WO2025189043A2 - Compositions de vaccin oral et procédés associés - Google Patents

Compositions de vaccin oral et procédés associés

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Publication number
WO2025189043A2
WO2025189043A2 PCT/US2025/018803 US2025018803W WO2025189043A2 WO 2025189043 A2 WO2025189043 A2 WO 2025189043A2 US 2025018803 W US2025018803 W US 2025018803W WO 2025189043 A2 WO2025189043 A2 WO 2025189043A2
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seq
cancer
viruses
phage
fold
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WO2025189043A3 (fr
WO2025189043A8 (fr
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Kirk SOLO
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Salvitus Inc
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Salvitus Inc
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Publication of WO2025189043A3 publication Critical patent/WO2025189043A3/fr
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
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    • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
    • A61K39/001195—Prostate specific membrane antigen [PSMA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/884—Vaccine for a specifically defined cancer prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
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    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
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    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
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    • C12N2770/16011—Caliciviridae
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    • C12N2770/00011—Details
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    • C12N2795/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2795/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates recombinant phage, probiotic vaccines and methods of using same.
  • 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.
  • 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.
  • CTL Cytotoxic T lymphocytes
  • 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.
  • probiotics 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.
  • recombinant phage e.g., filamentous
  • probiotic vaccines comprising a recombinant filamentous phage genome
  • therapeutic methods of using the invention recombinant phage and/or probiotic vaccines 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).
  • 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).
  • SlgA secretory IgA
  • SlgA contains a secretory component (SC) derived from polymeric Ig receptor (plgR) utilized for transcytosis through epithelial cells during secretion.
  • IgA 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.
  • GIT gastrointestinal tract
  • GALT gut-associated lymphoid tissue
  • 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).
  • the invention recombinant bacteriophage e.g, filamentous phage
  • IV intravenously
  • 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).
  • 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).
  • 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).
  • an invention probiotic vaccine e.g., via oral delivery, and the like
  • an invention recombinant bacteriophage e.g., via injection, and the like
  • 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.
  • 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.
  • the linker x is a single glycine.
  • the exogenous peptide dual-epitope is selected from SEQ ID NO:33- 51.
  • 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.
  • the coat protein is pill or pVIII.
  • 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, Betalipothr
  • 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.
  • the phage is M13KE or f88-4.
  • 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.
  • the exogenous peptide epitope is functionally expressed on a coat protein selected from the group consisting of: pill, pVI, pVH, pVIII and pIX.
  • the coat protein is pill.
  • the coat protein is pVIII.
  • 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,
  • 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.
  • the phage is M13KE.
  • the phage is f88 or f88-4.
  • the recombinant phage generates IgG and/or IgA antibodies (e.g., SigA) that bind to the exogenous peptide epitope.
  • a probiotic 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:
  • WGQPHGGG (SEQ ID NO:1), chronic wasting disease, PVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRN (SEQ ID NO:2), Norovirus,
  • NNYDPTEEIPAPLGTPDF SEQ ID NO:3
  • Norovirus
  • WIRNNF SEQ ID NO:4
  • Norovirus Norovirus
  • 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, NSELL
  • AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67), Folate Receptor/CD3, SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68), DLL3 Receptor, SPRMSGLLSQTxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:69), DLL3 Receptor,
  • KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), MEK1 , RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), MEK1 , QUYNLTLCELNGTDWL (SEQ ID NO:73), PRRS, and/or KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR
  • the exogenous peptide epitope is functionally expressed on a coat protein selected from the group consisting of: pill, pVI , pVII, pVIII and pIX.
  • the coat protein is pill.
  • the coat protein is pVIII.
  • the peptide sequence -TSGSGSGSGSGSGSG- is used as a linker between the coat protein and the exogenous peptide epitope.
  • 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 (Alphali
  • the 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 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.
  • 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.
  • the probiotic vaccine and/or recombinant phage generates both IgG and secretory IgA (SlgA) antibodies that bind to the exogenous peptide epitope.
  • compositions 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.
  • 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.
  • the coconut water further comprises a buffer.
  • the coconut water further compises a salt.
  • the composition has a pH of 7.0-8.0. In a particular embodiment, the composition has a pH of 7.5.
  • 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 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.
  • 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, lympho
  • 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), 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), SPRMSGL
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • a recombinant phage and/or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from:
  • 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).
  • 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).
  • 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).
  • 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).
  • 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 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).
  • 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).
  • 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).
  • secretive IgA is generated.
  • secretive IgA 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.
  • Figure 1A indicates the physiological effect of oral administration of the invention probiotic vaccine to produce slgA.
  • 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.
  • Figure 2B shows the resulting serum IgG levels of the formulations administered as set forth in Example 9.
  • 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.
  • Figure 5 shows the heterologous peptide epitope insertion site in the M13KE- SALV-0004 recombinant phage from Example 1.
  • Figure 6 shows the heterologous peptide epitope insertion site in the M13KE- SALV-0013 recombinant phage from Example 3.
  • a probiotic 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,
  • 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,
  • EGGVAMPGAEDDW SEQ ID NO:13
  • podoplanin PDPN
  • mucosal melanoma NSELLSLINDMPITNDQKKLMSNN
  • RSV f protein, NSELLSLINDMPITNDQKKLMSNNV
  • SEQ ID NO:15 RSV, f protein, NSELLSLINDMPITNDQKKLMSNNVQ
  • SEQ ID NO:16 RSV, f protein, NSELLSLINDMPITNDQKKLMSNNVQI
  • GFEIGISQEPFDP SEQ ID NO:18
  • Porcine Epidemic Diarrhea DGVCNGAAV
  • DGVCNGAAV SEQ ID NO:20
  • Porcine Epidemic Diarrhea ITGTPKPLEG
  • 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,
  • ALLEIASCLgYLSDNHILI SEQ ID NO:36
  • Prostate Cancer AGAFAPSAAVAgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:38), Prostate Cancer,
  • An intravenous administration with invention recombinant phage 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.
  • probiotic vaccine refers to a bacterium comprising a recombinant phage, such as those described herein.
  • the phrase “recombinant bacteriophage” refers to a phage having an exogenous peptide region encoded therein.
  • vacun 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.
  • a vaccine or a vaccine composition further comprises one or more immuno-adjuvant substances.
  • preventing generally means to avoid or minimize the onset or development of a disease or condition before its onset.
  • prevention encompasses "reducing the likelihood of occurrence of” or “reducing the likelihood of reoccurrence”.
  • 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.
  • a disease e.g., cancer, and the like
  • condition or symptoms of a disease or condition
  • an effective amount or “effective dose” as used herein is an amount which provides the desired effect.
  • 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.
  • an effective amount of the composition in terms of prevention, treatment, and/or reducing the risk, is an amount that is sufficient to induce a humoral and/or cell-mediated immune response directed against the disease/disorder.
  • 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).
  • 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.
  • 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.
  • 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.
  • the length of the exogenous peptide epitopes is in a range of 5-20 amino acids.
  • an exogenous peptide epitope 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.
  • 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.
  • 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
  • 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.
  • 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).
  • 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).
  • the linker x is 1 to 50 amino acids. In another embodiment, the linker x is a single glycine.
  • the exogenous peptide dual-epitope is selected from SEQ ID NO:33- 51.
  • 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.
  • 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
  • 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.
  • the phage is M13KE or f88-4.
  • 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”.
  • 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.
  • 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.
  • 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.
  • 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.
  • the length of “linker x” is from 1 to 50 amino acids.
  • the length of “linker x” is from 1 to 20 amino acids.
  • the length of “linker x” is from 1 to 10 amino acids.
  • the 1 to 20 amino acids are selected from Gly, Ala, Pro, Asn, Gin, Cys, Lys.
  • 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.
  • (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.
  • 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.
  • the single-epitope target sequences set forth as SEQ ID NOs:1-133 in WO2023/158883 and WO2024/178160 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.
  • the respective phage coat protein e.g., pill or pVIII
  • the phrase “recombinant phage genome” refers to a native phage genome that has been modified to contain nucleic acid encoding an exogenous peptide region.
  • 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.
  • fragment refers to a smaller peptide (e g., subset of amino acids) relative to the complete reference peptide sequence from which it is derived.
  • 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.
  • variants 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.
  • 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.
  • a variant will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid sequence identity.
  • 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.
  • 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 MEGALIGNTM (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.
  • 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.
  • 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)):
  • 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.
  • Glu may be substituted by Gin
  • Asp may be substituted by Asn.
  • the amino acid residues selected for modification are selected from the group consisting of: His, Glu, Asp, Cys, Lys and Tyr.
  • 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
  • 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.
  • 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.
  • invention probiotic vaccines comprising a recombinant phage and a bacterium infected with said recombinant phage.
  • 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.
  • F + cell F-factor
  • sex factor F-factor
  • 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.
  • 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.
  • 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 bifi
  • 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.
  • the probiotic bacteria is Escherichia coli Nissle 1917 or E. coli ER2738.
  • the probiotic bacteria is Escherichia coli Nissle 1917.
  • the probiotic bacteria is E. coli ER2738.
  • suitable phage systems 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.
  • phage T7 and phage 8 vector systems can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • donor bacteria e.g., probiotic bacteria and/or commensal bacteria
  • Probiotic bacteria 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.
  • 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.
  • 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.
  • Lactis 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.
  • 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.
  • 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.
  • Bacteroides thetaiotamicron e.g., Bl
  • B. fragilis e.g., ATCC 51477-B1 , B40-8, Bf-1
  • B. caccae e.g., phiHSCOI
  • 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
  • Pseudomonas aeruginosa e.g., phiKMV, PAK-P1 , LKD16, LKA1, delta, sigma-1 , J-l
  • Pseudomonas aeruginosa e.g., phiKMV, PAK-P1 , LKD16
  • 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.
  • endogenous bacterial cells may refer to non-pathogenic bacteria that are part of a normal internal ecosystem such as bacterial flora.
  • 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.
  • the bacteriophage of the present disclosure may target (e.g., specifically target) anaerobic bacterial cells.
  • 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,
  • 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, ITGT
  • AQRMTTQLLLLgYLSDNHILI (SEQ ID NO:48), Folate Receptor/CD3, SPRMSGLLSQTgAGNNWAKGHYTEGAELVD (SEQ ID NO:49), DLL3 Receptor, SPRMSGLLSQTgTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:50), DLL3 Receptor,
  • ALLEIASCLxAGNNWAKGHYTEGAELVD SEQ ID NO:52
  • Mucosal Melanoma ALLEIASCLxTFSVVPSPKVSDTVVEPYNATLSVHQLVE
  • Mucosal Melanoma ALLEIASCLxTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:54), Mucosal Melanoma,
  • AQRMTTQLLLLxYLSDNHILI (SEQ ID NO:67), Folate Receptor/CD3, SPRMSGLLSQTxAGNNWAKGHYTEGAELVD (SEQ ID NO:68), DLL3 Receptor, SPRMSGLLSQTxTFSVVPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:69), DLLS Receptor,
  • KKKPTPIQLNPAPAGSAVNG (SEQ ID NO:71), MEK1 , RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), MEK1 , QLIYNLTLCELNGTDWL (SEQ ID NO:73), PRRS, and/or KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR
  • the individual epitopes can be in reverse order (i.e., vice versa) than is depicted in the respective SEQ ID NOs.
  • 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.
  • the coat protein is pill.
  • the coat protein is pVIII .
  • 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.
  • 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, Betalipothrix
  • 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.
  • 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.
  • 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.
  • 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.
  • lytic bacteriophage may be used as delivery vehicles.
  • naturally lytic phage serve as cargo shuttles and do not inherently lyse target cells.
  • 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 (P
  • 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.
  • 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.
  • 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.
  • 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.
  • the exogenous peptide epitope is functionally expressed in gene pill of the M13 bacteriophage, in a particular embodiment, the bacteriophage is M13KE.
  • 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.
  • 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.
  • the exogenous peptide epitope is functionally expressed in gene pVIII of the f88 bacteriophage.
  • the bacteriophage is f88-4 (GenBank Accession # AF218363).
  • 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.
  • autoimmune diseases e.g., asthma
  • respiratory diseases e.g., asthma
  • neuroligical diseases e.g., infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like)
  • 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.
  • autoimmune diseases e.g., asthma
  • respiratory diseases e.g., asthma
  • neurological diseases e.g., infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like)
  • infectious diseases among other diseases (osteoporosis, elevated cholesterol, and the like)
  • an invention probiotic vaccine comprising a heterologous peptide selected from SEQ ID NOs:1-74 set forth herein.
  • 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.
  • autoimmune diseases e.g., asthma
  • respiratory diseases e.g., asthma
  • neurological diseases e.g., infectious diseases, among other diseases (osteoporosis, elevated cholesterol, and the like)
  • infectious diseases among other diseases (osteoporosis, elevated cholesterol, and the like)
  • 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.
  • 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).
  • the invention recombinant bacteriophage e.g, filamentous phage
  • IV intravenously
  • the invention recombinant bacteriophage 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).
  • 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).
  • 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, lympho
  • 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
  • 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),
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • EGGVAMPGAEDDW SEQ ID NO:13
  • 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).
  • PVAGAASAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRN SEQ ID NO:2
  • NNYDPTEEIPAPLGTPDF SEQ ID NO:3
  • 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),
  • NSELLSLINDMPITNDQKKLMSNNVQ SEQ ID NO:16
  • NSELLSUNDMPITNDQKKLMSNNVQI SEQ ID NO:17
  • 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:
  • 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/or LYYTSLSGDLLGFKNVSDGV (SEQ ID NO:32).
  • 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
  • Immunotherapy 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.
  • 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).
  • 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).
  • a recombinant phage and/or probiotic vaccine comprising at least one exogenous peptide epitope, or fragment or variant thereof, selected from KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR
  • the invention probiotic vaccine e.g., bacterial cells comprising invention recombinant phage
  • 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.
  • 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).
  • the probiotic bacteria may be delivered in vivo as tablets, capsules, powders and/or sachets containing the bacteria in freeze dried form.
  • the pharmaceutical carrier is a gelatin-based chewtype formulation (e.g., gummy or troches)
  • 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.
  • the gummy base is heated, mixed with other ingredients, and poured into molds, and cooled.
  • 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.
  • Klear GummyTM (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.
  • 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.
  • the probiotic vaccine and/or recombinant bacteriophage is administered into the axillary region as an intradermal injection.
  • the probiotic vaccine and/or recombinant bacteriophage is administered intravenously via injection or infusion.
  • the incorporation of invention recombinant bacteriophages into therapeutic formulations includes encapsulating them within a stabilizing substance.
  • various antimicrobial materials such as powders, semisolids and nanofibers can be produced, providing more options for effective delivery.
  • 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.
  • 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.
  • the invention compositions e.g., recombinant bacteriophage or probiotic vaccine
  • the pulmonary route e.g., 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.
  • 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.
  • a therapeutic dose e.g., either a single dose or multiple doses
  • the recombinant phage pharmaceutical compositions can be administered in two or more separate administrations (doses).
  • 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).
  • 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.
  • 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.
  • an appropriate carrier for administering the cells can be selected by one of skill in the art by routine techniques.
  • 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.
  • the invention probiotic vaccine and/or recombinant bacteriophage 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.
  • the suspension can comprise an invention probiotic vaccine-bacterium, coconut water, buffer and salt. It optionally further comprises a surfactant.
  • 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.
  • the pharmaceutical carrier is coconut water.
  • 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.
  • 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.
  • 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.
  • 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.
  • the coconut water further comprises a buffer.
  • the coconut water further comprises a salt.
  • the composition has a pH of 7.0-8.0. In a particular embodiment, the composition has a pH of 7.5.
  • 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.
  • 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.
  • 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.
  • 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
  • other therapeutic methodologies e.g., chemotherapy, and beam radiation therapy
  • a mammal preferably a human
  • a mammal preferably a human
  • probiotic vaccine cells in from about 0.05 mL to about
  • probiotic vaccine cells is performed at two-week intervals. In one embodiment, from about one to about five administrations of about 1x10 s /ml probiotic vaccine cells is performed at two-week intervals.
  • 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.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • diseases e.g., ulcerative colitis of the gut
  • 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 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).
  • 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.
  • this particular combination therapy can also be further combined with at least another therapeutic agent.
  • 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.
  • this particular combination therapy can also be further combined with at least another therapeutic agent.
  • 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.
  • probiotic vaccination and/or direct administration of invention recombinant bacteriophage (for vaccination or treatment) provided herein can be combined with other treatments.
  • 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.
  • chemotherapy can be combined safely with immunotherapy, with possibly additive or synergistic effects, as long as combinations are designed rationally.
  • 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.
  • a patient receiving an invention probiotic vaccination can be treated with any other treatments that are beneficial for the particular cancer.
  • 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.
  • a patient receiving an invention probiotic vaccination can be treated with bevacizumab (Avastin®) prior to, concurrently, or after probiotic vaccination.
  • 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.
  • M13KE RF I DNA was digested by Kpn 1/Eag I and then separated by agarose gel electrophoresis.
  • 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.
  • 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.
  • 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).
  • HNSCC non-small cell lung cancer
  • HNSCC head and neck squamous cell cancer
  • NMIBC non-muscle invasive bladder cancer
  • GEJ gastroesophageal junction
  • GEJ gastroesophageal junction
  • M113KE-AFP10 infected Nissle probiotic phage i.e., phage + E. coli
  • 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.
  • 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.
  • M13KE RF I DNA was digested by Kpn 1/Eag I and then separated by agarose gel electrophoresis.
  • 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:
  • 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. [161] 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.
  • 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
  • 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.
  • 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.
  • SALV-0013 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.
  • 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.
  • M13KE RF I DNA was digested by Kpn 1/Eag I and then separated by agarose gel electrophoresis.
  • 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.
  • 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 1.36x10 10 pfu/mL.
  • M113KE-SALV-0013 infected Nissle probiotic phage i.e., phage + E. coli
  • 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.
  • NNYDPTEEIPAPLGTPDF (SEQ ID NO:3)
  • WIRNNF SEQ ID NO:4.
  • WRSLYVIYEEGCNIVGVPS (SEQ ID NO:30), LHLDSCTDYNSYGRTGVGII (SEQ ID N0:31),
  • FLAEDALNTVgTFSVMPSPKVSDTVVEPYNATLSVHQLVE (SEQ ID NO:43), FLAEDALNTVgYLSDNHILI (SEQ ID NO:44),
  • ALLEIASCLxTFSVMPSPKVSDTWEPYNATLSVHQLVE SEQ ID NO:54
  • ALLEIASCLxYLSDNHILI SEQ ID NO:55
  • AGAFAPSAAVAxTFSVVPSPKVSDTVVEPYNATLSVHQLVE SEQ ID NO:57
  • AGAFAPSAAVAxTFSVMPSPKVSDTVVEPYNATLSVHQLVE SEQ ID NO:58
  • AGAFAPSAAVAxYLSDNHILI SEQ ID NO:59
  • 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), SPRMSGLLSQTxTFSVMPSPKVSDTWEPYNATLSVHQLVE (SEQ ID NO:70), KKKPTPIQLNPAPAGSAVNG (SEQ ID N0:71), RRRLNPAPAGSAVNGTSSAE (SEQ ID NO:72), QUYNLTLCELNGTDWL (
  • M13KE-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.
  • 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.
  • M13KE RF I DNA is digested by Kpn 1/Eag I and then separated by agarose gel electrophoresis.
  • Phage preparation [173] 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.
  • 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.
  • 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.
  • HPP High Pressure Pasteurization
  • 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.
  • 20ml aliquots from the 400ml were separated into 50ml test tubes and centrifuged at 3k rpm for 10 minutes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 20ml aliquots from the 400ml are separated into 50ml test tubes and centrifuged at 3k rpm for 10 minutes.
  • Probiotic Vaccine Gummy Formulations comprising SEQ ID NOs:1-74
  • the following protocol is conducted.
  • LB Luria Bertani
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1A 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.
  • 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.
  • Example 10 Orally Administered SALV-0013 Demonstrates Significant SlgA Response
  • 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.
  • 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.
  • N13 e.g., SALV-0013
  • results of this experiment demonstrate that the gummy formulation of N6G outperformed the oral gavage administration and represents a viable form of administration.
  • 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.
  • 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.
  • 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.
  • LNCaP FGC 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.
  • 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.
  • the therapeutic was created by filtering oral gavage through 0.2 micron filter.
  • 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.
  • Example 12 Construction of TNFA/RS10 & TNF/RSS10 & EH 4 Epitopecontaining Nlssle Probiotic Vaccine (f88-SALV-1001 ; f88-SALV-1002; and f88- SALV-1003):
  • VRS10, RSS10 and El 14 DNA were designed with codon optimization for E. coll, and chemically synthesized.
  • PAEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKAS f88-4-EI14 (signal peptide-El 14-p V///)
  • PAEGDDPAKA AFDSLQASA TEYIG YA WAMVVViVGA TiG/KLFKKFTSKA S
  • f88-4 vector 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.
  • F88-4 RF I DNA was digested by Hindlll/Pstl and then separated by agarose gel electrophoresis.
  • 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.
  • 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>:10 11 pfu/mL, for f88-4-RSS10 was 2.9x1012 pfu/mL, and for f88-4-E114 was 1.5x10 12 pfu/mL.

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Abstract

L'invention concerne des phages recombinants, des vaccins probiotiques et des agents thérapeutiques comprenant un phage recombinant ; et des procédés d'utilisation de ceux-ci associés.
PCT/US2025/018803 2024-03-07 2025-03-06 Compositions de vaccin oral et procédés associés Pending WO2025189043A2 (fr)

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