lactobacillus paracasei subsp. paracasei, strain 431 ; Lactobacillus rhamnosus, strain HN001 (DR20); Streptococcus salivarius, strain DSM 13084; Streptococcus thermophilus. See, e.g., Table 1 of Singh et al., Front Immunol. 2022; 13: 1002674; Published online 2022 Oct 3. doi: 10.3389 / fimmu.2022.1002674; which is incorporated herein by reference in its entirety for all purposes.

[0164]

[0090] In other embodiments, the invention recombinant bacteriophage of the present disclosure are contemplated herein to target bacteria other than Escherichia coli, including, without limitation, Bacteroides thetaiotamicron (e.g., Bl), B. fragilis (e.g., ATCC 51477-B1 , B40-8, Bf-1), B. caccae (e.g., phiHSCOI), B. ovatus (e.g., phiHSC02), Clostridium difficile (e.g., phiC2, phiCS, phiC6, phiCS, phiCD119, phiCD27), Klebsiella pneumoniae (e.g., KP01 K2, KI I, Kpn5, KP34, JDOOI), Staphylococcus aureus (e.g., phiNMI, SOalpha), Enterococcus faecalis (e.g., IME- EF1), Enterococcus faecium (e.g., ENB6, C33), and Pseudomonas aeruginosa (e.g., phiKMV, PAK-P1 , LKD16, LKA1, delta, sigma-1 , J-l).

[0165]

[0091] Thus, the bacteriophage of the present disclosure may target (e.g., specifically target) a bacterial ceil from any one or more of the foregoing genus and / or species of bacteria. Other bacterial cells and microbes may also be targeted.

[0166]

[0092] As used herein, "endogenous" bacterial cells may refer to non-pathogenic bacteria that are part of a normal internal ecosystem such as bacterial flora.

[0167]

[0093] In yet further embodiments, bacterial cells of the present disclosure are anaerobic bacterial cells (e.g., cells that do not require oxygen for growth). Anaerobic bacterial cells include facultative anaerobic cells such as, for example, Escherichia coli, Shewanella oneidensis and Listeria monocytogenes. Anaerobic bacterial cells also include obligate anaerobic cells such as, for example, Bacteroides and Clostridium species. In humans, for example, anaerobic bacterial cells are most commonly found in the gastrointestinal tract. Thus, the bacteriophage of the present disclosure may target (e.g., specifically target) anaerobic bacterial cells.

[0168] Recombinant Bacteriophage

[0169]

[0094] Also provided herein is a recombinant bacteriophage (e.g., filamentous) comprising a recombinant phage genome comprising a nucleic acid encoding a polypeptide comprising an exogenous peptide epitope, or an exogenous peptide dualepitope, or fragments or variants thereof, selected from the group consisting of: WGQPHGGG (SEQ ID NO:1), chronic wasting disease, PVAGAAIAAPLTGQQNSIDPWSMNNFVQAPGGEFTVSPRN (SEQ ID NO:2), Norovirus,

[0170] NNYDPTEEIPAPLGTPDF (SEQ ID NO:3), Norovirus, WIRNNF (SEQ ID NO:4), Norovirus, YLQRDISEM (SEQ ID NO:5), MUC1 ; pancreatic cancer, ELQRDISEM (SEQ ID NO:6), MUC1 ; pancreatic cancer, LEEPNRVQL (SEQ ID NOY), MUC1 ; pancreatic cancer, ATADLELAY (SEQ ID NO:8), Bacillus Calmette-Guerin, Liver Cancer, AQRMTTQLLLL (SEQ ID NO:9), folate receptor, Cancer, SLLMQITQC (SEQ ID NO:10), Chondro Sarcoma, DPQGVTCGAATLSAERV (SEQ ID NO:11), IL-12, IBD, VELMYPPPYYLGIGN (SEQ ID NO:12), CTLA-4,

[0171] EGGVAMPGAEDDVV (SEQ ID NO:13), podoplanin (PDPN), mucosal melanoma, NSELLSLINDMPITNDQKKLMSNN (SEQ ID NO:14), RSV, f protein, NSELLSLiNDMPITNDQKKLMSNNV (SEQ ID NO:15), RSV, f protein, NSELLSLINDMPITNDQKKLMSNNVQ (SEQ ID NO:16), RSV, f protein, NSELLSLINDMPITNDQKKLMSNNVQI (SEQ ID NO:17), RSV, f protein, GFEIGISQEPFDP (SEQ ID NO:18), Porcine Epidemic Diarrhea, SLPQDVTRC (SEQ ID NO: 19), Porcine Epidemic Diarrhea, DGVCNGAAV (SEQ ID NO:20), Porcine Epidemic Diarrhea, FDLDDGFYPISS (SEQ ID NO:21), Porcine Epidemic Diarrhea, ITGTPKPLEG (SEQ ID NO:22), Porcine Epidemic Diarrhea, SVYDPASGRVVQKR (SEQ ID NO:23), Porcine Epidemic Diarrhea, DQLPDVIPDYID (SEQ ID NO:24), Porcine Epidemic Diarrhea, PNRTGPSL (SEQ ID NO:25), Porcine Epidemic Diarrhea, YSNIGVCK (SEQ ID NO:26), Porcine Epidemic Diarrhea, NLLSHEQP (SEQ ID NO:27), Porcine Epidemic Diarrhea, ATAVIKTGTCPFSFDKLNNY (SEQ ID NO:28), Transmissible Gastroenteritis, PFSFDKLNNYLTFNKFCLSL (SEQ ID NO:29), Transmissible Gastroenteritis, WRSLYVIYEEGCNIVGVPS (SEQ ID NQ:30), Transmissible Gastroenteritis, LHLDSCTDYNIYGRTGVGII (SEQ ID NO:31), Transmissible Gastroenteritis, LYYTSLSGDLLGFKNVSDGV (SEQ ID NO:32), Transmissible Gastroenteritis, ALLEIASCLgAGNNWAKGHYTEGAELVD (SEQ ID NO:33), Mucosal Melanoma, ALLEIASCLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:34), Mucosal Melanoma,

[0172] ALLEIASCLgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:35), Mucosal Melanoma,

[0173] ALLEIASCLgYLSDNHILI (SEQ ID NO:36), Mucosal Melanoma / CD3,

[0174] AGAFAPSAAVAgAGNNWAKGHYTEGAELVD (SEQ ID NO:37), Prostate Cancer, AGAFAPSAAVAgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:38), Prostate Cancer,

[0175] AGAFAPSAAVAgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:39), Prostate Cancer,

[0176] AGAFAPSAAVAgYLSDNHILI (SEQ ID NO:40), Prostate Cancer / CD3, FLAEDALNTVgAGNNWAKGHYTEGAELVD (SEQ ID NO:41), Breast Cancer, FLAEDALNTVgTFSWPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:42), Breast Cancer,

[0177] FLAEDALNTVgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:43), Breast Cancer,

[0178] FLAEDALNTVgYLSDNHILI (SEQ ID NO:44), Breast Cancer / CD3,

[0179] AQRMTTQLLLLgAGNNWAKGHYTEGAELVD (SEQ ID NO:45), Folate Receptor, AQRMTTQLLLLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:46), Folate Receptor,

[0180] AQRMTTQLLLLgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:47), Folate Receptor,

[0181] AQRMTTQLLLLgYLSDNHILI (SEQ ID NO:48), Folate Receptor / CD3, SPRMSGLLSQTgAGNNWAKGHYTEGAELVD (SEQ ID NO:49), DLL3 Receptor, SPRMSGLLSQTgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:50), DLL3 Receptor,

[0182] SPRMSGLLSQTgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:51), DLL3 Receptor,

[0183] ALLEIASCLxAGNNWAKGHYTEGAELVD (SEQ ID NO:52), Mucosal Melanoma, ALLEIASCLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:53), Mucosal Melanoma, ALLEIASCLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:54), Mucosal Melanoma,

[0184] ALLEIASCLxYLSDNHILI (SEQ ID NO:55), Mucosal Melanoma / CD3,

[0185] AGAFAPSAAVAxAGNNWAKGHYTEGAELVD (SEQ ID NO:56), Prostate Cancer, AGAFAPSAAVAxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:57), Prostate Cancer,

[0186] AGAFAPSAAVAxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:58), Prostate Cancer,

[0187] AGAFAPSAAVAxYLSDNHILI (SEQ ID NO:59), Prostate Cancer / CD3, FLAEDALNTVxAGNNWAKGHYTEGAELVD (SEQ ID NO:60), Breast Cancer, FLAEDALNTVxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:61), Breast Cancer,

[0188] FLAEDALNTVxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:62), Breast Cancer,

[0189] FLAEDALNTVxYLSDNHILI (SEQ ID NO:63), Breast Cancer / CD3,

[0190] AQRMTTQLLLLxAGNNWAKGHYTEGAELVD (SEQ ID NO:64), Folate Receptor, AQRMTTQLLLLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:65), Folate Receptor,

[0191] AQRMTTQLLLLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:66), Folate Receptor,

[0192] AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67), Folate Receptor / CD3, SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68), DLL3 Receptor, SPRMSGLLSQTxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:69), DLLS Receptor,

[0193] SPRMSGLLSQTxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NQ:70), DLL3 Receptor,

[0194] KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), MEK1 , RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), MEK1 , QLIYNLTLCELNGTDWL (SEQ ID NO:73), PRRS, and / or KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR

[0195] I (SEQ ID NO:74) CDS.

[0196]

[0095] For each of the respective dual-epltope sequences set forth herein, that are separated by a single amino acid spacer (e.g. a “glycine”) or a spacer / llnker of multiple amino acids (“x”), the individual epitopes can be in reverse order (i.e., vice versa) than is depicted in the respective SEQ ID NOs.

[0197]

[0096] In particular embodiments of the recombinant phage, the exogenous peptide epitope is functionally expressed on a coat protein of the phage selected from the group consisting of: pill, pVI, pVII, pVI II and pIX. In a particular embodiment of the recombinant phage, the coat protein is pill. In another embodiment, the coat protein is pVIII . As used herein, the phrase “functionally expressed” refers to the expression of the exogenous peptide epitope, such that the epitope is displayed on the surface of the phage and can elicit an immune response.

[0198]

[0097] In further embodiments, the phage is selected from the group consisting of: filamentous phage, including, M13, fd, IKe, CTX-cp, Pfl, Pf2, Pf3, f1 , MKE, M13KE, type 8, type f88, f88-4, Myoviridae (Pl-like viruses; P2-like viruses; Mu-like viruses; SPOI-iike viruses; phiH-like viruses); Siphoviridae (A-like viruses, y-like viruses, Ti-like viruses; T5-like viruses; c2-like viruses; L5-like viruses; psiMI-like viruses; phiC31 -like viruses; N15-like viruses); Pooviridae (phi29-like viruses; P22-like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deitaiipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, fd-like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdeilomicrovirus, Chlamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus). In yet further embodiments, the phage is a filamentous phage selected from the group consisting of: M13, fd, IKe, CTX-rp, Pfl, Pf2, Pf3, f1 , MKE, M13KE, type 8, type f88, and f88-4.

[0199]

[0098] In a particular embodiment, the phage is M13KE. In yet a further embodiment, the phage is M13KE and the insertion site is between the pill signal peptide and the pill protein coding sequence as set forth in Figure 5. In another embodiment, the phage is f88 or f88-4. In yet a further embodiment, the phage is f88 or f88-4 and the insertion site is between the signal peptide and pVIII coding sequence as set forth in Example 12. In yet other embodiments, the recombinant phage generates IgG antibodies that bind to the exogenous peptide epitope.

[0200]

[0099] In particular embodiments of the present disclosure, nucleic acid encoding exogenous peptide epitopes is recombinantly combined into naturally- occurring, engineered (e.g., rationally engineered), or adaptively evolved bacteriophage for delivery to microbial cell populations, e.g., probiotic bacterial cells. A bacteriophage, or phage, is a virus that infects and replicates in bacteria. Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Bacteriophages replicate within bacteria following the injection of their genome into the cytoplasm and do so using either a lytic cycle, which results in bacterial cell lysis, or a lysogenic (non-lytic) cycle, which leaves the bacterial cell intact. The bacteriophages of the present disclosure are, in some embodiments, non-lytic (also referred to as lysogenic or temperate). Non-lytic phage may also include those that are actively secreted from infected cells in the absence of lysis, including, without limitation, filamentous phage such as, for example, f 1 , M13, M13KE, type 8, type f88, f88-4, fd, IKe, CTX-<p, Pfl, Pf2 and Pf3. Thus, after phage delivery of an exogenous peptide epitope into a bacterial cell, the bacterial cell may remain viable and able to stably maintain expression of antigenic epitope. Accordingly, in certain embodiments of the invention probiotic vaccine, the vaccine and / or bacteria continually produces lysogenic phage.

[0201]

[0100] In some embodiments, lytic bacteriophage may be used as delivery vehicles. When used with phagemid systems, naturally lytic phage serve as cargo shuttles and do not inherently lyse target cells.

[0202]

[0101] Examples of non-lytic bacteriophage for use in accordance with the present disclosure include, without limitation, those selected from the group consisting of: Myoviridae (Pl-like viruses; P2-iike viruses; Mu-iike viruses; SPOI-like viruses; phiH- iike viruses); Siphoviridae (A-like viruses, y-iike viruses, Tl-like viruses; T5-I ike viruses; c2-like viruses; L5-like viruses; psiMI-like viruses; phiC31-like viruses; N15-like viruses): Podoviridae (phi29-like viruses; P22-like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betaiipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, f1-like viruses, fd-like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus, Chlamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus). Such phages may be naturally occurring or engineered phages. In some embodiments, the bacteriophage is a coliphage (e.g., infects Escherichia coli). Those of skill in the art will readily understand that other bacteriophage may be used in accordance with the present disclosure.

[0102] In particular embodiments, the bacteriophages used herein are filamentous phages. Filamentous phages constitute a large family of bacterial viruses that infect many Gram-negative bacteria. Suitable well-known filamentous phages include those that infect Escherichia coll, such as, for example, f 1 , M13KE, type 8, type f88, f88-4, fd, Ike, and the like. Phages f 1 , M13, and fd have been used for filamentous phage display. Their genomes are more than 98% identical and their gene products are interchangeable.

[0203]

[0103] A unique aspect of filamentous phage assembly, in contrast to the assembly of many other bacteriophages, is that it is a secretory process. Incorporation of coat polypeptides into the growing phage occurs in the cytoplasmic membrane, and nascent phages are extruded from the cell as they assembly. The E. coli ceil does not lyse in this process. It is well-known to those of skill in the art that the five viral coat proteins (pill, pVI, pVII, pVIII and pIX) are inserted in the cytoplasmic membrane prior to their incorporation into phage particles. For example, the major part of pill is translocated across the membrane into the periplasm, while its C-terminal hydrophobic tail anchors the protein in the membrane.

[0204]

[0104] In a particular embodiment, the bacteriophage used to prepare an invention recombinant phage is an M 13 bacteriophage (e.g., M13KE, available from New Egiand Biolabs; Catalog #8101S). M 13 is a filamentous bacteriophage of the family Inoviridae and is composed of circular single- stranded DNA. M13 phages are about 900 nm long and 6-7 nm in diameter with 5 proteins. The minor coat protein, P3, attaches to the receptor at the tip of the F pilus of an Escherichia coli host cell. Thus, in a particular embodiment, the invention probiotic vaccines and their use in the invention methods for preventing, treating, and / or reducing the risk of disease comprise delivering to bacterial cells an invention recombinant M13 bacteriophage that is engineered to functionally express at least one exogenous peptide epitope selected from SEQ ID NOs:1-74 in one of its coat proteins selected from the group consisting of pill, pVI, pVII, pVIII and pIX. In a particular embodiment, the exogenous peptide epitope is functionally expressed in gene pill of the M13 bacteriophage, in a particular embodiment, the bacteriophage is M13KE. In yet a further embodiment, the phage is M13KE and the insertion site is between the pill signal peptide and the pill protein coding sequence as set forth in Figure 5.

[0205]

[0105] In another embodiment, the invention probiotic vaccines and their use in the invention methods for preventing, treating, and / or reducing the risk of disease comprise delivering to bacterial cells an invention recombinant f88 bacteriophage that is engineered to functionally express at least one exogenous peptide epitope selected from SEQ ID NOs:1-74 in one of its coat proteins selected from the group consisting of pill, pVi, pVII, pVill and pIX. In a particular embodiment, the exogenous peptide epitope is functionally expressed in gene pVIII of the f88 bacteriophage. In a particular embodiment, the bacteriophage is f88-4 (GenBank Accession # AF218363). In yet a further embodiment, the phage is f88 or f88-4 and the insertion site is between the signal peptide and pVIII coding sequence as set forth in Example 12.

[0206]

[0106] Also provided is a method of making the invention recombinant phage, comprising expressing a recombinant phage genome comprising a nucleic acid encoding a polypeptide comprising an exogenous peptide epitope, selected from the group consisting of SEQ ID NOs:1-74, in a bacterium.

[0207]

[0107] In other embodiments, the bacteriophages for use herein can be isolated from any environment where bacteria exist In some embodiments, the recombinant bacteriophages of the invention are isolated from (e.g., collected from, obtained from) stool or sewage, terrestrial or marine environments.

[0208] Therapeutic Methods

[0209]

[0108] Also provided herein, are methods of preventing, treating and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neuroligical diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like), comprising administering to a patient in need thereof, the invention probiotic vaccines and / or the invention recombinant bacteriophages comprising a heterologous peptide selected from SEQ ID NOs:1-74 set forth herein.

[0210]

[0109] In a particular embodiment, provided herein are methods of preventing, treating and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like), comprising administering to a patient in need thereof, an invention probiotic vaccine comprising a heterologous peptide selected from SEQ ID NOs:1-74 set forth herein. In another embodiment, provided herein are methods of preventing, treating and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like), comprising administering to a patient in need thereof, an invention recombinant bacteriophage comprising a heterologous peptide selected from SEQ ID NOs:1-74 set forth herein.

[0211]

[0110] Also provided herein, are methods of preventing, treating and / or reducing the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like), comprising administering to a patient in need thereof, an invention probiotic vaccine in combination with an invention recombinant bacteriophage, each of which comprise a heterologous peptide selected from SEQ ID NOs:1-74 set forth herein. In particular embodiments, the invention probiotic vaccines comprising invention recombinant bacteriophage, have demonstrated the ability to penetrate the mucous layer and generate a significant secretory IgA (SlgA) immune response allowing the body to respond to antigens and malignancies in the mucous membranes (see Figure 3; and Example 10). In other embodiments, the invention recombinant bacteriophage (e.g, filamentous phage), when delivered via injection and / or intravenously (IV), have demonstrated the ability to generate a significant IgA and IgG immune response (see Figure 2A and 2B; and Example 9). Accordingly, also provided herein are therapeutic methods comprising administering a combination of an invention probiotic vaccine (e.g., via oral delivery, and the like) with an invention recombinant bacteriophage (e.g., via injection, and the like).

[0212] Cancer

[0213]

[0111] As used herein, the term "cancer", or grammatical variations thereof, refers to a malignant neoplasm. In particular, the term "cancer" refers herein to any member of a class of diseases or disorders that are characterized by uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue through invasion or by implantation into distant sites by metastasis. Metastasis is defined as the stage in which cancer cells are transported through the bloodstream or lymphatic system. In particular embodiments, the cancer is selected from the group consisting of: multiple myeloma, epithelial cancer, epithelial ovarian cancer, mucosal melanoma, non-small cell lung cancer, melanoma, head and neck cancer, renal cell cancer, Hodgkin’s lymphoma, Cutaneous Squamous Cell Carcinoma, glioblastoma, esophageal cancer, gastric cancer, duodenal cancer, small intestinal cancer, appendiceal cancer, large bowel cancer, colon cancer, rectum cancer, colorectal cancer, anal cancer, pancreatic cancer, liver cancer, gallbladder cancer, spleen cancer, renal cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, breast cancer, pulmonary cancer, thyroid cancer, thymus cancer, brain cancer, nervous system cancer, gliomas, oral cavity cancer, skin cancer, blood cancer, lymphomas, eye cancer, bone cancer, bone marrow cancer, muscle cancer, nonsmall cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), urothelial carcinoma, non-muscle invasive bladder cancer [NMIBC]), colon or rectal cancer, esophageal or certain gastroesophageal junction (GEJ) carcinomas, cervical cancer, renal cell carcinoma (ROC), advanced endometrial carcinoma, cutaneous squamous cell carcinoma (cSCC), and / or triple-negative breast cancer (TNBC), and the like. In a particular embodiment, the cancer is multiple myeloma.

[0214]

[0112] In a particular embodiment, as set forth above, provided herein are methods of preventing, treating, and / or reducing the risk of cancer, comprising administering to a patient in need thereof, one or more of the probiotic vaccines provided herein; and / or one or more of the recombinant phage provided herein, wherein the exogenous peptide epitope is selected from the group consisting of: SEQ ID NOs: 5-10, 12, 13, and 33-72, and 74. in particular embodiments, the cancer is selected from the group consisting of: multiple myeloma, epithelial cancer, epithelial ovarian cancer, mucosal melanoma, non-small cell lung cancer, melanoma, head and neck cancer, renal cell cancer, Hodgkin's lymphoma, Cutaneous Squamous Cell Carcinoma, glioblastoma, esophageal cancer, gastric cancer, duodenal cancer, small intestinal cancer, appendiceal cancer, large bowel cancer, colon cancer, rectum cancer, colorectal cancer, anal cancer, pancreatic cancer, liver cancer, chondro sarcoma, gallbladder cancer, spleen cancer, renal cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, breast cancer, pulmonary cancer, thyroid cancer, thymus cancer, brain cancer, nervous system cancer, gliomas, oral cavity cancer, skin cancer, blood cancer, lymphomas, eye cancer, bone cancer, bone marrow cancer, muscle cancer, non- small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), urothelial carcinoma, non-muscle invasive bladder cancer [NMIBC]), colon or rectal cancer, esophageal or certain gastroesophageal junction (GEJ) carcinomas, cervical cancer, renal cell carcinoma (RCC), advanced endometrial carcinoma, cutaneous squamous cell carcinoma (cSCC), and / or triple-negative breast cancer (TNBC). In yet another embodiment, the cancer is selected from: pancreatic cancer, liver cancer, mucosal melanoma, and / or chondro sarcoma.

[0215]

[0113] Also provided herein is a method of preventing, treating, and / or reducing the risk of breast, bladder, pancreatic, liver, mucosal melanoma, chondro sarcoma, ovarian, and / or stomach cancer, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: YLQRDISEM (SEQ ID NO:5), ELQRDISEM (SEQ ID NO:6), LEEPNRVQL (SEQ ID NOY), ATADLELAY (SEQ ID NO:8), AQRMTTQLLLL (SEQ ID NO:9), SLLMQITQC (SEQ ID NO:10),

[0216] VELMYPPPYYLGIGN (SEQ ID NO:12), EGGVAMPGAEDDVV (SEQ ID NO:13), ALLEIASCLgAGNNWAKGHYTEGAELVD (SEQ ID NO:33), ALLEIASCLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:34), ALLEIASCLgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:35), ALLEIASCLgYLSDNHILI (SEQ ID NO:36), AGAFAPSAAVAgAGNNWAKGHYTEGAELVD (SEQ ID NO:37), AGAFAPSAAVAgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:38), AGAFAPSAAVAgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:39, AGAFAPSAAVAgYLSDNHILI (SEQ ID NO:40), FLAEDALNTVgAGNNWAKGHYTEGAELVD (SEQ ID NO:41), FLAEDALNTVgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:42), FLAEDALNTVgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:43), FLAEDALNTVgYLSDNHILI (SEQ ID NO:44), AQRMTTQLLLLgAGNNWAKGHYTEGAELVD (SEQ ID NO:45), AQRMTTQLLLLgTFSWPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:46), AQRMTTQLLLLgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:47), AQRMTTQLLLLgYLSDNHILI (SEQ ID NO:48), SPRMSGLLSQTgAGNNWAKGHYTEGAELVD (SEQ ID NO:49), SPRMSGLLSQTgTFSWPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:50), SPRMSGLLSQTgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:51), ALLEIASCLxAGNNWAKGHYTEGAELVD (SEQ ID NO:52), ALLEIASCLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:53), ALLEIASCLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:54), ALLEIASCLxYLSDNHILI (SEQ ID NO:55), AGAFAPSAAVAxAGNNWAKGHYTEGAELVD (SEQ ID NO:56), AGAFAPSAAVAxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:57), AGAFAPSAAVAxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:58), AGAFAPSAAVAxYLSDNHILI (SEQ ID NO:59), FLAEDALNTVxAGNNWAKGHYTEGAELVD (SEQ ID NQ:60), FLAEDALNTVxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID N0:61), FLAEDALNTVxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:62), FLAEDALNTVxYLSDNHILI (SEQ ID NO:63), AQRMTTQLLLLxAGNNWAKGHYTEGAELVD (SEQ ID NO:64), AQRMTTQLLLLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:65) AQRMTTQLLLLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:66), AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67), SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68), SPRMSGLLSQTxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:69), SPRMSGLLSQTxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:70), KKKPTPIQLNPAPAGSAVNG (SEQ ID N0:71), RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), and / or QLIYNLTLCELNGTDWL (SEQ ID NO:73).

[0217] In a certain aspect, the recombinant phage and / or probiotic vaccine comprising YLQRDISEM (SEQ ID NO:5), ELQRDISEM (SEQ ID NO:6), and / or LEEPNRVQL (SEQ ID NO:7), is used to prevent, treat, or reduce the risk of pancreatic cancer. In another aspect, the recombinant phage and / or probiotic vaccine comprising ATADLELAY (SEQ ID NO:8), is used to prevent, treat, or reduce the risk of liver cancer. In yet another aspect, the recombinant phage and / or probiotic vaccine comprising SLLMQITQC (SEQ ID NQ:10), is used to prevent, treat, or reduce the risk of chondro sarcoma. In yet another aspect, the recombinant phage and / or probiotic vaccine comprising VELMYPPPYYLGIGN (SEQ ID NO: 12) and / or EGGVAMPGAEDDW (SEQ ID NO: 13), is used to prevent, treat, or reduce the risk of mucosal melanoma, prostate cancer, pulmonary fibrosis, lung cancer, non-small cell lung cancer, and the like.

[0114] In particular embodiments, as set forth above, provided herein are methods of preventing, treating, and / or reducing the risk of mucosal melanoma, prostate cancer, pulmonary fibrosis, lung cancer, non-small cell lung cancer, and the like, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:

[0218] VELMYPPPYYLGIGN (SEQ ID NO:12) and / or EGGVAMPGAEDDW (SEQ ID NO:13).

[0219] Infectious Disease

[0220]

[0115] In another embodiment, as set forth above, provided herein is a method of preventing, treating, and / or reducing the risk of virus infection and / or chronic wasting disease manifestation, comprising administering, to a subject or patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: WGQPHGGG (SEQ ID NO:1).

[0221]

[0116] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Norovirus infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:

[0222] PVAGAASAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRN (SEQ ID NO:2), NNYDPTEEIPAPLGTPDF (SEQ ID NO:3), and / or

[0223] WIRNNF (SEQ ID NO:4).

[0224]

[0117] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Respiratory Syncytial Virus (RSV) infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: NSELLSLINDMPSTNDQKKLMSNN (SEQ ID NO:14), NSELLSLINDMPITNDQKKLMSNNV (SEQ ID NO:15),

[0225] NSELLSLINDMPITNDQKKLMSNNVQ (SEQ ID NO:16) and / or NSELLSUNDMPITNDQKKLMSNNVQI (SEQ ID NO:17).

[0118] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Porcine Epidemic Diarrhea viral infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:

[0226] GFEIGISQEPFDP (SEQ ID NO:18),

[0227] SLPQDVTRC (SEQ ID NO: 19), DGVCNGAAV (SEQ ID NO:20), FDLDDGFYPISS (SEQ ID NO:21), ITGTPKPLEG (SEQ ID NO:22), SVYDPASGRVVQKR (SEQ ID NO:23), DQLPDVIPDYID (SEQ ID NO:24), PNRTGPSL (SEQ ID NO:25), YSNIGVCK (SEQ ID NO:26), and / or NLLSHEQP (SEQ ID NO:27).

[0228]

[0119] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Transmissible Gastroenteritis viral infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:

[0229] ATAVIKTGTCPFSFDKLNNY (SEQ ID NO:28), PFSFDKLNNYLTFNKFCLSL (SEQ ID NO:29), VVRSLYVIYEEGCNIVGVPS (SEQ ID NO:30), LHLDSCTDYNIYGRTGVGII (SEQ ID NO:31), and / or LYYTSLSGDLLGFKNVSDGV (SEQ ID NO:32).

[0230]

[0120] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of Porcine Reproductive Respiratory Syndrome (PRRS) and / or disease manifestation, comprising administering, to a subject or patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: QLIYNLTLCELNGTDWL (SEQ ID NO:73) and / or

[0231] NNSSSSHLQLIYNLTICELNGTD (SEQ ID NO:80). immunotherapy

[0232]

[0121] Immunotherapy (Immuno-oncology therapy) refers to creating, facilitating and / or modulating a therapeutic immune response by causing the production of antibodies to selected targets, such as TNF, and the like. Accordingly, immunotherapy refers to engineering or generating a therapeutic immune response by causing the production of antibodies to the selected target.

[0233]

[0122] In another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of inflammatory bowel disease (IBD), comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from DPQGVTCGAATLSAERV (SEQ ID NO: 11), KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), and / or RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72). In yet another embodiment, provided herein are methods of preventing, treating, and / or reducing the risk of ankylosing spondylitis, primary sclerosing cholangitis and Takayasu’s arteritis, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from DPQGVTCGAATLSAERV (SEQ ID NO: 11), KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), and / or RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72). In yet another embodiment of the present invention, provided herein are methods of preventing, treating, and / or reducing the risk of inflammatory disease or type 1 diabetes, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR

[0234] I (SEQ ID NO:74).

[0235] Administration of Probiotic Vaccine

[0236]

[0123] The invention probiotic vaccine (e.g., bacterial cells comprising invention recombinant phage) and / or recombinant bacteriophage pharmaceutical compositions can be delivered or administered to a subject (e.g., a patient in need thereof having a cancer) by any suitable delivery route, including but not limited to the pulmonary, intramuscular, subcutaneous, intubation (endotracheal), or intraperitoneal route, as deemed most appropriate by attending medical personnel in light of all factors for a given subject. The subject may be any suitable subject, including but not limited to humans, primates, dogs, cats, horses, cattle, and the like. In one embodiment, the subject is a human subject.

[0237]

[0124] In some embodiments, live probiotic bacterial cells and / or recombinant bacteriophage may be administered orally, as the invention probiotic vaccine, via fermented dairy products and / or probiotic fortified foods. Exemplary delivery foods can be selected from: probiotic solution, gummy (e.g., gelatin-based), pill, yogurt, coconut water, pickled vegetables, fermented bean paste (e.g., tempeh, miso, doenjang), kefir, buttermilk or karnemelk, kimchi, pao cai, sauerkraut, soy sauce, zha cai). In other embodiments, the probiotic bacteria may be delivered in vivo as tablets, capsules, powders and / or sachets containing the bacteria in freeze dried form.

[0238]

[0125] In a particular embodiment, the pharmaceutical carrier is a gelatin-based chewtype formulation (e.g., gummy or troches), in this embodiment, the invention gummy is a semi-transparent, mild to no taste or smell, anhydrous gelatin gummy base derived from naturally sourced ingredients allowing for easy compounding of water-unstable APIs, or where stability of a drug is unknown. For this embodiment, the gummy base is heated, mixed with other ingredients, and poured into molds, and cooled. In one embodiment, the base has a relatively low working temperature when heated to form a pourable solution that forms a stable gelatin matrix when cooled having a desirable API load capacity. The invention gummy can be easily sweetened and flavored to preference.

[0239]

[0126] In a particular embodiment, commercially available Klear Gummy™ (SpecializedRx Products, MN) is used herein, which provides the convenience of a ready-to-use anhydrous gummy base allowing for an extended beyond-use dating (BUD) stability profile per USP. This Base has a slight translucent amber appearance and is ready for the addition of actives, sweetener, and preferred flavors.

[0240]

[0127] In other embodiments, the invention probiotic vaccine and / or recombinant bacteriophage can be delivered to a patient via injection, infusion, inoculation, direct surgical delivery, or any combination thereof, in some embodiments, the probiotic vaccine and / or recombinant bacteriophage is administered to a human in the deltoid region or axillary region. For example, the probiotic vaccine and / or recombinant bacteriophage is administered into the axillary region as an intradermal injection. In other embodiments, the probiotic vaccine and / or recombinant bacteriophage is administered intravenously via injection or infusion.

[0128] In particular embodiments, the incorporation of invention recombinant bacteriophages into therapeutic formulations includes encapsulating them within a stabilizing substance. Using this approach, various antimicrobial materials such as powders, semisolids and nanofibers can be produced, providing more options for effective delivery. Accordingly, various encapsulated phage formulations including spray and freeze-dried powders, emulsions and liposomes are contemplated herein; as set forth described in: Bulbake et al., Liposomal formulations in clinical use: An updated review. Pharmaceutics. 2017;9:12. doi: 10.3390 / pharmaceutics9020012; Esteban et al., Enhancement of the antimicrobial properties of bacteriophage-k via stabilization using oil-in-water nano-emulsions. Biotechnol. Prog. 2014;30:932-944. doi: 10.1002 / btpr.1898; which are each incorporated herein by reference in their entirety for all purposes.

[0241]

[0129] Also contemplated herein as a therapeutic formulation for the invention recombinant phage is immobilization, where phages are bound to substrate surfaces and / or encapsulation using either: emulsification, freeze-drying, spray-drying, liposome encapsulation and electrospinning, in which bacteriophages are coated / surrounded by certain stabilizing agents, providing protection against the external environment, as set forth in Rosner et al., Pharmaceuticals (Basel). 2021 Apr; 14(4): 359; Published online 2021 Apr 13. doi: 10.3390 / ph14040359; which is which is incorporated herein by reference in its entirety for all purposes.

[0242]

[0130] In other embodiments, the invention compositions (e.g., recombinant bacteriophage or probiotic vaccine) is administered via the pulmonary route. For example, freeze-drying and spray-drying techniques can be used to produce phage- coated powders, which can then also be incorporated into a cream for direct application, pill-form for oral application as well as incorporation into an inhaler system. See, e.g., Malik et al., Formulation, stabilization and encapsulation of bacteriophage for phage therapy. Adv. Colloid Interface Sci. 2017;249:100-133. doi: 10.1016 / j.cis.2017.05.014; which is incorporated herein by reference in its entirety for all purposes. In a particular embodiment of the invention, pulmonary administration comprises inhalation of a therapeutic dose (e.g., either a single dose or multiple doses) of an invention recombinant phage pharmaceutical composition set forth herein, such as by nasal, oral inhalation, or both. The recombinant phage pharmaceutical compositions can be administered in two or more separate administrations (doses). In one embodiment, the recombinant phage may be formulated as a dry powder (either atone, as a mixture, or in a dry biend with, for example, lactose) from a dry powder inhaler or as an aerosol spray (e.g., liquid droplets of a stable dispersion or suspension of the probiotic vaccine in a gaseous medium). See, w.g, Chang et al., Antimicrob. Agents Chemother. 2018;62:e01714-17. doi: 10.1128 / AAC.01714-17; which is incorporated herein by reference in its entirety for all purposes. Invention recombinant phage compositions delivered by aerosol may be deposited in the airways by gravitational sedimentation, inertial impaction, and / or diffusion. Any suitable device for generating the aerosol may be used, including but not limited to pressured meter inhalers (pMDI), nebulizers, vaporizers (g.e. via a vaping device; e-cigarette), dry powder inhalers (DPI), and soft-mist inhalers.

[0243]

[0131] In a particular embodiment, the invention recombinant phage are nebulized as set forth in Guellec et al., Viruses. 2023 Mar; 15(3): 602 (doi: 10.3390 / v15030602), which is incorporated herein by reference in its entirety for ail purposes. The phage ability to self-replicate in the presence of its target is contemplated herein to decrease the amount of administration and potentially leads to better compliance. The invention recombinant phage specificity limits the impact on the flora that participate in tissue homeostasis. Phages are generally considered safe, via different routes of administration, with a low incidence of adverse events.

[0244]

[0132] In other embodiments, an appropriate carrier for administering the cells can be selected by one of skill in the art by routine techniques. For example, the pharmaceutical carrier can be a buffered saline solution, e.g., cell culture media (e.g., LB media), and in another embodiment can include DMSO for preserving cell viability.

[0245]

[0133] In one embodiment, the invention probiotic vaccine and / or recombinant bacteriophage composition (e.g., a pharmaceutical composition) comprises a suspension further comprising a pharmaceutically acceptable aqueous carrier. The suspension can comprise a probiotic vaccine bacterium and / or a recombinant phage and a liquid carrier. The liquid carrier can be aqueous. The liquid carrier of the suspension can comprise either water, coconut water and / or LB medium; and optionally one or more excipients selected from the group consisting of buffer, tonicity adjusting agent, preservative, demulcent, viscosity modifier, osmotic agent, surfactant, antioxidant, alkalinizing agent, acidifying agent antifoaming agent, and colorant. For example, in one embodiment, the suspension can comprise an invention probiotic vaccine-bacterium, coconut water, buffer and salt. It optionally further comprises a surfactant. In some embodiments, the suspension consists essentially of or consists of coconut water, an invention probiotic vaccine bacterium and / or and invention recombinant phage suspended in the coconut water and buffer. The suspension can further contain an osmotic salt.

[0246]

[0134] In a particular embodiment, the pharmaceutical carrier is coconut water. In one embodiment, the invention probiotic vaccine-bacterium infected with said recombinant phage can remain in the coconut water medium that was used to grow respective probiotic vaccine bacterium, which in turn can be directly administered to the patient. In another embodiment, the invention probiotic vaccine-bacterium infected with said recombinant phage can be grown in a different medium and subsequently introduced into the coconut water as the final formulation, which in turn can be directly administered to the patient.

[0247]

[0135] Accordingly, also provided herein, is a composition comprising coconut water and a probiotic vaccine, wherein the probiotic vaccine comprises a recombinant bacteriophage, wherein the recombinant phage comprises at least one exogenous peptide epitope corresponding to SEQ ID NO: 1-74, or fragments or variants thereof. In certain embodiments, the coconut water further comprises one or more excipients selected from the group consisting of buffer, tonicity adjusting agent, preservative, demulcent, viscosity modifier, osmotic agent, surfactant, antioxidant, alkalinizing agent, acidifying agent antifoaming agent, and colorant. In a particular embodiment, the coconut water further comprises a buffer. In another embodiment, the coconut water further comprises a salt. In certain embodiments, the composition has a pH of 7.0-8.0. In a particular embodiment, the composition has a pH of 7.5.

[0248]

[0136] In another embodiment, it is also contemplated herein, that the invention probiotic vaccine-bacterium infected with said recombinant phage can remain in the LB medium that was used to grow respective probiotic vaccine bacterium, which in turn can be directly administered to the patient.

[0249]

[0137] In certain embodiments, the cells are administered in an infusible cry opreservation medium. The composition comprising the cells can include DMSO and hetastarch as cryoprotectants, Plasmalyte A and / or dextrose solutions and human serum albumin as a protein component.

[0250]

[0138] The quantity of probiotic vaccine and / or recombinant bacteriophage for administration to a patient as a cancer vaccine to effect the methods described herein and the most convenient route of such administration are based upon a variety of factors, as can the formulation of the vaccine itself. Some of these factors include the physical characteristics of the patient (e.g., age, weight, and sex), the physical characteristics of the tumor (e.g., location, size, rate of growth, and accessibility), and the extent to which other therapeutic methodologies (e.g., chemotherapy, and beam radiation therapy) are being implemented in connection with an overall treatment regimen. Notwithstanding the variety of factors to be considered for implementing the methods of the present invention to prevent, treat, and / or reduce the risk of acquiring cancer or infectious diseases, a mammal, preferably a human, can be administered with from about 5x106to about 2x108probiotic vaccine cells in from about 0.05 mL to about 2 mL solution (e.g., saline) in a single administration. In other embodiments, a mammal, preferably a human, can be administered with from about 1x103to about 1 x1015, 1x104to about 1 x1014, 1x105to about 1 x1013, 1x105to about 1 x1012, 1x105to about 1 x1011, 1x10sto about 1 x101°, 1x10sto about 1 x109, 1x10sto about 1 x108, 1x106to about 1 x1012, 1x106to about 1 x1011, 1x10sto about 1 x1010, 1x106to about

[0251] 1 x109, 1x10sto about 1 x108, probiotic vaccine cells in from about 0.05 mL to about

[0252] 2 mL solution (e.g., saline) in a single administration.

[0253]

[0139] Additional administrations can be carried out, depending upon the abovedescribed and other factors, such as the severity of tumor pathology In further embodiments, from about one to about five administrations of: about 1x104 / mi, about 1x105 / ml, about 1x106 / ml, about 1x10'7ml, about 1x108 / ml, about 1x109 / ml, about 1x1010 / ml, about 1x1011 / mi, about 1x1012 / ml, about 1x1013 / ml, about 1x1014 / mi, about 1x1015 / ml, probiotic vaccine cells is performed at two-week intervals. In one embodiment, from about one to about five administrations of about 1x10s / ml probiotic vaccine cells is performed at two-week intervals.

[0254] Coconut Water / native-Nissle 1917 Combination Embodiment

[0255]

[0140] In other embodiments, contemplated herein is a composition comprising a combination of coconut water and unmodified, native Nissle bacteria (e.g., Nissle 1917, the like). Other bacterium known for their probiotic effect can be used in leiu of, or in addition to, Nissle 1917 in combination with coconut water for drinking, or otherwise injesting. These coconut water / Nissle 1917, or coconut water / probiotic bacteria, combinations are useful herein to prevent, treat and / or reduce the risk of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), or other inflammatory conditions or diseases (e.g., ulcerative colitis of the gut), and the like.

[0256]

[0141] Accordingly, provided here is a method of preventing, treating, and / or reducing the risk of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), or other inflammatory conditions or diseases, said method comprising administering, to a patient in need thereof, an isolated composition comprising a combination of coconut water and unmodified, native Nissle bacteria (e.g., Nissle 1917, the like).

[0257] Combination Treatments

[0258]

[0142] Also contemplated herein is the use of a combination of more than one invention probiotic vaccine for a particular therapy to prevent, treat and / or reduce the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like), said method comprising administering to a patient in need thereof, more than one invention probiotic vaccine provided herein. In another embodiment, this particular combination therapy can also be further combined with at least another therapeutic agent.

[0259]

[0143] Also contemplated herein is the use of a combination of more than one invention recombinant phage for a particular therapy to prevent, treat and / or reduce the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like), said method comprising administering to a patient in need thereof, more than one invention recombinant phage provided herein. In another embodiment, this particular combination therapy can also be further combined with at least another therapeutic agent.

[0260]

[0144] Also contemplated herein is the use of a combination of both invention probiotic vaccines and recombinant phages for a particular therapy to prevent, treat and / or reduce the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like), said method comprising administering to a patient in need thereof, a combination of both an invention probiotic vaccine and a recombinant phage provided herein. In another embodiment, this particular combination therapy can also be further combined with at least another therapeutic agent.

[0261]

[0145] Also contemplated herein, the independent method of administering an invention probiotic vaccine for a particular therapy, or the independent method of administering a invention recombinant phage for a particular therapy, can also be combined in therapeutic methods with other therapeutic agents that also prevent, treat and / or reduce the risk of cancer, autoimmune diseases, respiratory diseases (e.g., asthma), neurological diseases, infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like), comprising administering to a patient in need thereof, an invention probiotic vaccine, recombinant phage, or both, in combination with another therapeutic agent. Accordingly, probiotic vaccination and / or direct administration of invention recombinant bacteriophage (for vaccination or treatment) provided herein can be combined with other treatments.

[0262]

[0146] For example, a patient receiving at least one invention probiotic vaccine and / or at least one recombinant bacteriophage, can also receive administration of chemotherapy, immuno-oncoiogy therapy, radiation, and / or surgical therapy before, concurrently, or after the direct and / or probiotic vaccination. Chemotherapy is used to shrink and slow cancer growth. Chemotherapy is recommended for numerous cancers after the initial surgery for cancer; however, sometimes chemotherapy is given to shrink the cancer before surgery. The number of cycles of chemotherapy treatment depends on the stage of the disease. Chemotherapy may neutralize antitumor immune response generated through vaccine therapy. In addition, chemotherapy can be combined safely with immunotherapy, with possibly additive or synergistic effects, as long as combinations are designed rationally. Examples of chemotherapeutic agents that can be used in treatments of patients with cancer include, but are not limited to, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, etoposide, gemcitabine, oxaliplatin, paclitaxel, TAXOL™, topotecan, and vinorelbine. In some embodiments, a patient receiving probiotic vaccination has already received chemotherapy, radiation, and / or surgical treatment for the gynecological or peritoneal cancer. Immunotherapy (Immuno-oncoiogy therapy) refers to creating, facilitating and / or modulating an immune response by causing the production of antibodies to selected targets, such as TNF, and the like targets. Immunotherapies for use in combination with the invention probiotic vaccines (or invention recombinant phage) include treatment with Keytruda® (pembrolizumab), Opdivo® (nivolumab), and the like.

[0263]

[0147] In addition to, or separate from chemotherapeutic treatment, a patient receiving an invention probiotic vaccination can be treated with any other treatments that are beneficial for the particular cancer. For example, a patient having ovarian, fallopian tube or peritoneal cancer, can be treated prior to, concurrently, or after the invention probiotic vaccination with a COX-2 inhibitor, as described, e.g., in Yu and Akasaki, WO 2005 / 037995. In another embodiment, a patient receiving an invention probiotic vaccination can be treated with bevacizumab (Avastin®) prior to, concurrently, or after probiotic vaccination.

[0264] EXAMPLES

[0265]

[0148] The following examples are provided to better illustrate the claimed disclosure and are not to be interpreted as limiting the scope of the disclosure. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the disclosure. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the disclosure.

[0266] Example 1 - Construction of PD-1 / AFP10 Epitope-containing Nissie Probiotic Vaccine (M13KE-SALV-0004):

[0267]

[0149] The mRNA sequence encoding the -AFPEDRSQPG- (SEQ ID NO:75; referred to herein as “AFP10” and “SALV-0004”) amino acid sequence was inserted between pill signal peptide of M13KE RF I DNA and pill protein coding sequence to construct M13KE-AFP10 RF I DNA, and the sequence information at the recombination insertion site within the M13KE phage is set forth in Figure 5.

[0268] Preparation of M13KE vector

[0269]

[0150] E.coli ER2738 in logarithmic growth phase was infected with M13KE phage. After culture, the bacteria were collected by centrifugation and used to extract plasmids by Axygen MidiPrep plasmid extraction kit. M13KE RF I DNA was further purified and then loaded into an agarose gel for QC.

[0270]

[0151] M13KE RF I DNA was digested by Kpn 1 / Eag I and then separated by agarose gel electrophoresis.

[0271] Construction of M13KE-SALV-0004 phages

[0272]

[0152] AFP10-epitope (SALV-0004; SEQ ID NO:75) encoding DNA was synthesized, digested by Kpn 1 / Eag I, and ligated with M13KE RF I DNA. The construct of AFP10- M13KE RF I DNA was transformed into E.coli ER2738 competent cells. After resuscitation, they infected E. coli ER2738 in logarithmic growth phase and cultured overnight at 32°C on TOP-Agar LB (containing IPTG / X-gal) plates. Results indicated that there were many blue spots on the plate with the transformation of recombinant M13KE-AFP10 phage into E.coli ER2738, while no blue spot was observed in control group. Several blue spots were selected and validated by sequencing. Results showed all clones had a correct construct corresponding to the correct mRNA sequence.

[0273] Phage preparation

[0274]

[0153] E.coli ER2738 in logarithmic growth phase was infected with M13KE phages having the correct AFP10 epitope sequence (SALV-0004; SEQ ID NO:75) inserted therein. After overnight culture, the culture supernatant was collected, glycerol was added to produce a 10% final concentration, and it was stored at -20°C.

[0275]

[0154] The above invention recombinant M13KE-SALV-0004 (AFP10) phage is used to infect the Nissle 1917 E. coii, and is used as a probiotic referred to herein as “N4” probiotic vaccine. This infected Nissle bacteria with the AFP10 epitope expressing bacteriophage (MK13E-SALV-0004) is then used as a probiotic oral vaccine for the patient to make anti-PD-1 antibodies against his / her cancer, such as gastric cancer, melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), urothelial carcinoma, non-muscle invasive bladder cancer [NMIBC]), colon or rectal cancer, esophageal or certain gastroesophageal junction (GEJ) carcinomas, cervical cancer, renal cell carcinoma (RCC), advanced endometrial carcinoma, cutaneous squamous cell carcinoma (cSCC), and / or triple-negative breast cancer (TNBC). M113KE-AFP10 infected Nissle probiotic phage (i.e., phage + E. coli) containing the above epitopes are produced. Oral administration of the M113KE- AFP10 infected Nissle probiotic is contemplated herein to generate both SlgA and IgG anti-PD-1 antibodies in the patient; whereas IV injection of the recombinant phage alone (separate from the probiotic bacteria) can generate IgA and IgG in the patient with reactivity toward PD-1 on the patient's human cancer ceils.

[0276] Example 2 - Construction of HER2 Epitope-containing Probiotic Vaccine (M13KE-SALV-0006 in EcN):

[0277]

[0155] Provided herein is a bacteriophage oral vaccine comprising a HER2 epitope functionally expressed on a M13KE phage that has infected Nissle 1917 E. coli bacterial cells. Specifically, HER2 epitopes are integrated into coat protein gene pill of MKE, fl and / or M13 bacteriophage producing a vaccination to overcome immunological tolerance against HER2. The carrier acts as an adjuvant and improves stability and B cell presentation of the epitopes.

[0278]

[0156] The mRNA sequence encoding the -ESFDGDPASNTAPLQPEQL- amino acid sequence (SEQ ID NO:76) was inserted between pill signal peptide of M13KE RF I DNA and pill protein coding sequence to construct M13KE-HER2 RF I DNA, as set forth in Example 3 below.

[0279] Preparation of M13KE vector

[0280] [1ST] E.coli ER2738 in logarithmic growth phase was infected with M13KE phage. After culture, the bacteria were collected by centrifugation and used to extract plasmids by Axygen MidiPrep plasmid extraction kit. M13KE RF I DNA was further purified and then loaded into an agarose gel for QC.

[0281]

[0158] M13KE RF I DNA was digested by Kpn 1 / Eag I and then separated by agarose gel electrophoresis.

[0282] Construction of recombinant M13KE-SALV-0006 phage

[0283]

[0159] HER2-epitope encoding DNA (SALV-0006; SEQ ID NO:76) was synthesized, digested by Kpn 1 / Eag I, and ligated with M13KE RF I DNA. The construct of HER2- M13KE RF I DNA was transformed into E.coli ER2738 competent cells. After resuscitation, they infected E. coli ER2738 in logarithmic growth phase and cultured overnight at 32°C on TOP-Agar LB (containing IPTG / X-gal) plates. Results indicated that there were many blue spots on the plate with the transformation of invention M13KE-HER2 phage into E.coli ER2738, while no blue spot was observed in control group. Several blue spots were selected and validated by sequencing. Results showed all clones had a correct construct corresponding to the mRNA sequence:

[0284] GAATCATTTGACGGAGATCCCGCTAGTAACACCGCGCCGCTGCAGCCGGAGCA GTTG.

[0285] Phage preparation

[0286]

[0160] E.coli ER2738 in logarithmic growth phase was infected with M13KE phages inserted with the correct HER2 epitope sequence (M 13 KE-SA LV-0006 phage; SEQ ID NO:76). After overnight culture, the culture supernatant was collected, added with glycerol of 10% final concentration, and stored at -20°C.

[0161] The above M13KE-HER2 phage is used to infect the Nissle 1917 E. coll, which has been identified as male and is used as a probiotic. The invention Nissle infected bacteria with the HER2 epitope (referred to herein as “N6” and comprising SEQ ID NO:76) expressing bacteriophage is non-lytic and continuously secretes (or pumps out) the SALV-0006 recombinant phage; and is thus used as a probiotic oral vaccine for the patient to continuously promote the production of anti-HER2 antibodies within a patient / subject against his / her cancer (e.g., breast, bladder, pancreatic, ovarian, and / or stomach cancer). M13KE-HER2 infected Nissle probiotic phage (e.g., phage + E. coll) containing the above epitopes are produced. Oral administration of the M13KE-HER2 infected Nissle probiotic phage is contemplated herein to generate secretory (SlgA) and IgG antibodies in the patient; whereas injection of the recombinant phage alone (separate from the probiotic bacteria) can generate IgG in the patient with reactivity toward HER2 on the patient's human cancer cells.

[0287] Example 3 - Construction of SALV-0013 Probiotic Vaccine:

[0288]

[0162] SALV-0013 corresponds to the peptide epitope SEQ ID NO:77 inserted into an MK13 phage as set forth above to form a recombinant bacteriophage MK13 / SALV- 0013. Bacteriophage MK13 / SALV-0013 was then infected into Nissle 1917 and ER2738 bacterial cells to form the “N13” and “ER13”, respectively, recombinant phage infected E. coli stocks.

[0289]

[0163] The mRNA sequence encoding the -ESKVDPSKAW- (SEQ ID NO:77; referred to herein as “SALV-0013”) amino acid sequence was inserted between pili signal peptide of M13KE RF I DNA and pill protein coding sequence to construct M13KE- SALV-0013 RF I DNA, and the sequence information at the recombination insertion site within the M13KE phage is set forth in Figure 6.

[0290] Preparation of M13KE vector

[0291]

[0164] E.coli TG1 / ER2738 in logarithmic growth phase was infected with M13KE phage. After culture, the bacteria were collected by centrifugation and used to extract plasmids by Axygen MidiPrep plasmid extraction kit. M13KE RF I DNA was further purified and then loaded into an agarose gel for QC.

[0292]

[0165] M13KE RF I DNA was digested by Kpn 1 / Eag I and then separated by agarose gel electrophoresis.

[0293] Construction of M13KE-SALV-0013 recombinant phage

[0166] SALV-0013-epitope (SEQ ID NO:77) encoding DNA was synthesized, digested by Kpn 1 / Eag I, and ligated with M13KE RF I DNA. The construct of SALV-0013- M13KE RF I DNA was transformed into E.coli TG1 competent cells. After resuscitation, they infected E. coli TG1 in logarithmic growth phase and cultured overnight at 32°C on TOP-Agar LB (containing IPTG / X-gai) plates. Results indicated that there were many blue spots on the plate with the transformation of recombinant M13KE-SALV-0013 phage into E.coli TG1 , while no blue spot was observed in control group. Several blue spots were selected and validated by sequencing. Results showed all clones had a correct construct corresponding to the correct mRNA sequence.

[0294] Phage preparation

[0295]

[0167] E.coli TG1 in logarithmic growth phase was infected with M13KE-SALV-0013 phages having the correct SALV-0013 epitope sequence inserted therein. After overnight culture, the culture supernatant was collected, glycerol was added to produce a 10% final concentration, and it was stored at -20°C. The titer was determined by gradient dilution, and was 1.36x1010pfu / mL.

[0296]

[0168] The above invention recombinant M13KE-SALV-0013 phage is used to infect the Nissle 1917 E. coil to generate an ivention probiotic vaccine, which can be used as an probiotic therapy. For example, this infected Nissle bacteria with the SALV- 0013 epitope expressing bacteriophage is then used as a probiotic oral vaccine for the patient to make anti-PMSA antibodies against his / her cancer, such as prostate cancer, melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), urothelial carcinoma, non-muscle invasive bladder cancer [NMIBC]), colon or rectal cancer, esophageal or certain gastroesophageal junction (GEJ) carcinomas, cervical cancer, renal cell carcinoma (RCC), advanced endometrial carcinoma, cutaneous squamous cell carcinoma (cSCC), and / or triple-negative breast cancer (TNBC), and the like. M113KE-SALV-0013 infected Nissle probiotic phage (i.e., phage + E. coli) containing the above epitopes are produced. Oral administration of the M113KE-SALV-00013 infected Nissle probiotic has been found to generate both SlgA and IgG anti-PSMA antibodies in animal studies set forth in the Examples; whereas IV injection or infusion of the recombinant phage alone (separate from the probiotic bacteria) can generate both IgA and IgG in the patient with reactivity toward PSMA on the subject’s human cancer cells. Example 4 - Construction of Specified Epitope-containing Nissie Probiotic Vaccine:

[0297]

[0169] The mRNA sequence encoding one or more than one of the amino acid sequences selected from:

[0298] WGQPHGGG (SEQ ID NO:1),

[0299] PVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRN (SEQ ID NO:2),

[0300] NNYDPTEEIPAPLGTPDF (SEQ ID NO:3),

[0301] WIRNNF (SEQ ID NO:4),

[0302] YLQRDISEM (SEQ ID NO:5),

[0303] ELQRDISEM (SEQ ID NO:6),

[0304] LEEPNRVQL (SEQ ID NO:7),

[0305] ATADLELAY (SEQ ID NO:8),

[0306] AQRMTTQLLLL (SEQ ID NO:9),

[0307] SLLMQITQC (SEQ ID NO: 10),

[0308] DPQGVTCGAATLSAERV (SEQ ID NO:11),

[0309] VELMYPPPYYLGIGN (SEQ ID NO:12),

[0310] EGGVAMPGAEDDW (SEQ ID NO: 13),

[0311] NSELLSLiNDMPITNDQKKLMSNN (SEQ ID NO:14),

[0312] NSELLSLINDMPITNDQKKLMSNNV (SEQ ID NO:15),

[0313] NSELLSLINDMPITNDQKKLMSNNVQ (SEQ ID NO:16),

[0314] NSELLSLINDMPITNDQKKLMSNNVQI (SEQ ID NO:17),

[0315] GFEIGISQEPFDP (SEQ ID NO:18),

[0316] SLPQDVTRC (SEQ ID NO: 19),

[0317] DGVCNGAAV (SEQ ID NO:20),

[0318] FDLDDGFYPISS (SEQ ID NO:21),

[0319] ITGTPKPLEG (SEQ ID NO:22),

[0320] SVYDPASGRVVQKR (SEQ ID NO:23),

[0321] DQLPDVIPDYID (SEQ ID NO:24),

[0322] PNRTGPSL (SEQ ID NO:25),

[0323] YSNIGVCK (SEQ ID NO:26),

[0324] NLLSHEQP (SEQ ID NO:27),

[0325] ATAVIKTGTCPFSFDKLNNY (SEQ ID NO:28),

[0326] PFSFDKLNNYLTFNKFCLSL (SEQ ID NO:29),

[0327] WRSLYVIYEEGCNIVGVPS (SEQ ID NO:30), LHLDSCTDYNSYGRTGVGII (SEQ ID N0:31),

[0328] LYYTSLSGDLLGFKNVSDGV (SEQ ID NO:32),

[0329] ALLEIASCLgAGNNWAKGHYTEGAELVD (SEQ ID NO:33),

[0330] ALLEIASCLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:34),

[0331] ALLEIASCLgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:35),

[0332] ALLEIASCLgYLSDNHILI (SEQ ID NO:36),

[0333] AGAFAPSAAVAgAGNNWAKGHYTEGAELVD (SEQ ID NO:37),

[0334] AGAFAPSAAVAgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:38),

[0335] AGAFAPSAAVAgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:39),

[0336] AGAFAPSAAVAgYLSDNHILI (SEQ ID NO:40),

[0337] FLAEDALNTVgAGNNWAKGHYTEGAELVD (SEQ ID N0:41),

[0338] FLAEDALNTVgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:42),

[0339] FLAEDALNTVgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:43), FLAEDALNTVgYLSDNHILI (SEQ ID NO:44),

[0340] AQRMTTQLLLLgAGNNWAKGHYTEGAELVD (SEQ ID NO:45),

[0341] AQRMTTQLLLLgTFSWPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:46),

[0342] AQRMTTQLLLLgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:47), AQRMTTQLLLLgYLSDNHILI (SEQ ID NO:48),

[0343] SPRMSGLLSQTgAGNNWAKGHYTEGAELVD (SEQ ID NO:49),

[0344] SPRMSGLLSQTgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:50):

[0345] SPRMSGLLSQTgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID N0:51), ALLEIASCLxAGNNWAKGHYTEGAELVD (SEQ ID NO:52),

[0346] ALLEIASCLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:53),

[0347] ALLEIASCLxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:54), ALLEIASCLxYLSDNHILI (SEQ ID NO:55),

[0348] AGAFAPSAAVAxAGNNWAKGHYTEGAELVD (SEQ ID NO:56),

[0349] AGAFAPSAAVAxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:57), AGAFAPSAAVAxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:58):AGAFAPSAAVAxYLSDNHILI (SEQ ID NO:59),

[0350] FLAEDALNTVxAGNNWAKGHYTEGAELVD (SEQ ID NO:60),

[0351] FLAEDALNTVxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID N0:61),

[0352] FLAEDALNTVxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:62), FLAEDALNTVxYLSDNHILI (SEQ ID NO:63),

[0353] AQRMTTQLLLLxAGNNWAKGHYTEGAELVD (SEQ ID NO:64), AQRMTTQLLLLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:65), AQRMTTQLLLLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:66), AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67), SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68), SPRMSGLLSQTxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:69), SPRMSGLLSQTxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:70), KKKPTPIQLNPAPAGSAVNG (SEQ ID N0:71), RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), QUYNLTLCELNGTDWL (SEQ ID NO:73), and / or KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR

[0354] I (SEQ ID NO:74) Is Inserted between pill signal peptide of M13KE RF I DNA and pill protein coding sequence to construct M13KE-SEQ ID NO: 1-74 RF I DNA; for example, M13KE-SEQ ID NO:1 RF I DNA; M13KE-SEQ ID NO:2 RF I DNA; M13KE- SEQ ID NO:3 RF I DNA, and the like.

[0355] Preparation of M13KE vector

[0356]

[0170] E.coli ER2738 in logarithmic growth phase is infected with M13KE phage. After culture, the bacteria is collected by centrifugation and used to extract plasmids by Axygen MidiPrep plasmid extraction kit. M13KE RF I DNA is further purified and then loaded into an agarose gel for QC.

[0357]

[0171] M13KE RF I DNA is digested by Kpn 1 / Eag I and then separated by agarose gel electrophoresis.

[0358] Construction of M13KE-SEQ ID NO: 1-74 phages

[0359]

[0172] Various epitope-encoding DNA encoding SEQ ID NOs:1-74 is synthesized, digested by Kpn 1 / Eag I, and ligated with M13KE RF I DNA. The construct of HER2- M13KE RF I DNA is transformed into E.coli ER2738 competent cells. After resuscitation, they infect E. coli ER2738 in logarithmic growth phase and are cultured overnight at 32°C on TOP-Agar LB (containing IPTG / X-ga!) plates. The results indicate that there are many blue spots on the plate with the transformation of the respective M13KE-SEQ ID NO: 1-74 into E.coli ER2738, while no blue spot is observed in the control group. Several blue spots are selected and validated by sequencing. Results show that all clones had a correct construct corresponding to the mRNA sequence encoding: SEQ ID NOs:1-74.

[0360] Phage preparation

[0173] E.coli ER2738 in logarithmic growth phase is infected with M13KE phage having the correct respective SEQ ID NO: 1-74 exogenous peptide epitope inserted therein. After overnight culture, the culture supernatant is collected; glycerol is added to produce a 10% final concentration, and stored at -20°C.

[0361]

[0174] The above respective M13KE-SEQ ID NO: 1-74 phages are used to infect the Nissle 1917 E. coli, and is used as a probiotic. This Nissle infected bacteria with the respective SEQ ID NO: 1-74 epitope expressing bacteriophage is then used as a probiotic oral vaccine for the patient to make anti-epitope antibodies against his / her cancer (such as multiple myeloma), or respective epitope target. M113KE-CD38 infected Nissle probiotic phage (i.g., phage + E. coli) containing the above epitopes are produced. Oral administration of the M113KE-SEQ ID NO:1-74 infected Nissle probiotic is contemplated herein to generate both SlgA and IgG anti-CD38 antibodies in the patient; whereas injection of the recombinant phage alone (separate from the probiotic bacteria) can generate IgG in the patient with reactivity toward the respective SEQ ID NO:1-74 epitope within the subject or patient sus as on the patient's human cancer cells.

[0362] Example 5 - Preparation of Oral Probiotic LB Formulation:

[0363]

[0175] From an Escherichia coli Nissle 1917 (EcN) starter batch, 2ml of concentrated Nissle was added to LB media in a 50ml flask of LB media and grown in a shaking incubator for 4 hours at 37° C and 140 rpm. Next, 20ul of invention recombinant phage particles (e.g., M13KE-SEQ ID NO:1-74) was added to the Nissle bacteria and grown for 4 hours at 37° C and 140 rpm. Next, the infected host bacteria media was added to 400ml of LB media and grown for 4 hours at 37° C and 140 rpm. Next, 20ml aliquots were separated into 50ml test tubes and centrifuged at 3k rpm for 10 minutes. Using a pipette, 2ml of supernatant was drawn and the remaining supernatant was poured back into the flask while retaining the pellet in the test tube. Next, the 2ml of supernatant was returned to the test tube and vortexed at 600 rpm until pellet was dissolved to produce a bacteriophage concentrate. Using a pipette, 2ml of the concentrated bacteriophage was drawn from the 50ml test tube and placed into 2ml test tubes for storage. The resulting concentrates containing the respective M13KE- SEQ ID NO: 1-74 were frozen at -20 ° C until ready for use. Example 6 - IV Therapy Formulation Preparation

[0364]

[0176] Starting with a 2ml concentrated invention probiotic recombinant bacteriophage obtained as described herein (e.g., M13KE-SEQ ID NO:1-74), 2m! of the concentrate was centrifuged at 10k rpm for 15 minutes. The supernatant was drawn into a 3ml syringe leaving the pellet in the test tube. From the syringe, remove the needle and attach a 0.2 micron Swinnex filter and push the supernatant through the filter and into a 2ml test tube. The resulting filtered supernatant containing the respective M 13KE- SEQ ID NO: 1-74 can be used as either an intravenous therapy or as a nasal therapy.

[0365] Example 7 - Preparation of Oral Probiotic Coconut Water Formulation:

[0366] A. Coconut Water Processing

[0367]

[0177] The objective of this methodology was to process coconut water in a manner that maintains maximum nutritional content for the Nissle 1917 host while eliminating other biologically active and negative factors. This was achieved through the following set of processing techniques: i. Low Temperature. The coconut water was not heated in this process, and is maintained at 10° C for the processing. ii. Filtration. As a first step, the raw coconut water was run through a double micron filter to remove particulates. iii. High Pressure Pasteurization (HPP). The coconut water was then bottled and subjected through the process of high-pressure pasteurization where the water was pressed to 85,000 PSI for 15 minutes.

[0368] B. Preparation of Coconut Water for Bacteriophage Growth

[0369]

[0178] Native coconut water has a pH value of about 5.0, which is inadequate for bacterial growth. i. pH Adjustment. The pH levels of the processed coconut water from step A were modified sodium bicarbonate to various non-native levels and their respective impact on the growth of Nissle 1917 bacteria was evaluated. It has been found the modifying coconut water pH levels in the range of 7.0-7.5 produces a significant increase in bacterial cell growth. C. Bacteria Growth in Coconut Water

[0370]

[0179] From an Escherichia coli Nissle 1917 (EcN) starter batch, 2ml of concentrated Nissle was added to the processed non-native coconut water from steb B above, in a 50ml flask of processed coconut water and grown in a shaking incubator for 4 hours at 37° C and 140 rpm. Next, 20ul of invention recombinant phage particles (e.g., SALV-0004, -0006, and -0013) was added to the Nissle bacteria / coconut water solution in the 50ml flask and grown for 4 hours at 37° C and 140 rpm. Next, the infected Nissle bacteria solution from the 50ml flask was added to 400ml of coconut water in a larger flask and grown for 4 hours at 37° C and 140 rpm. Next, 20ml aliquots from the 400ml were separated into 50ml test tubes and centrifuged at 3k rpm for 10 minutes. Using a pipette, 2ml of supernatant was drawn and the remaining supernatant was poured back into the flask with 400ml while retaining the pellet in the test tube. Next, the 2ml of supernatant was returned to the test tube and vortexed at 600 rpm until pellet was dissolved to produce a bacteriophage concentrate in coconut water. Using a pipette, 2ml of the concentrated bacteriophage was drawn from the 50ml test tube and placed into 2ml test tubes for storage. This resulting concentrate in coconut water was frozen at -20 °C until ready for use.

[0371]

[0180] It has been found that SALV-006 infected Nissle 1917 (“N6”) grows at a roughly equivalent rate in both coconut water and LB media. The plating depicted in an experiment comparing a 108dilution of both coconut water and LB broth media shows considerable and comparable plaque forming units for coconut water media when compared to LB media. The invention method of growing the invention probiotic bacterium therapeutic in coconut water advantageously leverages the coconut water as a delivery media to provide significant cost and logistical advantages over prior art methods.

[0372] Preparation of Probiotic Vaccines comprising SEQ ID NOs:1-74 in Coconut Water

[0373]

[0181] Likewise, to prepare recombinant Nissie 1917 (EcN) comprising the recombinant phage M13KE-SEQ ID NO:1-74 in coconut water the following protocl is conducted. From an Escherichia coli Nissle 1917 (EcN) starter batch, 2ml of concentrated Nissle is added to coconut water in a 50ml flask of coconut water and is grown in a shaking incubator for 4 hours at 37° C and 140 rpm. Next, 20ul of invention recombinant phage particles (e.g., M13KE-SEQ ID NO:1-74) is added to the Nissle bacteria / coconut water solution in the 50ml flask and is grown for 4 hours at 37° C and 140 rpm. Next, the infected Nissle bacteria solution from the 50ml flask is added to 400ml of coconut water in a larger flask and is grown for 4 hours at 37° C and 140 rpm. Next, 20ml aliquots from the 400ml are separated into 50ml test tubes and centrifuged at 3k rpm for 10 minutes. Using a pipette, 2ml of supernatant is drawn and the remaining supernatant is poured back into the flask with 400ml while retaining the pellet in the test tube. Next, the 2ml of supernatant is returned to the test tube and vortexed at 600 rpm until pellet is dissolved to produce a bacteriophage concentrate in coconut water. Using a pipette, 2ml of the concentrated bacteriophage is drawn from the 50ml test tube and placed into 2ml test tubes for storage. This resulting concentrate in coconut water is frozen at -20 °C until ready for use.

[0374] Example 8 - Preparation of Probiotic Vaccine Gummy Formulation for Oral Delivery

[0375]

[0182] To prepare Luria Bertani (LB) broth, 400 ml of distilled water was mixed with 10g of HIMEDIA Luria Bertani Broth (Miller) and mixed on a warming plate with magnetic mixer until 37°C. From this LB broth, 20 aliquots of therapy was produced sufficient to dose a 4 animal (e.g., rat) animal study arm three times with 2 doses for tittering and 6 spare doses.

[0376]

[0183] To inoculate the LB broth, thaw a 2ml vial of starter batch of the respective invention probiotic Nissle bacterium vaccine (e.g., SALV-0004 (N4)), SALV-0006 (N6), SALV-00013 (N 13)) at room temperature for combining into LB media. Once thawed the starter batch is briefly vortexed and added to the LB media. To grow the invention bacteria containing the invention recombinant bacteriaphage (SALV-0004, SALV- 0006, SALV-00013), the media infected with the starter batch is placed in a shaking incubator at 37°C and 140 rpm for 4-8 hours. The media will start to become cloudy after a few hours and will be nearly opaque after 5 hours.

[0377]

[0184] To prepare the gummy formulation, 250ml Welch's Grape Juice, 1 TBS Sugar, 2 tsp Agar AgarPowder are combined in a mixture and stirred on a hot plate until boiling for 2 minutes; then taken off the heat to start the cooling process. Using a pipette, place 1 ,5ml aliquots of the combined mixture into 5ml test tubes in pre-heated warming block.

[0378]

[0185] To prepare therapeutic aliquots for animal studies, take 20ml aliquots from each tube and centrifuge the aliqouts down at 3,500 rpm for 10 minutes. Remove 500 ul from each tube, drain supernatant, and put the respective 500 ul back into the respective tube. Vortex each tube until pellet is broken up. Take 500 ul of the liquified pellet using a pipette and add it to the 1.5ml of gummy preparation that is in the warming tray.

[0379]

[0186] To plate and mold the therapeutic gummy formulation, cap and shake each 5ml tube once the 500ul of liquified pellet has been added to the 1.5ml of gummy formulation. Next, briefly vortex to complete mixture and tap the tube to remove bubbles. Pour the mixture into a gummy mold and refrigerate until ready to distribute.

[0380] Preparation of Probiotic Vaccine Gummy Formulations comprising SEQ ID NOs:1-74

[0381]

[0187] Likewise, to prepare Probiotic Vaccine Gummy Formulations comprising SEQ ID NOs:1-74, the following protocol is conducted. To prepare Luria Bertani (LB) broth, 400 ml of distilled water is mixed with 10g of HIMEDIA Luria Bertani Broth (Miller) and mixed on a warming plate with magnetic mixer until 37°C. From this LB broth, 20 aliquots of therapy is produced.

[0382]

[0188] To innoculate the LB broth, a 2ml vial of starter batch of the respective invention probiotic Nissle bacterium vaccine (e.g., Nissle / M13KE-SEQ ID NO:1-74) is thawed at room temperature for combining into LB media. Once thawed the starter batch is briefly vortexed and added to the LB media. To grow the invention bacteria containing the invention recombinant bacteriaphage (e.g., M13KE-SEQ ID NO:1-74), the media infected with the starter batch is placed in a shaking incubator at 37°C and 140 rpm for 4-8 hours. The media will start to become cloudy after a few hours and will be nearly opaque after 5 hours.

[0383]

[0189] To prepare the gummy formulation, 250ml Welch’s Grape Juice, 1 TBS Sugar, 2 tsp Agar AgarPowder are combined in a mixture and stirred on a hot plate until boiling for 2 minutes; then taken off the heat to start the cooling process. Using a pipette, place 1.5ml aliquots of the combined mixture into 5ml test tubes in pre-heated warming block.

[0384]

[0190] To prepare therapeutic aliquots for animal studies, take 20ml aliquots from each tube and centrifuge the aliqouts down at 3,500 rpm for 10 minutes. Remove 500 ul from each tube, drain supernatant, and put the respective 500 ul back into the respective tube. Vortex each tube until pellet is broken up. Take 500 ul of the liquified pellet using a pipette and add it to the 1.5ml of gummy preparation that is in the warming tray.

[0191] To plate and mold the therapeutic gummy formulation, cap and shake each 5m! tube once the 500ui of liquified pellet has been added to the 1.5ml of gummy formulation. Next, briefly vortex to complete mixture and tap the tube to remove bubbles. Pour the mixture of the respective Nissle / M13KE-SEQ ID NO:1-74 into a gummy mold and refrigerate until ready to distribute.

[0385] Example 9 - Generation of targeted IgA and IgG response:

[0386]

[0192] In this example, oral gavage formulations of ER6, N6, N13 probiotic bacterium vaccines; a gummy formulation of N6G; and an IV preparation of invention recombinant phage obtained from the supernatents of an N6 batch, filtered as described herein, were prepared as described herein. These formulations were administered to rats either via oral gavage, oral gummy or intravenously.

[0387]

[0193] Figure 2A, left graph, shows that intravenous administration of “IV6” therapy corresponding to the invention recombinant phage MK13E-SALV-0006 sequence in an intravenous formulation showed statistically significant response for IgA across all time periods, following injection.

[0388]

[0194] Figure 2B, right graph, shows that by day 17 the blank phage (unmodified) and control group were the two lowest groups in terms of serum IgG antibody generation; and that the only IV injected formulation, IV6, had by far the highest statistically significant response observed for IgG on days 10 and 17.

[0389] Example 10 -- Orally Administered SALV-0013 Demonstrates Significant SlgA Response

[0390]

[0195] Figure 3 shows the overwhelming SlgA immune response of orally administered N13 (E coll. Nissle 1917 infected with recombinant phage M13KE-SALV- 0013), via an invention gummy formulation, in animal studies. The invention delivery mechanism represented by the “N” series of datapoints shows significant response vs. control when orally administered. Secretory IgA (SlgA) plays an important role in the protection and homeostatic regulation of intestinal, respiratory, and urogenital mucosal epithelia separating the outside environment from the inside of the body. The invention orally delivered Nissle 1917 hosted therapies (e.g., N13, N6, N4) demonstrated significantly higher levels of SlgA as compared to the control group. Additionally, the control blank phage did not elicit an SlgA response indicating that it is inert without modification. It is also notable that N13 (e.g., SALV-0013) with a length of only 10aa actually had the strongest S!gA response Indicating shorter 10aa sequences are able to produce significant secretory immune response.

[0391]

[0196] In addition, the results of this experiment demonstrate that the gummy formulation of N6G outperformed the oral gavage administration and represents a viable form of administration. These results also demonstrate the ability of the invention probiotic vaccine (e.g., SALV-0013) to cross the mucous membrane barrier and elicit an immune response when orally administered.

[0392]

[0197] As set forth below in Example 11 , when administered as a combination of both the oral formulation and an IV formulation, a significantly smaller tumor size was observed for SALV-0013 treated animals compared to control and similar oral / IV combinations of SALV-0004 and SALV-0006 in a preclinical prostate cancer xenograft mouse experiment.

[0393]

[0198] Accordingly, it has been found that an invention probiotic vaccine (SALV-0013) is administered through oral ingestion and is capable of penetrating the mucous membranes and delivering trillions of targeted epitopes thus inducing an immune response.

[0394] Exampie 11 - Prostate Cancer Xenograft Athymic Mouse Study:

[0395]

[0199] Athymic Nude Mice were implanted with the prostate carcinoma LNCaP FGC cell line. The LNCaP cell line is an epithelial cell line derived from a human prostate carcinoma. The FGC (fast growing colony) line, a derivative of the LNCaP cell line shares all the main characteristics, including its androgen dependence, described for the original LNCaP cultures; and is viewed as an industry standard. See, e.g, Steenbrugge et al., Urol Res. 1989;17(2):71-7. doi: 10.1007 / BF00262024.

[0396]

[0200] In this example, three invention recombinant phage were tested in an IV / oral combination therapy against a prostate cancer human cell line injected into immunocompromised mice. Oral and intravenous injection combinations of each of SALV-0004 (SEQ ID NO:75), SALV-0006 (SEQ ID NO:76), and SALV-0013 (SEQ ID NO:77), each at approximately 1.5 x 1O10pfu / ml, were administered as follows.

[0397]

[0201] Oral dosing was delivered via oral gavage at 0.25ml per day for three days. The IV dose was delivered via injection over ~1 minute at 0.15ml in a single dose on day one. For the IV therapeutic recombinant bacteriophage, the therapeutic was created by filtering oral gavage through 0.2 micron filter.

[0202] The mice were athymic as they did not have a developed thymus and thus were incapable of producing T cells that can be beneficial in fighting tumors. The results are shown in Figure 4 and indicate that a favorable anticancer response was obtained with each of SALV-0004, -0006, and -0013; whereas for the SALV-0013 (SEQ ID NO:77) arm in particular, the average tumor size was 42% smaller than the control group; while also demonstrating no mortality, no behavioral abnormalities, and no weight loss.

[0398] Example 12 - Construction of TNFA / RS10 & TNF / RSS10 & EH 4 Epitopecontaining Nlssle Probiotic Vaccine (f88-SALV-1001 ; f88-SALV-1002; and f88- SALV-1003):

[0399]

[0203] VRS10, RSS10 and El 14 DNA were designed with codon optimization for E. coll, and chemically synthesized. The mRNA sequence encoding the - VRSSSRTPSD- (SEQ ID NO:78; referred to herein as “VRS10” and “SA LV- 1001”); the -RSSSRTPSDK- (SEQ ID NO:79; referred to herein as “RSS10” and “SALV- 1002”); and the -EIRQAGRPNKPDSI- (SEQ ID NO:80; referred to herein as “EI14” and “SALV-1003”) amino acid sequence was inserted into 5' end of the f88-4 pVIII, between the signal peptide and pVIII coding sequence by restriction enzymes Hindlll / Pstl to construct f88-4-VRS10, F88-4-RSS10 and F88-4-EI 14 DNA, and the sequence information at the recombination insertion site within the f88-4 phage is as follows: f88-4-VRS10: (signa / pept / cte-VRS10-p Vi II)

[0400] MKKSL VLKA S VA VA TL VPMLSFA-VRSSSRTPSD-

[0401] PAEGDDPAKAAFDSLQASA TEYIG YA WAMVVV / VGA TIGIKLFKKFTSKA S f88-4-RSS10: (signal pepf / de-VRS10-p V7 / 7)

[0402] MKKSL VLKASVA VA TL VPMLSFA-RSSSRTPSDK-

[0403] PAEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKAS f88-4-EI14: (signal peptide-El 14-p V / / / )

[0404] MKKSL VLKASVA VA TLVPMLSFA-EIRQAGRPNKPDSI-

[0405] PAEGDDPAKA AFDSLQASA TEYIG YA WAMVVViVGA TiG / KLFKKFTSKA S

[0406] Preparation of f88-4 vector

[0204] E.coli ER2738 in logarithmic growth phase was infected with f88-4 phage. After culture, the bacteria were collected by centrifugation and used to extract plasmids by Axygen MidiPrep plasmid extraction kit. F88-4 RF I DNA was further purified and then loaded into an agarose gel for QC.

[0407]

[0205] F88-4 RF I DNA was digested by Hindlll / Pstl and then separated by agarose gel electrophoresis.

[0408] Construction of f88-4-SALV-1001, f88-4-SALV-1002 and f88-4-SALV-1003 phages

[0409]

[0206] VRS10-epitope (SALV-1001; SEQ ID NO:78), RSS10-epitope (S.ALV-1002; SEQ ID NO:79), and EI14-epitope (SALV-1003; SEQ ID NO:80) encoding DNA was synthesized, digested by Hindlll / Pstl, and ligated with f88-4 RF I DNA. The constructs of VRS10-f88-4 RF I DNA, RSS10-f88-4 RF I and El 14-f88-4 RF I DNA were each independently transformed into E.coli ER2738 competent cells. After resuscitation, they infected E. coll ER2738 in logarithmic growth phase and were cultured overnight at 32°C on TOP-Agar LB (containing IPTG / X-gal) plates. Results indicated that there were many blue spots on the plate with the transformation of recombinant f88-4- VRS10 phage, f88-4-RSS10 phage and f88~4~EI14 phage into E.coli ER2738, while no blue spot was observed in control group. Several blue spots were selected and validated by sequencing. Results showed all clones had a correct construct corresponding to the correct mRNA sequence.

[0410] Phage preparation

[0411]

[0207] E.coli ER2738 in logarithmic growth phase was infected with f88-4 phages having the correct VRS10 epitope sequence (SALV-1001 ; SEQ ID NO:78), RSS10 epitope sequence (SALV-1002; SEQ ID NO:79) o El 14 epitope sequence (SALV- 1003; SEQ ID NO:80) inserted therein. After overnight culture, the culture supernatant was collected, glycerol was added to produce a 10% final concentration, and it was stored at -20°C. The titer was determined by gradient dilution; and for f88-4-VRS10 was 8.6>:1011pfu / mL, for f88-4-RSS10 was 2.9x1012 pfu / mL, and for f88-4-E114 was 1.5x1012pfu / mL.

[0412]

[0208] The above invention recombinant f88-4- SALV-1001 (VRS10), f88-4-SALV- 1002 (RSS10) and f88-4-SALV-1003 (El 14) phage is used to infect the Nissle 1917 E. coli to create “N-f88-1001” (E coli. Nissle 1917 infected with recombinant phage f88- 4-SALV-1001), “N-f88-1002” (E coli. Nissle 1917 infected with recombinant phage f88- 4-SALV-1002), and “N-f88-1003” (E coli. Nissle 1917 infected with recombinant phage f88-4-SALV-1003), respectively; which are used as probiotic vaccines in the invention methods set forth herein.

[0413]

[0209] The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein can be applied to other embodiments without departing from the spirit or scope of the invention. Thus, it is to be understood that the description and drawings presented herein represent a presently preferred embodiment of the invention and are therefore representative of the subject matter which is broadly contemplated by the present invention. It is further understood that the scope of the present invention fully encompasses other embodiments that may become obvious to those skilled in the art and that the scope of the present invention is accordingly not limited.

Claims

CLAIMSWhat is claimed is:

1. A recombinant phage, comprising a recombinant phage genome comprising a nucleic acid encoding a polypeptide comprising an exogenous peptide dual-epitope, or fragments or variants thereof, wherein the exogenous peptide dual-epitope is selected from the group consisting of: SEQ ID NO:52-70; or wherein the exogenous peptide dual-epitope is represented by the formula: a first peptide epitope selected from SEQ ID NO:1-32 or 71-74; -linker x- ; a second peptide epitope selected from SEQ ID NO:1-32 or 71-74, wherein the first peptide epitope is different from the second peptide epitope.

2. The recombinant phage of claim 1 , wherein linker x is 1 to 50 amino acids.

3. The recombinant phage of claim 1 , wherein linker x is a single glycine.

4. The recombinant phage of claims 1-3, wherein the exogenous peptide dual-epitope is selected from SEQ ID NO:33-51.

5. The recombinant phage of claims 1-4, wherein the exogenous peptide epitope is functionally expressed on a coat protein of said phage selected from the group consisting of: pill, pVI, pVil, pVIII and piX.

6. The recombinant phage of claims 1-5, wherein the coat protein is pill or pVIII.

7. The recombinant phage of claims 1-6, wherein the phage is selected from the group of filamentous phage consisting of: M13, fd, IKe, CTX-q>, Pfl, Pf2, Pf3, f1 , MKE; M13KE; type 8; type f88; f88-4; Myoviridae (Pl-like viruses; P2-like viruses; Mu-like viruses; SPOI-like viruses; phiH- like viruses); Siphoviridae (A-like viruses, y- like viruses, Tl-like viruses; T5- like viruses; c2-like viruses; L5-like viruses; psiMI-like viruses; phiC31 -like viruses; N15-like viruses); Podoviridae (phi29-like viruses; P22- like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus);Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammaiipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, fd- like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus , Chlamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus).

8. The recombinant phage of claims 1-7, wherein the phage is a filamentous phage selected from the group consisting of: M13, fd, I Ke, CTX-(p, Pfl, Pf2, Pf3, f1 , MKE, M13KE, type 8, type f88, and f88-4.

9. The recombinant phage of claims 1-8, wherein the phage is M13KE or f88-4.

10. A recombinant filamentous phage, comprising a recombinant phage genome comprising a nucleic acid encoding a polypeptide comprising an exogenous peptide epitope, or fragments or variants thereof, selected from the group consisting of SEQ ID NOs:1-74.11 . The recombinant phage of claim 10, wherein the exogenous peptide epitope is functionally expressed on a coat protein of said phage selected from the group consisting of: pili, pVI, pVII, pVlli and pIX.

12. The recombinant phage of claims 10-11 , wherein the coat protein is pill or pVIII .

13. The recombinant phage of claims 10-12, wherein the phage is selected from the group of filamentous phage consisting of: M13, fd, IKe, CTX-cp, Pfl, Pf2, Pf3, f1 , MKE; M13KE; type 8; type f88; f88-4; Myoviridae (Pl-like viruses; P2-like viruses; Mu-like viruses: SPOI-like viruses; phi H-like viruses); Siphoviridae (A-like viruses, y- like viruses, Tl-iike viruses; T5-iike viruses; c2-like viruses; L5-iike viruses; psiMI-like viruses; phiC31 -like viruses; N15-like viruses); Podoviridae (ph!29-like viruses; P22- like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, Ml 3-like viruses, fd-like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus, Chlamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus).

14. The recombinant phage of claims 10-13, wherein the phage is a filamentous phage selected from the group consisting of: M13, fd, I Ke, CTX-cp, Pfl, Pf2, Pf3, f1 , MKE, M13KE, type 8, type f88, and f88-4.

15. The recombinant phage of claims 10-14, wherein the phage is M13KE or f88-4.

16. A probiotic vaccine, said vaccine comprising the recombinant phage of claims 1-15; and a bacterium infected with said recombinant phage.

17. The probiotic vaccine of claim 16, wherein the bacteria is selected from: E. coli Nissle 1917, E. coli ER2738, Bacillus amyloliquefaciens; Bacillus polyfermenticus, strain Bispan; Bifidobacterium animalis subsp. Lactis, strain BB-12; Bifidobacterium animalis subsp. Lactis, strain GPS1209; Bifidobacterium animalis subsp. Lactis, strain HN019 (DR1064); Bifidobacterium bifidum, strain BB-12; Bifidobacterium bifidum, strain Rosell-71 ; Bifidobacterium breve, strain M-16V; Bifidobacterium longum; Bifidobacterium thermophilum; Lactobacillus acidophilus, strain La-1 ; Lactobacillus brevis, strain HA-112; Lactobacillus fermentum, strain HA- 179; Lactobacillus helveticus, strain Lafti L10; Lactobacillus helveticus, strain Resell- 52; Lactobacillus paracasei, strain Lafti L26; Lactobacillus paracasei subsp. paracasei, strain 431 ; Lactobacillus rhamnosus, strain HN001 (DR20); Streptococcus salivarius, strain DSM 13084; Streptococcus thermophilus; Bacillus coagulans GBI-30, 6086, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis, Escherichia coli Nissle 1917, Lactobacillus acidophilus NCFM, Lactobacillus paracasei Stl 1 (or NCC2461), Lactobacillus johnsonii Lai (also referred to as Lactobacillus LCI, Lactobacillus johnsonii NCC533), Lactobacillus plantarum 299v, Lactobacillus reuteri ATCC 55730 (Lactobacillus reuteri SD2112), Lactobacillus reuteri Protectis (DSM 17938, daughter strain of ATCC 55730), Lactobacillus reuteri Prodentis (DSM 17938 / ATCC 55730 and ATCC PTA 5289 in combination), Lactobacillus rhamnosus GG, Saccharomyces boulardii, mixture of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, a mixture of Lactobacillus acidophilus NCFM and Bifidobacterium bifidum BB-12, a mixture ofLactobacillus acidophilus CL1285 and Lactobacillus easel LBC80R, a mixture of Lactobacillus plantarum HEAL 9 and Lactobacillus paracasei 8700:2, Lactobacillus bulgaricus, Lactococcus thermophiles and Lactobacillus bifidus.

18. The probiotic vaccine of ciaims 16-17, wherein the bacteria is F-factor positive; and seiected from E. coli Nissle 1917 or E. coli ER2738.

19. The probiotic vaccine of ciaims 16-18, wherein the probiotic vaccine generates both igG and secretory IgA (SlgA) antibodies that bind to the exogenous peptide epitope.

20. A composition comprising coconut water and a probiotic vaccine, wherein the probiotic vaccine comprises a recombinant bacteriophage, wherein the recombinant phage comprises at least one exogenous peptide epitope corresponding to SEQ ID NO: 1-74, or fragments or variants thereof.

21. The composition of claim 20, wherein the coconut water further comprises one or more excipients selected from the group consisting of buffer, tonicity adjusting agent, preservative, demulcent, viscosity modifier, osmotic agent, surfactant, antioxidant, alkalinizing agent, acidifying agent antifoaming agent, and colorant.

22. The composition of clams 20-21 , wherein the coconut water further comprises a buffer.

23. The composition of clams 20-22, wherein the coconut water further comprises a salt.

24. The composition of clams 20-23, wherein the composition has a pH of 7.0-8.0.

25. The composition of clams 20-24, wherein the composition has a pH of 7.5.

26. A method for preventing, treating, and / or reducing the risk of cancer, autoimmune disease, respiratory disease, neurological disease, and / or infectiousdisease, said method comprising administering to a patient in need thereof a combination of: a. an injectable composition comprising a recombinant phage or probiotic vaccine comprising at least one exogenous peptide epitope corresponding to SEQ ID NO: 1-74, or fragments or variants thereof; and b. an oral composition comprising a recombinant phage or probiotic vaccine comprising at least one exogenous peptide epitope corresponding to SEQ ID NO: 1-74, or fragments or variants thereof.

27. A method of preventing, treating, and / or reducing the risk of cancer, autoimmune disease, respiratory disease, neurological disease, and / or infectious disease, said method comprising administering, to a patient in need thereof, a probiotic vaccine of claims 16-19.

28. The method of claim 27, wherein the probiotic vaccine is administered orally.

29. The method of claims 27-18, further comprising administering a recombinant bacteriophage selected from claims 1-15.

30. The method of claim 29, wherein the recombinant phage is administered intravenously or intramuscularly.

31. The method of claims 26-30, wherein secretory IgA (SI gA) is generated.

32. The method of claims 26-31, wherein secretory IgA (SlgA) is generated in amount compared to a control, that is 2- to 100-fold, 2- to 90-fold, 2- to 80-fold, 2- to 70-fold, 2- to 60-fold, 2- to 50-fold, 2- to 40-fold, 2- to 30-fold, 2- to 20-fold, 2- to 10-fold, 2- to 9-fold, 2- to 8-fold, 2- to 7-fold, 2- to 6-fold, 2- to 5-fold, 2- to 4-fold, greater than a control.

33. A method of preventing, treating, and / or reducing the risk of cancer, comprising administering to a patient in need thereof, one or more of the probiotic vaccine of claims 16-19, or one or more of the recombinant phage of claims 1-15,wherein the exogenous peptide epitope is selected from the group consisting of: SEQ ID NOs: 5-10, 12, 13, and 33-72, and 74.

34. The method of claim 33, wherein the cancer is selected from the group consisting of: multiple myeloma, epithelial cancer, epithelial ovarian cancer, mucosal melanoma, non-small cell lung cancer, melanoma, head and neck cancer, renal cell cancer, Hodgkin’s lymphoma, Cutaneous Squamous Cell Carcinoma, glioblastoma, esophageal cancer, gastric cancer, duodenal cancer, small intestinal cancer, appendiceal cancer, large bowel cancer, colon cancer, rectum cancer, colorectal cancer, anal cancer, pancreatic cancer, liver cancer, chondro sarcoma, gallbladder cancer, spleen cancer, renal cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, breast cancer, pulmonary cancer, thyroid cancer, thymus cancer, brain cancer, nervous system cancer, gliomas, oral cavity cancer, skin cancer, blood cancer, lymphomas, eye cancer, bone cancer, bone marrow cancer, muscle cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), urothelial carcinoma, non-muscle invasive bladder cancer [NMIBC]), coion or rectal cancer, esophageal or certain gastroesophageal junction (GEJ) carcinomas, cervical cancer, renal cell carcinoma (RCC), advanced endometrial carcinoma, cutaneous squamous cell carcinoma (cSCC), and / or tripie-negative breast cancer (TNBC).

35. The method of claims 33-34, wherein the cancer is selected from: pancreatic cancer, liver cancer, and / or chondro sarcoma.

36. A method of preventing, treating, and / or reducing the risk of breast, bladder, pancreatic, liver, chondro sarcoma, ovarian, mucosal melanoma, prostate cancer, pulmonary fibrosis, lung cancer, non-small cell lung cancer, and / or stomach cancer, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:YLQRDISEM (SEQ ID NO:5),ELQRDISEM (SEQ ID NO:6),LEEPNRVQL (SEQ ID NO:7),ATADLELAY (SEQ ID N0:8),AQRMTTQLLLL (SEQ ID NO:9),SLLMQITQC (SEQ ID NO: 10),VELMYPPPYYLGIGN (SEQ ID NO:12),EGGVAMPGAEDDW (SEQ ID NO: 13),ALLEIASCLgAGNNWAKGHYTEGAELVD (SEQ ID NO:33),ALLEIASCLgTFSVVPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:34),ALLEIASCLgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:35),ALLEIASCLgYLSDNHILI (SEQ ID NO:36),AGAFAPSAAVAgAGNNWAKGHYTEGAELVD (SEQ ID NO:37),AGAFAPSAAVAgTFSVVPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:38),AGAFAPSAAVAgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:39),AGAFAPSAAVAgYLSDNHILI (SEQ ID NQ:40),FLAEDALNTVgAGNNWAKGHYTEGAELVD (SEQ ID NO:41),FLAEDALNTVgTFSVVPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:42),FLAEDALNTVgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:43),FLAEDALNTVgYLSDNHILI (SEQ ID NO:44),AQRMTTQLLLLgAGNNWAKGHYTEGAELVD (SEQ ID NO:45),AQRMTTQLLLLgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:46),AQRMTTQLLLLgTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:47),AQRMTTQLLLLgYLSDNHILI (SEQ ID NO:48),SPRMSGLLSQTgAGNNWAKGHYTEGAELVD (SEQ ID NO:49),SPRMSGLLSQTgTFSWPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:50),SPRMSGLLSQTgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:51),ALLESASCLxAGNNWAKGHYTEGAELVD (SEQ ID NO:52),ALLEIASCLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:53),ALLEIASCLxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:54),ALLEIASCLxYLSDNHILI (SEQ ID NO:55),AGAFAPSAAVAxAGNNWAKGHYTEGAELVD (SEQ ID NO:56),AGAFAPSAAVAxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:57),AGAFAPSAAVAxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:58),AGAFAPSAAVAxYLSDNHIU (SEQ ID NO:59),FLAEDALNTVxAGNNWAKGHYTEGAELVD (SEQ ID NO:60),FLAEDALNTVxTFSWPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:61),FLAEDALNTVxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:62),FLAEDALNTVxYLSDNHILI (SEQ ID NO:63),AQRMTTQLLLLxAGNNWAKGHYTEGAELVD (SEQ ID NO:64),AQRMTTQLLLLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:65),AQRMTTQLLLLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:66),AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67),SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68),SPRMSGLLSQTxTFSWPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:69),SPRMSGLLSQTxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:70),KKKPTPIQLNPAPAGSAVNG (SEQ ID N0:71),RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72),QLIYNLTLCELNGTDWL (SEQ ID NO:73), and / orKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGL NQRRI (SEQ ID NO:74) CD3.

37. The method of claim 36, wherein the recombinant phage and / or probiotic vaccine comprising YLQRDISEM (SEQ ID NO:5), ELQRDISEM (SEQ ID NO:6), and / or LEEPNRVQL (SEQ ID NO:7), is used to prevent, treat, or reduce the risk of pancreatic cancer.

38. The method of claim 36, wherein the recombinant phage and / or probiotic vaccine comprising ATADLELAY (SEQ ID NO:8), is used to prevent, treat, or reduce the risk of liver cancer.

39. The method of claim 36, wherein the recombinant phage and / or probiotic vaccine comprising SLLMQITQC (SEQ ID NO: 10), is used to prevent, treat, or reduce the risk of chondro sarcoma.

40. The method of claim 36, wherein the recombinant phage and / or probiotic vaccine comprising VELMYPPPYYLGIGN (SEQ ID NO:12) and / or EGGVAMPGAEDDW (SEQ ID NO:13), is used to prevent, treat, or reduce the risk of mucosal melanoma, prostate cancer, pulmonary fibrosis, lung cancer, and / or nonsmall cell lung cancer.

41. A method of preventing, treating, and / or reducing the risk of mucosal melanoma, prostate cancer, pulmonary fibrosis, lung cancer, non-small cell lung cancer, and the like, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:VELMYPPPYYLGIGN (SEQ ID NO:12) and / or EGGVAMPGAEDDW (SEQ ID NO:13).

42. A method of preventing, treating, and / or reducing the risk of virus infection and / or chronic wasting disease manifestation, comprising administering, to a subject or patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: WGQPHGGG (SEQ ID NO:1).

43. A method of preventing, treating, and / or reducing the risk of Norovirus infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:PVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRN (SEQ ID NO:2),NNYDPTEEIPAPLGTPDF (SEQ ID NO:3), and / orWIRNNF (SEQ ID NO:4).

44. A method of preventing, treating, and / or reducing the risk of Respiratory Syncytial Virus (RSV) infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:NSELLSLINDMPSTNDQKKLMSNN (SEQ ID N0:14),NSELLSUNDMPSTNDQKKLMSNNV (SEQ ID N0:15),NSELLSLiNDMPITNDQKKLMSNNVQ (SEQ ID N0:16), and / orNSELLSLINDMPITNDQKKLMSNNVQI (SEQ ID NO:17).

45. A method of preventing, treating, and / or reducing the risk of inflammatory bowel disease (IBD), comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from DPQGVTCGAATLSAERV (SEQ ID NO:11), KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), and / or RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72).

46. A method of preventing, treating, and / or reducing the risk of Porcine Epidemic Diarrhea viral infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:GFEIGISQEPFDP (SEQ ID NO:18),SLPQDVTRC (SEQ ID NO:19),DGVCNGAAV (SEQ ID NO:20),FDLDDGFYPISS (SEQ ID NO:21),ITGTPKPLEG (SEQ ID NO:22),SVYDPASGRVVQKR (SEQ ID NO:23),DQLPDVIPDYID (SEQ ID NO:24),PNRTGPSL (SEQ ID NO:25),YSNIGVCK (SEQ ID NO:26), and / orNLLSHEQP (SEQ ID NO:27).

47. A method of preventing, treating, and / or reducing the risk of Transmissible Gastroenteritis viral infection and / or disease manifestation, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:ATAVIKTGTCPFSFDKLNNY (SEQ ID NO:28),PFSFDKLNNYLTFNKFCLSL (SEQ ID NO:29),VVRSLYVIYEEGCNIVGVPS (SEQ ID NO:30),LHLDSCTDYNIYGRTGVGII (SEQ ID NO:31), and / orLYYTSLSGDLLGFKNVSDGV (SEQ ID NO:32).

48. A method of preventing, treating, and / or reducing the risk of Porcine Reproductive Respiratory Syndrome (PRRS) and / or disease manifestation, comprising administering, to a subject or patient in need thereof, a recombinant phage and / or probiotic vaccine, comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from: QLIYNLTLCELNGTDWL (SEQ ID NO:73).

49. A method of preventing, treating, and / or reducing the risk of inflammatory disease or type 1 diabetes, comprising administering, to a patient in need thereof, a recombinant phage and / or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR I (SEQ ID NO:74).

50. The methods of claims 26-49, wherein secretive IgA (SigA) is generated.

51. The method of claim 50, wherein secretive IgA (SigA) is generated in amount compared to a control, that is 2- to 100-fold, 2- to 90-fold, 2- to 80-fold, 2- to 70-fold, 2- to 60-fold, 2- to 50-fold, 2- to 40-fold, 2- to 30-fold, 2- to 20-fold, 2- to 10- fold, 2- to 9-fold, 2- to 8-fold, 2- to 7-fold, 2- to 6-fold, 2- to 5-fold, 2- to 4-fold, greater than a control.

52. The probiotic vaccine of claims 16-19, wherein the vaccine continually produces lysogenic phage.

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