Arthrospira platensis injection-free therapeutic delivery platform

Abstract

The present disclosure provides a non-injectable composition comprising recombinant Spirulina containing at least one exogenous therapeutic agent. The composition of the present disclosure can be used as a vaccine and / or a therapeutic drug. The present disclosure also provides a method for producing recombinant Spirulina containing at least one exogenous therapeutic agent, and a method of treatment. Provided herein is a non-injectable composition comprising recombinant Spirulina, wherein the recombinant Spirulina contains at least one exogenous therapeutic agent, prophylactic agent molecule, or a combination of two or more exogenous therapeutic or prophylactic agent molecules.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 870,478, filed July 3, 2019, U.S. Provisional Patent Application No. 62 / 937,995, filed November 20, 2019, and U.S. Provisional Patent Application No. 62 / 943,075, filed December 3, 2019, the entire contents of each of which are incorporated herein by reference.

[0002] Incorporation by reference to sequence listing The contents of the text file submitted electronically herewith are incorporated by reference in their entirety into this specification: Computer-readable format copy of the Sequence Listing (File name: LUBI-029_01WO_SeqList.ST25txt, Recording date: July 3, 2020, File size approximately 100 kilobytes).

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

[0004] background Non-injection administration of therapeutic agents is a convenient, portable, and inexpensive form of administration. While nasal and oral administration of therapeutic agents is commonly practiced, oral therapeutic agents are exposed to harsh conditions in the digestive tract and may degrade before they can be effective. Furthermore, these therapeutic agents are expensive to produce, and purification of the therapeutic agent, along with the development of compositions that protect the therapeutic agent from the digestive enzymes and low pH to which oral therapeutic agents are exposed after administration, are required. There is a need for cost-effective and stable compositions for non-injection administration. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention The present application addresses the problems of cost and exposure to degradation of therapeutic agents in the digestive tract, nasal passages, and respiratory tract by administering the therapeutic agent to a subject in Spirulina. Spirulina is a cyanobacterium that can persist in the digestive tract, nasal passages, and respiratory tract, thus protecting the encapsulated therapeutic agent until the Spirulina reaches its destination (e.g., the gastrointestinal tract). Furthermore, Spirulina is easily cultivated and harvested, grows rapidly, can be dried to avoid spoilage, and can be consumed raw. In fact, Spirulina is approved for human consumption and is commonly 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 or prophylactic molecule, or a combination of two or more exogenous therapeutic or prophylactic molecules. The exogenous therapeutic agent may be a compound produced by a microorganism or a plant. In particular, the exogenous therapeutic agent may be an antimicrobial compound or polypeptide. In some embodiments, the exogenous therapeutic or prophylactic molecule is a VHH and / or a lysin.

[0007] In some embodiments, the present disclosure provides a non-injection delivery composition comprising recombinant Spirulina, wherein the recombinant Spirulina comprises at least one therapeutic or prophylactic molecule, or a combination of two or more therapeutic or prophylactic molecules. In some embodiments, the therapeutic or prophylactic molecule is delivered to the gastrointestinal tract. In some embodiments, the therapeutic or prophylactic molecule is delivered to the nose. In some embodiments, the therapeutic or prophylactic molecule is delivered by breathing (inhalation). In some embodiments, the therapeutic or prophylactic molecule is delivered systemically. In some embodiments, the therapeutic or prophylactic molecule is delivered locally.

[0008] In some embodiments, the therapeutic or prophylactic molecule or combination of two or more therapeutic or prophylactic molecules is an endogenous Spirulina molecule, hi some embodiments, the endogenous Spirulina molecule is found in concentrations greater than those found in naturally occurring Spirulina.

[0009] In some embodiments, the therapeutic or prophylactic molecule or combination of two or more therapeutic or prophylactic molecules is exogenous to Spirulina, hi some embodiments, the exogenous molecules are produced by different bacteria, parasites, protozoa, viruses, phages, algae, animals, or plants.

[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, wherein at least one of the therapeutic or prophylactic agent molecules present in the combination is present in a higher copy number (e.g., 2x, 3x, 4x, 5x, 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 selected from the group consisting of 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, a F(ab)2, a reduced IgG (rIgG), a monospecific Fab2, a bispecific Fab2, a trispecific Fab3, a diabody, a bispecific diabody, a trispecific triabody, a minibody, a nanobody, an IgNAR, a V-NAR, an HcIgG, or a combination thereof.

[0012] In some embodiments, 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 peptide (GIP), immunomodulatory agent, immunosuppressant, peptide chemotherapeutic agent, antimicrobial peptide, magainin, NRc-3, NRC-7, buforin IIb, BR2, p16, Tat, TNF-alpha, and chlorotoxin.

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

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

[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 ameliorates a disease or disorder selected from the group consisting of celiac disease, type 1 diabetes, type 2 diabetes, cancer, an inflammatory disorder, a gastrointestinal disease, an autoimmune disease or disorder, an endocrine disorder, gastroesophageal reflux disease (GERD), an ulcer, high cholesterol, an inflammatory bowel disorder, irritable bowel syndrome, Crohn's disease, ulcerative colitis, constipation, a vitamin deficiency, an iron deficiency, and diarrhea.

[0017] In some embodiments, administering recombinant Spirulina to a subject treats, prevents, or ameliorates infectious diseases, such as acute respiratory distress syndrome (ARDS), pneumonia, pericarditis, stroke, and COVID-19.

[0018] In some embodiments, the infection is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection. In some embodiments, the bacteria causing the infection 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, Enterohemmorrhagic E. coli (EHEC), Enteroaggregative E. coli (EAEC), and Legionella.

[0019] In some embodiments, the virus causing the infection is selected from the group consisting of 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, rabies virus, Newcastle disease virus, white spot syndrome virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus.

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

[0021] In some embodiments, the parasite causing the infection is selected from the group consisting of Plasmodium, P. falciparum, P. malariae, P. ovale, P. vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, helminthic parasites: 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 a fusion protein.

[0023] In some embodiments, the recombinant Spirulina contains a nucleic acid encoding an exogenous polypeptide or a fragment thereof. In some embodiments, the nucleic acid sequence encoding at least one exogenous polypeptide or a fragment thereof is present in at least two copies, at least three copies, at least four copies, or at least five copies in the recombinant Spirulina. In some embodiments, the nucleic acid sequence encoding at least one exogenous polypeptide or a fragment thereof is present in at least two copies, at least three copies, at least four copies, or at least five copies in the recombinant Spirulina. In some embodiments, the nucleic acid sequence encoding at least one exogenous polypeptide or a fragment thereof is present in at least two copies, at least three copies, at least four copies, or at least five copies in a single molecule of exogenous polypeptide expressed in the recombinant Spirulina.

[0024] In some embodiments, at least one exogenous polypeptide or fragment thereof is present in 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 in a single molecule of exogenous polypeptide expressed in recombinant Spirulina.

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

[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 spacer sequences.

[0027] In some embodiments, within the exogenous polypeptide or fragment thereof, some copies of the exogenous polypeptide or fragment thereof are linked in tandem, with the remaining copies of the exogenous polypeptide or fragment thereof being separated by a spacer sequence. In some embodiments, the spacer sequence is between about 1 and 50 amino acids in length. In some embodiments, there is more than one spacer sequence within the exogenous polypeptide or fragment thereof. In some embodiments, the recombinant Spirulina comprises 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 comprises a carrier or chaperone protein. In some embodiments, the carrier protein is selected from the group consisting of maltose binding protein, hedgehog hepatitis virus-like particle, thioredoxin, and phycocyanin. In some embodiments, the fusion protein comprises a scaffold protein.

[0029] In some embodiments, at least one exogenous polypeptide is linked to a scaffold protein at the N-terminus, C-terminus, or within the body of the scaffold protein. In some embodiments, the scaffold protein is selected from the oligomerization domain of C4b-binding protein (C4BP), the cholera toxin b subunit, or the oligomerization domain of an extracellular matrix protein. In some embodiments, the 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 fragment thereof, wherein the at least one exogenous polypeptide or 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), where E is the at least one exogenous polypeptide or fragment thereof, SP is the scaffold protein, and n, n1, and n2 represent the number of copies of the at least one exogenous polypeptide or fragment thereof.

[0031] In some embodiments, the recombinant Spirulina comprises an anti-Campylobacter VHH. In some embodiments, the 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 increases Campylobacter shedding. In some embodiments, administration reduces 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-Clostridial toxin. In some embodiments, the Clostridium is C. difficile. In some embodiments, the VHH binds to Clostridium components A toxin, B toxin, or both. In some embodiments, the VHH comprises the amino acid sequence of any 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 the amino acid sequence of any of SEQ ID NOs: 40 to 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 a 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 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 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 disposed 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 In some embodiments, the multimer has an EC of greater than 10 μg / mL. 50 In some embodiments, the multimer has an EC of about 5 μg / mL to about 40 μg / mL. 50 In some embodiments, the multimer has an EC of between about 0.10 nM and about 100 nM. 50 In some embodiments, the multimer has an EC of between about 0.2 nM and about 55 nM. 50 In some embodiments, the binding affinity of the multimer is greater than the binding affinity of a multimer comprising fewer copies of an exogenous therapeutic agent or a combination of fewer copies of an exogenous therapeutic agent. In some embodiments, administering Spirulina comprising a multimeric exogenous therapeutic agent requires a lower dose of Spirulina for efficacy than administering Spirulina comprising a monomer of the same exogenous therapeutic agent.

[0039] In some embodiments, the recombinant Spirulina is A. amethystine, A. ardissonei, A. argentina, A. balkrishnanii, A. baryana, A. boryana, A. braunii, A. breviar ticulata, 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 In some embodiments, the recombinant Spirulina is selected from the group consisting of A. 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 non-injectable composition comprises a pharmaceutically acceptable excipient.

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

[0042] In some embodiments, the composition remains in the nasal cavity. In some embodiments, the composition remains in the upper respiratory tract. In some embodiments, the composition remains in the respiratory tract. In some embodiments, the composition remains in the nasal cavity, upper respiratory tract and / or respiratory tract for at least 5 minutes. In some embodiments, the composition remains in the nasal cavity, upper respiratory tract and / or respiratory tract 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, comprising administering to the subject a non-injection delivered composition of the present disclosure.

[0044] In some embodiments, administering the non-injection delivered composition reduces or prevents the onset of Campylobacter symptoms.

[0045] In some embodiments, administering the delivered composition reduces or prevents the onset of inflammation in a subject.

[0046] In some embodiments, the present disclosure provides a method of treating or preventing a C. difficile infection, the method comprising administering to a subject a non-injection-delivered composition of the present disclosure. In some embodiments, administering the non-injection-delivered composition reduces or prevents the onset of C. difficile symptoms. In some embodiments, the present disclosure provides a method of treating or preventing a malaria infection, the method comprising administering by inhalation or intranasally a composition of the present disclosure. In some embodiments, inhalation or intranasal administration of the composition reduces or prevents the onset of malaria symptoms.

[0047] In some embodiments, the present disclosure provides a method of treating or preventing a coronavirus infection, the method comprising administering a composition of the present disclosure by inhalation or intranasally, in some embodiments, inhalation or intranasal administration of the composition reduces or prevents the onset of coronavirus symptoms.

[0048] In some embodiments, the present disclosure provides a method of treating or preventing a malaria infection, the method comprising administering to a subject a non-injection-delivered composition of the present disclosure, hi some embodiments, administering the non-injection-delivered composition reduces or prevents the onset of malaria symptoms.

[0049] In some embodiments, the present disclosure provides a method of treating or preventing a coronavirus (e.g., SARS virus, SARS-CoV-2 virus) infection, comprising administering to a subject a non-injection-delivered composition of the present disclosure. In some embodiments, administering the non-injection-delivered composition reduces or prevents the onset of symptoms of the coronavirus infection (e.g., ARDS, inflammation).

[0050] In some embodiments, provided herein are methods of making the non-injection compositions described herein, comprising introducing at least one exogenous therapeutic agent into Spirulina.

[0051] In some embodiments, provided herein are methods for making the non-injection compositions described herein, comprising introducing a nucleic acid sequence encoding at least one exogenous therapeutic agent into Spirulina.

[0052] In some embodiments, provided herein is a non-injectable antigenic composition comprising recombinant Spirulina, wherein the recombinant Spirulina comprises at least one exogenous antigenic epitope, and wherein a nucleic acid sequence encoding the at least one exogenous antigenic epitope has been integrated into the Spirulina by homologous recombination.

[0053] In some embodiments, provided herein are non-injectable antigenic compositions prepared by a method comprising introducing a nucleic acid sequence encoding at least one exogenous antigenic epitope into Spirulina and integrating the nucleic acid sequence into Spirulina by homologous recombination. [Brief explanation of the drawings]

[0054] [Figure 1] Figures 1A-B show that oral Spirulina monomeric anti-Campylobacter VHH provides complete protection against Campylobacter infection in mice. Daily administration of oral gavage containing 10% Spirulina biomass (425 μg of monomeric VHH per dose) for 5 days halted the development of diarrhea in Campylobacter-infected mice (Panel A) and reduced Campylobacter shedding compared to controls (Panel B).

[0055] [Figure 2]Figures 2A-B demonstrate that Spirulina expressing trimeric anti-Campylobacter VHH has anti-inflammatory effects in Campylobacter-infected mice. Daily administration of 0.5% Spirulina biomass (19 μg trimeric VHH per dose) for 3 days reduces fecal lipocalin (Panel A), a marker of inflammation, and myeloid cell infiltration into the gastrointestinal lamina propria (Panel B).

[0056] [Figure 3] Figure 3A-B: Weight change and histology scores for mice pretreated with Spirulina and infected with C. jejuni. Mice were pretreated with one dose (left) or three doses (right) of Spirulina. Figure 3A. Mice were infected with 10 CFU of C. jejuni at time 0 and treated with PBS (infection), Spirulina strain SP651 (anti- C. jejuni), or SP257 (irrelevant VHH). Weight fluctuations represent weight change 72 hours post-infection. Figure 3B. Cecums from animals were examined at 72 hours post-infection and scored for histopathology.

[0057] [Figure 4] Figures 4A-C: Body weight change and pathogen shedding in mice pretreated with a single dose of Spirulina and infected with C. jejuni. Mice were pretreated with 1.33 mg of Spirulina and inoculated with 10 CFU of C. jejuni at time 0. Mice were then treated with PBS (infection), Spirulina SP651 (anti- C. jejuni VHH), or SP257 (irrelevant VHH). Figure 4A. Body weight change at 72 hours post-infection. Figure 4B. Pathogen shedding at 24 and 72 hours post-infection. C. Fecal lipocalin-2 (LCN2) levels and the percentage of myeloid cells infiltrating the lamina propria (PMN%) at 72 hours post-infection. LCN2 was measured by ELISA. Gr1+, CD11b+ myeloid cells infiltrating the lamina propria were identified by FACS.

[0058] [Figure 5] Figures 5A-B: Weight change and pathogen shedding in mice pretreated with protease-resistant VHH variants in Spirulina and infected with C. jejuni. Mice were pretreated with a single dose of varying concentrations of Spirulina-VHH and infected with 10 CFU of C. jejuni. Each row of data represents a different treatment strain (SP526, SP806, or SP651). Figure 5A. Weight change at 72 hours post-infection. Figure 5B. Pathogen shedding at 24 and 72 hours post-infection. Open circles represent uninfected control mice. Because SP526-treated and SP806-treated mice were treated simultaneously, the same uninfected and infected control groups were used.

[0059] [Figure 6] Figure 6: Inflammatory markers and leukocyte infiltration into the lamina propria in mice pretreated with Spirulina and infected with C. jejuni. Mice were pretreated with a single dose of various concentrations of Spirulina-VHH and infected with 10 CFU of C. jejuni. Each row of data represents a different treatment strain (SP526, SP806, or SP651). A) Fecal lipocalin-2 (LCN2) levels 72 hours post-infection. B) Gr1+, CD11b+ myeloid cells (PMN%) infiltrating the lamina propria were identified by FACS. Open circles represent uninfected control mice. Because SP526- and SP806-treated mice were treated simultaneously, the same uninfected and infected control groups were used.

[0060] [Figure 7] Figure 7: SP1182 construct as both a schematic structure and a ribbon structure.

[0061] [Figure 8]Figure 8: Sequence of the SP1182 construct. The VHH binds to the flagellin protein flaA from C. jejuni. CDR1, CDR2, and CDR3 are indicated above the corresponding segment of the VHH sequence. Mass spectrometry data of the intact protein indicates that the N-terminal methionine has been removed. Maltose-binding protein serves to increase the expression level and solubility of the fusion VHH, while the hexahistidine tag serves as an affinity tag for detection reagents. Two short, flexible linkers, GG and GSG, bridge the VHH to MBP and MBP to the hexahistidine tag, respectively.

[0062] [Figure 9] Figure 9: Bacterial shedding (CFU / g feces) measured in feces at 40 and 72 hours post-infection. C. jejuni-only mice received no treatment. Two-dose (24 and 48 hours post-infection) and three-dose (24, 36, and 48 hours post-infection) mice received 1.33 mg of the indicated Spirulina-VHH per dose.

[0063] [Figure 10] Figure 10: Lipocalin (LCN2) levels measured in feces at 72 hours post-infection. Uninfected and C. jejuni-only mice received no treatment. Two-dose (24 and 48 hours post-infection) and three-dose (24, 36, and 48 hours post-infection) mice received 1.33 mg of the indicated Spirulina-VHH per dose.

[0064] [Figure 11] Figures 11A-B demonstrate that encapsulation of anti-Campylobacter VHHs within Spirulina protects the polypeptide in a simulated gastric environment: the anti-Campylobacter VHHs in Spirulina can still be detected after overnight exposure (Panel A), and the Spirulina cells themselves remain intact (Panel B).

[0065] [Figure 12] Figure 12 demonstrates that anti-Campylobacter expressed in Spirulina is stable in dried biomass at high temperatures for extended periods. Each curve represents serial 1:5 dilutions of biomass resuspended in PBS, incubated in ELISA plate wells coated with flagellin antigen, and detected with an anti-His tag antibody. Results were normalized to the binding activity of purified VHH assayed simultaneously.

[0066] [Figure 13] Figure 13 demonstrates mouse weights after C. jejuni infection. On day 0, mice were weighed, infected with C. jejuni, and treated with the indicated Spirulina strains (SP257, SP526, SP742, and SP806). Mice were then weighed every two days post-infection, and the % weight change was calculated based on the initial weight.

[0067] [Figure 14] Figure 14 demonstrates C. jejuni shedding. Groups of mice were challenged with C. jejuni on day 0 and treated with the indicated Spirulina strains (SP257, SP526, SP742, SP806). Every two days post-infection, fecal samples were collected from each mouse and the average C. jejuni colony count (cfu) per 10 mg of feces was determined.

[0068] [Figure 15] Figure 15 demonstrates inflammatory biomarkers in C. jejuni-infected mice treated with Spirulina. Levels of two inflammatory biomarkers, lipocalin-2 (LCN2) (left) and myeloperoxidase (MPO) (right), were measured in fecal samples 11 days after infection and treatment with the indicated Spirulina strains (SP257, SP526, SP742, or SP806). Group numbers refer to the Spirulina strain used for treatment.

[0069] [Figure 16]Figure 16 demonstrates weight change in mice pretreated with Spirulina and infected with C. jejuni. Mice were pretreated with one dose (left) or three doses (right) of Spirulina. Mice were infected with 10 CFU of C. jejuni at time 0 and treated with PBS (infection), Spirulina strain SP651 (anti- C. jejuni), or SP257 (irrelevant VHH). Weight fluctuations represent weight change 72 hours post-infection.

[0070] [Figure 17] Figures 17A-C demonstrate weight change and pathogen shedding in mice pretreated with a single dose of Spirulina and infected with C. jejuni. Mice were pretreated with 1.2 mg of Spirulina, inoculated with 10 CFU of C. jejuni at time 0, and treated with PBS (infection), Spirulina SP651 (anti- C. jejuni VHH), or SP257 (irrelevant VHH). A, Weight change at 72 hours post-infection. B, Pathogen shedding at 24 and 72 hours post-infection. C, Fecal lipocalin-2 (LCN2) levels at 72 hours post-infection. D, Gr1+, CD11b+ myeloid cells infiltrating the lamina propria were identified by FACS.

[0071] [Figure 18] Figures 18A-B demonstrate weight change and pathogen shedding in mice pretreated with protease-resistant VHH variants in Spirulina and infected with C. jejuni. Mice were pretreated with a single dose of varying concentrations of Spirulina-VHH and infected with 10 CFU of C. jejuni. Each row of data represents a different treatment strain (SP526, SP806, or SP651). A) Weight change at 72 hours post-infection. B) Pathogen shedding at 24 and 72 hours post-infection. Open circles represent uninfected control mice. SP526-treated and SP806-treated mice were treated simultaneously, so the same uninfected and infected control groups were used.

[0072] [Figure 19] Figures 19A-B demonstrate inflammatory markers and leukocyte infiltration into the lamina propria in mice pretreated with Spirulina and infected with C. jejuni. Mice were pretreated with a single dose of various concentrations of Spirulina-VHH and infected with 10 CFU of C. jejuni. Each row of data represents a different treatment strain (SP526, SP806, or SP651). A, Fecal lipocalin-2 (LCN2) levels 72 hours postinfection. B, Gr1+, CD11b+ myeloid cells infiltrating the lamina propria were identified by FACS. Open circles represent uninfected control mice. Because SP526- and SP806-treated mice were treated simultaneously, the same uninfected and infected control groups were used.

[0073] [Figure 20] Figure 20 demonstrates chick weights after inoculation with C. jejuni. Birds were treated with no treatment (SP526, SP651), an irrelevant (SP257), or Spirulina (Campy) prior to inoculation with C. jejuni 81-176 and weighed at intervals.

[0074] [Figure 21] Figure 21 demonstrates quantitative Campylobacter colonization mitigated by Spirulina-expressing VHHs. Birds were treated as in Figure 12. At 72 hours post-inoculation with 10 CFU of Campylobacter, birds were euthanized and cecal contents were collected for quantitative bacterial load determination.

[0075] [Figure 22]Figures 22A-C show Spirulina expression constructs. A) Expression constructs designed for Spirulina expression. Indicated are the orientation of the VHH, the chaperone fusion partner used, and the oligomeric state of the final product. Selected Spirulina strains expressing anti-CfaE VHHs are reported by numbers preceded by the designation "SP". B) Molecular structure of the complement binding protein C4B heptamerization domain (PDB ID 4B0F). An intermolecular disulfide bond links the monomers to form the heptamer. C) Dimerization domain of the cAMP-dependent protein kinase type I alpha regulatory subunit used to express homodimeric VHHs. An intermolecular disulfide bond links the monomers to form the dimer.

[0076] [Figure 23] Figures 23A-C show VHH expression in Spirulina. A) Example of Spirulina expression of monomeric and dimeric VHHs as analyzed by Western blotting. B) Intermolecular disulfide bond formation in the dimerization domain is confirmed by SDS-PAGE gel under reducing (R) and non-reducing (NR) conditions. The sizes of the corresponding fragments and molecules are indicated. C) Expression of heteroheptameric VHHs targeting the attachment domain on F4+ and F18+ porcine ETEC.

[0077] [Figure 24] Figures 24A-B show: A) ELISA-based VHH activity of Spirulina strains binding to the F4+ adhesin apical domain, FaeG. Antibody titrations were measured by diluting total protein extracts from a starting concentration of 1000 μg / ml. Homodimeric and heteroheptameric constructs bind well to the antigen. B) ELISA-based VHH activity of Spirulina strains binding to the F18+ adhesin apical domain, FedF. Antibody titrations were measured by diluting total protein extracts from a starting concentration of 1000 μg / ml. The heteroheptameric construct binds well to the antigen, but VHHs raised against the F4+ adhesin show no binding to the F18+ adhesin.

[0078] [Figure 25-1] Figures 25A-C: A) Western blot demonstrating protein expression in dried Spirulina biomass. B) VHHs in Spirulina slurries from spray-dried (SD) and freeze-dried (FD) powders show comparable binding based on ELISA. C) Antigen binding efficiency of Spirulina expressing VHHs assessed using BLI-based kinetic measurements. Biotin-tagged FaeG was loaded onto a streptavidin biosensor and binding to Spirulina extracts was measured. [Figure 25-2] Same as above.

[0079] [Figure 26] Figures 26A-C. Gnotobiotic bacterial challenge study. A) Overview of oral gavage protocol using a gnotobiotic piglet model. B) Effect of administration of SP795 on gut bacterial load. C) Effect of SP795 and SP-1156 on bacterial shedding in K88-resistant piglets.

[0080] [Figure 27-1] Figures 27A-C show anti-norovirus Spirulina expression constructs. A) Expression constructs designed for Spirulina expression. The orientation of the VHH and chaperone fusion partners used with selected Spirulina strains expressing anti-CfaE VHHs are reported by the number preceded by the designation "SP." B) Protein expression in Spirulina strains is assessed by Western blotting. C) NI-NTA purified proteins from strains expressing VHHs are assayed by SDS-PAGE gel and Coomassie staining. The expected full-length fragment is indicated by a red box. [Figure 27-2] Same as above. [Figure 27-3] Same as above.

[0081] [Figure 28-1]Figures 28A-C show anti-norovirus VHH binding activity. A) ELISA-based VHH activity of Spirulina strains binding to the GII.4 HuNoV genotype capsid protrusion protein (P1). VHHs expressed by Spirulina and purified with Ni-NTA were titrated in a dilution series starting at 20 μg / ml. SP834 (Nano-26-MBP) shows good binding to the GII.4 P1 domain. B) ELISA-based VHH activity of Spirulina strains binding to the GII.10 HuNoV genotype capsid protrusion protein (P1). VHHs expressed by Spirulina and purified with Ni-NTA were titrated in a dilution series starting at 20 μg / ml. SP834 (Nano-26-MBP) shows good binding to the GII.10 P1 domain. C) ELISA-based VHH activity of Spirulina strains binding to the GI.1 HuNoV genotype capsid protrusion protein (P1). Spirulina-expressed and Ni-NTA-purified VHHs were titrated in a dilution series starting at 20 μg / mL. SP835 (Nano-94-TxnA), SP836 (Nano-94-MBP), and SP864 (Nano-94) show good binding to the GI.1 P1 domain. [Figure 28-2] Same as above.

[0082] [Figure 29-1] Figures 29A-B show an alternative neutralization assay. Plates were coated with porcine gastric mucin (PGM) and blocked with skim milk. GII.10 (2 μg / ml) or GI.1 VLPs (1 μg / ml) were preincubated with serially diluted samples for 1 hour at RT and then added to the plate. Bound VLPs were detected with the GI.1-specific biotinylated nanobody NB60 or GII.10 polyclonal serum. Antibodies were detected with the corresponding secondary antibodies (strep-HRP or anti-rabbit HRP). (A) VHHs expressed by Spirulina and purified with Ni-NTA exhibit HBGA-blocking properties in a similar range to the control. (B) VHHs expressed by Spirulina and purified with Ni-NTA exhibit HBGA-blocking properties comparable to the control. [Figure 29-2] Same as above.

[0083] [Figure 30] Figure 30A-B: Sequence alignment of Nano85 and K922, an anti-human norovirus (HuNoV) protrusion (P) domain antibody. The antibody CDRs are highlighted in blue. Amino acid positions that influence antigen binding are boxed. B) Structural analysis of the framework region amino acid differences between Nano85 and K922 based on the HuNoV GII.10 P domain-bound Nano85 structure (PDB ID 4X7E). The boxed amino acid side chains indicate the mutations incorporated into the loop-grafted Nano85. The Nano85 CDR3, which governs the antigen-binding interaction, is circled.

[0084] [Figure 31-1] Figures 31A-C: A) Western blot analysis of Spirulina strains transformed with native Nano85 fused to the C-terminal MBP (SP1371) and loop-grafted Nano85 fused to the C-terminal MBP (SP1372) demonstrates protein expression. B and C) Bacterially expressed native Nano85 (B) and loop-grafted Nano85 (C) demonstrate binding to recombinant P domains from various HuNoV GiI strains (GII.2, GII.4, and GII.17). [Figure 31-2] Same as above.

[0085] [Figure 32-1]Figure 32A-B: Binding kinetics and cross-reactivity of VHHs targeting bacterially expressed recombinant anti-human norovirus (HuNoV) P domains. (A) ELISA-based binding and cross-reactivity of various VHHs (Nano85 loop-grafted and Nano26) raised against the HuNoV genotype GII.10 protrusion (P) domain recombinantly expressed in a bacterial expression system, or various VHHs (VHH3.2, VHH4.1, and VHH5.4) raised against the GII.4 P domain recombinantly expressed in a bacterial expression system. Nano26 and Nano85 show broad cross-reactivity, while VHH3.2, VHH4.1, and VHH5.4 show no binding to the recombinant GII.17 P domain. (B) BLI-based binding kinetics of various VHHs (Nano85 loop-grafted and Nano26) raised against the HuNoV genotype GII.10 P domain or various VHHs (VHH3.2, VHH4.1, and VHH5.4) raised against the GII.4 P domain. 100 nM biotin-tagged recombinant GII.2 P domain was used as the antigen. The VHH concentration used to generate the binding kinetics is shown for each VHH. [Figure 32-2] Same as above.

[0086] [Figure 33] Figures 33A-B: ELISA-based binding and cross-reactivity of VHHs targeting the anti-human norovirus (HuNoV) P domain. A) ELISA-based binding is shown for VHHs raised against the HuNoV genotype GI.1 protruding (P) domain: Nano94, VHH10.4, VHH6.3, and VHH7.3. The VHHs tested exhibited binding EC50s ranging from 0.21 nM to 50.07 nM, with recombinant nano94-TxnA expressed in Spirulina exhibiting the weakest binding. B) Cross-reactivity of VHHs against the recombinant HuNoV GI.3 P domain. VHH7.3 exhibited cross-reactive binding to the GI.3 P domain.

[0087] [Figure 34]Figure 34A-B: Proteins expressed in Spirulina and purified with Ni-NTA were stable after freeze-drying by lyophilization. A) Binding activity of recombinant anti-norovirus VHHs expressed in Spirulina shows no loss of binding activity to the recombinant HuNoV GII.10 P domain after freeze-drying (SP833_lyo, SP834_Lyo, and SP1241_Lyo) when compared to purified proteins stored at 4°C after purification (SP833, SP834, and SP1241, respectively). ELISA-based binding observed as measured by EC50 is given in the accompanying table. B) Binding activity of recombinant anti-norovirus VHHs expressed in Spirulina shows no loss of binding activity to the recombinant HuNoV GI.1 P domain (SP835_lyo and SP864_Lyo) after freeze-drying when compared to purified proteins (SP835 and SP864, respectively) stored at 4°C after purification. ELISA-based binding observed as measured by EC50 is given in the accompanying table.

[0088] [Figure 35]Figure 35: VHHs targeting the anti-norovirus capsid protrusion domain (P) exhibit varying degrees of protease sensitivity, with the loop-grafted Nano85 targeting the GII genotype group exhibiting the highest resistance to chymotrypsin and trypsin. Bacterially expressed recombinant VHHs (1 μg total protein) were incubated 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 digestion buffer (1 mM Tris pH 8.0, 20 mM CaCl). Samples were incubated for 1, 2, or 4 hours. Protease sensitivity was assayed using ELISA-based binding. High-binding ELISA plates were coated with recombinant GII.2 P domain. The level of active VHH after protease digestion was determined by assessing VHH binding to antigen. The percentage of active VHHs after digestion was calculated as a ratio of activity from VHHs incubated with PBS.

[0089] [Figure 36-1] Figures 36A-C show the design, expression, and activity of anti-TNFα Spirulina expression constructs. A) ID34F, an anti-TNFα VHH, was designed as a monomer (SP865) and dimer (SP1030) for Spirulina expression. Expression was confirmed by Western blotting. B) VHH expressed in Spirulina exhibits binding activity to recombinant human TNFα on ELISA plates when high-affinity plates were coated with human TNFα and VHH in the form of Spirulina crude lysate was titrated in a dilution series starting at 20,000 μg / ml. C) Binding efficiency was calculated as EC50. Both the monomeric and dimeric forms of VHH bind equally well. [Figure 36-2] Same as above. [Figure 36-3] Same as above.

[0090] [Figure 37] Figure 37 shows an overview of the development and testing of antitoxin B (C. difficile) VHHs.

[0091] [Figure 38-1] Figure 38A-D: Western blot expression analysis of Spirulina strains expressing anti-TcdB VHHs 5D and E3 in various hybridization contexts. "ssPsbU" and "ssPsbP2" indicate the presence of putative thylakoid-targeting signal sequences at the N-terminus of the indicated proteins, derived from the cyanobacterial photosystem proteins PsbU and PsbP2, respectively. pAP205, pMS2, pQb, and PP7 are enhanced signal peptide dimers derived from the capsid proteins of RNA phages AP205, MS2, Qb, and PP7, respectively. CCMk2 indicates the Spirulina carboxysome shell protein CCMk2, circularly permuted to position the N- and C-termini facing outward to enable genetic fusion with the indicated VHHs. Trx indicates thioredoxin. "Tri" and "pent" represent the synthetically designed non-covalent multimers 1na0C3 and DHR5C5_G2, respectively. Single and dual VHHs were added to the multimers in the orientations indicated on the blot. SPs 744, 745, 746, and 747 are thiredoxin fusions with 5D and E3 in both N- and C-terminal orientations. [Figure 38-2] Same as above. [Figure 38-3] Same as above. [Figure 38-4] Same as above.

[0092] [Figure 39-1] Figure 39 and Table 1 demonstrate the efficacy of various anti-tcdB VHH constructs. [Figure 39-2] Figure 39 and Table 1 demonstrate the efficacy of various anti-tcdB VHH constructs.

[0093] [Figure 40] Figure 40 demonstrates a colorimetric assay testing anti-tcdB VHH constructs.

[0094] [Figure 41-1] Figures 41A-O demonstrate morphology and cytotoxicity assays testing anti-tcdB VHH constructs. Figures 28I-K and 28M-28O: Characterization of anti-TcdB neutralization potency of high-performing Spirulina strains in Vero cell rounding assays using both the 027 and 10463 forms of TcdB. Spirulina lysates were normalized to transgene mass and compared to titrations of toxin at high and low concentrations. Figure 28I: SP744: VHH 5D-Trx neutralization curve; Figure 28J: SP985: VHH 5D-d.PP7 VLP neutralization curve; Figure 28K: SP1087: Trx-trimer-VHH.5D neutralization curve; Figure 28M: SP1095: VHH.E3-Trx-trimer-VHH.5D neutralization curve; Figure 28N: SP977: VHH.5D-dMS2 VLP neutralization curve; Figure 28O: SP1091: Trx-pentamer-VHH.5D. Figure 28L: Characterization of the anti-TcdB neutralization potency of selected Spirulina strains in a Vero cell rounding assay using the 027 form of TcdB. Spirulina lysates were normalized to transgene mass and compared to a titration of toxin at high and low concentrations. The best performers are indicated by red ovals. [Figure 41-2] Same as above. [Figure 41-3] Same as above. [Figure 41-4] Same as above. [Figure 41-5] Same as above. [Figure 41-6] Same as above. [Figure 41-7] Same as above. [Figure 41-8] Same as above. [Figure 41-9] Same as above. [Figure 41-10] Same as above. [Figure 41-11] Same as above. [Figure 41-12] Same as above. [Figure 41-13] Same as above. [Figure 41-14] Same as above. [Figure 41-15] Same as above.

[0095] [Figure 42] Figure 42 demonstrates the binding strength of various VHH sequences to C. difficile TcdB toxin.

[0096] [Figure 43] Figure 43 demonstrates the binding strength of different VHH sequence combinations to C. difficile TcdB toxin.

[0097] [Figure 44-1] Figure 44 demonstrates the binding strength of the combination of 5D, E3 and 7F VHHs to C. difficile TcdB toxin both alone and in combination. [Figure 44-2] Same as above.

[0098] [Figure 45-1] Figures 45A-B demonstrate the effect of VHH concentration on binding to C. difficile TcdB toxin. Increasing the concentration of any single VHH sequence had little effect on efficacy, but surprisingly, increasing the concentration of the combination of VHH sequences showed a large increase in efficacy. [Figure 45-2] Same as above.

[0099] [Figure 46] Figure 46 shows the putative synergistic effect of different VHHs on C. difficile infection and signaling.

[0100] [Figure 47-1] Figures 47A-B: Two-way synergy between anti-TcdB VHHs. Scoring indicates cell rounding index as determined by visual inspection: 7 = normal, 1 = 100% rounded, 4 = 50% rounded. Scores of 5 and 6 fall on a gradient from 50% round to 100% normal, and scores of 3 and 2 fall on a similar gradient from 50% round to 100% round. [Figure 47-2] Same as above.

[0101] [Figure 48]Figure 48: Synergistic individual and two-way combinations of anti-TcdB VHHs measured in a Vero cell rounding assay using TcdB 027.

[0102] [Figure 49] FIG. 49 describes putative cocktails for preventing and / or treating C. difficile infection.

[0103] [Figure 50] Figure 50: Schematic representation of VHH hybridization with candidate scaffold partners. VHHs were selected based on our evaluation and published structure / function studies on TcdB.

[0104] [Figure 51] Figure 51 shows constructs for evaluation of rigid interdomain linkers.

[0105] [Figure 52-1] Figure 52 shows the crystal structure of VHH E3 co-crystallized with TcdB. [Figure 52-2] Same as above.

[0106] [Figure 53] Figure 53 shows exemplary sequences for engineering VHH.E3-like activity relative to other frameworks.

[0107] [Figure 54] Figure 54 shows the adherence values ​​for individual VHHs produced in Spirulina.

[0108] [Figure 55] Figure 55 demonstrates that the mixture of three Spirulina-expressing VHHs (5D+E3+7F) is substantially stronger than the individual components.

[0109] [Figure 56] Figure 56 shows the adhesion values ​​for a mixture of Spirulina produced VHHs.

[0110] [Figure 57] Figure 57 shows that a mixture of Spirulina-produced VHHs neutralizes high doses of TcdB.

[0111] [Figure 58] Figure 58 shows how the present disclosure can be used to rapidly discover custom antibodies for oral delivery.

[0112] [Figure 59] Figure 59 shows maximized strain cross-reactivity. Comparison of the domains in FlaA targeted by LMN-101 from the Navy (NCBIC) C. jejuni database (>10,000 sequences) showed that 79% of the sequences share at least 75% homology, suggesting that the cross-reactivity of this one lead VHH may extend to 79% of Campylobacter strains.

[0113] [Figure 60] Figure 60 shows a proposed model for the prevention of C. difficile in mice.

[0114] [Figure 61] FIG. 61 shows an exemplary double-blind, placebo-controlled study to evaluate the safety and tolerability of LMN-101.

[0115] [Figure 62] FIG. 62 shows an exemplary double-blind, placebo-controlled study to evaluate the safety and prophylactic activity of LMN-101 against C. jejuni CG8421 (human challenge strain).

[0116] [Figure 63]Figure 63 shows the results of a cell lysis assay for both E. coli and Spirulina-expressed proteins. Log-phase cultures of C. difficile, OD600=1, were treated with the indicated concentrations of lysin. Cell lysis was measured over time by a decrease in optical density. The Spirulina-expressed lysin is biologically active.

[0117] [Figure 64] Figure 64: Effect of rigid linkers on VHH 5D neutralizing activity. The assay has a numerical readout ranging from 1 (completely detached and dying) to 7 (normal).

[0118] [Figure 65] Figure 65: Overview of Spirulina stability assay.

[0119] [Figure 66] Figure 66: Aqueous stability study of SP1308, MBP-5HVZ-VHH 5D. Lysates were incubated in medium for 4 hours.

[0120] [Figure 67] Figure 67: Aqueous stability study of SP1312, MBP-5HVZ-VHH E3. Lysates were incubated in medium for 4 hours.

[0121] [Figure 68] Figure 68: Aqueous stability study of SP1308+SP1312+SP1313. Lysates were incubated in media for 4 hours.

[0122] [Figure 69] Figure 69A-B: Aqueous stability of VHHs. Aqueous stability of VHHs was measured at 12 hours using a VERO cell, cell rounding assay. Panel A) shows neutralizing activity. Panel B) shows cell rounding assay.

[0123] [Figure 70]Figure 70: Overview of the gnotobiotic pig model to evaluate the effect of anti-TcdB VHHs on C. difficile infection.

[0124] [Figure 71-1] Figure 71A-B: Clinical data: Piglet III treated with 3-VHH combination + / - lysin. A) Diarrhea burden among animals experimentally infected with Clostridium difficile strain 027. B) Diarrhea burden among individual animals. Animals were treated from day -1 until the end of the study with PBS (negative control), wild-type Spirulina (negative control), or Spirulina containing three different anti-TcdB VHHs (Mix 1), or Spirulina containing the same three VHHs and anti-Clostridial lysin (Mix 2). [Figure 71-2] Same as above.

[0125] [Figure 72] Figure 72: Overview of Monash mouse CDI model studies of anti-TcdB VHHs.

[0126] [Figure 73] Figure 73A-B: Prophylactic activity of anti-TcdB VHHs with or without C. difficile-specific lysin in a mouse model of CDI. Mice were treated daily with the indicated Spirulina biomass, or with vancomycin as a positive control, starting on day -1 and continuing through day 4. Mice were inoculated with pandemic 027 C. difficile on day 0. A) Effect on weight loss associated with CDI. B) Effect on survival. C) Effect on C. difficile spore shedding. (Dashed line is limit of detection.)

[0127] [Figure 74]Figure 74: ELISA titration curves of SP1182 extracts prepared in buffers of various pH. Each binding curve represents a four-fold serial dilution of protein extract from Spirulina biomass (μg / mL) resuspended at different pH. Each curve was internally normalized to 1. Results are the average of two replicates.

[0128] [Figure 75] Figure 75: Western blot gel analysis of SP1182 extracts prepared in buffers of various pH. Lanes represent 600-fold dilutions of clarified Spirulina extracts from Spirulina biomass resuspended at 50 mg / mL and extracted for 60 minutes in buffers of different pH.

[0129] [Figure 76] Figure 76: Western blot of gut phase digestion of dried Spirulina-VHH biomass. Spray-dried Spirulina-VHH from SP806 was incubated in SIF for the times indicated. All incubation times are shown in minutes or overnight (ON). The experiment was performed twice at different time points (left and right panels). Intact biomass (pellet) was analyzed in parallel with the release sample (supernatant). Samples were run on a Western blot and detected with an anti-VHH antibody. The arrow indicates the expected band size for the full-length VHH protein.

[0130] [Figure 77] Figure 77: Western blot of in vitro gut phase digestion of SP1182 drug substance. Dried Spirulina biomass was incubated in SIF for the times indicated. Intact biomass (pellet) was analyzed in parallel with the release sample (supernatant). Samples were run on a Western blot and detected with an anti-VHH antibody. The red box indicates the band of VHH (aa682).

[0131] [Figure 78]Figure 78: Western blot of in vitro gut phase digestion of aa682. Purified aa682 was incubated in simulated intestinal fluid for the times indicated. Samples were run on a Western blot and detected with an anti-VHH antibody.

[0132] [Figure 79] Figure 79: SDS-PAGE analysis of gastric phase digestion of dried Spirulina biomass. Spray-dried Spirulina biomass (containing trimeric VHH, SP806) was incubated in SGF for the indicated times or overnight (O / N). Intact biomass (pellet) was analyzed in parallel with the release sample (supernatant). Samples were run on an SDS-PAGE gel and analyzed by Coomassie staining (upper gel) and Western blot (lower gel). Proteins were detected by anti-VHH antibody on Western blot. The black box highlights the band corresponding to the VHH.

[0133] [Figure 80] Figure 80: Western blot of gastric phase digestion of SP1182 drug substance. Dried Spirulina was incubated in SGF for the indicated times or overnight (O / N). Intact biomass (pellet) was analyzed in parallel with the release sample (supernatant). Samples were run on a Western blot and detected with an anti-VHH antibody. The red box indicates the band of VHH (aa682).

[0134] [Figure 81] Figure 81: Serum IgG response to maltose binding protein (MBP) on day 14. Serum was diluted as indicated. PO = oral administration; IN = intranasal administration. Pos. cont = positive control of hyperimmune serum in 3-fold serial dilutions starting at 1 / 200.

[0135] [Figure 82]Figure 82: Serum IgG response to NANP on day 14. Serum was diluted as indicated. PO = oral administration; IN = intranasal administration. Pos. cont = positive control of hyperimmune serum in 3-fold serial dilutions starting at 1 / 200. Groups 2 and 3 show IgG antibody production by day 14.

[0136] [Figure 83] Figure 83: Serum IgG response to NANP on day 27. Serum was diluted as indicated. PO = oral administration; IN = intranasal administration. Pos. cont = positive control of hyperimmune serum in 3-fold serial dilutions starting at 1 / 200. Groups 2 and 3 show IgG antibody production by day 27.

[0137] [Figure 84] Figure 84: Serum IgG response to NANP on day 41. Serum was diluted as indicated. PO = oral administration; IN = intranasal administration. Pos. cont = positive control of hyperimmune serum in 3-fold serial dilutions starting at 1 / 200. Groups 2 and 3 show IgG antibody production by day 41.

[0138] [Figure 85] Figure 85: Serum IgG response to NANP on day 56. Serum was diluted as indicated. PO = oral administration; IN = intranasal administration. Pos. cont = positive control of hyperimmune serum in 3-fold serial dilutions starting at 1 / 200. Groups 2 and 3 show IgG antibody production by day 56.

[0139] [Figure 86] Figure 86: Serum IgG response to NANP on day 69. Serum was diluted as indicated. PO = oral administration; IN = intranasal administration. Pos. cont = positive control of hyperimmune serum in 3-fold serial dilutions starting at 1 / 200. Groups 2 and 3 show IgG antibody production by day 69.

[0140] [Figure 87] Figure 87: Survival of vaccinated mice after challenge with P. falciparum. DETAILED DESCRIPTION OF THE INVENTION

[0141] Detailed Description The present disclosure teaches packaging exogenous therapeutic or prophylactic molecules into prokaryotic algae and then administering them to a subject by non-injection means. In some embodiments, the recombinant prokaryotic algae are edible and can serve as an edible composition for delivering the payload expressed in the algae. In the case of polypeptide therapeutic or prophylactic molecules (e.g., antibodies, antigens, etc.), the expression level of the exogenous polypeptide is 10-100 times higher in the Spirulina delivery system of the present disclosure 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 for making 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 compositions or biological systems may vary. It should also be understood that the terminology used herein is merely for the purpose of describing certain exemplary embodiments and is not intended to be limiting. The terms used herein generally have their ordinary meanings in the art in the context of this disclosure and in the specific context in which each term is used. Specific terms are discussed below or elsewhere herein to provide practitioners with additional guidance regarding the description of the compositions and methods of the present disclosure and how to make and use them. The scope and meaning of any term's use will be apparent from the specific context in which the term is used. As such, the definitions set forth herein are intended to provide exemplary guidance in identifying specific embodiments of the present disclosure, without limiting them solely to specific compositions or biological systems.

[0144] In accordance with long-standing patent law convention, the terms "a," "an," and "the," when used in this application, including the claims, refer to "one or more" unless expressly 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 to a disease or disorder, or that can be used to treat a disease or disorder described herein.

[0146] The term "antigen," as used herein, refers to a protein or peptide that binds to a receptor on an immune cell in a human or animal and induces an immune response. Antigens can be derived from infectious microorganisms, including viruses, bacteria, parasites, or fungi, or they can be tumor antigens or autoantigens associated with autoimmune diseases.

[0147] The term "antigenic epitope," as used herein, refers to a short amino acid sequence, for example, a sequence of about 4 to 1,000 amino acids, of an antigen that is recognized by and binds to a receptor on an immune cell in a human or animal, thereby inducing an immune response. The antigenic epitopes of the present disclosure are derived from the above-mentioned antigens.

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

[0149] Non-injectable therapeutic compositions Provided herein is a non-injectable composition comprising recombinant Spirulina, wherein the Spirulina has been engineered to contain at least one exogenous therapeutic agent or 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 "prophylactic agent" refers to any molecule that can be used to prevent the onset of a disease or disorder in a subject.

[0150] Delivery of therapeutic or prophylactic molecules encapsulated in spirulina without injection offers several advantages. One of these advantages is increased resistance of the encapsulated therapeutic or prophylactic molecule to proteolytic degradation. For example, when delivered orally, encapsulation in spirulina protects the therapeutic or prophylactic molecule from enzymes and gastrointestinal conditions, thereby allowing delivery of the therapeutic agent to the portion of the gastrointestinal tract that digests the spirulina cells and releases the therapeutic or prophylactic molecule. In some embodiments, orally delivered compositions of the present disclosure remain (e.g., remain substantially intact) at a pH of about 1.3 to about 8.0. In some embodiments, orally delivered compositions of the present disclosure remain in the oral cavity. In some embodiments, orally delivered compositions of the present disclosure remain in the stomach. In some embodiments, orally delivered compositions remain in the small intestine and / or large intestine. In some embodiments, orally delivered compositions remain in the colon. In some embodiments, orally delivered compositions remain 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 composition may remain in the gastrointestinal conditions or simulated gastric environment for about 5 minutes to about 1 day. In some embodiments, the orally delivered composition remains in the gastrointestinal conditions or simulated gastric environment 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, or about 24 hours. In some embodiments, the orally delivered composition remains in the gastrointestinal conditions or simulated gastric environment overnight. [Added paragraph regarding nasal and respiratory tract]

[0151] In some embodiments, compositions delivered without injection of the present disclosure remain (e.g., remain substantially intact) at a pH of about 5.0 to about 8.0. In some embodiments, compositions delivered without injection of the present disclosure remain (e.g., remain substantially intact) at a pH of about 5.5 to about 6.5. In some embodiments, compositions delivered without injection of the present disclosure remain in the oral cavity. In some embodiments, compositions delivered without injection of the present disclosure remain in the nose. In some embodiments, compositions delivered without injection remain in the pharynx. In some embodiments, compositions delivered without injection remain in the trachea. In some embodiments, compositions delivered without injection remain in the bronchi. In some embodiments, compositions delivered without injection remain in the lungs. In some embodiments, compositions delivered without injection remain in the alveoli. In some embodiments, compositions delivered without injection remain in the airways. In some embodiments, compositions delivered without injection remain 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 may remain in nasal, airway, or simulated airway conditions for about 5 minutes to about 1 day. In some embodiments, the composition delivered without injection may remain in nasal, airway, or simulated airway conditions 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, or about 24 hours. In some embodiments, the composition delivered without injection may remain in nasal, airway, or simulated airway conditions overnight. In some embodiments, the composition delivered non-by injection is an extract of recombinant Spirulina biomass.

[0152] Another advantage of the non-injection delivery compositions of the present disclosure is their stability during storage. In some aspects, the non-injection delivery compositions of the present disclosure are stable at elevated temperatures (e.g., temperatures higher than room temperature). In some embodiments, the non-injection delivery compositions of the present disclosure are stable at 42°C. In some embodiments, the non-injection delivery compositions of the present disclosure are stable at 42°C for about 1 day to 5 years. In some embodiments, the non-injection delivery compositions of 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 non-injection delivery compositions of the present disclosure are stable at 42°C for 1 month or 3 months. In some embodiments, the non-injection delivery compositions of the present disclosure are stable at room temperature (e.g., about 20°C to about 29°C). In some embodiments, the non-injection delivered compositions of the present disclosure are stable at 27° C. In some embodiments, the non-injection delivered compositions of the present disclosure are stable at 27° C. for about 1 day to 5 years. In some embodiments, the non-injection delivered compositions of 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 non-injection delivered compositions of the present disclosure are stable at 27° C. for 1 month or 3 months.

[0153] Treatment drugs Any exogenous (i.e., non-Spirulina) therapeutic or prophylactic molecule suitable for administration without injection can be used in the compositions and methods of the present disclosure. In some embodiments, the therapeutic or prophylactic molecule is a small molecule. In some embodiments, the therapeutic or prophylactic molecule is a polypeptide or fragment thereof. In some embodiments, the recombinant Spirulina comprises a mixture of therapeutic agents, including a mixture of polypeptides or fragments thereof, a mixture of small molecules, or a mixture of prophylactic molecules and / or polypeptides or fragments thereof and small molecules.

[0154] In some embodiments, the therapeutic or prophylactic molecule is a small molecule produced by a cell. In some embodiments, the small molecule is produced by a microorganism, such as a bacterium, a virus, a fungus, or a 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, malacidins, penicillins, streptomycins, polymyxins, colistins, circulins, bacillomycins, mycobacillins, fungistatins, tannins, terpenoids, saponins, alkaloids, flavonoids, polyphenols, saponins, chloroquine, quinine, amodiaquine, hydroxychloroquine, metronidazole, tinidazole, iodoquinol, paromomycin, metronidazole, and tinidazole, or combinations thereof.

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

[0157] In some aspects, the present disclosure does not include compositions or methods of Spirulina comprising an antigen, antigenic epitope, or fragment thereof. In some embodiments, the present disclosure does not include the subject matter of PCT / US2019 / 032998, filed May 17, 2019. In some embodiments, the present disclosure does not include compositions or methods for eliciting or increasing an immune response in a subject. In some embodiments, the present disclosure does not include compositions or methods for eliciting or increasing the production of antibodies or fragments thereof against exogenous polypeptides 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 selected from the group consisting of, but 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, a F(ab)2, a reduced IgG (rIgG), a monospecific Fab2, a bispecific Fab2, a trispecific Fab3, a diabody, a bispecific diabody, a trispecific triabody, a minibody, a nanobody, an IgNAR, a V-NAR, an HcIgG, or a combination thereof.

[0159] In some embodiments, the therapeutic peptide is an antibacterial agent that is effective against bacteria, including, but not limited to, E. coli, enterotoxigenic E. coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophages, RNA bacteriophages (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 syndrome virus, Coronavirus, SARS virus, MERS virus, SARS-CoV-2 virus, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmo dium, P.falciparum, P.malariae, P.ovale, P.vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, Parasitic helminth: 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.or combinations thereof relating to, resulting from, or treating or preventing infection by any microorganism.

[0160] In some embodiments, the exogenous polypeptide is an antigen or autoantigen. In some embodiments, the autoantigen is associated with 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 associated with an autoimmune disease.

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

[0163] In some embodiments, compositions of the present disclosure comprise recombinant Spirulina comprising at least one polypeptide, antigen, or antigenic epitope from a virus, including, but not limited to, bacteriophage, RNA bacteriophage (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 syndrome virus, coronavirus, MERS virus, SARS virus, and 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 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 from a coronavirus. In some embodiments, the compositions of the present disclosure include recombinant Spirulina containing at least one polypeptide, antigen, or antigenic epitope from 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 from a parvovirus, such as canine parvovirus. In some embodiments, the compositions of the present disclosure include recombinant Spirulina SP673 or SP678.

[0164] In some embodiments, compositions of the present disclosure comprise recombinant Spirulina comprising at least one polypeptide or fragment thereof that binds to a virus or portion thereof, including, but not limited to, bacteriophage, RNA bacteriophage (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 syndrome virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus.

[0165] In some embodiments, the recombinant Spirulina comprises a polypeptide or fragment thereof that binds to a Norovirus polypeptide or antigen. In some embodiments, the recombinant Spirulina comprises a polypeptide or fragment thereof that binds to a Norovirus P domain. In some embodiments, the polypeptide or 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 a Norovirus P domain. In some embodiments, the recombinant Spirulina comprises a polypeptide or fragment thereof that binds to the GII genotype, the G1 genotype, or the G11.10 genotype. In some embodiments, the recombinant Spirulina comprises a polypeptide or fragment thereof that binds to a polypeptide from two or more Norovirus genotypes. In some embodiments, the recombinant Spirulina comprises a VHH comprising a Nano85 nanobody, a Nano26 nanobody, a Nano94 nanobody, a K922 antibody, or a modified sequence or fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH comprising Nano85 and / or a loop-grafted modification thereof. In some embodiments, the VHH comprises an amino acid sequence of any one of SEQ ID NOs: 40-79 or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or 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 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 fragment thereof that binds to an anti-Clostridial toxin or a fragment thereof.In some embodiments, the recombinant Spirulina comprises a VHH comprising Nano85 and / or a loop-grafted modification 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 VHH comprising Nano85 and / or a loop-grafted modification thereof 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 recombinant Spirulina comprising at least one antigenic epitope 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 pilus 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 pilus polypeptide, or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that binds to an adhesin, or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a VHH that binds to a polypeptide from two or more adhesins. In some embodiments, the recombinant Spirulina comprises a polypeptide, or a fragment thereof, that binds to the F4+ adhesin domain FaeG or the F18+ adhesin domain FedF. In some embodiments, the recombinant Spirulina comprises a polypeptide, or a fragment thereof, that binds to one or more of adhesins K88 (also referred to as F4), K99 (F5), 987P (F6), F41, and F18, or modifications or fragments thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or fragment thereof that binds to K88. In some embodiments, the recombinant Spirulina comprises a polypeptide or fragment thereof that binds to an ETEC polypeptide or antigen or 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 fragment thereof that binds to an ETEC polypeptide or antigen or 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 fragment thereof that binds to an ETEC polypeptide or fragment thereof, or a monomer, dimer, or heptamer of an ETEC polypeptide or antigen or 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-Clostridial toxin. In some embodiments, the Clostridium is C. difficile. In some embodiments, the recombinant Spirulina comprises a VHH that binds to an anti-Clostridial toxin. In some embodiments, the polypeptide or fragment thereof binds to Clostridial components A toxin, B toxin, or both. In some embodiments, the polypeptide is a VHH comprising an amino acid sequence of any of SEQ ID NOs: 5-17 or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or fragment thereof that binds to a Clostridial antigen or fragment thereof, or an anti-Clostridial toxin or fragment thereof, fused with a chaperone polypeptide. In some embodiments, multiple copies of the recombinant Spirulina comprise a polypeptide or fragment thereof that binds to a Clostridial antigen or fragment thereof, or an anti-Clostridial toxin or fragment thereof, fused 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 fragment thereof that binds to a Clostridial antigen or fragment thereof, or an anti-Clostridial toxin or 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 Campylobacter or an 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 fragment thereof that binds to a Campylobacter polypeptide or antigen or 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 fragment thereof that binds to a Campylobacter polypeptide, antigen, or 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 a 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 a 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 a 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 fragment thereof is a VHH comprising the amino acid sequence of any one of SEQ ID NOs: 26-31 or a fragment thereof. In some embodiments, the recombinant Spirulina comprises a polypeptide or 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 chaperone polypeptide is maltose binding protein (MBP) or thioredoxin A (TxnA). In some embodiments, the recombinant Spirulina comprises multiple copies of a polypeptide or 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 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, the at least one exogenous polypeptide expressed in Spirulina is included in the exogenous antigen. In some embodiments, the exogenous antigen is a natural antigen. For example, recombinant Spirulina may express the entire circumsporozoite protein containing one or more antigenic epitopes, or a portion or domain of the circumsporozoite protein containing one or more antigenic epitopes. In this case, the exogenous antigen is considered to be a natural antigen. Other examples of natural antigens that can be expressed in Spirulina to prepare oral antigenic compositions include influenza virus hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins.

[0173] In addition to immunogenic epitopes, the present disclosure provides structures and / or ligands for stimulating the innate immune system (e.g., by engineering epitopes into VLP structures). The innate immune system can be activated by the inherent adjuvant-like properties of VLPs and / or by adjuvants added to vaccine compositions. In some embodiments, these structures and / or ligands that stimulate 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 may 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 fragment thereof is present in a 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 comprises one or more polypeptides or fragments thereof attached to one or more scaffold polypeptides. In some embodiments, a fusion protein comprises one or more polypeptides or fragments thereof attached to one or more chaperone polypeptides. In some embodiments, a fusion protein comprises a tag for separation and / or purification (e.g., a 6xHis tag). In some embodiments, a fusion protein comprises one or more targeting signals or polypeptides. In some embodiments, a fusion protein comprises one or more VHH sequences as fusions with one or more chaperone polypeptides. In some embodiments, a fusion protein comprises one or more VHH sequences as fusions with one or more chaperone polypeptides and one or more scaffold polypeptides.

[0174] In some embodiments, the exogenous antigen epitopes may be derived from different antigens that activate different types of immunity (e.g., innate immunity, cellular immunity, or humoral immunity). In some embodiments, the 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, the one or more exogenous antigen epitopes are present in a fusion protein with a viral protein (e.g., coronavirus spike protein). In some embodiments, the one or more exogenous antigen epitopes are present in a fusion protein with a viral protein (e.g., coronavirus spike protein), with one epitope at either end. In some embodiments, the one or more exogenous antigen epitopes are a B cell epitope fused to one end of the 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 molecules. In some embodiments, the compositions of the present disclosure comprise recombinant Spirulina comprising a combination of therapeutic and / or prophylactic molecules. In some embodiments, the oral compositions of the present disclosure comprise recombinant Spirulina comprising multiple copies of one therapeutic or prophylactic agent and at least one other therapeutic or prophylactic 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 molecule. In some embodiments, the compositions of the present disclosure comprise at least one VHH and at least one other therapeutic or prophylactic 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 molecules are enzymes. In some embodiments, the enzymes are hydrolases. In some embodiments, the hydrolases cleave cell walls. In some embodiments, the hydrolases target bonds in peptidoglycan. In some embodiments, the hydrolases include, but are not limited to, lysins, phage lysins, cytolysins, egg lysins, hemolysins, NK lysins, streptolysins, autolysins, LytC amidases, LytD glucosaminidases, N-acetylmuramolyl-L-alanine amidases, polypeptides comprising or consisting of one or more catalytic domains from a lysin or autolysin, or combinations and / or fragments thereof.

[0177] fusion proteins In some aspects of the present disclosure, therapeutic or prophylactic drug molecules may be present in Spirulina as part of a complex. In some embodiments, Spirulina contains multiple copies of one or more therapeutic and / or prophylactic drug molecules in a complex. In some embodiments, Spirulina contains a combination of one or more therapeutic and / or prophylactic drug molecules in a complex. In some embodiments, Spirulina contains one or more therapeutic and / or prophylactic drug molecules in a fusion protein.

[0178] In some embodiments, Spirulina comprises one or more therapeutic and / or prophylactic molecules in a complex containing a linker. In some embodiments, the construct inserted into recombinant Spirulina comprises a linker. In some embodiments, the polypeptide expressed from recombinant Spirulina comprises 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 is not limited to, a chaperone protein, a targeting protein, a scaffold, an oligomerization domain, an enzyme, or a lysin. 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), or PA10 lin25.

[0179] In some embodiments, at least one exogenous polypeptide is expressed in Spirulina as a fusion protein, where the fusion protein forms a three-dimensional structure (sometimes referred to herein as a "particle"). In some embodiments, the fusion protein that forms the three-dimensional structure may contain 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 with multiple modular domains. An example of this fusion protein is woodchuck hepadnavirus core antigen (WHcAg) engineered with a B-cell antigen in 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) that has been engineered to be a tandem dimer with an antigen at its N-terminus and a fragment of Salmonella flagellin at its C-terminus, thus combining an immunogenic epitope with an innate immune system stimulator to act as an endogenous adjuvant that self-assembles into a three-dimensional structure with two functional domains displayed on its surface. β) In some embodiments, recombinant Spirulina can express two heterologous polypeptides. For example, recombinant Spirulina can express one gene encoding a tandem RNA bacteriophage capsid protein dimer with an N-terminal antigenic structure and a second gene encoding an identical capsid dimer but with 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 expressing a gene encoding one RNA phage particle genetically linked to a viral capsid protein or polypeptide, such as WHcAg, and a second gene carrying a native viral protein. This avoids steric clashes that can occur when all particles have bulky hybrid partners attached. The particles formed in this example can self-assemble to form further higher-order structures.

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

[0181] In some embodiments, recombinant Spirulina contains a fusion protein comprising at least one exogenous polypeptide and a viral protein capable of forming a virus-like particle (VLP). In these embodiments, the exogenous polypeptide is expressed in Spirulina as a protein polymer particle, such as a virus-like particle (VLP). VLPs mimic the overall structure of a virus particle by retaining the three-dimensional structure of the virus, but do not contain infectious materials. VLPs have the ability to stimulate B cell and T cell-mediated responses. When expressed in a heterologous system such as Spirulina, viral proteins can spontaneously form VLPs. Thus, in some embodiments, at least one exogenous antigen epitope is fused with a VLP-forming viral protein. When expressed in Spirulina, this fusion protein forms a VLP.

[0182] In some embodiments, tethering an exogenous polypeptide to a VLP-forming viral protein (or other protein that forms a tertiary structure) allows for the expression of hundreds of monomeric proteins per VLP (e.g., 180-240 monomeric proteins per VLP when using hepatitis VLPs). This allows for 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 after the amino acid sequence of the viral protein (attachment of the viral protein at the N-terminus of the antigen or epitope) or before 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 a viral protein. Alternatively, a region of a 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 it is said that at least one exogenous polypeptide is included in or present within a VLP, it refers to a fusion protein comprising at least one exogenous polypeptide as 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 from various viruses. Exemplary capsid proteins that can be used in the VLPs of the present disclosure include capsid proteins from viruses of the Hepadnaviridae family, papillomaviruses, picornaviruses, caliciviruses, rotaviruses, and reoviruses. In some embodiments, viral proteins that can be used to form VLPs expressing the polypeptides, antigens, or antigenic epitopes of the present disclosure include Hepadnaviridae core antigen (HBcAg). An exemplary HBcAg that can be used in the present disclosure is woodchuck hepadnavirus core antigen (WHcAg) from woodchuck hepadnavirus (also referred to herein as woodchuck hepatitis virus).

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

[0185] In some embodiments, the recombinant Spirulina comprises a fusion protein comprising at least one exogenous polypeptide, antigen, or antigenic epitope and a scaffold protein. The term "scaffold protein," as used herein, refers to a protein that acts as a docking protein and facilitates interaction between two or more proteins. For example, a fusion protein comprising at least one exogenous polypeptide and a scaffold protein can facilitate binding of the exogenous polypeptide to a receptor on a cell. In some embodiments, the exogenous polypeptide is tethered 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 scaffold function can be replaced by inserting at least one polypeptide into that region. For example, in recombinant Spirulina containing multiple copies of an exogenous polypeptide and a scaffold protein, the exogenous antigen epitope and scaffold protein may be 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) (where E is the exogenous polypeptide, SP is the scaffold protein, and n, n1, and n2 represent the number of copies of the exogenous polypeptide). It is understood that recombinant Spirulina may contain more than one exogenous polypeptide and one or more scaffold proteins, where the multiple exogenous polypeptides and scaffold proteins may be arranged in the various patterns described above.

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

[0187] Exemplary scaffold proteins include the oligomerization domain of C4b-binding protein (C4BP), the cholera toxin b subunit, or the oligomerization domain of an extracellular matrix protein. In some embodiments, a scaffold protein used in an oral antigenic composition of the present disclosure comprises a sequence 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 exogenous polypeptide multimer binds to a target or target molecule with higher affinity than a monomer or smaller multimer. For example, a heptameric VHH can bind to a target with higher affinity than a dimer of the same exogenous polypeptide. In some embodiments, the multimer is a heteromer. In some embodiments, different components of the heteromer bind to different targets or target molecules.

[0189] The recombinant Spirulina present in the non-injectable compositions 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 a fusion protein as described above, wherein the exogenous polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of at least one exogenous polypeptide per single molecule of exogenous antigen. In some embodiments, the recombinant Spirulina expresses an exogenous polypeptide, wherein the exogenous polypeptide comprises 1-5, 2-5, 2-4, 3-6, 3-8, or 4-5 copies of at least one exogenous polypeptide per single molecule of exogenous antigen. In some embodiments, the recombinant Spirulina comprises 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 exogenous antigen. In some embodiments, the recombinant Spirulina expresses an exogenous polypeptide, wherein the exogenous polypeptide comprises 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, 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-injectable compositions of the present disclosure may contain multiple copies of a nucleic acid sequence encoding at least one exogenous polypeptide. The multiple copies of the nucleic acid sequence encoding at least one exogenous polypeptide may be integrated into the genome of the Spirulina or may be present on one or more vectors introduced into the Spirulina. In some embodiments, the recombinant Spirulina contains 2 to 100 copies of a nucleic acid sequence 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 a nucleic acid sequence encoding at least one exogenous polypeptide 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 a nucleic acid sequence encoding at least one exogenous polypeptide integrated into its genome or present on one or more vectors. In some embodiments, the recombinant Spirulina comprises 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 a nucleic acid sequence encoding at least one exogenous polypeptide integrated into its genome or present on one or more vectors. In some embodiments, the recombinant Spirulina comprises 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, or 30-60 copies of a nucleic acid sequence encoding at least one exogenous polypeptide integrated into its genome or present on one or more vectors.

[0191] In some embodiments, multiple copies of at least one exogenous polypeptide are linked in tandem, i.e., the first copy is immediately followed by the second copy without any amino acid separation, the second copy is immediately followed by the third copy, and so on. In some embodiments, when recombinant Spirulina contains more than one exogenous polypeptide, each polypeptide can be similarly linked in tandem with other antigenic epitopes. For example, in a 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, where 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, multiple copies of at least one exogenous polypeptide present in a protein can be separated by a spacer sequence. In some embodiments, multiple copies of an 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 an 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 more than two copies of an exogenous polypeptide are present, it is understood that some copies can be linked in tandem and some copies can be separated by a spacer sequence. For example, in a recombinant Spirulina containing multiple copies of E1 as at least one exogenous polypeptide, multiple copies of this epitope can be separated as follows: (E1)xS-(E1)y, (E1)(E1)xS-(E1)y, (E1)xS-(E1)yS-(E1)z. where S represents a 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 identical or different.

[0193] In embodiments in which the recombinant Spirulina comprises a protein comprising more than one exogenous polypeptide, a first exogenous polypeptide can be separated from the 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 of the copies can be linked in tandem with other polypeptides, while some copies can be separated by spacer sequences; alternatively, all copies of one polypeptide can be linked in tandem, followed by a spacer sequence, followed by all copies of a second polypeptide, etc. For example, in recombinant Spirulina containing E1 and E2 as exogenous polypeptides, the two polypeptides can be arranged as follows: (E1)xS-(E2)y, (E2)xS-(E1)y, (E1)xS-(E2)yS(E1)zS-(E2)v, (E1)xS-(E2)y(E1)z, (E1)xS-(E2)yS-(E1)z, (E2)xS-(E1)y(E2)z, etc., where v, x, y, and z represent the number of copies of the polypeptides.

[0194] In some embodiments, recombinant Spirulina may contain one or more exogenous polypeptides and multiple copies thereof in the arrangement pattern just described, i.e., without being part of or fused to 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 antigenic epitopes, where the exogenous polypeptides, antigens, and / or antigenic epitopes and their multiple copies (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 antigenic epitopes, where the exogenous antigenic epitopes and their multiple copies (if present) can be arranged in the various patterns described above within the fusion protein. In some other embodiments, recombinant Spirulina may comprise a fusion protein comprising a VLP-forming viral protein, a trimer-forming protein, and / or a scaffolding protein and one or more exogenous polypeptides, antigens, and / or antigenic epitopes, where the exogenous polypeptides, antigens, and / or antigenic epitopes and multiple copies thereof (if present) can be arranged in the various patterns described above within the fusion protein.

[0196] Non-injection compositions provided by the present disclosure include recombinant Spirulina, wherein the recombinant Spirulina comprises at least one exogenous polypeptide, small molecule, antigen, or epitope, as any of those described above.

[0197] Spirulina The non-injectable compositions of the present disclosure comprise recombinant Spirulina in a non-viable form. These non-viable Spirulina containing expressed exogenous polypeptides, small molecules, antigens, or epitopes 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 antigenic epitope is prepared by drying a live culture of recombinant Spirulina. Drying methods include heat drying, such as drying in an oven; air drying, spray drying, lyophilization, or freeze-drying. Thus, in some embodiments, the non-injectable compositions of the present disclosure comprise dried recombinant Spirulina biomass containing at least one exogenous polypeptide, antigen, or at least one exogenous antigenic epitope described herein.

[0198] As used herein, "Spirulina" is synonymous with "Arthrospira." The non-injectable compositions of the present disclosure may include any one of the following Spirulina species: 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 Dosages As used herein, the terms "oral composition" or "orally delivered composition" include compositions administered or delivered to the gastrointestinal tract (e.g., compositions administered orally, via a feeding tube to the stomach, etc.). Any suitable region of the gastrointestinal tract can be targeted by the compositions of the present disclosure.

[0200] In some aspects, the compositions of the present disclosure are administered via the respiratory tract. 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 lyophilized powder or powder resuspended in liquid.

[0201] In some embodiments, the compositions of the present disclosure are formulated for administration via the respiratory tract.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 contain 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 solution, 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, non-specific stimulators of the immune response, known as adjuvants, can be used to enhance the immunogenicity of certain compositions. Exemplary adjuvants include water-in-oil (W / O) emulsions composed of mineral oil and surfactants from the mannide monooleate family (e.g., the MONTANIDE™ class of adjuvants) and flagellin adjuvants.

[0204] In some embodiments, compositions of the present disclosure comprise between about 0.1% and about 5% total Spirulina biomass. In some embodiments, compositions of the present disclosure comprise between about 1 mg and about 50 mg of exogenous antigenic epitopes per gram of dry Spirulina biomass. In some embodiments, compositions of the present disclosure comprise 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 antigenic 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 compositions can be used to prevent a disease or disorder in a subject. In some embodiments, the compositions can be used to prevent the onset of a disease or disorder in a subject. In some embodiments, the compositions can be used to reduce the severity of a disease or disorder in a subject. In some embodiments, the compositions can be used to prevent or delay the recurrence of a disease in a subject. In some embodiments, the compositions can be used to treat, prevent, or delay the recurrence of cancer in a subject.

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

[0207] In some embodiments, provided herein are methods for inducing an immune response in a subject in need thereof, comprising administering any of the compositions described herein to the subject. Without wishing to be bound by theory, it is expected that administering a composition of the present disclosure to a subject will cause at least one exogenous antigen epitope to 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 a composition 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 weekly, once every two weeks, monthly, etc. In some embodiments, the compositions of the present disclosure are administered to a subject for about one day to about one year. In some embodiments, the compositions of the present disclosure are administered to a subject for about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, one month, two months, three months, four months, five months, or more. In some embodiments, the compositions of the present disclosure are administered on consecutive days. In some embodiments, the compositions of the present disclosure are administered on non-consecutive days. In some embodiments, the compositions of the present disclosure are administered once daily. In some embodiments, the compositions of the present disclosure are administered multiple times per day. In some embodiments, the compositions of the present disclosure are administered twice daily, three times daily, four times daily, or more than twice daily. 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 a meal. In some embodiments, the compositions of the present disclosure are administered when the subject is in a fasting state.

[0209] The compositions of the present disclosure can be administered according to a schedule, for example, a priming dose of the antigenic composition, followed by one or more booster doses of the antigenic composition. In some embodiments, the first booster 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 booster 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 booster dose of the antigenic composition can be administered after the first booster dose and at any time from about 3 months to about 10 years after the priming dose. In some embodiments, the second booster dose of the antigenic composition can be administered after the first booster 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 immunoglobulin or low levels of specific immunoglobulin are detected in the subject's serum and / or other bodily fluids after the second booster dose, a third booster dose can be administered as needed.

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

[0211] The compositions of the present disclosure can be used to treat and / or prevent or reduce the severity of a disease or disorder. In some embodiments, the disease or disorder is selected from the group including, 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, high cholesterol, inflammatory bowel disorders, irritable bowel syndrome, Crohn's disease, ulcerative colitis, constipation, and diarrhea.

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

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

[0214] In some embodiments, the compositions described herein can be used to induce an immune response against, treat, and / or reduce the severity of infections 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 syndrome 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 infection caused by IHNV.

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

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

[0218] In some embodiments, the compositions described herein can be used to induce an immune response against, treat, and / or reduce the severity of infections 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 may be administered to insects, including but not limited to, Plasmodium, Trypanosoma, Toxoplasma, Giardia, and Leishmania, Cryptosporidium, parasitic helminths: Trichuris spp. (whipworms), Enterobius spp. (pinworms), Ascaris spp. (roundworms), Ancylostoma spp. and Necatro spp. (hookworms), Strongyloides spp. (threadworms), Dracunculus spp. (guinea worms), Onchocerca spp. and Wuchereria spp. (heartworms), Taenia spp., Echinococcus spp., and Diphyllobothrium spp. (human and animal tapeworms), Fasciola spp. (liver flukes), and Schistosoma The compounds can be used to induce an immune response against and / or reduce the severity of infections caused by parasites, including Schistosoma spp. (Schistosomal).

[0220] In some embodiments, the compositions described herein can be used to induce an immune response against and / or reduce the severity of an infection 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 an infection caused by a 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 compositions can be used to induce an immune response against and / or reduce the severity of infections 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 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 immune responses against autoantigens. In some embodiments, the compositions can be used to induce immune responses 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 respiratory tract (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 easily determined by those skilled in the art, for example, by first identifying the effective dose for eliciting a preventive or therapeutic effect. The dosage can be determined through animal testing. Non-limiting examples of animals used to test the effectiveness of vaccines include guinea pigs, hamsters, ferrets, chinchillas, mice, and cotton rats. Although test animals may not be natural hosts for infectious agents, they are still useful for testing various aspects of disease. For example, any of the above animals can be administered with the composition of the present disclosure, for example, recombinant Spirulina containing VLPs containing polypeptides.

[0226] In some embodiments, administering a composition of the present disclosure reduces the burden of infectious agents. In some embodiments, administering a composition of the present disclosure reduces the establishment of infectious agents. In some embodiments, administering a composition of the present disclosure reduces the shedding of infectious agents (e.g., viral shedding). In some embodiments, administering a composition of the present disclosure reduces the shedding of infectious agents. In some embodiments, administering a composition of the present disclosure increases shedding over a period of time (e.g., 24 hours), and then decreases shedding thereafter (e.g., at 72 hours). In some embodiments, administering a composition reduces the expression of a biomarker. In some embodiments, the biomarker is a marker of inflammation.

[0227] In some embodiments, administering a composition of the present disclosure neutralizes or blocks the activity of the target. In some embodiments, administering a composition of the present disclosure neutralizes or blocks 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% of the activity of the target.

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

[0229] Methods for making non-injectable compositions Methods for producing the non-injectable compositions described herein are provided. The non-injectable composition methods include introducing a nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigenic epitope into Spirulina. In some embodiments, the non-injectable composition methods include introducing a polypeptide, antigen, and / or antigenic epitope into Spirulina. In some embodiments, the non-injectable composition methods include introducing a small molecule into Spirulina.

[0230] Any suitable means for transforming Spirulina can be used in the present disclosure. An exemplary method for transforming Spirulina to express heterologous proteins is described in U.S. Patent No. 10,131,870, which is incorporated herein by reference in its entirety.

[0231] In some embodiments, the method for producing a non-injection composition includes introducing into Spirulina cells an expression vector having a nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigenic epitope. 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 antigenic 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 antigenic 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 antigenic epitope is under the control of an inducible promoter.

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

[0233] In some embodiments, vectors carrying homology arms and nucleic acid sequences encoding at least one exogenous polypeptide, antigen, and / or antigenic epitope can be introduced into Spirulina using electroporation, preferably in the presence of a suitable osmotic stabilizer.

[0234] Before introducing the vector into Spirulina, the Spirulina can be cultured in any suitable medium for growing cyanobacteria, such as SOT medium. SOT medium contains 1.68 g of NaHCO3, 50 mg of K2HPO4, 250 mg of NaNO3, 100 mg of K2504, 100 mg of NaCl, 20 mg of MgSO4.7H2O, 4 mg of CaCl2.2H2O, 1 mg of FeSO4.7H2O, 8 mg of Na2EDTA.2H2O, 0.1 mL of A5 solution, and 99.9 mL of distilled water. A5 solution contains 286 mg of H3BO3, 217 mg of MnSO4.5H2O, 22.2 mg of ZnSO4.7H2O, 7.9 mg of CuSO4.5H2O, 2.1 mg of Na2MoO4.2H2O, and 100 mL of distilled water. Cultures are grown at temperatures higher than room temperature (e.g., 25–37 °C) with shaking (e.g., 100–300 rpm) under continuous illumination (e.g., 20–2,000, 50–500, or 100–200 μmol photons m ). -2 s -1 The optical density at 750 nm can be measured at a predetermined threshold (e.g., OD 750 Growing cells can be harvested once the pH reaches 0.3-2.0, 0.5-1.0, or 0.6-0.8. The harvested cells can be centrifuged and then concentrated by resuspending in a pH balancer and salt solution. The pH balancer can be any suitable buffer that maintains the pH of the medium between 6 and 9, 6.5 and 8.5, or 7 and 8 while maintaining Spirulina viability. Suitable pH balancers include HEPES, HEPES-NaOH, sodium or potassium phosphate buffer, and TES. The salt solution can be between 50 mM and 500 mM, 100 mM and 400 mM, or 200 mM and 300 mM NaCl. In some embodiments, 1-50 mL of a 1-100 mM pH balance can be used to neutralize the pH.

[0235] Cells collected by centrifugation can be washed with an osmotic stabilizer and, if necessary, a salt solution (e.g., 0.1-100 mM NaCl, 1-50 mL). Any volume of culture can be concentrated by centrifugation. In some embodiments, 5-500 mL of culture can be centrifuged. The osmotic stabilizer can be any type of osmotic balancer that stabilizes the integrity of Spirulina cells during electroporation. In some embodiments, the osmotic stabilizer can be a sugar (e.g., 0.1-25% w / v), such as glucose or sucrose. In some embodiments, the osmotic stabilizer can be a simple polyol (e.g., 1-25% v / v), including glycerin, glycerol, or glycerol. In some embodiments, the osmotic stabilizer can be a polyether (e.g., 0.1-20% w / v), including polyethylene glycol (PEG), poly(oxyethylene), or poly(ethylene oxide) (PEO). The 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 in place of or in addition to the osmotic stabilizer.

[0236] A vector containing homology arms and a nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigenic epitope can be introduced into Spirulina cells cultured as described above and washed with an osmotic stabilizer. Electroporation can be used to introduce the vector.

[0237] Electroporation can be performed in a 0.1 cm, 0.2 cm, or 0.4 cm electroporation cuvette at 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 performed at 4.5 kV / cm, 50 μF, and 100 mΩ.

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

[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 antigen-encoding nucleic acid sequence can be inserted or cloned. The vector can contain one or more specific sequences that allow recombination into a specific, desired site on the Spirulina chromosome. These specific sequences can be homologous to sequences present in wild-type Spirulina. The vector system can contain a single vector or plasmid, or two or more vectors or plasmids, some of which increase the efficiency of targeted mutagenesis or transposition. The choice of vector generally depends on the compatibility of the vector with the Spirulina cells into which it will be introduced. The vector can contain a reporter gene, such as green fluorescent protein (GFP), which can be fused in-frame with one or more of the encoded antigen epitopes or can be expressed separately. The vector can also contain a positive selection marker, such as an antibiotic resistance gene, that can be used to select suitable transformants. The vector may also contain 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 makes it possible to identify cells that have been successfully transformed with the vector.

[0240] In some embodiments, the vector comprises one or two homology arms that are homologous to DNA sequences in the Spirulina genome adjacent to the targeted locus. The sequences of the homology arms can be partially or completely complementary to regions of the Spirulina genome adjacent to the targeted locus.

[0241] The homologous arms may be of any length that allows site-specific homologous recombination. The homologous arms may be of any length between about 2000 bp and 500 bp. For example, the homologous arms may 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 may be of any length between about 2000 bp and 500 bp. For example, each of the two homologous arms may be about 2000 bp, about 1500 bp, about 1000 bp, or about 500 bp.

[0242] The targeted locus of the Spirulina genome is modified by homologous recombination using a portion of the vector flanked by one homologous arm or two homologous arms. The modification can be a change in the length of the targeted locus, including a deletion or addition of nucleotides. The addition or deletion can be of any length. The modification can also change the sequence of nucleotides within the targeted locus without changing its 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 to the mouse 7Mice were inoculated with Campylobacter jejuni. Spirulina was transfected with a vector expressing a monomeric VHH antibody targeting Campylobacter. After growing the Spirulina to allow expression of the monomeric VHH antibody, the Spirulina was dried and Campylobacter-infected mice were administered 200 μl of PBS and 10% Spirulina biomass (13 mg) by daily gavage for 5 days. 13 mg of Spirulina contains 425 μg of monomeric VHH per dose. As controls, mice were administered daily gavage of 1) PBS, 2) wild-type Spirulina, or 3) Spirulina expressing an unrelated VHH.

[0245] As shown in Figure 1A, 100% of Campylobacter-infected mice treated with either one of the control treatments developed diarrhea. In contrast, none of the mice administered Spirulina expressing monomeric anti-Campylobacter VHHs developed diarrhea. Furthermore, at 7 days postinoculation, mice treated with Spirulina expressing monomeric anti-Campylobacter VHHs demonstrated a 4-log reduction in Campylobacter shedding (Figure 1B).

[0246] Example 2 Spirulina expressing trimeric VHH Oral Spirulina-VHH has anti-inflammatory activity in Campylobacter infection. 8Mice were inoculated with Campylobacter jejuni. Spirulina was transfected with a vector expressing a trimeric VHH antibody targeting Campylobacter. After growing the Spirulina to allow expression of the trimeric VHH antibody, the Spirulina was dried and Campylobacter-infected mice were administered 400 μl of PBS + 0.5% Spirulina biomass (1.3 mg) by 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 unrelated 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 with controls; indeed, fecal lipocalin in these treated mice closely resembled that in uninfected mice. Furthermore, Figure 2B demonstrates that treatment of infected mice with Spirulina expressing trimeric anti-Campylobacter VHH prevented myeloid cell infiltration into the gastrointestinal lamina propria.

[0248] Example 3 Protective effect of Spirulina-VHH in mice challenged with C. jejuni strain 81-176 Test substance:

[0249] Spirulina strain SP257 (unrelated 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 preventive efficacy of anti-C. jejuni VHHs expressed in Spirulina [Giallourou et al.]. Spirulina strains expressing either the VHH FlagV6 (SP526), ​​a protease-resistant form of FlagV6 (FlagV6-F23) (SP806), or an unrelated 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 before treatment. On day 0, 10 8 Mice were administered an inoculum of C. jejuni strain 81-176. Food and water were available 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 inoculation with C. jejuni. Mice were divided into four different groups: 13.3 mg of Spirulina-VHH (670 mg / kg) containing an unrelated VHH; 13.3 mg of anti-Campylobacter VHH (SP651) on a trimeric scaffold; 13.3 mg of anti-Campylobacter VHH (SP737) on a pentameric scaffold; · Control group treated with PBS. Compared to infected controls treated with PBS, all mice treated with Spirulina-VHH demonstrated nonspecific flushing of C. jejuni in the feces at 24 hours, followed by reduced 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 a dose 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), 5% Spirulina-VHH powder w / v resuspended in PBS, administered by gavage 1.5 hours before inoculation; administration of three 400 μL doses (equivalent to 1.33 mg of Spirulina-VHH per dose) of SP561, a 0.5% Spirulina-VHH powder w / v resuspended in PBS, administered by gavage 1.5 hours before and 24 and 48 hours after inoculation; administration of a single 400 μL dose (equivalent to 1.33 mg of Spirulina-VHH per dose) of SP257, a 0.5% Spirulina-VHH powder w / v of irrelevant VHH resuspended in PBS, administered by gavage 1.5 hours before and 24 and 48 hours after inoculation; administration of three 400 μL doses (equivalent to 1.33 mg of Spirulina-VHH per dose) of SP257, a 0.5% Spirulina-VHH powder w / v of irrelevant VHH resuspended in PBS, gavaged 1.5 hours before and 24 and 48 hours after inoculation; · Control mice administered PBS gavage. There were five mice in each experimental group. Three days after Campylobacter inoculation, control-infected mice (PBS gavage) showed significant weight loss compared with uninfected mice (Figure 3A). Infected mice treated with SP257 showed a similar weight loss. In contrast, infected mice treated according to both dosing regimens with SP651, which expresses an anti-Campylobacter binding protein on a trimeric scaffold, showed weight gain that was comparable to or significantly better than uninfected mice (Figure 3A).

[0256] Ceca from all animals were examined at necropsy 72 hours after infection. Tissue sections were processed and scored on a scale of 0 to 24 by a histopathologist in a blinded fashion. 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 received scores significantly lower than infected controls and similar to uninfected controls (Figure 3B). These results suggested that Spirulina itself had a positive impact on reducing histopathology in 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 the anti- C. jejuni VHH strain compared to Spirulina (SP257) expressing an unrelated VHH.

[0259] Mice were administered a single 400 μL dose containing 1.33 mg of Spirulina-VHH in PBS 1.5 hours prior to infection with C. jejuni. Four cohorts, each containing 5 mice, were treated as follows: Uninfected, infected and treated with PBS gavage, infected and treated with SP257 by gavage, infected and treated with SP651 by gavage. Treatment with this single prophylactic dose of Spirulina containing anti-Campylobacter VHH was sufficient to significantly accelerate Campylobacter flushing at 24 h postinfection and reduce Campylobacter shedding at 72 h postinfection, as measured by fecal Campylobacter CFU (Fig. 4B). Postinfection inflammation was measured by fecal lipocalin levels and flow cytometry quantification of myeloid cell infiltration in the cecal lamina propria. Campylobacter infection caused a significant increase in both inflammatory biomarkers (Fig. 4C). This increase was prevented by a single prophylactic dose of SP651 (expressing an anti-Campylobacter binding VHH), but not by a prophylactic dose of SP257 (expressing an unrelated VHH) (Fig. 4). As in the previous experiment, the weight loss caused by Campylobacter infection was also prevented by a prophylactic dose of SP651. (Fig. 4A). However, in this experiment, Spirulina expressing an unrelated VHH also suppressed the infection-associated weight loss, further suggesting a possible nutritional benefit of Spirulina itself. Importantly, Spirulina expressing an unrelated VHH had no effect on biomarkers of inflammation or myeloid cell infiltration in the cecal lamina propria.

[0260] In the third experiment, single doses of increasing 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 were compared: SP526 and SP806. SP526 exhibits high expression levels of anti-C. jejuni FlagV6 VHH, while SP806 expresses a protease-resistant form of FlagV6 (FlagV6-F23) containing two mutations in the VHH that have been reported to confer resistance to chymotrypsin (Hussack et al. 2014). These results were also retrospectively compared to the efficacy of SP651 in a previous experiment.

[0261] 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 1.5 h before infection with C. jejuni. Measurement of body weight fluctuations showed that, as in the previous experiment, Campylobacter caused a weight gain defect at 72 h postinfection. Treatment with each of the three Spirulina-VHH strains inhibited 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 showed that, as in the previous experiment, all three Spirulina-VHH strains accelerated Campylobacter flushing at 24 h post-treatment and reduced long-term shedding at 72 h post-treatment (Figure 5B). Again, VHH expression level and protease resistance independently increased efficacy, with both SP526 and SP806 exhibiting an MED of 0.399 mg (20 mg / kg) in this assay.

[0263] Biomarkers of inflammation—fecal lipocalin and myeloid cell infiltration into the cecal lamina propria—showed that, as in the previous experiment, all three strains 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 cell infiltration into the lamina propria. The MED for all three strains was 0.399 mg (20 mg / kg), with partial efficacy at 0.133 mg (6.7 mg / kg).

[0264] Cecums 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 groups that demonstrated significant reduction in histopathology compared to infected controls were treated with 1.33 mg (67 mg / kg) of SP526. Below this dose, or in groups treated with different Spirulina-VHHs (SP806 or SP651), the positive effects of treatment were determined using other metrics of efficacy (i.e., bacterial shedding, inflammatory biomarkers, etc.).

[0265] Conclusion: Administration of all Spirulina-VHH strains expressing the anti-C. jejuni VHH FlagV6 produced favorable results for the treatment of C. jejuni-infected mice with a single dose of 1.33 or 0.399 mg of Spirulina-VHH. Compared to untreated mice, these mice gained more weight and had reduced levels of inflammatory markers.

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

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

[0268] Example 4 Effect of postchallenge treatment with anti-Campylobacter Spirulina VHH in mice challenged with C. jejuni CG8421 strain The SP1182 construct is depicted in Figures 7 and 8. This fusion protein contains the camelin VHH FLAGV6-F23, which binds to the flagellin protein flaA from C. jejuni. The SP1182 fusion protein does not contain a targeting protein and therefore remains in the cytoplasm of Spirulina cells.

[0269] A murine Campylobacter challenge experiment was performed to test the efficacy of orally delivered SP1182 administered as a treatment modality.

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

[0271] Fecal Campylobacter shedding was measured at 40 and 72 hours post-infection. At 40 hours, there was a significant (p<0.05) burst of Campylobacter clearance 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 shedding. 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 closely resembled the effect of a single pre-vaccination (prophylactic) dose of SP1182.

[0272] Ceca from all animals were examined at necropsy 72 hours after infection. Tissue sections were processed and scored on a scale of 0 to 24 by a histopathologist in a blinded fashion. 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. No treatment group demonstrated a reduction in histopathology, and all treatment groups received scores similar to those of C. jejuni-infected controls.

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

[0274] Example 6 Polypeptides expressed in Spirulina are stable over time in dry biomass To test the effect of long-term storage of dried biomass on polypeptide stability, Spirulina expressing monomeric anti-Campylobacter VHHs was spray-dried and stored for 1) 1 month at 27°C, 2) 3 months at 27°C, 3) 1 month at 42°C, or 4) 3 months at 42°C. At various time points, VHHs were purified from Spirulina and tested for binding activity. As shown in Figure 12, no decrease in the biological activity of the anti-Campylobacter VHHs was observed upon long-term incubation at elevated temperatures.

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

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

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

[0278] Spirulina strain SP257 (expressing unrelated VHHs)

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

[0280] A mouse model of C. jejuni infection developed at the Institute Research in Biomedicine in Switzerland was used to evaluate the efficacy of prophylactic treatment with Spirulina expressing an anti-C. jejuni VHH. Several Spirulina strains expressing either the anti-C. jejuni VHH FlagV6, a protease-resistant form of FlagV6 (FlagV6-F23) (Hussack et al. 2014; Riazi et al. 2013), or an unrelated 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 Mice were given an inoculum of C. jejuni, strain 81-176.

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

[0282] A single 1.2 mg dose of Spirulina was used to determine the efficacy of the anti-C. jejuni VHH strain (SP651) compared to Spirulina expressing an unrelated VHH (SP257). Mice were administered a single 400 μL dose containing 1.2 mg of Spirulina in PBS 1.5 hours before infection with C. jejuni. Compared to untreated infected mice, mice administered anti-C. jejuni Spirulina demonstrated better weight gain, increased shedding at 24 hours followed by a decrease at 72 hours, and reduced levels of inflammatory biomarkers (Figures 17A-C). Spirulina containing the unrelated VHH had little to no effect on shedding and inflammatory biomarker reduction.

[0283] Single doses of increasing dilutions of Spirulina were tested to determine the critical 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 the anti-C. jejuni FlagV6 VHH, and SP806 was identical to SP651 except that SP806 contained two mutations in the VHH that reportedly confer resistance to chymotrypsin (Hussack et al. 2014). 1.5 h 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, with varying degrees of decreased efficacy at the 0.36 mg and 0.12 mg doses. Mice treated with SP526 showed the best weight gain, while SP806, at intermediate dose concentrations, reduced shedding at 72 hours (Figure 18A-C). All three strains significantly reduced levels of inflammatory biomarkers at the 0.36 mg dose (Figure 19A-B), but the protease-resistant strain (SP806) produced the greatest reduction in both lipocalin-2 levels and lamina propria-infiltrating myeloid cells. At the 0.12 mg dose of spirulina, all strains behaved similarly to the C. jejuni-only control, suggesting that this amount was below the effective therapeutic dose.

[0284] In summary, all Spirulina strains expressing the anti-C. jejuni VHH FlagV6 yielded positive results for treatment of C. jejuni-infected mice with a single dose of 1.2 mg of Spirulina-VHH: these mice gained more weight and had reduced levels of inflammatory markers compared to untreated mice.

[0285] In these experiments, no adverse events were observed up to the highest biomass dose administered. The drug material was well tolerated and no signs of toxicity were observed.

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

[0287] Spirulina strain SP257 (unrelated VHH)

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

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

[0290] The efficacy of orally delivered Spirulina-VHH in blocking colonization of the chicken intestine was investigated. A chicken model of C. jejuni intestinal colonization was used to evaluate the preventive efficacy of anti-C. jejuni VHHs expressed in Spirulina. Spirulina strains expressing a monomeric anti-Campylobacter VHH (SP526), ​​a homotrimeric multimeric VHH (SP651), or an unrelated VHH (SP257) were tested. Strains were cultured and spray-dried at a 3% biomass concentration in 2% trehalose. This experiment was designed to evaluate the therapeutic efficacy of different Spirulina strains with respect to their ability to block gastrointestinal colonization by C. jejuni, a highly prevalent pathogen in commercial flocks and a major cause of human foodborne illness.

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

[0292] Birds showed normal weight gain without any defects unrelated to Campylobacter inoculation or prophylactic treatment (Figure 20). Cecal colony counts were used to assess bacterial load. Cecal colonization by Campylobacter was significantly reduced after pretreatment with Spirulina SP651, a strain expressing a homotrimeric anti-Campylobacter FlagV6. Treatment with SP257, expressing an unrelated VHH, and SP526, expressing a monomeric VHH FlagV6, resulted in a reduction in Campylobacter colonization, albeit to a small extent, compared to no Spirulina treatment (Figure 21).

[0293] Example 9 ETEC therapeutic agent: Spirulina-expressing anti-adherence VHH Enterotoxigenic Escherichia coli (ETEC) is one of the causative agents of diarrhea in children in developing countries and traveler's diarrhea in people traveling to ETEC-endemic areas. According to the WHO, this pathogen causes over 200 million illnesses and approximately 500,000 deaths annually worldwide. Diarrhea caused by ETEC has long-term effects on young patients, including stunted growth, reduced intellectual ability, and associated long-term economic disadvantage. The detrimental effects of ETEC pathogenesis necessitate effective postinfection preventative treatments or preventative therapies such as passive immunization. The two main virulence factors in ETEC infection that can be targeted by vaccine development or preventative treatments are enterotoxins and colonization factors (CFs) or fimbriae. Enterotoxins are directly involved in the development of diarrhea after bacterial colonization of gastrointestinal epithelial cells. On the other hand, ETEC CFs facilitate the organism's colonization of the small intestine and subsequently lead to the expression of enterotoxins near mucosal cells, resulting in diarrhea.

[0294] We developed a therapeutic agent based on single-domain camelid antibodies (VHHs) that targets the ETEC pilus tip domain and inhibits bacterial binding to host intestinal epithelial cells, thus blocking bacterial colonization. VHHs were obtained by immunizing llamas with the pilus tip attachment protein CfaE or screened against the same antigen from a yeast-based synthetic library. VHHs that exhibited higher antigen binding and bacterial inhibition in hemagglutination or cell-based assays were engineered as monomers, dimers, trimers, tetramers, pentamers, and heptamers for Spirulina expression and displayed on nanoparticles. Chaperone proteins, such as maltose-binding protein (MBP), thioredoxin A (TxnA), and neutrophil gelatinase-associated lipocalin (LCN), can be used to increase heterologous protein solubility, resulting in higher protein expression levels of therapeutic VHHs in Spirulina.

[0295] Spirulina strains expressing anti-CfaE VHHs exhibit good binding activity to the CFA / I pilus tip attachment domain. Increased multimeric state of VHHs corresponds to increased binding activity in ELISA.

[0296] Example 10 Porcine ETEC therapeutic agent: Spirulina expressing anti-adherence VHH Enterotoxigenic Escherichia coli (ETEC) is the leading cause of diarrhea in piglets. Infection with ETEC in newborn pigs can induce diarrhea during the first one or two weeks of the postweaning period, usually resulting in dehydration, reduced weight gain, and death. Economic problems in the swine industry make postweaning diarrhea and the causative agent ETEC an economically significant disease in the swine industry. The primary virulence factors in ETEC strains are adhesins expressed as part of the fimbriae (pili) structure. The most common adhesins found in porcine ETEC are adhesins K88 (also called F4), K99 (F5), 987P (F6), F41, and F18, of which K88 and F18 are most frequently found in the swine industry.

[0297] We have developed a system to cost-effectively produce 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 via passive immunization without the need for purification or expensive preservatives and delivery methods to protect the gastrointestinal tract, allowing the therapeutics to be incorporated as part of the animal feed.

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

[0299] 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 strains is assessed using ELISA, in which the antigen is coated onto a high-binding plate and crude cell lysates of the antibody-expressing Spirulina strains are titrated at dilutions. We expressed monomeric, dimeric, and heteroheptameric anti-adhesin VHHs in Spirulina (Figures 22A-C). VHH binding activity against the antigen by ELISA indicates that the VHHs are active in Spirulina crude lysates. F4 + and F18 + Heteropentameric constructs expressing VHHs targeting the adhesin were synthesized using F4 + Adhesin domains FaeG and F18 + It binds to both the adhesin domain FedF (Fig. 23A-C).

[0300] Example 11 VHHs targeting ETEC fimbriae domains inhibit bacterial binding in a gnotobiotic piglet model ETEC K88ac, an ETEC strain causing postweaning diarrhea in piglets +We designed VHHs targeting the pilus domain of the strain. These VHHs were expressed in Spirulina as homodimers (SP795) and heteroheptamers (SP1156) (Figure 22A). Spirulina biomass was dried and protein expression was confirmed (Figure 25A). VHHs in Spirulina slurries from spray-dried and freeze-dried powders show comparable binding based on ELISA (Figure 25B). The antigen-binding efficiency of Spirulina-expressed VHHs was further evaluated using BLI-based kinetic measurements (Figure 25C).

[0301] Table 2 shows the total VHH expression per mass of dried Spirulina biomass as assessed using Western blot. Binding strength was assessed using ELISA EC50, and KD was measured from BLI-based kinetic measurements. The level of active VHH was determined by comparing the binding activity observed from Spirulina biomass with that of purified proteins. Table 2 [Table 2-1] [Table 2-2]

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

[0303] Furthermore, ETEC K88ac, an ETEC strain that causes postweaning diarrhea in piglets, + (F4 + a) VHHs targeting the fimbria domain of strains affect bacterial load in gnotobiotic piglets. Surgically delivered gnotobiotic piglets were treated with wild-type or therapeutic VHH-expressing Spirulina powder slurry by oral gavage twice daily from day 0 onwards. Then, one day later, the piglets were fed 10 10The K88(F4ac)-susceptible piglets were challenged with ETEC (Figure 26A). K88(F4ac)-susceptible piglets were administered 0.5 g of Spirulina biomass in 10 ml of diluted VH795 Spirulina, SP1156 Spirulina, or wild-type Spirulina in water. K88(F4ac)-resistant piglets were administered Spirulina containing either SP795 or SP1156 VHH by oral gavage twice daily from day 0 onwards.

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

[0305] High bacterial doses caused even resistant piglets to develop symptoms. K88-resistant piglets were maintained for 4 days and bacterial shedding was assessed by fecal swabbing. Piglets treated with Spirulina strain SP1156 showed a reduction in bacterial load after challenge (Figure 26C). Although these piglets were symptomatic, they were still healthy enough to discontinue treatment after challenge and divert them to a different study.

[0306] Example 12 Norovirus therapeutics: Spirulina-expressing anti-norovirus capsid protrusion domain VHH Human norovirus (HuNoV) is one of the most important causative agents of gastroenteritis, with approximately one-fifth of all acute infections attributed to this virus. HuNoV is the leading cause of acute gastroenteritis. According to a study examining the burden of diarrheal disease 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 contains a gene encoding the viral capsid protein (VP1). Based on sequence variation in the gene encoding the capsid (VP1), noroviruses are classified into various genotype groups (GI-GVII). Genotype groups are further divided into genotypes. The most prominent genotype groups isolated from recent incidents of human infection are genotype groups GI, GII, and GIV, of which more than 25 genotypes have been identified. The most frequently occurring genotypes in recent HuNoV outbreaks are GI.1, GII.4, and GII.10.

[0307] Orally delivered single-domain antibodies (VHHs)-based prophylactic therapeutics will be developed. This approach combines the favorable VHH properties that make these classes of antibodies suitable for oral delivery (such as high solubility, increased pH stability, and resistance to enzymatic degradation) with Spirulina-based oral delivery of therapeutics. VHHs targeting viral capsid proteins, some of which degrade viral particles upon binding to neutralize infectious virus, will be engineered for expression in Spirulina.

[0308] To enable oral delivery of protein therapeutics against HuNoV for gastrointestinal protection via passive immunization without the need for therapeutic purification or expensive preservatives and delivery methods, we will develop multivalent camelid single-domain antibodies (VHHs) targeting the viral capsid protein. The VHHs will be engineered for Spirulina expression as monomers with or without chaperone proteins such as maltose-binding protein (MBP) or thioredoxin A (TxnA) to increase the solubility of the heterologous protein. The expression constructs will be engineered to contain affinity tags.

[0309] Determine the expression level of the protein of interest by Western blotting using a combination of anti-tag or anti-VHH primary and appropriate secondary antibodies. ELISA is used to assess the binding activity of the protein expressed in the Spirulina strains by coating the antigen onto a high-binding plate and titrating crude cell lysates of the antibody-expressing Spirulina strains at various dilutions.

[0310] We expressed monomeric anti-HuNoV capsid protrusion protein VHHs in Spirulina with or without chaperone fusion partners (Figures 27A-C). ELISA-based binding assays demonstrate that Spirulina-expressed VHHs are active in Spirulina crude lysates (Figures 28A-C). Furthermore, VHHs purified from Spirulina crude lysates exhibit the expected viral capsid degradation and block viral binding to tissue biopsies mimicking the intestinal environment (Figures 29A-B).

[0311] Example 13 Development of VHHs for treating norovirus infection To generate a novel VHH (Nano85) targeting norovirus, we modified an anti-human norovirus (HuNoV) protruding (P) domain antibody by grafting the binding region of Nano85 onto the framework of the K922 antibody (SEQ ID NO: 18), which is known to be resistant to gastrointestinal proteases and allows for increased expression in Spirulina (Figure 30). A construct containing unmodified Nano85 with a C-terminal maltose-binding protein (MPB) (SP1371) and a construct containing modified Nano85 with a C-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, and GII.17) (Figures 31B and 31C). The purified protein also shows measurable binding to unrelated antigens, including the Campylobacter flagellin protein FlaA, the porcine ETEC adhesin protein FaeG, and the human ETEC fimbrial adhesin domain CfaE.

[0312] Furthermore, we investigated the binding kinetics and cross-reactivity of VHH sequences targeting various recombinant anti-human P domains. Nano26 (SEQ ID NO: 73) and Nano85 (SEQ ID NO: 71) exhibited broad cross-reactivity, whereas VHH3.2, VHH4.1, and VHH5.4 showed no 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.17P domains. [Table 3] Table 4: BLI-based binding kinetics for HuNoV GII.2 P domain [Table 4-1] [Table 4-2]

[0313] Additionally, the binding and cross-reactivity of VHHs Nano94 (SEQ ID NO: 75), VHH10, VHH6.3, and VHH7.3 targeting the human norovirus (HuNoV) P domain were evaluated. The VHHs tested exhibited binding EC50s ranging from 0.21 nM to 50.07 nM, with recombinant nano94-TxnA expressed in Spirulina exhibiting the weakest binding (Figure 33A). VHH7.3 exhibited cross-reactive binding to the GI.3 P domain (Figure 33B).

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

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

[0316] The protease susceptibility of various anti-human norovirus P domain VHH constructs was assessed by incubating 1 μg of bacterially expressed recombinant VHH 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 digestion buffer (1 mM Tris pH 8.0, 20 mM CaCl2) for 1, 2, or 4 hours. Protease susceptibility was measured using ELISA-based binding as shown in Figure BB6. Loop-grafted Nano85 exhibits the highest protease resistance compared to recombinant Nano85 and the others tested. VHH3.2, VHH4.1, and VHH5.4 are resistant to chymotrypsin, while showing varying susceptibility to trypsin.

[0317] Example 14 Inflammatory bowel disease therapeutics: 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 relapsing disease that tends to be progressive. IBD treatments include anti-inflammatory drugs, immunosuppressants, and anti-TNFα biologics. 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. Increased TNFα accumulation causes chronic inflammation and subsequent damage to intestinal epithelial cells. The latest anti-TNFα biologic therapeutics under investigation include infliximab, adalimumab, golimumab, and certolizumab. Given the chronic nature of IBD, oral administration of biologics is ideal for patient comfort, ease of treatment, willingness to adhere to prescription regimens, and cost. However, biologics developed for IBD are currently delivered intravenously or subcutaneously due to physical barriers that preclude oral delivery of biologics. These challenges include the instability of protein-based therapeutics in the GI tract, extreme pH environments, and high enzymatic activity in the GI tract.

[0318] Single-domain llama antibodies (VHHs) possess properties that make them suitable for oral delivery. VHHs retain binding specificity and potency comparable to traditional IgG antibodies. The small size and rigid structural properties of VHHs, as well as their solubility, ease of expression, and stability in the GI environment, make them suitable for oral-based therapeutics. Given these properties, VH Squared has developed a VHH (V565) that can bind 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 (Figures 36A-C). 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. Binding activity of proteins expressed in Spirulina strains was assessed using ELISA, in which antigen was coated onto high-binding plates and crude cell lysates of antibody-expressing Spirulina strains were titrated at various dilutions. Both monomeric and dimeric forms of VHHs showed good binding to recombinant human TNF-α.

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

[0321] For trimers and pentamers, thioredoxin was always used as the scaffold. Some were designed as homomultimers (e.g., Trx-Trimer-VHH), some as homomultivalent constructs (e.g., E3.VHH-Trx-TRIMER-E3.VHH), and some as heteromultivalent constructs (e.g., E3.VHH-Trx-TRIMER-5D.VHH).

[0322] Constructs containing VHH.5D are expressed at higher levels than those with VHH.E3. Certain heteromultivalent constructs are expressed at higher levels when E3 is N-terminal as opposed to 5D (Figures 38A-C).

[0323] Constructs were evaluated for their neutralizing activity against tcdB in vitro (Figure 39). Vero cells (African green monkey epithelial cells) were exposed to a range of doses of tcdB with or without Spirulina extract containing VHHs. Biological effects were measured in two ways: first, a colorimetric reagent that reacts linearly with healthy metabolic cells was used for quantitative measurements (Figure 40); and second, visual microscopy was used to assess the degree of "rounding," i.e., the extent to which Vero cells, which are normally attached and angular, detach from the plastic substrate and appear round (Figures 41A-O). These methods are generally consistent, although the visual rounding assay was consistently more sensitive.

[0324] result

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

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

[0327] iii. In general, 5D-containing constructs are more abundantly expressed and demonstrate more potent neutralizing activity.

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

[0329] SP1095, heterobifunctional trimeric construct, E3_Trx_TRI_5D

[0330] SP747, monomeric Trx_5D

[0331] SP1087, trimeric construct Trx_TRI_5D

[0332] The in vitro efficacy was slightly weaker because

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

[0334] SP1091, pentameric construct Trx_PENT_5D It was.

[0335] A VHH-5E (SEQ ID NO: 7) construct was also constructed. While VHH.5E-containing constructs performed more strongly than those with VHH.E3, the most potent construct on a per-molar basis was a trimer containing both VHH.E3 and VHH.5D. Potency generally followed expression level, with the most effective / potent construct being VHH.E3-Trx-trimer-VHH.5D, which was expressed at only approximately 0.1% of total protein, more potent than the second most potent extract, Trx-VHH.5D, which was expressed at approximately 2% of total protein. Spirulina extracts without VHHs did not exhibit any inherent neutralizing activity.

[0336] The three or four best-performing strains will be expanded and propagated in bioreactors and spray-dried. Furthermore, next-generation constructs will be designed and new strains will be constructed (e.g., marker-free versions of this strain, native Arthrospira thioredoxin, heteromultimers with 5D, and new Tufts VHHs directed against the RBD). Animal studies will also be initiated using these hit strains: 1) Mouse Model I: Lyras / Australia; 2) Mouse Model II: Guerrant / Virginia; 3) Swine Model: Tzipori / Tufts.

[0337] Example 16 VHH combinations show synergistic increases in binding to C. difficile toxins The binding strength of various VHHs, alone and in combination, to C. difficile TcdB toxin was tested. VHHs were produced in E. coli and tested in vitro. Figure 42 shows the binding strength of VHHs 5D (SEQ ID NO: 5), E3 (SEQ ID NO: 6), 7F (SEQ ID NO: 7), alone and in combination. 13 Figure 4 shows the binding strength of VHH 5D, E3, 7F, 2D (SEQ ID NO: 65), and 5E (SEQ ID NO: 7) to TcdB at various concentrations. 5D VHH shows the highest binding, while 2D shows the lowest binding. Figure 43 shows the binding strength of different combinations of VHH 5D, E3, 7F, 2D, and 5E. Figure 44 shows the binding strength of VHH 5D, E3, and 7F alone and in combination. Figures 45A-B show the binding strength of VHH 5D, E3, and 7F alone and in combination at different concentrations. Increasing the concentration of individual VHHs did not significantly increase efficacy. In contrast, higher concentrations of combined VHHs (i.e., VHH cocktails) showed a surprising increase in efficacy with increasing concentration.

[0338] The increased efficacy of VHH combinations can be explained by the different targets of different VHHs. For example, as shown in Figure 46, VHHs may act at different points in the process of the TcdB signaling pathway. VHH E3 blocks receptor binding, VHH5D blocks pH-dependent pore formation, and VHH 7F blocks autocatalysis and may block the GTD site. This explains the synergistic effect of VHH cocktails over the effect of single VHHs. Table 6: Anti-TcdB VHH [Table 6-1] [Table 6-2]

[0339] Bacterial lysates of VHHs constructed in fusion with maltose-binding protein (MBP) in the MBP-VHH orientation (except for 5D, which was used as a Spirulina lysate expressing PP7 particles decorated with VHH 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. VHHs were tested against three concentrations of TcdB 027 type as indicated.

[0340] Example 17 Anti-TcdB (Clostridium difficile toxin B) VHH produced in Spirulina Multimerization of single-domain antibodies on a single polypeptide chain increases avidity and, in many cases, biological activity. While multi-VHH single polypeptides have been produced in E. coli, expression in Spirulina has proven extremely difficult. Recently, the crystal structure of the entire TcdB protein (approximately 300 kDa) bound to three VHHs (VHHs 5D, E3, and 7F) was solved. See Figure 52, which shows TcdB bound to E3. Each VHH binds to distinct domains that are spatially distant from each other. Two of the three domains possess essential biological activities identified during the intoxication process, and the combined VHHs appear to disrupt the structural changes required for these functions. The third domain binds to a domain associated with target cell membrane localization in the cognate toxin. Each VHH has previously been shown to naturally possess some degree of toxin-neutralizing activity.

[0341] A single polypeptide containing three VHHs would be sterically disadvantaged in either binding all three epitopes on one toxin or binding distinct 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 merely additive effects. Using bacterially expressed proteins, mixtures of two VHHs from a panel of 10 species were tested, independently confirming that VHHs E3, 5D, and 7F were particularly active when mixed with each other in two-member mixtures or with several other VHHs with lower activity. Subsequent experiments using 3-fold, 4-fold, and 5-fold mixtures of the 10 VHHs found that the maximum neutralizing activity was the same for all combinations containing E3, 5D, and 7F, the simplest being the combination of these three together.

[0342] To maximize the accumulation of biologically active VHHs in Spirulina, each of the three VHHs was engineered into a hybrid construct with known solubility- or folding-optimized (chaperone) parameters. Spirulina lysates containing individual constructs containing E3, 5D, or 7F were assayed for TcdB neutralization activity, both individually (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 >1000-fold greater neutralization activity than any single VHH lysate. Complete neutralization of TcdB was observed at toxin concentrations much higher than those seen in human clinical isolates, using VHH concentrations well below those expected 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 can be present in recombinant Spirulina to further increase the efficacy of orally delivered therapeutic agents. The effectiveness of multidrug cocktails has been demonstrated for numerous organisms, including M. tuberculosis, where therapeutic agents targeting cell wall synthesis, replication and transcription, energy metabolism, and translation can be combined to target different parts of the pathogen's life cycle (Figure 49). Similarly, targeting different aspects of C. difficile receptor activation and the cell membrane can increase the efficacy of orally delivered therapeutic agents. To demonstrate this, recombinant Spirulina is produced that expresses one or more VHHs that bind to the C. difficile S-layer, one or more VHHs that neutralize toxin B, and a polypeptide such as a lysin to attack the cell membrane (see Figure 50).

[0344] Example 19 Lysin expressed from Spirulina is active PlyCD and the catalytic domain fragment PlyCD1-174 have previously been expressed in E. coli and shown to be bactericidal in vitro and in vivo. To determine whether the phage-derived anti-Clostridial cell wall-digesting lysin PlyCD 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 cytolysis assay. Figure 63 shows the cytolysis assay results for both the E. coli-expressed protein and the Spirulina-expressed protein. The lysin expressed in Spirulina is catalytically active.

[0345] Example 20 Effect of linker on neutralizing capacity of anti-TcdB VHH sequences A range of constructs were generated containing different rigid linkers linking a series of transgenes encoding anti-TcdB VHH 5D 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 the computationally designed dimer.

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

[0348] Example 21 Stability of Spirulina constructs in water and drinking fluids Recombinant Spirulina can be administered orally, and adding VHHs to drinking water will significantly increase the dose of VHHs deliverable to animals. To test the stability and activity of VHHs kept at room temperature in various buffers palatable to mice, rats, or pigs, 1 mg / mL Spirulina lysate was mixed with water, 50 mM phosphate pH 7.4, 5% sucrose, 5% nonfat milk (NFM), sucrose + phosphate, or sucrose + milk (Figure 65). Western blots were performed at 0, 1, 2, 3, and 4 hours. TcdB neutralization assays were 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 neutralization potency was observed at either the 4- or 12-hour time points in either aqueous medium (Figures 66 and 67). Similar results were obtained for the individual VHHs 5D and E3, and for the three-way synergistic combination of 5D, E3, and 7F (Figure 68).

[0350] Example 22 Study of C. difficile protection in a gnotobiotic pig model A gnotobiotic pig model was used to study the effect of Spirulina expressing anti-TcdB VHHs on protection from C. difficile challenge (Figure 70). In this study, pigs were divided into four groups as follows:

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

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

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

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

[0355] At 5 days of age, the animals were 6 Animals were infected with C. diff. UK6 BI / NAP1 / 027. Animals were treated three times daily for five days starting on day -0 post-infection. After treatment, animals were evaluated for clinical outcomes, survival, fecal spore shedding, and GIT histology.

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

[0357] Example 23 Study of the effect of prophylactic administration of anti-TcdB VHH against C. difficile infection in the Monash mouse CDI model Mice received an antibiotic cocktail in their drinking water from days -11 to -4. From days -4 to 0, mice received cefaclor alone and were infected with C. difficile on day 0. From days -1 to 4, mice received Spirulina (3x VHH mix, or 3x VHH mix + lysin), PBS, or vancomycin by oral gavage once daily. During this period, mice were monitored daily for weight, diarrhea, activity, and appearance, and feces were collected (Figure 72). Administration of the anti-TcdB VHH mix reduced 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 the 3x VHH mix + PlyCD lysin achieved 100% survival, comparable to vancomycin (Figure 73B). Finally, administration of the 3x VHH mix + lysin reduced fecal C. difficile spore shedding by >2 logs (Figure 73C).

[0358] Example 24 Effect of pH on the release of VHHs from LMN-101 Therapeutic VHHs encapsulated within Spirulina biomass were not released into gastric fluid-mimicking buffers. Bioencapsulation also prevented enzymatic degradation of VHHs under simulated gastric digestion conditions. To analyze the effect of low pH on VHH release from Spirulina biomass, dried Spirulina-VHH biomass was resuspended in buffers of different pHs. Spray-dried Spirulina-VHH biomass used for LMN-101 (strain SP1182) was resuspended at 50 mg / mL in citrate-phosphate buffers ranging from pH 3 to pH 7 and incubated at room temperature for 60 minutes with gentle agitation. The resuspended biomass was clarified by centrifugation at 14,000 RPM for 1 minute in a refrigerated microcentrifuge. The clarified extract was used in an ELISA-based binding assay using recombinant C. jejuni flagellin to determine the amount of aa682 present. High-binding ELISA plates were coated with the antigen, and SP1182 extracts were assayed as 4-fold serial dilutions in PBS supplemented with 0.05% Tween®-20 and 5% nonfat dry 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 extracts corresponded to the amount of aa682 extracted at each pH. Calculated EC50 values ​​indicated comparable 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 was reduced by 50% when Spirulina biomass was extracted with pH 4 buffer. In contrast, extracts prepared with pH 3 buffer demonstrated relatively less binding activity. The EC50 of extracts from biomass resuspended at pH 3 suggested that 40-fold less aa682 was released compared to the release in pH 7 buffer. To evaluate the effect of pH on VHH stability and activity, purified aa682 was incubated in pH 3 buffer, and the integrity of the VHH was assessed by an ELISA-based binding assay as described above. No measurable loss of binding was observed due to exposure to low pH buffers (data not shown). Table 7: EC50 for SP1182 biomass resuspended in buffers of various pH values [Table 7]

[0360] To further demonstrate that the differences in binding activity were the result of differences in VHH concentrations, clarified Spirulina extracts were also assayed using a capillary electrophoresis immunoassay. Clarified extracts were prepared as described above. VHHs were 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 in low pH stomach-like conditions, VHHs may remain encapsulated within the Spirulina biomass and be protected from the harsh environment of the stomach until they transition to the higher pH conditions of the small intestine.

[0362] Example 25 Phase 1 clinical trial of LMN-101 A phase 1 safety and tolerability study was conducted in healthy volunteers using a single Spirulina strain, LMN-101 (SP1182), engineered to express a binding protein that inhibits C. jejuni (CG8421) infection. Part A of the study involved 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 of three dose levels of LMN-101: 300 mg, 1000 mg, or 3000 mg (Figures 61 and 62). Wild-type Spirulina was used as the 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 indicated no significant systemic absorption. This indicates that spirulina can pass through the stomach and deliver VHHs to the gastrointestinal tract. Orally delivered LMN-101 was safe and well tolerated at doses up to 3000 mg 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 in simulated intestinal fluid (SIF): 50 mM citrate-phosphate buffer, pH 7.0, 164 mM NaCl, 85 mM NaHCO3, 3 mM CaCl2, and 1 mg / L pancreatin containing 10 mM porcine bile extract, incubated at 37°C. The integrity of intact anti-Campylobacter binding proteins was determined by Western blot. In two independent experiments using dried anti-Campylobacter Spirulina-VHH biomass (strain SP806) expressing trimeric VHH, it was observed that more than 80% of the binding protein was 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). The fully intact released binding protein did not accumulate to measurable levels in the simulated intestinal fluid in any case, indicating that its proteolytic cleavage rate was faster than its release rate. The detection limit in this experiment was approximately 20% recovery of released intact anti-Campylobacter binding protein. Consistent with this interpretation, purified anti-Campylobacter binding protein spiked directly into simulated intestinal fluid had a proteolytic cleavage half-life of less than 5 minutes (Figure 78).

[0364] The rapid release in the simulated intestinal environment suggests that aa682 is released in the proximal small intestine and is available to bind to Campylobacter in that environment, and the rapid degradation of aa682 suggests that detectable levels remain in the fecal content.

[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 VHHs 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 the digested Spirulina VHH biomass demonstrated that the Campylobacter binding proteins expressed within this biomass were 50% intact for 120 minutes (Figure 79). The analysis was repeated using Spirulina VHHs present in LMN-101 (strain SP1182) under otherwise identical conditions. Western blotting demonstrated that the Campylobacter binding proteins expressed within this biomass were 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 intranasal administration of a Spirulina extract containing the malaria antigen NANP would demonstrate an IgG response to NANP. Mice were further analyzed for survival of malaria infection.

[0367] Mice were immunized with PfCSP-VLPs (SP648 - a malaria vaccine based on the NANP repeat region of P. falciparum CSP fused into a virus-like particle) or empty VLPs (SP79). Mice were assigned to six groups (5 mice / group) and treated as shown in Table 8. Table 8 [Table 8] PfCSP-VLP whole biomass resuspended in PBS - oral administration (PO) PfCSP-VLP whole biomass resuspended in PBS - intranasal administration (IN) PfCSP-VLP extract - intranasal administration (IN) Empty VLP whole biomass resuspended in PBS - oral administration (PO) Empty VLP whole biomass resuspended in PBS - intranasal administration (IN) Empty VLP extract - intranasal administration (IN)

[0368] Both groups 5 and 6 underwent a repriming period on the day groups 1–4 received their boosts. Group 5 was not treated at the time groups 1–4 received their first boosts. When groups 1–4 received their second boosts, group 5 was "reprimed" with intranasal administration of PfCSP-VLP extract, followed by one oral boost of PfCSP Spirulina biomass. When groups 1–4 received their first boosts, group 6 was "reprimed" with intranasal administration of PfCSP-VLP extract, followed by two oral boosts of PfCSP Spirulina biomass. This repriming was performed to determine whether the number of boosts administered to mice affected IgG production. Group 3 received three oral boosts, group 6 received two oral boosts, and group 5 received only one 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. NANP, an antigen coated on a plate, was covered with mouse serum containing various amounts of antibodies specific to the NANP antigen, and then covered with a secondary antibody conjugated to horseradish peroxidase (HRP). As an indirect method for measuring the amount of antibodies specific to NANP present in each serum sample, a substrate was added in the presence of hydrogen peroxide. ELISA was performed using serial dilutions of serum from each animal to determine the lowest amount of serum that could still produce a positive response to the antigen.

[0371] result

[0372] The results shown in Figures 81-86 demonstrate serum IgG responses 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 is the degree of absorbance value from the plate reader. A positive response is one that approximates the amount of IgG found in the positive control hyperimmune serum. The dilution of the hyperimmune serum differs from the dilution for the experimental groups because the hyperimmune serum is very potent and does not require a larger amount to detect IgG.

[0373] The data in Figure 76 (day 14) measure the serum IgG response to a different substrate (MBP) as a control. Because the NANP protein is fused to MBP, it is important that the serum does not become reactive with MBP. Figure 76 shows that there is no IgG response to MBP at 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 respond to NANP with IgG production by day 28, but not by day 14 as seen here. In contrast, intranasal administration results in a fairly robust serum IgG response to NANP by just day 14.

[0375] As shown in Figures 81-86, serum IgG production in mice in Group 3 was more uniform than that in Group 2. This may reflect differences between administration of the extract and the resuspended biomass. The extract is a homogenous solution, while the resuspended biomass is not, and therefore, mice within a given group may receive different amounts of Spirulina.

[0376] Furthermore, mice inoculated with the extract were readily exposed to vaccine antigens, whereas mice administered Spirulina biomass may not be exposed as efficiently or uniformly. Encapsulation of vaccine antigens within Spirulina may not be an important component of intranasally administered vaccines, as opposed to orally administered vaccines, where vaccine protection is crucial for crossing the stomach.

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

[0378] Malaria Challenge

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

[0380] Some mice appeared to be protected from challenge despite having a lower detectable serum IgG response. This indicates that other factors, including other types of antibody responses, play a role in the immune response. The data presented here examine only serum IgG—mice also produce serum IgA and IgM. Furthermore, analysis of fecal samples would provide information on mucosal IgA, an indicator of a good mucosal response. However, in general, high serum IgG titers indicate protection from challenge, and indeed, since it is difficult to protect mice from malaria, the 50% protection observed in group 2 is quite good. Therefore, the demonstrated protection of up to 80% is surprising.

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

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

[0383] Embodiment 2. The non-injection delivered composition of embodiment 1, wherein the therapeutic or prophylactic molecule is delivered to the gastrointestinal tract.

[0384] Embodiment 3. The non-injection delivered composition of embodiment 1, wherein the therapeutic or prophylactic molecule is delivered systemically.

[0385] Embodiment 4. The composition for delivery without injection of any of embodiments 1 to 3, wherein the therapeutic or prophylactic molecule is an endogenous spirulina molecule.

[0386] Embodiment 5. The non-injection delivered composition of embodiment 4, wherein the endogenous Spirulina molecule is found in a concentration higher than that found in naturally occurring Spirulina.

[0387] Embodiment 6. The non-injection delivered composition of any of embodiments 1 to 3, wherein the therapeutic or prophylactic molecule is exogenous to Spirulina.

[0388] Embodiment 7. The non-injection delivered composition of embodiment 6, wherein the exogenous molecules are produced by different bacteria or plants.

[0389] Embodiment 8 The non-injection delivered composition of embodiment 7, wherein the exogenous therapeutic agent is malacidine.

[0390] Embodiment 9. The composition for delivery without injection of embodiment 6, wherein the exogenous molecule is a polypeptide or a fragment thereof.

[0391] Embodiment 10. The composition for delivery without injection of embodiment 9, wherein the exogenous polypeptide is an antibody or a fragment thereof.

[0392] Embodiment 11. The composition for delivery without injection of embodiment 10, wherein the antibody or fragment thereof is selected from the group consisting of 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, a scFv, an Fc, an Fv, a Fab, a F(ab)2, a reduced IgG (rIgG), a monospecific Fab2, a bispecific Fab2, a trispecific Fab3, a diabody, a bispecific diabody, a trispecific triabody, a minibody, an IgNAR, a V-NAR, an HcIgG, or a combination thereof.

[0393] Embodiment 12. The non-injection delivered composition of 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 peptide (GIP), immunomodulatory agents, immunosuppressants, peptide chemotherapeutic agents, antimicrobial peptides, magainin, NRc-3, NRC-7, buforin IIb, BR2, p16, Tat, TNF-alpha, and chlorotoxin.

[0394] Embodiment 13. The delivered composition of embodiment 9, wherein the exogenous polypeptide is an antigen or epitope.

[0395] Embodiment 14. The composition for delivery without injection of embodiment 13, wherein the antigen or epitope is derived from an infectious microorganism, a tumor antigen, or an autoantigen associated with an autoimmune disease.

[0396] Embodiment 15. The non-injection delivered composition of any of embodiments 1 to 14, wherein a disease or disorder is prevented, treated, or ameliorated by administering recombinant Spirulina to a subject.

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

[0398] Embodiment 17. The non-injection delivered composition of embodiment 15, wherein the infectious disease is treated, prevented, or ameliorated by administering recombinant Spirulina to a subject.

[0399] Embodiment 18. The composition delivered without injection of embodiment 17, wherein the infection is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection.

[0400] Embodiment 19. The composition delivered without injection of embodiment 18, wherein the bacteria causing the infection 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, heliobacter, Bacillus anthracis, ETEC, EHEC, EAEC, and Legionella.

[0401] Embodiment 20. The composition for delivery without injection of embodiment 18, wherein the virus causing the infection 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 syndrome virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus.

[0402] Embodiment 21. The composition delivered without injection of embodiment 18, wherein the fungus causing the infection is selected from the group consisting of Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma.

[0403] Embodiment 22. A composition delivered without injection according to embodiment 18, wherein the parasite causing the infection 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.

[0404] Embodiment 23. A composition for delivery without injection according to any of embodiments 9 to 22, wherein the exogenous polypeptide or fragment thereof is present in a fusion protein.

[0405] Embodiment 24. A composition for delivery without injection according to any of embodiments 9 to 22, wherein the recombinant Spirulina comprises a nucleic acid encoding an exogenous polypeptide or a fragment thereof.

[0406] Embodiment 25. The composition for delivery without injection of embodiment 24, wherein the nucleic acid sequence encoding at least one exogenous polypeptide or fragment thereof is present in the recombinant Spirulina in at least two copies, at least three copies, at least four copies, or at least five copies.

[0407] Embodiment 26. The composition for delivery without injection of any of embodiments 24 to 25, wherein the nucleic acid sequence encoding at least one exogenous polypeptide or fragment thereof is present in the recombinant Spirulina in 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.

[0408] Embodiment 27. The composition for delivery without injection of embodiment 25, wherein at least one exogenous polypeptide or fragment thereof is present in at least two copies, at least three copies, at least four copies, or at least five copies in a single molecule of the exogenous polypeptide expressed in the recombinant Spirulina.

[0409] Embodiment 28. The composition for delivery without injection of embodiment 25 or 27, wherein at least one exogenous polypeptide or a fragment thereof is present in 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 in a single molecule of the exogenous polypeptide expressed in the recombinant Spirulina.

[0410] Embodiment 29. A composition for delivery without injection according to any of embodiments 25 or 27-28, wherein copies of the exogenous polypeptide are linked in tandem within the molecule of the exogenous polypeptide.

[0411] Embodiment 30. A composition for delivery without injection according to any of embodiments 25 or 27-28, wherein within the molecule of the exogenous polypeptide or fragment thereof, copies of the exogenous polypeptide or fragment thereof are separated by a spacer sequence.

[0412] Embodiment 31. A composition for delivery without injection according to any of embodiments 25 to 30, wherein some copies of the exogenous polypeptide or fragment thereof are linked in tandem within the molecule of the exogenous polypeptide or fragment thereof, and the remaining copies of the exogenous polypeptide or fragment thereof are separated by spacer sequences.

[0413] Embodiment 32. The composition for delivery without injection of embodiment 30 or 31, wherein the spacer sequence is between about 1 and 50 amino acids in length.

[0414] Embodiment 33. A composition for delivery without injection according to any of embodiments 30 to 32, wherein there is more than one spacer sequence within the molecule of the exogenous polypeptide or fragment thereof.

[0415] Embodiment 34. The non-injection delivered composition of any one of embodiments 9 to 34, wherein the recombinant Spirulina comprises at least two, at least three, at least four, or at least five different exogenous polypeptides or fragments thereof.

[0416] Embodiment 35. A composition for delivery without injection according to any one of embodiments 23 to 34, wherein the fusion protein comprises a carrier protein.

[0417] Embodiment 36. The composition for delivery without injection of embodiment 35, wherein the carrier protein is selected from the group consisting of maltose binding protein, hedgehog hepatitis virus-like particle, thioredoxin, and phycocyanin.

[0418] Embodiment 37. A composition for delivery without injection according to any one of embodiments 23 to 36, wherein the fusion protein comprises a scaffold protein.

[0419] Embodiment 38. The composition for delivery without injection of embodiment 37, wherein at least one exogenous polypeptide is linked to the scaffold protein at the N-terminus or C-terminus or within the body of the scaffold protein.

[0420] Embodiment 39. The composition for delivery without injection of embodiment 37 or 38, wherein the scaffold protein is selected from the oligomerization domain of C4b binding protein (C4BP), the cholera toxin b subunit, or the oligomerization domain of an extracellular matrix protein.

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

[0422] Embodiment 41. The composition for delivery without injection of any of embodiments 37 to 40, wherein the fusion protein comprises multiple copies of at least one exogenous polypeptide or fragment thereof, and the at least one exogenous polypeptide or 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), where E is the at least one exogenous polypeptide or fragment thereof, SP is the scaffold protein, and n, n1, and n2 represent the number of copies of the at least one exogenous polypeptide or fragment thereof.

[0423] Embodiment 42. A composition for delivery without injection according to any of embodiments 9 to 42, wherein the recombinant Spirulina comprises an anti-Campylobacter VHH.

[0424] Embodiment 43. The composition delivered without injection of embodiment 42, wherein the Campylobacter is C. jejuni.

[0425] Embodiment 44. A composition for delivery without injection according to any of embodiments 42 to 43, wherein the VHH binds to a Campylobacter component.

[0426] Embodiment 45. A composition for delivery without injection according to embodiment 44, wherein the VHH binds to flagellin.

[0427] Embodiment 46. A composition delivered without injection according to any of embodiments 42 to 45, wherein administration increases Campylobacter shedding.

[0428] Embodiment 47. A composition delivered without injection according to any of embodiments 42 to 46, wherein administration reduces the level of a biomarker.

[0429] Embodiment 48. The composition for delivery without injection of embodiment 47, wherein the biomarker is an inflammatory biomarker.

[0430] Embodiment 49. A composition for delivery without injection according to any of embodiments 9 to 42, wherein the recombinant Spirulina comprises a VHH that binds to an anti-Clostridial toxin.

[0431] Embodiment 50. The composition delivered without injection of embodiment 49, wherein the Clostridium is C. difficile.

[0432] Embodiment 51. A composition delivered without injection according to any one of embodiments 48 to 49, wherein the VHH binds to Clostridium component A toxin or B toxin.

[0433] Embodiment 52. A composition for delivery without injection according to any of embodiments 49 to 51, wherein the VHH comprises an amino acid sequence of any of SEQ ID NOs: 5 to 10.

[0434] Embodiment 53. A composition for delivery without injection of any of embodiments 1 to 52, wherein the therapeutic or prophylactic molecule is a monomer.

[0435] Embodiment 54. A composition for delivery without injection of any of embodiments 1 to 52, wherein the therapeutic or prophylactic molecule is a multimer.

[0436] Embodiment 55. The composition for delivery without injection of embodiment 54, wherein the therapeutic or prophylactic molecule is a trimer.

[0437] Embodiment 56. A composition for delivery without injection according to any of embodiments 54 to 55, wherein the multimer is a heteromer.

[0438] Embodiment 57. A composition for delivery without injection according to any of embodiments 54 to 55, wherein the multimer is a homomer.

[0439] Embodiment 58. A composition for delivery without injection according to any of embodiments 54 to 57, wherein the multimers are disposed in nanoparticles.

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

[0441] Embodiment 60. The composition for delivery without injection of embodiment 59, wherein the binding affinity of the multimer is greater than the binding affinity of the monomer or dimer.

[0442] Embodiment 61. EC of multimers greater than 5 μg / mL 50 61. The composition for delivery without injection of embodiment 60, comprising:

[0443] Embodiment 62. EC of multimers greater than 10 μg / mL 50 62. The composition for delivery without injection of embodiment 61, comprising:

[0444] Embodiment 63. The EC of the multimer is about 5 μg / mL to about 40 μg / mL. 50 62. The orally delivered composition of embodiment 61, comprising:

[0445] Embodiment 64. The composition delivered without injection of any of embodiments 59 to 63, wherein the binding affinity of the multimer is greater than the binding affinity of a multimer comprising fewer copies of the exogenous therapeutic agent or a combination of fewer copies of the exogenous therapeutic agent.

[0446] Embodiment 65. The non-injection delivered composition of any 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. amethystine, A. ardissonei, A. argentina, A. balkrishnanii, A. baryana, A. boryana, A. braunii, A. breviarti culata, 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. A composition for delivery without injection according to any one of embodiments 1 to 66, wherein the recombinant Spirulina is non-viable.

[0449] Embodiment 68. The non-injection delivery composition of 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 comprises a pharmaceutically acceptable excipient.

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

[0452] Embodiment 71. The composition delivered without injection of embodiment 70, which remains in the gastrointestinal tract or simulated gastric environment for at least 5 minutes.

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

[0454] Embodiment 73. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a non-injection delivered composition of any one of embodiments 1 to 72 or 87 to 92.

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

[0456] Embodiment 75. The method of embodiment 74, wherein the infection is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection.

[0457] Embodiment 76. The method of embodiment 75, wherein the bacteria causing the infection 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.

[0458] Embodiment 77. The method of embodiment 75, wherein the virus causing the infection is selected from the group consisting of bacteriophages, RNA bacteriophages (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 syndrome virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus.

[0459] Embodiment 78. The method of embodiment 75, wherein the fungus causing the infection 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 infection 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.

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

[0462] Embodiment 81. A method of treating or preventing a Campylobacter infection, comprising administering to a subject a non-injection delivered composition of any of embodiments 1 to 72.

[0463] Embodiment 82. The method of embodiment 81, wherein administering a composition delivered without injection reduces or prevents the onset of Campylobacter symptoms.

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

[0465] Embodiment 83. A method for treating or preventing a C. difficile infection, comprising administering to a subject a composition delivered without injection described in any of embodiments 1 to 72.

[0466] Embodiment 84. The method of embodiment 83, wherein administering a composition delivered without injection reduces or prevents the onset of symptoms due to C. difficile.

[0467] Embodiment 85. The method of any one of embodiments 81 to 84, wherein administering a composition that is delivered without injection reduces or prevents the occurrence of diarrhea in the subject.

[0468] Embodiment 86. A composition or method for delivery without injection of any one of embodiments 1 to 85, wherein the therapeutic or prophylactic molecule is not an antigen or epitope.

[0469] Embodiment 88. A non-injection delivered composition or method according to any of the preceding embodiments, wherein a synergistic effect is achieved by administering two or more different recombinant Spirulina comprising different exogenous polypeptides or antigens or fragments thereof.

[0470] Embodiment 89. The composition or method for non-injection delivery of embodiment 88, wherein each of the different recombinant Spirulina administered comprises a different VHH.

[0471] Embodiment 90. A non-injection delivered composition or method according to any of the preceding embodiments, wherein a synergistic effect is achieved by administering recombinant Spirulina comprising a different exogenous polypeptide or antigen or fragment thereof.

[0472] Embodiment 91. A composition or method for delivery without injection according to embodiment 90, wherein the recombinant Spirulina comprises two or more different VHH sequences.

[0473] Embodiment 92. A composition or method for delivery without injection according to any of embodiments 88 to 92, wherein the recombinant Spirulina comprises a lysin.

[0474] Embodiment 93. A composition or method for delivery without injection according to any of embodiments 88 to 92, wherein the recombinant Spirulina comprises a lysin and an exogenous polypeptide.

[0475] Embodiment 94. A composition or method for delivery without injection according to embodiment 94, wherein the recombinant Spirulina comprises a lysin and a VHH.

[0476] Embodiment 95. A composition or method for delivery without injection according to any of the previous embodiments, wherein the composition is administered orally.

[0477] Embodiment 96. A non-injection delivered composition or method according to any of the previous embodiments, wherein the composition is delivered to the respiratory tract.

[0478] Embodiment 97. The composition or method for delivery without injection described in embodiment 88, wherein the composition is delivered by inhalation or intranasally.

[0479] Embodiment 98. A composition or method delivered without injection according to any of the previous embodiments, wherein the composition is Spirulina biomass.

[0480] Embodiment 99. A composition or method for delivery without injection according to any of the previous embodiments, wherein the composition is delivered as an extract of Spirulina biomass.

[0481] Embodiment 100. A composition or method for delivery without injection according to any of the previous embodiments, wherein the composition is delivered as a purified composition obtained from Spirulina biomass. 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 and for all purposes the following patent publications and applications: US 10,131,870, US 62 / 672,891 filed May 17, 2018, and PCT / US2019 / 032998 filed May 17, 2019.

[0483] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. The present invention provides, for example, the following items. (Item 1) A composition for delivery without injection comprising recombinant Spirulina, wherein said recombinant Spirulina comprises at least one therapeutic or prophylactic molecule. (Item 2) 10. The orally delivered composition of claim 1, wherein the therapeutic or prophylactic molecule is delivered to the gastrointestinal tract, respiratory tract, or nasal cavity. (Item 3) 3. The non-injection delivery composition of claim 1 or 2, wherein the therapeutic or prophylactic molecule is exogenous to Spirulina. (Item 4) 4. The non-injection delivery composition according to item 3, wherein the exogenous molecule is a polypeptide or a fragment thereof. (Item 5) 5. The non-injection delivered composition according to item 3 or 4, wherein the exogenous polypeptide is an antibody or a fragment thereof. (Item 6) 6. The composition for non-injection delivery according to item 5, wherein the antibody or fragment thereof is a VHH. (Item 7) 4. The non-injection delivery composition according to item 3, wherein the exogenous polypeptide is an antigen or epitope. (Item 8) 8. The non-injection delivery composition according to any one of items 1 to 7, wherein the recombinant Spirulina is administered to a subject to prevent, treat, or ameliorate a disease or disorder. (Item 9) 9. The non-injection-delivered composition of any one of items 1 to 8, wherein the recombinant Spirulina is administered to a subject to treat, prevent, or ameliorate an infectious disease. (Item 10) 10. The non-injection delivered composition of item 9, wherein the infection is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection. (Item 11) 11. The non-injectionable composition of claim 10, wherein the bacteria causing the infection 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) 11. The composition for delivery without injection of item 10, wherein the virus causing the infection 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 syndrome virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus. (Item 13) 11. The non-injection delivered composition of claim 10, wherein the fungus causing the infection is selected from the group consisting of Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma. (Item 14) 11. The composition for delivery without injection according to item 10, wherein the parasite causing the infection 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. (Item 14) 10. The non-injection delivered composition of any preceding item, wherein the exogenous polypeptide or fragment thereof is present in a fusion protein. (Item 15) The non-injection-delivered composition of any preceding item, wherein the recombinant Spirulina comprises a nucleic acid encoding the exogenous polypeptide or a fragment thereof. (Item 16) 2. The non-injection-delivered composition of any preceding item, wherein the recombinant Spirulina comprises an anti-Campylobacter VHH. (Item 17) 17. The non-injection-delivered composition of claim 16, wherein the Campylobacter is C. jejuni. (Item 18) 18. The composition for non-injection delivery according to item 16 or 17, wherein said VHH binds to a Campylobacter component. (Item 19) 19. The non-injectionable composition of item 18, wherein the VHH binds to flagellin. (Item 20) 20. The non-injection-delivered composition of any of items 16 to 19, wherein administration increases Campylobacter shedding. (Item 21) 21. The non-injection delivered composition of any of items 16 to 20, wherein administration reduces the level of a biomarker. (Item 22) 22. The non-injection delivered composition of claim 21, wherein the biomarker is an inflammatory biomarker. (Item 23) 2. The non-injection delivery composition of any preceding item, wherein the recombinant Spirulina comprises a VHH that binds to an anti-Clostridial toxin. (Item 24) 24. The non-injection-delivered composition according to item 23, wherein the Clostridium is C. difficile. (Item 25) 25. The composition for delivery without injection according to any one of items 23 to 24, wherein said VHH binds to Clostridium component A toxin or B toxin. (Item 26) 26. The composition for non-injection delivery according to any of items 23 to 25, wherein the VHH comprises an amino acid sequence of any of SEQ ID NOs: 5 to 17. (Item 27) 10. The non-injection delivered composition of any preceding item, wherein the therapeutic or prophylactic agent molecule is a monomer. (Item 28) 27. The non-injectionable composition of any of items 1 to 26, wherein the therapeutic or prophylactic molecule is a multimer. (Item 29) 29. The non-injectionable composition of claim 28, wherein the multimer is a heteromer. (Item 30) 30. The non-injectionable composition of claim 29, wherein the multimer is a homomer. (Item 31) 60. The non-injectionable composition of item 59, wherein the binding affinity of the multimer is greater than the binding affinity of the monomer or dimer. (Item 32) 32. The non-injection-delivered composition of any of items 28 to 31, wherein the binding affinity of the multimer is greater than the binding affinity of a multimer comprising fewer copies of the exogenous therapeutic agent or a combination of fewer copies of the exogenous therapeutic agent. (Item 33) 10. The non-injectionable composition of any preceding item, further comprising a lysin. (Item 34) The recombinant Spirulina is A.amethystine, A.ardissonei, A.argentina, A.balkrishnanii, A.baryana, A.boryana, A.braunii, A.breviarticu. lata, 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 non-injection-delivered composition of any preceding item, wherein the recombinant Spirulina is non-viable. (Item 36) The non-injection delivery composition of any preceding item, wherein the recombinant Spirulina is dried, spray-dried, freeze-dried, or lyophilized. (Item 37) 2. The non-injection delivery composition of any preceding item, wherein the recombinant Spirulina is delivered as an extract. (Item 38) 2. The non-injection delivery composition of any preceding item, wherein the recombinant Spirulina is administered orally. (Item 39) The non-injection delivery composition of any preceding item, wherein the recombinant Spirulina is administered to the respiratory tract. (Item 40) 40. The non-injection delivery composition of item 39, wherein the recombinant Spirulina is administered intranasally. (Item 41) 1. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject the non-injection delivered composition of any one of the preceding items. (Item 42) 42. The method of item 41, wherein the disease or disorder is an infectious disease. (Item 43) 43. The method of claim 42, wherein the infection is a bacterial infection, a viral infection, a fungal infection, or a parasitic infection. (Item 44) 44. The method of item 43, wherein the bacteria causing the infection 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) 44. The method of claim 43, wherein the virus causing the infection 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 syndrome virus, coronavirus, MERS virus, SARS virus, and SARS-CoV-2 virus. (Item 46) 44. The method of claim 43, wherein the fungus that causes the infection is selected from the group consisting of Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma. (Item 47) Item 44. The method according to item 43, wherein the parasite causing the infection 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

1. A composition for oral delivery comprising three recombinant Spirulina, each of the three recombinant Spirulina expressing a different VHH molecule or antigen-binding fragment thereof that binds to C. difficile toxin, wherein the VHH molecule or antigen-binding fragment thereof is a) amino acid residues 26-32 (CDR1), 52-57 (CDR2), and 98-116 (CDR3) of SEQ ID NO:5; b) amino acid residues 26-32 (CDR1), 52-56 (CDR2), and 98-100 (CDR3) of SEQ ID NO:6; and c) amino acid residues 26-32 (CDR1), 52-56 (CDR2), and 98-105 (CDR3) of SEQ ID NO: 13 A composition comprising:

2. The composition of claim 1 , wherein the oral delivery is delivery to the gastrointestinal tract.

3. The composition of claim 1 or claim 2, wherein the VHH molecule or its antigen-binding fragment is present in a fusion protein.

4. The VHH molecule or antigen-binding fragment thereof further comprises: a) an amino acid sequence consisting of amino acid residues 2 to 126 of SEQ ID NO:5; b) an amino acid sequence consisting of amino acid residues 2 to 110 of SEQ ID NO:6; and c) an amino acid sequence consisting of amino acid residues 2 to 115 of SEQ ID NO: 13 The composition according to any one of claims 1 to 3, comprising:

5. The composition of claim 1 , wherein at least one of the VHH molecules or antigen-binding fragments thereof is fused to maltose binding protein (MBP).

6. The composition of any one of claims 1 to 5, further comprising a lysin, wherein at least one of the VHH molecules or antigen-binding fragments thereof is fused to a maltose binding protein (MBP).

7. The composition of claim 1 further comprising a lysin.

8. The recombinant Spirulina is 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. giantean, 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. setcellii, A. skujae, A. spirulinoides f. tenuis, A. spirulinoides, A. subsalsa, A. subtilissima, A.

8. The composition of any of claims 1 to 7, wherein the antibacterial agent is selected from the group consisting of A. tenuis, A. tenuissima, and A. versicolor.

9. 9. The composition of any one of claims 1 to 8, wherein the recombinant Spirulina is non-viable.

10. 10. The composition of any of claims 1 to 9, wherein the recombinant Spirulina is dried, spray dried, freeze dried, or lyophilized.

11. 11. The composition of any of claims 1 to 10, wherein the recombinant Spirulina is spray dried.

12. The composition of claim 6, 7, or any one of claims 8 to 11 dependent on claim 6 or 7, wherein the lysin is PlyCD.

13. The composition of claim 12, wherein the lysin is PlyCD1-174.

14. An orally delivered composition comprising: i) three non-viable recombinant Spirulina, each of the three non-viable recombinant Spirulina expressing a different VHH molecule or antigen-binding fragment thereof that binds to C. difficile toxin, wherein the VHH molecule or antigen-binding fragment thereof is a) amino acid residues 26-32 (CDR1), 52-57 (CDR2), and 98-116 (CDR3) of SEQ ID NO:5; b) amino acid residues 26-32 (CDR1), 52-56 (CDR2), and 98-100 (CDR3) of SEQ ID NO:6; and c) amino acid residues 26-32 (CDR1), 52-56 (CDR2), and 98-105 (CDR3) of SEQ ID NO: 13; Three non-viable recombinant Spirulina, including: ii) lysin and A composition comprising:

15. The VHH molecule or antigen-binding fragment thereof further comprises: a) an amino acid sequence consisting of amino acid residues 2 to 126 of SEQ ID NO:5; b) an amino acid sequence consisting of amino acid residues 2 to 110 of SEQ ID NO:6; and c) an amino acid sequence consisting of amino acid residues 2 to 115 of SEQ ID NO: 13 15. The composition of claim 14, comprising:

16. A composition according to any preceding claim for use in a method of treating an infection in a subject.

17. 17. The composition of claim 16, wherein the infection is a C. difficile infection.

18. 17. The composition of claim 16, wherein administration of the composition reduces or prevents the onset of symptoms due to C. difficile.

19. 15. The composition of claim 14 for use in a method of treating an infection in a subject.

20. 20. The composition of claim 19, wherein the infection is a C. difficile infection.

21. 20. The composition of claim 19, wherein administration of the composition reduces or prevents the onset of symptoms due to C. difficile.

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