Arthrospira platensis non-parenteral therapeutic delivery platform

Abstract

To provide a composition for non-parenteral administration of a therapeutic, such as oral, intranasal, or airway administration, which is capable of protecting the therapeutic from degradation after administration, in particular from degradation caused by digestive enzymes and low pH to which the therapeutic is subjected after oral administration.SOLUTION: The present disclosure provides non-parenteral compositions comprising a recombinant Spirulina comprising at least one exogenous therapeutic. Compositions of the present disclosure can be used as vaccines and / or therapeutic drugs. The present disclosure also provides methods of making recombinant Spirulina comprising at least one exogenous therapeutic, and methods of treatment. A non-parenteral composition comprising a recombinant Spirulina, wherein the recombinant Spirulina comprises at least one exogenous therapeutic, a prophylactic agent molecule, or a combination of two or more exogenous therapeutics or prophylactic agent molecules, is provided herein.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 870,478, filed Jul. 3, 2019; U.S. Provisional Patent Application No. 62 / 937,995, filed Nov. 20, 2019; and U.S. Provisional Patent Application No. 62 / 943,075, filed Dec. 3, 2019, each of which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Sequence Listing The content of the text file electronically submitted together with this specification is incorporated herein by reference in its entirety: a copy of the computer - readable format of the sequence listing (file name: LUBI - 029_01WO_SeqList.ST25txt, recording date: Jul. 3, 2020, file size: about 100 kilobytes).

[0003] Field The present disclosure is directed to non - parenteral therapeutic compositions. In particular, the present disclosure provides oral, nasal, and respiratory (inhaled) compositions comprising recombinant Spirulina comprising one or more exogenous therapeutic agents.

Background Art

[0004] Background Non - parenteral administration of therapeutic agents is a convenient, portable, and inexpensive form of administration. Nasal and oral administration of therapeutic agents are commonly practiced, but oral therapeutic agents are exposed to harsh conditions in the gastrointestinal tract and may decompose before exerting their effects. Further, these therapeutic agents are costly to produce, and there is a need for the development of compositions that protect the therapeutic agents from digestive enzymes and low pH to which oral therapeutic agents are exposed after administration, along with the purification of the therapeutic agents. There is a need for cost - effective and stable compositions for non - parenteral administration.

Summary of the Invention

Means for Solving the Problems

[0005] Summary of the Invention This application solves problems related to costs and exposure to degradation of therapeutic agents in the gastrointestinal tract, nasal passages, and airways by administering the therapeutic agent to a subject in Spirulina. Spirulina is a cyanobacterium that can persist in the gastrointestinal tract, nasal passages, and airways, and thus the encapsulated therapeutic agent is protected until Spirulina reaches its destination (e.g., within the gastrointestinal tract). Furthermore, Spirulina can be easily cultivated, harvested, grows rapidly, can be dried to avoid spoilage, and can be consumed raw. Indeed, Spirulina is approved for human consumption and is generally consumed as a dietary supplement.

[0006] Provided herein is a non-injectable composition comprising recombinant Spirulina, wherein the recombinant Spirulina comprises at least one exogenous therapeutic agent, prophylactic agent molecule, or a combination of two or more exogenous therapeutic agents or prophylactic agent molecules. The exogenous therapeutic agent can be a compound produced by a microorganism or a plant. In particular, the exogenous therapeutic agent can be an antimicrobial compound or a polypeptide. In some embodiments, the exogenous therapeutic agent or prophylactic agent molecule is a VHH and / or a lysin.

[0007] In some embodiments, the disclosure provides a composition comprising recombinant Spirulina that is delivered without injection, wherein the recombinant Spirulina comprises at least one therapeutic agent or prophylactic agent molecule, or a combination of two or more therapeutic agents or prophylactic agent molecules. In some embodiments, the therapeutic agent or prophylactic agent molecule is delivered to the gastrointestinal tract. In some embodiments, the therapeutic agent or prophylactic agent molecule is delivered to the nose. In some embodiments, the therapeutic agent or prophylactic agent molecule is delivered by respiration (inhalation). In some embodiments, the therapeutic agent or prophylactic agent molecule is delivered systemically. In some embodiments, the therapeutic agent or prophylactic agent molecule is delivered locally.

[0008] In some embodiments, the therapeutic or prophylactic agent molecule or a combination of two or more therapeutic or prophylactic agent molecules is an endogenous Spirulina molecule. In some embodiments, the endogenous Spirulina molecule is found at a higher concentration than that found in naturally occurring Spirulina.

[0009] In some embodiments, the therapeutic or prophylactic agent molecule or a combination of two or more therapeutic or prophylactic agent molecules is exogenous to Spirulina. In some embodiments, the exogenous molecule is one produced by a different bacterium, parasite, protozoan, virus, phage, alga, animal, or plant.

[0010] In some embodiments, the combination contains two or more therapeutic or prophylactic agent molecules that are endogenous to Spirulina. In some embodiments, the combination contains two or more therapeutic or prophylactic agent molecules that are exogenous to Spirulina. In some embodiments, the combination contains two or more therapeutic or prophylactic agent molecules that are a mixture of endogenous and exogenous to Spirulina. In some embodiments, the combination contains two or more therapeutic or prophylactic agent molecules, and at least one of the therapeutic or prophylactic agent molecules present in the combination is present in a greater copy number (e.g., 2-fold, 3-fold, 4-fold, 5-fold, or more) than another therapeutic or prophylactic agent molecule.

[0011] In some embodiments, the exogenous molecule is a polypeptide or a fragment thereof. In some embodiments, the exogenous polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is a full-length antibody, a monospecific antibody, a bispecific antibody, a trispecific antibody, an antigen-binding region, a heavy chain, a light chain, a VHH, a VH, a VL, a CDR, a variable domain, an scFv, an Fc, an Fv, a Fab, an F(ab) 2 , a reduced IgG (rIgG), a monospecific Fab 2 , a bispecific Fab 2 , a trispecific Fab3 selected from the group consisting of diabody, bispecific diabody, trispecific triabody, minibody, nanobody, IgNAR, V-NAR, HcIgG, or combinations thereof.

[0012] In some embodiments, the exogenous polypeptide is insulin, C-peptide, amylin, interferon, hormone, receptor, receptor agonist, receptor antagonist, incretin, GLP-1, glucose-dependent insulinotropic polypeptide (GIP), immunomodulatory agent, immunosuppressive agent, peptide chemotherapeutic agent, antimicrobial peptide, magainin, NRc-3, NRC-7, buforin IIb, BR2, p16, Tat, TNF alpha selected from the group consisting of, and chlorotoxin.

[0013] In some embodiments, the exogenous polypeptide is an antigen or epitope. In some embodiments, the antigen or epitope is derived from an infectious microorganism, tumor antigen or autoantigen associated with an autoimmune disease.

[0014] In some embodiments, the exogenous polypeptide is a catalytic enzyme such as a lysin that cleaves cell walls or a fragment thereof.

[0015] In some embodiments, the recombinant Spirulina contains a combination of one or more different antibodies or antibody fragments. In some embodiments, the recombinant Spirulina contains a combination of one or more different VHHs. In some embodiments, the recombinant Spirulina contains a combination of one or more different antibodies or antibody fragments and one or more polypeptides. In some embodiments, the recombinant Spirulina contains a combination of one or more different VHHs and one or more polypeptides. In some embodiments, the recombinant Spirulina contains a combination of one or more different VHHs and one or more lysin polypeptides.

[0016] In some embodiments, administering recombinant Spirulina to a subject prevents, treats, or improves a disease or disorder. In some embodiments, the disease or disorder is selected from the group consisting of celiac disease, type 1 diabetes, type 2 diabetes, cancer, inflammatory disorders, gastrointestinal diseases, autoimmune diseases or disorders, endocrine disorders, gastroesophageal reflux disease (GERD), ulcers, high cholesterol, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, constipation, vitamin deficiency, iron deficiency, and diarrhea.

[0017] In some embodiments, administering recombinant Spirulina to a subject treats, prevents, or improves an infectious disease. In some embodiments, the infectious disease causes disorders such as acute respiratory distress syndrome (ARDS), pneumonia, endocarditis, stroke, and COVID-19.

[0018] In some embodiments, the infectious disease is a bacterial infection, viral infection, fungal infection, or parasitic infection. In some embodiments, the bacteria that cause the infectious disease are selected from the group consisting of E. coli, enterotoxigenic E. coli (ETEC), Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Helicobacter, Bacillus anthracis, enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), and Legionella.

[0019] In some embodiments, the virus causing the infectious disease is bacteriophage, RNA bacteriophage (e.g., MS2, AP205, PP7 and Qβ), coronavirus, infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus (DengeVirus), selected from the group consisting of rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus.

[0020] In some embodiments, the fungus causing the infectious disease is selected from the group consisting of Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma.

[0021] In some embodiments, the parasite causing the infectious disease is selected from the group consisting of Plasmodium, P. falciparum, P. malariae, P. ovale, P. vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, parasitic worms (helminthic parasite): Trichuris spp., Enterobius spp., Ascaris spp., Ancylostoma spp. and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp. and Wuchereria spp., Taenia spp., Echinococcus spp., and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp.

[0022] In some embodiments, the exogenous polypeptide or fragment thereof is present in the fusion protein.

[0023] In some embodiments, the recombinant Spirulina contains a nucleic acid encoding an exogenous polypeptide or fragment thereof. In some embodiments, there are at least 2 copies, at least 3 copies, at least 4 copies, or at least 5 copies of a nucleic acid sequence encoding at least one exogenous polypeptide or fragment thereof in the recombinant Spirulina. In some embodiments, there are 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 8 copies, 10 copies, 15 copies, 20 copies, 25 copies, 30 copies, 40 copies, or 50 copies of a nucleic acid sequence encoding at least one exogenous polypeptide or fragment thereof in the recombinant Spirulina. In some embodiments, there are at least 2 copies, at least 3 copies, at least 4 copies, or at least 5 copies of at least one exogenous polypeptide or fragment thereof in a single molecule of the exogenous polypeptide expressed in the recombinant Spirulina.

[0024] In some embodiments, there are 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 8 copies, 10 copies, 15 copies, 20 copies, 25 copies, 30 copies, 40 copies, or 50 copies of at least one exogenous polypeptide or fragment thereof in a single molecule of the exogenous polypeptide expressed in the recombinant Spirulina.

[0025] In some embodiments, within the molecule of the exogenous polypeptide or fragment thereof, copies of the exogenous polypeptide are linked tandemly.

[0026] In some embodiments, within the molecule of the exogenous polypeptide or fragment thereof, copies of the exogenous polypeptide or fragment thereof are separated by a spacer sequence.

[0027] In some embodiments, within the molecule of an exogenous polypeptide or a fragment thereof, a part of the copy of the exogenous polypeptide or the fragment thereof is tandemly linked, and the remaining copies of the exogenous polypeptide or the fragment thereof are separated by a spacer sequence. In some embodiments, the spacer sequence is between about 1 amino acid in length and 50 amino acids in length. In some embodiments, more than one spacer sequence is present within the molecule of the exogenous polypeptide or the fragment thereof. In some embodiments, the recombinant Spirulina contains at least two, at least three, at least four, or at least five different exogenous polypeptides or fragments thereof.

[0028] In some embodiments, the fusion protein contains a carrier or a chaperone protein. In some embodiments, the carrier protein is selected from the group consisting of maltose-binding protein, hepatitis B virus-like particles, thioredoxin, and phycocyanin. In some embodiments, the fusion protein contains a scaffold protein.

[0029] In some embodiments, at least one exogenous polypeptide is linked to a scaffold protein at the N-terminus or C-terminus of the scaffold protein, or within the body thereof. In some embodiments, the scaffold protein is selected from the oligomerization domain of C4b-binding protein (C4BP), cholera toxin b subunit, or the oligomerization domain of an extracellular matrix protein. In some embodiments, at least one exogenous polypeptide and the scaffold protein are separated by about 1 to about 50 amino acids.

[0030] In some embodiments, the fusion protein comprises multiple copies of at least one exogenous polypeptide or a fragment thereof, and at least one exogenous polypeptide or a fragment thereof and the scaffold protein are arranged in any one of the following patterns: (E)n-(SP), (SP)-(E)n, (SP)-(E)n-(SP), (E)n1-(SP)-(E)n2, (SP)-(E)n1-(SP)-(E)n2, and (SP)-(E)n1-(SP)-(E)n2-(SP) (in the pattern, E is at least one exogenous polypeptide or a fragment thereof, SP is the scaffold protein, and n, n1, and n2 represent the number of copies of at least one exogenous polypeptide or a fragment thereof).

[0031] In some embodiments, the recombinant Spirulina comprises an anti-Campylobacter VHH. In some embodiments, Campylobacter is C. jejuni. In some embodiments, the VHH binds to a Campylobacter component. In some embodiments, the VHH binds to flagellin. In some embodiments, administration results in increased Campylobacter excretion. In some embodiments, administration results in a decrease in the level of a biomarker. In some embodiments, the biomarker is an inflammatory biomarker.

[0032] In some embodiments, the recombinant Spirulina comprises a VHH that binds to an anti-Clostridium toxin. In some embodiments, Clostridium is C. difficile. In some embodiments, the VHH binds to the A toxin or the B toxin or both, which are Clostridium components. In some embodiments, the VHH comprises an amino acid sequence of any one of SEQ ID NOs: 5-17 or a fragment thereof.

[0033] In some embodiments, the recombinant Spirulina comprises a VHH that binds to the norovirus P domain. In some embodiments, the VHH comprises an amino acid sequence of any one of SEQ ID NOs: 40-79 or a fragment thereof.

[0034] In some embodiments, the recombinant Spirulina comprises a VHH that binds to a malaria polypeptide. In some embodiments, the recombinant Spirulina comprises a malaria antigen. In some embodiments, the malaria antigen is circumsporozoite protein (CSP). In some embodiments, the malaria antigen comprises at least one NANP repeat. In some embodiments, the recombinant Spirulina comprises a nucleotide sequence encoding a malaria antigen. In some embodiments, the recombinant Spirulina comprises an amino acid sequence comprising a malaria antigen. In some embodiments, the recombinant Spirulina comprises a molecule of any one of SEQ ID NOs: 26-31. In some embodiments, the recombinant Spirulina comprising a malaria antigen or VHH is administered intranasally. In some embodiments, an extract of the recombinant Spirulina comprising a malaria antigen or VHH is administered intranasally.

[0035] In some embodiments, the therapeutic or prophylactic agent molecule is a monomer.

[0036] In some embodiments, the therapeutic or prophylactic agent molecule is a multimer.

[0037] In some embodiments, the therapeutic or prophylactic agent molecule is a trimer. In some embodiments, the therapeutic or prophylactic agent molecule is a pentamer. In some embodiments, the therapeutic or prophylactic agent molecule is a heptamer. In some embodiments, the multimer is a heteromer. In some embodiments, the multimer is a homomer. In some embodiments, the multimer is arranged in a nanoparticle. In some embodiments, the multimer binds to a target or target molecule with high affinity. In some embodiments, the binding affinity of the multimer is greater than the binding affinity of the monomer or dimer.

[0038] In some embodiments, the multimer has an EC 50 greater than 5 μg / mL. In some embodiments, the multimer has an EC 50 greater than 10 μg / mL. In some embodiments, the multimer has an EC of about 5 μg / mL to about 40 μg / mL 50It has. In some embodiments, the multimer has an EC between about 0.10 nM and about 100 nM 50 It has. In some embodiments, the multimer has an EC between about 0.2 nM and about 55 nM 50 It has. In some embodiments, the binding affinity of the multimer is greater than the binding affinity of multimers containing fewer copies of the exogenous therapeutic agent or combinations of fewer copies of the exogenous therapeutic agent. In some embodiments, administration of Spirulina containing the multimer exogenous therapeutic agent results in a lower dose of Spirulina for efficacy than administration of Spirulina containing the monomer of the same exogenous therapeutic agent.

[0039] In some embodiments, the recombinant Spirulina is selected from the group consisting of A. amethystine, A. ardissonei, A. argentina, A. balkrishnanii, A. baryana, A. boryana, A. braunii, A. breviarticulata, A. brevis, A. curta, A. desikacharyiensis, A. funiformis, A. fusiformis, A. ghannae, A. gigantean, A. gomontiana, A. gomontiana var. crassa, A. indica, A. jenneri var. platensis, A. jenneri Stizenberger, A. jenneri f. purpurea, A. joshii, A. khannae, A. laxa, A. laxissima, A. laxissima, A. leopoliensis, A. major, A. margaritae, A. massartii, A. massartii var. indica, A. maxima, A. meneghiniana, A. miniata var. constricta, A. miniata, A. miniata f. acutissima, A. neapolitana, A. nordstedtii, A. oceanica, A. okensis, A. pellucida, A. platensis, A. platensis var. non-constricta, A. platensis f. granulate, A. platensis f. minor, A. platensis var. tenuis, A. santannae, A. setchellii, A. skujae, A. spirulinoides f. tenuis, A. spirulinoides, A. subsalsa, A. subtilissima, A. tenuis, A. tenuissima, and A. versicolor. In some embodiments, the recombinant Spirulina is non-viable. In some embodiments, the recombinant Spirulina is dried, spray-dried, freeze-dried, or lyophilized.

[0040] In some embodiments, the injectable-free composition comprises a pharmaceutically acceptable excipient.

[0041] In some embodiments, the composition remains in the gastrointestinal tract or a simulated gastric environment. In some embodiments, the composition remains in the gastrointestinal tract or a simulated gastric environment for at least 5 minutes. In some embodiments, the composition remains in the gastrointestinal tract or a simulated gastric environment overnight.

[0042] In some embodiments, the composition remains in the nasal cavity. In some embodiments, the composition remains in the upper airway. In some embodiments, the composition remains in the airway. In some embodiments, the composition remains in the nasal cavity, upper airway, and / or airway for at least 5 minutes. In some embodiments, the composition remains in the nasal cavity, upper airway, and / or airway overnight.

[0043] In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition delivered without injection according to the present disclosure.

[0044] In some embodiments, by administering the composition delivered without injection, the onset of symptoms due to Campylobacter is reduced or prevented.

[0045] In some embodiments, by administering the delivered composition, the onset of inflammation in the subject is reduced or prevented.

[0046] In some embodiments, the present disclosure provides a method for treating or preventing Clostridioides difficile infection, the method comprising administering to a subject a composition that is delivered without injection of the present disclosure. In some embodiments, by administering the composition that is delivered without injection, the onset of symptoms due to Clostridioides difficile is reduced or prevented. In some embodiments, the present disclosure provides a method for treating or preventing malaria infection, the method comprising administering the composition of the present disclosure by inhalation or intranasally. In some embodiments, by inhalation or intranasal administration of the composition, the onset of symptoms due to malaria is reduced or prevented.

[0047] In some embodiments, the present disclosure provides a method for treating or preventing coronavirus infection, the method comprising administering the composition of the present disclosure by inhalation or intranasally. In some embodiments, by inhalation or intranasal administration of the composition, the onset of symptoms due to coronavirus is reduced or prevented.

[0048] In some embodiments, the present disclosure provides a method for treating or preventing malaria infection, the method comprising administering to a subject a composition that is delivered without injection of the present disclosure. In some embodiments, by administering the composition that is delivered without injection, the onset of symptoms due to malaria is reduced or prevented.

[0049] In some embodiments, the present disclosure provides a method for treating or preventing coronavirus (e.g., SARS virus, SARS-CoV-2 virus) infection, the method comprising administering to a subject a composition that is delivered without injection of the present disclosure. In some embodiments, by administering the composition that is delivered without injection, the onset of symptoms of coronavirus infection (e.g., ARDS, inflammation) is reduced or prevented.

[0050] In some embodiments, provided herein is a method of making an injection-free composition described herein, the method comprising the step of introducing at least one exogenous therapeutic agent into Spirulina.

[0051] In some embodiments, provided herein is a method of making an injection-free composition described herein, the method comprising the step of introducing a nucleic acid sequence encoding at least one exogenous therapeutic agent into Spirulina.

[0052] In some embodiments, provided herein is an injection-free antigenic composition comprising recombinant Spirulina, wherein the recombinant Spirulina comprises at least one exogenous antigenic epitope and a nucleic acid sequence encoding the at least one exogenous antigenic epitope is incorporated into Spirulina by homologous recombination.

[0053] In some embodiments, provided herein is an injection-free antigenic composition prepared by a method comprising the step of introducing a nucleic acid sequence encoding at least one exogenous antigenic epitope into Spirulina and the step of incorporating the nucleic acid sequence into Spirulina by homologous recombination. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0141] Detailed Description The present disclosure teaches packaging an exogenous therapeutic or prophylactic agent molecule in a prokaryotic alga and then administering it to a subject by means other than injection. In some embodiments, the recombinant prokaryotic alga is edible and can function as an edible composition for delivering a payload expressed in the alga. In the case of polypeptide therapeutic or prophylactic agent molecules (e.g., antibodies, antigens, etc.), the expression level of the exogenous polypeptide in the Spirulina delivery system of the present disclosure is 10 to 100 times higher compared to other systems.

[0142] Provided herein are non-injectable compositions comprising recombinant Spirulina containing at least one exogenous therapeutic or prophylactic agent molecule, methods of making the same, and uses thereof.

[0143] Before describing certain embodiments in detail, it should be understood that the present disclosure is not limited to specific compositions or biological systems and that the compositions or biological systems can vary. It should also be understood that the terminology used herein is for the purpose of describing only certain exemplary embodiments and is not intended to be limiting. The terms used herein generally have their ordinary meanings in the context of the present disclosure and in the specific context in which each term is used. To provide additional guidance to practitioners regarding the compositions and methods of the present disclosure and the manner of their making and use, certain terms are considered below or elsewhere in this specification. The scope and meaning of any use of the terms will become apparent from the specific context in which the terms are used. As such, the definitions set forth herein are not intended to be limited to specific compositions or biological systems, but rather are intended to provide exemplary guidance in the identification of specific embodiments of the present disclosure.

[0144] In accordance with longstanding patent law convention, the terms "a", "an", and "the", as used in this application, including in the claims, refer to "one or more" unless clearly indicated otherwise. By way of example, "an antigenic epitope" means one epitope or more than one epitope.

[0145] As used herein, the term "antigenic composition" refers to a preparation that, when administered to a subject, induces a protective immune response that confers immunity against a disease or disorder, or a preparation that can be used to treat a disease or disorder described herein.

[0146] As used herein, the term "antigen" refers to a protein or peptide that binds to a receptor of an immune cell in a human or animal and induces an immune response. An antigen can be derived from an infectious microorganism, including a virus, bacterium, parasite, or fungus, or an antigen can be a tumor antigen or a self - antigen associated with an autoimmune disease.

[0147] As used herein, the term "antigenic epitope" refers to a short amino acid sequence of an antigen, for example, a sequence of about 4 - 1000 amino acids, that is recognized and bound by a receptor of an immune cell in a human or animal and induces an immune response. The antigenic epitopes of the present disclosure are derived from the above - mentioned antigens.

[0148] As used herein, the term "subject" refers to a vertebrate or invertebrate, including mammals, birds, fish, insects, and amphibians. Subjects include humans as well as other primates, including non-human primates such as chimpanzees and other apes and monkey species. Subjects include farm animals such as cows, sheep, pigs, goats, and horses; domesticated mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds including poultry such as chickens, turkeys, and other game birds, ducks, geese, wild birds, and game birds; and aquatic animals such as fish, shrimp, and crustaceans.

[0149] Therapeutic composition not by injection Provided herein is a composition not by injection, comprising recombinant Spirulina, wherein the Spirulina is engineered to contain at least one exogenous therapeutic agent or a fragment thereof. As used herein, the term "therapeutic agent" refers to any molecule that can be used to treat a disease or disorder and / or has a therapeutic effect in a subject. As used herein, the term "preventive agent" refers to any molecule that can be used to prevent the onset of a disease or disorder in a subject.

[0150] There are several advantages to delivering therapeutic or prophylactic molecules encapsulated in Spirulina without the need for injection. One of these advantages is increased resistance of the encapsulated therapeutic or prophylactic molecule to proteolysis. For example, when delivered orally, encapsulation in Spirulina protects the therapeutic or prophylactic molecule from enzymes and gastrointestinal conditions, thereby enabling delivery of the therapeutic to the portion of the gastrointestinal tract that digests Spirulina cells and release of the therapeutic or prophylactic molecule. In some embodiments, the orally delivered compositions of the present disclosure remain intact (e.g., substantially intact) at a pH of about 1.3 to about 8.0. In some embodiments, the orally delivered compositions of the present disclosure remain intact in the oral cavity. In some embodiments, the orally delivered compositions of the present disclosure remain intact in the stomach. In some embodiments, the orally delivered compositions remain intact in the small intestine and / or large intestine. In some embodiments, the orally delivered compositions remain intact in the colon. In some embodiments, the orally delivered compositions remain intact in a simulated gastric environment. In some embodiments, the simulated gastric environment has an acidic pH and contains pepsin. In some embodiments, the simulated gastric environment has a pH of about 3.0 and about 2000 U / mL of pepsin. In some embodiments, the orally delivered compositions can remain intact for about 5 minutes to about 1 day under gastrointestinal conditions or in a simulated gastric environment. In some embodiments, the orally delivered compositions remain intact for about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 24 hours under gastrointestinal conditions or in a simulated gastric environment. In some embodiments, the orally delivered compositions remain intact overnight under gastrointestinal conditions or in a simulated gastric environment. [Paragraphs related to the nose and respiratory tract added]

[0151] In some embodiments, the composition delivered without injection of the present disclosure remains (e.g., remains substantially intact) at a pH of about 5.0 to about 8.0. In some embodiments, the composition delivered without injection of the present disclosure remains (e.g., remains substantially intact) at a pH of about 5.5 to about 6.5. In some embodiments, the composition delivered without injection of the present disclosure remains in the oral cavity. In some embodiments, the composition delivered without injection of the present disclosure remains intranasally. In some embodiments, the composition delivered without injection remains in the pharynx. In some embodiments, the composition delivered without injection remains in the trachea. In some embodiments, the composition delivered without injection remains in the bronchi. In some embodiments, the composition delivered without injection remains in the lungs. In some embodiments, the composition delivered without injection remains in the alveoli. In some embodiments, the composition delivered without injection remains in the airway. In some embodiments, the composition delivered without injection remains in a simulated nasal and / or airway environment. In some embodiments, the simulated nasal environment has a pH of about 5 to about 7. In some embodiments, the simulated nasal environment has a pH of about 5.5 to about 6.5. In some embodiments, the simulated airway environment has a pH of about 7 to about 8. In some embodiments, the simulated airway environment has a pH of about 7.3 to about 7.5. In some embodiments, the composition delivered without injection can remain for about 5 minutes to about 1 day in nasal conditions, airway conditions, or a simulated airway environment. In some embodiments, the composition delivered without injection can remain for about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 24 hours in nasal conditions, airway conditions, or a simulated airway environment. In some embodiments, the composition delivered without injection can remain overnight in nasal conditions, airway conditions, or a simulated airway environment. In some embodiments, the composition delivered without injection is an extract of recombinant Spirulina biomass.

[0152] Another advantage of the compositions delivered without injection according to the present disclosure is stability during storage. In some embodiments, the compositions delivered without injection according to the present disclosure are stable at elevated temperatures (e.g., temperatures higher than room temperature). In some embodiments, the compositions delivered without injection according to the present disclosure are stable at 42°C. In some embodiments, the compositions delivered without injection according to the present disclosure are stable at 42°C for about 1 day to 5 years. In some embodiments, the compositions delivered without injection according to the present disclosure are stable at 42°C for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the compositions delivered without injection according to the present disclosure are stable at 42°C for 1 month or 3 months. In some embodiments, the compositions delivered without injection according to the present disclosure are stable at room temperature (e.g., about 20°C to about 29°C). In some embodiments, the compositions delivered without injection according to the present disclosure are stable at 27°C. In some embodiments, the compositions delivered without injection according to the present disclosure are stable at 27°C for about 1 day to 5 years. In some embodiments, the compositions delivered without injection according to the present disclosure are stable at 27°C for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the compositions delivered without injection according to the present disclosure are stable at 27°C for 1 month or 3 months.

[0153] Therapeutic agent Any exogenous (i.e., non-Spirulina) therapeutic or prophylactic agent molecule suitable for non-injection administration can be used in the compositions and methods of the present disclosure. In some embodiments, the therapeutic or prophylactic agent molecule is a small molecule. In some embodiments, the therapeutic or prophylactic agent molecule is a polypeptide or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a mixture of polypeptides or fragments thereof, a mixture of therapeutic agents including a mixture of small molecules, or a mixture of prophylactic agent molecules and / or polypeptides or fragments thereof and small molecules.

[0154] In some embodiments, the therapeutic or prophylactic drug molecule is a small molecule produced by a cell. In some embodiments, the small molecule is produced by a microorganism such as a bacterium, virus, fungus, or parasite. In some embodiments, the small molecule is produced by a plant.

[0155] In some embodiments, the small molecule has an antimicrobial effect. In some embodiments, the small molecule has an antifungal effect. In some embodiments, the small molecule has an antiviral effect. In some embodiments, the small molecule has an antiparasitic effect. In some embodiments, the small molecule is selected from the group consisting of, but not limited to, antibiotics, marasine, penicillin, streptomycin, polymyxin, colistin, circulin, bacillomycin, mycobacillin, fungistatin, tannin, terpenoid, saponin, alkaloid, flavonoid, polyphenol, saponin, chloroquine, quinine, amodiaquine, hydroxychloroquine, metronidazole, tinidazole, iodoquinol, paromomycin, metronidazole and tinidazole, or combinations thereof. rifenol, saponin, chloroquine, quinine, amodiaquine, hydroxychloroquine, metronidazole, tinidazole, iodoquinol, paromomycin, metronidazole and tinidazole, or combinations thereof.

[0156] In some embodiments, the exogenous therapeutic or prophylactic agent molecule is a polypeptide or a fragment thereof. In some embodiments, the polypeptide or prophylactic agent molecule is, but is not limited to, a receptor, agonist, hormone, neurotransmitter, secreted polypeptide, tethered polypeptide, transcription factor, antimicrobial peptide, chemokine, cytokine, proprotein, preproprotein, interferon, antibody, neuropeptide, antigen, epitope derived from an antigen, autoantigen, secretin, G-protein coupled receptor, opioid peptide, cell surface protein, cytoplasmic protein, mitochondrial protein, cell signaling protein, insulin, C-peptide, amylin, interferon, hormone, receptor, receptor agonist, receptor antagonist, incretin, GLP-1, glucose-dependent insulinotropic polypeptide (GIP), immunomodulatory agent, immunosuppressive agent, peptide chemotherapeutic agent, antimicrobial peptide, magainin, NRc-3, NRC-7, buforin IIb, BR2, p16, Tat, TNF alpha, and chlorotoxin, or a combination thereof selected from the group consisting of.

[0157] In some aspects, the present disclosure does not include compositions or methods of Spirulina containing an antigen, antigen epitope, or fragment thereof. In some embodiments, the present disclosure does not include the subject matter of PCT / US2019 / 032998 filed on May 17, 2019. In some embodiments, the present disclosure does not include compositions or methods for eliciting or enhancing an immune response in a subject. In some embodiments, the present disclosure does not include compositions or methods for eliciting or enhancing the production of an antibody or fragment thereof against an exogenous polypeptide contained in Spirulina.

[0158] In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is, but is not limited to, a full-length antibody, a monospecific antibody, a bispecific antibody, a trispecific antibody, an antigen-binding region, a heavy chain, a light chain, a VHH, a VH, a VL, a CDR, a variable domain, an scFv, an Fc, an Fv, a Fab, an F(ab) 2 , reduced IgG (rIgG), monospecific Fab2 、 bispecific Fab 2 、 trispecific Fab 3 、 diabody, bispecific diabody, trispecific tribody, minibody, nanobody, IgNAR, V-NAR, HcIgG, or a combination thereof.

[0159] In some embodiments, the therapeutic peptide includes, but is not limited to, E. coli, enterotoxigenic E. coli (ETEC), Bacillus anthracis, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophage, RNA bacteriophage (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, SARS virus, MERS virus, SARS-CoV-2 virus, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, P. falciparum, P. malariae, P. ovale, P. vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, parasitic worms: Trichuris spp., Enterobius spp., Ascaris Relates to, is derived from, or treats or prevents an infectious disease caused by any microorganism, including spp., Ancylostoma spp., and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp., and Wuchereria spp., Taenia spp., Echinococcus spp., and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp., or combinations thereof.

[0160] In some embodiments, the exogenous polypeptide is an antigen or autoantigen. In some embodiments, the autoantigen is related to an autoimmune disease or disorder. In some embodiments, the autoantigen is a tumor antigen. In some embodiments, the exogenous polypeptide binds to an antigen or autoantigen.

[0161] In various embodiments, the compositions of the present disclosure comprise recombinant Spirulina comprising at least one exogenous polypeptide (e.g., a portion or fragment thereof, or an antigenic variant thereof) derived from an infectious microorganism, a tumor antigen, or an autoantigen related to an autoimmune disease.

[0162] In some embodiments, the composition comprises recombinant Spirulina comprising at least one exogenous antigenic epitope derived from an infectious microorganism such as a virus, bacterium, parasite, or fungus. The infectious microorganism can be a microorganism that causes an infectious disease in a human or animal species, such as livestock, poultry, and fish.

[0163] In some embodiments, the compositions of the present disclosure include, but are not limited to, bacteriophages, RNA bacteriophages (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus (IHNV), parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, human immunodeficiency virus (HIV), influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, MERS virus, SARS virus, and recombinant Spirulina containing at least one polypeptide, antigen, or antigenic epitope derived from a virus, including the SARS-CoV-2 virus. In some embodiments, the compositions of the present disclosure include recombinant Spirulina containing at least one polypeptide, antigen, or antigenic epitope derived from IHNV. In some embodiments, the compositions of the present disclosure include recombinant Spirulina SP105 or SP113. In some embodiments, the compositions of the present disclosure include recombinant Spirulina containing at least one polypeptide, antigen, or antigenic epitope derived from the coronavirus. In some embodiments, the compositions of the present disclosure include recombinant Spirulina containing at least one polypeptide, antigen, or antigenic epitope derived from the SARS-CoV-2 virus. In some embodiments, the oral compositions of the present disclosure include recombinant Spirulina containing at least one polypeptide, antigen, or antigenic epitope derived from parvovirus, e.g., canine parvovirus. In some embodiments, the compositions of the present disclosure include recombinant Spirulina SP673 or SP678.

[0164] In some embodiments, the compositions of the present disclosure include, but are not limited to, bacteriophages, RNA bacteriophages (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus (IHNV), parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, human immunodeficiency virus (HIV), influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, MERS virus, SARS virus, and recombinant Spirulina comprising at least one polypeptide or a fragment thereof that binds to a virus or a part thereof, including SARS-CoV-2 virus.

[0165] In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to a norovirus polypeptide or antigen. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to the norovirus P domain. In some embodiments, the polypeptide or the fragment thereof is a VHH. In some embodiments, the recombinant Spirulina comprises a VHH that binds to a norovirus polypeptide. In some embodiments, the recombinant Spirulina comprises a VHH that binds to the norovirus P domain. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to genotypes GII, G1, or G11.10. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to polypeptides from two or more norovirus genotypes. In some embodiments, the recombinant Spirulina comprises a VHH that includes the Nano85 nanobody, the Nano26 nanobody, the Nano94 nanobody, the K922 antibody, or a modified sequence or fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that includes Nano85 and / or its loop grafted type modification. In some embodiments, the VHH comprises an amino acid sequence of any one of SEQ ID NOs: 40 to 79 or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to a norovirus polypeptide or antigen, or a fragment thereof, as a fusion with a chaperone polypeptide. In some embodiments, the recombinant Spirulina comprises multiple copies of a polypeptide or a fragment thereof that binds to a norovirus polypeptide or antigen, or a fragment thereof, as a fusion with a chaperone polypeptide. In some embodiments, the chaperone polypeptide is maltose-binding protein (MBP) or thioredoxin A (TxnA). In some embodiments, the recombinant Spirulina comprises a monomer, dimer, or heptamer of a polypeptide or a fragment thereof that binds to an anti-Clostridium toxin or a fragment thereof.In some embodiments, the recombinant Spirulina comprises a VHH comprising Nano85 and / or its loop grafted modifications as a fusion with a chaperone polypeptide. In some embodiments, the chaperone polypeptide is maltose binding protein (MBP) or thioredoxin A (TxnA). In some embodiments, the recombinant Spirulina comprises multiple copies of a VHH comprising Nano85 and / or its loop grafted modifications as a fusion with a chaperone polypeptide. In some embodiments, the recombinant Spirulina is SP833, SP834, SP835, SP864, SP1241, SP1371 or SP1372.

[0166] In some embodiments, the composition comprises a recombinant Spirulina comprising at least one antigenic epitope derived from bacteria including, but not limited to, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, E. coli (including pathogenic E. coli), and Legionella.

[0167] In some embodiments, the recombinant Spirulina comprises a polypeptide that binds to an ETEC polypeptide or antigen or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide that binds to a fimbrial polypeptide or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that binds to an ETEC polypeptide. In some embodiments, the recombinant Spirulina comprises a VHH that binds to a fimbrial polypeptide or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that binds to an adhesion or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that binds to polypeptides derived from two or more adhesions. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to the F4+ adhesion domain FaeG or the F18+ adhesion domain FedF. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to one or more of the adhesions K88 (also referred to as F4), K99 (F5), 987P (F6), F41, and F18 or a modification or fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to K88. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to an ETEC polypeptide or antigen or a fragment thereof as a fusion with a chaperone polypeptide. In some embodiments, the chaperone polypeptide is maltose-binding protein (MBP) or thioredoxin A (TxnA). In some embodiments, the recombinant Spirulina comprises multiple copies of a polypeptide or a fragment thereof that binds to an ETEC polypeptide or antigen or a fragment thereof as a fusion with a chaperone polypeptide. In some embodiments, the recombinant Spirulina comprises a monomer, dimer, or heptamer of a polypeptide or a fragment thereof that binds to an ETEC polypeptide or a fragment thereof, or a monomer, dimer, or heptamer of an ETEC polypeptide or antigen or a fragment thereof. In some embodiments, the dimer or heptamer is a homodimer or homoheptamer.In some embodiments, the dimer or heptamer is a heterodimer or heteroheptamer. In some embodiments, the recombinant Spirulina is SP795 or SP1156.

[0168] In some embodiments, the recombinant Spirulina comprises a polypeptide that binds to an anti-Clostridium toxin. In some embodiments, Clostridium is C. difficile. In some embodiments, the recombinant Spirulina comprises a VHH that binds to an anti-Clostridium toxin. In some embodiments, the polypeptide or a fragment thereof binds to the A toxin or B toxin, or both, which are Clostridium components. In some embodiments, the polypeptide is a VHH comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 17 or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a Clostridium antigen or a fragment thereof, or a polypeptide or a fragment thereof that binds to an anti-Clostridium toxin or a fragment thereof as a fusion with a chaperone polypeptide. In some embodiments, multiple copies of the recombinant Spirulina comprise a Clostridium antigen or a fragment thereof, or a polypeptide or a fragment thereof that binds to an anti-Clostridium toxin or a fragment thereof as a fusion with a chaperone polypeptide. In some embodiments, the chaperone polypeptide is maltose-binding protein (MBP) or thioredoxin A (TxnA). In some embodiments, the recombinant Spirulina comprises a monomer, dimer, or heptamer of a Clostridium antigen or a fragment thereof, or a polypeptide or a fragment thereof that binds to an anti-Clostridium toxin or a fragment thereof. In some embodiments, the dimer or heptamer is a homodimer or homoheptamer. In some embodiments, the dimer or heptamer is a heterodimer or heteroheptamer. In some embodiments, the recombinant Spirulina is SP744, SP977, SP985, SP1087, SP1091, or SP1095.

[0169] In some embodiments, the recombinant Spirulina comprises a polypeptide that binds to a Campylobacter polypeptide or antigen or a fragment thereof. In some embodiments, the Campylobacter is C. jejuni. In some embodiments, the recombinant Spirulina comprises a polypeptide that binds to a flagellin component. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to a flagellin polypeptide or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to flaA or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that binds to a Campylobacter polypeptide or antigen or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that binds to a flagellin polypeptide. In some embodiments, the recombinant Spirulina comprises a VHH that binds to flaA or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to a Campylobacter or antigen or a fragment thereof as a fusion with a chaperone polypeptide. In some embodiments, the chaperone polypeptide is maltose-binding protein (MBP) or thioredoxin A (TxnA). In some embodiments, the recombinant Spirulina comprises multiple copies of a polypeptide or a fragment thereof that binds to a Campylobacter polypeptide or antigen or a fragment thereof as a fusion with a chaperone polypeptide. In some embodiments, the recombinant Spirulina comprises a monomer, dimer, trimer, pentamer, or heptamer of a polypeptide or a fragment thereof that binds to a Campylobacter polypeptide, antigen, or a fragment thereof. In some embodiments, the dimer, trimer, pentamer, or heptamer is a homodimer, homotrimer, homopentamer, or homoheptamer. In some embodiments, the dimer, trimer, pentamer, or heptamer is a heterodimer, heterotrimer, heteropentamer, or heteroheptamer. In some embodiments, the recombinant Spirulina is SP526, SP651, SP742, or SP806.

[0170] In some embodiments, the recombinant Spirulina comprises a polypeptide that binds to a malaria polypeptide or antigen or a fragment thereof. In some embodiments, the malaria is P. falciparum. In some embodiments, the recombinant Spirulina comprises a polypeptide that binds to the circumsporozoite protein (CSP) or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to a polypeptide comprising one or more NANP repeats. In some embodiments, the recombinant Spirulina comprises a VHH that binds to a malaria polypeptide or antigen or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that binds to the CSP polypeptide. In some embodiments, the recombinant Spirulina comprises a VHH that binds to a polypeptide comprising one or more NANP repeats. In some embodiments, the recombinant Spirulina comprises a malaria antigen. In some embodiments, the malaria is P. falciparum. In some embodiments, the recombinant Spirulina comprises the circumsporozoite protein (CSP) or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide comprising one or more NANP repeats. In some embodiments, the polypeptide or a fragment thereof is a VHH comprising the amino acid sequence of any one of SEQ ID NOs: 26 to 31 or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or a fragment thereof that binds to a malaria or antigen or a fragment thereof as a fusion with a chaperone polypeptide. In some embodiments, the chaperone polypeptide is maltose-binding protein (MBP) or thioredoxin A (TxnA). In some embodiments, the recombinant Spirulina comprises a plurality of copies of a polypeptide or a fragment thereof that binds to a malaria polypeptide or antigen or a fragment thereof as a fusion with a chaperone polypeptide. In some embodiments, the recombinant Spirulina comprises a monomer, dimer, trimer, pentamer, or heptamer of a polypeptide or a fragment thereof that binds to a malaria polypeptide, antigen, or a fragment thereof.In some embodiments, the dimer, trimer, pentamer, or heptamer is a homodimer, homotrimer, homopentamer, or homoheptamer. In some embodiments, the dimer, trimer, pentamer, or heptamer is a heterodimer, heterotrimer, heteropentamer, or heteroheptamer. In some embodiments, the recombinant Spirulina is SP648, SP803, or SP856.

[0171] In some embodiments, at least one exogenous polypeptide is expressed by itself in Spirulina, i.e., the polypeptide is not fused to another protein.

[0172] In some embodiments, at least one exogenous polypeptide expressed in Spirulina is included in an exogenous antigen. In some embodiments, the exogenous antigen is a native antigen. For example, the recombinant Spirulina can express the entire circumsporozoite protein containing one or more antigenic epitopes, or a part or domain of the circumsporozoite protein containing one or more antigenic epitopes. In this case, the exogenous antigen is regarded as a native antigen. Other examples of native antigens that can be expressed in Spirulina to prepare an oral antigenic composition include the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of influenza virus.

[0173] The present disclosure provides structures and / or ligands for stimulating the innate immune system in addition to immunogenic epitopes (e.g., by engineering an epitope into a VLP structure). The innate immune system can be activated by adjuvant-like properties inherent to the VLP and / or an adjuvant added to the vaccine composition. In some embodiments, these structures and / or ligands for stimulating the innate immune system include, but are not limited to, fragments of Salmonella flagellin, fliC, human and mouse TNF-alpha, and human and mouse CD40 ligand. In some embodiments, the exogenous polypeptide is a fusion protein. For example, in some embodiments, recombinant Spirulina can express a fusion protein comprising at least one exogenous polypeptide and a portion of another protein such as a viral protein or a scaffold protein. In some embodiments, the exogenous polypeptide or a fragment thereof is present in the fusion protein. In some embodiments, the fusion protein is a fusion of two or more polypeptides or fragments thereof. In some embodiments, the fusion protein is one or more polypeptides or fragments thereof attached to one or more scaffold polypeptides. In some embodiments, the fusion protein is one or more polypeptides or fragments thereof attached to one or more chaperone polypeptides. In some embodiments, the fusion protein comprises a tag (e.g., a 6×His tag) for isolation and / or purification. In some embodiments, the fusion protein comprises one or more targeting signals or polypeptides. In some embodiments, the fusion protein comprises one or more VHH sequences as a fusion with one or more chaperone polypeptides. In some embodiments, the fusion protein comprises one or more VHH sequences as a fusion with one or more chaperone polypeptides and one or more scaffold polypeptides.

[0174] In some embodiments, the exogenous antigen epitopes can be derived from different antigens that activate different types of immunity (e.g., innate immunity, cellular immunity, or humoral immunity). In some embodiments, one or more exogenous antigen epitopes derived from different antigens are derived from at least one B cell antigen and at least one T cell antigen. In some embodiments, one or more exogenous antigen epitopes are present in a fusion protein with a viral protein (e.g., a coronavirus spike protein). In some embodiments, one or more exogenous antigen epitopes are present in a fusion protein with a viral protein (e.g., a coronavirus spike protein), and one epitope is at either end. In some embodiments, one or more exogenous antigen epitopes are a B cell epitope fused to one end of a viral protein and a T cell epitope fused to the other end of the viral protein.

[0175] In some embodiments, the compositions of the present disclosure comprise recombinant Spirulina comprising multiple copies of one or more therapeutic and / or prophylactic drug molecules. In some embodiments, the compositions of the present disclosure comprise recombinant Spirulina comprising a combination of therapeutic and / or prophylactic drug molecules. In some embodiments, the oral compositions of the present disclosure comprise recombinant Spirulina comprising multiple copies of one therapeutic or prophylactic drug and at least one other therapeutic or prophylactic drug molecule. In some embodiments, the compositions of the present disclosure comprise recombinant Spirulina comprising at least one antibody and at least one other therapeutic or prophylactic drug molecule. In some embodiments, the compositions of the present disclosure comprise at least one VHH and at least one other therapeutic or prophylactic drug molecule. In some embodiments, the compositions of the present disclosure comprise at least one VHH, and a polypeptide. In some embodiments, the compositions of the present disclosure comprise at least one VHH, and a lysin polypeptide.

[0176] In some embodiments, the one or more therapeutic and / or prophylactic agent molecules are enzymes. In some embodiments, the enzyme is a hydrolase. In some embodiments, the hydrolase cleaves the cell wall. In some embodiments, the hydrolase targets the bonds in peptidoglycan. In some embodiments, the hydrolase includes, but is not limited to, lysin, phage lysin, cytolysin, ovocleidin, hemolysin, NK-lysin, streptolysin, autolysin, an LytC amidase, an LytD glucosaminidase, an N-acetylmuramoyl-L-alanine amidase, a polypeptide comprising or consisting of one or more catalytic domains derived from lysin or autolysin, or combinations and / or fragments thereof.

[0177] Fusion protein In some aspects of the present disclosure, the therapeutic or prophylactic agent molecule may be present as part of a complex in Spirulina. In some embodiments, Spirulina comprises a plurality of copies of one or more therapeutic and / or prophylactic agent molecules in the complex. In some embodiments, Spirulina comprises a combination of one or more therapeutic and / or prophylactic agent molecules in the complex. In some embodiments, Spirulina comprises one or more therapeutic and / or prophylactic agent molecules in a fusion protein.

[0178] In some embodiments, Spirulina comprises one or more therapeutic and / or prophylactic drug molecules in a complex containing a linker. In some embodiments, the construct inserted into recombinant Spirulina contains a linker. In some embodiments, the polypeptide expressed from recombinant Spirulina contains a linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker attaches two or more VHH sequences. In some embodiments, the linker attaches one or more VHH sequences to another polypeptide. In some embodiments, the other polypeptide is selected from, but not limited to, chaperone proteins, targeting proteins, scaffolds, oligomerization domains, enzymes, or lysins. In some embodiments, the linker is a helix 1 linker (SEQ ID NO: 19), a helix 2 linker (SEQ ID NO: 20), a helix 4 linker (SEQ ID NO: 21), a PA5 linker (SEQ ID NO: 22), a PA10 linker (SEQ ID NO: 25).

[0179] In some embodiments, at least one exogenous polypeptide is expressed as a fusion protein in Spirulina, where the fusion protein forms a three-dimensional structure (sometimes referred to herein as a "particle"). In some embodiments, the fusion protein forming the three-dimensional structure can include multiple functional domains and one or more exogenous polypeptides. Such fusion proteins can be engineered in several ways. In some embodiments, the fusion protein is a single polypeptide having multiple modular domains. An example of such a fusion protein is the woodchuck hepatitis virus core antigen (WHcAg) engineered with a B cell antigen at the major insertion region / spike position and a T cell epitope at the C-terminus. Another example is an RNA bacteriophage (i.e., MS2, PP7, AP205 or Q) engineered to form a tandem dimer using an antigen at the N-terminus and a fragment of Salmonella flagellin at the C-terminus, thus combining an immunogenic epitope and a stimulant of the innate immune system to act as an endogenous adjuvant that self-organizes into a three-dimensional structure with two functional domains displayed on the surface. β) In some embodiments, the recombinant Spirulina can express two heterologous polypeptides. For example, the recombinant Spirulina can express one gene encoding a tandem RNA bacteriophage capsid protein dimer having an N-terminal antigenic structure, and a second gene encoding a capsid dimer that is identical but has an adjuvant such as Salmonella flagellin at the C-terminus. These two nearly identical polypeptides expressed in Spirulina can cooperatively form three-dimensional mosaic particles, where the two polypeptides contribute to the "tiling" that forms the VLP capsid. Another example of this is to express a gene encoding one of the RNA phage particles genetically linked to a viral capsid protein or polypeptide such as WHcAg, and a second gene having a native viral protein. This makes it possible to avoid the steric collisions that can occur when a bulky hybrid partner is attached to all particles. The particles formed in this example can self-assemble and form further higher-order structures.

[0180] In some embodiments, the recombinant Spirulina comprises a fusion protein comprising at least one exogenous polypeptide and a trimerization domain of a specific protein that naturally exists as a trimer. Exemplary proteins comprising a trimerization domain are described below. For example, the HA protein from influenza virus (either the entire external domain or the minimal stem region) naturally forms trimers, and the interface between monomeric subunits is thought to be an important immunodominant epitope. The fusion protein (F protein) from respiratory syncytial virus (RSV) is an absolute trimer. Similarly, tumor necrosis factor alpha (TNFα) and the ligand of CD40 (CD40L) are absolute trimers. Recombinant Spirulina comprising a fusion protein comprising at least one exogenous antigenic epitope and the trimerization domain of any of these proteins is encompassed by the present disclosure. In an exemplary embodiment, the inventors genetically linked the WHcAg monomer with several coiled-coil domains to facilitate trimer formation. Both of these facilitate trimer formation, and the spike domain of WHcAg positions bulky domains such as influenza HA away from potential steric interference. The inventors used a trimerization-inducing derivative of the Saccharomyces cerevisiae transcription factor GCN4, a parallel trimeric coiled-coil, and related structures based on CGN4 with mutations derived from the HIV GP41 trimer structure. The inventors genetically linked these two trimers to WHcAg and several RNA bacteriophages using linker sequences of various lengths.

[0181] In some embodiments, the recombinant Spirulina comprises a fusion protein comprising at least one exogenous polypeptide and a viral protein capable of forming virus-like particles (VLPs). In these embodiments, the exogenous polypeptide is expressed in Spirulina as a protein macromolecular particle such as a virus-like particle (VLP). VLPs mimic the overall structure of virus particles by retaining the three-dimensional structure of the virus and do not contain infectious material. VLPs have the ability to stimulate responses mediated by B cells and T cells. Viral proteins can spontaneously form VLPs when expressed in a heterologous system such as Spirulina. Thus, in some embodiments, at least one exogenous antigenic epitope is fused to the VLP-forming viral protein. This fusion protein forms VLPs when expressed in Spirulina.

[0182] In some embodiments, by tethering an exogenous polypeptide to a VLP-forming viral protein (or other protein that forms a tertiary structure), it becomes possible to express hundreds of monomeric proteins per VLP (for example, when using hepatitis VLPs, 180 to 240 monomeric proteins per VLP). This enables the expression of billions of VLPs per cell. In some embodiments, an exogenous polypeptide is tethered to a VLP-forming viral protein. In some embodiments, an exogenous antigenic epitope is tethered to a VLP-forming viral protein at the C-terminus or N-terminus of the viral protein. That is, the amino acid sequence of the polypeptide is placed behind the amino acid sequence of the viral protein (attachment of the viral protein at the N-terminus of the antigen or epitope), or in front of the amino acid sequence of the viral protein (attachment of the viral protein at the N-terminus of the antigen or epitope). In some other embodiments, an exogenous antigenic epitope is inserted into a VLP-forming viral protein. For example, at least one exogenous polypeptide can be inserted between two adjacent amino acid residues of the viral protein. Alternatively, a region of the viral protein that is not required for VLP formation can be replaced by inserting at least one exogenous polypeptide into that region. Throughout this disclosure, when at least one exogenous polypeptide is described as being included in or present within a VLP, it refers to a fusion protein comprising at least one exogenous polypeptide described herein and a VLP-forming viral protein.

[0183] Viral proteins that can be used to form the polypeptide-containing VLPs of the present disclosure include capsid proteins of various viruses. Exemplary capsid proteins that can be used in the VLPs of the present disclosure include capsid proteins of viruses derived from Hepadnaviridae, papillomavirus, picornavirus, calicivirus, rotavirus, and reovirus. In some embodiments, the viral protein that can be used to form VLPs that express the polypeptides, antigens or antigen epitopes of the present disclosure includes the Hepadnaviridae core antigen (HBcAg). An exemplary HBcAg that can be used in the present disclosure is the woodchuck hepatitis virus core antigen (WHcAg) derived from the woodchuck hepatitis virus (also referred to herein as the woodchuck hepatitis virus).

[0184] In some embodiments, the recombinant Spirulina comprises a fusion protein comprising at least one exogenous therapeutic agent and a protein that forms a trimer. In some embodiments, the trimer-forming protein is derived from an RNA bacteriophage or Helicobacter pylori. In some embodiments, the trimer-forming protein is the Helicobacter pylori ferritin protein. At least one exogenous polypeptide, antigen, or antigen epitope can be attached to the C-terminus or N-terminus, or can be attached within the body of the protein that forms the trimer. In some embodiments, these proteins that form trimers include, but are not limited to, GCN4 polypeptides or fragments, mutants or variants thereof derived from S. cerevisiae and / or HIV.

[0185] In some embodiments, the recombinant Spirulina comprises a fusion protein comprising at least one exogenous polypeptide, antigen, or antigen epitope and a scaffold protein. As used herein, the term "scaffold protein" refers to a protein that acts as a docking protein and facilitates the interaction between two or more proteins. For example, a fusion protein comprising at least one exogenous polypeptide and a scaffold protein may facilitate the binding of the exogenous polypeptide to a receptor on the cell surface. In some embodiments, the exogenous polypeptide is ligated to the scaffold protein at the C-terminus or N-terminus of the scaffold protein. In some other embodiments, the exogenous polypeptide is inserted into the scaffold protein (e.g., within the body of the scaffold protein). For example, at least one exogenous polypeptide can be inserted between two adjacent amino acid residues of the scaffold protein. Alternatively, a region of the scaffold protein that is not required for the scaffold function can be replaced by inserting at least one polypeptide into that region. For example, in a recombinant Spirulina comprising multiple copies of an exogenous polypeptide and a scaffold protein, the exogenous antigen epitope and the scaffold protein can be arranged in any one of the following patterns: (E)n-(SP), (SP)-(E)n, (SP)-(E)n-(SP), (E)n 1 -(SP)-(E)n 2 , (SP)-(E)n 1 -(SP)-(E)n 2 , and (SP)-(E)n 1 -(SP)-(E)n 2 -(SP) (in the pattern, E is an exogenous polypeptide, SP is a scaffold protein, and n, n 1 , and n 2 represent the number of copies of the exogenous polypeptide). It is understood that the recombinant Spirulina may comprise more than one exogenous polypeptide and one or more scaffold proteins, and the multiple exogenous polypeptides and the scaffold protein can be arranged in the various patterns described above.

[0186] In some embodiments, the recombinant Spirulina can comprise a fusion protein comprising at least one exogenous polypeptide, a scaffold protein, a VLP-forming viral protein, and / or a trimer-forming protein. In these embodiments, at least one exogenous polypeptide can be tethered or inserted into one or more of the above scaffold proteins, and a fusion protein comprising a scaffold protein and at least one exogenous polypeptide can be tethered or inserted into a VLP-forming viral protein and / or a trimer-forming protein.

[0187] Exemplary scaffold proteins include the oligomerization domain of C4b-binding protein (C4BP), cholera toxin b subunit, or the oligomerization domain of an extracellular matrix protein. In some embodiments, the scaffold protein used in the oral antigenic compositions of the present disclosure comprises a sequence derived from the oligomerization domain of C4BP selected from the group consisting of: SAGAHAGWETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL (SEQ ID NO: 1) , WVIPEGCGHVLAGRKVMQCLPNPEDVKMALEVYKLSLEIELLEIQRDKARDPAMD (SEQ ID NO: 2), WEYAEGCEQVVKGKKLMQCLPTPEEVRLALEVYKLYLEIQKLELQKDEAKQA (SEQ ID NO: 3), and WVVPAGCEQVIAGRELTQCLPSVEDVKMALELYKLSLEIELLELQKDKAKKSTLESPL (SEQ ID NO: 4)

[0188] In some embodiments, the exogenous polypeptide binds to a target or target molecule. In some embodiments, the multimer of the exogenous polypeptide binds to the target or target molecule with a higher affinity than the monomer or a smaller multimer. For example, a heptameric VHH can bind to the target with a higher affinity than a dimer of the same exogenous polypeptide. In some embodiments, the multimer is a heteromer. In some embodiments, the different components of the heteromer bind to different targets or target molecules.

[0189] The recombinant Spirulina present in the non-injection composition of the present disclosure may contain multiple copies of at least one exogenous polypeptide. In some embodiments, the recombinant Spirulina expresses an exogenous polypeptide or the fusion protein described above, wherein the exogenous polypeptide contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of at least one exogenous polypeptide per single molecule of the exogenous antigen. In some embodiments, the recombinant Spirulina expresses an exogenous polypeptide, wherein the exogenous polypeptide contains 1-5, 2-5, 2-4, 3-6, 3-8, or 4-5 copies of at least one exogenous polypeptide per single molecule of the exogenous antigen. In some embodiments, the recombinant Spirulina contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 copies of at least one exogenous polypeptide per single molecule of the exogenous antigen. In some embodiments, the recombinant Spirulina expresses an exogenous polypeptide, wherein the exogenous polypeptide contains 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, 5-10, 5-15, 5-20, 5-25, 5-30, 5-40, 5-50, 10-25, 10-50, 10-60, 15-30, 15-45, 15-60, 20-50, 20-60, 20-70, 25-50, 25-60, 30-60, or 2-100 copies of at least one exogenous polypeptide epitope per single molecule of the exogenous polypeptide. In some embodiments, the recombinant Spirulina cells may contain thousands of copies of at least one exogenous polypeptide (e.g., by expressing the corresponding nucleic acid sequence in the cell by one or more vectors or by integration into the Spirulina genome).

[0190] The recombinant Spirulina present in the non-injection composition of the present disclosure may contain multiple copies of nucleic acid sequences encoding at least one exogenous polypeptide. The multiple copies of nucleic acid sequences encoding at least one exogenous polypeptide may be integrated into the genome of Spirulina or may be present on one or more vectors introduced into Spirulina. In some embodiments, the recombinant Spirulina contains 2 to 100 copies of nucleic acid sequences encoding at least one exogenous polypeptide. In some embodiments, the recombinant Spirulina contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of nucleic acid sequences encoding at least one exogenous polypeptide that is integrated into its genome or present on one or more vectors. In some embodiments, the recombinant Spirulina contains 1 to 5, 2 to 5, 2 to 4, 3 to 6, 3 to 8, or 4 to 5 copies of nucleic acid sequences encoding at least one exogenous polypeptide that is integrated into its genome or present on one or more vectors. In some embodiments, the recombinant Spirulina contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 copies of nucleic acid sequences encoding at least one exogenous polypeptide that is integrated into its genome or present on one or more vectors. In some embodiments, the recombinant Spirulina contains 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, 1 to 50, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 40, 5 to 50, 10 to 25, 10 to 50, 10 to 60, 15 to 30, 15 to 45, 15 to 60, 20 to 50, 20 to 60, 20 to 70, 25 to 50, 25 to 60, or 30 to 60 copies of nucleic acid sequences encoding at least one exogenous polypeptide that is integrated into its genome or present on one or more vectors.

[0191] In some embodiments, at least one exogenous polypeptide of multiple copies is linked in tandem, i.e., the second copy follows immediately after the first copy without being separated by any amino acids, and the third copy follows immediately after the second copy, and so on. In some embodiments where the recombinant Spirulina contains more than one exogenous polypeptide, the individual polypeptides can similarly be linked in tandem with other antigenic epitopes. For example, in recombinant Spirulina containing E1 and E2 as exogenous polypeptides, these two polypeptides can be linked in tandem as follows: (E1E2)x, (E2E1)x, (E1)x(E2)y, (E1)x(E2)y(E1)z, (E2)x(E1)y(E2)z. Here, x, y, and z represent the number of copies of the polypeptide. Similar arrangement patterns are contemplated for more than two exogenous polypeptides.

[0192] In some embodiments, at least one exogenous polypeptide of multiple copies present within a protein can be separated by a spacer sequence. In some embodiments, multiple copies of the exogenous polypeptide can be separated by a spacer sequence of about 1 to about 50 amino acids. For example, in some embodiments, multiple copies of the exogenous polypeptide can be separated by a spacer sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 amino acids. In these embodiments, when there are more than two copies of the exogenous polypeptide, it is understood that some copies can be linked in tandem and some copies can be separated by a spacer sequence. For example, in recombinant Spirulina containing multiple copies of E1 as at least one exogenous polypeptide, multiple copies of this epitope can be separated as follows: (E1)x-S-(E1)y, (E1)(E1)x-S-(E1)y, (E1)x-S-(E1)y-S-(E1)z. Here, S represents the spacer sequence, and x, y, and z represent the number of copies of the exogenous polypeptide. When multiple spacer sequences are present, the lengths and / or amino acid sequences of these sequences may be the same or different.

[0193] In embodiments where the recombinant Spirulina contains a protein comprising more than one exogenous polypeptide, the first exogenous polypeptide can be separated from other polypeptide epitopes by a spacer sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 amino acids. When multiple copies of each exogenous polypeptide are present, some copies can be linked tandemly with other polypeptides while some copies can be separated by spacer sequences; alternatively, all copies of one polypeptide can be linked tandemly followed by a spacer sequence followed by all copies of a second polypeptide, and so on. For example, in recombinant Spirulina containing E1 and E2 as exogenous polypeptides, the two polypeptides can be arranged as follows: (E1)x-S-(E2)y, (E2)x-S-(E1)y, (E1)x-S-(E2)y-S(E1)z-S-(E2)v, (E1)x-S-(E2)y(E1)z, (E1)x-S-(E2)y-S-(E1)z, (E2)x-S-(E1)y(E2)z, etc. Here, v, x, y, and z represent the number of copies of the polypeptide.

[0194] In some embodiments, the recombinant Spirulina can contain one or more exogenous polypeptides and multiple copies thereof in the arrangement patterns described immediately above, i.e., without becoming part of or fusing with another protein.

[0195] In some embodiments, the recombinant Spirulina comprises a fusion protein comprising a VLP-forming viral protein or trimer-forming protein and one or more exogenous polypeptides, antigens, and / or antigen epitopes, wherein the exogenous polypeptides, antigens, and / or antigen epitopes and multiple copies thereof (if present) can be arranged in the various patterns described above within the fusion protein. In some other embodiments, the recombinant Spirulina can comprise a fusion protein comprising a scaffold protein and one or more exogenous polypeptides, antigens, and / or antigen epitopes, wherein the exogenous antigen epitopes and multiple copies thereof (if present) can be arranged in the various patterns described above within the fusion protein. In some other embodiments, the recombinant Spirulina can comprise a fusion protein comprising a VLP-forming viral protein, a trimer-forming protein, and / or a scaffold protein, and one or more exogenous polypeptides, antigens, and / or antigen epitopes, wherein the exogenous polypeptides, antigens, and / or antigen epitopes and multiple copies thereof (if present) can be arranged in the various patterns described above within the fusion protein.

[0196] The non-injectable composition provided by the present disclosure comprises recombinant Spirulina, wherein the recombinant Spirulina comprises at least one exogenous polypeptide, small molecule, antigen or epitope as described above.

[0197] Spirulina The non-injectable compositions of the present disclosure contain recombinant Spirulina in a non-viable form. These non-viable Spirulina containing the expressed exogenous polypeptide, small molecule, antigen or epitope are then administered to a subject to elicit an immune response in the subject. In some embodiments, non-viable recombinant Spirulina containing at least one exogenous polypeptide, antigen, or at least one exogenous antigen epitope is prepared by drying a live culture of recombinant Spirulina. Drying methods include heat drying, e.g., drying in an oven; air drying, spray drying, freeze drying, or freeze-drying. Thus, in some embodiments, the non-injectable compositions of the present disclosure comprise a dried biomass of recombinant Spirulina containing at least one exogenous polypeptide, antigen, or at least one exogenous antigen epitope described herein.

[0198] As used herein, "Spirulina" is synonymous with "Arthrospira". The non-injectable compositions of the present disclosure can include any one of the following species of Spirulina: A. amethystine, A. ardissonei, A. argentina, A. balkrishnanii, A. baryana, A. boryana, A. braunii, A. breviarticulata, A. brevis, A. curta, A. desikacharyiensis, A. funiformis, A. fusiformis, A. ghannae, A. gigantean, A. gomontiana, A. gomontiana var. crassa, A. indica, A. jenneri var. platensis, A. jenneri Stizenberger, A. jenneri f. purpurea, A. joshii, A. khannae, A. laxa, A. laxissima, A. laxissima, A. leopoliensis, A. major, A. margaritae, A. massartii, A. massartii var. indica, A. maxima, A. meneghiniana, A. miniata var. constricta, A. miniata, A. miniata f. acutissima, A. neapolitana, A. nordstedtii, A. oceanica, A. okensis, A. pellucida, A. platensis, A. platensis var. non-constricta, A. platensis f. granulate, A. platensis f. minor, A. platensis var. tenuis, A. santannae, A. setchellii, A. skujae, A. spirulinoides f. tenuis, A. spirulinoides, A. subsalsa, A. subtilissima, A. tenuis, A. tenuissima, and A. versicolor.

[0199] Pharmaceutical Compositions and Administration As used herein, the terms "oral composition" or "composition for oral delivery" include compositions administered or delivered to the gastrointestinal tract (e.g., orally, compositions administered to the stomach by a feeding tube, etc.). Any suitable region of the gastrointestinal tract can be targeted by the compositions of the present disclosure.

[0200] In some embodiments, the compositions of the present disclosure are administered via the airway. In some embodiments, the compositions of the present disclosure are administered by inhalation. In some embodiments, the compositions of the present disclosure are administered intranasally. In some embodiments, the compositions of the present disclosure are administered by nebulizer, inhaler, or mist. In some embodiments, the compositions of the present disclosure are delivered as a lyophilized powder resuspended in a powder or liquid.

[0201] In some embodiments, the compositions of the present disclosure are formulated for administration via the airway. In some embodiments, the compositions of the present disclosure are formulated for administration by inhalation. In some embodiments, the compositions of the present disclosure are formulated for intranasal administration. In some embodiments, the compositions of the present disclosure are formulated for administration by nebulizer, inhaler, or mist.

[0202] In some embodiments, the compositions of the present disclosure may include one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. In some embodiments, the pharmaceutically acceptable excipient is sodium bicarbonate.

[0203] In some embodiments, the compositions of the present disclosure may include an adjuvant. As is known in the art, the immunogenicity of a particular composition can be enhanced by using a non-specific stimulator of the immune response known as an adjuvant. Exemplary adjuvants include water-in-oil (W / O) emulsions composed of mineral oil and a surfactant from the mannide monooleate family (e.g., adjuvants of the MONTANIDE™ class) and flagellin adjuvants.

[0204] In some embodiments, the compositions of the present disclosure include from about 0.1% to about 5% total Spirulina biomass. In some embodiments, the compositions of the present disclosure include from about 1 mg to about 50 mg of exogenous antigen epitopes per gram of dry Spirulina biomass. In some embodiments, the compositions of the present disclosure include at least about 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, 750 mg, 1 mg, 5 mg, 10 mg, or 50 exogenous antigen epitopes per gram of dry Spirulina biomass.

[0205] Use of the Composition In some embodiments, the compositions of the present disclosure can be used to reduce the severity of a disease or disorder in a subject in need thereof. In some embodiments, the composition can be used to prevent a disease or disorder in a subject. In some embodiments, the composition can be used to prevent the onset of a disease or disorder in a subject. In some embodiments, the composition can be used to reduce the severity of a disease or disorder in a subject. In some embodiments, the composition can be used to prevent or delay the recurrence of a disease in a subject. In some embodiments, the composition can be used to treat, prevent, or delay the recurrence of cancer in a subject.

[0206] The compositions of the present disclosure can be used as a vaccine. In some embodiments, the composition can be used to induce an immune response in a subject. For example, the composition can be used to induce an immune response against an infectious microorganism, a tumor antigen, or a self-antigen.

[0207] In some embodiments, provided herein is a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject any of the compositions described herein. Without wishing to be bound by theory, it is expected that when the compositions of the present disclosure are administered to a subject, at least one exogenous antigen epitope will be recognized by the subject's immune cells, such as T cells or B cells, thereby activating an immune response against the exogenous antigen epitope. In some embodiments, administration of the compositions described herein can induce a humoral immune response and / or a cellular immune response.

[0208] The compositions of the present disclosure can be administered daily, weekly, twice a week, once every two weeks, monthly, etc. In some embodiments, the compositions of the present disclosure are administered to a subject over a period of about 1 day to about 1 year. In some embodiments, the compositions of the present disclosure are administered to a subject for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months or longer. In some embodiments, the compositions of the present disclosure are administered for consecutive days. In some embodiments, the compositions of the present disclosure are administered for non-consecutive days. In some embodiments, the compositions of the present disclosure are administered once a day. In some embodiments, the compositions of the present disclosure are administered multiple times a day. In some embodiments, the compositions of the present disclosure are administered 2 times a day, 3 times a day, 4 times a day, or more. In some embodiments, the compositions of the present disclosure are administered continuously (e.g., via a feeding tube). In some embodiments, the compositions of the present disclosure are administered with food. In some embodiments, the compositions of the present disclosure are administered when the subject is fasting.

[0209] The compositions of the present disclosure can be administered according to a schedule, for example, a priming dose of the antigenic composition can be administered, followed by one or more additional immunizing doses of the antigenic composition. In some embodiments, the first additional immunizing dose of the antigenic composition can be administered at any time from about 2 weeks to about 10 years after the priming dose. In some embodiments, the first additional immunizing dose of the antigenic composition can be administered at any time from about 2 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, or 5 years after the priming dose. The second additional immunizing dose of the antigenic composition can be administered after the first additional immunizing dose and at any time from about 3 months to about 10 years after the priming dose. In some embodiments, the second additional immunizing dose of the antigenic composition can be administered after the first additional immunizing dose and at about 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, or 5 years after the priming dose. If no specific immunoglobulins are detected or the levels of specific immunoglobulins detected are low in the serum and / or other body fluids of the subject after the second additional immunizing dose, a third additional immunizing dose can be administered as needed.

[0210] In some embodiments, to prime the immune response of a subject, a composition other than the compositions of the present disclosure can be administered prior to the administration of the present composition. In these embodiments, the methods of the present disclosure include administering a composition other than the present antigenic composition as a priming dose, followed by administering the present composition in one or more additional immunizing doses.

[0211] The compositions of the present disclosure can be used to treat and / or prevent a disease or disorder or reduce its severity. In some embodiments, the disease or disorder is selected from the group consisting of, but not limited to, type 1 diabetes, type 2 diabetes, cancer, inflammatory disorders, gastrointestinal diseases, autoimmune diseases or disorders, endocrine disorders, gastroesophageal reflux disease (GERD), ulcers, hypercholesterolemia, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, constipation, and diarrhea.

[0212] The compositions of the present disclosure can be used as vaccines against diseases or infections caused by viruses, bacteria, parasites, or fungi, or to treat and / or prevent them or reduce their severity.

[0213] In some embodiments, the composition can be used as a vaccine against infectious diseases such as tetanus, diphtheria, pertussis, pneumonia, meningitis, campylobacteriosis, mumps, measles, rubella, polio, influenza, hepatitis, chickenpox, malaria, toxoplasmosis, giardiasis, or leishmaniasis, or to treat and / or reduce their severity.

[0214] In some embodiments, the compositions described herein can be used to induce an immune response against, treat, and / or reduce the severity of infectious diseases caused by viruses including, but not limited to, bacteriophages, RNA bacteriophages (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus (IHNV), parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, SARS virus, MERS virus, and SARS-CoV-2 virus.

[0215] In some embodiments, the compositions described herein can be used to induce an immune response against, treat, and / or reduce the severity of an infectious disease caused by IHNV.

[0216] In some embodiments, the compositions described herein can be used to induce an immune response against an infection caused by a parvovirus, such as canine parvovirus, and / or to reduce the severity thereof.

[0217] In some embodiments, the compositions described herein can be used to induce an immune response against an infection caused by a coronavirus, such as ARDS, COVID-19, and / or to reduce the severity thereof.

[0218] In some embodiments, the compositions described herein can be used to induce, treat, and / or reduce the severity of an immune response against an infection caused by bacteria including, but not limited to, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella.

[0219] In some embodiments, the compositions described herein include, but are not limited to, Plasmodium, Trypanosoma, Toxoplasma, Giardia, and Leishmania, Cryptosporidium, parasitic worms: Trichuris spp. (whipworms), Enterobius spp. (pinworms), Ascaris It can be used to induce an immune response against, and / or reduce the severity of, infections caused by parasites including spp. (roundworms), Ancylostoma spp. and Necatro spp. (hookworms), Strongyloides spp. (threadworms), Dracunculus spp. (Guinea worms), Onchocerca spp. and Wuchereria spp. (filarial worms), Taenia spp., Echinococcus spp., and Diphyllobothrium spp. (human and animal tapeworms), Fasciola spp. (liver flukes) and Schistosoma spp. (blood flukes).

[0220] In some embodiments, the compositions described herein can be used to induce an immune response against, and / or reduce the severity of, infections caused by Plasmodium. In some embodiments, the compositions of the present disclosure can be used to induce an immune response against, and / or reduce the severity of, infections caused by Plasmodium selected from the group consisting of P. falciparum, P. malariae, P. ovale and P. vivax.

[0221] In some embodiments, the compositions described herein can be used to induce an immune response against, and / or reduce the severity of, infections caused by fungi including, but not limited to, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma. In some embodiments, the composition can be used to induce an immune response against, and / or reduce the severity of, infections caused by Candida albicans or Candida auris.

[0222] In some embodiments, the compositions described herein can be used to induce an immune response against tumor antigens. In some embodiments, the compositions can be used to induce an immune response against tumor antigens expressed in cancer cells including, but not limited to, breast cancer cells, colon cancer cells, brain cancer cells, pancreatic cancer cells, lung cancer cells, cervical cancer cells, uterine cancer cells, prostate cancer cells, ovarian cancer cells, melanoma cancer cells, lymphoma cancer cells, myeloma cancer cells, and leukemic cancer cells.

[0223] In some embodiments, the compositions described herein can be used to induce an immune response against autoantigens. In some embodiments, the compositions can be used to induce an immune response against autoantigens associated with autoimmune diseases including, but not limited to, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), celiac disease, inflammatory bowel disease, Hashimoto's disease, Addison's disease, Graves' disease, type 1 diabetes, autoimmune thrombocytopenic purpura (ATP), idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), Crohn's disease, multiple sclerosis, and myasthenia gravis.

[0224] In some embodiments, the compositions of the present disclosure are administered orally. In some embodiments, the compositions of the present disclosure are administered via the airway (e.g., intranasally or by inhalation). In some embodiments, the compositions of the present disclosure are administered as Spirulina biomass. In some embodiments, the compositions of the present disclosure are administered as lyophilized Spirulina biomass. In some embodiments, the compositions of the present disclosure are administered as an extract of Spirulina biomass.

[0225] The dosage of the composition can be readily determined by one of ordinary skill in the art, for example, by first identifying an effective dosage to elicit a prophylactic or therapeutic effect. The dosage can be determined from animal tests. A non-limiting list of animals used to test the effectiveness of the vaccine includes guinea pigs, hamsters, ferrets, chinchillas, mice, and cotton rats. The test animals may not be natural hosts for the infectious agent, but can still be useful for testing various aspects of the disease. For example, the composition of the present disclosure, such as a recombinant Spirulina containing VLPs comprising a polypeptide, can be dosed to any of the above animals.

[0226] In some embodiments, administration of the composition of the present disclosure results in a decrease in the load of the infectious agent. In some embodiments, administration of the composition of the present disclosure results in a decrease in the establishment of the infectious agent. In some embodiments, administration of the composition of the present disclosure results in a decrease in the excretion of the infectious agent (e.g., viral excretion). In some embodiments, administration of the composition of the present disclosure results in a decrease in the excretion of the infectious agent. In some embodiments, administration of the composition of the present disclosure results in an increase in excretion over a period (e.g., 24 hours), followed by a decrease in excretion thereafter (e.g., at the 72-hour time point). In some embodiments, administration of the composition results in a reduction in the expression of a biomarker. In some embodiments, the biomarker is a marker of inflammation.

[0227] In some embodiments, administration of the composition of the present disclosure neutralizes or blocks the activity of the target. In some embodiments, administration of the composition of the present disclosure neutralizes or blocks the activity of the target by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%.

[0228] Furthermore, human clinical trials can be conducted to enable those skilled in the art to determine the preferred effective dosage for humans. Such clinical trials are routine and well-known in the art. The effective dosage can be estimated from the dose-response curves derived from in vitro tests, animal tests, and / or clinical trials.

[0229] Method for producing a composition not administered by injection Provided is a method for producing a composition not administered by injection. The method for producing a composition not administered by injection includes the step of introducing into Spirulina a nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigen epitope. In some embodiments, the method for producing a composition not administered by injection includes the step of introducing into Spirulina a polypeptide, antigen, and / or antigen epitope. In some embodiments, the method for producing a composition not administered by injection includes the step of introducing into Spirulina a small molecule.

[0230] Any suitable means for transforming Spirulina can be used in the present disclosure. Exemplary methods for transforming Spirulina to express a heterologous protein are described in U.S. Patent No. 10,131,870, which is hereby incorporated by reference in its entirety.

[0231] In some embodiments, a method of making a composition that does not rely on injection involves introducing an expression vector having a nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigen epitope into Spirulina cells. In some embodiments, the vector is not integrated into the Spirulina genome. In some embodiments, the vector is a high-copy or high-expression vector. In some embodiments, the nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigen epitope is under the control of a strong promoter. In some embodiments, the nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigen epitope is under the control of a constitutive promoter. In some embodiments, the nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigen epitope is under the control of an inducible promoter.

[0232] In some embodiments, a method of making a composition involves introducing a vector having homology arms and a nucleic acid sequence encoding at least one exogenous polypeptide antigen and / or antigen epitope into Spirulina cells (e.g., by homologous recombination).

[0233] In some embodiments, a vector having homology arms and a nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigen epitope can be introduced into Spirulina using electroporation. Electroporation is preferably performed in the presence of a suitable osmotic stabilizer.

[0234] Prior to introducing the vector into Spirulina, Spirulina can be cultured in any suitable medium for growing cyanobacteria, such as SOT medium. SOT medium contains NaHCO 3 1.68 g, K 2 HPO 4 50 mg, NaNO 3 250 mg, K 2 50 4100 mg, NaCl 100 mg, MgSO 4 .7H 2 O, 20 mg, CaCl 2 .2H 2 O 4 mg, FeSO 4 .7H 2 O 1 mg, Na 2 EDTA.2H 2 O 8 mg, A 5 solution 0.1 mL, and 99.9 mL of distilled water. A 5 solution is H 3 BO 3 286 mg, MnSO 4 .5H 2 O)217 mg, ZnSO 4 .7H 2 O 22.2 mg, CuSO 4 .5H 2 O 7.9 mg, Na2MoO 4 .2H 2 O 2.1 mg, and 100 mL of distilled water. Cultivation can be carried out while shaking at a temperature higher than room temperature (for example, 25 - 37 °C) (for example, 100 - 300 rpm) under continuous illumination (for example, 20 - 2,000, 50 - 500, or 100 - 200 μmol of photons m -2 s -1 ). The optical density at 750 nm is a predetermined threshold value (for example, OD 750Once it reaches 0.3 - 2.0, 0.5 - 1.0, or 0.6 - 0.8, the growing cells can be harvested. The harvested cells can be centrifuged and then the volume can be concentrated by resuspending them in a pH balancer and a salt solution. The pH balancer can be any suitable buffer that maintains the pH of the medium between pH 6 and 9, between pH 6.5 and 8.5, or between pH 7 and 8 while maintaining the viability of Spirulina. Suitable pH balancers include HEPES, HEPES - NaOH, sodium phosphate or potassium phosphate buffer, and TES. The salt solution can be NaCl at a concentration between 50 mM and 500 mM, between 100 mM and 400 mM, or between 200 mM and 300 mM. In certain embodiments, 1 - 100 mM of pH balance 1 - 50 mL can be used to neutralize the pH.

[0235] The cells collected by centrifugation can be washed with an osmotic stabilizer and, optionally, a salt solution (e.g., 0.1 - 100 mM NaCl, 1 - 50 mL). Any amount of the culture can be concentrated by centrifugation. In certain embodiments, 5 - 500 mL of the culture can be centrifuged. The osmotic stabilizer can be any type of osmotic balancer that stabilizes the integrity of Spirulina cells during electroporation. In certain embodiments, the osmotic stabilizer can be a sugar such as glucose or sucrose (e.g., 0.1 - 25 w / v%). In certain embodiments, the osmotic stabilizer can be a simple polyol including glycerin, glycerine, or glycerol (e.g., 1 - 25 v / v%). In certain embodiments, the osmotic stabilizer can be a polyether including polyethylene glycol (PEG), poly(oxyethylene), or poly(ethylene oxide) (PEO) (e.g., 0.1 - 20 w / v%). PEG or PEO can have any molecular weight from 200 to 10,000, from 1000 to 6000, or from 2000 to 4000. In certain embodiments, a pH balancer or buffer can be used instead of or in addition to the osmotic stabilizer.

[0236] Vectors having the same arm and nucleic acid sequences encoding at least one exogenous polypeptide, antigen, and / or antigen epitope can be cultured as described above and introduced into Spirulina cells washed with an osmotic stabilizer. Electroporation can be used to introduce the vector.

[0237] Electroporation can be carried out in an electroporation cuvette of 0.1 cm, 0.2 cm or 0.4 cm, between 0.6 kV / cm and 10 kV / cm, between 2.5 kV / cm and 6.5 kV / cm, or between 4.0 kV / cm and 5.0 kV / cm; between 1 μF and 100 μF, between 30 μF and 70 μF, or between 45 μF and 55 μF; and between 10 mΩ and 500 mΩ, between 50 mΩ and 250 mΩ, or between 90 mΩ and 110 mΩ. In some embodiments, electroporation can be carried out at 4.5 kV / cm, 50 μf, and 100 mΩ.

[0238] After electroporation, the cells can be grown in the presence of one or more antibiotics selected based on the resistance conferred through successful transformation with the plasmid. The culture after electroporation can be carried out with a reduced illumination level (e.g., 5 - 500, 10 - 100, or 30 - 60 μmol photons m -2 s -1 )). The culture can also be carried out with shaking (e.g., 100 - 300 rpm). The level of antibiotic in the medium can be between 5 μg / mL and 100 μg / mL. The culture after electroporation can be continued for 1 - 5 days or longer. The successful transformants identified by antibiotic resistance can be selected on plates supplemented with 0.1 - 2.0 μg of the appropriate antibiotic or in 5 - 100 mL of SOT medium over a period of one week to one month.

[0239] The vector used in the method can be a plasmid, bacteriophage, or viral vector into which at least one exogenous polypeptide, antigen, and / or nucleic acid sequence encoding an antigen can be inserted or cloned. The vector can contain one or more specific sequences that enable recombination at a specific desired site in the chromosome of Spirulina. These specific sequences can be homologous to sequences present in wild-type Spirulina. The vector system can include a single vector or plasmid, two or more vectors or plasmids, some of which can increase the efficiency of targeted mutagenesis or translocation. The selection of the vector generally depends on the compatibility of the vector with the Spirulina cells into which it is introduced. The vector can contain a reporter gene such as green fluorescent protein (GFP), and the reporter gene can also be fused in-frame with one or more of the encoded antigen epitopes or can be expressed separately. The vector can also include a positive selection marker such as an antibiotic resistance gene that can be used to select appropriate transformants. The vector can also include a negative selection marker such as the type II thioesterase (tesA) gene or the Bacillus subtilis structural gene (sacB). The use of a reporter or marker enables the identification of cells that have been successfully transformed with the vector.

[0240] In some embodiments, the vector contains one or two homologous arms that are homologous to the DNA sequence of the Spirulina genome adjacent to the targeted locus. The sequence of the homologous arm can be partially or completely complementary to the region of the Spirulina genome adjacent to the targeted locus.

[0241] The homologous arms can be of any length that enables site-specific homologous recombination. The homologous arms can be of any length between about 2000 bp and 500 bp. For example, the homologous arms can be about 2000 bp, about 1500 bp, about 1000 bp, or about 500 bp. In some embodiments having two homologous arms, the lengths of the homologous arms may be the same or different. Thus, each of the two homologous arms can be of any length between about 2000 bp and 500 bp. For example, each of the two homologous arms can be about 2000 bp, about 1500 bp, about 1000 bp, or about 500 bp.

[0242] A targeted locus in the Spirulina genome is modified by homologous recombination by a part of the vector adjacent to one homologous arm or flanked by two homologous arms. The modification can be a change in the length of the targeted locus, including nucleotide deletions or nucleotide additions. The addition or deletion can be of any length. Also, due to the modification, the nucleotide sequence within the targeted locus can change without a change in length. The targeted locus can be any part of the Spirulina genome, including coding regions, non-coding regions, and regulatory sequences.

Example

[0243] (Example 1) Oral Spirulina-VHH provides complete protection against Campylobacter Spirulina expressing monomeric VHH

[0244] 10 in mice 7Campylobacter jejuni was inoculated. A vector expressing a monomeric VHH antibody targeting Campylobacter was transfected into Spirulina. After growing Spirulina to enable the expression of the monomeric VHH antibody, Spirulina was dried and 200 μl of PBS and 10% Spirulina biomass (13 mg) were administered to Campylobacter-infected mice by daily gavage for 5 days. 13 mg of Spirulina contains 425 μg of monomeric VHH per dose. As a control, mice were administered daily gavage of 1) PBS, 2) wild-type Spirulina, and 3) Spirulina expressing an irrelevant VHH.

[0245] As shown in Figure 1A, 100% of Campylobacter-infected mice treated with any one of the control treatments presented diarrhea. In contrast, none of the mice administered Spirulina expressing monomeric anti-Campylobacter VHH presented diarrhea. Furthermore, at the time point 7 days after inoculation, mice treated with Spirulina expressing monomeric anti-Campylobacter VHH demonstrated a 4-log reduction in Campylobacter excretion (Figure 1B).

[0246] (Example 2) Spirulina expressing trimeric VHH Oral Spirulina-VHH has anti-inflammatory activity in Campylobacter infection. In mice, 10 8Mice were inoculated with Campylobacter jejuni. A vector expressing a trimeric VHH antibody targeting Campylobacter was transfected into Spirulina. After growing Spirulina to enable the expression of the trimeric VHH antibody, the Spirulina was dried and 400 μl of PBS + 0.5% Spirulina biomass (1.3 mg) was administered to Campylobacter-infected mice via gavage daily for 3 days. 1.3 mg of Spirulina contains 19 μg of trimeric VHH per dose. As a control, mice were administered daily gavage of Spirulina expressing an irrelevant VHH.

[0247] As shown in Figure 2A, the expression of fecal lipocalin, a marker of inflammation, was reduced in mice treated with Spirulina expressing trimeric anti-Campylobacter VHH compared to the control, and in fact, fecal lipocalin in these treated mice closely resembled that of uninfected mice. Furthermore, Figure 2B demonstrates that treatment of infected mice with Spirulina expressing trimeric anti-Campylobacter VHH prevents the infiltration of myeloid cells into the lamina propria of the gastrointestinal mucosa.

[0248] (Example 3) Preventive effect of Spirulina-VHH in mice challenged with C. jejuni strain 81-176 Test substances:

[0249] Spirulina SP257 strain (irrelevant VHH)

[0250] Spirulina SP526 strain (anti-C. jejuni VHH FlagV6)

[0251] Spirulina SP651 strain (anti-C. jejuni VHH FlagV6)

[0252] A mouse model of C. jejuni infection was used to evaluate the prophylactic efficacy of anti-C. jejuni VHH expressed in Spirulina [Giallourou et al]. Spirulina strains expressing either VHH FlagV6 (SP526), protease-resistant FlagV6 (FlagV6-F23) (SP806), or an irrelevant VHH (SP257) were tested. Biomass was prepared by spray-drying a 4% Spirulina-VHH biomass resuspension in a solution containing 2% trehalose.

[0253] To prepare for C. jejuni infection, 21-day-old C57BL / 6 female mice were treated with vancomycin 48, 24, and 12 hours prior to treatment. On day 0, inoculum of 10 8 C. jejuni strain 81-176 resuspended in PBS was administered to the mice. Food and water were provided ad libitum throughout the study.

[0254] To determine how well mice tolerated Spirulina-VHH administered by gavage, a high-dose three-dose regimen was tested. Spirulina-VHH was resuspended in PBS and 400 μL of the slurry was delivered by oral gavage 90 minutes before and 24 and 48 hours after C. jejuni inoculation. Mice were divided into four different groups: · 13.3 mg of Spirulina-VHH (670 mg / kg) containing an irrelevant VHH, · 13.3 mg of anti-Campylobacter VHH (SP651) on a trimer scaffold, · 13.3 mg of anti-Campylobacter VHH (SP737) on a pentamer scaffold, · A control group treated with PBS. Compared with the infection control group treated with PBS, all mice treated with Spirulina-VHH demonstrated non-specific flushing of C. jejuni into feces at the 24-hour time point, followed by a reduction in bacterial load at 48 and 72 hours (data not shown). No adverse events were observed in any of the mice at this dose.

[0255] To identify the dosing regimen of Spirulina-VHH that confers a specific anti-Campylobacter effect, mice were tested as follows: · Administration of a single 400 μL dose of SP561 (equivalent to 13.3 mg of Spirulina-VHH per dose), a 5% Spirulina-VHH powder w / v resuspended in PBS, by gavage 1.5 hours before inoculation; · Administration of three 400 μL doses of SP561 (equivalent to 1.33 mg of Spirulina-VHH per dose), a 0.5% Spirulina-VHH powder w / v resuspended in PBS, by gavage 1.5 hours before, and 24 and 48 hours after inoculation; · Administration of a single 400 μL dose of SP257 (equivalent to 1.33 mg of Spirulina-VHH per dose), a 0.5% Spirulina-VHH powder w / v of irrelevant VHH resuspended in PBS, by gavage 1.5 hours before, and 24 and 48 hours after inoculation; · Administration of three 400 μL doses of SP257 (equivalent to 1.33 mg of Spirulina-VHH per dose), a 0.5% Spirulina-VHH powder w / v of irrelevant VHH resuspended in PBS, by gavage 1.5 hours before, and 24 and 48 hours after inoculation; · Control mice received PBS gavage. There were five mice in each experimental group. Three days after Campylobacter inoculation, the sham-infected mice (PBS gavage) showed significant weight loss compared to the uninfected mice (Figure 3A). The infected mice treated with SP257 showed a similar weight loss. In contrast, the infected mice treated according to both dosing regimens with SP651, which expresses an anti-Campylobacter binding protein on a trimeric scaffold, showed weight gain equivalent to or significantly better than that of the uninfected mice (Figure 3A).

[0256] The ceca from all animals were examined at necropsy 72 hours after infection. Tissue sections were processed and scored blindly by a histopathologist on a scale of 0 - 24. Each section was evaluated for submucosal edema, crypt hyperplasia, goblet cell depletion, epithelial integrity, mucosal mononuclear cell infiltration, and submucosal PMN and mononuclear cell infiltration. Animals treated with either SP651 or SP257 obtained scores significantly lower than those of the infected controls and closer to those of the uninfected controls (Figure 3B). These results suggested that Spirulina itself had a positive effect on reducing the histopathology of C. jejuni-infected animals. Without wishing to be bound by theory, this effect may be due to the inherent health benefits of Spirulina (i.e., Spirulina is considered a superfood).

[0257] Spirulina-VHH was well tolerated and no adverse effects were observed in mice treated with the highest dose of 13.3 mg of Spirulina-VHH.

[0258] In a second experiment, a single 1.33 mg dose of Spirulina-VHH (SP651) was used to determine the efficacy of an anti-C. jejuni VHH strain compared to Spirulina (SP257) expressing an irrelevant VHH.

[0259] One and a half hours before infection with C. jejuni, mice were administered a single 400 μL dose containing 1.33 mg of Spirulina-VHH in PBS. Four cohorts, each containing five mice, were treated as follows: · Non-infected, · Infected and treated with gavage of PBS, · Infected and treated with gavage of SP257, · Infected and treated with gavage of SP651. Treatment with this single prophylactic dose of Spirulina containing anti-Campylobacter VHH was sufficient to significantly accelerate the clearance of Campylobacter at 24 hours post-infection, as measured by fecal Campylobacter CFU, and was sufficient to reduce Campylobacter excretion at 72 hours post-infection. (Figure 4B). Post-infection inflammation was measured by fecal lipocalin levels and by flow cytometry quantification of myeloid cell infiltration in the cecal lamina propria. Campylobacter infection caused a significant increase in both inflammatory biomarkers (Figure 4C). This increase was prevented by a single prophylactic dose of SP651 (expressing anti-Campylobacter binding VHH), but not by a prophylactic dose of SP257 (expressing an irrelevant VHH) (Figure 4). Also, as in the previous experiment, the weight loss caused by Campylobacter infection was prevented by a prophylactic dose of SP651. (Figure 4A). However, in this experiment, Spirulina expressing an irrelevant VHH also suppressed infection-related weight loss. This further suggests the potential nutritional benefits of Spirulina itself. Importantly, Spirulina expressing an irrelevant VHH had no effect on either the inflammatory biomarker or the myeloid cell infiltration biomarker in the cecal lamina propria.

[0260] In the third experiment, single doses of serial dilutions of Spirulina-VHH were tested to determine the minimum effective dose (MED) of Spirulina-VHH required to observe positive results. Two Spirulina-VHH strains: SP526 and SP806 were compared. SP526 showed high expression levels of anti-C. jejuni FlagV6 VHH, and SP806 expressed a protease-resistant FlagV6 (Fl agV6-F23) containing two mutations in the VHH reported to confer resistance to chymotrypsin (Hussack et al. 2014). These results were also compared retrospectively with the efficacy of SP651 in the previous experiment.

[0261] One hour and thirty minutes prior to infection with C. jejuni, mice were administered a single 400 μL dose containing 1.33 mg, 0.399 mg or 0.133 mg of Spirulina-VHH (SP526, SP806 or SP651) in PBS. Measurement of body weight fluctuations, as in the previous experiment, showed that Campylobacter caused a lack of weight gain at 72 hours post-infection. Treatment with each of the three Spirulina-VHH strains suppressed this loss at the 1.33 mg dose (Figure 5A). In this assay, the minimum effective dose (MED) for SP526 was 0.133 mg (6.7 mg / kg), the MED for SP806 was 0.399 mg (20 mg / kg), and the MED for SP651 was 1.33 mg (67 mg / kg).

[0262] Measurement of fecal Campylobacter CFU, as in the previous experiment, showed that all three Spirulina-VHH strains accelerated Campylobacter flushing at 24 hours post-treatment and reduced long-term excretion at 72 hours post-treatment (Figure 5B). Again, in this case, VHH expression levels and protease resistance independently increased efficacy, and both SP526 and SP806 showed an MED of 0.399 mg (20 mg / kg) in this assay.

[0263] Inflammatory biomarkers - fecal lipocalin and myeloid cell infiltration into the cecal lamina propria - showed that all three strains, as in the previous experiment, suppressed intestinal inflammation after Campylobacter infection (Figure 6). The protease-resistant strain (SP806) resulted in a significant reduction in both lipocalin-2 levels and myeloid cells infiltrating the lamina propria. The MED for all three strains was 0.399 mg (20 mg / kg), and partial efficacy was observed at 0.133 mg (6.7 mg / kg).

[0264] The ceca from all animals were examined at necropsy 72 hours after infection. Tissue sections were processed and scored by a histopathologist in a blinded manner as previously described. Only the groups that demonstrated a significant reduction in histopathology compared to the infection control were treated with 1.33 mg (67 mg / kg) of SP526. Below this dose, or in groups treated with different Spirulina-VHH (SP806 or SP651), the positive effect of the treatment was determined using other measures of efficacy (i.e., bacterial excretion, inflammatory biomarkers, etc.).

[0265] Conclusion: Administration of all Spirulina-VHH strains expressing anti-C. jejuni VHH FlagV6 resulted in favorable outcomes for the treatment of C. jejuni-infected mice with a single dose of 1.33 or 0.399 mg of Spirulina-VHH. These mice had better weight gain and lower levels of inflammatory markers compared to untreated mice.

[0266] No adverse events were observed up to the highest biomass dose administered in these experiments. This drug material was well tolerated, and no signs of toxicity were observed.

[0267] The most significant new observation made using the Grassi model is that the minimum effective dose was 0.399 mg of dried Spirulina-VHH. A single oral dose administered by gavage nutrition 90 minutes before Campylobacter inoculation was sufficient to prevent infection-related weight loss, to reduce Campylobacter fecal excretion on day 3, and to maintain control (baseline) levels of both molecular and cellular measures of intestinal inflammation related to infection (fecal calprotectin and myeloid cell infiltration into the intestinal mucosa lamina propria).

[0268] (Example 4) Effect of post-challenge treatment with anti-Campylobacter Spirulina-VHH in mice challenged with C. jejuni strain CG8421 The SP1182 construct is described in FIGS. 7 and 8. This fusion protein is a camelid VH that binds to the flagellin protein flaA from C. jejuni containing H FLAGV6-F23. Since the SP1182 fusion protein does not contain a targeting protein, it remains in the cytoplasm of Spirulina cells.

[0269] A mouse Campylobacter challenge experiment was conducted to test the efficacy of orally delivered SP1182 administered in treatment modalities.

[0270] Twenty-one-day-old C57BL / 6 mice were subjected to a 48-hour vancomycin conditioning regimen and then challenged with 10 8 CFU of C. jejuni CG8421 (in PBS). Food and water were available ad libitum throughout the test. Three cohorts, each containing five mice, were treated as follows beginning 24 hours after Campylobacter challenge: · Two treatment doses of 67 mg / kg of SP1182 at 24 and 48 hours post-challenge, · Three treatment doses of 67 mg / kg of SP1182 at 24, 36, and 48 hours post-challenge, · Two doses of 67 mg / kg of wild-type Spirulina (SP3) at 24 and 48 hours · Three doses of 67 mg / kg of wild-type Spirulina (SP3) at 24, 36, and 48 hours.

[0271] Fecal Campylobacter excretion was measured at 40 and 72 hours post-infection. At 40 hours, there was a significant (p<0.05) burst of Campylobacter elimination only in the three-dose cohort that received SP1182 at 36 hours (Figure 9). At 72 hours post-infection, there was a significant (p<0.05) reduction in fecal Campylobacter excretion. Furthermore, there was a significant (p<0.05) reduction in fecal lipocalin (a measure of inflammation) only in the cohort of mice that received three doses of SP1182 (Figure 10). Overall, these results were very similar to the effects of a single pre-inoculation (prophylactic) dose of SP1182.

[0272] Ceca from all animals were examined at necropsy 72 hours after infection. Tissue sections were processed and scored blindly by a histopathologist on a scale of 0 - 24. Each section was evaluated for submucosal edema, crypt hyperplasia, goblet cell depletion, epithelial integrity, mucosal mononuclear cell infiltration, and submucosal PMN and mononuclear cell infiltration. There was no treatment group that showed a reduction in histopathology, and all treatment groups received scores similar to the C. jejuni-infected control.

[0273] (Example 5) Encapsulation by Spirulina protects polypeptides in the stomach To demonstrate the protective effect of Spirulina on polypeptides, Spirulina was transfected with anti-Campylobacter VHH and expressed. These Spirulina were incubated overnight in a simulated gastric environment (pH 3; pepsin at 2,000 U / ml). Samples were collected at 0 minutes, 5 minutes, 60 minutes, and overnight. As shown in Figure 11A, the VHH protein encapsulated within Spirulina could be detected after overnight treatment, while that of the purified VHH could not be detected after a 5-minute exposure to the simulated gastric environment. Figure 11B shows microscopic images of Spirulina expressing anti-Campylobacter VHH at time 0 and overnight, and the Spirulina maintained their integrity in the simulated gastric environment.

[0274] (Example 6) The polypeptide expressed in Spirulina is stable in the dry biomass for a long time To test the effect of long-term storage of the dry biomass on polypeptide stability, Spirulina expressing monomeric anti-Campylobacter VHH was spray-dried and stored at 1) 1 month, 27 °C, 2) 3 months, 27 °C, 3) 1 month, 42 °C, or 4) 3 months, 42 °C. At various time points, the VHH was purified from the Spirulina and tested for binding activity. As shown in Figure 12, no decrease in the biological activity of the anti-Campylobacter VHH was observed due to long-term incubation at high temperature.

[0275] (Example 7) Preclinical efficacy of multiple doses in mice challenged with Campylobacter Test substance:

[0276] Spirulina SP651 strain (expressing anti-C. jejuni VHH FlagV6)

[0277] Spirulina strain SP806 (expressing anti-C. jejuni VHH FlagV6-F23)

[0278] Spirulina strain SP257 (expressing an irrelevant VHH)

[0279] Spirulina strain SP526 (expressing anti-C. jejuni VHH FlagV6)

[0280] Using a mouse model of C. jejuni infection developed at the Institute Research in Biomedicine in Switzerland, the efficacy of prophylactic treatment with anti-C. jejuni VHH-expressing Spirulina was evaluated. Several Spirulina strains expressing either anti-C. jejuni VHH FlagV6, a protease-resistant form of FlagV6 (FlagV6-F23) (Hussack et al. 2014; Riazi et al. 2013), or an irrelevant VHH were tested. Biomass was prepared by spray drying a 3% Spirulina biomass resuspension in a solution containing 2% trehalose. To prepare for C. jejuni infection, 21-day-old C57BL / 6 mice were treated with vancomycin 48 - 12 hours before treatment. On day 0, 10 8 of C. jejuni, strain 81-176 inoculum was administered to the mice.

[0281] To determine how well mice tolerated Spirulina administered by gavage, two dosing regimens were tested: 1) a single 400 μL dose of 5% Spirulina powder w / v resuspended in phosphate buffered saline (PBS) (equivalent to 12 mg of Spirulina per dose), given by gavage 1.5 h before inoculation; 2) three 400 μL doses of 0.5% Spirulina powder w / v resuspended in PBS (equivalent to 1.2 mg of Spirulina per dose), given by gavage 1.5 h before, and 24 and 48 h after inoculation. Under both regimens, infected mice treated with Spirulina (either SP257 or SP651) showed weight gain similar to that of the uninfected control group (Figure 16). Since no adverse effects were observed in mice treated with the highest dose of 12 mg of Spirulina, Spirulina was considered to be well tolerated.

[0282] The efficacy of an anti-C. jejuni VHH strain (SP651) was determined using a single 1.2 mg dose of Spirulina, compared to Spirulina expressing an irrelevant VHH (SP257). Mice were given a single 400 μL dose containing 1.2 mg of Spirulina in PBS 1.5 h before infection with C. jejuni. Compared to untreated infected mice, mice administered anti-C. jejuni Spirulina demonstrated good weight gain, an increase in excretion at 24 h followed by a decrease at 72 h, and a reduction in the levels of inflammatory biomarkers (Figure 17A–C). Spirulina containing the irrelevant VHH had little or no effect on excretion and reduction of inflammatory biomarkers.

[0283] The single - dose incremental dilution of Spirulina was tested to determine the limiting amount of Spirulina required to observe positive results. Three Spirulina strains expressing different types of anti - C. jejuni FlagV6 VHH were compared for efficacy. Notably, SP526 was selected for its high expression level of anti - C. jejuni FlagV6 VHH, and SP806 was identical to SP651 except that it contained two mutations (Hussack et al. 2014) of the VHH that have been reported to confer resistance to chymotrypsin. One hour and a half before infection with C. jejuni, mice were administered a single 400 μL dose containing 1.2 mg, 0.36 mg, or 0.12 mg of Spirulina in PBS. All three strains showed good efficacy at the 1.2 mg dose and varying degrees of reduced efficacy at the 0.36 mg and 0.12 mg doses. Mice treated with SP526 showed the best weight gain, while SP806 reduced excretion at the intermediate dose concentration at the 72 - hour time point (Figures 18A - C). All three strains significantly reduced the levels of inflammation biomarkers at the 0.36 mg dose (Figures 19A - B), but the protease - resistant strain (SP806) resulted in the greatest reduction in both lipocalin - 2 levels and mucosal lamina propria - infiltrating myeloid cells. At the 0.12 mg dose of Spirulina, all strains behaved the same as the C. jejuni - only control, suggesting that this amount was below the effective treatment dose. In short, all Spirulina strains expressing anti - C. jejuni VHH FlagV6 produced positive results for the treatment of C. jejuni - infected mice with a single dose of 1.2 mg of Spirulina - VHH. Compared to untreated mice, these mice had better weight gain and lower levels of inflammatory markers.

[0284] In these experiments, no adverse events were observed up to the highest biomass dose administered. This drug material was well - tolerated and no signs of toxicity were observed.

[0285]

[0286] (Example 8) Effect of Spirulina-VHH in chickens challenged with C. jejuni 81-176 strain Test substance:

[0287] Spirulina SP257 strain (irrelevant VHH)

[0288] Spirulina SP526 strain (anti-C. jejuni VHH FlagV6)

[0289] Spirulina SP651 strain (anti-C. jejuni VHH FlagV6)

[0290] The effectiveness of orally delivered Spirulina-VHH in blocking colonization of the chicken intestine was investigated. Using a chicken model of C. jejuni intestinal colonization, the preventive effectiveness of anti-C. jejuni VHH expressed in Spirulina was evaluated. Spirulina strains expressing monomeric anti-Campylobacter VHH (SP526), Spirulina strains expressing homotrimeric multimeric VHH (SP651), or Spirulina strains expressing irrelevant VHH (SP257) were tested. The strains were cultured and spray-dried at a biomass concentration of 3% in 2% trehalose. This experiment was designed to evaluate the therapeutic effectiveness of different Spirulina strains with respect to their ability to block gastrointestinal colonization by C. jejuni, which is very frequently present in commercial flocks and a major cause of human food poisoning.

[0291] The study animals were 14-day-old SPF Leghorn mixed-sex chicks. A dose of 13.3 mg of Spirulina-VHH (150 mg / kg) in 200 μL of PBS was administered to 10 8One hour before challenge inoculum of C. jejuni 81-176 strain, it was administered by oral gavage. Chicks were randomly assigned to negative control group, positive control group and treatment group, raised in an isolation breeding unit, and allowed to freely consume standard feed and water. Two days after isolation, the chicks were treated with one dose by gavage of PBS or Spirulina suspended in PBS. One hour later, the chickens were inoculated with 10 8 CFU of C. jejuni 81-176 by gavage or sham-inoculated with PBS. Body weights were measured at 24, 48 and 72 hours post-inoculation. At 72 hours, the chickens were euthanized and cecal contents were aseptically collected for quantitative assessment of C. jejuni colonization.

[0292] Normal weight gain without deficits unrelated to Campylobacter inoculation or prophylactic treatment was observed in the chickens (Figure 20). Cecal colony counts were used to evaluate bacterial load. Cecal colonization by Campylobacter was significantly reduced after pretreatment with Spirulina SP651, a strain expressing anti-Campylobacter FlagV6 in a homotrimeric configuration. Treatment with SP257 expressing an irrelevant VHH, and SP526 expressing monomeric VHH FlagV6 resulted in a slight reduction in Campylobacter colonization compared to no Spirulina treatment (Figure 21).

[0293] (Example 9) ETEC Therapeutic: Spirulina-expressing anti-adhesion VHH Enterotoxigenic Escherichia coli (ETEC) is one of the causative agents of diarrhea in children in developing countries and traveler's diarrhea in persons traveling to regions where ETEC is endemic. According to the WHO, this pathogen is responsible for more than 200 million illnesses and approximately 500,000 deaths worldwide annually. Diarrhea caused by ETEC has long-term effects on young patients, including stunted growth, reduced intellectual fitness, and associated long-term economic disadvantages. Due to the adverse effects of ETEC etiology, effective prophylactic measures after infection, or prophylactic therapies such as passive immunization, are needed. Two major virulence factors in ETEC infections targeted by vaccine development or prophylactic treatment are enterotoxins and colonization factors (CFs) or fimbriae. Enterotoxins are directly involved in the development of diarrhea after bacterial colonization of the gastrointestinal enterocytes. On the other hand, ETEC CFs facilitate the easy colonization of the organism in the small intestine and then cause the expression of enterotoxins near mucosal cells, resulting in diarrhea.

[0294] The inventors have developed a therapeutic agent based on single-domain camelid antibodies (VHHs) that targets the ETEC fimbrial tip domain, inhibits the binding of bacteria to host enterocytes, and thus blocks bacterial colonization. The VHHs are obtained by immunizing llamas with the fimbrial tip adhesin protein CfaE or screened against the same antigen from a yeast-based synthetic library. VHHs showing higher antigen binding and bacterial inhibition in hemagglutination or cell-based assays are designed as monomers, dimers, trimers, tetramers, pentamers, heptamers for Spirulina expression and presented on nanoparticles. Chaperone proteins such as maltose-binding protein (MBP), thioredoxin A (TxnA), and neutrophil gelatinase-associated lipocalin (LCN) can be used to increase the solubility of heterologous proteins, resulting in higher protein expression levels of the therapeutic VHHs in Spirulina.

[0295] The Spirulina strain expressing anti-CfaE VHH shows good binding activity to the adhesion domain at the tip of CFA / I fimbriae. The increase in the multimeric state of VHH corresponds to the increase in binding activity in ELISA.

[0296] (Example 10) Porcine ETEC Therapeutic: Spirulina-Expressed Anti-Adhesion VHH Porcine enterotoxigenic Escherichia coli (ETEC) is the leading cause of diarrhea in piglets. Infection with ETEC in neonatal pigs can induce diarrhea during the first 1 or 2 weeks of the post-weaning period, resulting in, usually, dehydration, reduced weight gain, and death. The economic problems in the pig industry make post-weaning diarrhea and the causative agent ETEC economically significant diseases in the swine industry. The main virulence factors in ETEC strains are adhesins expressed as part of the fimbriae (pili) structure, and the most commonly seen in porcine ETEC are adhesin K88 (also called F4), K99 (F5), 987P (F6), F41, and F18, among which K88 and F18 are the most frequently seen in the swine industry.

[0297] The inventors have developed a system for cost-efficient production of multivalent camelid single-domain antibodies (VHHs) targeting virulence factors in K88 and F18 on the Spirulina platform, enabling the oral delivery of protein therapeutics to livestock by passive immunity without the need for expensive purification, storage, and delivery methods to protect the gastrointestinal tract. The therapeutic can be incorporated as part of the animal feed.

[0298] The inventors designed VHHs targeting ETEC virulence factors important for binding to host cells for Spirulina expression as monomers, dimers, and heptamers. To achieve higher protein expression levels of therapeutic VHHs in Spirulina, chaperone proteins such as maltose-binding protein (MBP) or thioredoxin A (TxnA) were used to increase heterologous protein solubility. Expression constructs with affinity tags were designed to facilitate downstream protein expression, purification, and ELISA assays.

[0299] The expression levels of the target proteins were determined by Western blotting using a combination of anti-tag or anti-VHH primary and appropriate secondary antibodies. The binding activity of the proteins expressed in Spirulina strains was evaluated using ELISA, where the antigen was coated on high-binding plates and the crude cell lysates of the antibody-expressing Spirulina strains were titrated with dilutions. The inventors expressed monomeric, dimeric, and heteroheptameric anti-adhesin VHHs in Spirulina. (Figs. 22A - C). The VHH binding activity to the antigen by ELISA indicates that the VHHs are active as Spirulina crude lysates. F4 + and F18 + The hetero-pentameric constructs expressing VHHs targeting the F4 + adhesin domain FaeG and F18 + both bind to the adhesin domain FedF. (Figs. 23A - C).

[0300] (Example 11) VHHs targeting the ETEC fimbrial domain inhibit bacterial binding in the neonatal piglet model ETEC K88ac, an ETEC strain that causes post-weaning diarrhea in piglets +VHHs targeting the fimbrial domain of the strain were designed. These VHHs were expressed in Spirulina as homodimers (SP795) and heteroheptamers (SP1156). (Figure 22A). The Spirulina biomass was dried and protein expression was confirmed. (Figure 25A). VHHs in Spirulina slurries from spray-dried and freeze-dried powders showed equivalent binding based on ELISA. (Figure 25B). The antigen-binding efficiency of Spirulina-expressed VHHs was further evaluated using kinetic measurements based on BLI. (Figure 25C).

[0301] Table 2 shows the total VHH expression per mass of dried Spirulina biomass evaluated using Western blot. Binding strength was evaluated using ELISA EC50 and KD was measured from kinetic measurements based on BLI. The level of active VHH was determined by comparing the observed activity from Spirulina biomass with the binding activity by purified protein. Table 2

Table 2-1

Table 2-2

[0302] The level of active protein in SP1156 was determined to be 0.5%, while the active level in SP795 was determined from 1.4%.

[0303] Furthermore, VHH targeting the fimbrial domain of the ETEC K88ac + (F4 + ac) strain, which causes post-weaning diarrhea in piglets, affects the bacterial load in Notobiotok piglets. Surgically delivered Notobiotok piglets were treated with wild-type or therapeutic VHH-expressing Spirulina powder slurries by oral gavage twice a day starting from day 0. Then, one day later, the piglets were 10Challenged with ETEC. (Figure 26A). 0.5 g of Spirulina biomass was administered to K88 (F4ac)-sensitive piglets with 10 ml of diluted VH795 Spirulina, SP1156 Spirulina, or wild-type Spirulina aqueous solution. For K88 (F4ac)-resistant piglets, Spirulina containing either SP795 or SP1156 VHH was administered by oral gavage twice a day after day 0.

[0304] On day 1, both K88-sensitive and K88-resistant piglets showed signs and symptoms of infection at 12 - 18 hours post-infection. Due to the too high bacterial dose used, sensitive piglets had to be euthanized on day 2. K88-sensitive piglets were necropsied due to severe symptoms, and intestinal samples were assayed for bacterial load. Piglets treated with therapeutic Spirulina powder containing SP1156 VHH showed a decrease in bacterial load in all tissues assayed. (Figure 26B).

[0305] A high bacterial dose causes symptoms even in resistant piglets. K88-resistant piglets were maintained for 4 days, and bacterial excretion was evaluated by taking fecal swabs. Piglets treated with the Spirulina SP1156 strain showed a decrease in bacterial load after challenge. (Figure 26C). These piglets showed symptoms but were still healthy enough to stop treatment after challenge and divert them to different studies.

[0306] (Example 12) Norovirus Therapeutic: Spirulina-expressed Anti-Norovirus Capsid Protrusion Domain VHH Human norovirus (HuNoV) is one of the most important causative agents of gastroenteritis associated with approximately one-fifth of all acute infectious diseases thought to be caused by this virus. HuNoV is a major causative agent of acute gastroenteritis. According to a study examining the burden of diarrheal diseases in the United States, HuNoV infections result in approximately 2 million outpatient visits, 800 deaths, 70,000 hospitalizations, and nearly 400,000 emergency department visits per year in the United States. According to the CDC, HuNoV is a leading cause of foodborne illness. HuNoV is a single-stranded RNA virus whose genome has a gene encoding the viral capsid protein (VP1). Based on the sequence diversity of the gene encoding the capsid (VP1), noroviruses are classified into various genogroups (GI - GVII). Genogroups are further divided into genotypes. The most prominent genogroups isolated from recent incidents of human infection are genogroups GI, GII, and GIV, among which more than 25 genotypes have been identified. The genotypes most frequently seen in recent HuNoV pandemics are GI.1, GII.4, and GII.10.

[0307] A prophylactic therapeutic agent based on a single-domain antibody (VHH) for oral delivery will be developed. This approach combines suitable VHH properties (such as high solubility, increased pH stability, and resistance to enzymatic degradation) that make these classes of antibodies suitable for oral delivery, with Spirulina-based oral delivery of the therapeutic agent. A VHH that targets the viral capsid protein and partially degrades the viral particles upon binding to neutralize infectious virus is designed for expression in Spirulina.

[0308] To enable passive immunization to orally deliver a protein therapeutic against HuNoV that protects the gastrointestinal tract without the need for expensive storage and delivery methods for therapeutic purification, a multivalent camelid single domain antibody (VHH) targeting the viral capsid protein will be developed. VHHs are designed for monomeric expression in Spirulina with or without the use of chaperone proteins such as maltose binding protein (MBP) or thioredoxin A (TxnA) to increase the solubility of heterologous proteins. Expression constructs are engineered to have affinity tags.

[0309] The expression level of the protein of interest is determined by Western blotting using a combination of anti-tag or anti-VHH primary and appropriate secondary antibodies. The binding activity of the protein expressed in the Spirulina strain is evaluated using ELISA, where the antigen is coated on a high-binding plate and the crude cell lysate of the antibody-expressing Spirulina strain is titrated with dilutions.

[0310] We expressed monomeric anti-HuNoV capsid protruding protein VHHs in Spirulina with or without a chaperone fusion partner (Figures 27A - C). ELISA-based binding assays indicate that Spirulina-expressed VHHs are active as Spirulina crude lysates. (Figures 28A - C). Furthermore, VHHs purified from Spirulina crude lysates show expected viral capsid degradation and block virus binding to tissue biopsy materials mimicking the intestinal environment. (Figures 29A - B).

[0311] (Example 13) Development of VHHs for treating norovirus infections To create a novel VHH (Nano85) targeting norovirus, the binding region of Nano85 was grafted onto the framework of the K922 antibody (SEQ ID NO: 18), which is known to be resistant to gastrointestinal proteases and enable increased expression in Spirulina, thereby modifying an anti-human norovirus (HuNoV) protrusion (P) domain antibody. (Figure 30). Constructs containing unmodified Nano85 with an N-terminal maltose-binding protein (MPB) (SP1371) and modified Nano85 with an N-terminal MPB (SP1372) were expressed in Spirulina. (Figure 31A). Furthermore, SP1371 and SP1372 bind to various recombinant P domains derived from different human norovirus GII strains (GII.2, GII.4, GII.17). (Figures 31B and 31C). The purified proteins also show measurable binding to irrelevant antigens including Campylobacter flagellin protein FlaA, porcine ETEC adhesin protein FaeG, and human ETEC fimbrial adhesin domain CfaE.

[0312] Furthermore, the binding kinetics and cross-reactivity of VHH sequences targeting various recombinant anti-human P domains were studied. Nano26 (SEQ ID NO: 73) and Nano85 (SEQ ID NO: 71) show broad cross-reactivity, while VHH3.2, VHH4.1, and VHH5.4 do not show binding to the GII.17 P domain. (Figures 32A - B). Table 3: ELISA-based binding to HuNoV GII.2, GII.3, GII.4, GII.4, GII.10, and GII.17 P domains. [Table 3] Table 4: Binding kinetics based on BLI to the HuNoV GII.2 P domain [Table 4-1] [Table 4-2]

[0313] In addition, the binding and cross-reactivity of VHH Nano94 (SEQ ID NO: 75), VHH10, VHH6.3, and VHH7.3 targeting the anti-human norovirus (HuNoV) P domain were evaluated. The VHHs tested showed binding EC50 values in the range of 0.21 nM to 50.07 nM, with the recombinant nano94-TxnA expressed in Spirulina showing the weakest binding. (Figure 33A). VHH7.3 had cross-reactive binding to the GI.3 P domain. (Figure 33B).

[0314] Table 5: EC50 values from ELISA-based binding to HuNoV GI.1 and GI.3

Table 5

[0315] To generate effective Spirulina expressing anti-human norovirus VHHs, the stability of recombinant Spirulina upon lyophilization was determined. Constructs from SP833, SP834, SP835, SP864, and SP1241 were lyophilized and tested for stability. (Figures 33A - B). Comparison of the stability of lyophilized protein with that of purified protein stored at 4°C showed no loss of binding activity.

[0316] The protease sensitivity of various anti-human norovirus P domain VHH constructs was determined by incubating 1 μg of recombinant VHH expressed in bacteria in digestion buffer (1 mM Tris pH 8.0, 20 mM CaCl 2)It was evaluated by incubating with 20 μL of chymotrypsin (0.1 mg / mL or 0.01 mg / mL) or trypsin (0.01 mg / mL or 0.001 mg / mL) in [the medium] for 1 hour, 2 hours, or 4 hours. Protease susceptibility was measured using ELISA-based binding as shown in Figure BB6. Loop grafted Nano85 showed the highest protease resistance compared to recombinant Nano85 and others tested. VHH3.2, VHH4.1, and VHH5.4 showed resistance to chymotrypsin, while showing various susceptibilities to trypsin.

[0317] (Example 14) Therapeutic agent for inflammatory bowel disease: Anti-TNF alpha VHH expressed in Spirulina Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract. IBD, including Crohn's disease and ulcerative colitis, is a recurrent disease that tends to be progressive. IBD treatments include anti-inflammatory drugs, immunosuppressive drugs, and anti-TNFα biologic agents. Tumor necrosis factor alpha (TNF-α) is a cytokine involved in inflammation. In chronic IBD, TNFα accumulates in the lamina propria of the gastrointestinal mucosa. The increased accumulation of TNFα causes chronic inflammation and subsequent damage to intestinal epithelial cells. Current anti-TNFα biologic therapies under investigation include infliximab, adalimumab, golimumab, and certolizumab. Given the chronic nature of IBD, oral administration of biologic agents would be ideal with respect to patient comfort, ease of treatment, compliance with the prescription regimen, and cost. However, biologic agents developed for IBD are currently delivered intravenously or subcutaneously due to physical barriers that render oral delivery of biologic agents ineffective. These challenges include the instability of protein-based therapeutic agents in the GI tract, extreme pH environments, and high enzymatic activity in the GI tract.

[0318] Single-domain llama antibodies (VHHs) have properties that make them suitable for oral delivery. VHHs retain binding specificity and potency equivalent to those of conventional IgG antibodies. The small size, rigid structural nature, solubility, ease of expression, and stability in the GI environment of VHHs make them suitable for oral-based therapeutics. In view of these properties, VH Squared developed a VHH (V565) that can bind to TNFα and can be used for the management of IBD by oral delivery.

[0319] Anti-TNF-α VHHs derived from VH squared were expressed as monomers and dimers. (Figs. 36A - C). The expression levels of anti-TNF-α VHHs were determined by Western blotting using a combination of anti-tag or anti-VHH primary and appropriate secondary antibodies. The binding activity of the proteins expressed in the Spirulina strain was evaluated using ELISA, in which the antigen was coated on a high-binding plate and the crude cell lysates of the antibody-expressing Spirulina strain were titrated with dilutions. VHHs in both monomeric and dimeric forms showed good binding to recombinant human TNF-α.

[0320] (Example 15) Clostridium difficile toxin B (tcdB)-specific VHHs in Spirulina Anti-tcdB VHHs 5D (SEQ ID NO: 5) and E3 (SEQ ID NO: 6) were constructed on various scaffolds and expressed in Spirulina. (Fig. 37). The scaffolds include thioredoxin (Trx) from E. coli, virus-like particles (MS2, Q β , PP7, and AP205) derived from several RNA phages, as well as computationally designed trimers and pentamers.

[0321] For trimers and pentamers, thioredoxin was always used as the scaffold structure. Some are designed as homo-oligomers (e.g., Trx-Trimer-VHH), some as homo-multivalent constructs (e.g., E3.VHH-Trx-TRIMER-E3.VHH), and some as hetero-multivalent constructs (e.g., E3.VHH-Trx-TRIMER-5D.VHH).

[0322] Constructs containing VHH.5D express at higher levels than those with VHH.E3. Certain hetero-multivalent constructs express at higher levels when E3 is at the N-terminus as opposed to 5D. (Figs. 38A - C).

[0323] The constructs were evaluated in vitro for their neutralizing activity against tcdB. (Fig. 39). Vero cells (African green monkey epithelial cells) were exposed to a range of doses of tcdB with or without Spirulina extract containing VHH. Biological effects were measured in two ways: First, a colormetric reagent that reacts linearly with healthy metabolic cells was used for quantitative measurement (Fig. 40), and second, light microscopy was used to assess the degree of "rounding", i.e., the extent to which Vero cells, which are normally adherent and flattened, detach from the plastic substrate and appear round. (Figs. 41A - O). These methods generally agreed, but the visible rounding assay was consistently more sensitive.

[0324] Results

[0325] i. B5.2, B13.6 VHH (Canada) do not neutralize when expressed on VLPs.

[0326] ii. Tuft VHH E3 and 5D both demonstrate neutralizing activity.

[0327] iii. Generally, 5D-containing constructs express more abundantly and demonstrate more potent neutralizing activity.

[0328] iv. The following strains showed the highest in vitro activity:

[0329] SP1095, heterodimeric trimer construct, E3_Trx_TRI_5D

[0330] SP747, monomeric Trx_5D

[0331] SP1087, trimer construct Trx_TRI_5D

[0332] Those with slightly weaker potency in vitro were

[0333] SP985, RNA phage VLP PP7 hybridized to VHH 5D

[0334] SP1091, pentamer construct Trx_PENT_5D as follows.

[0335] The VHH-5E (SEQ ID NO: 7) construct was also constructed. The VHH.5E-containing constructs functioned stronger than those with VHH.E3, but based on the per mole criterion, the most potent was the trimer containing both VHH.E3 and VHH.5D. Potency generally followed the expression level, but the most effective / potent construct was the VHH.E3-Trx-trimer-VHH.5D, which was expressed at only about 0.1% of the total protein and was more potent than Trx-VHH.5D, the second most potent extract, which was expressed at about 2% of the total protein. The Spirulina extract without VHH did not exhibit intrinsic neutralizing activity.

[0336] Three or four of the best performing strains will be expanded for bioreactor and spray drying. Additionally, next generation constructs will be designed and new strains (e.g., markerless versions of the present strain, native Arthrospira thioredoxin, heteromultimers with 5D, and new tuft VHHs directed to RBD) will be constructed. Also, animal studies will be initiated using the present hit strain: 1) mouse model I: Lyras / Australia; 2) mouse model II: Guerrant / Virginia; 3) pig model: Tzipori / Tufts.

[0337] (Example 16) Combinations of VHHs show a synergistic increase in binding to C. difficile toxin The binding strengths of various VHHs to C. difficile TcdB toxin, alone and in combination, were tested. The VHHs were produced in E. coli and tested in vitro. Figure 42 shows the binding strengths of VHH 5D (SEQ ID NO: 5), E3 (SEQ ID NO: 6), 7F (SEQ ID NO: 13), 2D (SEQ ID NO: 65) and 5E (SEQ ID NO: 7) alone, at various concentrations, to TcdB. VHH 5D shows the greatest binding and 2D shows the least binding. Figure 43 shows the binding strengths of different combinations of VHH 5D, E3, 7F, 2D and 5E. Figure 44 shows the binding strengths of VHH 5D, E3 and 7F alone and in combination. Figures 45A - B show the binding strengths of VHH 5D, E3 and 7F alone and in combination at different concentrations. Increasing the concentration of VHH alone increased the efficacy little. In contrast, higher concentrations of combined VHHs (i.e., VHH cocktails) showed a surprising increase in efficacy with increasing concentration.

[0338] The increased effectiveness of the VHH combinations can be explained by different targets of different VHHs. For example, as shown in Figure 46, the VHHs can act at different points in the process of the TcdB signaling pathway. VHH E3 can block receptor binding, VHH5D can block pH-dependent pore formation, VHH 7F can block autoproteolytic reactions, and may block the GTD site. This explains the synergistic effect of the VHH cocktail that outperforms the effect of a single VHH. Table 6: Anti-TcdB VHH

Table 6-1

Table 6-2

[0339] Bacterial lysates of VHHs constructed by fusion with maltose-binding protein (MBP) in the orientation of MBP-VHH (used as Spirulina lysates expressing PP7 particles decorated with VHH 5d, except for 5D) were used at the concentrations shown in Figures CC1 and CC2. Individual VHHs were used at 100 ng / ml, and two combinations were used at 50 ng / ml each for a total VHH concentration of 100 ng / ml. The VHHs were tested against three concentrations of TcdB type 027 as shown.

[0340] (Example 17) Anti-TcdB (Clostridium difficile toxin B) VHH produced in Spirulina Multimerization of single-domain antibodies in a single polypeptide chain increases binding affinity and often also increases biological activity. Single polypeptides of multimeric VHHs have been produced in E. coli, but have proven very difficult to express in Spirulina. Recently, the crystal structure of the entire TcdB protein (ca. 300 kDa) with three VHHs (VHH 5D, E3 and 7F) bound was solved. See Figure 52 showing TcdB bound to E3. Each VHH bound to a separate domain that was spatially far apart from each other. Two of the three domains had essential biological activities identified during the intoxication process, and it became clear that the bound VHHs disrupted the structural changes necessary for these functions. The third domain bound to a domain associated with targeting to the target cell membrane in the homologous toxin. Each VHH has previously been shown to have some natural toxin-neutralizing activity.

[0341] A single polypeptide containing three VHHs is sterically disadvantaged either to bind to all three epitopes on one toxin or to distinct, different epitopes on multiple toxin molecules. Given that they individually demonstrated neutralizing activity, a simple mixture of the three VHHs would have neutralizing activity that exceeds a mere additive effect. Using proteins expressed in bacteria, mixtures of two VHHs from a 10-member panel were tested, and it was independently confirmed that VHH E3, 5D and 7F were particularly active when mixed with each other in 2-member mixtures or when mixed with some other VHHs with lower activity. Continuing with 3-fold, 4-fold and 5-fold mixtures of the 10 VHHs, it was found that the maximum neutralizing activity was the same using any combination containing E3, 5D and 7F, and the simplest one was the one combining these three.

[0342] Each of the three VHHs was engineered into a hybrid construct with known solubility optimization parameters or folding optimization parameters (chaperones) to maximize the accumulation of bioactive VHHs in Spirulina. Spirulina lysates containing individual constructs containing E3, 5D, or 7F were assayed for TcdB neutralizing activity separately (Figure 54) and in various combinations containing all three VHHs (Figures 55 and 56). Surprisingly, the combination of lysates containing all three VHHs appeared to have a neutralizing activity >1000-fold higher than any single VHH lysate. Complete neutralization of TcdB at toxin concentrations far higher than those seen in human clinical isolates was seen at VHH concentrations well below those predicted to be obtained after human administration (Figure 57).

[0343] (Example 18) Administration of VHHs and Other Therapeutic Molecules In addition to different VHHs, other therapeutic agents may be present in recombinant Spirulina to further increase the efficacy of the orally delivered therapeutic. The effect of multi-drug cocktails has been demonstrated for a very large number of organisms, including M. tuberculosis, and by combining therapeutic agents that target cell wall synthesis, replication and transcription, energy metabolism, and translation, different parts of the pathogen life cycle can be targeted (Figure 49). Similarly, targeting C. difficile receptor activation and different aspects of the cell membrane can increase the efficacy of the orally delivered therapeutic. To demonstrate this, recombinant Spirulina is produced that expresses one or more VHHs that bind to the S-layer of C. difficile, one or more VHHs that neutralize toxin B, and polypeptides such as lysins for attacking the cell membrane (see Figure 50).

[0344] (Example 19) Lysins Expressed from Spirulina are Active PlyCD and the catalytic domain fragment PlyCD1-174 have been previously expressed in E. coli and shown to be bactericidal in vitro and in vivo. To determine whether the phage-derived lysin PlyCD, which digests the Clostridium cell wall and is expressed from Spirulina, is active, the genes for PlyCD and PlyCD1-174 were inserted into Spirulina under the control of the cpc600 promoter, and expression was confirmed by Western blot. Various concentrations were tested in a standard cell lysis assay. Figure 63 shows the cell lysis assay results for both the protein expressed in E. coli and the protein expressed in Spirulina. The lysin expressed in Spirulina is catalytically active.

[0345] (Example 20) Effect of linker on the neutralizing ability of anti-TcdB VHH sequences A series of transgenes encoding anti-TcdB VHH 5D were used to generate various constructs containing different rigid linkers that bind to chaperone partners. (Figure 51). The specific constructs tested in this experiment are listed in Figure 59.

[0346] The control strain uses a flexible (GGS)x linker between 5D and a computationally designed dimer.

[0347] Figure 64 demonstrates neutralization data for strains expressing numerous linkers that bind 5D to MBP, and for a single strain with an IgA-derived linker that binds 5D to the PP7 VLP.

[0348] (Example 21) Stability of Spirulina constructs in water and drinking fluids Recombinant Spirulina can be administered orally, and the addition of VHH to drinking water will significantly increase the dose of VHH that can be delivered to animals. To test the stability and activity of VHH held at room temperature in various palatable buffers for mice, rats or pigs, 1 mg / mL of Spirulina lysate was mixed into water, 50 mM phosphate pH 7.4, 5% sucrose, 5% non-fat milk (NFM), sucrose + phosphate, or sucrose + milk. (Figure 65). Western blots were performed at 0, 1, 2, 3 and 4 hours. The TcdB neutralization assay was performed at 0 and 4 hours.

[0349] Western blotting showed that the abundance of the his-tagged protein did not decrease over time. No decrease in TcdB neutralizing potency was observed in any of the aqueous media, either at 4 hours or at 12 hours. (Figures 66 and 67). Similar results were obtained for the individual VHH 5D and E3, as well as for the three synergistic combinations of 5D, E3 and 7F. (Figure 68).

[0350] (Example 22) Study on the Defense Against Clostridium difficile in the Notobiotically Colonized Pig Model To study the effect of Spirulina expressing anti-TcdB VHH on protection from Clostridium difficile challenge, the notobiotically colonized pig model was used. (Figure 70). In this study, pigs were divided into 4 groups as follows:

[0351] Group 1 (2 pigs) - infected, untreated (or sham-capsule treated)

[0352] Group 2 (2 pigs) - infected, wild-type Spirulina treated

[0353] Group 3 (4 pigs) - infected, Spirulina mix #1: 3×VHH

[0354] Group 4 (4 pigs) - Infection, Spirulina mix #2: 3×VHH + PlyCD lysin.

[0355] At 5 days of age, the animals were inoculated with 10 6 of C. diff. UK6 BI / NAP1 / 027. After infection, starting on day -0 and for 5 days, the animals were treated three times a day. After treatment, the animals were measured for clinical metrics, survival, fecal spore shedding, and GIT histology.

[0356] Figures 71A - B show that after day 4, animals in both Group 3 and Group 4 demonstrated a reduced incidence of diarrhea compared to infected animals treated with wild - type Spirulina or PBS.

[0357] (Example 23) Study on the effect of prophylactic administration of anti - TcdB VHH against Clostridium difficile infection in the Monash mouse CDI model Mice were administered an antibiotic cocktail in drinking water from day -11 to day -4. From day -4 to day 0, mice were administered cefaclor alone and infected with C. difficile on day 0. From day -1 to day 4, mice were administered Spirulina (3×VHH mix, or 3×VHH mix + lysin), PBS, or vancomycin once a day by orogastric gavage. During this period, the mice were monitored daily for body weight, diarrhea, activity, and appearance, and feces were collected. (Figure 72). Administration of the anti - TcdB VHH mix reduced the body weight loss associated with C. difficile infection. (Figure 73A). Mice treated with VHH alone had improved survival compared to mice treated with wild - type Spirulina, and mice treated with 3×VHH mix + PlyCD lysin achieved 100% survival equivalent to vancomycin. (Figure 73B). Finally, administration of 3×VHH mix + lysin reduced C. difficile spore shedding in feces by >2 log. (Figure 73C).

[0358] (Example 24) Effect of pH on the release of VHH from LMN - 101 The therapeutic VHH encapsulated within Spirulina biomass is not released into gastric juice mimicking buffer. Bioencapsulation also prevented enzymatic degradation of VHH under simulated gastric digestion conditions. To analyze the effect of low pH on the release of VHH from Spirulina biomass, dried Spirulina-VHH biomass was resuspended in buffers of different pH. The spray-dried Spirulina-VHH biomass used for LMN-101 (SP1182 strain) was resuspended at 50 mg / mL in citrate-phosphate buffer in the range of pH 3 to pH 7 and incubated at room temperature for 60 minutes with gentle stirring. The resuspended biomass was clarified by centrifugation at 14,000 RPM for 1 minute in a cooled microcentrifuge. The clarified extract was used in a binding assay based on ELISA using recombinant C. jejuni flagellin to determine the amount of aa682 present. Antigen was coated on a high-binding ELISA plate and the SP1182 extract was assayed as a 4-fold serial dilution in PBS supplemented with 0.05% Tween®-20 and 5% skim dried milk. Bound aa682 was detected using a mouse anti-His tag primary antibody and a goat anti-mouse-HRP secondary antibody.

[0359] In this ELISA, the relative binding activity of the extract corresponded to the amount of aa682 extracted at each pH. The calculated EC50 values showed equivalent amounts of aa682 binding activity when Spirulina biomass was resuspended in pH 5, pH 6 and pH 7 buffer solutions (Figure 74 and Table 7). The amount of binding activity decreased by 50% when Spirulina biomass was extracted with pH 4 buffer. In contrast, extracts prepared with pH 3 buffer demonstrated a relatively small amount of binding activity. The EC50 of the extract from biomass resuspended at pH 3 suggested that 1 / 40th of the aa682 was released compared to release in pH 7 buffer. To evaluate the effect of pH on the stability and activity of VHH, purified aa682 was incubated in pH 3 buffer and the integrity of VHH was evaluated by an ELISA-based binding assay as described above. No measurable loss of binding due to exposure to low pH buffer was observed (data not shown). Table 7: EC50 for SP1182 biomass resuspended in buffers of various pH [Table 7]

[0360] To further demonstrate that the differences in binding activity are a result of differences in VHH concentration, clarified Spirulina extract was also assayed using capillary electrophoresis immunoassay. The clarified extract was prepared as described above. VHH was detected using a mouse anti-His tag primary antibody (Genscript) and an HRP-conjugated anti-mouse secondary antibody (ProteinSimple). The amount of VHH protein released from Spirulina biomass increased with increasing pH, with the least VHH observed at pH 3 (Figure 75).

[0361] These results suggest that under gastric-like conditions of low pH, VHH could remain encapsulated within the Spirulina biomass and be protected from the harsh gastric environment until it migrates to the higher pH conditions of the small intestine.

[0362] (Example 25) Phase 1 Clinical Trial of LMN-101 The first-phase safety and tolerability study was conducted in healthy volunteers using LMN-101 (SP1182), a single Spirulina strain engineered to express a binding protein that inhibits C. jejuni (CG8421) infection. Part A of the study was an open-label oral administration of a single 3000 mg dose of LMN-101. Part B was a randomized, double-blind, placebo-controlled, dose-escalation study at three dose levels of LMN-101: 300 mg, 1000 mg, or 3000 mg. (Figures 61 and 62). Wild-type Spirulina was used as a control. In Part B, healthy volunteers orally ingested one of these three dose levels of LMN-101 or placebo three times daily for 28 days. No significant adverse events were reported. Additionally, pharmacokinetic data showed no significant systemic absorption. This indicates that Spirulina can pass through the stomach and deliver the VHH to the gastrointestinal tract. Orally delivered LMN-101 was safe and well tolerated at doses of 3000 mg or less TID for 28 days, and no significant adverse events attributable to LMN-101 were observed.

[0363] (Example 26) In vitro stability of Spirulina-VHH biomass in simulated intestinal fluid To model the intestinal delivery phase, dried Spirulina-VHH biomass was incubated at 37 °C in simulated intestinal fluid (SIF): 50 mM citrate-phosphate buffer, pH 7.0, 164 mM NaCl, 85 mM NaHCO 3 , 3 mM CaCl 2, and incubated in 1 mg / L pancreatin containing 10 mM porcine bile extract. The integrity of the intact anti-Campylobacter binding protein was determined by Western blot. In two independent experiments using the dried biomass of Spirulina-VHH (strain SP806) expressing trimeric VHH, more than 80% of the binding protein was observed to be released from the biomass within 5 minutes and more than 95% within 30 minutes (Figure 76). In a similar experiment using Spirulina-VHH present in LMN-101 (strain SP1182), more than 95% of the binding protein was released within 5 minutes (Figure 77). In both cases, the fully intact released binding protein did not accumulate to measurable levels in simulated intestinal fluid. This indicates that the rate of proteolytic cleavage was faster than the rate of its release. The detection limit in this experiment was approximately 20% recovery of the intact anti-Campylobacter binding protein released. Consistent with this interpretation, the purified anti-Campylobacter binding protein added directly to simulated intestinal fluid had a half-life of proteolytic cleavage of less than 5 minutes (Figure 78).

[0364] Rapid release in the simulated intestinal environment suggests that aa682 is released in the proximal small intestine and is available for binding to Campylobacter in that environment. Rapid degradation of aa682 suggests that detectable levels remain in fecal contents.

[0365] (Example 27) In vitro stability of Spirulina-VHH biomass in simulated gastric fluid Spirulina biomass protects Campylobacter-binding proteins during passage through the harsh environment of the stomach. Dried biomass of anti-Campylobacter Spirulina-VHH was incubated in simulated gastric fluid (SGF): 10 mM citrate-phosphate buffer, pH 3.5, 94 mM NaCl, 13 mM KCl, and 2,000 units / mL pepsin, incubated at 37°C. Western blotting of digested Spirulina-VHH biomass demonstrated that the Campylobacter-binding protein expressed within this biomass was 50% intact after 120 minutes (Figure 79). Analysis was repeated using Spirulina-VHH present in LMN-101 (strain SP1182) but otherwise under the same conditions. Western blotting demonstrated that the Campylobacter-binding protein expressed within this biomass was 20% intact after 120 minutes (Figure 80).

[0366] (Example 28) Intranasal administration of SP648 elicits antibody production in a mouse model Mice were tested to determine whether intranasal administration of Spirulina expressing the malaria antigen NANP, or an extract of Spirulina containing the malaria antigen NANP, demonstrated an IgG response to NANP. Mice were further analyzed for survival from malaria infection.

[0367] Mice were immunized with PfCSP-VLP (a malaria vaccine based on the NANP repeat region of P. Falciparum CSP fused within SP648-virus-like particles) or empty VLP (SP79). Mice were assigned to 6 groups (5 mice / group) and treated as shown in Table 8. Table 8

Table 8

[0368] Both groups 5 and 6 had a re-priming period on the day when groups 1 - 4 were boosted. When groups 1 - 4 were first boosted, group 5 was not treated. When groups 1 - 4 were given their second boost, group 5 was "re-primed" by intranasal administration of PfCSP-VLP extract, and then boosted once by oral administration of PfCSP Spirulina biomass. When groups 1 - 4 were given their first boost, group 6 was "re-primed" by intranasal administration of PfCSP-VLP extract, and then boosted twice by oral administration of PfCSP Spirulina biomass. This re-priming was done to determine whether the number of boosts given to the mice affected IgG production. Group 3 was given 3 oral boosts, group 6 was given 2 oral boosts, and group 5 was given only 1 oral boost.

[0369] IgG measurement

[0370] Serum was collected on days 14, 27, 41, 56 and 69 as shown in Table 8. The amount of IgG produced in different groups was measured by indirect ELISA. The plate was coated with the antigen NANP and covered with mouse sera containing various amounts of antibodies specific for the NANP antigen, and then covered with a secondary antibody conjugated to horseradish peroxidase (HRP). As an indirect method of measuring how much antibody specific for NANP is present in each serum sample, a substrate was added in the presence of hydrogen peroxide. ELISA was performed using serial dilutions of the sera of each animal to detect the minimum amount of serum that could still produce a positive response to the antigen.

[0371] Results

[0372] The results shown in Figures 81 - 86 indicate that serum IgG responds to NANP at various time points after administration of the malaria vaccine or control as outlined above. Measurement of the IgG response to maltose-binding protein (MBP) served as a control. The Y-axis for each relates to the degree of absorbance value from the plate reader. A positive response is close to the amount of IgG seen in the hyperimmune serum which is the positive control. Since the hyperimmune serum is very strong and requires less amount to detect IgG, the dilution of the hyperimmune serum is different from that for the experimental groups.

[0373] The data in Figure 76 (day 14) measures the serum IgG response to different substrates (MBP) as a control. Since the NANP protein fuses with MBP, it is important that the serum does not react with MBP. Figure 76 shows that there is no IgG response to MBP on day 14 after vaccination with the malaria vaccine tested here. Similar results were obtained for the other days tested (data not shown).

[0374] Previous reports have demonstrated that after oral administration, mice produce IgG in response to NANP, not by day 14 as seen here, but by day 28. In contrast, intranasal administration results in a fairly robust serum IgG response to NANP by day 14.

[0375] As shown in FIGS. 81 - 86, serum IgG production in the mice of Group 3 was more uniform than that of Group 2. This could reflect the difference between administration of the extract and administration of the resuspended biomass. The extract is a homogeneous solution, whereas the resuspended biomass is not, and thus mice within a given group may ingest different amounts of Spirulina.

[0376] Furthermore, mice inoculated with the extract were readily exposed to the vaccine antigen, whereas mice administered Spirulina biomass may not have been exposed to the vaccine antigen as efficiently or uniformly. Encapsulation of the vaccine antigen within Spirulina may not be an important component of an intranasally administered vaccine, in contrast to an orally administered vaccine where protection of the vaccine as it traverses the stomach is important.

[0377] Importantly, nasal administration of the extract elicits a stronger and more uniform response than administration of Spirulina biomass, whether administered orally or intranasally.

[0378] Challenge in malaria

[0379] FIG. 87 shows the survival rates of various groups after challenge with P. Falciuparum.

[0380] Some mice appear to be protected from challenge despite having a lower detectable serum IgG response. This indicates that other elements, including other types of antibody responses, play a role in the immune response. The data presented here examines only serum IgG - mice also produce serum IgA and IgM. Additionally, analysis of fecal samples will yield information regarding mucosal IgA, an indicator of a good mucosal response. However, in general, high serum IgG titers indicate protection from challenge, and indeed, it is difficult to protect mice from malaria, so the 50% protection observed in the second group is quite good. Therefore, the demonstrated protection of up to 80% is remarkable.

[0381] Examples of non - limiting embodiments of the present disclosure The embodiments of the subject matter disclosed herein may be beneficial alone or in combination with one or more other embodiments. Without limiting the foregoing description, specific non - limiting embodiments of the present disclosure are presented below. As will be apparent to those skilled in the art upon reading the present disclosure, each of the individually numbered embodiments may be used or combined with any of the individually numbered embodiments described above or below. This is intended to support all such combinations of embodiments and is not limited to the combinations of embodiments explicitly presented below.

[0382] Embodiment 1. A composition delivered without injection, comprising recombinant Spirulina, wherein the recombinant Spirulina comprises at least one therapeutic or prophylactic drug molecule.

[0383] Embodiment 2. The composition delivered without injection according to Embodiment 1, wherein the therapeutic or prophylactic drug molecule is delivered to the gastrointestinal tract.

[0384] Embodiment 3. The composition delivered without injection according to Embodiment 1, wherein the therapeutic or prophylactic drug molecule is delivered systemically.

[0385] Embodiment 4. A composition delivered without injection as described in any of Embodiments 1 to 3, wherein the therapeutic or prophylactic drug molecule is an endogenous Spirulina molecule.

[0386] Embodiment 5. A composition delivered without injection as described in Embodiment 4, wherein the endogenous Spirulina molecule is found at a higher concentration than that found in naturally occurring Spirulina.

[0387] Embodiment 6. A composition delivered without injection as described in any of Embodiments 1 to 3, wherein the therapeutic or prophylactic drug molecule is exogenous to Spirulina.

[0388] Embodiment 7. A composition delivered without injection as described in Embodiment 6, wherein the exogenous molecule is produced by a different bacterium or plant.

[0389] Embodiment 8. A composition delivered without injection as described in Embodiment 7, wherein the exogenous therapeutic drug is marasitide.

[0390] Embodiment 9. A composition delivered without injection as described in Embodiment 6, wherein the exogenous molecule is a polypeptide or a fragment thereof.

[0391] Embodiment 10. A composition delivered without injection as described in Embodiment 9, wherein the exogenous polypeptide is an antibody or a fragment thereof.

[0392] Embodiment 11. The antibody or fragment thereof is a full-length antibody, a monospecific antibody, a bispecific antibody, a trispecific antibody, an antigen-binding region, a heavy chain, a light chain, a VHH, a VH, a VL, a CDR, a variable domain, an scFv, an Fc, an Fv, a Fab, an F(ab) 2 , reduced IgG (rIgG), a monospecific Fab 2 , a bispecific Fab 2 , a trispecific Fab 3、A composition delivered without injection as described in embodiment 10, selected from the group consisting of diabodies, bispecific diabodies, trispecific tribodies, minibodies, IgNAR, V-NAR, HcIgG, or combinations thereof.

[0393] Embodiment 12. A composition delivered without injection as described in embodiment 9, wherein the exogenous polypeptide is selected from the group consisting of insulin, C-peptide, amylin, interferon, hormone, receptor, receptor agonist, receptor antagonist, incretin, GLP-1, glucose-dependent insulinotropic polypeptide (GIP), immunomodulatory agent, immunosuppressive agent, peptide chemotherapeutic agent, antimicrobial peptide, magainin, NRc-3, NRC-7, buforin IIb, BR2, p16, Tat, TNF alpha, and chlorotoxin.

[0394] Embodiment 13. A composition delivered as described in embodiment 9, wherein the exogenous polypeptide is an antigen or an epitope.

[0395] Embodiment 14. A composition delivered without injection as described in embodiment 13, wherein the antigen or epitope is derived from an infectious microorganism, a tumor antigen or a self-antigen associated with an autoimmune disease.

[0396] Embodiment 15. A composition delivered without injection as described in any of embodiments 1 to 14, wherein administering recombinant Spirulina to a subject prevents, treats, or improves a disease or disorder.

[0397] Embodiment 16. A composition delivered without injection as described in embodiment 15, wherein the disease or disorder is selected from the group consisting of type 1 diabetes, type 2 diabetes, cancer, inflammatory disorder, gastrointestinal disease, autoimmune disease or disorder, endocrine disorder, gastroesophageal reflux disease (GERD), ulcer, hypercholesterolemia, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, constipation, vitamin deficiency, iron deficiency, and diarrhea.

[0398] Embodiment 17. A composition delivered without relying on the injection described in Embodiment 15, wherein the recombinant Spirulina is administered to a subject and an infectious disease is treated, prevented, or improved.

[0399] Embodiment 18. A composition delivered without relying on the injection described in Embodiment 17, wherein the infectious disease is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection.

[0400] Embodiment 19. The bacteria causing the infectious disease are selected from the group consisting of E. coli, enterotoxigenic E. coli (ETEC), Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, heliobacteter, Bacillus anthracis, ETEC, EHEC, EAEC, and Leg ionella, and the composition is delivered without relying on the injection described in Embodiment 18.

[0401] Embodiment 20. The virus causing the infectious disease is selected from the group consisting of bacteriophage, RNA bacteriophage (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus, and the composition is delivered without relying on the injection described in Embodiment 18.

[0402] Embodiment 21. A composition delivered without injection as described in Embodiment 18, wherein the fungus causing the infectious disease is selected from the group consisting of Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma.

[0403] Embodiment 22. A composition delivered without injection as described in Embodiment 18, wherein the parasite causing the infectious disease is selected from the group consisting of Plasmodium, P. falciparum, P. malariae, P. ovale, P. vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, parasitic worms: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostoma spp. and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp. and Wuchereria spp., Taenia spp., Echinococcus spp., and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp.

[0404] Embodiment 23. A composition delivered without injection as described in any of Embodiments 9 to 22, wherein the exogenous polypeptide or fragment thereof is present in a fusion protein.

[0405] Embodiment 24. A composition delivered without injection as described in any of Embodiments 9 to 22, wherein the recombinant Spirulina contains a nucleic acid encoding an exogenous polypeptide or fragment thereof.

[0406] Embodiment 25. A composition delivered without injection as described in Embodiment 24, wherein there are at least 2 copies, at least 3 copies, at least 4 copies, or at least 5 copies of the nucleic acid sequence encoding at least one exogenous polypeptide or fragment thereof in the recombinant Spirulina.

[0407] Embodiment 26. A composition delivered without injection as described in any of Embodiments 24 to 25, wherein there are 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 8 copies, 10 copies, 15 copies, 20 copies, 25 copies, 30 copies, 40 copies, or 50 copies of a nucleic acid sequence encoding at least one exogenous polypeptide or a fragment thereof in recombinant Spirulina.

[0408] Embodiment 27. A composition delivered without injection as described in Embodiment 25, wherein there are at least 2 copies, at least 3 copies, at least 4 copies, or at least 5 copies of at least one exogenous polypeptide or a fragment thereof in a single molecule of the exogenous polypeptide expressed in recombinant Spirulina.

[0409] Embodiment 28. A composition delivered without injection as described in Embodiment 25 or 27, wherein there are 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 8 copies, 10 copies, 15 copies, 20 copies, 25 copies, 30 copies, 40 copies, or 50 copies of at least one exogenous polypeptide or a fragment thereof in a single molecule of the exogenous polypeptide expressed in recombinant Spirulina.

[0410] Embodiment 29. A composition delivered without injection as described in any of Embodiments 25 or 27 to 28, wherein copies of the exogenous polypeptide are tandemly linked within the molecule of the exogenous polypeptide.

[0411] Embodiment 30. A composition delivered without injection as described in any of Embodiments 25 or 27 to 28, wherein copies of the exogenous polypeptide or a fragment thereof are separated by a spacer sequence within the molecule of the exogenous polypeptide or a fragment thereof.

[0412] Embodiment 31. A composition delivered without injection according to any of Embodiments 25 to 30, wherein within the exogenous polypeptide or a fragment thereof, a part of the copy of the exogenous polypeptide or a fragment thereof is tandemly linked, and the remaining copies of the exogenous polypeptide or a fragment thereof are separated by a spacer sequence.

[0413] Embodiment 32. A composition delivered without injection according to Embodiment 30 or 31, wherein the spacer sequence is between about 1 amino acid length and 50 amino acid lengths.

[0414] Embodiment 33. A composition delivered without injection according to any of Embodiments 30 to 32, wherein more than one spacer sequence is present within the exogenous polypeptide or a fragment thereof.

[0415] Embodiment 34. A composition delivered without injection according to any one of Embodiments 9 to 34, wherein the recombinant Spirulina contains at least two, at least three, at least four, or at least five different exogenous polypeptides or fragments thereof.

[0416] Embodiment 35. A composition delivered without injection according to any one of Embodiments 23 to 34, wherein the fusion protein contains a carrier protein.

[0417] Embodiment 36. A composition delivered without injection according to Embodiment 35, wherein the carrier protein is selected from the group consisting of maltose-binding protein, hepatitis C virus-like particles, thioredoxin, and phycocyanin.

[0418] Embodiment 37. A composition delivered without injection according to any one of Embodiments 23 to 36, wherein the fusion protein contains a scaffold protein.

[0419] Embodiment 38. A composition delivered without injection according to Embodiment 37, wherein at least one exogenous polypeptide is linked to a scaffold protein at the N-terminus or C-terminus of the scaffold protein, or within the body.

[0420] Embodiment 39. A composition delivered without injection according to Embodiment 37 or 38, wherein the scaffold protein is selected from the oligomerization domain of C4b-binding protein (C4BP), cholera toxin b subunit, or the oligomerization domain of an extracellular matrix protein.

[0421] Embodiment 40. A composition delivered without injection according to any one of Embodiments 37 to 39, wherein at least one exogenous polypeptide and the scaffold protein are separated by about 1 to about 50 amino acids.

[0422] Embodiment 41. The fusion protein contains multiple copies of at least one exogenous polypeptide or a fragment thereof, and at least one exogenous polypeptide or a fragment thereof and the scaffold protein are arranged in one of the following patterns: (E)n-(SP), (SP)-(E)n, (SP)-(E)n-(SP), (E)n1-(SP)-(E)n2, (SP)-(E)n1-(SP)-(E)n2, and (SP)-(E)n1-(SP)-(E)n2-(SP) (in the pattern, E is at least one exogenous polypeptide or a fragment thereof, SP is the scaffold protein, and n, n1, and n2 represent the number of copies of at least one exogenous polypeptide or a fragment thereof). A composition delivered without injection according to any one of Embodiments 37 to 40.

[0423] Embodiment 42. A composition delivered without injection according to any one of Embodiments 9 to 42, wherein the recombinant Spirulina contains an anti-Campylobacter VHH.

[0424] Embodiment 43. A composition delivered without injection according to Embodiment 42, wherein Campylobacter is C. jejuni.

[0425] Embodiment 44. A composition in which the VHH binds to a Campylobacter component and is delivered without relying on the injection described in any of Embodiments 42 to 43.

[0426] Embodiment 45. A composition in which the VHH binds to flagellin and is delivered without relying on the injection described in Embodiment 44.

[0427] Embodiment 46. A composition in which Campylobacter excretion increases upon administration and is delivered without relying on the injection described in any of Embodiments 42 to 45.

[0428] Embodiment 47. A composition in which the level of a biomarker decreases upon administration and is delivered without relying on the injection described in any of Embodiments 42 to 46.

[0429] Embodiment 48. A composition in which the biomarker is an inflammatory biomarker and is delivered without relying on the injection described in Embodiment 47.

[0430] Embodiment 49. A composition in which recombinant Spirulina contains a VHH that binds to an anti-Clostridium toxin and is delivered without relying on the injection described in any of Embodiments 9 to 42.

[0431] Embodiment 50. A composition in which Clostridium is C. difficile and is delivered without relying on the injection described in Embodiment 49.

[0432] Embodiment 51. A composition in which the VHH binds to A toxin or B toxin, which is a Clostridium component, and is delivered without relying on the injection described in any one of Embodiments 48 to 49.

[0433] Embodiment 52. A composition in which the VHH contains any of the amino acid sequences of SEQ ID NOs: 5 to 10 and is delivered without relying on the injection described in any of Embodiments 49 to 51.

[0434] Composition delivered without injection according to any one of Embodiments 1 to 52, wherein the therapeutic or prophylactic drug molecule is a monomer.

[0435] Embodiment 54. A composition delivered without injection according to any one of Embodiments 1 to 52, wherein the therapeutic or prophylactic drug molecule is a multimer.

[0436] Embodiment 55. A composition delivered without injection according to Embodiment 54, wherein the therapeutic or prophylactic drug molecule is a trimer.

[0437] Embodiment 56. A composition delivered without injection according to any one of Embodiments 54 to 55, wherein the multimer is a heteromer.

[0438] Embodiment 57. A composition delivered without injection according to any one of Embodiments 54 to 55, wherein the multimer is a homomer.

[0439] Embodiment 58. A composition delivered without injection according to any one of Embodiments 54 to 57, wherein the multimer is disposed in nanoparticles.

[0440] Embodiment 59. A composition delivered without injection according to any one of Embodiments 54 to 57, wherein the multimer binds to a target or target molecule with high affinity.

[0441] Embodiment 60. A composition delivered without injection according to Embodiment 59, wherein the binding affinity of the multimer is greater than the binding affinity of the monomer or dimer.

[0442] Embodiment 61. The multimer has an EC 50 greater than 5 μg / mL. A composition delivered without injection according to Embodiment 60.

[0443] Embodiment 62. The multimer has an EC 50 greater than 10 μg / mL. A composition delivered without injection according to Embodiment 61.

[0444] Embodiment 63. An orally delivered composition according to Embodiment 61, having an EC of the multimer of about 5 μg / mL to about 40 μg / mL. 50 The composition according to Embodiment 61, having an EC of the multimer of about 5 μg / mL to about 40 μg / mL.

[0445] Embodiment 64. A composition delivered without injection according to any one of Embodiments 59 to 63, wherein the binding affinity of the multimer is greater than the binding affinity of a multimer containing a smaller number of copies of an exogenous therapeutic agent or a combination of a smaller number of copies of an exogenous therapeutic agent.

[0446] Embodiment 65. A composition delivered without injection according to any one of Embodiments 59 to 64, wherein administering Spirulina containing a multimeric exogenous therapeutic agent results in a lower dose of Spirulina for efficacy than administering Spirulina containing a monomer of the same exogenous therapeutic agent.

[0447] Embodiment 66. The recombinant Spirulina is a composition delivered without injection as described in any one of Embodiments 1 to 65, selected from the group consisting of A. amethystine, A. ardissonei, A. argentina, A. balkrishnanii, A. baryana, A. boryana, A. braunii, A. breviarticulata, A. brevis, A. curta, A. desikacharyiensis, A. funiformis, A. fusiformis, A. ghannae, A. gigantean, A. gomontiana, A. gomontiana var. crassa, A. indica, A. jenneri var. platensis, A. jenneri Stizenberger, A. jenneri f. purpurea, A. joshii, A. khannae, A. laxa, A. laxissima, A. laxissima, A. leopoliensis, A. major, A. margaritae, A. massartii, A. massartii var. indica, A. maxima, A. meneghiniana, A. miniata var. constricta, A. miniata, A. miniata f. acutissima, A. neapolitana, A. nordstedtii, A. oceanica, A. okensis, A. pellucida, A. platensis, A. platensis var. non-constricta, A. platensis f. granulate, A. platensis f. minor, A. platensis var. tenuis, A. santannae, A. setchellii, A. skujae, A. spirulinoides f. tenuis, A. spirulinoides, A. subsalsa, A. subtilissima, A. tenuis, A. tenuissima, and A. versicolor.

[0448] Embodiment 67. The recombinant Spirulina is a composition delivered without injection as described in any one of Embodiments 1 to 66, which is not alive.

[0449] Embodiment 68. A composition delivered without injection according to any one of Embodiments 1 to 67, wherein the recombinant Spirulina is dried, spray-dried, freeze-dried, or lyophilized.

[0450] Embodiment 69. A composition delivered without injection according to any one of Embodiments 1 to 68, wherein the oral composition contains a pharmaceutically acceptable excipient.

[0451] Embodiment 70. A composition delivered without injection according to any one of Embodiments 1 to 69, which remains in the gastrointestinal tract or a simulated gastric environment.

[0452] Embodiment 71. A composition delivered without injection according to Embodiment 70, which remains in the gastrointestinal tract or a simulated gastric environment for at least 5 minutes.

[0453] Embodiment 72. A composition delivered without injection according to Embodiment 71, which remains in the gastrointestinal tract or a simulated gastric environment overnight.

[0454] Embodiment 73. A method for treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition delivered without injection according to any one of Embodiments 1 to 72 or 87 to 92.

[0455] Embodiment 74. The method according to Embodiment 73, wherein the disease or disorder is an infectious disease.

[0456] Embodiment 75. The method according to Embodiment 74, wherein the infectious disease is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection.

[0457] Embodiment 76. The method according to Embodiment 75, wherein the bacterium causing the infectious disease is selected from the group consisting of E. coli, enterotoxigenic E. coli (ETEC), Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella.

[0458] Embodiment 77. The method according to Embodiment 75, wherein the virus causing the infectious disease is selected from the group consisting of bacteriophage, RNA bacteriophage (e.g., MS2, AP205, PP7, and Qβ), infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus.

[0459] Embodiment 78. The method according to Embodiment 75, wherein the fungus causing the infectious disease is selected from the group consisting of Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma.

[0460] Embodiment 79. The method according to embodiment 75, wherein the parasite causing the infectious disease is selected from the group consisting of Plasmodium, P. falciparum, P. malariae, P. ovale, P. vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, parasitic worms: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostoma spp. and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp. and Wuchereria spp., Taenia spp., Echinococcus spp., and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp.

[0461] Embodiment 80. The method according to embodiment 73, wherein the disease or disorder is selected from the list consisting of celiac disease, type 1 diabetes, type 2 diabetes, cancer, inflammatory disorders, gastrointestinal diseases, autoimmune diseases or disorders, endocrine disorders, gastroesophageal reflux disease (GERD), ulcers, high cholesterol, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, constipation, and diarrhea.

[0462] Embodiment 81. A method for treating or preventing Campylobacter infection, comprising administering to a subject a composition delivered without relying on the injection described in any of embodiments 1 to 72.

[0463] Embodiment 82. The method according to embodiment 81, wherein the onset of symptoms caused by Campylobacter is reduced or prevented by administering a composition delivered without relying on injection.

[0464] Embodiment 83. The method according to any of embodiments 81 to 82, wherein the onset of inflammation in a subject is reduced or prevented by administering a composition delivered without relying on injection.

[0465] Embodiment 83. A method for treating or preventing C. difficile infection, comprising administering to a subject a composition that is delivered without relying on the injection described in any one of Embodiments 1 to 72.

[0466] Embodiment 84. The method according to Embodiment 83, wherein the onset of symptoms caused by C. difficile is reduced or prevented by administering a composition that is delivered without relying on injection.

[0467] Embodiment 85. The method according to any one of Embodiments 81 to 84, wherein the onset of diarrhea in the subject is reduced or prevented by administering a composition that is delivered without relying on injection.

[0468] Embodiment 86. The composition or method according to any one of Embodiments 1 to 85, wherein the therapeutic or prophylactic molecule is neither an antigen nor an epitope and is delivered without relying on the injection described.

[0469] Embodiment 88. The composition or method according to any of the foregoing embodiments, wherein a synergistic effect is exerted by administering two or more different recombinant Spirulinas containing different exogenous polypeptides or antigens or fragments thereof, and is delivered without relying on the injection described.

[0470] Embodiment 89. The composition or method according to Embodiment 88, wherein each of the different recombinant Spirulinas administered contains a different VHH and is delivered without relying on the injection described.

[0471] Embodiment 90. The composition or method according to any of the foregoing embodiments, wherein a synergistic effect is exerted by administering a recombinant Spirulina containing different exogenous polypeptides or antigens or fragments thereof, and is delivered without relying on the injection described.

[0472] Embodiment 91. The composition or method according to Embodiment 90, wherein the recombinant Spirulina contains two or more different VHH sequences and is delivered without relying on the injection described.

[0473] Embodiment 92. A composition or method in which recombinant Spirulina is delivered without injection as described in any of Embodiments 88 to 92, and which contains lysin.

[0474] Embodiment 93. A composition or method in which recombinant Spirulina is delivered without injection as described in any of Embodiments 88 to 92, and which contains lysin and an exogenous polypeptide.

[0475] Embodiment 94. A composition or method in which recombinant Spirulina is delivered without injection as described in Embodiment 94, and which contains lysin and a VHH.

[0476] Embodiment 95. A composition or method in which the composition is orally administered and is delivered without injection as described in any of the preceding embodiments.

[0477] Embodiment 96. A composition or method in which the composition is delivered to the airway and is delivered without injection as described in any of the preceding embodiments.

[0478] Embodiment 97. A composition or method in which the composition is delivered by inhalation or intranasally and is delivered without injection as described in Embodiment 88.

[0479] Embodiment 98. A composition or method in which the composition is Spirulina biomass and is delivered without injection as described in any of the preceding embodiments.

[0480] Embodiment 99. A composition or method in which the composition is delivered as an extract of Spirulina biomass and is delivered without injection as described in any of the preceding embodiments.

[0481] Embodiment 100. A composition or method in which the composition is delivered as a purified composition obtained from Spirulina biomass and is delivered without injection as described in any of the preceding embodiments. References: · Giallourou et al. A novel mouse model of Campylobacter jejuni enteropathy and diarrhea. PLoS Pathog. 2018 Mar; 14(3): e1007083. · Riazi et al. Pentavalent Single-Domain Antibodies Reduce Campylobacter jejuni Motility and Colonization in Chickens. PLoS One. 2013; 8(12): e83928.

[0482] Incorporation by reference This patent application incorporates by reference in its entirety the following patent publications and applications for all purposes: US10,131,870, US62 / 672,891 filed on May 17, 2018, and PCT / US2019 / 032998 filed on May 17, 2019.

[0483] All references, papers, publications, patents, patent publications, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. However, any reference to any reference, paper, publication, patent, patent publication, and patent application cited herein is not an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country of the world, nor should they be taken as such. The present invention provides, for example, the following items. (Item 1) A composition delivered without injection, comprising recombinant Spirulina, wherein the recombinant Spirulina comprises at least one therapeutic or prophylactic drug molecule. (Item 2) The composition for oral delivery according to Item 1, wherein the therapeutic or prophylactic drug molecule is delivered to the gastrointestinal tract, airway, or nasal cavity. (Item 3) The composition delivered without relying on the injection according to item 1 or 2, wherein the therapeutic or prophylactic drug molecule is exogenous to Spirulina. (Item 4) The composition delivered without relying on the injection according to item 3, wherein the exogenous molecule is a polypeptide or a fragment thereof. (Item 5) The composition delivered without relying on the injection according to item 3 or 4, wherein the exogenous polypeptide is an antibody or a fragment thereof. (Item 6) The composition delivered without relying on the injection according to item 5, wherein the antibody or a fragment thereof is a VHH. (Item 7) The composition delivered without relying on the injection according to item 3, wherein the exogenous polypeptide is an antigen or an epitope. (Item 8) The composition delivered without relying on the injection according to any one of items 1 to 7, wherein a disease or disorder is prevented, treated, or improved by administering the recombinant Spirulina to a subject. (Item 9) The composition delivered without relying on the injection according to any one of items 1 to 8, wherein an infectious disease is treated, prevented, or improved by administering the recombinant Spirulina to a subject. (Item 10) The composition delivered without relying on the injection according to item 9, wherein the infectious disease is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection. (Item 11) The composition delivered without injection according to item 10, wherein the bacterium causing the infectious disease is selected from the group consisting of E. coli, enterotoxigenic E. coli (ETEC), Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, helicobacter, Bacillus anthracis, ETEC, EHEC, EAEC, and Legionella. (Item 12) The composition delivered without injection according to item 10, wherein the virus causing the infectious disease is selected from the group consisting of bacteriophage, RNA bacteriophage (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus. (Item 13) The composition delivered without injection according to item 10, wherein the fungus causing the infectious disease is selected from the group consisting of Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma. (Item 14) The composition delivered without injection according to item 10, wherein the parasite causing the infectious disease is selected from the group consisting of Plasmodium, P. falciparum, P. malariae, P. ovale, P. vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, parasitic worms: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostoma spp. and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp. and Wuchereria spp., Taenia spp., Echinococcus spp., and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp. (Item 14) The composition delivered without injection according to any of the preceding items, wherein the exogenous polypeptide or a fragment thereof is present in a fusion protein. (Item 15) The composition delivered without injection according to any of the preceding items, wherein the recombinant Spirulina contains a nucleic acid encoding the exogenous polypeptide or a fragment thereof. (Item 16) The composition delivered without injection according to any of the preceding items, wherein the recombinant Spirulina contains an anti-Campylobacter VHH. (Item 17) The composition delivered without injection according to item 16, wherein the Campylobacter is C. jejuni. (Item 18) The composition delivered without injection according to item 16 or 17, wherein the VHH binds to a Campylobacter component. (Item 19) The composition delivered without injection according to item 18, wherein the VHH binds to flagellin. (Item 20) A composition that is delivered without injection as described in any of items 16 to 19, and that increases Campylobacter excretion upon administration. (Item 21) A composition that is delivered without injection as described in any of items 16 to 20, and that reduces the level of a biomarker upon administration. (Item 22) A composition that is delivered without injection as described in item 21, wherein the biomarker is an inflammatory biomarker. (Item 23) A composition that is delivered without injection as described in any of the preceding items, wherein the recombinant Spirulina contains a VHH that binds to an anti-Clostridium toxin. (Item 24) A composition that is delivered without injection as described in item 23, wherein Clostridium is C. difficile. (Item 25) A composition that is delivered without injection as described in any one of items 23 to 24, wherein the VHH binds to A toxin or B toxin, which is a Clostridium component. (Item 26) A composition that is delivered without injection as described in any of items 23 to 25, wherein the VHH contains an amino acid sequence of any one of SEQ ID NOs: 5 to 17. (Item 27) A composition that is delivered without injection as described in any of the preceding items, wherein the therapeutic or prophylactic drug molecule is a monomer. (Item 28) A composition that is delivered without injection as described in any of items 1 to 26, wherein the therapeutic or prophylactic drug molecule is a multimer. (Item 29) A composition that is delivered without injection as described in item 28, wherein the multimer is a heteromer. (Item 30) A composition that is delivered without injection as described in item 29, wherein the multimer is a homomer. (Item 31) The composition delivered without injection according to item 59, wherein the binding affinity of the multimer is greater than the binding affinity of the monomer or dimer. (Item 32) The composition delivered without injection according to any one of items 28 to 31, wherein the binding affinity of the multimer is greater than the binding affinity of a multimer containing a smaller number of copies of an exogenous therapeutic agent or a combination of a smaller number of copies of exogenous therapeutic agents. (Item 33) The composition delivered without injection according to any one of the preceding items, further comprising lysin. (Item 34) The recombinant Spirulina is a composition delivered without injection according to any of the above items, selected from the group consisting of A. amethystine, A. ardissonei, A. argentina, A. balkrishnanii, A. baryana, A. boryana, A. braunii, A. breviarticulata, A. brevis, A. curta, A. desikacharyiensis, A. funiformis, A. fusiformis, A. ghannae, A. gigantean, A. gomontiana, A. gomontiana var. crassa, A. indica, A. jenneri var. platensis, A. jenneri Stizenberger, A. jenneri f. purpurea, A. joshii, A. khannae, A. laxa, A. laxissima, A. laxissima, A. leopoliensis, A. major, A. margaritae, A. massartii, A. massartii var. indica, A. maxima, A. meneghiniana, A. miniata var. constricta, A. miniata, A. miniata f. acutissima, A. neapolitana, A. nordstedtii, A. oceanica, A. okensis, A. pellucida, A. platensis, A. platensis var. non-constricta, A. platensis f. granulate, A. platensis f. minor, A. platensis var. tenuis, A. santannae, A. setchellii, A. skujae, A. spirulinoides f. tenuis, A. spirulinoides, A. subsalsa, A. subtilissima, A. tenuis, A. tenuissima, and A. versicolor. (Item 35) The recombinant Spirulina is a composition delivered without injection according to any of the above items, which is not alive. (Item 36) A composition in which the recombinant Spirulina is delivered without relying on injection as described in any of the preceding items, and is dried, spray-dried, freeze-dried, or lyophilized. (Item 37) A composition in which the recombinant Spirulina is delivered as an extract without relying on injection as described in any of the preceding items. (Item 38) A composition in which the recombinant Spirulina is orally administered without relying on injection as described in any of the preceding items. (Item 39) A composition in which the recombinant Spirulina is administered to the respiratory tract without relying on injection as described in any of the preceding items. (Item 40) A composition in which the recombinant Spirulina is administered intranasally without relying on injection as described in item 39. (Item 41) A method for treating or preventing a disease or disorder in a subject in need thereof, comprising the step of administering to the subject a composition delivered without relying on injection as described in any one of the preceding items. (Item 42) The method according to item 41, wherein the disease or disorder is an infectious disease. (Item 43) The method according to item 42, wherein the infectious disease is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection. (Item 44) The method according to item 43, wherein the bacteria causing the infectious disease are selected from the group consisting of E. coli, enterotoxigenic E. coli (ETEC), Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella. (Item 45) The method according to item 43, wherein the virus causing the infectious disease is selected from the group consisting of bacteriophage, RNA bacteriophage (e.g., MS2, AP205, PP7, and Qβ), infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, white spot disease virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus. (Item 46) The method according to item 43, wherein the fungus causing the infectious disease is selected from the group consisting of Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma. (Item 47) The method according to item 43, wherein the parasite causing the infectious disease is selected from the group consisting of Plasmodium, P. falciparum, P. malariae, P. ovale, P. vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, parasitic helminths: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostoma spp., and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp., and Wuchereria spp., Taenia spp., Echinococcus spp., and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp.

Claims

[Claim 1] The invention described in this specification.

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